Abstract
Y. D. Teng*†‡
*Department of Neurosurgery, Harvard Medical School, Boston, MA, USA
†Department of Physical Medicine and Rehabilitation, Harvard Medical School, Boston, MA, USA
†Division of SCI Research, Veterans Affairs Boston Healthcare System, Boston, MA, USA
Based on the pluripotency or multipotency of developmental cells, it was initially hypothesized that the biology of neural stem cells (NSCs) makes them ideally and uniquely suited to reconstructing the damaged central nervous system (CNS) through cell replacement. Emerging evidence, however, increasingly suggests that stem cells may repair the CNS through multimechanistic strategies that are often concurrent (i.e., functional multipotency of stem cells). They may serve not only as tissue engineering units of cellular reconstitution but also as vectors for the delivery of molecules. Analyzing results of our recent studies in which biodegradable polymer seeded with human NSCs (hNSCs) or mesenchymal stromal cells (hMSCs) was applied for both investigative and therapeutic purposes, I propose to first discuss how retrievable and drug-releasing polymer implants containing stem cells may hold significant promise for providing a broad range of insight regarding essential neurological mechanisms required for repairing the adult mammalian spinal cord after injury. I will present data elucidating molecular events underlying rapid loss of donor cells in acutely injured spinal cord and how counteracting strategies proved effective in rodent and nonhuman primate models of spinal cord injury (SCI). Additionally, data obtained by adopting similar stem cell biology plus genetic reduction of reactive gliosis will be examined for understanding the role of distal spinal cord adaptation in the process of invoking neuroplasticity and functional recovery post-SCI and the critical etiology of motor neuron diseases. In the summary note, I will navigate three subcortical circuits that are essential for defining the “recovery neurobiology” of the adult mammalian spinal cord, using outcomes from a newly completed study in which locomotion is restored by a peripheral nerve-mediated sensory rebuild. Our findings may provide a multimodal approach to help formulate therapeutic strategies achieving clinically meaningful improvements for traumatic SCI and other neurodegenerative conditions.
J. Silver
Department of Neurosciences, School of Medicine, Case Western Reserve University, Cleveland, OH, USA
The ultimate goal of the Silver lab is to understand the basic biology that underlies regeneration failure in the adult spinal cord and then use this knowledge to develop strategies to maximally overcome the lack of regeneration after cord injury in order to promote functional repair. Although highly controversial from its inception, the Silver lab was one of the very first to suggest that overtly growth-repulsive environments, whose function was to actively turn axons away from improper trajectories during embryogenesis, might reappear in the injured central nervous system (CNS) and block the attempt of severed axons to regrow. One of the most interesting families of inhibitory extracellular matrix molecules, the lectican family of sulfated proteoglycans and, in particular, the chondroitin sulfate proteoglycans (CSPGs), were first discovered by the Silver lab in the early 1990s to be involved in creating such developmental as well as regenerative glial boundaries. But why did axons sometimes turn away from but other times become entrapped within substrates occupied by proteoglycans? In 2009 and 2011, nearly two decades after CSPGs had first been implicated in regeneration failure, his lab was a major collaborator in the discovery of the first neuronal receptors for CSPGs that bind specifically to the sugar chains of proteoglycans and mediate this newly appreciated entrapment phenomenon. Indeed, two members of the prosynaptic, leukocyte antigen- related (LAR) family of receptor protein tyrosine phosphatases (RPTPs), which upregulate in growth cones when they encounter proteoglycans are the major receptors on neurons that are involved with the overly adhesive properties of this family of extracellular matrix (ECM) inhibitors. The lab has now generated small peptides, administered simply by subcutaneous injection, that block these receptors on damaged neurons in the lesioned spinal cord. There is great excitement in the lab that the peptides are even more highly effective than chondroitinase in releasing axons from proteoglycan-mediated entrapment. Behavioral recovery, particularly after administration of the peptide that blocks the sigma (RPTPσ) member of this receptor family, is especially impressive. Indeed, this novel, easily injectable, small peptide inhibitor allows unprecedented levels of functional recovery following spinal cord injury, presenting a potential new avenue of noninvasive treatment for paralysis. Over the years, many other labs around the world have substantiated Dr. Silver's pioneering ideas and are showing, with the use of a variety of proteoglycan- or proteoglycan receptor-modifying techniques, coupled with various other strategies for enhancing the intrinsic growth response of neurons, that they can foster a good measure of functional plasticity as well as frank regeneration in a wide variety of CNS injury models.
R. Zorec*†
*Celica Biomedical Center, Ljubljana, Slovenia
†Laboratory of Neuroendocrinology–Molecular Cell Physiology, Institute of Pathophysiology, Faculty of Medicine, University of Ljubljana, Ljubljana, Slovenia
In this presentation, Professor Zorec will outline the different vesicle properties of astrocytes and how movement of their vesicles can be regulated in physiologic and pathologic conditions. He will also discuss how vesicle traffic is the target of new medicines, such as Fingolimod, a novel drug to treat remitting and relapsing multiple sclerosis, modulating Ca2+ homeostasis, and how astrocyte swelling in cytotoxic edema can be controlled.
Astrocytes are an abundant type of glial cells in the brain. They form tight connections with synapses (tripartite synapse), contain glycogen, and provide for homeostasis in the central nervous system (CNS). Astrocytes contain vesicles, whose content is released into extracellular fluid by regulated exocytosis. Exocytosis in astrocytes is slower than in neurons, suggesting that astrocytes play an important role in the slow processes in the brain, making them ideal signal integrators. One of the many steps involved in exocytosis is delivery of the vesicle to plasma membrane sites, where vesicles dock and fuse with the plasma membrane. This creates an aqueous channel called a “fusion pore,” which connects the vesicle's lumen to the extracellular space and determines gliotransmitter secretion, which modulates synaptic transmission in normal and pathological conditions. Therefore, astrocytes are no longer considered subservient to neurons, but are now known to be active partners in cell-to-cell communication. Their involvement in this communication is a focus of new research into new drugs to treat a range of neurologic diseases, including trauma, Alzheimer's disease, multiple sclerosis, neuroinflammation, intelligence deficiency, neurodegeneration, and autism.
S. A. Acosta,* N. Tajiri,* K. Shinozuka,* H. Ishikawa,* S. Song,†‡§ P. R. Sanberg,*¶ J. Sanchez-Ramos,†‡§ Y. Kaneko,* and C. V. Borlongan*
*Center of Excellence for Aging and Brain Repair, Department of Neurosurgery and Brain Repair, University of South Florida Morsani College of Medicine, Tampa, FL, USA
†James Haley Veterans Affairs Medical Center, Tampa, FL, USA
†Department of Neurology, University of South Florida Morsani College of Medicine, Tampa, FL, USA
§Department of Molecular Pharmacology and Physiology, University of South Florida Morsani College of Medicine, Tampa, FL, USA
¶Office of Research and Innovation, University of South Florida, Tampa, FL, USA
Traumatic brain injury (TBI) is associated with neuroinflammation, and sensory–motor, learning and memory impairments. Cell-based therapies are a promising intervention to regulate the hostile milieu typically present in neurotrauma. In tandem, the stimulation/mobilization of endogenous stem/progenitor cells from the bone marrow through granulocyte colony-stimulating factor (G-CSF) poses as an attractive intervention for chronic TBI. In the present in vivo study, we tested the potential of a combined therapy of human umbilical cord blood cells (hUCB) and G-CSF to ameliorate the progressive secondary effects of chronic TBI. Four groups of rats were treated with saline alone, G-CSF + saline, hUCB + saline, or hUCB + G-CSF at acute stages, 7 days post-TBI. Eight weeks after TBI, rats were euthanized and the brain harvested for histopathological analysis. Hippocampal cell loss, neuro inflammatory response, and neurogenesis were analyzed using immunohistochemistry along with unbiased stereology techniques. Results revealed that rats exposed to chronic TBI + saline exhibited widespread neuroinflammation, impaired endogenous neurogenesis in the dentate gyrus and subventricular zone, and severe hippocampal cell loss. hUCB mono therapy suppressed neuroinflammation, nearly normalized the neurogenesis, and reduced hippocampal cell loss compared to saline alone. G-CSF monotherapy produced partial/short-lived benefits characterized by low levels of neuroinflammation, a modest neurogenesis, and a moderate reduction of hippocampal cell loss. On the other hand, combined therapy of hUCB + G-CSF displayed synergistic effects that robustly dampened neuroinflammation, while enhancing endogenous neurogenesis and reducing hippocampal cell loss. These results suggest that combined treatment rather than monotherapy appears optimal for abrogating histopathological and motor impairments in chronic TBI.
Research supported by Department of Defense W81XWH-11–1–0634.
Z. Aljuboori,*†1 X. Zeng,*†1 I. Han,*†‡ A. E. Ropper,*† D. Yu,*† J. E. Anderson,*† E. Ifedigbo,§# A. M. K. Choi,§# and Y. D. Teng*†¶
*Department of Neurosurgery, Harvard Medical School/Brigham and Women's Hospital Boston, MA, USA
†Division of Spinal Cord Injury Research, VA Boston Healthcare System, Boston, MA, USA
†Department of Neurosurgery, Cha University, Gyeonggi-do, Republic of Korea
§Division of Pulmonary and Critical Care Medicine, Harvard Medical School/Brigham and Women's Hospital Boston, MA, USA
¶Department of PM&R, Harvard Medical School/Spaulding Rehabilitation Hospital, Boston, MA, USA
1These authors provided equal contribution to this work.
#Present address: Department of Medicine, Weill Cornell Medical Center, New York, NY, USA
Previously, we demonstrated that carbon monoxide (CO) inhalation dose-dependently spares epicenter motor neurons (MNs) and white matter in a rat model of compression spinal cord injury (SCI), resulting in significantly improved hindlimb function largely through impeding secondary inflammatory events (Han IB et al., SfN Press Conference 2012). Since spinal cord interneurons (INs) play important roles in locomotor pattern generation (LPG) by relaying and coordinating supraspinal and peripheral neural signals to enable pace making, we reasoned that CO treatment might also protect INs and tested our hypothesis by investigating whether there was any effect of 500 ppm CO ventilatory exposure, the most potent dose identified, on sparing INs at different levels of the spinal cord compared with controls that received room air (RA) inhalation in a replication study. Moderate compression injury was produced by static loading of 35 g weight at T9–10 for 5 min. Four groups of conscious rats were randomly housed inside whole body ventilation chambers that were supplied with 100, 250, and 500 ppm CO or control RA (n = 7/group), respectively. The rats first underwent 2 h CO or RA treatment ~4 h post-SCI and, thereafter, 1 h inhalation therapy daily for 12 consecutive days. Locomotor function recovery was evaluated using the Basso, Beattie, and Bresnahan (BBB) scale: spinal cord tissue samples were analyzed for measurement of lesion volume, white matter sparing, and MN and IN survival. Our neural counting outcomes are based on established methods of digital camera lucida mapping of Rexed laminae and immunocytochemistry (ICC). We found that SCI rats treated with 500 ppm CO inhalation demonstrated significant sparing of interneurons (p < 0.05, Student t test; n = 7/group) that parallel with the markedly improved hindlimb locomotor recovery (p < 0.05, repeated measure ANOVA) and reduced inflammatory responses, compared to control treatment of room air ventilation. Furthermore, the CO treatment significantly mitigated loss of INs around the lesion epicenter, relative to the RA control group. Our ongoing quantitative analyses of both inhibitory and excitatory INs as determined by ICC of glutamate decarboxylase 67 (GAD67) and choline acetyltransferase (ChAT), respectively, plus mitogen-activated protein kinase (MAPK) signaling factors will provide data on their correlative contribution to the therapeutic benefits of CO. The results suggest that INs surviving around T9–10 levels may also play a critical role in CO-triggered locomotion recovery in rats post SCI, and INs may be an effective therapeutic target for developing neural therapies for SCI.
Sponsored by CIMIT under US Army Medical Research Acquisition Activity Cooperative Agreement DAMD-17–02–2–0006 and VA RR&D.
A. E.-E. Aly,* B. T. Harmon,* K. Dines,† O. Sesenoglu-Laird,† L. Padegimas,† M. J. Cooper,† and B. L. Waszczak*
*Department of Pharmaceutical Sciences, Northeastern University, Boston, MA, USA
†Copernicus Therapeutics, Inc., Cleveland, OH, USA
Glial cell line-derived neurotrophic factor (GDNF) gene therapy is a promising therapeutic approach for Parkinson's disease (PD) since it could provide a renewable source of GDNF within the brain. However, translating this potential into clinical reality will require developing a safe, effective, noninvasive means of delivery to the brain. Current approaches rely on viral vectors to carry the gene into cells, and most require direct intracranial injection. The immunogenicity of viral vectors and the invasiveness of surgical intervention are serious limitations. We are investigating the intranasal route of administration of polyethylene glycol (PEG)-ylated polylysine (PEG-CK30) DNA nanoparticles (NPs) encoding GDNF. These NPs, developed by Copernicus Therapeutics, Inc., compact a single molecule of expression plasmid, have a minimum size of 10 nm, and provide a nonimmunogenic, nonviral vector for central nervous system gene therapy. We have shown that DNA NPs expressing either enhanced green fluorescent protein (eGFP) alone (pCG), or eGFP linked with hGDNF (pUGG), transfect cells in vitro and brain cells in vivo.
The first goal of this study was to assess transfection in the brain after intranasal administration of Copernicus' hGDNF DNA NPs in rats. GDNF expression was determined by ELISA 7 days posttreatment in a series of rostral–caudal brain slabs. Results showed significant increases in GDNF expression throughout the brain of rats given intranasal naked pGDNF or pGDNF NPs relative to controls. To determine which cell type(s) were transfected, double-label immunohistochemistry (IHC) was done on brain sections from rats given intranasal pUGG NPs. Most of the eGFP-positive cells observed at 7 days posttreatment were adjacent and abluminal to cell staining for rat endothelial cell antibody (RECA-1), suggesting preferential transfection of pericytes. The second goal of this study was to test whether intranasal delivery of pGDNF could protect dopamine cells in the rat 6-hydroxydopamine (6-OHDA) model of PD. Rats were given intranasal saline, naked pGDNF, or pGDNF NPs 7 days prior to receiving a unilateral 6-OHDA lesion. Three weeks later, rotational behavior to 5 mg/kg d-amphetamine was assessed, followed by sacrifice. Ipsilateral rotation was significantly reduced in rats treated with both naked pGDNF and pGDNF NPs. In addition, tyrosine hydroxylase (TH) IHC revealed significant increases in dopamine cell counts and increased TH staining density in the substantia nigra and striatum on the 6-OHDA-lesioned side of pGDNF-treated rats. For all measures, pGDNF NPs provided greater neuroprotection than naked pGDNF.
Collectively, these results demonstrate that intranasal administration of Copernicus' hGDNF expression plasmid leads to transfection and expression of the encoded protein in rat brain and significant protection of dopamine neurons in the 6-OHDA model. This approach offers a new, noninvasive, and nonviral strategy for arresting disease progression in early stage PD.
Supported by the Michael J. Fox Foundation and a Tier 1 Grant from Northeastern University.
A. Antoine,* M. Bastawrous,* S. A. Acosta,* M. M. Pabon,* D. G. Hernandez-Ontiveros,* N. Tajiri,* P. R. Sanberg,*† Y. Kaneko,* and C. V. Borlongan*
*Center of Excellence for Aging and Brain Repair, Department of Neurosurgery and Brain Repair, University of South Florida Morsani College of Medicine, Tampa, Florida, USA
†Office of Research and Innovation, University of South Florida, Tampa, Florida, USA
The development of sensory–motor and cognitive diseases is considered one of the long-term consequences of traumatic brain injury (TBI). Notably, Parkinson's disease (PD), which is characterized by a gradual degeneration of the nigrostriatal dopaminergic neurons, is one of the age-related diseases that long-term TBI survivors are predisposed to. However, preclinical studies on the pathophysiological changes in substantia nigra (SN) after chronic TBI are lacking. In the present in vivo study, we examined the pathological link between PD-associated dopaminergic neuronal loss and chronic TBI.
Brain tissues of rats were harvested 60 days post-TBI. Stereology was performed on brain sections immunostained with tyrosine hydroxylase (TH), an enzyme required for the synthesis of dopamine in neurons, and a synuclein, a presynaptic protein that plays a role in synaptic vesicle recycling, both key players in PD pathology.
There was a significant decrease in the TH-positive expression in the surviving dopaminergic neurons of the SN pars compacta relative to sham control. Also, significant increments of expression of aggregated a synuclein were detected in the ipsilateral SN compared with the contralateral SN in TBI animals compared to the sham control.
Chronic TBI reduces the TH-positive cell survival in the SN pars compacta accompanied by a synuclein overexpression, thus resulting in a PD-like pathology in chronic TBI, which can be an indication of the long-term damage seen in victims of TBI.
N. Ashki,* A. Chan,* Y. Qin,* W. Wang,* M. Kiyohara,† L. Lin,† J. Braun,†‡ M. Wadehra,† and L. K. Gordon*
*Department of Ophthalmology, Jules Stein Eye Institute, Los Angeles, CA, USA
†Department of Pathology and Laboratory Medicine, David Geffen School of Medicine at UCLA, Los Angeles, CA, USA
†Department of Molecular and Medical Pharmacology, David Geffen School of Medicine at UCLA, Los Angeles, CA, USA
Corneal neovascularization (NV) is a sight-threatening condition often associated with infection, inflammation, prolonged contact lens use, corneal burns, and acute corneal graft rejection. Macrophages recruited to the cornea release nitric oxide (NO) and superoxide anion (O2-), which react together to form the highly toxic molecule peroxynitrite (ONOO-). The role of ONOO- in upregulating multiple angiogenic factors in cultured human corneal limbal epithelial (HCLE) cells was investigated.
HCLE cells were incubated with 500 μM of ONOO- donor for various times. Vascular endothelial growth factor-A (VEGF-A), basic fibroblast growth factor (bFGF), and hypoxia-inducible factor-1α (HIF-1α) were investigated via Western blot, and RT-PCR was performed for VEGF. Functional assays using human umbilical vein endothelial cells (HUVECs) used conditioned media from ONOO- exposed HCLE cells. Secreted VEGF in the conditioned media was detected and analyzed using an enzyme-linked immunosorbent assay (ELISA).
Increased angiogenic factors were observed as early as 4 h following HCLE cell exposure to ONOO-. HIF-1α expression was seen at 4, 6, and 8 h post-ONOO- exposure (p < 0.05). bFGF expression was elevated at 4 h and peaked at 8 h following treatment with ONOO- (p < 0.005). Increased VEGF-A gene expression was observed at 6 and 8 h post-ONOO- treatment. Functional assays using conditioned media showed increased HUVEC migration and tube formation.
Exposure of HCLE cells to ONOO- augments production of multiple angiogenic factors. Therefore, exposure to ONOO- could be one contributor to cause corneal neovascularization.
Future studies will focus on validating the role of ONOO- in pathologic corneal neovascularization in vivo, investigating potential neural involvement in such processes, and identifying the mechanisms through which reactive nitrogen/oxygen species (RNS/ROS) regulate HIF-1α expression.
M. J. Benskey,* N. C. Kuhn,* S. Mishra,* N. M. Kanaan,* C. E. Sortwell,* C. Jiang,† and F. P. Manfredsson*
*Department of Translational Science and Molecular Medicine, Michigan State University College of Human Medicine, Grand Rapids, MI, USA
†Center for Proteomics and Systems Biology, University of Texas, Health Science Center, Houston, TX, USA
Parkinson disease (PD) is characterized by the progressive loss of midbrain nigrostriatal dopaminergic (DAergic) neurons and the presence of proteinacious inclusions known as Lewy bodies. Lewy bodies are highly enriched in the protein α-synuclein (α-syn). In addition, mutations or multiplications of the gene encoding α-syn result in familial forms of PD. Thus, α-syn has been proposed to directly contribute to DAergic cell loss in PD. The predominant theory posits that α-syn- mediated PD pathology arises due to a toxic gain of function. Accordingly, current therapeutic strategies are centered on eliminating the protein from DAergic neurons. In contrast, we hypothesize that α-syn is not the primary toxic species in PD; rather, α-syn aggregation produces pathology by effectively decreasing the pool of soluble α-syn available to the cell. To test the hypothesis that loss of functional α-syn leads to DAergic degeneration, adult male rats received unilateral stereotaxic injections of recombinant adeno-associated virus (rAAV) expressing short hairpin RNA (shRNA) directed toward α-syn or a scrambled control shRNA into the substantia nigra pars compacta (SNpc). Administration of α-syn shRNA resulted in loss of tyrosine hydroxylase immunoreactive (THir) cells in the SNpc, whereas the scrambled shRNA had no effect. The loss of THir cells in the SNpc following α-syn shRNA was found to be both dose and time dependent, occurring as early as 14 days postinjection. To further confirm that loss of α-syn produces dopaminergic neurodegeneration, the ability of wild-type human α synuclein (WT α-syn) or a nonaggregatable mutant form of α-syn (α-synC6) to rescue α-syn shRNA-induced neurodegeneration was explored. The nonaggregatable form of α-syn was produced by inserting six cysteine residues into the primary amino acid sequence of α-syn, increasing intramolecular interactions and stabilizing the protein in a partially folded conformation that renders the amyloidogenic portion of the protein inaccessible. The α-synC6 mutant decreases the ability of α-syn to aggregate and thus ensures that the protein will remain in the accessible soluble pool of protein. Adult male rats received unilateral stereotaxic injections of rAAV expressing either WT α-syn, α-synC6, or a green fluorescent protein (GFP) control 1 month prior to administration of the α-syn shRNA. Stereological cell counts 1 month following the α-syn shRNA administration in animals receiving rAAV-GFP or rAAV-WT α-syn showed a significant loss of THir neurons in the ipsilateral SNpc. In contrast, animals that were injected with α-synC6 showed a partial rescue of THir neurons in the injected SNpc. These results show that loss of functional α-syn is toxic to nigrostriatal dopaminergic neurons.
S. Brahmachari,*†‡ X. Mao,*†‡ S. S. Karuppagounder,*†‡ Y. Lee,*†‡ V. L. Dawson,*†‡§¶# H. S. Ko,*†** and T. M. Dawson*†#**
*Neuroregeneration, Institute for Cell Engineering, Johns Hopkins University School of Medicine, Baltimore, MD, USA
†Department of Neurology, Johns Hopkins University School of Medicine, Baltimore, MD, USA
†Adrienne Helis Malvin Medical Research Foundation, New Orleans, LA, USA
§Stem Cell Programs, Institute for Cell Engineering, Johns Hopkins University School of Medicine, Baltimore, MD, USA
¶Department of Physiology, Johns Hopkins University School of Medicine, Baltimore, MD, USA
#Solomon H. Snyder Department of Neuroscience, Johns Hopkins University School of Medicine, Baltimore, MD, USA
**Diana Helis Henry Medical Research Foundation, New Orleans, LA, USA
α-Synuclein is a major component of Lewy body and Lewy neurites, the common pathological hallmarks of Parkinson's disease (PD) and α-synucleinopathies. However, the mechanisms by which accumulation of α-synuclein causes toxicity are poorly understood. Recently, Abelson murine leukemia viral oncogene homolog (c-Abl) kinase activation, a key indicator of oxidative stress, and its role in parkin inactivation and subsequent accumulation of parkin substrates, have been implicated in PD. In the present study, we hypothesize that c-Abl activation resulting from a mutant (A53T) α-synuclein-related oxidative stress, critically regulates α-synuclein toxicity. Time-course biochemical studies in A53T α-synuclein transgenic mice expressing α-synuclein constitutively in the brain clearly show an increase in c-Abl activation and α-synuclein oligomerization with aging. Our aggregation studies confirm that c-Abl promotes aggregation of α-synuclein. To examine the effect of c-Abl activation on α-synuclein pathology in vivo, we have generated tetracycline (Tet)-controllable breakpoint cluster region (Bcr)-Abl-A53T α-synuclein bigenic mice by using a prion protein (PrP)-Tet driver. Biochemical and survival analysis using these mice indicate that Bcr-Abl overexpression strongly promotes α-synuclein aggregation as well as significantly shortens the lifespan of the bigenic mice, and these changes are prevented by doxycycline, which suppresses Bcr-Abl transgene expression. To examine if c-Abl is necessary for α-synuclein-mediated toxicity, we have generated conditional c-Abl knockout (KO)-A53T α-synuclein double mutant mice with neuronal knockdown of c-Abl in the brain. Biochemical and survival analysis reveal that c-Abl knockdown markedly reduces aggregation of α-synuclein and prolongs the survival of double mutant mice. In addition, to examine the role of c-Abl in α-synuclein-mediated dopamine (DA)-neuronal loss, we induced α-synuclein overexpression in tetracycline-responsive promotor (TetP)-A53T α-synuclein responder mice by unilateral stereotaxic injection of recombinant adeno-associated virus tet-activator (rAAV-tTA) virus into the substantia nigra pars compacta (SNpc). Biochemical and stereological analysis following 6 months of injection reveal that increased c-Abl activity is associated with dramatic loss of DA neurons. To examine if c-Abl is required for α-synuclein-mediated DA neuronal loss, we have injected rAAV-rTA into the SNpc of conditional c-Abl KO-TetP-A53T α-synuclein double mutant mice for stereological and biochemical studies that will be performed at 6 months postinjection. Taken together, our studies describe a novel mechanism underlying α-synuclein-mediated toxicity. Moreover, our study provides a strong rational for screening different c-Abl inhibitors to inhibit the toxic consequences of α-synuclein. In addition, the mouse models that we have generated for the above studies may provide further insights of any novel pathological modification of α-synuclein contributing to its toxicity.
A. N. Bubak,* D. E. Redmond Jr.,† J. D. Elsworth,† R. H. Roth,† T. J. Collier,§ K. B. Bjugstad,¶ B. C. Blanchard,¶ and J. R. Sladek Jr.¶
*Neuroscience Program, University of Colorado-Denver Anschutz Medical Campus, Denver, CO, USA
†Psychiatry and Neurosurgery, Yale University School of Medicine, New Haven, CT, USA
†Pharmacology, Yale University School of Medicine, New Haven, CT, USA
§Center of Excellence in Parkinson's Disease Research, Michigan State University, Grand Rapid, MI, USA
¶Neurology and Pediatrics, University of Colorado-Denver Anschutz Medical Campus, Denver, CO, USA
The selective destruction of dopaminergic (DAergic) neurons in the nigrostriatal pathway by the neurotoxin 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP) serves as a significant tool in Parkinson's disease (PD) research. Administration of MPTP induces signature Parkinsonian symptoms including delayed movement initiations, freezing, resting tremor, and others, which have been shown through extensive studies in our laboratories to be reversed following bilateral fetal ventral mesencephalic grafts in the striatum of the African green monkey. We and others have reported a significant upregulation of endogenous tyrosine hydroxylase-positive neurons in the striatum of MPTP-treated animals (~140% increase). This population may represent a compensatory mechanism to replenish DA in the system. The aim of the current research was to investigate further the potential compensatory role of this population by tracking changes in cell numbers as behavioral function deteriorates in monkeys rendered Parkinsonian via MPTP administration. Furthermore, we hypothesized that relieving PD symptoms and replenishing DA in the striatum via fetal ventral mesencephalic grafts would reduce the number of upregulated neurons. By categorizing Parkinsonian animals into groups based on symptoms (asymptomatic, mild/moderate, and severe), we discovered that the upregulated endogenous population becomes significantly elevated at the onset of mild/moderate symptoms and remains elevated at a constant level in severely symptomatic individuals. Asymptomatic monkeys retained levels similar to untreated, control animals. Severely symptomatic monkeys with functionally successful transplants returned to untreated, control levels for the endogenous cell population, while severely symptomatic monkeys with unsuccessful transplants (i.e., grafts that failed to contain DA donor cells) retained the elevated cell numbers similar to severe, sham-operated subjects. Collectively, these results indicate a close relationship between symptom severity and endogenous cell numbers perhaps suggesting a potential compensatory effect in the form of delayed progression of symptoms. If this population represents an endogenous compensatory mechanism for DA deprivation as seen in PD, then future studies to influence these cells could be an attractive target in developing new, less invasive therapies for PD.
Supported by an Academic Enrichment Grant from the University of Colorado School of Medicine, The Axion Research Foundation, and 5PO1NS044281.
T. Bezdudnaya, V. Marchenko, and M. A. Lane
Drexel University College of Medicine, Philadelphia, PA, USA
High cervical spinal cord injury (SCI) results in significant respiratory dysfunction and contributes to morbidity and mortality. Spontaneous recovery of respiratory function is observed in many clinical and experimental studies highlighting the potential for neuroplasticity and giving an opportunity for therapeutic interventions to enhance intrinsic recovery mechanisms. Most studies to date have focused on functional and anatomical changes within the spinal cord after SCI. Much less known are what changes occur at the supraspinal level (brainstem). In the current study, we were interested in the neuroplastic changes of the supraspinal respiratory network at the caudal medullary level acutely after SCI. Lateral hemisection at C2 cervical segment (C2Hx) of the spinal cord has been used as an animal model of SCI. Decerebrate, unanesthetized, and artificially ventilated rats were used for all experiments. We electrophysiologically mapped the location of inspiratory and expiratory neurons ipsilateral to C2Hx using extracellular recording of single and multiunit activity within following coordinates: 1–2.4 mm from midline and 1 to −1.8 mm rostral and caudal from calamus scriptorius, at depths 1–3.6 mm from surface. Additionally, bilateral phrenic activity was recorded to monitor respiratory function and determine the respiratory phase of the recorded cells. Maps of respiratory activity were constructed and compared for control and injured rats after 4 h following C2Hx. We show 1) partial recovery of ipsilateral phrenic activity acutely ~4 h after C2Hx and 2) reorganization of active inspiratory and expiratory sites acutely after C2Hx. Reorganization includes an overall decrease in respiratory activity particularly in the most caudal part of the medulla and an increase in the proportion of expiratory phase activity within the rostral part of the map. Moreover, we observed an increase in respiratory activity within the reticular nuclei after C2Hx. This is the first experimental evidence for acute neuroplastic changes within the supraspinal respiratory network following cervical SCI. Future studies will investigate reorganization of the supraspinal respiratory network contralateral to C2Hx and the impact of this supraspinal neuroplasticity on phrenic function and breathing after cervical SCI.
C. R. Cashman,*†‡ R. Mi,† and A. Höke†‡
*MSTP/MD-PhD Program, Johns Hopkins University, Baltimore, MD, USA
†Department of Neurology, Johns Hopkins University, Baltimore, MD, USA
†Department of Neuroscience, Johns Hopkins University, Baltimore, MD, USA
While acute regeneration in the peripheral nervous system is quite successful, regeneration in the chronic setting is much more limited, largely due to Schwann cell atrophy. During regeneration, Schwann cells provide trophic and structural support for regenerating nerve fibers. However, Schwann cells may also die from chronic stress or loss of axonal contact. Thus, if an axon has not reached its target at the time of Schwann cell atrophy, it is unlikely to do so. This limit is especially relevant to large animals, such as primates, whereby an axon may need to regrow over a meter. Over such a distance, when the rate of axon growth is 1 mm/day, it may take years for an axon to reach the distal portion of the nerve, at which point many of the supporting Schwann cells will have already been lost. To overcome this limit of regeneration, we propose a supraphysiological solution, whereby neurons injected within a nerve may extend axons to form end organ specializations and serve as postsynaptic targets of endogenously regenerating fibers to form a relay from the central nervous system to the end organ. In this situation, axon extension may occur at multiple points simultaneously, thereby reducing the time of regeneration to be within the period of a viable Schwann cell population. Initial studies include determination of optimal cell preparation, cell survival, axon extension, and synaptogenic potential of the transplanted cells. To this end, one of the tibial nerves of an immunosuppressed Sprague–Dawley rat was transected 7 days prior to cell transplant. Transplanted cells were either derived mouse motor neurons with a genetically encoded homeobox protein 9:green fluorescent protein (Hb9:GFP) reporter of motor neuron fate or E14 primary ventral horn cells from a rat with a thymus cell antigen (Thy1.2):GFP genetically encoded reporter of neuron fate. Motor neuron purity of both populations of cells was determined to be about 33% of all cells. Cells were injected into the denervated distal stump of the rat tibial nerve and survival assessed at 1 and 3 weeks posttransplantation. Survival of mouse motor neurons was determined by immunofluorescent (IF) staining to be 1.5% and 4.6% at 1 and 3 weeks, respectively, while rat motor neurons had improved survival at 1 and 3 weeks (3.3% and 15.7%, respectively). Axons were also observed to project distally. Three weeks after injection, the soleus muscle was also collected to determine neuromuscular junction (NMJ) formation ability. No NMJs were detected by IF. In vitro IF staining suggests that the injected neurons are capable of forming glutamatergic synapses, which is ideal for postsynaptic targets of regenerating, endogenous fibers. Additional studies will focus on the NMJ formation assay after 2 months, as well as repair of the host tibial nerve to form the relay after endogenous regeneration. Relay connection will be assayed with IF staining as well as electrophysiological and pharmacological characterization.
J. N. Chang,*† H. Hatic,* E. Shaw,† V. Ravindranath,† B. A. Citron*†
*Laboratory of Molecular Biology, Research and Development, Department of Veterans Affairs, Bay Pines VA Healthcare System, Bay Pines, FL, USA
†Department of Molecular Medicine, University of South Florida Morsani College of Medicine, Tampa, FL, USA
†Centre for Neuroscience, Indian Institute of Science, Bangalore, India
Traditional medicines have been used for millenia, but the efficacy and mechanisms are largely unknown. The root of the Withania somnifera plant has been used to treat several disorders, including neurodegeneration, and likely acts by increasing antioxidant capacities. We sought to determine whether this extract can protect cultured neurons from an in vitro injury that mimics a traumatic brain injury. Neuronal cultures were plated on silastic membranes and treated with 20 μg of W. somnifera root extract. A brief pressure pulse produced a momentary biaxial stretch injury of the neurons to mimic the rotational forces that occur following a traumatic brain injury. Neuronal health was evaluated by annexin and propidium iodide (PI) staining, monitoring lactose dehydrogenase activity, and measuring neuronal processes. Pretreatment with W. somnifera root extract produced a significant decrease in annexin and PI staining following traumatic injury as well as a decrease in released lactose dehydrogenase activity. Measurement of neuronal processes indicated that the treatment benefited both the number and length of processes extending from neurons following traumatic injury. Preliminary data suggests that the protection is not conferred through the nuclear factor, erythroid 2-like 2 (Nrf2) antioxidant pathway. In summary, W. somnifera extract was able to protect neurons from model traumatic brain injury in vitro. We are currently in the process of measuring protein changes that may give insight into the underlying molecular processes responsible for the neuroprotection observed in this system.
Research supported by The Department of Veterans Affairs (Veterans Health Administration, Office of Research and Development, Biomedical Laboratory Research and Development), The Bay Pines Foundation, and the Florida Department of Health James and Esther King Biomedical Research Program.
G. Chen, Z. Guom, L. Zhang, Z. Wu, Y. Chen, and F. Wang
Department of Biology, The Huck Institutes of Life Sciences, Pennsylvania State University, University Park, PA, USA
Brain injury results in loss of neurons and activation of glial cells, a phenomenon called reactive gliosis. Gliosis is also widely associated with spinal cord injury, stroke, glioma, and neurodegenerative disorders such as Alzheimer's disease (AD). Currently, there is no effective way to reverse gliosis after brain injury or neurodegeneration. Here we demonstrate that reactive glial cells can be directly reprogrammed into functional neurons in vivo in stab-injured or AD mouse model brain. We injected retrovirus encoding a single neural transcription factor neuronal differentiation 1 (NeuroD1) into adult mouse cortex to infect reactive glial cells that are activated by stab injury. In 3–7 days after viral injection, NeuroD1-infected cells adopted a neuronal morphology and were immunopositive for neuronal markers doublecortin (DCX), neuronal class III β tubulin, and neuronal nuclei (NeuN). Using astrocytic promoter glial fibrillary acidic protein (GFAP) or neuron–glia antigen 2 (NG2)-promoter to drive NeuroD1 expression, we demonstrated that both astrocytes and NG2 cells were converted into neurons in vivo and in vitro. Interestingly, astrocytes were reprogrammed into glutamatergic neurons, while NG2 cells were reprogrammed into glutamatergic and γ-aminobutyric acid (GABA)ergic neurons after NeuroD1 expression. Cortical slice recordings revealed both spontaneous and evoked synaptic responses in NeuroD1-converted neurons, suggesting the integration of converted neurons into local neural circuits. Using a transgenic mouse model for AD, we demonstrated that NeuroD1 converted reactive glial cells into functional neurons in 7- to 14-month-old AD model mice. Moreover, NeuroD1 also converted cultured human astrocytes into functional neurons. Our studies demonstrate that reprogramming reactive glial cells into functional neurons in injured or diseased brain may provide a potential new approach for brain repair.
K.-Y. Chen,* C. Hui Li,* C.-C. Wu,† Y. H. Chen,* S.-C. Hsueh,* T.-H. Hsieh,* and Y.-H. Chiang*†
*Graduate Institute of Neural Regenerative Medicine, College of Medical Science and Technology, Taipei Medical University, Taipei, Taiwan
†Department of Neurosurgery, Taipei Medical University Hospital, Taipei, Taiwan
Parkinson's disease (PD) is one of the most common neurodegenerative disorders with motor symptoms. Exercise training maybe feasible for motor rehabilitation and recovery of dopaminergic neurons in the stratum and substantia nigra. In this study, we aim to explore whether voluntary physical exercise can improve motor behavior function and induce cellular plasticity of Sprague–Dawley rats with PD. Exercise group rats were exposed to voluntary wheel running 2 weeks before administration of 6-hydroxydopamine (6-OHDA) into the median forebrain bundle (MFB) and then to intermittent running for 4 weeks. The sedentary group was not exposed to the running wheel. Bromodeoxyuridine (BrdU) was injected to label the proliferating cells, enabling observation of cellular plasticity. The gait analysis and amphetamine-induced rotation test were performed to investigate the motor behavior of PD rats. In gait analysis, exercise PD rats exhibited changes in gait patterns. In the exercise group, stride length on the contralateral side was decreased, and also the gait cycle was increased. The sedentary group showed significantly decreased walking speed and step/stride length and increased base of support. Apomorphine-induced rotation behavior showed reduced numbers of turn in the exercise group at 4 weeks. Tyrosine hydroxylase (TH)-positive neurons in the striatum and substantia nigra pars compacta (SNpc) were significantly increased in the exercise group compared to the sedentary group. Furthermore, BrdU-positive cells also showed an increase in the exercise group indicating that exercise could induce cellular plasticity. Our results demonstrated that voluntary exercise could ameliorate motor behavior and also induce cellular plasticity in PD rats, providing therapeutic value for treatment of Parkinson's disease patients.
P. Chen,* S. Jergova,* F. Nasirinezhad,* N. Pathak,* C. Gordon,* J. Imperial,† B. M. Olivera,† S. Gajavelli,* and J. Sagen*
*Miami Project to Cure Paralysis, University of Miami, Miami, FL, USA
†Biology, University of Utah, Salt Lake City, UT, USA
Chronic pain following spinal cord injury (SCI) is a challenging clinical problem with few effective treatments. The development of novel pharmacological agents and treatment approaches are essential. There are several selective and potent peptides produced by marine cone snails that have promising effects on severe pain. A synthetic peptide ω-conotoxin, MVIIA, (marketed as Prialt®) is FDA approved, and the n-methyl d-aspartate (NMDA) antagonist peptide conantokin G (Con G) has undergone phase I clinical trials. However, the use of these peptides are limited by both the necessary and inconvenient intrathecal route of administration and the emergence of untoward side effects when administered as single peptide dosing for chronic pain. Previously, we found that the two peptides are significantly antinociceptive when injected intrathecally in rat pain models and synergistically alleviated SCI pain when combined in approximately a 2.5:1 ratio without adverse side effects. We therefore designed combined MVIIA and ConG gene constructs with genetic engineering in ratios 1:1, 1:2, and 1:3 and evaluated their analgesic effect in an SCI chronic pain model. ConG cDNA was amplified to introduce Bgl II restriction sites. The Bgl II flanked ConG insert was ligated in frame into MVIIA/pGEM®T vector linearized with Bgl II. This approach yielded constructs with various ratios of MVIIA: ConG genes. Colonies with ratios 1:1, 1:2, and 1:3 were selected by BamHI and SaII amplified and subcloned into adeno-associated virus (AAV) plasmids to produce AAV–nConGMVIIA–enhanced green fluorescent protein (EGFP)–Woodchuck hepatitis virus post-transcriptional regulatory element (WPRE) plasmids (and AAV–EGFP–WPRE as control plasmid). The recombinant AAV viruses were produced by Miami Project Viral Vector Core. Supernatant from cells transfected by nConGMVIIA showed a positive signal for MVIIA using fluorophore-linked immunosorbent assay (FLISA), suggesting successful production and release of peptide. Spinal cord clip compression injury was used to induce pain-related behavior in rats, and tactile and cold allodynia were evaluated weekly. At 3–5 weeks postinjury when pain-related behavior was clearly established, animals were injected with the AAV MVIIA/ConG vectors or control AAV vector intraspinally into lumbar dorsal horn. Attenuation of tactile and cold allodynia was observed by 2–3 weeks postinjection with gradual improvement of behavioral outcomes toward preinjury levels by 8–9 weeks post-SCI. In contrast, allodynia persisted in animals receiving control vector. No effects on motor impairment were observed in any vector-treated groups, with comparable Basso, Beattie, and Bresnahan (BBB) locomotor scores over the experiment duration. Our findings suggest that engineered conopeptide constructs encoding synergistic analgesic agents have the potential to alleviate SCI-induced pain.
Grant/other support: Craig H. Neilsen Foundation Award #190848.
Y.-H. Chiang,*†‡§ K.-Y. Chen,*† T.-H. Hsieh,† Y.-W. Yu,*† C.-C. Wu,† and J.-W. Lin§
*Translational Research Laboratory, Cancer Center, Taipei Medical University Hospital, Taipei, Taiwan
†Graduate Institute of Neural Regenerative Medicine, College of Medical Science and Technology, Taipei Medical University, Taipei, Taiwan
†Department of Neurosurgery, Taipei Medical University Hospital, Taipei, Taiwan
§Department of Surgery, College of Medicine, Taipei Medical University, Taipei, Taiwan
Traumatic brain injury (TBI) is a global problem for which mild TBI (mTBI) makes up a majority of the cases. mTBI produces a broad range of short- and long-term cognitive, behavioral, and emotional impairments. Currently, no clear pharmaceutical-based therapies have been established to prevent the ensuing neurological dysfunctions and to manage the secondary pathological events after mTBI. In this study, we investigated the neuroprotective effect of glucose-dependent insulinotropic polypeptide (GIP) delivered via a subcutaneous micro-osmotic pump in an animal model of mTBI. We further investigated its effect on the learning, memory, motor, and somatosensory functions by using the Morris water maze test, adhesive removal test, and gait analysis. In the Morris water maze test, GIP/mTBI rats displayed an improvement in memory as early as 7 days postlesion when compared to the sham-treatment group. With regard to locomotor and somatosensory functions, compared to the sham- treatment group, our results indicated that GIP/mTBI rats exhibited increased walking speed and decreased time spent in the adhesive removal test around 7 days postlesion. The mTBI rats with GIP treatment showed a positive pattern on walking speed in gait and sensory measurement in the first week of observation. Mechanisms of GIP treatment were also analyzed with sulforhodamine B (SRB) assay and Western blot analysis. Results showed a neuroprotective effect in the in vitro study as decreased cell death under hypoxia was observed in cultures treated with GIP. Western blot analysis showed signal transducer and activator of transcription 3 (Stat3) activation was involved in the neuroprotective effect of GIP. In conclusion, GIP demonstrates neuroprotective effects in a mTBI model both in vitro and in vivo via the Stat3 signaling pathway.
S.-Y. Chou,*†‡ Y.-C. Chen,* and P.-S. Hsieh*
*Department of Physiology and Biophysics, National Defense Medical Center, Taipei, Taiwan
†Graduate Institute of Neural Regenerative Medicine, Taipei Medical University, Taipei, Taiwan
†Center for Neurotrauma and Neuroregeneration, Taipei Medical University, Taipei, Taiwan
The hypothalamus is the integration center for body energy homeostasis. The long-term levels of high blood glucose in type II diabetes mellitus (DM) lead to chronic inflammation and variant neuronal degeneration such as retina degeneration, dementia, and Alzheimer's disease.
Type 5 C-C motif chemokine ligand [CCL5/RANTES (regulated upon activation, normally T-expressed, and presumably secreted)] and activation of its receptor, CCR5, have a strong correlation in the inflammation of the peripheral adipose tissue and blood glucose utility in type II DM. The role and mechanism of CCL5/CCR5 signaling in the hypothalamus remains unclear. Herein, C57BL/6 mice were used as control wildtype (WT) mice, and the experimental group consisted of whole body knockout CCR5 mice. Experimental mice at 13 weeks old were separately housed at 22°C or 4°C for 1 week to investigate the role of hypothalamic CCR5 signaling in energy homeostasis and adaptive thermogenesis. An increased body weight and daily food consumption was found in CCR5 knockout mice at 13 weeks old, which paralleled higher blood glucose, insulin, and leptin levels under 22°C housing. The expression of the insulin signaling substrates—insulin receptor substrate protein 1 and 2 (IRS1 and IRS2)—and the mitochondria energy-generating enzymes—uncoupling proteins 1 and 2 (UCP1 and UCP2)—were lower in CCR5-/- mice hypothalamus with quantitative RT-PCR analysis. Increased hypothalamic adenosine monophosphate (AMP)-activated protein kinase a (AMPKa) phosphorylation was found in CCR5-/- mice. The 4°C cold stress housing increased body energy consumption. Increased food intake in both the WT and CCR5 KO mice but reduced insulin and leptin levels in the blood of CCR5 KO mice were observed. Primary cultured hypothalamic neurons treated with CCL5 induced a membrane translocation of the insulin-responsive glucose transporter type 4 (GLUT4) within 10~15 min. The above findings suggest that hypothalamic CCR5 might play a negative regulatory role in energy expenditure.
B. A. Citron,*† R. F. Mervis,‡§ S. K. Foley,‡§ P. Hanna,‡§ L. Rachmany,¶ A. Shaer,¶ V. Rubovitch,¶ C. G. Pick,¶ and J. N. Chang*†
*Laboratory of Molecular Biology, Research and Development, Department of Veterans Affairs, Bay Pines VA Healthcare System, Bay Pines, FL, USA
†Department of Molecular Medicine, University of South Florida Morsani College of Medicine, Tampa, FL, USA
†NeuroStructural Research Laboratories, Inc., Tampa, FL, USA
§Center of Excellence for Aging and Brain Repair, Department of Neurosurgery and Brain Repair, University of South Florida Morsani College of Medicine, Tampa, FL, USA
¶Department of Anatomy and Anthropology, Sackler School of Medicine, Tel Aviv University, Tel Aviv, Israel
Worldwide, the incidence of traumatic brain injury (TBI) is approximately 0.5% per year in the general population, while there has been a 15% prevalence among the deployed military forces. The vast majority of TBIs are mild, yet they frequently do result in significant negative effects on brain function. Our lab has uncovered molecular mechanisms, in particular, certain inflammatory-responsive regulatory factors, involved in the health of neurons. We employed a closed-head system involving a weight drop-induced impact and rotational injury and have measured behavioral, mRNA, and protein changes. We obtained cognitive improvements by tertiary butylhydroquinone (tBHQ) treatment after injury that activates the transcription factor, nuclear factor erythroid 2-related factor 2 (Nrf2), and have identified pathway changes involved in the neuroprotection. Here we describe the changes seen in dendritic complexity after tBHQ treatment and TBI in mice. To determine alterations in connectivity, brain samples were stained with the Golgi method to quantitatively assess dendritic branching and spine morphologies and densities. Dendritic complexity, determined by several different measures including branch point analysis, was higher in the untreated TBI brains compared to sham injured or to brains from mice exposed to TBI plus treatment. This may indicate a preferential loss of the least connected neurons in the injured, untreated mice. Through these experiments, we seek to develop effective treatment strategies to combat the secondary effects of traumatic brain injury.
This study was supported by the Department of Veterans Affairs (Veterans Health Administration, Office of Research and Development, Biomedical Laboratory Research and Development), The Bay Pines Foundation, and the Florida Department of Health James and Esther King Biomedical Research Program.
A. T. Crane,* K. D. Fink,*† K. A. Grace,* A. K. Antcliff,* A. C. Neale,* R. Wyse,* J. Rossignol,*‡ and G. L. Dunbar*§
*Central Michigan University, Program in Neuroscience, Mount Pleasant, MI, USA
†INSERM, University of Nantes, Nantes, France
†Central Michigan University, College of Medicine, Mount Pleasant, MI, USA
§Field Neurosciences Institute, Saginaw, MI, USA
Huntington's disease (HD) is an autosomal dominant neurodegenerative disorder caused by an expansion of cytosine–adenine–guanine (CAG) repeats of the huntingtin gene. Adult-onset symptoms of HD begin with cognitive and motor deficits as a result of degeneration within the caudate and putamen. As the disease progresses, degeneration spreads throughout the brain, and the ability to care for one's self is lost, eventually leading to a premature death. At present, only palliative treatments for the phenotypic symptoms exist. With the generation of induced pluripotent stem cells (iPSCs) from somatic tissue, the use of these cells for a cell-replacement strategy has gained interest for treatment of such neurodegenerative disorders as HD. Adenovirus-generated iPSCs, developed in our lab so they do not integrate known oncogenes, have previously been transplanted into the striata of both healthy rats and rats given 3-nitropropionic acid (3-NP), which causes symptoms analogous to HD. Although we have shown that these iPSCs survive and differentiate into region-specific neurons, as well as promote recovery of 3-NP-induced motoric deficits, it is currently unknown whether these cells will confer similar benefits in a transgenic model of HD. In the current study, iPSCs were characterized in vitro through flow cytometry and RT-PCR, as well as their potential to differentiate into medium-spiny neurons of the striatum. Hoechst-labeled iPSCs were then transplanted into the 51 CAG transgenic rat model of HD (tgHD) at 12 months of age, prior to the onset of motor deficits. Wildtype or tgHD animals were tested every other week on the accelerating rotarod and open field for 20 weeks following transplantation or sham surgery. The animals were then perfused and their brains removed for histological analysis. Contrary to previous findings, transplanted iPSCs were not observed 20 weeks following transplantation, but rather, an increase in inflammation surrounding the transplant site was evident. These results suggest that our iPSCs may be model sensitive and that further exploration of the possible mechanisms for cell survival in transgenic animals is needed to delineate the extent to which iPSC-induced recovery can be generated.
Funding was provided by the John G. Kulhavi Professorship in Neuroscience and the Field Neurosciences Institute (to GLD).
I. De La Peña, S. A. Acosta, M. M. Pabon, A. Yoo, D. G. Hernandez, M. Staples, P. Pantcheva, C. Tamboli, S. Tamboli, K. Duncan, D. Lozano, M. Bastawrous, A. Antoine, N. Tajiri, Y. Kaneko, and C. Borlongan
Center of Excellence for Aging and Brain Repair, Department of Neurosurgery and Brain Repair, University of South Florida Morsani College of Medicine, Tampa, FL, USA
Neuroprotective effects of granulocyte colony-stimulating factor (G-CSF) have been shown in animal models of ischemia. In light of these findings, administration of G-CSF may reduce hemorrhagic transformation (HT) associated with delayed treatment of tissue plasminogen activator (tPA) therapy in experimental models of stroke. The primary aim of this study is to demonstrate that administration of G-CSF reduces HT after a delayed tPA treatment in experimental models of stroke. Another aim is to provide a mechanism of action underlying G-CSF's therapeutic benefits against delayed tPA-induced HT. Sprague–Dawley rats subjected to middle cerebral artery occlusion (MCAO) were given saline (control) or tPA (10 mg/kg, IV) at 5 h after reperfusion (delayed tPA treatment group). Effects of G-CSF (300 μg/kg, IV) alone or delayed tPA in tandem with G-CSF on infarct volume and extent of intracerebral hemorrhage as well as neurological and behavioral outcomes were observed 24 h and 3 days in rats subjected to MCAO. For the mechanism-driven studies, immunohistochemical analyses for endothelial progenitor cell (EPC) markers using cluster of differentiation 34 and vascular endothelial growth factor receptor 2 (CD34 and VEGFR2) were performed in the ischemic brain region of stroke animals at 3 days after MCAO following the last behavioral tests. Western blots were also performed to measure the degree of angiogenesis, vasculogenesis, and vascular remodeling using appropriate phenotypic markers. Administration of G-CSF reduced HT after a delayed tPA treatment in experimental models of stroke. Moreover, neurological outcomes were improved in G-CSF-treated MCAO rats subjected to delayed tPA treatment. Immunohistochemistry and Western blots revealed increased expression of angiogenesis and vasculogenesis markers in the ischemic brain region of stroke animals at 3 days after MCAO. In conclusion, G-CSF reduces HT associated with a delayed tPA treatment in experimental models of stroke. The mechanism of action may involve G-CSF-induced recruitment of EPCs from the bone marrow to the ischemic brain. Mobilized EPCs in turn may maintain cerebrovascular integrity via angiogenesis, vasculogenesis, or vascular remodeling. Altogether, these results indicate the potential therapeutic value of G-CSF in preventing HT during delayed tPA therapy.
This study was supported by the NIH NINDS 5R01NS071956–02.
D. Dolezalova,*†‡ M. Hruska-Plochan,*†‡ L. Goldstein,§¶ and M. Marsala*†‡
*Department of Anesthesiology, University of California at San Diego, La Jolla, CA, USA
†Stem Cell Program, University of California at San Diego, La Jolla, CA, USA
†Institute for Genomic Medicine, University of California at San Diego, La Jolla, CA, USA
§Department of Cellular and Molecular Medicine, University of California at San Diego School of Medicine, San Diego, CA, USA
¶Department of Neurosciences, University of California at San Diego School of Medicine, San Diego, CA, USA
Regeneration and replacement of neurons and glia that undergo cell death due to progressive neurodegenerative disorders and/or central nervous system (CNS) injury are the main goals of all stem cell-based therapies. However, well-defined and clinically relevant methods for the generation of neural stem cells (NSCs) from human embryonic stem cells (hESCs) as well as their safety in vivo remain to be defined. The aim of the present study was to develop an in vitro technique for isolation of NSCs from pluripotent hESC lines. The potency and safety of generated cell lines were probed by their differentiation potential both in vitro and in vivo and by spinal grafting into immunodeficient rats.
Three independent cell lines of pluripotent hESCs (H9, UCSF4, UCSF4.3) were expanded and induced to form neural rosettes. Morphologically distinct populations of proliferating NSCs were harvested from the periphery of neural rosettes and further expanded in the presence of fibroblast growth factor 2 (FGF2) as a solo mitogen. NSCs were expanded up to at least 40 passages while being periodically characterized by flow cytometry for pluripotency and NSC-specific markers [Nanog, sex-determining region Y box 2 (Sox2), Sox1, paired box 6 (Pax6), nestin, glial fibrillary acidic protein (GFAP), cluster of differentiation 24 (CD24), CD44, CD184, CD271]. To evaluate safety, NSCs were grafted spinally into immunodeficient rats and the presence and phenotype of grafted cells analyzed from 2 weeks up to 6 months using human-specific antibodies.
Newly generated cell lines of NSCs showed homogenous morphology upon extensive propagation in vitro. Established lines displayed consistent expression of markers typical for NSCs (>75% of nestin, Sox1, Sox2, and Pax6 expression) and no residual expression of pluripotency transcription factor Nanog. A stable karyotype as well as the ability to differentiate into glial cells and functional neurons was seen in vitro. After transplantation in vivo, cells integrate into host tissue and show morphology and markers typical for mature neurons and/or astrocytes such as doublecortin (DCX), neuronal nuclei (NeuN), and/or GFAP. Grafted cells showed moderate Ki67 immunoreactivity, which was quantitatively similar to the endogenous pool of proliferating Ki67+ precursors. Importantly, no tumor formation or the appearance of any aberrant cell type formation (rosettes or malignant cell clusters) was noted. This isolation protocol can be effectively used to generate high numbers of transplantable NSCs from pluripotent hESCs, which demonstrate a favorable safety profile and can potentially be used in future human clinical trials.
This study was supported by CIRM DR1–01471.
M. Duffy,* J. MacKeigan,† F. Manfredsson,* S. G. Lampe,* N. Kuhn,* C. Kemp,* and C. Sortwell*
*Michigan State University, Grand Rapids, MI, USA
†Van Andel Research Institute, Grand Rapids, MI, USA
While there are many pharmaceutical options available to attenuate motor symptoms of Parkinson's disease (PD), no treatments currently exist to halt or slow the progression of nigrostriatal degeneration, and many existing treatments exacerbate dyskinesias after prolonged use. In order to accelerate new developments for PD treatment, many studies seek to reposition drugs with a known safety profile in humans. Previous studies in our lab have shown that fasudil, a Rho kinase [Rho-associated, coiled coil-containing protein kinase 1 (ROCK)] inhibitor, provides neuro protection from recombinant adeno-associated virus (rAAV) α-synuclein-mediated toxicity. However, the mechanism behind fasudilmediated neuroprotection remains unknown. Recent studies have shown α-synuclein to be a direct mediator of neuroinflammation via upregulation of phagocytic microglia.
ROCK regulates microglial polarization and motility. The present study aimed to investigate the mechanism behind fasudil-mediated neuroprotection, which we hypothesized to be attenuation of microglial polarization and motility via ROCK inhibition. Nigral tissue sections from rAAV α-syn-injected animals treated orally with either a neuroprotective high-dose fasudil chow (25 mg/kg/day, n = 6), low-dose fasudil chow (10 mg/kg/day, non-neuroprotective, n = 4), or control chow (n = 7) were immunofluorescently double labeled for tyrosine hydroxylase (TH), a marker for dopamine neurons, and ionized calcium-binding adapter molecule 1 (Iba-1), a general marker for microglia that has been shown to be upregulated during microgliosis. Tissue sections were analyzed using near infrared imaging to quantify Iba-1 signal intensity, and immunofluorescence was used to analyze qualitative changes in microglial number and morphology. High-dose fasudil administration resulted in a significant decrease in Iba-1 signal intensity in the α-syn substantia nigra (SN), intact SN, and tectum (used as a control), compared to equivalent regions in the control and low fasudil groups, suggesting that fasudil attenuates α-synmediated microgliosis. Future studies will investigate levels of cluster of differentiation 68 (CD68), a marker specific to phagocytic microglia. These findings, along with previous findings from our lab, demonstrate that fasudil may protect SN dopamine neurons against α-syn-mediated inflammation via inhibition of ROCK, ultimately attenuating microglial motility. Given that orally administered fasudil has an established safety profile in humans and is, to our knowledge, the first orally available drug to provide neuroprotection in the α-syn model of PD, it demonstrates potential for development as an effective therapeutic agent to slow progressive nigrostriatal degeneration in Parkinson's disease.
Supported by the Michael J. Fox Foundation for Parkinson's Research (JPM/CES) and the Michigan State University Neuroscience Program T32 NS44928 (CLS).
J. Ehrhart,* D. Darlington,† C. D. Sanberg,* N. Kuzmin-Nichols,* and J. Tan†‡
*Saneron CCEL Therapeutics, Inc., Tampa, FL, USA
†Rashid Laboratory for Developmental Neurobiology, Silver Child Development Center, University of South Florida Morsani College of Medicine, Tampa, FL, USA
†Center of Excellence for Aging and Brain Repair, Department of Neurosurgery and Brain Repair, University of South Florida Morsani College of Medicine, Tampa, FL, USA
Alzheimer's disease (AD) is the most common progressive age-related dementia and the fourth major cause of disability and mortality in the elderly population. The disease is characterized by deposition of β-amyloid plaques and neurofibrillary tangles in the brain. Current treatments of AD are less than efficacious in terms of significantly slowing or halting the underlying pathophysiological progression of the disease. Modulation by cell therapy may be a new promising disease-modifying therapy. Recently, we showed a reduction in amyloid-β (Aβ) levels/β-amyloid plaques and associated astrocytosis following a single infusion of human umbilical cord blood cells (hUCBCs). Our current study extended our previous findings by examining cognition via (1) the rotarod test, (2) a 2-day version of the radial-arm water maze test, and (3) a subsequent observation in an open pool platform test to characterize the effects of monthly peripheral HUCBC infusion (1 × 106 cells/ml) on the double transgenic presenilin amyloid precursor protein (PSAPP) AD mouse model from 6 to 12 months of age. We show that hUCBC therapy correlates with decreased (1) cognitive impairment, (2) Aβ levels/β-amyloid plaques, (3) amyloidogenic APP processing, and (4) reactive microgliosis after a treatment of 6 or 10 months. We also followed the biodistribution of hUCBCs in the AD-like transgenic PSAPP mouse and nontransgenic Sprague–Dawley rats. hUCBCs were injected into the tail veins (IV) of mice or rats at a single dose of 1 × 106 or 2.2 × 106 cells, respectively. Tissues were harvested at 24 h, 7 days, and 30 days after injection. For determination of hUCBC distribution, tissues from both species were subjected to total DNA isolation and PCR amplification of the gene for human glycerol-3-phosphate dehydrogenase. Our results show a relatively similar biodistribution and retention of hUCBCs in both mouse and rat organs. hUCBCs were broadly detected both in the brain and several peripheral organs, including liver, kidney, and bone marrow, of both species, starting within 7 days and continuing up to 30 days post-transplantation. No hUCBCs were recovered in the peripheral circulation, even at 24 h post-transplantation. As such, this report lays the groundwork for hUCBC therapy as a potentially novel alternative to oppose AD at the disease-modifying level and suggests that the IV route of administration can be a viable method of administration of these cells for the treatment of neurodegenerative diseases.
M. S. Eksi,* Z. O. Toktas,† Y. Bayri,† and D. Konya†
*University of California at San Francisco, San Francisco, CA, USA
†Bahcesehir University Medical School, Istanbul, Turkey
†Marmara University Medical School, Istanbul, Turkey
The peripheral nervous system is responsible for mediating autonomic functions, conducting sensorial input from the environment to the central nervous system, and relaying the motor response from the central nervous system to muscles and joints. Peripheral nerve injury has a high morbidity ratio. Injury in both the macro- and microenvironment needs to be considered while planning the treatment. Nerve function can be evaluated with electrodiagnostic tests. Many electrodiagnostic tests have been used for monitoring the central nervous system, especially the response of the somatosensorial cortex to peripheral nerve injury. But functional magnetic resonance imaging studies are scant in literature.
Our aim was to evaluate the functional magnetic resonance imaging (fMRI) changes in the somatosensorial cortex after sciatic injury.
With Institutional Review Board approval, we used 10 similar weighted and sized male rats. After peritoneal anesthesia, sciatic nerve injury was performed with meticilous surgical precision in five rats' right hindpaws and in five rats' left hindpaws. During the experiment, the rats were followed in the same room within different cages. During fMRI, electrical stimulation of the same amplitude was given under general anesthesia.
All rats' somatosensorial cortices were active before the surgery. Post-op second day, this number decreased to nine rats; on post-op 15th day, there were only two rats with active somatosensorial cortices. Finally, on post-op 30th day, we observed no activation in the presumed cortical area. Somatosensorial cortex metabolic activation level decreased due to disruption of sensorial inputs from injured peripheral nerves. In ongoing future experiments, the functional status of the somatosensorial cortex will be monitored after surgical repair of the injured peripheral nerve.
L. Feng,* A. Sharma,† D. F. Muresanu,† R. Patnaik,§ Z. R. Tian,¶ and H. S. Sharma†
*Department of Neurology, Bethune International Peace Hospital, Shijiazhuang, Hebei Province, China
†Surgical Sciences, Anesthesiology and Intensive Care Medicine, Uppsala University Hospital, Uppsala, Sweden
†Clinical Neurosciences, University of Medicine and Pharmacy, Cluj-Napoca, Romania
§Biomedical Engineering, Indian Institute of Technology, Banaras Hindu University, Varanasi, India
¶Department of Chemistry and Biochemistry, University of Arkansas, Fayetteville, AR, USA
Spinal cord injury (SCI) results in deficiency of neurotrophic factors like brain-derived neurotrophic factor (BDNF) and insulin-like growth factor-1 (IGF-1) in the plasma and/or in the spinal cord. Since exogenous application of stem cells, for example, mesenchymal stem cells (MSCs), in SCI often results in improvement of functional and cellular recovery after trauma, it is quite likely that stem cells could also elevate the levels of BDNF and IGF-1 in the spinal cord as well as in the plasma after successful therapy.
In the present investigation, we injected 1 million MSCs intravenously 2 days before SCI was produced in rats by making a longitudinal incision of the rats' spinal cord on the T10–11 segments and measured BDNF and IGF-1 levels in plasma and the cord 12 h or 24 h after insult. In addition, we also delivered MSCs labeled to titanium dioxide (TiO2) nanowires under identical conditions in SCI to see whether nanodelivery of MSCs is more potent in attenuating SCI-induced pathophysiology and functional recovery.
Our observations show that MSCs alone given before SCI are able to attenuate SCI-induced functional recovery and cord pathology following 12 h post-trauma but were not successful 24 h after injury. On the other hand, nanodelivery of MSCs was able to thwart cord pathology and improve functional recovery 12 and 24 h after SCI, indicating that nanodrug delivery for MSCs has superior neuroprotective effects in SCI. Moreover a significant increase in the BDNF and IGF-1 levels was seen in the plasma and cord that was well maintained up to 12 h after SCI in the MSC-treated group. Nanodelivery of MSCs resulted in 200% to 250% elevated levels of BDNF and IGF-1 in the plasma and cord even 24 h after SCI. These findings are the first to suggest that nanowired delivery of MSCs is able to enhance neurotrophic factor levels in the cord and plasma after SCI for longer periods of time following injury, and this could be one of the key factors in stem cell-induced neuroprotection in SCI.
E. G. Ferrazoli,* T. T. Schwindt,† and H. Ulrich‡
*Department of Neurology and Neurosurgery, Federal University of São Paulo, São Paulo, SP, Brazil
†Department of Cellular and Developmental Biology, Institute of Biomedical Sciences, University of São Paulo, São Paulo, Brazil
†Department of Biochemistry, University of São Paulo, São Paulo, Brazil
Purigenic receptor P2X7, ligand-gated ion channel-7 (P2X7R) inhibition has been suggested as strategy for prevention of neuronal cell death. Following another mechanism, peroxisome proliferator-activated receptor-g coactivator-1 a (PGC-1a), a protein involved in the control of cellular bioenergetics, has also been attributed with neuroprotective properties. We have evaluated the effects of Brilliant Blue-G (BBG), an antagonist of the P2X7R, and fenofibrate, an activator of PGC-1a, in reverting cell death of dopaminergic neurons in a rat model of Parkinson's disease. For this purpose, unilateral hemisphere lesions of the nigrostriatal pathway of adult male Sprague–Dawley rats were induced by stereotactic injection of 6-hydroxydopamine (6-OHDA). One week after lesion, the animals presented rotational behavior when challenged with apomorphine. Treatment with BBG had a beneficial functional effect. Animals treated with 6-OHDA and who received BBG (n = 6) during 7 days at a 50mg/kg dose showed a statistically significant decrease in the number of rotations per minute (13 to 4, p < 0.05), whereas animals receiving only saline did not reveal any significant improvement in rotational tests (11 to 8, p > 0.05). In agreement, levels of regeneration of dopaminergic neurons in BBG-treated animals, as revealed by anti-tyrosine hydroxylase staining, were twice as high as those observed for the saline-treated control group. Lesioned animals, which had received 0.2% fenofibrate in their diet for 14 days (n = 5), did not show any reduction in apomorphine-induced rotations (11 to 14, p > 0.05) when compared to animals fed with common diets (n = 5) (12 to 17, p > 0.05). Accordingly, in the fenofibrate group, there was no evidence of recovery of the dopaminergic circuitry. In conclusion, BBG is a promising tool for developing novel strategies in the therapy of Parkinson's disease.
Supported by FAPESP and CNPq funding agencies, Brazil.
W. J. Freed, J. Chen, A. Kindberg, R. Bendriem, C. E. Spivak, C. Lupica and C.-T. Lee
Intramural Research Program, National Institute on Drug Abuse, NIH/DHHS, Baltimore, MD, USA
The cerebral cortex, or neocortex, is the most complex component of the central nervous system and is highly susceptible to cerebral trauma and adverse developmental events. While the neocortex is similarly organized in all mammals, the complexity of the human cortex greatly exceeds that of any other animal. Cerebral cortical development involves sequential specification of forebrain cortical progenitor cells to various specific subtypes of neurons, forming the layered cortical structure. An in vitro model of the human neocortex, which preserves the essential spatial and temporal features of human cortical development has the potential to provide new avenues for drug discovery and treatment of neurological disorders.
Human pluripotent stem cells (hPSCs), including both human embryonic stem cells (hESCs) and induced pluripotent stem cells (hiPSCs), can be directed to form specific human cell types and organ systems in vitro. Although there are prior protocols for generating human neurons and cortical structures from hPSCs, these do not replicate the inside-out sequential feature of human cortical layer formation. Our aim is to develop an in vitro model of the human neocortex that preserves the essential layered cortical structure.
We employed a strategy of allowing embryoid body (EB) formation, including blocking of fibroblast growth factor 2 (FGF2) and dual-SMAD [small body size (sma) and mothers against decapentaplegic (MAD) homologs] signaling, and preserving hPSC aggregates. We hypothesized that this would promote the exit of hPSCs from the pluripotent state and enhance neural conversion, thus efficiently generating structures resembling the human neocortex. These aggregates were expanded in FGF2 for 1week and subsequently allowed to differentiate in low-attachment conditions for 6 weeks to form three-dimensional structures. Alternatively, the aggregates could be broken up into small colonies in the presence of the Rho-associated, coiled-coil containing protein kinase (ROCK) inhibitor Y-27632 and differentiated for 3 weeks in attachment-promoting conditions to form two-dimensional structures.
Ninety percent of colonies formed rosettes at day 16 when FGF2 and dual-SMAD signaling were blocked during the EB stage. In addition, treatment with FGF2 and dual-SMAD inhibitors created large paired box 6 positive (Pax6+) (dorsal forebrain progenitor marker) neural rosettes with diameters greater than 300 μm. Following differentiation, these efficiently formed organized neocortical structures. At week 2 of the neocortical specification period, neuroepithelial structures positive for the forebrain markers Pax6 and brain factor 1 (BF1) were observed. After 6 weeks, neurons positive for neuronal class III beta tubulin and Reelin, a marker for the earliest-generated subplate neurons, were located superficially to Pax6+ neuroepithelial cells. Thus, the three-dimensional structures developed an organized layered formation resembling that of the normal human cortex.
The two-dimensional structures followed a temporal pattern of development replicating cortical neurogenesis. Deep-layer chicken ovalbumin upstream promoter-transcription factor (COUP-TF) interacting protein 2 (CTIP2+) neurons appeared during the first week of differentiation, while upper-layer special adenine-thymine (AT)-rich sequence-binding protein 2 (SATB2+) neurons formed by 3 weeks. The resulting cortical neurons demonstrated the capacity to generate action potentials.
The three-dimensional model was used to examine the developmental effects of cocaine. These structures were highly sensitive to disruption by cocaine, which inhibited the proliferation of Pax6+ neuroepithelial cells while causing premature neuronal differentiation.
The current in vitro models mimic the spatial and temporal sequence of events in human neocortical development, thereby providing an efficient and inexpensive means of modeling the human cortex in vitro. These techniques can be used to develop unique strategies for mechanistic and translational studies of neocortical development and disruption of cerebral cortical organization. For example, these model systems could be used to efficiently test chemical agents for potential toxicity for the human cortex or for testing drugs to alleviate or prevent traumatic brain injury.
Research supported by the IRP of NIDA, NIH.
M. Gao and L. Zhao
Department of Neurosurgery, State University of New York, Upstate Medical University, Syracuse, NY, USA.
Stem cell factor (SCF) and granulocyte colony-stimulating factor (G-CSF) are the essential hematopoietic growth factors to regulate hematopoietic stem cell survival, growth, and differentiation. Accumulating evidence has revealed that SCF and G-CSF also contribute to neuronal plasticity. We have recently demonstrated that SCF in combination with G-CSF (SCF+G-CSF) increases axonal sprouting, dendritic branching, and synaptogenesis in the brain of chronic stroke. However, the mechanism underlying the SCF+G-CSF-induced neuronal network reorganization remains poorly understood. Neurite outgrowth is the initial process for neurons to build the neuronal networks. Here we have determined the involvement of mitogen-activated protein kinase kinase/extracellular signal regulated kinase/tumor protein 53 (MEK/ERK/p53) signaling in SCF+G-CSF-regulated neurite outgrowth using primary neuronal cultures. We observed that SCF+G-CSF enhanced neurite extension by activating MEK/ERK signaling. In addition, SCF+G-CSF displayed a synergistic effect on upregulation of p53 gene expression, and MEK/ERK signaling inhibitors significantly blocked the SCF+G-CSF-induced increase of p53 gene expression. Knocking down the translation of p53 by p53 small interfering RNAs (siRNAs), the SCF+G-CSF-induced enhancement of neurite outgrowth was eliminated. These data suggest that MEK/ERK/p53 signaling is required for SCF+G-CSF-promoted neurite outgrowth. This study demonstrates a novel role for p53, which is distinct from its proapoptotic effects on supporting neurite outgrowth in the setting of SCF+G-CSF treatment.
This study was supported by The National Institutes of Health, National Institute of Neurological Disorders and Stroke (NINDS), R01 NS060911.
X. Gao and J. Chen
Spinal Cord and Brain Injury Research Group, Stark Neuroscience Research Institute, Department of Neurosurgery, Indiana University, Indianapolis, IN, USA
Traumatic brain injury (TBI) causes significant cell death and tissue lesion in the neocortex, leaving many patients with substantial motor disability and cognitive impairment. At present, there are no clinically demonstrated FDA-approved drug therapies for treatment of TBI patients that reduce the neurological injuries.
The discovery that somatic mammalian cells can be epigenetically reprogrammed to induced pluripotent stem cells (iPSCs) through the exogenous expression of octamer binding transcription factor 4 (Oct4), sex-determining region Y box 2 (Sox2), Krüppel-like factor 4 (Klf4), and c-Myc (OSKM) has demonstrated a new way for cell-replacement therapy in regenerative medicine. This novel technology has opened new therapeutic opportunities to generate stem cells in any area for cell replacement therapy in a number of disorders. In the case of TBI, there is an abundance of proliferating glia located around the injury area. These glia represent a potential cellular reservoir for restoration of brain function following injury if they could be reprogrammed into iPSCs in vivo.
Here we show that retroviral-mediated transcription factor expression in the reactive glial cells cooperatively reprogrammed mainly the microglia into induced pluripotent stem cells (iPSCs) in the adult neocortex following TBI. These iPSCs further differentiated into neural stem cells, expressing nestin and forming neural tube-like structures in the injury area. These neural stem cells derived from glia reprogrammed iPSCs further differentiated into neurons. These new neurons developed typical neuronal morphology, sequentially expressed neuron markers, and exhibited function with action potentials. These results indicate that we were able to reprogram reactive glia into iPSCs in vivo and produce new neurons in the cortex following traumatic brain injury. This study suggests a novel strategy for brain repair through reprogramming reactive glia resident in the injury area.
R. Gonzalez,* I. Garitaonandia,* A. Ostrowska,* T. Abramihina,* G. Wambua,* M. Poustovoitov,* A. Noskov,* A. Crain,† C. R. S. McEntire,† T. Chu,* L. C. Laurent,§ J. D. Elsworth,¶ E. Y. Snyder,† D. E. Redmond,‡¶ and R. Semechkin*
*International Stem Cell Corporation, Carlsbad, CA, USA
†Sanford-Burnham Medical Research Institute, La Jolla, CA, USA
†Axion Research Foundation, Hamden, CT, USA
§Department of Reproductive Medicine, UC San Diego, La Jolla, CA, USA
¶Departments of Psychiatry and Neurosurgery, Yale University School of Medicine, New Haven, CT, USA
Neural cell transplantation has attracted considerable interest as a promising therapeutic alternative for patients with Parkinson's disease (PD). Clinical studies have shown that grafted fetal neural tissue can achieve considerable biochemical and clinical improvements in PD. However, the source of fetal tissue grafts is limited and ethically controversial. Human parthenogenetic stem cells offer a good alternative because they are derived from unfertilized oocytes without destroying viable human embryos and can be used to generate an unlimited supply of neural stem cells for transplantation. We have previously reported that human parthenogenetic stem cell-derived neural stem cells (hpNSCs) successfully engraft, survive long term, and increase brain dopamine levels in rodent and nonhuman primate models of PD. Here we report the interim results of an ongoing long-term (12-month) transplantation study of hpNSCs in 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP)-lesioned African green monkeys with moderate to severe clinical Parkinsonian symptoms. hpNSCs manufactured under current good manufacturing practice (cGMP) conditions were injected bilaterally into the striatum and substantia nigra of immunosuppressed MPTP-lesioned African green monkeys with moderate to severe clinical Parkinsonian symptoms. Behavioral changes and motor movements were evaluated against sham vehicle control based on a Parkinsonian summary score (parkscore). Additionally, necropsy, histopathology, and biodistribution analysis were performed to determine the safety profile of the implanted NSCs.
D. G. Hernandez-Ontiveros, N. Tajiri, S. A. Acosta, M. M. Pabon, S. Kazutaka, I. Hiroto, Y. Kaneko, P. C. Bickford and C. V. Borlongan
Center of Excellence for Aging and Brain Repair, Department of Neurosurgery and Brain Repair, University of South Florida Morsani College of Medicine, Tampa, FL, USA
Intense military conflict worldwide urges the need for valuable clinical treatment to wounded soldiers whose impacted brains suffer multiple head injuries; if left untreated, they may lead to progressive neurodegeneration, chronic neuroinflammation, and cell death. Fatty acid translocase/cluster of differentiation 36 (FAT/CD36), a scavenger receptor of modified low-density lipoproteins (mLDLs) found in splenocytes and monocytes has been implicated in lipid metabolism, atherosclerosis, oxidative stress, the inflammatory response after stroke, and some neurodegenerative diseases. CD36 may play a key pathological role in mediating the neuroinflammatory response in a rat model of traumatic brain injury (TBI). Preliminary data implicating splenic CD36 expression after TBI and data suggesting the inhibitory action of the soluble receptor of advanced glycation end products (sRAGE) blocking CD36-mediated uptake of mLDL in various cell types have helped us to characterize pathological alterations in the acute and chronic TBI stages. Adult Sprague–Dawley rats underwent TBI using a controlled cortical impact injury model (CCI). Controls were age-matched rats receiving sham surgery. Rats from both groups were euthanized at 1, 2, 7, and 60 days postsurgery, their brains removed, processed for protein analysis and immunohistochemistry against CD36, monocyte chemotactic protein 1 (MCP-1), and ionized calcium-binding adapter molecule 1 (Iba-1) for microglia. We observed brain colocalization of CD36, MCP-1, and Iba-1 on impact cortical area, significant increases of CD36 and MCP-1 positive cells in the ipsi versus contra hemispheres of TBI versus sham groups, but no significant increases of Iba-1-expressing cells over time. Immunoblotting studies support overexpression of CD36 in the brain and spleen at acute post-TBI time points versus sham-treated animals. Our supporting data suggests that increased CD36 expression in spleen and brain may contribute to the inflammatory response after TBI.
Y.-C. Hsieh,* N.-F. Chi,† J.-S. Jeng,† H.-J. Lin,§ C.-J. Hu,† S.-C. Tang,† L.-M. Lien,¶ G.-S. Peng,# Y.-R. Chen,** F.-I. Hsieh,** and H.-Y. Chiou**
*PhD Program for Neural Regenerative Medicine, College of Medical Science and Technology, Taipei Medical University, Taipei, Taiwan
†Department of Neurology, Taipei Medical University Hospital and Shuang Ho Hospital, Taipei, Taiwan
†Stroke Center and Department of Neurology, National Taiwan University Hospital, Taipei, Taiwan
§Department of Neurology, Chi-Mei Medical Center, Tainan, Taiwan
¶Department of Neurology, Shin Kong Wu Ho-Su Memorial Hospital, Taipei, Taiwan
#Department of Neurology, Tri-Service General Hospital, Taipei, Taiwan
**School of Public Health, College of Public Health and Nutrition, Taipei Medical University, Taipei, Taiwan
Stroke is the third most common cause of death and the leading cause of adult disability in Taiwan. The majority of strokes are ischemic. Excessive oxidative stress is a major critical challenge in the pathogenesis of stroke. The redox-sensitive transcription factor nuclear factor erythroid 2-related factor 2 (Nrf2), targeted with Kelch domain-like erythroid cell-derived protein with cap ‘n’ collar (CNC) homology (ECH)-associated protein (Keap1), plays a key role in orchestrating cellular antioxidant defenses and maintaining redox homeostasis. Cells have evolved endogenous defense mechanisms against sustained oxidative stress, and Nrf2 regulates antioxidant response element (ARE)-mediated expression of detoxifying and antioxidant enzymes. Thus, diminishing Nrf2/ARE activity might contribute to increased oxidative stress and mitochondrial dysfunction in the vasculature leading to endothelial dysfunction and abnormal angiogenesis observed in ischemic stroke patients. The purpose of this study is to investigate the association between the candidate genes on Nrf2-Keap1 pathway and ischemic stroke risk.
A total of 493 ischemic stroke patients, confirmed by computerized tomographic (CT) scan and/or magnetic resonance imaging (MRI) and 493 age- and gender-matched healthy controls will be recruited in our study. Genotyping of the candidate genes on the Nrf2-Keap1 pathway, including NRF2, KEAP1, nicotinamide adenine dinucleotide (phosphate) [NAD(P)H] dehydrogenase, quinone 1(NQO1), and Sulfiredoxin 1(SRXN1) will be conducted using iPLEX Gold assay MassARRAY (Sequenom, Inc., San Diego, CA, USA). NQO1 rs10517 polymorphism showed a significant correlation with susceptibility of ischemic stroke. Compared with study subjects who carried less than one risk genotype of NRF2, KEAP1, NQO1, and SRXN1, those with two and greater than three risk genotypes of NRF2, KEAP1, NQO1, and SRXN1 had 1.4- and 1.8-fold risk of ischemic stroke, respectively, revealing a significant dose-response relationship between the number of risk genotypes of these genes and the risk of ischemic stroke.
Our findings support that susceptible genes in redox pathway were significantly associated with increased risk of ischemic stroke.
K. Iwatsuki,* T. Yoshimine,* Y. Sankai,† F. Tajima,† M. Umegaki,* Y.-I. Ohnishi,* M. Ishihara,* K. Ninomiya,* and T. Moriwaki*
*Department of Neurosurgery, Osaka University Medical School, Osaka, Japan
†Center for Cybernics Research, University of Tsukuba, Ibaraki, Japan
†Department of Rehabilitation Medicine, Wakayama Medical University, Wakayama, Japan
The efficacy of olfactory mucosa autograft (OMA) for chronic spinal cord injury has been reported. New activity in response to voluntary effort has been documented by electromyography (EMG), but the emergence of motor evoked potential (MEP) reflecting electrophysiological conductivity in the central nervous system, including the corticospinal pathway, after OMA, and the best indications for OMA, have not been clarified. Here, we report the emergence of MEPs after OMA and offer recommendations for appropriate indications based on the presence of involuntary muscle spasm (IMS). We used analysis of MEP to examine the efficacy of OMA for patients with complete paraplegia due to chronic spinal cord injury. To clarify the indications for OMA, we investigated the association of IMS and efficacy of OMA.
Four patients, three men and one woman, were enrolled. The mean age of the cases was 30.3 ± 9.5 years (range, 19 to 40 years). All four cases were American Spinal Injury Association (ASIA) grade A. The mean duration from injury to OMA was 95.8 ± 68.2 months (range, 17 to 300 months). Samples of olfactory mucosa were removed, cut into smaller pieces, and grafted into the sites of spinal cord lesions after laminectomy. Postoperative subcutaneous fluid collection, postoperative meningitis, postoperative nosebleed, postoperative infection in the nasal cavity, impaired olfaction, neoplastic tissue overgrowth at the autograft site, new sensory disturbance, and involuntary muscle spasm were investigated as safety issues. Improvements in ASIA grade, variations in ASIA scores, EMG, somatosensory evoked potentials (SSEPs), and improved urological function were evaluated as efficacy indicators. There were no serious adverse events in this series. In two of the four cases, an improvement in motor function below the level of injury was recognized. In one, the motor score was 50 until 16 weeks after surgery, and it increased to 52 from 20 weeks after surgery. In the other, the motor score was 50 until 20 weeks after surgery, and it increased to 52 at 24 weeks after surgery with a further increase to 54 at 48 weeks after surgery. The emergence of MEPs was recognized in the latter case at 96 weeks after surgery. The other two cases had no improvement in ASIA motor score. Both of the cases that showed improvements in the ASIA motor scores exhibited relative IMS compared with those who had no ASIA motor score recovery.
We recognized the emergence of MEPs in a case with complete paraplegia due to chronic spinal cord injury after OMA. Motor recovery may be associated with IMS in patients who demonstrate restored function.
D. Jarocha,* O. Milczarek,† S. Kwiatkowski,† and M. Majka*
*Department of Transplantation, Polish-American Institute of Pediatrics, Jagiellonian University School of Medicine, Cracow, Poland
†Department of Children Surgery, Polish-American Institute of Pediatrics, Jagiellonian University School of Medicine, Cracow, Poland
Cell therapy is an evolving modality for spinal cord injury (SCI) patients. A 15-year-old girl with total spinal cord interruption at the Th2–Th3 level was enrolled to experimental cell therapy of a single dose of autologous bone marrow-derived cells (BMCs) and multiple doses of mesenchymal stem cells (MSCs) with intense neurorehabilitation including physiotherapy. The patient exhibited Th1 sensation level and paraplegia with sphincter palsy and was without the ability to fix the trunk, at admission, 3 months after SCI. The girl was scored American Spinal Injury Association (ASIA) A. Neurophysiology examination [electromyography and electronystagmogram (EMG and ENG)] showed bilateral axonal damage of both motor and sensory neural fibers with no motor unit potential and peripheral motor nerve conduction of lower extremities. The standard therapy did not bring any improvement.
Autologous BMCs were injected intravenously (3.2×109) and intraspinally (0.5×109) 10 weeks after the SCI followed by four rounds of MSCs, at 1 month (13×106), 5 months (36.5×106), 8 months (36×106), and 1 year later (36×106). There were neither complications after any of the transplantation procedures nor any side effects during 1.5 years of therapy follow-up.
We observed continuous improvement seen as a decrease in the sensation level from Th1 through Th6–Th7, L1 to L2/3 after four MSC infusion rounds. Ability to control the trunk was fully restored. Muscle strength at the left lower extremity improved from plegia to deep paresis (1° in Lovett scale). Ability to move her lower extremities against gravity supported by the movements in her quadriceps was restored. EMG and ENG examination demonstrated slightly decreased bilateral surface sensation at the level of Th8/Th9 and L1/L2. The patient's minimal motor unit potential of lower extremities was restored with predominance on the left side. Peripheral motor nerve conduction of the lower extremities became almost normal. Bladder and rectal bulb filling sensation was restored as well as the partial sphincter function. The patient gained the ability to stand in a standing frame and supported walking in hips and knee ortheses. Her ASIA score changed from A through B, B/C, C to C/D after four MSC infusions, with the point sum change from 112 through 152, 187, 210 to 216. Quality of life in the spinal cord independence measure (SCIM) scale increased from 33, through 50, 56, and 63 to 67 points. The hip muscle's strength is increasing.
Our results support the notion about using MSC transplantation for the treatment of SCI.
C. Jia, J. Li, H. Wang and H. Wang
Department of Chemistry, Chemical Biology and Biomedical Engineering, Stevens Institute of Technology, Hoboken, NJ, USA
Tissue-engineered constructs have shown great promise to replace the need of autografts, which are restricted from a wide application as a result of its limited availability and high donor site-associated morbidity. In consideration of the fact that cells in most natural tissues obtain their nutrients and oxygen or remove metabolic byproducts within 200 μm of the nearest vasculature, prompt vascularization becomes crucial for both in vitro tissue formation and in vivo survival of tissue-engineered constructs after implantation. Despite the diverse endeavors, it remains a big challenge to create a functional vasculature structure in the constructs. In recognition of the challenge together with the attempt to develop a controllable vascularization approach, we therefore propose a templateguided strategy to form microvascular networks (5–10 μm in diameter). To obtain the templates, two methods were developed, that is, nearfield electrostatic prototyping and microetching and used to fabricate biodegradable microfiber scaffolds with two distinct patterns: 1) wellorganized and 2) random microfiber networks. Mouse endothelial cells were cultured onto both microfiber networks, which initially served as a template to support the growth of endothelial cells and then degraded to form an empty endothelium lumen. Immunostaining for CD31 and tight junctions [occludin/vascular endothelial (VE)-cadherin] revealed the proper formation of cell–cell connection. The formation of luminal structure was confirmed by confocal microscopy. Further assembly of such microfiber-guided microvascular networks into three-dimensional tissue constructs using our layer-by-layer approach led to the formation of vascularized skin grafts.
V. Joers,*† K. Dilley,* S. Rahman,* C. Jones,*† J. Shultz,* and M. E. Emborg*†‡
*Wisconsin National Primate Research Center, University of Wisconsin-Madison, Madison, WI, USA
†Neuroscience Training Program, University of Wisconsin-Madison, Madison, WI, USA
†Department of Medical Physics, University of Wisconsin-Madison, Madison WI, USA
Cardiac sympathetic neurodegeneration and dysautonomia are found in sporadic and familial cases of Parkinson's disease (PD) and are currently proposed as prodromal signs of PD. Due to the lack of animal models to study these symptoms and to test neuroprotective strategies, we developed a nonhuman primate model of cardiac dysautonomia. Animals were treated with systemic 6-hydroxydopamine (6-OHDA) and showed decreased cardiac uptake of the radioligand (C11)MHED (meta-hydroxyephedrine; an epinephrine analogue), suggesting cardiacsympathetic denervation as well as decreased circulating catecholamines. Here we report the postmortem characterization of the model. Ten adult rhesus monkeys (5–9 years old; 6–10 kg) were used in this study. Five animals received 50 mg/kg of 6-OHDA IV, and five were used as age-matched controls. Three months post neurotoxin, the animals were euthanized by transaortic perfusion of phosphate-buffered saline (PBS) followed by 4% paraformaldehyde (PFA). Hearts were postfixed for 24–48 h in 4% PFA and preserved further with 70% ethanol. Hearts were cut in transverse orientation into 4 mm sections and blocked in paraffin. Adrenals were postfixated in 10% neutral buffered formalin (NBF), trimmed and blocked in paraffin. All tissue was sectioned on a standard rotary microtome at 5 μm thick. Cardiac tissue was processed by immunohistochemistry to visualize tyrosine hydroxylase (TH; catecholaminergic marker), protein gene protein 9.5 (PGP9.5; pan-neuronal marker), human leukocyte antigen (HLA-DR; inflammatory marker), nitrotyrosine (oxidative stress marker), and α-synuclein (neuronal protein accumulated in PD), and counterstained with hematoxylin. Adrenal glands were stained for TH and aromatic L-amino acid decarboxylase (AADC). Quantification of immunoreactivity (ir) was performed by an investigator blinded to the treatment groups using NIH ImageJ software [for cardiac bundles and adrenals, area above threshold (AAT) and optical density (OD)] and MBF Biosciences Stereo Investigator (for cardiac fibers, area fraction fractionator probe). Sympathetic cardiac nerve bundle analysis showed a significant reduction in global cardiac TH-ir (OD p = 0.009; AAT p = 0.009) in 6-OHDA-treated animals compared to controls with an average 28% and 34% deficit, respectively. The global TH-ir fiber density was significantly reduced (70%) in 6-OHDA animals compared to controls (p = 0.001). Quantification of PGP9.5-ir cardiac fibers showed a 47% deficit in 6-OHDA monkeys compared to controls and correlated with TH-ir fiber area (R2= 0.9079; p< 0.0001). Semiquantitative evaluation of HLA-DR and nitrotyrosine immunostaining with a novel rating scale did not show significant changes 3 months post-toxin. Cardiac nerve α-synuclein-ir was reduced in 6-OHDA treated monkeys, although aggregated proteinase-K-resistant α-synuclein was not observed in epicardial nerve bundles. In the adrenal medulla, 6-OHDA monkeys had significant reduced TH-ir (OD p =0.009; AAT p = 0.005) and AADC-ir (AAT p =0.013). Our results confirm that systemic 6-OHDA dosing to nonhuman primates induces cardiac sympathetic neurodegeneration and decreased catecholamine production by the adrenal medulla and suggests that this model can be used as a platform to evaluate disease-modifying strategies aiming to induce peripheral neuroprotection.
B. Karain,* D. Xu,† J. A. Bellone,† R. E. Hartman,† and W-X. Shi*†
*Department of Basic Sciences, Loma Linda University Health Schools of Medicine, Pharmacy, and Behavioral Health, Loma Linda, CA, USA
†Department of Pharmaceutical and Administrative Sciences, Loma Linda University Health Schools of Medicine, Pharmacy, and Behavioral Health, Loma Linda, CA, USA
†Department of Psychology, Loma Linda University Health Schools of Medicine, Pharmacy, and Behavioral Health, Loma Linda, CA, USA
The rat globus pallidus (GP) is homologous to the primate globus pallidus externus. Studies in rats anesthetized with injectable anesthetics suggest that GP neurons can be functionally classified into subtypes, and each plays a unique role in Parkinson's disease. In this study, we examined the electrophysiology of GP neurons using the inhalational anesthetic isoflurane, which offers more constant and easily regulated levels of anesthesia than injectable anesthetics. We also tested whether subtypes of GP neurons respond differently to dopamine depletion. GP neurons were recorded extracellularly in isoflurane or ketamine-anesthetized rats. Cortical local field potentials were simultaneously recorded to determine the functional connectivity of individual GP neurons to the cortex. According to studies in ketamine-anesthetized rats, GP neurons can be divided into Type I and Type II. Spikes in Type I neurons have a negative initial phase, whereas those in Type II cells have a positive initial phase. In this study, all GP neurons recorded in isoflurane-anesthetized rats fired Type II spikes. This lack of Type I cells is apparently not due to the use of isoflurane since all GP neurons recorded in ketamine-anesthetized rats in this study also fired Type II spikes. Our evidence further suggests that the spike shape depends critically on the recording electrode and is not reliable in distinguishing Type I and Type II neurons. Thus, while all GP cells fired Type II spikes when recorded with our routinely used small-tip (<1 μm), high-impedance electrodes, more than half of GP neurons displayed Type I spikes when large-tip (>2 μm), low-impedance electrodes were used. GP neurons can also be classified as positively coupled (PC), negatively coupled (NC), or uncoupled (UC) based on their functional connectivity to the cortex. All three subtypes were found under isoflurane anesthesia. However, NC cells were relatively fewer than PC and UC cells. NC cells also fired faster and showed reduced firing variability compared to PC and UC cells. Lesions of dopamine neurons using 6-hydroxydopamine (6-OHDA) increased NC cells and decreased UC cells. Although the number of PC cells was unchanged, their phase relationship to the cortex was significantly altered. In control rats, cortical activity led GP activity in 56% of PC cells. In lesioned animals, cortical activity led GP activity in 91% of PC cells. If PC and NC cells are coupled to the cortex through the subthalamic nucleus and striatum, respectively, these results suggest that dopamine depletion alters information transmission through both nuclei. To summarize, PC, NC, and UC cells are present in isoflurane-anesthetized rats. These cells differ from each other not only in functional connectivity to the cortex, but also in firing rate, firing regularity, and responses to 6-OHDA lesions. These results support the suggestion that PC, NC, and UC GP neurons play different roles in the pathophysiology of Parkinson's disease.
S-H. Kim,* A. J. Smith,* B. Giunta,† and R. D. Shytle*
*Center of Excellence for Aging and Brain Repair, Department of Neurosurgery and Brain Repair, University of South Florida Morsani College of Medicine, Tampa, FL
†Neuroimmunology Laboratory, Department of Psychiatry and Behavioral Neurosciences, University of South Florida Morsani College of Medicine, Tampa, FL
HIV-associated neurocognitive disorder (HAND) caused by neuronal loss is common among HIV-infected patients. Tat protein, transactivator of transcription, is released from HIV-1-infected cells and is the key factor that causes neuronal dysregulation. Glutathione (GSH) is the major antioxidant in the brain. It acts as a substrate to neutralize hydroxyl radicals via glutathione peroxidase (GPx) and detoxifies xenobiotics by acting as a substrate for glutathione-S-transferase (GST). In this study, HIV-1 Tat-induced oxidative stress was measured and the therapeutic effect of MSM, a naturally occurring organic sulfur compound, was assessed in murine neuroblastoma 2a (N2a) cells.
To accomplish the hypothesis, recombinant HIV-1 Tat protein and MSM were coadministered to N2a neuronal cells. Cell survival rate [3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay], reactive oxygen species (ROS), and nitric oxide (NO) release were measured to assess Tat-induced oxidative stress. In addition, GSH, GPx, and GST were measured to determine the antioxidant effect of MSM on disrupted redox homeostasis.
HIV-1 Tat administration leads to elevated ROS and NO levels but did not show significant differences in cell survival rate compared to untreated cells. Also, Tat decreased GSH levels, GPx, and GST activities. MSM treatment decreased ROS and NO production, but did not change cell survival rate either. MSM treatment also replenished GSH levels, GPx, and GST activities compared to Tat-treated cells.
These results suggest that HIV-1 Tat disturbs redox homeostasis in N2a cells by inducing ROS and NO production but did not lead to cell death in this study. In addition, MSM showed a neuroprotective effect against Tat-induced oxidative stress by boosting antioxidant capacity in neuronal cells by replenishing glutathione levels and related enzyme activities. Further studies will be needed to characterize the functional consequences of this redox stabilization with MSM.
BG is supported by NIMH/NIH grant (1R01MH098737–02) (PI). Recombinant HIV-1 Tat protein was obtained from NIH reagent aid program.
A. A. Kindberg,* C.-T. Lee,* R. M. Bendriem,* M. P. Williams,* B. K. Harvey,† C. T. Richie,† and W. J. Freed*
*Cellular Neurobiology Research Branch, Intramural Research Program, National Institute on Drug Abuse, National Institutes of Health (NIH), Department of Health and Human Services (DHHS), Baltimore, MD, USA
†Optogenetics and Transgenic Technology Core, Intramural Research Program, National Institute on Drug Abuse, National Institutes of Health (NIH), Department of Health and Human Services (DHHS), Baltimore, MD, USA
The practical utility of human pluripotent stem cells (hPSCs) for clinical applications depends on our ability to control their differentiation to specific somatic cell types, such as mesodiencephalic dopaminergic (mDA) neurons. Through analysis of copy number variations (CNVs) on chromosome 17 in a series of hPSC lines, including one trisomy 17 line, we identified duplications at 17q21.31, containing the Wingless-related mouse mammary tumor virus integration site 3 (WNT3)-WNT9B gene cluster, in hPSCs with enhanced mDA differentiation. In order to examine the roles of CNVs at 17q21.31/WNT3-WNT9B in the differentiation of mDA neurons, we employed lentiviral vectors to overexpress WNT3 and WNT9B, or to silence WNT3 and WNT9B expression through delivery of short hairpin RNAs (shRNAs) at various stages of mDA differentiation.
hPSC lines with amplified WNT3 and WNT9B exhibited three properties that differed from those of control hPSC lines: (i) enhanced proliferation in the undifferentiated state, indicated by increased percentages of bromodeoxyuridine (BrdU)-labeled octamer-binding transcription factor 3/4 positive (OCT3/4+) cells; (ii) accelerated loss of pluripotency, as indicated by rapid decreases in OCT3/4+ cells after basic fibroblast growth factor (bFGF) withdrawal; and (iii) accelerated mDA differentiation, indicated by earlier expression of LIM homeobox transcription factor 1 α (LMX1A), an essential transcription factor in mDA differentiation, and more rapid development of tyrosine hydroxylase positive (TH+) dopaminergic neurons.
In hPSCs with amplified WNT3-WNT9B, WNT3 knockdown reversed the enhanced proliferation in the undifferentiated state and reversed enhanced mDA differentiation. In contrast, WNT9B knockdown reversed the accelerated loss of pluripotency in WNT3–WNT9B amplified hPSCs. Conversely, WNT3 overexpression in control hPSC lines caused increases in undifferentiated proliferation and mDA differentiation, while WNT9B overexpression caused an acceleration of departure from pluripotency.
WNT3 and WNT9B act mainly via the β-catenin-dependent (canonical) and the Rho/C-Jun N-Terminal Kinase (JNK) (noncanonical) signaling pathways, respectively. Therefore, we tested the effects of DKK-1, an inhibitor of canonical β-catenin signaling, and SP600125, an inhibitor of noncanonical Rho/JNK signaling. DKK-1 treatment, but not SP600125, reversed both the enhanced undifferentiated proliferation and accelerated mDA differentiation in cells with amplified WNT3/WNT9B. LMX1A is known to be upregulated by WNT/β-catenin signaling and coordinates induction of the mDA phenotype through several downstream transcription factors including muscle segment homeobox (MSH) homeobox 1 (MSX1), nuclear receptor related 1 (NURR1), and paired-like homeodomain 3 (PITX3). DKK-1 treatment, but not SP600125, during late differentiation decreased the expression of mDA markers, including TH, neuronal class III β tubulin, and LMX1A, and its downstream transcription factors. WNT3-knockdown in abnormal chromosome 17 lines decreased LMX1A expression and differentiation of mDA neurons, while overexpression of WNT3 in control hPSCs resulted in the opposite effect, enhancing mDA differentiation.
Taken together, these results suggest that amplification of WNT3 and WNT9B have multiple, distinct roles in the differentiation of mDA neurons from hPSCs. Enhanced noncanonical Rho/JNK pathway signaling via WNT9B promotes early differentiation and departure from pluripotency, while enhanced canonical/β-catenin signaling through WNT3 accelerates subsequent mDA specification via upregulation of LMX1A.
Research supported by the IRP of NIDA, NIH, DHHS.
M. Kodali,*†‡ B. Hattiangady,*†‡ J. Watanabe,* X. Rao,*†‡ and A. K. Shetty*†‡
*Institute for Regenerative Medicine, Texas A&M Health Science Center College of Medicine, Temple, TX, USA
†Research Service, Olin E. Teague Veterans' Medical Center, Temple, TX, USA
†Department of Molecular and Cellular Medicine, Texas A&M Health Science Center College of Medicine, College Station, TX, USA
Traumatic brain injury (TBI) occurs when a sudden trauma causes a closed or a penetrating head injury. Multiple previous reports have documented the immediate behavioral consequences, brain plasticity, or recovery, after TBI. While clinical reports imply that an incidence of mild to moderate TBI can lead to long-term behavioral complications, such as cognitive impairment and depression, such lasting behavioral alterations have not been critically assessed in animal models. Therefore, in this study, we performed rigorous neurobehavioral studies 8 months after the induction of a unilateral, moderate controlled cortical impact (CCI) TBI using a mouse model. Adult C57BL/6 male mice (n = 10) received a unilateral impact on a cortical region overlying the hippocampus and located between the bregma and lambda at 2 mm lateral to the midline, using a 3-mm impactor tip set to deliver a deformation of 0.8 mm depth with a velocity of 4.5 m/s and a dwell time of 250 ms. An additional group of age-matched mice (n = 9) received sham-TBI surgery and served as controls. Animals were subjected to a battery of behavioral tests 8 months after the TBI/sham surgery to elucidate alterations in cognitive and mood function. A hippocampus-dependent water maze test revealed the presence of spatial learning and memory deficits in mice receiving TBI, which was evidenced through increased swim path lengths to reach the submerged platform and decreased swim path efficiency over training sessions and a lack of preference to the platform quadrant in the probe test conducted 24 h after the last learning session. Comparison of swim speeds between groups did not reveal any motor dysfunction in mice with TBI however. Analysis with an alternative behavioral test that involves no stress (object location test) confirmed the hippocampus-dependent spatial memory dysfunction in mice receiving TBI, which was exemplified by a lack of preference of mice toward an object that was displaced to a novel location. Characterization using a novel object recognition test (a memory test that critically requires the integrity of the perirhinal cortex) in addition demonstrated the presence of hippocampus-independent memory dysfunction in mice receiving TBI, which was typified by a lack of preference toward the novel object vis-a-vis the familiar object. Furthermore, mice receiving TBI failed to discriminate the reorganized object positions in an object in place test, which requires normal circuit function between the hippocampus, the perirhinal cortex, and the medial prefrontal cortex. Additionally, mice receiving TBI exhibited impairments in pattern separation function (which requires maintenance of dentate neurogenesis at normal levels) in a behavioral test examining their ability for discriminating distinct familiar objects placed on a different floor pattern. Investigation of mood function using forced swim and novelty suppressed feeding tests revealed increased depressive-like behavior in mice that underwent TBI. This was epitomized by an increased amount of time spent in immobility (floating) in the forced swim test and greater latency values to eat food in a novelty suppressed feeding test. Immunohistochemical studies of brain tissues are currently underway to ascertain the structural changes underlying these cognitive and mood impairments. Collectively, our wide-ranging neurobehavioral analyses provide novel evidence that an episode of moderate trauma impacting the cerebral cortex over and around the hippocampus can induce lasting hippocampus-dependent and hippocampus-independent memory impairments as well as depression.
A. L'Huillier* and Y. Shi*†
*Child Health Institute, Rutgers University, New Brunswick, NJ, USA
†Institute of Health Science, Chinese Academy of Science, Shanghai, China
Mesenchymal stem cells (MSCs) are tissue resident, multipotent stem cells capable of self-renewal and differentiation into a number of cell types, including adipocytes, chondrocytes, and osteoblasts, and have been noted for their immunosuppressive characteristics. We have previously identified that while cytokine-stimulated mouse MSCs primarily owe their immunosuppressive capabilities to the expression of inducible nitric oxide synthase (iNOS), human MSCs achieve this through the expression of indoleamine 2,3 dioxygenase (IDO). Through these mechanisms, MSCs help to reduce inflammation, preventing secondary damage and allowing tissue repair to occur. IDO is thought to exert its suppressive effects via tryptophan depletion, but recent evidence suggests that IDO-derived metabolites of the kynurenine pathway may exert direct effects on lymphocytes. We report here that human MSCs that have been activated with cytokines reduce cluster of differentiation 25 (CD25) expression on T-cells and potently inhibit lymphocyte proliferation induced by anti-CD3 activation in an IDO-dependent manner, as evidenced by the reversal of these observations with the addition of the IDO-specific inhibitor 1-MT. L-kynurenine, the product of IDO catalysis, similarly reduces CD25 expression and proliferation of T-cells in a dose-dependent manner. While we did not observe an increase in the proportion of regulatory T-cells (Tregs) with L-kynurenine treatment alone, L-kynurenine combined with transforming growth factor (TGF)-β1 induced a much higher proportion of Tregs compared to TGF-β1 treatment alone. Additionally, kynurenine must be present during T-cell receptor (TCR) activation to exert its effects. While all T-cells lost CD25 expression with high doses of L-kynurenine, Tregs preferentially gained CD25 expression with low doses that correspond to doses that enhance the Treg population, suggesting differential effects of IDO and L-kynurenine on these subsets of T-cells. By reducing inflammation and with its downstream metabolites known to exert effects as neurotransmitters, MSC-derived L-kynurenine represents a potential key player in neural repair.
J. Lebkowski,* R. Fessler,† L. Jones,† G. Steinberg,§ S. McKenna,¶ D. Apple,# and E. Wirth III*
*Asterias Biotherapeutics, Inc., Menlo Park, CA, USA
†Rush University Medical Center, Chicago, IL, USA
†Craig Neilsen Foundation, Encino, CA, USA
§Stanford University, Stanford, CA, USA
¶Santa Clara Valley Medical Center, Santa Clara, CA, USA
#Shepherd Center, Atlanta, GA, USA
Spinal cord injury (SCI) produces numerous clinical sequelae, including impaired limb function, spasticity, autonomic dysfunction, thromboses, increased infections, decubitus ulcers, and chronic pain, which can significantly impact quality of life and be life threatening.
In animal models, transplantation of oligodendrocyte progenitors (OPCs) into the lesion site elicits repair through multiple mechanisms. Methods have now been developed to differentiate human embryonic stem cells (hESCs) into OPCs under current good manufacturing practice (cGMP) at scales suitable for clinical development. These OPCs are referred to as AST-OPC1 [formerly Geron OPC1 (GRNOPC1)]. Preclinical studies using AST-OPC1 have shown that these cells survive, migrate throughout the injury site, reduce parenchymal cavitation, induce the presence of myelinated fibers in the injury site, and improve locomotor activity. No toxicity, tumors, or allodynia were induced by AST-OPC1.
A phase 1 clinical trial assessing the safety of AST-OPC1 was initiated in subjects with neurologically complete T3–T11 thoracic spinal cord injury (SCI). Subjects enrolled in the trial consented to two protocols: 1) the primary protocol under which subjects were followed for 1 year, and 2) a long-term follow-up protocol under which subjects are being followed for an additional 14 years. Five patients were enrolled from October 2010 to November 2011. All subjects were administered a low dose of 2 × 106 AST-OPC1 in a separate dedicated surgical procedure within 14 days of injury using a syringe positioning device designed to facilitate controlled dose delivery. Subjects received a low dose of tacrolimus for 46 days, which was tapered and eventually discontinued at day 60. The primary endpoint of the study was safety, with the secondary endpoint being neurological function. Safety was assessed with respect to AST-OPC1 itself, the procedure to deliver the cell product and the transient immunosuppression used subsequent to implantation. Multiple magnetic resonance images (MRIs) and neurological exams were performed during the first year of study to assess safety of AST-OPC1.
To date, all five patients have been followed for over 2 years. There have been no serious adverse events related to AST-OPC1, tacrolimus, or the injection procedure. There were five adverse events (AEs) judged to be possibly related to AST-OPC1. One of these AEs was a brief mild elevation of body temperature. The remaining four AEs all occurred in one subject and were primarily neuropathic pain reported as a burning sensation in the trunk and lower extremities. Pain of this type and distribution is also a common complication of SCI. Regarding the delivery procedure or use of tacrolimus, all possibly related adverse events were assessed as grade 1 or 2. Serial MRI scans indicate that lesion cavity formation at the AST-OPC1 injection sites was substantially reduced through 2 years of follow-up in four of five subjects. In addition, there were no reports of abnormal cyst formation or enlarging masses at the injection sites on MRI scans. There were no unexpected changes in neurological function. The data to date suggest that AST-OPC1 can be safely administered to patients in the subacute period after spinal cord injury. Future plans include dose escalation and inclusion of subjects with cervical injuries where the anatomy and outcome measures provide a superior opportunity to measure potential activity of the cells.
C.-T. Lee, A. A. Kindberg, R. M. Bendriem, T. Drgon, B. S. Mallon, G. R. Uhl, and W. J. Freed
Intramural Research Program, National Institute on Drug Abuse, NIH/DHHS, Baltimore, MD, USA
Self-renewal and differentiation capacities of human pluripotent stem cells (hPSCs) provide the foundation for numerous potential cell-based therapies. Prolonged culture of hPSCs can lead to chromosomal abnormalities, including major changes, such as trisomy of chromosomes 12, 17, and/or X, as well as more subtle genetic abnormalities. These can affect various properties of hPSC lines. We examined associations between copy number variations (CNVs) and hPSC mesodiencephalic dopaminergic (mDA) differentiation in hPSCs.
Five karyotypically normal human embryonic stem cell (hESC) lines (BG01, 02, 03, ES02, 04) and one karyotypically abnormal trisomy 17 line (BG01V2) were characterized for morphology, proliferation, cell survival, growth factor dependence, and ability to differentiate into mDA neurons. All hESC lines displayed similar survival rates, normal morphology, and well-defined colony boundaries. BG01V2 and BG03 showed enhanced proliferation, evidenced by increased percentages of octamer-binding transcription factor 3/4 positive (OCT3/4+) cells labeled by bromodeoxyuridine (BrdU). Following basic fibroblast growth factor (bFGF) withdrawal, BG01V2 and BG03 lost pluripotency more rapidly than the other cell lines. This suggests that an enhanced disposition for departure from pluripotency distinguishes BG01V2 and BG03 from the characteristic in vitro behavior of transformed or tumorigenic hESC variants, despite their increased rates of proliferation.
BG01V2 and BG03 also displayed more rapid mDA differentiation, as evidenced by expression of LIM homeobox transcription factor 1 a (LMX1A), muscle segment homeobox (MSH) homeobox 1 (MSX1), tyrosine hydroxylase (TH), and neuronal class III b tubulin. Since BG01V2 contains an extra copy of chromosome 17, enhanced differentiation of BG03 might also be linked to extra genetic material on chromosome 17. We therefore developed an a priori list of 103 genes potentially related to mDA differentiation and focused on the seven genes located on chromosome 17. Principal component analysis of hybridization intensity data from Affymetrix 6.0 arrays were used to search for evidence of CNVs on chromosome 17. A genomic duplication located between 41.3 and 42.5 MB at 17q21.31 was identified in BG03. This CNV encompasses the WNT3–WNT9 genes, two of the seven a priori chromosome 17 candidates for involvement with mDA differentiation. Additional copies of WNT3 and WNT9B in both BG01V2 and BG03 were confirmed by qPCR, and expression of both WNT3 and WNT9B mRNAs was increased during mDA differentiation.
We therefore screened 10 additional hESC and eight human induced pluripotent stem cell (hiPSC) lines for extra copies of WNT3/WNT9B, by using 14 probes across the N-ethylmaleimide-sensitive factor (NSF), WNT3, WNT9B, and golgi soluble NSF attachment protein (SNAP) receptor complex member 2 (GOSR2) region of chromosome 17. Two hESC lines, CT3 and SA01, showed increased levels of both WNT3 and WNT9B DNA, while hESC lines H1 and SA02 and hiPSC line BC1 showed increased WNT3 DNA only. Similar to BG01V2 and BG03, CT3 exhibited enhanced proliferation on the undifferentiated state in the presence of bFGF, accelerated loss of pluripotency after bFGF removal, and highly efficient mDA induction.
Therefore, amplifications of small regions of chromosome 17 containing either the WNT3 or WNT9B genes occur frequently in hPSCs. These amplifications can have the paradoxical effect of simultaneously accelerating in vitro proliferation, departure from pluripotency, and specific mDA differentiation. Detailed analysis of relatively minor genetic alterations and their functional significance at a cellular level is thus an important consideration in the potential use of hPSCs for therapeutic purposes.
Research supported by the IRP of NIDA, NIH.
J.-Y. Lee,* B. Grimmig,* A. Flowers,* C. D. Sanberg,† C. Hudson,† and P. C. Bickford*‡
*Center of Excellence for Aging and Brain Repair, Department of Neurosurgery and Brain Repair, University of South Florida Morsani College of Medicine, Tampa, FL, USA
†Natura Therapeutics, Inc., Tampa, FL, USA
†James A. Haley Veterans Hospital, Research Service, Tampa, FL, USA
Aging is associated with a decline in stem cell proliferation. We have previously shown that NT-020 (a patented proprietary formulation from Natura Therapeutics, Inc.) can improve cognitive function and increase proliferation of neural progenitors in aging rats. In this study, we examined the neurogenic niche of young and aged rats to determine if treatment with NT-020 regulated oxidative stress response pathways in neurons, astrocytes, and microglia in the dentate gyrus subgranular zone (SGZ) and the subventricular zone (SVZ), two neurogenic-rich areas of the brain. Localization of double-labeled cells—Neuron [neuronal nuclei (NeuN)], microglia [ionized calcium-binding adapter molecule 1 (IBA-1)], astrocyte [glial fibrillary acidic protein (GFAP)]with β-catenin, heme oxygenase 1 (HO-1) or nuclear factor erythroid 2-related factor 2(Nrf2)—were immunohistochemically observed in fixed tissue from adult and aged F344 rats. The primary antisera were polyclonal antibodies raised in rabbit against NeuN, IBA-1, GFAP, doublecortin (DCX), and monoclonal antibodies raised in mouse against β-catenin, HO-1, Nrf-2. The primary antiserum was diluted 1:500. The secondary antisera were incubated in a 1:500 dilution of Alexa488 anti-mouse, Alexa555 anti-rabbit IgG. The sections were coverslipped with mounting vectorshield with 4′,6-diamidino-2-phenylindole (DAPI) and then examined and photographed using a Zeiss confocal microscope to verify double labeling of immunostains. We randomly selected three sections per animal for counting for each condition. Labeling for Nrf2 and β-catenin was quantified only when located in the nuclear compartment, as these are transcription factors, and this location in the nucleus is consistent with activation of the transcription factor. Interestingly, we observed an increase in immunopositive labeling of β-catenin, HO-1, and Nrf2 in all subsets of cell types in both young and aged rats in the SGZ following NT-020 treatment. In NeuN-positive cells, there was a basal increase in nuclear β-catenin in the aged rats that was not observed in DCX-labeled cells, microglia, or astrocytes. The results suggest that NT-020 activates oxidative stress response pathways showing that Nrf2 and β-catenin translocate to the nucleus. One downstream effector of Nrf2, HO-1, was also increased. This indicates that antioxidant response pathways and stem cell pluripotency pathways are activated by NT-020, which may be involved in the mechanism by which they increase neurogenesis in aged rats. Companion studies of gene and protein expression are ongoing.
Conflict of interest: PCB is cofounder of Natura Therapeutics, Inc.
Y. Li*†
*Center for Stem Cell and Regenerative Medicine, University of Texas Health, Houston, TX, USA
†Department of Pediatric Surgery, University of Texas Medical School at Houston, Houston, TX, USA
Regenerative medicine, such as stem cells, has been considered as promising to repair or replace diseased or injured tissues, including numerous central nervous system diseases and injuries. The movement of these applications is slow and unsatisfying because of the lack of autologous, tissue-specific, and engrafting multipotent stem cells (SCs). Although embryonic and induced pluripotent stem cells (iPSCs) have been used in field studies, challenges still remain besides the ethical and technical procedures. Thus, detection of a new type of SCs, with easy accessibility, improved migration and neuronal differentiation potential, and with low tendency of tumor formation and immune rejection is needed.
My team has over 10 years of experience in the study of adult stem cells, specifically in muscle-derived stem cells. We recently discovered and identified a novel population of dedifferentiated stem cells (deSCs) from murine skeletal muscles. These deSCs can be isolated based on the Cre/Lox-β-galactosidase system that we used in our previous report. The group of specific β-galactosidase versus LacZ-positive stem cells expressed myogenic SC [paired box 7 (Pax7) and stem cell antigen 1 (Sca1)] as well as pluripotency SC markers [octamer-binding transcription factor 4 (Oct4), Nanog, sex-determining region Y box 2 (Sox2), stage-specific embryonic antigen 1 (Ssea1), chemokine C-X-C motif receptor 4 (Cxcr4), muscle segment homeobox (MSH) homeobox 1 (Msx1), and nestin]. With a time-lapse motility assay, it was confirmed that deSCs accumulated longer distances compared to control cells within the same time period, indicating the natural migration ability. These deSCs can form neurospheres spontaneously in neuronal stem cell mediums, and they also can differentiate into neuron-like cells once cultured in neuron differentiation medium. Interestingly, deSCs keep myogenic memory and can form myotubes once the neurospheres are placed into the muscle medium. These findings suggest that these deSCs are a new type of muscle-derived SCs with partial pluripotency and neurogenic differentiation, have myogenic memory and natural strong migration, and can be beneficial for the application in regenerative medicine for motor nerve or neural-muscle-related injuries and diseases. We will evaluate if these deSCs are an ideal candidate for motor neuron synapses and reinnervation of injured skeletal muscles in our future studies.
L. López-Velázquez, D. L. Haus, E. Gold, G. A. Lacuesta, J. Bustos, D. Su, H. Perez, A. J. Anderson, and B. J. Cummings
Sue & Bill Gross Stem Cell Research Center, University of California, Irvine, CA, USA
Traumatic brain injury (TBI) is an alteration in brain function caused by an external force. An estimated 1.7 million head injuries occur every year in the US. Around 40% of TBI patients suffer long-term disabilities in cognition, sensation, movement, and emotion. Following the initial TBI, secondary brain injury progresses for days and weeks, thus offering a window of opportunity for therapeutic interventions, and stem cell therapy is an alternative for neuroprotection. However, preclinical testing depends on the selection and characterization of appropriate animal models. We suggest that 2 months post-TBI is the minimum period needed to evaluate human cell transplant efficacy and safety. Only 10% of TBI literature has evaluated post-TBI ≤2 months. Few papers that evaluated functional outcome at a minimum of 2 months post-TBI reported deficits. A 2-month survival and assessment period would allow sufficient time for differentiation and integration of human neural stem cells with the host. The aim of the present study was to evaluate long-term deficits on emotional and cognitive behaviors in a controlled cortical injury (CCI) model of TBI in athymic nude (ATN) rats. ATN rats are immunodeficient, which is an opportunity to use human stem cells for transplantation without any thought for rejection. Male ATN rats were anesthetized; a craniotomy was performed over the left cortex. After the craniotomy, CCI was delivered to the left parietal cortex (2.5 mm depth and 4.5 m/s velocity). Nine rats were sham injured. Nine weeks postinjury, sham and injured rats were trained and tested in novel place recognition (NPR) and novel object recognition (NOR). The results showed significant differences between sham and TBI rats in NPR but not in NOR. In NPR, TBI rats show less preference for the novel place. Ten weeks postinjury, sham and injured animals were tested on the elevated plus maze (EPM). Results showed that TBI rats presented antianxiety behaviors; animals spent significantly more time in the open arms than Sham rats. Thirteen weeks postinjury, animals performed acquisition and reversal of the Morris Water Maze task (MWM). TBI rats showed significant deficits in spatial acquisition and reversal. TBI rats in the probe test presented deficits in the reference memory. Sixteen weeks postinjury, sham and TBI rats were trained in conditioning taste aversion (CTA). There are no significant differences between sham and TBI rats in CTA. In conclusion, TBI rats showed deficits in NPR, MWM, and EPM after 2 months postinjury, which could indicate that these tasks can be used to evaluate long-term deficits in TBI rats and evaluate long-term cell transplant efficacy.
Supported by CIRM-ETA.
A. R. Massensini,*†‡ C. J. Medberry,*§ F. Nicholls,*† W. Ling,*† S. F. Badylak,*§¶ and M. Modo*†§
*McGowan Institute for Regenerative Medicine, University of Pittsburgh, Pittsburgh, PA, USA
†Department of Radiology, University of Pittsburgh, Pittsburgh, PA, USA
†Universidade Federal de Minas Gerais, Department of Physiology and Biophysics, Belo Horizonte, Brazil
§Department of Bioengineering, University of Pittsburgh, Pittsburgh, PA, USA
¶Department of Surgery, University of Pittsburgh, Pittsburgh, PA, USA
Stroke remains the leading cause of adult disability worldwide, but there are currently no therapies that afford a replacement of lost brain tissue. Use of bioactive materials composed of extracellular matrix (ECM) has proved to be a promising strategy for the constructive remodeling of other tissue and organ systems (e.g., urinary bladder). For example, ECM composed of porcine urinary bladder matrix (UBM) can offer both structural support and a mixture of bioactive molecules that promote tissue repair. Applications of this bioactive scaffold approach to treat stroke lesions requires that two technical challenges must be overcome: 1) delivery of an appropriate volume of material into the brain to fill the cavity without increasing intracranial pressure and 2) retaining a sufficiently high concentration of ECM material within the cavity. Herein, we show how magnetic resonance images (MRIs) can be used to guide the site and volume of injection using a novel stereotactic surgical approach that injects ECM material through one burr hole, while draining extracellular fluid (ECF) from the cavity through another. Additionally, different concentrations of ECM are evaluated for their ability to gel inside the cavity while retaining structural integrity.
A rat model of stroke that occludes the middle cerebral artery for 70 min created a lesion cavity detectable on T2-weighted MRIs. MRIs were used to locate and measure infarct volume. Stereotactic coordinates for the burr holes for ECM injection and ECF drainage were determined from these MRIs. Based on MRIs and drainage of ECF, it was possible to achieve injections of volume equivalent to the size of the lesion that resulted in a complete coverage of the cavity with a bioscaffold. However, retention of the ECM bioscaffold (as determined by collagen-1 staining) was dependent on the amount of gelation. At 2 mg/ml ECM, no bioscaffold was retained within the cavity, whereas at 3 mg/ml, there was good coverage of a scaffold within the lesion, but leakage of ECM into the host striatum was also evident. At 4 mg/ml, there was good gelation throughout the cavity with no more leakage of material into the host tissue, as was the case for 8 mg/ml. The density of the ECM scaffold was much higher and appeared structurally distinct with sharp boundaries around the lesion 1 day postinjection with the 8 mg/ml ECM. Evidence of host cell infiltration [based on 4′,6-diamidino-2-phenylindole positive (DAPI+) cells inside the scaffold] was evident at all concentrations.
In conclusion, we have developed a method that allows for the complete coverage of a stroke lesion cavity with an ECM bioscaffold. These experiments will form the basis for a regenerative medicine approach for the stroke-damaged brain.
O. Milczarek,* D. Jarocha,† K. Zdzislaw,* S. Kwiatkowski,* and M. Majka†
*Department of Children Surgery, Polish–American Institute of Pediatrics, Jagiellonian University School of Medicine, Cracow, Poland
†Department of Transplantation, Polish–American Institute of Pediatrics, Jagiellonian University School of Medicine, Cracow, Poland
Cell therapy is shown in preclinical studies to bring short- and long-term and sensomotor improvement after neonatal brain damage. The purpose of the study was to assess the safety and potential benefits of a cell therapy in encephalopathy.
Four children (two boys and two girls) aged 6 months to 2 years with a hypoxia-derived encephalopathy were enrolled into cell therapy consisting of autologous bone marrow nucleated cell (BMNC) and mesenchymal stem cell (MSC) transplantation with intense neurological and neuropsychological rehabilitation.
Three children experienced hypoxia and ischemia of the central nervous system during labor, while one at the age of 8 months experienced sudden hypoxia for an unexplained reason, followed by sepsis in the second day. Neuropsychological examination at admission revealed that three of the children did not react to any stimuli of the Polish children development DSR scale and experienced regular epileptic seizures.
BMNCs were injected once intravenously (1 × 109) and via lumbar puncture (LP) (0.5 × 109) 6 months after the injury, and MSCs were injected twice via LP (2.5 × 107) a month and 4 months later.
Efficacy was assessed based on neurodevelopmental improvement evaluated using the Polish children DSR scale.
There were neither serious complications of the transplantation procedures nor any side effects of the cell therapy during 10 months follow-up. Two children experienced temperature and a c-reactive protein (CRP) level increase 2 days after MSC infusion.
Four to five weeks after BMNC infusion, the first sensomotor improvement was noticed. Three children moved their heads in reaction to the bell sound, a grimace on their face, and an attempt to cry appeared. Two weeks after MSC infusion, the first cognitive improvement appeared, including all of the children trying to lead the eye after a moving object, more intensely after a familiar person, and an emotional reaction to familiar faces. Two children made first attempts to grab the object and an attendant's hands. Two months later, a reaction to light signal appeared in all children, and in two cases, restoration of the swallowing reflex appeared, and the nutritional probe was extracted. In two cases, the number of seizures considerably decreased after the first MSC infusion. The second MSC infusion caused further sensomotor improvement, including two children who acquired the ability of holding the head and neck with the active muscle action. The cognitive improvement included two children now being able to open their hands, and they regained the ability to squeeze hands on the objects. Reduction of the number of seizures is now visible in all epileptic children. Our results show potential benefit of using cell therapy for encephalopathy patients.
V. Mishra,*†‡ B. Hattiangady,*†‡ A. B. Robbins,§ B. Shuai,*†‡ M. R. Moreno,§ D. J. Prockop,* and A. K. Shetty*†‡
*Institute for Regenerative Medicine, Texas A&M Health Sciences Center College of Medicine, Temple, TX, USA
†Research Service, Olin E. Teague Veterans' Medical Center, Central Texas Veterans Health Care System, Temple, TX, USA
†Department of Molecular and Cellular Medicine, Texas A&M University, College Station, TX, USA
§Department of Biomedical Engineering, Texas A&M University, College Station, TX, USA
Treating blast shock wave-induced mild traumatic brain injury (mTBI) is a challenging problem in combat casualty care, as magnetic resonance image (MRI) scans and other conventional imaging investigations do not normally reveal explicit brain damage in the early postinjury period. Yet clinical studies show that such mTBI can lead to disorders such as memory impairments, depression, and/or posttraumatic stress disorder several years after a mTBI incident. Furthermore, animal studies have mainly focused on examining behavioral consequences of exposure to blast shock waves (BSWs) in early postexposure periods, and some studies have even suggested spontaneous recovery within a few months after the exposure. Therefore, using C57BL/6 male mice, we rigorously examined long-term alterations in cognitive and mood function following an exposure of their heads to BSWs. For inducing BSW exposure, each mouse was first anesthetized, then wrapped in flexible Kevlar and placed within a Schedule 80 PVC container designed to leave only the head exposed. The mouse was then restrained at the end of the shock tube apparatus. The head was then exposed once to BSWs by controlled rupture of a Mylar membrane, which separated the tube from a high-pressure chamber. Compressed air was employed as the working fluid. The blast overpressure experienced by the animals was 11.79 ± 0.10 psi. An additional group of age-matched mice served as sham exposure controls, which were anesthetized and placed near the shock tube to receive the sound of the rupturing Mylar membrane, but not BSWs. Animals were subjected to a battery of behavioral tests 6–8 months after the exposure to elucidate the alterations in cognitive and mood function. Animals exposed to BSWs showed no impairments in water maze (spatial) learning but exhibited hippocampus-dependent spatial memory dysfunction. This was evidenced in the probe test through a lack of preference for swimming in a quadrant of the pool where the platform was placed during learning sessions (i.e., platform quadrant, PQ) vis-a-vis other quadrants, which is in sharp contrast to control animals showing a highest level of preference to the PQ. Interestingly, animals receiving BSWs did not show impairments in another relatively simpler hippocampus-dependent object location test. Investigation with a novel object recognition test (a memory test critically dependent upon the integrity of the perirhinal cortex), however, demonstrated their inability to discriminate between novel and familiar objects, implying the occurrence of hippocampus-independent recognition memory dysfunction following exposure to BSWs. Furthermore, animals exposed to BSWs also showed memory dysfunction in the “object in place” test, which requires normal circuit function between the medial prefrontal cortex, perirhinal cortex, and hippocampus. This was typified by their failure to discriminate reorganized object positions. Moreover, mice exposed to BSWs displayed impairments in pattern separation function (which requires normal levels of dentate neurogenesis) in a behavioral test that examined their ability for discriminating distinct familiar objects placed on a different floor pattern. Additionally, mice exposed to BSWs showed clear signs of mood dysfunction in two different tests of depression, a forced swim test, and a novelty suppressed feeding test. This was evidenced through their increased tendencies to exhibit floating (immobility) in a forced swim test and increased latencies to eat food in a novelty suppressed feeding test following 24 h of fasting, which are clear signs of lack of motivation. Additional studies on cellular and molecular alterations in brain tissues are currently in progress to understand the pathology underlying cognitive and mood impairments in animals exposed to BSWs. Taken together, our rigorous behavioral analyses underscore that a solitary exposure to BSWs is sufficient to cause long-term changes in functions such as memory and mood.
F. J. Nicholls,*† W. Ling,* G. Ferrauto,† A. R. Massensini,*§ D. D. Castelli,† S. Aime,† and M. Modo*
*University of Pittsburgh, Pittsburgh, PA, USA
†King's College London, London, UK
†University of Torino, Torino, Italy
§Universidade Federal de Minas Gerais, Belo Horizonte, Brazil
Noninvasive in vivo monitoring of regenerative medicine approaches for tissue and organ reconstruction will be an important technological development to ensure the safety and efficacy of such strategies. However, the involvement of multiple cell types complicates in vivo monitoring that is dependent on a single contrast mechanism. Herein we explore the use of paramagnetic chemical exchange saturation transfer (ParaCEST) magnetic resonance image (MRI) contrast agents to specifically visualize two transplanted cell populations used for an in situ regenerative medicine approach for the treatment of the stroke-damaged brain. In order to promote the long-term survival of transplanted cells within the lesion, vascularization is vital. One potential avenue to achieve this would be through cotransplantation of neural stem cells with endothelial cells. The ability to simultaneously monitor both populations in vivo is important to correlate the survival and distribution of each cell type with eventual outcome measures, such as neovascularization and behavioral recovery. In this study, human neural stem cells (NSCs) and human brain endothelial cells (ECs) were labeled using ParaCEST agents Eu-HPDO3A and Yb-HPDO3A, respectively. For cellular incorporation of the agents, electroporation and pinocytosis were compared for uptake efficiency and cellular effects. Both methods affected acute cell viability, and ECs were found to be much more resilient than NSCs. Electroporation resulted in a higher intracellular concentration and was therefore taken forward to prepare cells for transplantation. Labeled cells were transplanted into the lesion cavity of a rat stroke model, and the two cell populations could be independently visualized 24 h later and overlaid onto the T2-weighted anatomical image to show cell distribution. These results provide proof of principle that the simultaneous noninvasive imaging of two populations of cells in the brain is feasible. The development of noninvasive imaging will be a key technology to ensure the efficient translation of tissue engineering from bench to bedside, and ParaCEST MRI is a promising platform to further develop for this purpose.
W. Niu,* T. Zang,* R. Bachoo,† and C.-L. Zhang*
*Department of Molecular Biology, University of Texas Southwestern Medical Center at Dallas, TX, USA
†Department of Neurology, University of Texas Southwestern Medical Center at Dallas, TX, USA
Brain injury leads to irreversible neuronal loss and the formation of a glial scar. This scar is initially beneficial for restricting secondary damage but ultimately inhibitory for neural regeneration. Strategies remodeling the scar-forming glial cells might enhance regeneration and promote functional recovery after neural damage. We examined the possibility of converting reactive astrocytes, a major cell type forming the glial scar, to neurons in the adult brain.
The reactive astrocytes were specifically targeted by lentivirus-mediated gene expression under the human glial fibrillary acidic protein (GFAP) promoter. Our in vivo screens showed that sex-determining region Y box 2 (SOX2) alone can induce the appearance of neuroblasts (also called neuronal precursors) within the adult or even aged mouse striatum. Fate mapping showed that these induced neuroblasts were generated from the virus-transduced striatal astrocytes but not any other cells in the striatum or from the endogenous neurogenic niche. Based on morphology, cellular proliferation, and marker expression, the converted neuroblasts resemble the endogenous ones. Most interestingly, the induced neuroblasts become functionally mature when additional signaling molecules, such as brain-derived neurotrophic factor (BDNF), BDNF/Noggin, or valproic acid (VPA), were applied. Electrophysiological recordings demonstrate that astrocyte-converted neurons can integrate into the local neuronal network.
In summary, a protocol was successfully developed for converting resident astrocytes in a step-wise fashion to neuroblasts and functionally mature neurons. This proof-of-concept study on in vivo reprogramming might have great implications in regenerative medicine by using endogenous scar-forming glial cells.
This work was supported by The American Heart Association (09SDG2260602), The Whitehall Foundation Award (2009–12–05), The Welch Foundation Award (I-1724), The Ellison Medical Foundation Award (AG-NS-0753–11), and NIH Grants (1DP2OD006484 and R01NS070981).
*Center of Excellence for Aging and Brain Repair, Department of Neurosurgery and Brain Repair, University of South Florida Morsani College of Medicine, Tampa, FL, USA
†Department of Psychiatry and Behavioral Neurosciences, University of South Florida Morsani College of Medicine, Tampa, FL, USA
Stroke is a major health problem that affects both males and females, yet many preclinical studies have only tested experimental therapeutics in male stroke animals. Current statistics reveal that more women in the US suffer from stroke than men, and the morbidity and mortality is higher in women. Stroke affects women at a later age compared to males, which is likely due to a decrease in hormonal levels since the incidence in female stroke victims increases at the menopausal stages. In ovariectomized rats, there is more severe stroke deficits than those with intact ovaries due to ovariectomy-induced reductions in progesterone and estrogen, which respectively exert antiedema and perivascular stabilizing effects against ischemic brain injury. A major component of stroke pathology that has been recently recognized is the reduced capacity of stem cell regeneration, which may be subject to gender-mediated alterations. The overarching hypothesis of this study is the recognition that gender- dependent stroke outcomes are magnified during postpartum depression, directly advancing our understanding of gender as a key comorbidity factor to the disease pathology and treatment of stroke. The underlying mechanisms of gender-mediated stroke deficits are not well understood, and with our long-standing interest in stem cells, we therefore are testing the effects of gender-associated hormonal shifts inherent in postpartum depression on neurogenesis after stroke. In the in vitro analysis, cultured male- and female-derived endothelial progenitor cells (EPCs) were compared to each other when exposed to ambient and oxygen glucose deprivation (OGD) conditions as a model of stroke. For the in vivo part of the study, we will identify postpartum depressed and postpartum nondepressed rats and characterize the effects of postpartum depression on stroke outcome. The rats with postpartum depression will be identified based on high levels of corticosterone (CORT; indicating a hormonal shift) and behavioral signs of depression (neglect of pups and no interest in nursing). In vitro preliminary data demonstrated that under ambient conditions both maleand female-derived EPCs did not differ in viability, stemness, proliferation, and migration. Interestingly, when EPCs were exposed to OGD, the cultured female-derived cells showed an increased resistance to OGD, but male-derived cells displayed better proliferation and migration levels. Understanding the genotypic and phenotypic differences of dimorphisms, likely to be reflected in the gender of stem cell donors, will further optimize the safety and efficacy of stem cell use for stroke. These findings directly impact on advancing our knowledge on stem cell-based therapeutics for stroke and postpartum depression.
This work is supported by USF Women's Health Collaborative Grant.
P. Pantcheva, I. De La Peña, S. A. Acosta, M. M. Pabon, A. Yoo, D. G. Hernandez-Ontiveros, M. Staples, C. Tamboli, S. Tamboli, K. Duncan, D. Lozano, M. Bastawrous, A. Antoine, N. Tajiri, Y. Kaneko, and C. V. Borlongan
Center of Excellence for Aging and Brain Repair, Department of Neurosurgery and Brain Repair, University of South Florida Morsani College of Medicine, Tampa, FL, USA
Granulocyte colony-stimulating factor (G-CSF) promotes brain neurogenesis and improves functional outcome in animal models following stroke through mechanisms that influence apoptotic pathways and suppress edema formation. Tissue plasminogen activator (tPA) is currently the most effective treatment for acute ischemic stroke but leads to edema formation and hemorrhagic transformation when given to patients beyond its therapeutic time window. Based on these observations, we hypothesized that a combination of G-CSF with tPA will attenuate the risk of hemorrhagic transformation associated with delayed administration of tPA and improve the safety and impact of tPA. An in vitro study was performed to investigate the tPA toxicity in cultured endothelial progenitor cells and to examine the effects of G-CSF on tPA-induced blood–brain barrier (BBB) breakdown and endothelial progenitor cell death. In vitro, G-CSF decreased oxygen-glucose deprivation-induced BBB permeability and endothelial progenitor cell injury. In vitro results support the protective action of G-CSF under ischemic conditions and suggest a reduced risk of hemorrhagic transformation in vivo. Administration of G-CSF in delayed tPA treatment holds potential therapeutic value.
C. Perez,* S. Jergova,* S. Gajavelli,* L. Imperial,† B. Olivera,† and J. Sagen*
*Miami Project, University of Miami, Miami, FL, USA
†University of Utah, Salt Lake City, UT, USA
Current long-term pharmacotherapy for chronic pain has great limitations. The identification of alternative approaches and new therapeutic targets is essential. Cannabinoid (CB) receptor agonists have emerged as potential therapeutic targets, as robust antinociceptive effects have been reported in various pain models. However, most of the available drugs that interact with CB receptors are derived from cannabis and considered clinically unacceptable for long-term therapy due to psychoactive side effects. Therefore, specific compounds interacting with CB receptors without aversive side effects are of clinical interest. The venoms of marine snail genus Conus are a natural source of various peptides (conopeptides) with potent analgesic effects, some of them already FDA approved. In this study, we evaluated the ability of several Conus venom extracts to interact with the CB1 receptor.
The venom extracts of six Conus species were analyzed in vitro. Human embryonic kidney 293 (HEK293) cells expressing CB1 receptors were treated with venom extracts for 30 min, and the rate of CB1 receptor internalization was analyzed by immunofluorescence. Results showed the highest rate of CB1 internalization in HEK293 cells after treatment with venoms of C. textile (C. tex.) and C. miles (C. mil.). High-performance liquid chromatography (HPLC) fractions (seven per species) of these venoms were subsequently analyzed using a similar approach. Based on the analysis, C. tex. fraction 5 and C. mil. fraction 4 with the highest CB1 agonist activity was evaluated in a formalin test. IT injection of the C.tex. and C. mil. fractions reduced flinching/licking behavior during the second phase of the formalin test. The results indicate the presence of CB1 agonists within the Conus venom extract and their potential analgesic effects. To identify the nature of CB1 agonists, the C.tex. and C. mil. fractions were treated with proteolytic enzymes. The effect of treatment on CB1 internalization was subsequently evaluated. Results showed that proteolytic treatment attenuated the ability of the venom fraction to induce CB1 receptor internalization. The third generation of individual fractions of C. tex. and C. mil. were analyzed. Based on the analysis, fractions C. tex. 22 and C. mil. 17 have the highest percentage of internalization compared to the other fraction. Currently, such fractions are being treated with proteolytic enzymes. The final peptide compounds of screened venoms with CB1 receptor affinity could subsequently be used as a new analgesic agent in recombinant cell or gene therapy approaches.
Supported by NS51667.
R. Periasamy, A. L. Fuchs, S. Seiler, H. R. Widmer, and S. Di Santo
Department of Neurosurgery Research Laboratory and Regenerative Neuroscience Cluster, University of Bern and University Hospital Bern, Switzerland
Recent observations suggest that impairment of vascular tissue in the brain is involved in the pathogenesis of neurodegenerative disorders. It is known that endothelial progenitor cells (EPCs) play an important role in revascularization and regeneration of several tissues. Hence, in the present study, we investigated whether EPCs can promote brain microvascular endothelial cell functions and addressed the type of factors and signaling pathways that may be involved. For the preparation of EPC-derived conditioned medium (EPC-CM), mononuclear cells were isolated from the peripheral blood of healthy human donors and consequently cultured under hypoxic conditions (1.5% O2) to stimulate the secretion of growth factors. Cultures from rat brain endothelial cells (rBCEC4 cell line) were incubated with EPC-CM for 2 days. Cell viability was assessed by using Presto Blue. Serial dilutions, molecular weight fractionation, and heat inactivation experiments were performed to narrow down key effectors of EPC-CM-mediated effects. The specific AKT inhibitor LY294002 and the extracellular signal-regulated kinase (ERK) inhibitor PD98059 were used to analyze for the involvement of these signaling pathways in the effects of EPC-CM. We observed that rBCEC4 viability was significantly increased following incubation with EPC-CM. This effect was maintained up to five times dilution of EPC-CM. Heat inactivation and molecular weight fractionation abolished the effects of EPC-CM. Furthermore, inhibition of the AKT and ERK pathways suppressed the EPC-CM-dependent increase of rBCEC4 viability. In summary, our findings demonstrate that EPC-derived paracrine factors substantially promote viability of brain microvascular cells by activating AKT and ERK signaling cascades. Furthermore, the activity of EPC-CM to support brain microvascular cell viability likely relies on the concerted action of a variety of proteinaceous factors with different molecular weights.
C. Pernia,*† B. Tobe,* A. Crain,* A. Winquist,* and E. Snyder*†
*Del E. Webb Center for Neuroscience, Aging, and Stem Cell Research, Sanford Burnham Medical Research Institute, La Jolla, CA, USA
†Sanford-Burnham Graduate School of Biomedical Sciences, Sanford Burnham Medical Research Institute, La Jolla, CA, USA
Bipolar 1 disorder (BD1) is a psychiatric illness that affects 2.6% of the adult population in the US and is characterized by severe fluctuations between manic and depressive episodes. One of the foremost therapeutics for BD1 is lithium, which interestingly is not efficacious for any other major psychiatric disorder. Lithium is a potent mood stabilizer, but clinicians reluctantly prescribe it due to its serious medical side effects. How and where in the brain lithium acts to treat BD1 has yet to be elucidated. Psychiatric disorders have historically been difficult to study at the cellular and molecular level because of the lack of relevant animal behavioral models and the ethical dilemma of studying human subjects. We hypothesized that studying the lithium response pathway in mixed neuronal cultures derived from human BD1-induced pluripotent stem cells (iPSCs) would reveal novel insights into the mechanism of action of lithium in BD1 and, in conjunction with expression data of lithium-sensitive targets, indicate regions of the human brain involved with BD1. This study utilizes a comparative proteomic approach, sensitive to posttranslational modifications, combined with human iPSC technologies for understanding the therapeutic mechanism of lithium on BD1. Our in vitro screen identified at least 15 proteins that are effected by lithium treatment in human BD1-derived neurons, which we cross-referenced with brain structure microarray data from the Allen Brain Atlas to identify areas of the brain where lithium's therapeutic action, and perhaps BD1 pathology, could be most pertinent. Four brain areas were identified by their high expression levels of these proteins of interest, which we compared to the expression profiles of other brain areas postulated to be associated with BD1. Our findings substantiate previous human functional magnetic resonance imaging (fMRI) studies' assertions of specific brain areas being implicated in BD1, and identifies new areas that have yet to be systematically studied in BD1, and also provides a list of novel BD1 drug targets for future research.
K. Pollock,* H. Stewart,* W. Cary,* H. Nelson,* C. Nacey,* K. Pepper,* K. D. Fink,* W. Gruenloh,* G. Annett,* T. Tempkin,† V. Wheelock,† and J. A. Nolta*
*Stem Cell Program and Institute for Regenerative Cures, University of California Davis Health Systems, Sacramento, CA, USA
†Department of Neurology, University of California Davis Health Systems, Sacramento, CA, USA
Huntington's disease (HD) is an autosomal dominant disorder, caused by an expanded cytosine–adenine–guanine (CAG) trinucleotide repeat that causes a progressive degeneration of neurons in the putamen and caudate nucleus. Survival and function of striatal neurons is dependent on brain-derived neurotrophic factor (BDNF), and levels of this trophic factor are significantly reduced in HD patients. Recently, strategies aimed at BDNF restoration have become a leading candidate for the treatment of HD. Transplantation of adult stem cells, such as bone marrow-derived mesenchymal stem/stromal cells (MSCs), show considerable therapeutic promise through stimulation of endogenous neuronal growth, decreased neuronal apoptosis, regulation of inflammation, and the secretion of trophic factors. MSCs are readily available, easily expanded in vitro, have immunomodulatory properties, and can be easily engineered to overproduce trophic factors. Previously, in animal models of HD, it has been shown that allogeneic transplantations of MSCs can significantly delay the onset of behavioral abnormalities and reduce the severity of neuropathological changes in both transgenic and toxic lesion models of HD. In a pivotal proof-of-concept study, transgenic mice showed significant behavioral and neuropathological sparing following the intrastriatal administration of MSCs engineered to overexpress BDNF.
The aim of the current study is to test the preclinical safety and efficacy of human MSCs, genetically engineered to produce BDNF, for the treatment of HD. Our developmental candidate is allogeneic human MSCs engineered, using a lentiviral vector, to secrete BDNF (MSC/BDNF). Our product combines the beneficial effects of MSC administration with the benefits of sustained BDNF production. These proof-of-concept and safety studies are being conducted in support of HD-Cell, a planned future phase I clinical trial designed to examine the safety and potential efficacy of MSC/BDNF. The current studies focus on the optimal transduction coefficients, the level of BDNF produced by our developmental candidate, the karyotypic stability of the candidate, the safety profile following transplantation in immune deficient mice, cell retention in vivo, and efficacy following transplantation in the YAC128 transgenic mouse model of HD, as investigational new drug (IND)-enabling studies for the FDA, in support of a future planned phase I clinical trial.
Support for this project was provided by California Institute for Regenerative Medicine (CIRM) grants no. TR1–01257 (Nolta) and DR2–05415 (Wheelock/Nolta), NIH Director's transformative award 1R01GM099688 (Nolta), and philanthropic donors from the HD community, including the Roberson family and Team KJ.
J.-F. Poulin,* J. Zou,* J. Drouin-Ouellet,† F. Cicchetti,‡§ and R. B. Awatramani*
*Department of Neurology, Northwestern University, Evanston, IL, USA
†John van Geest Centre for Brain Repair, University of Cambridge, Cambridge, UK
†Centre de Recherche du CHU de Québec (CHUQ), Axe Neurosciences, Québec, QC, Canada
§Département de Psychiatrie et Neurosciences, Université Laval, Québec, QC, Canada
The midbrain dopaminergic neurons are involved in diverse physiological pathways including motor control, learning, cognition, and reward behaviors. In the last 30 years, several lines of evidence have suggested that there may be additional levels of dopamine heterogeneity that may be even more meaningful than their classification into three anatomically defined clusters. In order to assess the extent of midbrain dopamine diversity, we used a high-throughput gene expression platform based on microfluidic dynamic arrays to evaluate the expression of 96 genes in single dopamine cells. This approach revealed the existence of multiple genetically distinct populations of dopamine neurons with distinct molecular signatures. One of these populations is defined by high expression of aldehyde dehydrogenase 1 family, member A1 (Aldh1a1) and low expression of calbindin 1 (Calb1). This population is localized in the ventral tier of the substantia nigra. Moreover, Aldh1a1 expressing neurons of the substantia nigra are more vulnerable in the 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP) model of Parkinson's disease. This gene might provide additional clues to the vulnerability of a subset of dopamine neurons to Parkinson's disease, as well as provide an avenue for an earlier diagnostic test for the disease.
B. H. Priddy,* S. Jergova,* S. Gajavelli,* J. Imperial,† B. Olivera,† and J. Sagen*
*Neurological Surgery, The Miami Project to Cure Paralysis, Miami, FL, USA
†Department of Biology, University of Utah, Salt Lake City, UT, USA
Current treatments for chronic pain are limited and usually not very efficacious. Most treatments are limited to opioids and nonsteroidal anti-inflammatory drugs. Unfortunately, their regular usage is limited by both central nervous system (CNS) and autonomic-mediated adverse side effects; these problems combined with the low efficacy of pharmacotherapies imply the need for alternatives and new therapeutic targets. Researchers have implicated voltage-gated ion channels as contributing mediators of pathological pain. Local anesthetics such as lidocaine are of interest as sodium channel (NaV) antagonists; however, they are also problematic because of their short-duration activity, off-target systemic effects, and lack of specificity and discrimination for NaV subtypes. This study focused on a unique NaV antagonist, μ-conotoxin SbIIIA peptide from the marine snail Conus striatus. Its sequence homology to known NaV 1.8 antagonist SIIIA and its placement in the μ-conotoxin family make us believe it to be a highly specific tetrodotoxin-resistant (TTX-r) NaV 1.8 channel antagonist. A viral construct was designed, and SbIIIA cDNA was cloned, amplified, a secretion signal sequence was added (ssPAM), and then SbIIIA was ligated to adeno-associated virus 2/8 (AAV2/8) hybrid serotype. ssPAM-SbIIIA in AAV-2/8 was then sequenced, and the sequence matched that of the expected theoretical sequence. We also cloned in an internal ribosomal entry site-enhanced green fluorescent protein (IRES-eGFP) in frame after the stop codon of SbIIIA to visualize transfection. In order to determine the long-term effect of this construct in animal models, Sprague–Dawley male rats were prepared by performing direct intradorsal root ganglia (DRG) injections of either AAV-2/8 GFP (vehicle control) or AAV-2/8 ssPAM-SbIIIA viral vector (treatment). At 4 and 6 weeks, there were no abnormal changes in responses to innocuous tactile stimuli in either treatment group. There was a modest reduction in sensitivity to noxious heat stimuli in animals receiving SbIIIA. After 6 weeks, all rats were challenged with 5% formalin in their right hindpaw. Direct DRG injection of the SbIIIA viral vector led to reduced flinching and pain-like responses at multiple intervals implying an analgesic effect. If SbIIIA is a specific antagonist of NaV channels, then local AAV-mediated delivery of SbIIIA could serve as an efficacious analgesic with minimal side effects by segmentally targeting the DRG peripheral and central distribution of affected nerves. Furthermore selective gene therapy using this construct could ameliorate ectopic pathological NaV neuropathic pain conditions, that is, diabetic neuropathy or HIV neuropathy, while at the same time reducing or eliminating the need for repeated administration of analgesics and their systemic effects.
Supported by NIH Grant NS72769, Craig T. Neilsen Foundation, and Buoniconti Fund.
J. E. Quintero,* J. A. Gurwell,† J. T. Slevin,† G. A. Gerhardt,*†‡ and C. G. Van Horne*‡
*Department of Anatomy and Neurobiology, University of Kentucky, Lexington, KY, USA
†Department of Neurology, University of Kentucky, Lexington, KY, USA
†Department of Neurosurgery, University of Kentucky, Lexington, KY, USA
In Parkinson's disease (PD), the substantia nigra undergoes a loss of dopaminergic cells and cell function that, in part, manifests into the outward symptoms of PD. One avenue of intense efforts to treat this disease involves the delivery of neurotrophic factors to restore dopaminergic cell function. A potential source of these neurotrophic factors are Schwann cells from the peripheral nervous system. After injury, Schwann cells release a host of growth factors including glial-derived neurotrophic factor (GDNF), nerve growth factor (NGF), brain-derived neurotrophic factor (BDNF), and neurotrophin-3 (NT-3). We have begun a pilot study to examine the safety and feasibility of implanting an autologous peripheral nerve graft into the substantia nigra of PD patients undergoing deep brain stimulation (DBS) surgery. Multistage, DBS surgery targeting the subthalamic nucleus was performed using standard procedures. After the DBS leads were implanted, a section of sural nerve (approximately 5 mm in length) containing Schwann cells was excised and unilaterally delivered, using a custom-designed cannula, into the area of the substantia nigra. Adverse events were continuously monitored. Subjects have undergone or will undergo a Unified Parkinson's Disease Rating Scale (UPDRS) evaluation before surgery and at 1, 3, 6, 9, and 12 months after surgery. We have successfully completed peripheral nerve graft surgery in five of five participants. Immediate, postoperative magnetic resonance scans did not indicate evidence of abnormal tissue disruption. Participants who have completed 6 months in the study reported comparable adverse effects to standard DBS surgery. In addition, UPDRS Part III scores off medication/off stimulation remained mostly unchanged after 6 months (36 ± 8 baseline vs. 33 ± 9, mean ± SD; N = 4), but two of the participants under 60 years old showed an 11-point improvement. Scores on medication and on stimulation improved by six points (16 ± 11 baseline vs. 10 ± 7 6 months), and daily levodopa equivalents decreased from a mean of 844 ± 691 mg at baseline to zero after 6 months. During the initial months of the study, we have observed a limited number of adverse events along with some improvements on UPDRS evaluations, but ongoing assessments will help gauge the safety and feasibility of implanting peripheral nerve tissue in conjunction with DBS surgery and the potential benefits these grafts may provide.
Support provided by University of Kentucky start-up funds (CVH) and the National Center for Advancing Translational Sciences, through grant UL1TR000117.
A. J. Schwab, M.-E. A. Barabas, A. K. Smith, C. L. Stucky, and A. D. Ebert
Medical College of Wisconsin, Milwaukee, WI, USA
Less prominent features of Parkinson's disease (PD) are nonmotor symptoms, which are generally underappreciated and can severely impact quality of life. Pain in PD is one of the most common nonmotor symptoms and is typically associated with motor function loss, but as many as 43% of Parkinson's patients show pain prior to the onset of motor symptoms. Nociceptors are primary pain-sensing afferent neurons and are typically lightly myelinated or unmyelinated. The long axons of the unmyelinated nociceptors may be particularly vulnerable to cellular metabolism deficits or toxic insults. We hypothesize that disease processes in PD are not confined to dopamine neurons but may include morphological changes and sensitization in sensory neurons, which may underlie the pain involved with PD. In order to study sensory neuron function in a human system, we utilized human induced pluripotent stem cell (iPSC) technology. We obtained commercially available mutant leucine-rich repeat kinase 2 (LRRK2; G2019S) and α-synuclein (SNCA) triplication familial PD iPSCs and generated a sporadic PD iPSC line using the Sendai viral reprogramming method. Upon PCR and immunocytochemical verification of reprogramming, we followed previously published differentiation protocols to generate peripherin-positive sensory neurons from the three PD iPSCs lines as well as unaffected control iPSC lines. Additional immunocytochemical analysis for neurotrophic receptor kinase 1 (NTRK1) and transient receptor potential vanilloid 1 (TRPV1) confirmed a proportion of the sensory neurons as nociceptors. Live cell calcium imaging demonstrated that the sensory neurons in culture were indeed functional as the majority responded to potassium. Further analysis revealed morphological alterations in regard to microtubule disruption and axonal spheroids in sporadic and LRRK2 sensory neurons. This is the first demonstration of axonal spheroids in sensory neurons of Parkinson's disease suggesting that disease processes are not confined to dopaminergic systems. Further analysis will investigate the composition of the axonal spheroids as well as testing whether LRRK2 inhibition rescues morphological and functional changes. Since nonmotor symptoms affect a large proportion of PD patients, characterizing sensory neurons from PD iPSC lines may provide insight into the underlying mechanisms of nonmotor symptoms in PD, thereby aiding therapeutic development.
A. Sharma,* D. F. Muresanu,† R. Patnaik,† H. Huang,§ Z. R. Tian,¶ H. Möessler,# and H. S. Sharma*
*Surgical Sciences, Anesthesia and Intensive Care Medicine, Uppsala University Hospital, Uppsala, Sweden
†Clinical Neurosciences, University of Medicine and Pharmacy, Cluj-Napoca, Romania
†Biomedical Engineering, Indian Institute of Technology, Banaras Hindu University, Varanasi, India
§Department of Neurosurgery, Beijing Rehabilitation Centre, Neuroscience Institute of Taishan Medical University, Beijing, China
¶Chemistry and Biochemistry, University of Arkansas, Fayetteville, AR, USA
#Ever Neuro Pharma, Oberburgau, Austria
Concussive head injury (CHI) is caused by a variety of external and internal factors leading to serious behavioral dysfunctions and brain pathology. Thus, CHI requires urgent neuroprotection strategies using potential drugs in combination with neuroregenerative approaches to reduce the consequences of CHI in patients.
Since Cerebrolysin is a balanced combination of several neurotrophic factors, we compared nanodrug delivery of Cerebrolsyin with several other neurotrophic factors either alone or in combination using nanobiotechnology for effective therapeutic approaches in a rat model of CHI.
In present investigations, we showed that a multiple combination of neurotrophic factors derived from neurons, for example, brain-derived neurotrophic factor (BDNF), glial-derived neurotrophic factor (GDNF), nerve growth factor (NGF), or ciliary neurotrophic factor (CNTF), when administered exogenously following CHI (250 to 500 μg/kg/min for 30 min) 2 to 4 h after injury, and the animals were followed for 12 h, induced remarkable restoration of neuronal, glial, and endothelial cell structure and functions compared to the untreated injured groups. However, a significantly improved result is obtained when administering Cerebrolysin (250 or 500 μl/kg/min for 30 min) under identical conditions following CHI. Interestingly, the therapeutic efficiency of these neurotrophic factors and Cerebrolysin is further enhanced when they are administered using titanium dioxide (TiO2) nanowired technology. The central nervous system (CNS) pathology after trauma is not affected by TiO2 nanowires alone. Moreover, the nanowired drug delivery of phenoxybenzamine, propranolol, or tumor necrosis factor-α (TNF-α) did not induce any neuroprotection indicating that any drug could not become neuroprotective just because they are delivered through nanowired technology. These observations suggest that nanowired delivery of suitable drugs is the need of the hour to induce better neuroprotection in the clinic following CHI.
H. S. Sharma,* D. Muresanu,† A. Nozari,† R. Patnaik,§ H. Mössler,¶ and A. Sharma*
*Surgical Sciences, Anesthesiology and Intensive Care Medicine, Uppsala University Hospital, Uppsala, Sweden
†Clinical Neurosciences, University of Medicine and Pharmacy, Cluj-Napoca, Romania
†Anesthesia and Critical Care, Massachusetts General Hospital, Boston, MA, USA
§Biomaterials, Indian Institute of Technology, Banaras Hindu University, Varanasi, India
¶Ever NeuroPharma, Oberburgau, Austria
Military personnel engaged in combat operations in the desert areas are often exposed to silicon dioxide (SiO2) nanoparticles (NPs) from the sand, as well as carbon (C), copper (Cu), or silver (Ag) NPs from gunfire, missile explosion, or blast injuries. NP intoxication can worsen neurological injury after cardiac arrest (CA) via an aggravated blood–brain barrier (BBB) disruption, edema formation, and oxidative injury. Therapeutic interventions aimed at mitigating the oxidative and the NP-induced postresuscitation injuries can, therefore, improve the neurological outcome.
CA was induced in rats using apneic asphyxia for 3–4 min. Return of spontaneous circulation (ROSC) was obtained after 4–5 min of no-flow CA by standard cardiopulmonary resuscitation (CPR) (Katz et al., 1995. JCBFM 15: 1032–1039). The NP group was pretreated with SiO2, Cu, or Ag NPs (50–60 nm, 50 mg/kg, IP/day) for 7 days prior to the experiment. Animals were allowed to survive 4 h or 8 h after ROSC.
CA resulted in a 10- to 14-fold increase in the BBB breakdown, three-to fourfold increase in brain edema, and four- to sixfold increase in astrocytic activation and myelin damage in the NP-treated animals. Treatment with 5 ml/kg dose of Cerebrolysin 60 to 120 min after ROSC significantly reduced CA induced brain pathology in non-NP animals, but 10–15 ml/kg was required for comparable neuroprotection in NP-treated rats.
These observations are the first to suggest that NP-intoxication exacerbates CA-induced brain pathology. An almost two- to threefold increase in the dose of the neuroprotective drug Cerebrolysin is needed to achieve comparable neuroprotection in NP-treated animals after CA. This indicates that Cerebrolysin could be used to restore brain pathology and recovery following CA.
V. M. Spruance,* D. E. Sanchez,† K. M. Negron,* T. Bezdudnaya,* E. J. Gonzalez-Rothi,† G. B. Grossl,† B. E. O'Steen,† P. J. Reier,† and M. A. Lane*
*Drexel University College of Medicine, Department of Neurobiology and Anatomy, Philadelphia, PA, USA
†University of Florida College of Medicine, Department of Neuroscience, Gainesville, FL, USA
Cervical spinal cord injuries (SCIs) result in devastating functional consequences, including severe respiratory dysfunction. This is largely due to the disruption of phrenic circuitry, which controls the diaphragm. Though spontaneous, functional recovery of respiratory systems has been demonstrated in both laboratory and clinical settings, this recovery is limited, and life-threatening deficits remain. Considering that over 50% of all SCIs occur at the cervical level, and that respiratory complications remain one of the main causes of mortality among these patients, there is a clear need for novel, therapeutic approaches to improving respiratory function following injury.
Previous work has suggested that prephrenic interneurons are involved in the neural plasticity that occurs after injury and is associated with partial functional recovery. Therefore, this neuronal population represents a potential target for therapeutic strategies. The transplantation of fetal spinal cord (FSC) tissue, inherently rich in interneuronal progenitors, could provide an additional substrate for neuronal plasticity and facilitate the formation of novel pathways that will restore input to the diaphragm. Additionally, we hypothesize that pairing FSC transplantation with intermittent exposure to hypoxia—shown previously to enhance respiratory neuroplasticity—will optimize host and graft connectivity and neuronal function via Hebbian principles.
Adult, female Sprague–Dawley rats received C3/C4 contusion injures (Infinite Horizons Device, 200 kDa) and injections of E13.5 FSC suspension directly into the lesion cavity at 1 week postinjury. One month following transplantation, a pseudorabies virus (PRV) tracer was applied either to the diaphragm or directly into the transplant. Animals were perfuse-fixed 72 h later. A second group of animals underwent terminal diaphragmatic electromyograms (EMGs) in lieu of tracing experiments. Results of the PRV studies have demonstrated connectivity between host and transplanted tissues, with transplanted neurons integrating into circuits that extend throughout the thoracic spinal cord and into the brain stem. Though variability exists in the degree of host–transplant connectivity between animals, certain neuroanatomical structures are consistently associated with the new transplant circuitry (including the raphe nucleus, locus coeruleus, and reticular formations). EMG data reveals improved, ipsilateral, hemidiaphragm function under eupneic conditions as well as in response to a respiratory challenge. Ongoing studies are investigating the nature of the contribution of transplanted cells to respiratory recovery and whether the combined use of intermittent hypoxia can optimize host–transplant connectivity.
A. K. Srivastava,*† S. K. Gross,† C. A. Bulte,*† N. J. Maragakis,† and J. W. M. Bulte*†
*Russell H. Morgan Department of Radiology and Radiological Science, Division of MR Research, The Johns Hopkins University School of Medicine, Baltimore, MD, USA
†Institute for Cell Engineering, The Johns Hopkins University School of Medicine, Baltimore, MD, USA
†Department of Neurology, The Johns Hopkins University School of Medicine, Baltimore, MD, USA
Amyotrophic lateral sclerosis (ALS) is a fatal neurodegenerative disease characterized by selective loss of motor neurons in the motor cortex, brainstem, and spinal cord. Intraspinal transplantation of stem cells is being investigated as a possible treatment for ALS. Transplantation of stem cells may help in the repairing of damaged motor neurons. To assess long-term risks and benefits of stem cell therapy, monitoring the fate of engrafted cells is very crucial. Using two imaging modalities, computed tomography (CT) and bioluminescence imaging (BLI), we noninvasively investigated survival of intraspinal transplanted neural stem cells (NSCs) during the course of disease progression in transgenic G93A mutated superoxide dismutase [SOD1(G93A)] ALS mice. Allogeneic firefly luciferase-expressing NSCs, isolated from transgenic Friend leukemia virus B (FVB) mouse brain, were transplanted bilaterally (100,000 cells in 2 μl/injection) into the cervical spinal cord ventral gray matter of 64-day-old B6SJL-Tg(SOD1*G93A)1Gur/J ALS mice (n = 9) and control B6SJLF1/J wildtype mice (n = 6) via laminectomy. Mice were immunosuppressed by using FK506/rapamycin (1 mg/kg IP) daily. BLI and CT were performed for several weeks post-transplantation using an in vivo imaging system (IVIS) Spectrum CT system. Two- and three-dimensional images generated by coregistering BLI and CT images confirmed a deposit of cells at the site of the targeted injection in the spinal cords of mice. BLI showed that transplanted cells did not excessively proliferate and did not form tumors. The survival of transplanted cells decreased in ALS mice as the disease progressed despite immunosuppression. A significant decline (p < 0.01) in the BLI signal was observed as mice entered into the symptomatic stage (84 days of age) At the end stage (126 days of age) of the disease, BLI signal was not detectable, indicating the cells were not surviving. Contrary to ALS mice, in wild-type mice, transplanted cells survived well throughout the study period. By serial monitoring of cell survival, we observed unreported differences between symptomatic ALS and control mice. These findings indicate that ALS mice exhibit a hostile microenvironment toward engrafted NSCs, mandating further improvement for NSC therapy in ALS.
K. G. Stanford,* J. D. Odum,* A. D. Rorie,* R. S. Rogers,* J. L. Wheatley,* P. C. Geiger,* H. Nishimune,† and J. A. Stanford*
*Department of Molecular and Integrative Physiology, University of Kansas Medical Center, Kansas City, KS, USA
†Department of Anatomy and Cell Biology, University of Kansas Medical Center, Kansas City, KS, USA
Protecting neuromuscular junction (NMJ) innervation is a therapeutic goal for slowing the rapid progression of motor neuron degeneration in amyotrophic lateral sclerosis (ALS). The G93A mutated superoxide dismutase 1 (SOD1-G93A) rat models ALS in that they exhibit a presymptomatic stage followed by neuromuscular denervation, progressive muscle weakness and atrophy, and finally paralysis. We trained SOD1-G93A rats to perform a unilateral isometric strength-training task to determine if muscle strength training protects against NMJ denervation in the trained forelimb. Specifically, water-restricted rats were trained to press an operandum at a force of 20 g to receive a water reward. This force requirement is approximately 33% of maximum voluntary force in this task for rats. After testing rats daily for approximately 2 months (until ~5.5 months of age), trained and untrained forelimb muscles were harvested for tissue analysis. Muscle contraction increases the activity of adenosine monophosphate (AMP)-activated protein kinase (AMPK), and so we measured AMPK and its activated form, phosphorylated AMPK (pAMPK). We also analyzed the autophagy marker microtubule-associated protein 1A/1B-light chain 3 (LC3) and NMJ innervation. Our results revealed that even at end stage, SOD1-G93A rats maintained force output. Strength training significantly increased pAMPK (only in the SOD1-G93A rats) and LC3, and protected against NMJ denervation in trained muscles. In fact, in the faster progressing SOD1-G93A rats, innervation in the trained and untrained forelimbs was 74 ± 4% and 17 ± 11%, respectively. These results are the first to demonstrate robust neuroprotective effects of nonaerobic resistance training in ALS. They also suggest that AMPK activation may have a protective role against NMJ denervation in ALS.
M. Staples,* Y. Kaneko,* N. Tajiri,* T. B. Freeman,* H. Van Loveren,* S. U. Kim,† and C. V. Borlongan*
*Center of Excellence for Aging and Brain Repair, Department of Neurosurgery and Brain Repair, University of South Florida Morsani College of Medicine, Tampa, FL, USA
†Department of Neurology, University of British Columbia, Vancouver, Canada
Presently, there is little data on treatment strategies that target the repair of the blood–brain barrier (BBB) in metastatic brain cancer. In fact, restoration of the BBB through angiogenesis and vasculogenesis is believed to aid the formation of metastatic brain cancers. Here we propose a paradigm-shifting approach, whereby we demonstrate that continued neglect of BBB contributes to the formation of brain metastatic cancers of melanoma. Subsequently, we will demonstrate that bone marrow-derived endothelial progenitor cell (BMEPC) therapy can repair the BBB by augmenting angiogenesis and vasculogenesis, which ultimately results in the attenuation of developing metastatic brain cancers. To determine the detrimental effects of a leaky BBB, B16F10 melanoma mice are evaluated at three time points (days 3, 7, and 21) and compared to controls. Western immunoblotting, immunohistochemistry, and PCR are used to examine metastatic brain cancer growth, BBB leakage, and inflammation. To determine the potential for BMEPC therapy, we evaluated the efficacy of grafted BMEPCs to repair BBB damage and ameliorate brain inflammation. Cell transplantation therapy occurred at acute and delayed time points to represent varying stages of metastasis and determine the optimal timing of cell transplant. Preliminary data showed that transplantation of BMEPCs repaired the damaged BBB associated with metastatic brain cancers of melanoma. Such BBB restoration appeared to attenuate brain inflammation and suppress tumor growth by preventing the entrance of inflammatory factors from the systemic circulation. This study characterizes the immunological and inflammatory responses associated with a leaky BBB that contribute to the development of brain metastatic cancers of melanoma. The demonstration of BBB repair through augmented angiogenesis and vasculogenesis represents a novel therapeutic approach to metastatic brain cancers of melanoma.
G. K. Steinberg,*†‡ D. Kondziolka,§¶ N. E. Schwartz,*†‡ L. Wechsler,§¶ D. Lunsford,§¶ M. L. Coburn,*†‡ J. B. Billigen,§¶ H. Keren-Gill,*†‡ M. McGrogan,# C. Case,# K. Mori,# and E. W. Yankee#
*Department of Neurosurgery, Stanford University, Stanford, CA, USA
†Department of Neurology, Stanford University, Stanford, CA, USA
†Stanford Stroke Center, Stanford University, Stanford, CA, USA
§Department of Neurological Surgery, University of Pittsburgh, Pittsburgh, PA, USA
¶Department of Neurology, University of Pittsburgh, Pittsburgh, PA, USA
#SanBio, Inc., Mountain View, CA, USA
No treatment exists to restore lost brain function after stroke. Animal studies demonstrate that brain transplantation of SB623, a human bone marrow-derived stromal cell with transient transfection of Notch-1 gene, after experimental stroke can improve neurologic outcome. This clinical study is the first North American trial of intraparenchymal transplantation of bone marrow-derived cell therapy for chronic stroke patients.
This is a two-center (Stanford University and the University of Pittsburgh) open-label safety and dose escalation feasibility study. Stereotactic transplantation is targeted to the subcortical peri-infarct area. Inclusion criteria include 18–75 years old, 6–60 months postsubcortical middle cerebral artery (MCA) ischemic stroke, modified Rankin Scale (mRS) 3–4 and National Institutes of Health (NIH) Stroke Scale (NIHSS)<7. Safety endpoints include World Health Organization (WHO) toxicity scale, magnetic resonance images (MRIs) and clinical follow-up to 2 years. The primary efficacy endpoint is European Stroke Scale (ESS) at 6 months; secondary efficacy measures are ESS, NIHSS, Fugl–Meyer, mRS, cognitive scores up to 2 years, and fludeoxyglucosepositron emission tomography (FDG-PET) at 6 months.
Eighteen patients (33–75 years old; 7–36 months poststroke) have been treated (six each with 2.5 M, 5 M, and 10 M cells). Follow-up is currently 6 months in 15 patients, 9 months in 12 patients, and 12 months in 9 patients. There were four adverse events related to the surgery (seizure, asymptomatic subdural hematoma, pneumonia, urinary tract infection) but not to the cells. Cytokine levels, human leukocyte antigen (HLA) antibody levels, and peripheral blood mononuclear cell (PBMC) function did not change from baseline. Three measures of efficacy (NIHSS, ESS, Fugl–Meyer) all show a statistically significant improvement at 6 months after treatment. Two patients showed remarkable improvement in their motor (2) and language function (1) within 24 h of surgery, effects which have been sustained during follow-up (12 and 3 months). These were the only two patients with new fluid attenuated inversion recovery (FLAIR) lesions [diffusion weighted imaging (DWI) neg] in the motor cortex that resolved at 2 months.
Intraparenchymal transplantation of human modified bone marrowderived stromal cells in chronic stroke patients is safe, feasible, and shows significant neurologic improvement at 6 months following treatment. Larger studies and longer follow-up are being initiated to further assess clinical efficacy.
R. Sullivan,* N. Tajiri,*† D. Travis,* Y. Kaneko,* and C. V. Borlongan*
*Center of Excellence for Aging and Brain Repair, Department of Neurosurgery and Brain Repair, University of South Florida Morsani College of Medicine, Tampa, FL, USA
†School of Physical Therapy and Rehabilitation Sciences, University of South Florida Morsani College of Medicine, Tampa, FL, USA
Epilepsy is a debilitating neurological disorder affecting approximately 2% of the world's population. Epilepsy may be caused by brain insults, including traumatic brain injury, stroke, encephalitis, tumor, and infection, or it may arise from mutations of ion channels or neurotransmitters involved in brain homeostasis. Golgi complex fragmentation is a pathological feature of several neurodegenerative diseases, including Parkinson's disease, Alzheimer's disease, and amyotrophic lateral sclerosis. However, the molecular mechanisms by which Golgi complex fragmentation may aggravate neuronal function to contribute to the pathology of epilepsy have not been investigated. Primary rat neuron cells (PRNCs) were cultured with kainate (KA) for 3 days at 37°C without antibiotics. Cell viability, mitochondrial activity, the expression of Golgi apparatus proteins [Golgi phosphoprotein of 130 KDa (GPP130) and formimidoyltransferase cyclodeaminase; G58/FTCD], and Golgi complex fragmentation/dispersal were assessed by calcein, 3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT), Western blot, and immunocytochemistry analysis, respectively. PRNC viability and mitochondrial activity are significantly decreased with KA administration in a dose-dependent manner; the estimated IC50 values for cell viability and mitochondrial activity are 5.89 ± 0.185 μM and 2.79 ± 0.515 μM, respectively. Topiramate, a common antiepileptic treatment, prevented the neuronal degradation normally seen with KA administration, indicating that this study reliably demonstrates an experimental epilepsy model in vitro. After treatment of PRNCs with 5 μM KA, we observed Golgi complex fragmentation/dispersal without microtubule disassembly. Reelin, an extracellular matrix glycoprotein, is localized to the Golgi complex. The Golgi–lysosome–endoplasmic reticulum (ER) network plays an important role in reelin glycosylation, its proteolytic processing, and the synthesis of proteoglycans, which are key components of the extracellular matrix. Separation of reelin from this network is associated with Golgi fragmentation/dispersal. Reduction of secreted reelin (320 kDa fragment) contributes to the pathogenesis of neurological disorders such as epilepsy. Our findings revealed that Golgi complex fragmentation modified the expression patterns of reelin leading to neuronal aggravation in an experimental epilepsy model. In the present study, we observed that the administration of KA attenuates neuronal function by causing fragmentation/dispersal of the Golgi apparatus. Golgi complex fragmentation damages a sophisticated network of biochemical reactions involved in energy production and macromolecular biosynthesis. KA-induced Golgi complex fragmentation decreases the physiological function of reelin, which has been implicated in the pathogenesis and progression of a number of neurodegenerative diseases. Thus, Golgi complex fragmentation and reelin dysregulation may be considered as a disease biomarker as well as a therapeutic target for epilepsy.
N. Tajiri,* S. A. Acosta,* M. M. Pabon,* A. Yoo,* I. De La Pena,* Y. Kaneko,* S. Tamir,† Y. Landesman,† S. Shacham,† and C. V. Borlongan*
*Center of Excellence for Aging and Brain Repair, Department of Neurosurgery and Brain Repair, University of South Florida Morsani College of Medicine, Tampa, FL, USA
†Karyopharm Therapeutics Inc., Natick, MA, USA
Exportin 1 (XPO1/CRM1) plays prominent roles in the regulation of nuclear protein export and was recently shown to be overexpressed in central nervous system (CNS) lesions in rats following traumatic brain injury (TBI) and to regulate neuronal apoptosis following brain injury. Selective inhibitors of nuclear export (SINE) are small molecule, orally bioavailable, drug-like inhibitors of XPO1. SINE possess potent anticancer properties in animal models and are currently being tested in clinical trials for cancer patients. TBI is associated with a progressive secondary cell death characterized by a massive neuroinflammatory response regulated by nuclear receptors. SINE restore and increase nuclear localization of antiinflammatory and neuroprotective proteins, including nuclear factor of κ light polypeptide gene enhancer in B-cells inhibitor (IκB) and nuclear factor erythroid 2-related factor 2 (Nrf2). The present study was designed to assess whether SINE similarly reduced TBI-induced neuroinflammationrelated signaling within the nucleus, which should reduce disease pathology. Cultured primary rat neuronal cells were incubated initially for 2 h with SINE [10 μM Karyopharm Therapeutics-350 (KPT-350) or 10 μM KPT-335] or vehicle, followed by tumor necrosis factor (TNF)-α (20 ng/ml) for 48 h, with subsequent analysis of cell viability, cellular enzymatic activity, and inflammation- and nuclear-based immunocytochemistry. SINE were found to significantly counteract TNF-α-induced loss of cell viability compared to vehicle-treated primary rat neuronal cells (p < 0.05). SINE also increased XPO1, AKT, and forkhead box P1 (FOXP1) nuclear expression, brain-derived neurotrophic factor (BDNF) expression, and retained nuclear factor of κ light polypeptide gene enhancer in B-cells (NF-κB) protein within the nuclei. In an animal model of moderate TBI, adult Sprague–Dawley rats underwent controlled cortical impact injury or sham surgery, followed by oral administration of KPT-350 (5 mg/kg or 7 mg/kg 2 h later and once a day thereafter over the next 4 days) to test for effects of XPO1 inhibition on TBI-induced behavioral deficits and histological pathology. TBI animals treated with KPT-350 exhibited significantly better motor coordination and balance in the rotarod test and motor asymmetry tests, with up to twofold improvement as early as 4 h after initial treatment, which was sustained over the 18-day postinjury assessment period, compared to vehicle treatment (p < 0.05). Preliminary data also showed reduction of cell death in the peri-impact cortical areas in KPT-350-treated animals. In summary, oral administration of KPT-350 postinjury effectively ameliorated behavioral and histological deficits in TBI animals. Altogether, these findings support the potential of KPT-350 as a novel antineuroinflammatory therapy for TBI.
D. K. Thakor
United States Patent and Trademark Office, Alexandria, VA, USA
Intellectual property is becoming increasingly important in the life sciences, not only in industry but also in academia, and it is therefore advisable for researchers to gain a basic understanding of the patent system. This presentation will describe what patents are, what can be patented, and what determines patentability from the perspective of the US patent statutes, which require novelty, nonobviousness, written description, enablement, and distinct claiming of the invention. The Leahy–Smith America Invents Act, which changed US patent practice in 2012 and 2013, most notably by shifting patentability criteria from “first-to-invent” to “first-to-file,” will also be discussed. This presentation will also address the ways that patents can be used as a commodity when granted and even still under application and thus why they are so valuable. Particular attention will be paid to opportunities for university innovation and technology transfer, which have been exponentially increasing since the Bayh–Dole act of 1980 that enabled US universities to license or sell intellectual property developed using federal funding. The goal of this presentation is to give researchers a new perspective on the patent system for application to their own research, such that they can maximize the value of their studies by protecting and exploiting the intellectual property that they develop.
B. T. D. Tobe,*† A. M. Crain,* A. M. Winquist,* B. Calabrese,† M. Sidor,§ M. Brandel,* C. Duerr,* C. Pernia,* L. Dorsett,* M. McCarthy,† C. McClung,† M. Niepel,¶ M. Wada,# Y. Inoue,# N. Yamashita,# J. Li,¶ S. Haggarty,¶ P. Sorger,¶ C. Shamu,¶ R. L. Sidman,¶ L. M. Brill,* I. Singec,* S. Halpain,† Y. Goshima,# and E. Y. Snyder*
*Sanford-Burnham Medical Research Institute, La Jolla, CA, USA
†Veterans Administration Medical Center–La Jolla, Department of Psychiatry, La Jolla, CA, USA
†Division of Biological Sciences, University of California–San Diego, La Jolla, CA, USA
§University of Pittsburgh, Department of Psychiatry, Pittsburgh, PA, USA
¶Harvard Medical School, Boston, MA, USA
#Yokohama City University School of Medicine, Yokohama City, Japan
It has become common to make human-induced pluripotent stem cells (hiPSCs) from patients with monogenic conditions. However, the greatest challenge for “Disease-in-a-Dish” modeling is approaching complex, polygenic, multifactorial disorders, the underlying pathophysiological molecular mechanisms for which are poorly understood. Strategies for such conditions have been allusive yet could help advance the field of disease modeling in general. Neuropsychiatric disorders are a prototype for such conditions. Hence, not only are they a critical category of diseases in their own right, but strategies that prove fruitful for them may have broader applicability for other complex conditions. Of such conditions, bipolar disease (BPD), a highly lethal illness, is attractive and unique in that 50% of patients respond to lithium chloride (LiCl). Indeed, LiCl responsiveness is pathognomonic. Critically, LiCl “mechanism-of-action” in BPD is unknown; however, were its target(s) to be identified, LiCl could offer a molecular “handle” for discerning underlying mechanisms and deriving better treatments (LiCl has unacceptable side effects and a dangerously narrow safety index). Therefore, as proof of concept, we have begun to show how exploiting a known functional agent in hiPSCs, even one whose action is unknown in a particular disease context, such as LiCl in BPD patients, might provide a molecular “can opener” for revealing heretofore unrecognized underlying pathophysiological mechanisms for such a disorder, potentially allowing better drugs and therapies to be discovered. By proteomic analysis of neurons generated from hiPSCs derived from LiCl-responsive BPD patients, we have discovered an unanticipated LiCl target—“CRMP2” (“Collapsin response mediator protein 2”)—that, mechanistically, not only helps establish BPD as a heritable neuro-developmental disorder and not only potentially ties it to such neuro-degenerative disorders as the “tauopathies” but is eminently “druggable” (based on kinase modification). We have discovered in vitro (validated preliminarily in vivo in animal models and possibly in postmortem human specimens) that LiCl alters kinases that govern the phosphorylation state of CRMP2, which, in turn, regulates its association with cytoskeletal elements and ion channels, hence modulating neural connectivity, neuronal activity, and tubulin/tau deposition. Phosphorylated CRMP2 (p-CRMP2) becomes dissociated from tau and tubulin and leads to neurite retraction. p-CRMP2 is likely excessively high in LiCl-responsive BPD. We have been mapping the intracellular pathways to and from this molecule. Drugs, including repurposed drugs, that lower its phosphorylated form are potentially widely therapeutic, providing a basis not only for better psychotropic agents but possibly for prophylactic agents against some acquired neurodegenerative conditions. More broadly, this type of investigation might represent a strategy for using hiPSCs to unravel complex pathophysiological mechanisms and develop more effective drugs if there is a functional, molecular (ideally, therapeutic) “hook.”
H. Ulrich
Department of Biochemistry, Institute of Chemistry, University of São Paulo, Brazil
Bradykinin (BK) and related kinins are released into the plasma or interstitial fluid after proteolytic cleavage of kininogens. BK actions mediated through kinin-B2 receptors do not only participate in inflammation and blood pressure regulation but have also been described for neurotransmission and neuromodulation. Novel functions for BK and its receptor were obtained using neural stem and progenitor cells isolated from fetal rat or mouse brain (E14 or E12.5, respectively) as in vitro models whose differentiation into neural and glial phenotypes closely resembles events occurring during cortex development. Functional kinin-B2 receptors and liberation of BK into the culture medium suggested the existence of an autocrine loop participating in neural differentiation. The presence of the kinin-B2 receptor antagonist HOE-140 resulted in reduced neurogenesis and enhanced gliogenesis of neurospheres obtained from embryonic telencephalon. As an underlying mechanism of inhibition of neurogenesis, we found that migration of neural progenitors was largely restricted in the presence of the inhibitor. These results were confirmed in migration and differentiation assays with neurospheres obtained from kinin-B2 receptor knockout mice. Neurogenic and neuroprotective features of BK in neurodegenerative diseases were further characterized in vitro and in vivo. Effects exerted by 6-hydroxydopamine, such as cell death and inhibition of migration and neurogenesis of neurospheres induced to differentiation into dopaminergic neurons, were abolished by BK. Moreover, degeneration of dopaminergic neurons, as measured by a decrease in tyrosine hydroxylase staining, and clinical symptoms, such as apomorphine-induced rotations, in a rat model of Parkinson's disease were mostly reversed following a single BK injection. Neuroprotective properties of BK were also observed in an in vitro model of ischemic neuronal cell death. Following perfusion of hippocampal slices with n-methyl d-aspartate (NMDA) for induction of cytotoxicity, BK application reverted the loss of functional pyramidal neuron responses as well as the induction of apoptosis. Neuroprotective actions mediated by kinin-B2 receptors were abolished in the presence of a kinin-B1 receptor agonist. As BK is converted to the B1 receptor agonist des-Arg9-BK by carboxypeptidases, present in different tissues including the brain, the herein reported results provide a mechanism for the neuroprotective effects exerted by BK, despite deleterious effects reported in disease states. Overall, stable B2 receptor agonists or protease inhibitors resulting in an increase of extracellular BK accumulation are promising tools for brain tissue repair.
Supported by grants from FAPESP and CNPq, Brazil.
N. Vardjan,*† M. Kreft,*†‡ and R. Zorec*†
*Celica Biomedical Center, Ljubljana, Slovenia
†Laboratory of Neuroendocrinology-Molecular Cell Physiology, Institute of Pathophysiology, Faculty of Medicine, University of Ljubljana, Ljubljana, Slovenia
†Biotechnical Faculty, University of Ljubljana, Ljubljana, Slovenia
The morphology of astrocytes, likely regulated by cyclic adenosine monophosphate (cAMP), determines the structural association between astrocytes and the synapse, consequently modulating synaptic function in health and disease. β-Adrenergic receptors (β-AR), which increase cytosolic cAMP concentration ([cAMP]i) may affect cell morphology. However, the real-time dynamics of β-AR-mediated cAMP signaling in single live astrocytes and its effect on cell morphology have not been studied. We used the fluorescence resonance energy transfer (FRET)-based cAMP biosensor exchange protein directly activated by cAMP (Epac1) to study time-dependent changes in [cAMP]i while morphological changes in primary rat astrocytes were monitored by real-time confocal microscopy. Stimulation of β-AR by adrenaline, noradrenaline, and isoprenaline, a specific agonist of β-AR, rapidly increased [cAMP]i (~15 s). The FRET signal response, mediated via β-AR, was faster than in the presence of forskolin (twofold) and dibutyryl-cAMP (>35-fold), which directly activate adenylyl cyclase and Epac1, respectively, likely due to slow entry of these agents into the cytosol. Oscillations in [cAMP]i have not been recorded, indicating that cAMP-dependent processes operate in a slow time domain. Most Epac1-expressing astrocytes revealed a morphological change upon β-AR activation and attained a stellate morphology within 1 h. The morphological changes exhibited a bell-shaped dependency on [cAMP]i, indicating that maximal morphological changes are limited to an optimal (narrow) range of [cAMP]i. The rapid 5–10% decrease in cell cross-sectional area and the 30–50% increase in cell perimeter are likely due to withdrawal of the cytoplasm to the perinuclear region and the appearance of protrusions on the surface of astrocytes. Astrocyte processes ensheath neurons, so β-AR/cAMP-mediated morphological restructuring can modify the geometry of the extracellular space, affecting synaptic, neuronal, and astrocyte functions in normal and pathological conditions.
K. Vojnits,*† H. Pan,*† X. Mu,† W. Xu,† Y. Tang,† C. Cox,*† and Y. Li*†
*Department of Pediatric Surgery, University of Texas Medical School at Houston, Houston, TX, USA
†Center for Stem Cell and Regenerative Medicine, University of Texas Health Science Center at Houston, Houston, TX, USA
†University of Pittsburgh, School of Medicine, Pittsburgh, PA, USA
More than 60 million people worldwide are affected by degenerative disorders that cannot be cured to date. Regenerative medicine and stem cell research with the promise of complete organ restoration could change the treatment paradigm for these currently incurable diseases, such as varieties of central nervous system diseases and injuries. However, application of regenerative medicine is limited by the lack of autologous, nonembryonic multipotent stem cells (SCs). Hence, new SCs with easy accessibility, improved migration, and neuronal differentiation potential and with a low tendency of tumor formation and immune rejection are needed. Recently, we identified a novel population of dedifferentiated stem cells (deSCs) from muscles that, as we hypothesize, can fulfill these criteria. A conditional transgenic model dependent on the Cre/Lox-β-galactosidase system was used in this study that can specifically tag deSCs based on the activation of the marker gene, β-galactosidase, for example, LacZ. Cre and Lox- β-galactosidase cells were implanted into the gastrocnemius muscles of adult severe combined immunodeficient (SCID) mice. Three weeks later, a laceration injury was created, and 4 days after injury, β-galactosidase-positive deSCs were isolated and populated. Our results showed that deSCs had stemness features, remarkable migration, myogenic and neurogenic differentiation ability. They were characterized by the expression of several pluripotency and stem cell markers [octamer-binding transcription factor 4 (Oct4), Nanog, sex-determining region Y box 2 (Sox2), stage-specific embryonic antigen 1 (Ssea1), chemokine C-X-C motif receptor 4 (Cxcr4), muscle segment homeobox (Msh) homeobox homolog 1 (Msx1), stem cell antigen 1 (Sca1), paired box 7 (Pax7), and nestin]; and data from time-lapse motility assay confirmed that deSCs accumulated over a longer distance compared to control cells within the same time period. Also, they highly expressed migration markers both at the protein and mRNA levels. Moreover, deSCs could easily and effectively form neurospheres, floating in suspension in neural stem cell media, while the control primary myoblasts showed no sign of forming these structures. The induced neurospheres represented neural phenotypes expressing nestin, 2′,3′-cyclic nucleotide 3′ phosphodiesterase (CNPase) and neurofilament, medium polypeptide (Nefm), as analyzed by immunostaining and real-time qPCR. DeSCs-induced neurospheres, after replating into laminin/polyornithine-coated monolayer culture in neural differentiation media, differentiated into the three major neural lineages (neurons, astrocytes, and oligodendrocytes) as they expressed microtubule-associated protein 2 (Map2), β-tubulin III, glial fibrillary acidic protein (Gfap), nestin, and oligodendrocyte transcription factor 1/2 (Olig1/2) protein and mRNA also. These findings suggest that upon muscle injury, a new type of multipotent and highly proliferative stem cell can be isolated from adult mammalian skeletal muscles. Besides their abilities of great migration and multipotent status, these cells can also overcome germ lineage restrictions and differentiate into multiple neuronal lineages in vitro. Further work is planned to elucidate deSC survival, migration, and neuronal/myogenic differentiation capacity in vivo to prove their benefits in application of regenerative medicine for motor neurons or neural-muscle related injuries and diseases.
T. M. Vorobyeva, S. P. Kolyadko, O. G. Berchenko, E. O. Zaitseva, D. A. Bevzyuk, and N. A. Levicheva
State Institute of Neurology, Psychiatry and Narcology, National Academy of Medical Sciences of Ukraine, Kharkiv, Ukraine
In the early 1960s, it was found that pharmaceutical substances, regardless of their chemical structure, demonstrated biological activity in ultralow doses, providing the optimal setting for tuning perceptions of environment signals and changes in homeostasis under normal and pathological conditions (Tatarenko et al., 1962). Kramova and Vorobyova (1962) proved that the initial reaction to a novel ultralow dose stimulus mobilizes the body's physiological, visceral, immunological, and neurotransmitter systems as a universal adaptive response to stress. These ideas about the effects of ultralow doses resonate with the phenomenon of hormesis (according to Paracelsus), when miniscule doses of strong poisons may be effective therapeutic substances (pharmacological inversion). Although hormesis is an unusual phenomenon, it points to the existence of a secondary mechanism, biological, yet unknown, which changes the perception processes (Kaznacheev and Trofimov, 1968). Presently, an alternative method of pharmacotherapy is transplantation of allo- and xenogeneic embryonic neural tissues, which are genetically programmed for regenerative potential by the principle of autotrophy (protein nucleotide). The relative regenerative properties of embryonic brain tissue for allo- and xenoimplantation have been demonstrated, and sufficient data have been accumulated to draw a conclusion about its merits and possibilities. These properties include activation of compensatory and regenerative processes, which inhibit apoptosis of neurons and glial cells in damaged areas of the brain, migration of various substances from implanted neuronal tissue to the impaired brain area, facilitation of synaptogenesis, and activation of neurotrophin and growth factor production in recipient cells, which are inherent in the neuroprotective effects and the decline in the inflammatory processes of the damaged cerebral area. Thus, embryonic tissue transplants promote anatomical/physiological reconstruction of the damaged brain tissue. Researchers believe that the hippocampus is the most favorable area for transplanting embryonic neural tissue and neuronal cells. Hippocampal tissue contains “adult” multipotent stem cells and neuronal cells, so the process of neurogenesis is possible in case of damage or under normal physiological conditions. Of particular interest in regenerative therapy is the use of embryonic tissues that produce neurotransmitters similar to those existing at the frontal areas of the brain, the amygdala complex, structures of the extrapyramidal system, and the epiphysis. In the intimate mechanisms of the therapeutic effects of the regenerative properties of the central nervous system (CNS), brain-specific proteins participate in new roles as neuropeptides, or regulators of neuroimmune processes. In this regard, some effectors in the regulation of regenerative processes are brain-specific protein S100 (Ashmarin, Shtark, and Ephstein, 1992), tumor necrosis factors, and biologically active substances of the medicinal leech, which can perform the hormesis function. Hormesis functions upon activation of regenerative processes by neurotransplantation, as shown by our research, can elicit information contained in brain biopotentials, potentials that can be recorded by an electroencephalograph and stored for later use by the recipient.
D. R. Wakeman,* V. B. Mattis,† C. Tom,† H. B. Dodiya,* S. Y. Yeung,† A. H. Tran,† K. Bernau,§ L. Ornelas,† A. Sahabian,† J. Reidling,† D. Sareen,† L. M. Thompson,† C. N. Svendsen,† and J. H. Kordower*
*Rush University Medical Center, Chicago, IL, USA
†Regenerative Medicine Institute, Cedars-Sinai Medical Center, Los Angeles, CA, USA
†University of California-Irvine, Irvine, CA, USA
§University of Wisconsin-Madison, Madison, WI, USA
Assessing the long-term efficacy of human stem cell transplantation in genetic rodent models of disease has been complicated by the significant immune rejection that occurs in vivo. Human xenografts require immunosuppression for their viability. The immunosuppressive regimens are expensive, have associated morbidity, as well as off-target effects in the central nervous system (CNS) that can confound interpretations. To circumvent long-term immunosuppression of xenografts, a neonatal desensitization technique was previously described (Kelly et al., 2009), allowing for long-term survival of grafted human neuronal cells to nonimmunosuppressed rats. Here we extended the neonatal tolerance approach to outbred and inbred immune-intact mice, including transgenic N171–82Q Huntington's disease (HD) mice and assessed whether neonatal tolerance could prevent the rapid rejection of human xenografts. We transplanted human neural progenitor cells (hNPCs) derived from induced pluripotent stem cells (iPSCs), embryonic stem cells (ESCs), or fetal cortex into the striatum of neonatal mice as well as adult mice that had been “desensitized” with a prior IP injection of the same cell type and examined tolerance to neural xenografts. In contrast to neonatal rats, neonatal and adult tolerized mice displayed rapid immune rejection to xenografts derived from all three NPC sources. Owing to the poor survival of the grafts, mice with neonatal desensitization prior to adult xenotransplantation in HD mice, pre- or postsymptomatically treated with either fetal or ESC-derived NPCs displayed no statistically significant behavioral improvement in functional motor skills (hindlimb clasp and rotarod) or extended survival time compared to wild-type (WT) littermates. These results demonstrate that neonatal desensitization with iPSC-, hESC- or fetal-derived NPCs in multiple mouse strains does not render animals immunologically privileged to xenografts at early or adult stages. The desensitization technique first reported in rats, therefore, is not at present a viable methodology in mice.
H. Wang,* C. Jia,* J. Li,* L. Wang,* X. Chen,† Q. Liu,† and W. Lau‡
*Department of Chemistry, Chemical Biology and Biomedical Engineering, Stevens Institute of Technology, Hoboken, NJ, USA
†Center for Biomaterials, Sichuan University, Chengdu, China
†3D Biotek, Hillsborough, NJ, USA
Maintenance of desired cell phenotype is always essential in functional tissue regeneration, which is at least equally, if not more important, when it comes to in vitro creation of implantable tissue constructs using a tissue engineering approach. Increasing evidence has demonstrated that in tissue engineering, the scaffolds not only provide the cells with a temporary support for attachment and growth, but also instruct the cells toward their fate. In contrast to a tremendous knowledge base on the effect of biochemical factors, limited understanding has been made in correlating the physical characteristics of scaffolds with cellular responses. In this regard, there is a great need to encode the regulatory effect of physical features of scaffolds on cell phenotypic expression, which can better guide the scaffold design for functional tissue formation. Ideally, tissue-engineering scaffolds should maximally recapture the physicochemical properties of native tissues. The establishment of a variety of new fabrication technologies, such as electrospinning and three-dimensional printing, offers us the versatile capabilities of incorporating the hierarchical and complex features of the native extracellular matrix (ECM), especially on a micro-/nanoscale, into scaffold design. This talk will first summarize the recent scaffold fabrication technologies developed in our lab and then elaborate on their potential utility in fabrication of multiscale biomimetic scaffolds to regulate the cellular responses and tissue formation.
J.-Y. Wang,* K.-F. Huang,† L.-Y. Yang,* and W.-C. Hsu‡
*Graduate Institutes of Medical Sciences, Taipei Medical University, Taipei, Taiwan
†Department of Neurosurgery, Bhuddist Tzu Chi General Hospital, Taipei Branch, Taipei, Taiwan
†Department of Ophthalmology, Bhuddist Tzu Chi General Hospital, Taipei Branch, Taipei, Taiwan
Surgical brain trauma (SBT) is unavoidable during many neurosurgical procedures intrinsically linked to postoperative neurological deficits. There is as yet no clinically effective strategy for neuroregeneration. The purpose of this study was to evaluate the effects of collagen–glycosaminoglycan (CG) matrix scaffolds in a rat model of surgical brain trauma. Sprague–Dawley male rats (weighing 300–350 g) were randomly divided into three groups: 1. Sham 2. Trauma 3. Trauma with CG matrix implantation. Postoperative assessment at various time periods (day 1, day 3, day 7, day 14, day 21, and day 28) included evaluation of neurological scoring [by modified neurological severity score (mNSS)], histological as well as immunohistochemical staining, and enzyme-linked immunosorbent assay (ELISA) for measurement of the tissue concentrations of growth factors in areas of the parietal cortex in all groups after sacrifice of animals at various time periods.
Implantation of porous biodegradable CG matrix after SBT improved sensorimotor functional outcomes as shown by the mNSS at various time points. The numbers of proliferative (Ki67 positive) and differentiated migratory [doublecortin (DCX) positive] cells timedependently increased after surgery both in the intramatrix zone (IMZ) and lesion boundary zone (LBZ). Therefore, the CG scaffold facilitated proliferation, differentiation, and migration of endogenous neural precursor cells. Immunohistochemical staining for smooth muscle actin (SMA) and cluster of differentiation 31 (CD31) also indicated neovascularization after implantation of CG scaffolds in LBZ and IMZ following implantation of CG scaffolds. In addition, the tissue concentrations of vascular endothelial growth factor (VEGF), platelet-derived growth factor (PDGF), and basic fibroblast growth factor (bFGF) revealed a sustained increase in both zones up to 28 days following implantation of CG scaffold.
We conclude that CG scaffolds provide a microenvironment to facilitate neovascularization and neurogenesis after surgical brain trauma.
Supported in part by a grant from the National Science Council NSC102–2321-B-038 -003 to JY Wang.
S. N. Williams,* E. D. Haim,* N. Tajiri,* D. G. Hernandez-Ontiveros,* A. Frisina-Deyo,* P. R. Sanberg,*† C. V. Borlongan,* and S. Garbuzova-Davis*†‡
*Center of Excellence for Aging and Brain Repair, Department of Neurosurgery and Brain Repair, University of South Florida Morsani College of Medicine, Tampa, FL, USA
†Department of Molecular Pharmacology and Physiology, University of South Florida Morsani College of Medicine, Tampa, FL, USA
†Department of Pathology and Cell Biology, University of South Florida Morsani College of Medicine, Tampa, FL, USA
Vascular injury after ischemic stroke occurs in a time-dependent manner and may be categorized as acute (minutes to hours), subacute (hours to days), or chronic (days to months). Following such an ischemic insult, cerebral vascular perturbations can lead to blood–brain barrier (BBB) damage. Despite intensive research into BBB complications from ischemic stroke, the bulk of these studies have focused on the acute poststroke stage and the cerebral hemisphere of initial ischemic insult. Recently, we demonstrated BBB alterations in brain areas remote from the initial ischemic lesion at the subacute (7 days) ischemic stage in a transient middle cerebral artery occlusion (tMCAO) rat model. BBB breakdown was mainly characterized by damaged endothelial cells containing numerous autophagosomes, pericyte degeneration, and perivascular edema in addition to vascular leakage in both the ipsilateral and contralateral hemispheres. While microvascular damage is closely associated with BBB impairment accompanied by endothelial autophagosome accumulation in remote brain microvessels at subacute ischemia, the cerebral endothelial cell condition in chronic ischemic stroke until now remains not well understood. The aim of this study was to evaluate capillary endothelial cell damage by autophagosome formation in chronic tMCAO rats. A specific focus was analyzing microvessel endothelium integrity in association with BBB permeability in the contralateral cerebral hemisphere, an area with remote brain structures not directly affected by ischemia. tMCAO was performed in Sprague–Dawley adult male rats using the intraluminal filament technique with 60 min reperfusion. tMCAO and control rats were euthanatized 30 days after reperfusion and assayed for quantitative analysis of Evans Blue (EB) extravasation into the brain parenchyma, and processed for immunohistochemistry using Beclin-1 to detect the autophagic response within capillary endothelial cells in discrete areas of the striatum and motor cortex in both hemispheres. Results demonstrated significantly higher EB levels in ipsilateral and contralateral hemispheres versus controls. A significantly elevated EB level was determined in the ipsilateral compared to contralateral hemisphere. Beclin-1 immunoexpression was significantly upregulated in ipsilateral lateral (L), dorsal (D), and ventral (V) striatum areas of tMCAO rats compared to controls. In the contralateral striatum, a significant increase of Beclin-1 fluorescent expression was determined in area V, although elevated protein expressions were seen in medial (M), L, and D areas versus controls. In the motor cortex, a significant increase of Beclin-1 immunoexpression in capillary endothelium was determined in M2 and M1 areas in ipsilateral hemisphere. Overexpression of Beclin-1 in contralateral motor cortex was noted in the M2 area. Of note, more extensive Beclin-1 upregulation was shown in the ipsilateral M1 than in M2 or contralateral M1 or M2 areas. Additionally, immunofluorescence revealed expansion of Beclin-1 in numerous capillaries of analyzed brain structures in both hemispheres. This upregulation of Beclin-1 in capillary endothelium in ipsilateral and contralateral striatum and motor cortex microvessels in chronic post-tMCAO may indicate excessive autophagosome accumulation, potentially leading to the endothelium damage and vascular leakage. Since autophagy plays an important role in the balance between cell survival and cell death, a better understanding of specific molecular mechanism(s) associated with the autophagosomal pathway will likely provide pivotal insights into poststroke endothelial cell impairment.
Supported by the NIH (1RO1NS071956–01A1) and the James and Esther King Biomedical Research Program (1KG01–33966).
A. E. Willing, J. Newcomb, C. Gemma, P. R. Sanberg, and P. C. Bickford
Center of Excellence for Aging and Brain Repair, Department of Neurosurgery and Brain Repair, University of South Florida Morsani College of Medicine, Tampa, FL, USA
Human umbilical cord blood (HUCB) cells transplanted directly into the central nervous system (CNS) of adult rats do not survive well long term even when aggressive immune suppression is used (Walczak et al., J. Neurosci. Res., 76:244; 2004). Further, when transplanted into the nonobese diabetic/severe combined immunodeficient (NOD/SCID) mouse, which has no competent lymphocytes that could induce rejection, the HUCB cells only survived short term (Walczak et al., Brain Res. Bull., 74:155; 2007), suggesting the innate immune system (myeloid cells) plays a significant role in graft rejection. Systemic HUCB injection suppresses the brain inflammatory response after stroke (Vendrame et al., Stem Cells Dev., 14:595; 2005) and induces anti-inflammatory changes in peripheral immune organs (Vendrame et al., Exp. Neurol., 199:191; 2006). Against this background, we postulated that preconditioning with IV administration of the HUCB cells may suppress the graft rejection of direct hippocampal (HC) transplantation by modulating the innate immune system of the rat. We compared hippocampal graft survival (3 weeks post-HUCB HC transplant) and blood immune cell profile between young adult Fisher 344 rats (normal control), aging rats with a HC HUCB transplant only, or aging rats that received one of three different protocols of IV HUCB administration prior to the HC transplants (single IV injection 7 days prior to HC transplant; three IV injections on consecutive days 7 days prior to HC transplant; three IV injections at weekly intervals prior to HC transplant). No surviving grafts were found in those animals that received hippocampal transplants only. The only group that had surviving cells was the group that received IV HUCB cells on three consecutive days 7 days prior to HC transplants. When we examined the white blood cell profile, the only consistent change was a significant decrease in the monocyte population (p < 0.01). These results suggest that HUCB cell-induced immune suppression is a function of the interaction of HUCB cells with the innate immune system of the host. Further, this effect is either time-limited since the three injections at weekly intervals failed to protect the subsequent HC transplant or a threshold must be reached to observe CNS protection.
Supported in part by the NIA (5R01AG20927–5). AEW is a consultant and PRS a cofounder of Saneron CCEL Therapeutics, Inc. AEW and PRS are inventors on cord blood patents licensed to Saneron.
F. Xiao,* M. Juliano,*† L. L. Stone,* H. Mihalko,* D. Vinodkumar,*† M. Suresh,* C. Nan,* N. Kuzmin-Nichols,† C. D. Sanberg,† P. R. Sanberg,§ A. Grande,*† and W. C. Low*†
*Department of Neurosurgery, University of Minnesota, Minneapolis, MN, USA
†Stem Cell Institute, University of Minnesota, Minneapolis, MN, USA
†Saneron CCEL, Tampa, FL, USA
§Center of Excellence for Aging and Brain Repair, Department of Neurosurgery and Brain Repair, University of South Florida Morsani College of Medicine, Tampa, FL, USA
Over 750,000 individuals in the US suffer a stroke each year. For those with ischemic stroke, only 5% are eligible to be treated with tissue plasminogen activator (tPA) because of the narrow therapeutic time window of 3–6 h after injury. Previously, our group demonstrated that a cluster of differentiation 34 (CD34) negative fraction of human nonhematopoietic umbilical cord blood stem cells (nh-UCBSCs) could be administered 48 h after injury, and the infarct volume was reduced by 50% as well as significant amelioration of neurological deficits in rats with cerebral ischemia. In the present study, we explored possible mechanisms of action using next generation RNA sequencing (RNAseq) analysis to quantify changes in the brain transcriptome in rats with ischemic brain injury and following nh-UCBSC therapy. Ischemic animals were treated with nh-UCBSCs 2 days after injury and sacrificed for extraction of brain mRNA for RNAseq analysis 5 days after nh-UCBSC treatment. Over 21,000 transcripts were interrogated, and among these, we found approximately 170 transcripts that were significantly elevated after ischemia compared with control brains and also significantly downregulated after nh-UCBSC treatment. Detailed analysis of these altered transcripts revealed that the vast majority were associated with activation of the immune system following cerebral ischemia and normalization of these transcripts following nh-UCBSC therapy. Major changes include activation of infiltrating macrophage, neutrophils, and T-cells and upregulation of Toll-like receptor 4 (TLR4) ligands and endothelial cell adhesion molecule [intercellular (I)CAM1] involved in immune cell infiltration after ischemia. These changes were all normalized by 5 days after nh-UCBSC treatment. These results suggest that nh-UCBSCs protect the brain following ischemic injury by downregulating the aberrant activation of innate and adaptive immune responses.
Disclaimer: PRS is a cofounder of Saneron CCEL Therapeutics, Inc. PRS and WCL are inventors on cord blood patents licensed to Saneron.
C. Yang, J. He, Y.-X. Zhang, T. Lou, and W.-M. Duan
Department of Anatomy, Capital Medical University, Beijing, China
We have recently demonstrated that adeno-associated virus serotype 9 (AAV9) mediated either human erythropoietin or green fluorescent protein (GFP) gene delivery into rat striatum results in a robust and long-term transgene expression, leading to protection of dopaminergic neurons in the substantia nigra in a rat model of Parkinson's disease. In the present study, we examined whether intracranial injections of AAV9-GFP can achieve long-term GFP gene expression in the monkey brain and host inflammatory and immune responses to GFP transduction. Ten microliters of AAV9-GFP with a titer of 1.8 × 1013 vg ml−1 were stereotactically injected into the right putamen of rhesus monkeys with a 10-μl Hamilton microsyringe. The monkeys were sacrificed 1 year after viral injections, and monkey brains were prepared for histological examinations and immunofluorescent staining. Inflammatory and immune responses to AAV-GFP in the putamen were immunocytochemically examined by assessing the levels of major histocompatibility complex (MHC) class I and class II antigen expression and infiltration of the cluster of differentiation (CD) 4 and CD8 T lymphocytes and accumulation of activated microglia and astrocytes. GFP gene expression was robust, and numerous GFP-positive cells were widespread in the right putamen. A number of GFP-positive cells resembled well-developed neurons with several processes. GFP-positive cells were also colocalized with neuronal nuclei (NeuN), confirming that GFP-positive cells are neurons. Immunofluorescent staining showed that a number of neurons in the ipsilateral substantia nigra pars compacta were double labeled for tyrosine hydroxylase and GFP, suggesting that there was GFP retrograde transduction. In addition, immunofluorescent staining showed that there was accumulation of activated microglia and astrocytes, increased levels of MHC class I and class II antigen expression and infiltration of a few numbers of CD4 and CD8 T lymphocytes in GFP transduction areas. These inflammatory cells were likely localized around blood vessels. Perivascular cuffs were also sometimes observed. Our results suggest that although AAV9-mediated GFP gene expression is robust and widespread in injected putamen for 1 year, there are ongoing chronic inflammatory and immune responses to GFP transduction.
Supported by grants from the Chinese Ministry of Science and Technology, Beijing Natural Science Foundation and Beijing Institute for Brain Disorders.
A. Yoo, S. A. Acosta-Perez, M. M Pabon, D. G. Hernandez-Ontiveros, I. De La Pena, N. Tajiri, and C. V. Borlongan
Center of Excellence for Aging and Brain Repair, Department of Neurosurgery and Brain Repair, University of South Florida Morsani College of Medicine, Tampa, FL, USA
Stroke is caused by a blocked blood vessel or bleeding in the brain and is a major factor of death in many nations, and even noncritical strokes lead to a high reduction in the quality of life. To date, only one drug, tissue plasminogen activator (tPA), is effective in stroke but with a very restricted therapeutic window. Exploring novel treatments for stroke is an urgent clinical need, especially for stroke patients who do not benefit from tPA (i.e., incomplete reperfusion). Assessment of therapeutic agents requires a clinically pertinent stroke model. The middle cerebral artery (MCA) is a prominent artery that supplies blood to the brain. Therefore, we produced an MCA occlusion (MCAo) with incomplete reperfusion as a model to replicate this subset of stroke patients. Briefly, adult Sprague–Dawley rats (9 weeks, 250 ± 20g) were anesthetized with isoflurane under a mixture of oxygen/nitrous oxide, then, we employed laser Doppler for assessment of baseline cerebral blood flow at 0 h. After making a midline incision in the neck, the external carotid artery (ECA) was tightly ligated and a 4–0 monofilament nylon suture (20 mm) was inserted from the bifurcation of the common carotid artery (CCA) to the left internal carotid artery (ICA). To prevent bleeding, the common carotid artery and left internal carotid artery were tightly ligated with silk. Laser Doppler was again performed during suture placement to confirm successful occlusion. After 1 h occlusion, rats were reanesthetized as described above, and then the filament was removed and the CCA ligated, thereby allowing only an incomplete reperfusion from the contralateral CCA, verified by another laser Doppler measurement. We conducted neurological tests at 1 h, 24 h, and 72 h, and then euthanized the animals for histological evaluation of cerebral infarcts. For detection of striatal and cortical infarction, we performed TTC (2,3,5-triphenyl-2H-tetrazolium chloride) staining 3 days after MCAo surgery. Consequently, these brain infarcted animals exhibited significant neurological deficits at 1 h, 24 h, and 72 h. Based on these results, our team established a model of focal stroke with incomplete reperfusion analogous to the histopathological alterations seen in stroke patients who do not benefit from thrombolytic therapy, which should allow testing of novel therapeutics for this specific stroke population.
D. Yu,*† X. Zeng,*† Z. Aljuboori,*† J. E. Anderson,*† and Y. D. Teng*†‡
*Neurosurgery, Harvard Medical School/Brigham and Women's Hospital, Boston, MA, USA
†Division of SCI Research, Veteran Affairs Boston Healthcare System, Boston, MA, USA
†PM&R, Harvard Medical School/Spaulding Rehabilitation Hospital, Boston, MA, USA
We reported previously that peripheral nerve anastomosis (i.e., neurotization) post-thoracolumbar hemisection spinal cord injury (SCI) reroutes sensorimotor signals that reactivates locomotor pattern generation (LPG) in rats (Konya et al., 2008). We have now tested our hypothesis that peripheral nerve rerouting may enable reactivation of LPG after thoracolumbar contusion. Female Sprague–Dawley (S-D) rats (220–235g) received T13–L1 contusion (mild: 10 g × 12.5 mm; moderate: 10g × 25mm). T12 intercostal nerve was anastomosed to L3 nerve root (n = 14/group) or sham-operated (n = 8/group) either 1 week or 13 weeks after either mild or moderate SCI. For chronic SCI, neurotization was done 10 days following human mesenchymal stem cell (hMSC) injection (4 × 50 k cells/μl; 1 μl injection at 1 mm rostral and caudal to the epicenter, respectively, and 2 μl into the injury site; n = 8 for each SCI group). Behavior tests were performed for 8–12 weeks following neurotization. Also used were multimodal analyses of neural tracing and ethynyldeoxyuridine (EdU)-labeling of endogenous neural stem cell proliferation (50 mg/kg, q.d. × 5 days, IP), plus electrophysiological and neuropharmacological assessments. Our data demonstrates that subacute T12–L3 neurotization markedly improves hindlimb ambulation and other coordinated functions. Moreover, there are discernible propriospinal and somatic sensory improvements. Neurotization after hMSC transplantation also restores locomotion in rats with a chronic contusion. Mechanistic analyses show neurotization-triggered reorganization of neural circuits, increased propriospinal projection connectivity across the injury site, and critical rules of serotonin neurotransmission. Therefore, peripheral nerve neurotization builds unique neural circuits for LPG reanimation after thoracolumbar contusion, the second most common type of clinical SCI.
F. Yue,* P. Chan,* H. Wan,† R. Weeks,† R. Grondin,† and Z. Zhan‡
*Department of Neurobiology, Beijing Institute of Geriatrics, Xuanwu Hospital of Capital Medical University, Beijing, China
†WinconTheracells Biotechnologies Co., LTD, Nanning, Guangxi, China
†Department of Anatomy and Neurobiology, University of Kentucky College of Medicine, Lexington, K Y, USA
Cognitive and motor declines are two major geriatric problems often coexisting in older adults. Also, mild cognitive impairment (MCI) is frequently observed in patients with Parkinson's disease (PD) in addition to motor dysfunction. Amelioration of cognitive deficits either in patients with PD or in senescence has proven problematic in the clinic due, at least in part, to a poor understanding of the mechanism underlying PD-MCI and age-associated cognitive declines. Thus, a better characterization of the similarities and differences between age-associated and disease-related cognitive impairments should help better design therapeutic strategies tailored to restore cognitive functions in either the elderly or PD patients. In the present study, 18 cynomolgus monkeys were trained and tested on a delayed matching-to-position (DMTP) and delayed matching-to-sample (DMTS) task, including six middle-aged, six aged, and six 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP)-treated animals. After a 3-month break, cognitive testing was resumed in all animals both before and after a 4-day oral L-dopa administration. Here we are reporting that 1) delay-dependent declines in percentage accuracy were seen on both tasks in normal (MPTP naive) animals while only seen on the DMTP task in MPTP-treated animals, 2) animals with dopamine deficiency had a greater difficulty learning and performing on the DMTS task compared to normal animals, 3) L-dopa treatment did not improve performance on either task in any animals, but rather led to a worsening in performance on the DMTP task in normal middle-aged and MPTP-treated animals, and 4) cognitive declines correlated with age only on the DMTS but not on the DMTP task. Our results suggest that cognitive impairments seen in animals with MPTP-induced dopamine deficiency differ from those seen in aged animals.