Abstract
M. E. Emborg*†
*Preclinical Parkinson's Research Program, Wisconsin National Primate Research Center, University of Wisconsin-Madison, Madison, WI, USA
†Department of Medical Physics, University of Wisconsin-Madison, Madison, WI, USA
In a relay race, team members take turns participating in a portion of a race. Success depends on each person performing to the best of his or her abilities. Translational research is like a relay race, in which scientists build upon other scientists' results, ultimately benefitting patients by creating and selecting better treatments. Every scientist, like every racer, is responsible for the quality of his or her contribution, in this case, to the scientific enterprise. In this context, it is humbling to realize the high rate of attrition during the clinical translation of neurological treatments. During the 2017 Presidential Symposium, we will have an opportunity to analyze factors affecting the translational process of candidate neurological therapies and discuss strategies to improve the outcomes.
J. Kimmelman
STREAM Research Group, Department of Biomedical Ethics, McGill University, Montreal, QC, Canada
The vast majority of neurological drugs put into clinical development fail to demonstrate safety and efficacy—failure rates are especially high for neurodegenerative disorders like Parkinson's disease. These failures exact heavy burdens on patients, research systems, and society in general. Does the high rate of attrition in neurological drug development reflect inevitable uncertainties in the process of clinical development, or are these failures due to correctable factors in the way we conduct preclinical and clinical research? In this presentation, I argue that both explanations are at play. I also suggest that both explanations also harbor important lessons for how we might reorganize and refine the way pre-clinical and clinical research in neurology is organized and conducted. I close by outlining a set of recommendations for limiting the burdens of failure in neurological drug development, maximizing prospects of success, and capitalizing on failed drug development trajectories.
S. A. Acosta, V. A. Guedes, J.-Y. Lee, 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
Stroke is the number one cause of disability in the adult population and the fourth leading cause of death in the US. Currently, the therapeutic interventions are limited with only one FDA-approved drug for ischemic stroke, namely, tissue plasminogen activator or tPA. Recent novel findings revealed that the cerebral endothelium secretes molecules, namely, the inflammation-associated vasculome. In the present study, we evaluated the therapeutic effect of endothelial progenitor cells (EPCs) on the inflammation-associated stroke vasculome in vitro and in vivo. Briefly, in vitro, human endothelial cells (HEN6) were prepared and grown for 10 days. qRT-PCR analysis of the expression of specific stroke vasculome genes revealed that under ambient condition, basal levels of Brahma (BRM), nuclear factor of κ light polypeptide gene enhancer in B-cell inhibitor (IκB), forkhead box F1 (FOXF1), and inter-α-trypsin inhibitor heavy chain family member 5 (ITIH-5) could be detected (compared to nonendothelial NT2N cells acting as control), but following oxygen-glucose deprivation (OGD), there were significant elevations in all four inflammation-associated stroke vasculome genes (p < 0.05 vs. respective level of each gene in ambient condition). Interestingly, coculture of HEN6 with human EPCs (1:1 ratio) during the OGD treatment significantly blocked the elevations of BRM, IKB, and FOXF1 (p < 0.05 vs. respective level of each gene in OGD condition), but not ITIH-5 (p > 0.05). Next, employing knockdown/antisense technology, silencing the inflammation-associated stroke vasculome gene, IκB, as opposed to scrambled knockdown, blocked the EPC-mediated protection of HEN6 against OGD (p < 0.05 vs. OGD or OGD + IκB knockdown). In vivo, rats received injection of intracerebral human EPCs (300,000 cells) or vehicle (saline) into the striatum and cortex 4 h after ischemic stroke. Motor and neurological functions were assessed at baseline, after ischemic stroke on day 0, day 1, day 3, day 7, and day 30 after human EPC transplantation. Elevated body swing test, forelimb akinesia, and paw grasp test revealed a significant amelioration of stroke-hEPC animals at all time points compared to stroke-vehicle animals (p < 0.01). Rotarod test showed a significant progressive amelioration on motor function of stroke-hEPC animals relative to stroke-vehicle animals up to day 30 (p < 0.01). At 7 days after transplantation, quantification of the fluorescent staining intensity in the cortex and striatum revealed a significant upregulation of the endothelial marker rat endothelial cell antigen 1 (RECA1) and a downregulation of the stroke-associated vasculome BRM, IKB, FOXF-1, ITIH-5, and plasma membrane calcium-transporting ATPase 2 (PMCA2) in the ipsilateral side of the cortex and striatum of stroke-hEPC animals compared to the stroke-vehicle animals (p < 0.0001). In sum, coculture and intracerebral transplantation of human EPCs show therapeutic benefits possibly by interacting and modulating the inflammatory vasculome associated with stroke injury.
Research supported by NIH R21 1R21NS094087-01.
S. A. Acosta, 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
Stroke is the primary cause of severe long-term disabilities in the adult population in the US. Stroke alters functional and structural neuroplasticity, which contributes to neuronal death and cognitive dysfunction. High levels of cognitive ability at an early age have been implicated in neuroprotection in adulthood, characterized by a decreased incidence of cognitive impairments and neurodegenerative diseases. A similar neuroplasticity-based neuroprotective mechanism may influence the neuropathology of stroke after stem cell treatment; therefore, we investigated whether transplantation with human bone marrow stem cells (hBMSCs) in adult smart rats (fast learners) exposed to stroke could produce a robust therapeutic effect and stem cell mobilization compared to slow learners and/or untrained rats. Briefly, to test this hypothesis, we analyzed the cognitive function of adult Sprague–Dawley rats prior to stroke using radial arm water maze. After training, rats were divided into fast learners and slow learners as a measure of high cognitive ability and poor cognitive ability, respectively, and then animals were subjected to ischemic stroke. Animals were transplanted intravenously with 2 × 106 hBMSCs or vehicle (saline) at 3 h after ischemic stroke. Motor, cognitive (radial arm water maze), and neurological tests were evaluated at day 1, day 3, and day 7 after stroke. Results from immunofluorescence revealed a significant increased mobilization of human nuclei-positive (HUNU+) cells in the hilus and dentate gyrus of stroke-hBMSC fast learners compared to stroke-hBMSC slow learners, stroke-hBMSC untrained rats, and stroke-vehicle groups. Meanwhile, relative fluorescence intensity showed significant increments in nestin and N-methyl-D-aspartate receptor subtype 2B (NMDAR2B) (neuroplasticity markers) in the striatum, subventricular zone (SVZ), and dentate gyrus of stroke-hBMSC fast learners compared to stroke-hBMSC slow learners, stroke-hBMSC untrained rats, and stroke-vehicle groups. Moreover, quantification of the relative expression of NMDAR modulators revealed a significantly increased expression of vascular endothelial growth factor (VEGF+) and von Willebrand factor (WF+) cells in the striatum, dentate gyrus, hilus, and CA1 area of stroke-hBMSC fast learners relative to stroke-hBMSC slow learners, stroke-hBMSC untrained rats, and stroke-vehicle groups. These findings indicate that higher cognitive function may positively influence the neuroplasticity and stem cell mobilization after stroke.
Support: C.V.B. is supported by NIH NINDS 1R01NS071956-01, NIH NINDS 1 R21 NS089851-01, DOD TATRC W81XWH-11-1-0634, James and Esther King Foundation for Biomedical Research Program 1KG01-33966, SanBio Inc., Celgene Cellular Therapeutics, KMPHC, and NeuralStem Inc.
J. D. Anderson,* J. Rossignol,†‡ G. L. Dunbar,‡ D. Yavagal,§ S. EL-Andaloussi,¶# J. Lehtio,** and J. A. Nolta*
*Stem Cell Program, Department of Internal Medicine, University of California Davis, Davis, CA, USA
†College of Medicine, Central Michigan University, Mount Pleasant, MI, USA
‡Field Neurosciences Institute for Restorative Neuroscience, Central Michigan University, Mount Pleasant, MI, USA
§University of Miami School of Medicine, University of Miami, Miami, FL, USA
¶Department of Laboratory Medicine, Karolinska Institute, Stockholm, Sweden
#Department of Physiology, Anatomy and Genetics, University of Oxford, Oxford, UK
**Clinical Proteomics, Department of Oncology-Pathology, Karolinska Institute, Stockholm, Sweden
Mesenchymal stem cells (MSCs) mediate functional recovery in animal models of neurological diseases such as ischemic stroke and traumatic brain injury via secretion of therapeutic factors such as growth factors and cytokines. Recently, a new cell-to-cell communication system has been characterized that is mediated by the extracellular secretion and uptake of small lipid-bound vesicles called exosomes. Our group demonstrated that MSC-derived exosomes function as paracrine effectors of neurovascular remodeling and reduces inflammatory cytokines and induces functional recovering in a rat model of ischemic stroke. Our group has also identified numerous proteins packaged into MSC exosomes that potentially mediate these observations. We used high-resolution isoelectric focusing coupled liquid chromatography tandem mass spectrometry (HiRIEF LC-MS/MS), a novel, unbiased high-throughput proteomics approach to comprehensively characterize the proteinaceous contents of MSC exosomes derived from in vivo-like conditions (hypoxia and serum starvation). In total, we identified 1,927 proteins in MSC exosomes, representing, to our knowledge, the first time their proteome has been comprehensively assessed. Multilayered analyses identified several putative effectors that modulate neurite outgrowth, axonal guidance, and oxidative stress. Furthermore, we demonstrate that MSC exosomes are capable of being modified to be enriched for therapeutic factors such as microRNAs (miRNAs) and taken up by target cell populations within 1 h. Taken together, these studies provide functional and mechanistic insight into the use of MSC exosomes as a putative therapeutic for neurological diseases such as ischemic stroke.
M. Assadian,* A. Thomas,* R. De Souza,* I. Backstorm,* A. Brown,* E. Ouellet,* S. Garg,* G. Tharmarajah,* K. Marshall,* S. Chang,* T. Leaver,* A. Wild,* P. Deng,†‡ D. J. Segal,‡ J. A. Nolta,† K. D. Fink,† R. J. Taylor,* and E. Ramsay*
*Precision NanoSystems Inc., Vancouver, BC, Canada
†Stem Cell Program and Institute for Regenerative Cures, University of California, Davis Health Systems, Sacramento, CA, USA
‡Genome Center, MIND Institute, Department of Biochemistry and Molecular Medicine, University of California, Davis, CA, USA
Advances in the gene-editing arena, specifically with clustered regularly interspaced short palindromic repeats (CRISPR)–CRISPR-associated protein-9 nuclease (Cas9), have pushed the demand for efficiently delivering payloads even further. Of the tools available, developments in the field of lipid nanoparticles (LNPs) have allowed for the reliable and efficient delivery of CRISPR components, both in research and clinical settings. Here we bridge that gap by describing the development of an LNP delivery system for CRISPR components, robustly manufactured with clinical-grade materials using microfluidic technology at scales for screening applications, in vitro experiments, and research in animals. We describe the use of LNPs for highly efficient encapsulation and delivery of payloads, such as small interfering RNA (siRNA), messenger RNA (mRNA), and plasmid. In this proof of concept, we show that representative small RNAs, mRNAs, and plasmids can be successfully delivered to primary neurons. LNPs manufactured to encapsulate various nucleic acids can do so with high efficiency, encapsulating more than 95% of the payload, minimizing payload loss. Transfection efficiency of the LNPs is >95%, quantified using a fluorescent dye. The biological endpoint assays used to determine the accessibility of the payloads delivered varies for siRNA, mRNA, and plasmid. Using doses of 1 μg/ml of media, we achieved >90% knockdown with siRNA delivery, >90% of the primary neurons are green fluorescent protein positive (GFP+) with GFP mRNA delivery, and >60% of the primary neurons are GFP+ with GFP plasmid delivery. The LNPs are well tolerated, such that 5X the required doses have no observable cytotoxicity. We show that the LNPs can also be used to deliver payloads into various regions of the animal brain. The localized injections into the cortex and the striatum are well tolerated and have extensive distribution. These validation studies provide suitable insights in establishing strategies for efficiently delivering CRISPR components into primary cultures and into the animal. The use of LNPs can be extrapolated to CRISPR components with a simple change in payload. We have editing efficiencies associated with delivering guide RNAs (gRNAs) to Cas9-expressing cells, as well as simultaneously delivering Cas9 mRNA and gRNAs to cells.
S. Bäck,* K. A. Trychta,* C. Richie,* M. J. Henderson,† and B. K. Harvey*
*Intramural Research Program, National Institute on Drug Abuse, National Institutes of Health, Baltimore, MD, USA
†National Center for Advancing Translational Sciences, NIH, Bethesda, MD, USA
Endoplasmic reticulum (ER) stress is observed in a wide range of neurodegenerative diseases and is linked to the intracellular accumulation of misfolded proteins. Many chaperones important for proper protein folding carry a C-terminal Lys-Asp-Glu-Leu (KDEL) or KDEL-like sequence, which mediates ER retention by interaction with trans-membrane Golgi-localized KDEL receptors. The binding of KDEL ligand to receptor in the Golgi network induces a signaling cascade that results in the formation of a retrogradely transported vesicle bringing the KDEL-containing protein back to the ER. The increase in demand for chaperones during ER stress would suggest that a pathway aimed at keeping these proteins in the ER may also be regulated by ER stress. We therefore carried out transcriptional analysis of the three mammalian KDEL receptor genes (KDELR1, KDELR2, and KDELR3) in a human neuronal cell line and rat primary cortical neurons treated with thapsigargin and tunicamycin, chemical inducers of ER stress. We found a robust upregulation of KDELR2 and KDELR3 in response to ER stress. TRANScription FACtor database (TRANSFAC) analysis of the promoter from all three genes revealed putative binding sites for X-box-binding protein 1 (XBP1) and activating transcription factor 6 (ATF6)—two transcription factors activated during ER stress. Using transgenic tools, we found that KDELR2 and KDELR3 were highly responsive to increased XBP1 activity. Inhibition of ER stress-induced XBP1 splicing was able to attenuate the upregulation of KDELR2 and KDELR3, further supporting our observation that XBP1 is capable of upregulating KDEL receptor expression. Overall, our results identify the KDEL receptors as novel ER stress-responsive genes. Considering their function as a retrieval mechanism for chaperones and other proteins important for the maintenance of protein folding and ER homeostasis, targeting or augmenting the KDEL receptor pathway may have therapeutic value in diseases characterized by an accumulation of misfolded protein and ER stress.
T. R. Brickler, A. Hazy, K. Gresham, B. Okeyere, X. Wang, and M. H. Theus
Department of Biomedical Sciences and Pathobiology, Virginia-Maryland Regional College of Veterinary Medicine, Blacksburg, VA, USA
Arteriogenesis is the most important adaptive process known to occur following vascular injury throughout the body. Unlike angiogenesis (the long-term sprouting of new capillary networks), arteriogenesis refers to the immediate growth/enlargement of preexisting arterioles, which are a principal delivery route for oxygen, nutrients, and potential therapeutic agents. Recent studies have established that pial arteriole anastomosis or collaterals develop during three distinct phases: early embryonic proliferation of the vascular plexus at E15.5-E18.5, postnatal pruning at P1-P21, followed by maturation of the pruned collaterals from P21 into adulthood. While having greater numbers of collaterals that form during development can prevent progressive tissue damage in adulthood, tissue reperfusion can also be enhanced by the postinjury process of arteriogenesis. Analysis of the mechanisms regulating arteriole collateralization has important implications for therapies aimed at promoting neurovascular repair following traumatic brain injury (TBI). Murine studies have provided significant insight into collateral development, including strain differences, and its acute hemodynamics in response to environmental stimuli. However, limited methods exist to visualize the morphological development and remodeling in postnatal and juvenile mice that are fast, efficient, and economically feasible. Although changes in the collateral circulation have been extensively studied in cerebral ischemia, our understanding of collateral formation and remodeling is limited in the context of brain trauma. Consistent with previous findings, vessel painting shows that postnatal collateral vessel numbers are reduced in CD1 mice from P1 to P21, while adult numbers remain at P21 levels, indicating that collateral pruning is complete by 21 days postnatal. However, cortical trauma using the moderate controlled cortical impact (CCI) injury model in juvenile P21 mice caused a significant increase in diameter of the pial arterioles ipsilateral and contralateral to the injury site at 4 days post-CCI injury and maintained their increase in size at 14 days after CCI. This effect was abolished in adult mice, suggesting the potential to reform collateral arteriole branches remains after pruning and is complete in the juvenile cerebral collateral circulation, the mechanism(s) of which are lost during the maturation stage. Furthermore, juvenile mice that displayed a significant increase in blood flow restoration to the area of injury that occurred as soon as 2 days after injury and at 4 and 14 days after CCI had a significantly smaller lesion volume compared to adult mice. This correlated with a significant increase in motor recovery and memory using the rotarod and novel object recognition behavioral tests. A novel approach to look at the gene expression profiles and epigenetic profiles of endothelial cells after trauma between adult and juvenile mice was employed to begin to elucidate the factors and mechanisms that regulate arteriogenesis. These data highlight a novel healing response that occurs following TBI in juvenile mice that is not exhibited in adult mice.
T. J. Collier,*† L. Lapidus,‡ C. E. Sortwell,*† C. Justman,§ P. Lansbury,§¶ and K. L. Paumier#
*Department of Translational Science and Molecular Medicine, Michigan State University, Grand Rapids, MI, USA
†Hauenstein Neuroscience Center, Mercy Health Saint Mary's Hospital, Grand Rapids, MI, USA
‡Department of Physics and Astronomy, Michigan State University, East Lansing, MI, USA
§Lysosomal Therapeutics, Inc., Cambridge, MA, USA
¶Center for Neurologic Diseases, Brigham and Women's Hospital, Department of Neurology, Harvard Medical School, Cambridge, MA, USA
#Department of Neurology, Washington University, Saint Louis, MO, USA
The pathology of Parkinson's disease (PD) is characterized by the formation of intraneuronal inclusions called Lewy bodies and Lewy neurites, which are composed of misfolded, fibrillar α-synuclein (α-syn). One therapeutic strategy to slow disease progression is to reduce the accumulation of toxic species by preventing the native/monomeric form of α-syn from misfolding and aggregating. Previous work from our laboratory suggests that tricyclic antidepressants (TCAs) reduce neurodegeneration in a preclinical toxin model of parkinsonism. In a retrospective analysis of data from an early cohort of patients with PD, TCAs were specifically identified as the class of antidepressant medications associated with a significant delay in the need for dopaminergic therapy. Together, these findings, and others, support the notion that TCAs have disease-modifying potential within an existing framework of established safety. Using in vitro aggregation and kinetics assays, our current studies demonstrate that the TCA nortriptyline (NOR) directly binds to monomeric α-syn at physiologically relevant concentrations and reduces the rate of aggregation eightfold by enhancing reconfiguration of the monomer. In addition, NOR inhibits the accumulation and subsequent aggregation of α-syn in several in vivo models including transgenic Drosophila and mice, and the rat preformed fibril α-synucleinopathy model. These findings suggest that NOR, a compound with established safety and efficacy for the treatment of depression, may slow progression of α-syn pathology associated with synucleinopathies, including PD, by directly binding to monomeric α-syn, thereby inhibiting formation of toxic conformations of the protein and preserving its normal functions.
M. R. Cookson
Laboratory of Neurogenetics, National Institute on Aging, NIH, Bethesda, MD, USA
Many genes and genetic variants have been convincingly shown to be associated with Parkinson's disease (PD). These generally fall into two categories—amino acid-changing variants that segregate with inherited PD in families and a mixture of coding and noncoding variants associated with risk of sporadic PD at the population level. A few key genes, including leucine-rich repeat kinase 2 (LRRK2), contain variants that fall into both categories, which nominates the LRRK2 chromosomal region as a pleomorphic risk locus. The major question to be addressed in this talk is how we can interpret this complex genetic information in the context of mechanisms that affect cell biology. My lab has been using a range of large-scale unbiased approaches to address the specific problem of the normal function and mutation-induced dysfunction of LRRK2. We have shown that LRRK2 physically interacts with many other proteins in the cell, including two that are also nominated as risk factor genes for PD, Rab-7-like protein 1 (Rab7L1) and cyclin G-associated kinase (GAK). The significance of this association is that both proteins are involved in vesicular trafficking and Rabs, in general, are direct kinase substrates of LRRK2, suggesting a direct and important mechanistic relationship, especially since some mutations in LRRK2 enhance kinase activity. I will also present recent proteomics data that suggest some outputs of altered LRRK2 activity in the context of knockout mice, which nominate proteostasis as a major outcome of loss of LRRK2.
S. E. Counts,*†‡§ S. C. Kelly,*‡ R. B. Weinberg,* and J. S. Beck*
*Department of Translational Science and Molecular Medicine, Michigan State University, Grand Rapids, MI, USA
†Department of Family Medicine, Michigan State University, Grand Rapids, MI, USA
‡Cell and Molecular Biology Program, Michigan State University, Grand Rapids, MI, USA
§Hauenstein Neurosciences Center, Mercy Health Saint Mary's Hospital, Grand Rapids, MI, USA
We previously reported that cortical expression levels of chaperones and proteases involved in the mitochondrial unfolded protein response (mtUPR) are upregulated in familial and sporadic Alzheimer's disease (AD), suggesting sustained activation of this protective proteostasis pathway. However, whether mtUPR activation is evident earlier in disease and the functional consequences of this sustained response are unknown. To measure mtUPR activation during the progression of AD, we performed quantitative PCR (qPCR) and Western blotting for several mtUPR markers in frozen temporal cortex samples from subjects who died with a clinical diagnosis of no cognitive impairment (NCI), mild cognitive impairment (MCI), or mild AD (n = 8/group). To study sustained mtUPR activation in vitro, we mimicked mitochondrial proteostatic stress by transfecting differentiated human hNT neurons with a mutant form of ornithine transcarbamylase (OTC), which activates the mtUPR. Cultures were (1) probed for mtUPR gene expression using qPCR, (2) cotreated with MitoTracker and LysoTracker dyes to analyze subcellular mitochondrial dynamics, or (3) evaluated for cell survival. Transcripts encoding the mtUPR chaperone heat shock protein 60 (HSP60) and protease YME1-like 1 (YME1L1) were upregulated by 50%-60% in MCI and AD compared to NCI (p < 0.01), whereas protein levels for the mtUPR chaperone DnaJ homolog subfamily A member 3, mitochondrial (DNAJA3)/HSP40, and protease caseinolytic mitochondrial matrix peptidase proteolytic subunit (CLPP) were also upregulated by 50%-60% in MCI and AD (p < 0.01). In vitro studies revealed that mutant OTC-transfected hNT neurons displayed an upregulation of mtUPR genes (e.g., HSP60, DNAJA3) and then, paradoxically, underwent cell death compared to wild-type OTC-transfected neurons (p < 0.01, via LIVE/DEAD assay). Further analysis revealed that cell death was preceded by increased mitochondrial fission, mitophagy, and caspase 2 activation. Sustained activation of the normally protective mtUPR may trigger a unique neuronal cell death effector pathway during the early stages of AD.
A. T. Crane,*† J. Mikkila,* J. Brown,* V. Savanur,* J. Voth,* P. Swaminathan,* H. Hewitt,*‡ F. Xiao,*‡ D. Carlson,§ S. Fahrenkrug,*§ and W. C. Low*‡
*Stem Cell Institute, University of Minnesota, Minneapolis, MN, USA
†Minnesota Craniofacial Research Training Program, University of Minnesota, Minneapolis, MN, USA
‡Department of Neurosurgery, University of Minnesota, Minneapolis, MN, USA
§Recombinetics Inc., St. Paul, MN, USA
Blastocyst complementation is an emerging technique in regenerative medicine that can provide an alternative source of human fetal tissue. In an effort to obtain genuine human fetal midbrain dopamine (mDA) progenitors suitable for transplantation, transcription factors necessary for mDA development need to be knocked out in porcine embryos, and then human stem cells can be injected into the blastocyst, which are then transferred to a surrogate gilt. Through normal development, the injected stem cells can potentially fill the niche left by transcription factor knockout and develop into mDA neurons. Our previous study demonstrated that complementation of paired-like homeodomain 3 (Pitx3) knockout porcine embryos with human induced pluripotent stem cells or human umbilical cord blood stem cells was insufficient to generate human cells within the substantia nigra. This could potentially be due to two main factors: first, human cells were unable to respond to the porcine microenvironment and were outcompeted by the host tissue, or it is possible that the niche created by the late onset of Pitx3 expression in the porcine ventral mesencephalon is too late in development for human cells to occupy. To address these factors, the current study injected green fluorescent protein-positive (GFP+) porcine blastomeres into LIM homeobox transcription factor 1 α (Lmx1a)/Pitx3 knockout porcine embryos. Lmx1a is expressed earlier in mDA development, while allogeneic porcine blastomeres are better suited to incorporate within the porcine embryo. Complimented porcine fetuses were extracted at embryonic day 30 and were processed for immunohistochemistry. GFP+ cells were observed throughout the brain of all complimented porcine fetuses, particularly in the alar/basal boundary of the developing midbrain. Closer investigation of the ventral mesencephalon revealed immature dopaminergic progenitors as well as tyrosine hydroxylase+ dopaminergic neurons with fibers in the medial forebrain bundle. Through confocal fluorescence microscopy, GFP+ cells were observed not to colocalize with dopaminergic precursors. The GFP+ cells have a radial glia morphology with fibers running perpendicular to the ventricular space. Results from these studies suggest that compatibility of donor cells for blastocyst complementation is an important factor. Furthermore, successful complementation in the ventral mesencephalon will require greater manipulation of the niche. Future studies will interrogate the transcriptome of the porcine embryo to identify genes necessary for early incorporation as well as knockout of the earliest genes in the specification of the midbrain.
M. G. Crowley,* V. A. Guedes,* M. G. Liska,* L. Gelineau,* S. A. Acosta,* J.-Y. Lee,* M. Provenzano,† I. Antonucci,† L. Stuppia,† C. Cao,‡ 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
†Laboratory of Molecular Genetics, DISPUTer, School of Medicine and Health Sciences, “G. d'Annunzio” University, Chieti-Pescara, Italy
‡Department of Pharmaceutical Sciences, College of Pharmacy, University of South Florida, Tampa, FL, USA
Traumatic brain injury (TBI) may lead to long-term disability or death. Treatment options are very limited, thereby necessitating the development of novel therapies. Coffee has been suggested to possess neuroprotective properties in animal models and epidemiological studies. Here we investigated the possible protective effect of nanosized coffee (nanocoffee) against inflammation-induced cell death in vitro and in vivo, using a well-established TBI model. Mouse cortical neurons (E18) in culture were treated with tumor necrosis factor-α (TNF-α; 10 ng/ml) alone or in combination with nanocoffee for 24 h. Cell viability was measured immediately thereafter using a calcein AM (acetoxymethyl) assay. Sprague–Dawley rats (n=20) were submitted to the controlled cortical impact model of TBI. Animal motor function was evaluated before injury (baseline), hours after the TBI surgery (day 0), and at days 1 and 3 post-TBI using the elevated body swing test (EBST), forelimb akinesia test, and paw grasp test. Rats received an intrajugular vein injection of nanocoffee 3 h after the injury. At day 3, rats were perfused with 4% paraformaldehyde, and the brain tissue was processed for histology and immunohistochemistry. Nanocoffee significantly protected against TNF-α-induced cell death as revealed by an increase in cell viability when it was added to the cell culture (p < 0.05, one-way ANOVA). In vivo, we observed a TBI-induced motor impairment as shown by EBST, paw grasp, and forelimb akinesia (p < 0.0001 vs. baseline, for all tests). We also found a significant effect of the nanocoffee treatment at days 0, 1, and 3 for EBST (p < 0.05, 0.001, 0.001, respectively), and at day 1 (p < 0.01) and day 3 (p < 0.01) for paw grasp and forelimb akinesia. Statistical analysis for behavior tests was performed using two-way ANOVA followed by Bonferroni's test. The present study shows that nanocoffee displays neuroprotective properties in in vitro and in vivo models of TBI. Brain tissue is currently being processed to evaluate nanocoffee effects on TBI-induced cell death and inflammation markers. Future research might determine the applicability of nanocoffee as a form of prevention from and postinjury treatment of the devastating effects of TBI.
C.V.B. is supported by NIH NINDS 1R01NS071956-01, NIH NINDS 1 R21 NS089851-01, DOD TATRC W81XWH-11-1-0634, James and Esther King Foundation for Biomedical Research Program 1KG01-33966, SanBio Inc., Celgene Cellular Therapeutics, KMPHC, and NeuralStem Inc.
P. Deng,*† J. A. N. M. Halmai,* A. Komarla,*† T. Nguyen,* S. Burk,*, J. Carter,* S. Carter,* J. A. Aprile,* M. Cheng,* J. R. Gutierrez,* D. J. Segal,† J. A. Nolta,* and K. D. Fink*‡
*Stem Cell Program and Institute for Regenerative Cures, University of California, Davis Health Systems, Sacramento, CA, USA
†Genome Center, MIND Institute, and Department of Biochemistry and Molecular Medicine, University of California, Davis, CA, USA
‡Department of Neurology, University of California Davis Heath Systems, Sacramento, CA, USA
Huntington's disease (HD) is an autosomal dominant neurodegenerative disorder caused by an abnormal expansion of CAG repeats encoding a polyglutamine sequence in the N-terminal region of the HD gene, huntingtin. It has been suggested that postnatal reduction of mutant huntingtin through protein interference or conditional gene knockout could prove to be an effective therapy for patients suffering from HD. Our previous work established allele-specific targeting and silencing by designing transcription activator-like effectors (TALEs) to target single-nucleotide polymorphisms (SNPs) in the mutant allele with a Krüppel-associated box (KRAB) domain to promote transcriptional repression. We have demonstrated that TALEs significantly reduce mutant Huntingtin (muHTT) at the RNA and protein level without affecting the healthy Htt expression in both patient-derived HD fibroblasts and mouse primary YAC128 neurons. Currently, our group has validated the efficacy of lipid nanoparticles (LNPs) as a delivery vehicle for TALEs in the YAC128 HD mouse. LNPs have been previously shown to have strong potential as a delivery vehicle for biopharmaceuticals with minimal in vivo toxicity and immunogenicity. Our preliminary work has demonstrated high biodistribution (>10 mm3) following unilateral single injections into the mouse striata and robust on-target gene silencing of muHtt with the LNP-TALE complex (p=0.012). In the present study, our group examined alternative routes of administration, therapeutic dosing, toxicity, and functional rescue following delivery of LNP-TALE. We interrogated alternative routes of administration through single intrastriatal, intracerebroventricular, or injection into the corpus callosum with LNP-TALE to identify an efficacious and minimally invasive means to deliver our therapeutic. Previously, we have observed nonsignificant tumor protein p53 (Trp53) activation [injected: F(4,15) = 2.053, p=0.156; contralateral: F(2,8)=0.108, p=0.899] following striatal injection via qPCR. We have probed for altered immunological response in the central nervous system (CNS) following LNP-TALE injection following escalating dose of our therapeutic via immunohistochemistry. Finally, we measured functional rescue through mutant protein clearance and phenotypic behavior restoration. This study builds on our previous work of the potential of TALE as a powerful, personalized gene therapy for individuals suffering from Huntington's disease and seeks to identify a robust in vivo delivery mechanism.
Support for this project was provided by a NIH NIGMS Predoctoral Fellowship T32GM099608 (P.D.), NIH NRSA Postdoctoral Fellowship F32NS090722 (K.D.F.), the California Institute for Regenerative Medicine (CIRM) DR2-05415 (J.A.N.), Graduate Fellowships (NSF GRFP 2011116000, NIH T32-GM008799, NSF GROW 201111600, T32-HL086350), a NIH Director's Transformative Award 1R01GM099688 (J.A.N.), A Stewart's and Dake Family Gift (K.D.F.), HELP4HD International, and philanthropic donors from the HD community, including the Roberson family and Team KJ.
P. Deng,*† J. A. N. M. Halmai,* A. Komarla,*† T. Nguyen,* S. Burk,* J. A. Aprile,* M. Cheng,* J. Carter,* S. Carter,* J. R. Gutierrez,* D. J. Segal,† J. A. Nolta,* and K. D. Fink*‡
*Stem Cell Program and Institute for Regenerative Cures, University of California, Davis Health Systems, Sacramento, CA, USA
†Genome Center, MIND Institute, and Department of Biochemistry and Molecular Medicine, University of California, Davis, CA, USA
‡Department of Neurology, University of California, Davis Heath Systems, Sacramento, CA, USA
Our team focuses on genetically linked neurological disorders such as cyclin-dependent kinase-like 5 (CDKL5) deficiency (a rare pediatric intractable epilepsy), Angelman syndrome, and Huntington's disease, in addition to heat shock proteins linked to protein aggregation and genes implicated in brain cancer that are targetable with gene-editing molecules. Our research is focused on two major goals: (1) the therapeutic application of transcription activator-like effector (TALE) and clustered regularly interspaced short palindrome repeats (CRISPR) to modify gene expression in genetically linked neurological diseases, and (2) the development of delivery systems that can safely and efficaciously target the central nervous system (CNS) with our novel gene-targeting platforms. These goals specifically address (A) how to deliver these potent therapeutics to maximize the biodistribution in the CNS, (B) how to increase the specificity of each construct to limit off-target effects, and (C) how to minimize the immune response to increase safety and the therapeutic potential of this approach. We have validated several transcriptional repressors [Krüppel-associated box (KRAB) and Friend of GATA protein 1 (FOG1)] in our Huntington's and glioblastoma models using both TALE- and CRISPR-guided systems. We have also validated transcriptional activation using domain VP64 in CDKL5 deficiency and for heat shock protein DnaJ heat shock protein family (Hsp40) member B6 (DNAJB6). We have validated the therapeutic potential of these gene-modifying strategies in human and mouse neuronal cell lines as well as in transgenic mouse models of disease. We are now focused on optimizing delivery modalities to efficiently deliver both TALE and CRISPR therapeutics in vivo. Our lab is evaluating viral-mediated delivery (AAV), stem cell-based delivery (genetically engineered mesenchymal stem cells), and lipid nanoparticle delivery (LNP). We have demonstrated significant allele-specific knockdown of the mutant Huntingtin allele in the YAC128 mouse brain following in vivo injections of an LNP-encapsulated TALE fused to a transcriptional repressor. Our preliminary work has demonstrated high biodistribution following unilateral single injections into the mouse striata and robust on-target gene silencing of mutant Huntingtin with the LNP–TALE complex. Our group is evaluating the immunological profile in the CNS following LNP–TALE or CRISPR injection of our therapeutic via immunohistochemistry (IHC) and qPCR. These studies build on our previous work of the potential of TALE and CRISPR/CRISPR-associated protein-9 nuclease (Cas9) as a powerful, personalized gene therapy for individuals suffering from Huntington's disease, Angelman syndrome, and CDKL5 deficiency.
This work was supported by a National Institute of Health National Research Service Award Postdoctoral Fellowship (F32NS090722) (K.D.F.), NIH NIGMS Predoctoral Fellowship 1T32GM099608 (P.D.), National Institute of Health Director's Transformative Award (1R01 GM099688) (J.A.N.), National Institute of Health (1R01GM097073) (D.J.S.), California Institute for Regenerative Medicine (CIRM DR2-05415) (J.A.N.), A Stewart's and Dake Family Gift (K.D.F.), Help4HD International, and philanthropic donors from the HD community, including the Roberson family and Team KJ. The authors declare no conflicts of interest.
J. Ehrhart,* P. R. Sanberg,† and S. Garbuzova-Davis†
*Saneron CCEL Therapeutics, 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
Amyotrophic lateral sclerosis (ALS) is a fatal neurodegenerative disease that affects the motor neurons in the brain and spinal cord. This disease is characterized by progressive muscle atrophy leading to paralysis and eventual death within 3-5 years after diagnosis. Currently, treatment options for ALS are mainly supportive due to the complexity of various factors underlying this disease. Cell therapy might be a promising new treatment for ALS. The mononuclear cell fraction from human umbilical cord blood (MNC hUCB) has been shown to improve disease outcomes in animal models of ALS by providing neuroprotection for motor neurons. The plasma fraction from cord blood has also been found to have promising therapeutic benefits for various disorders. Recently, we showed reduced apoptosis of ALS peripheral blood-derived mononuclear cells by incubation with cord blood plasma (CBP) in vitro. However, the mechanism of how CBP is eliciting these effects is unknown. The aim of this current study was to determine CBP contents. Various cytokines and growth factors within CBP were investigated. Additionally, the trophic potential of CBP was determined by culturing MNC hUCB cells in media supplemented with either autologous CBP, commercially available human control plasma and serum, or the traditional serum supplement of fetal bovine serum (FBS). Results showed a significant reduction in the levels of a majority of the proinflammatory cytokines assayed. Also, CBP contains significantly higher amounts of all growth factors investigated, specifically vascular endothelial growth factor (VEGF). These findings suggest that CBP not only has potential as a stand-alone therapy but also might be a supportive diluent for combined CBP/MNC hUCB cell infusion in ALS patients.
Disclaimer: J.E. is the Director of Research and Development for Saneron CCEL Therapeutics Inc. S.G.-D. is a consultant for, and P.R.S is a cofounder of, Saneron. J.E., P.R.S., and S.G.-D. are inventors on a patent/disclosure (15/250,239): Plasma Derived From Human Umbilical Cord Blood for the Treatment of Neurodegenerative Disorders.
D. J. Eve,*† G. Steiner,* A. Mahendrasah,* C. Kurien,* A. Thomson,* D. Falco,* 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 Neurosurgery and Brain Repair, University of South Florida Morsani College of Medicine, Tampa, FL, USA
Amyotrophic lateral sclerosis (ALS) is a fatal disease characterized by motor neuron degeneration in the brain and spinal cord. Accumulating evidence has demonstrated structural and functional alterations in the blood–spinal cord barrier (BSCB) in ALS, and this barrier damage likely represents an additional pathogenic disease mechanism. Moreover, microhemorrhages as a consequence of the impaired microvessel integrity associated with BSCB damage were indicated in the spinal cord in patients and in animal models of ALS. Repairing the BSCB may therefore be a potential therapeutic strategy for treating ALS. The aim of this study was to determine whether potential BSCB repair by intravenous transplantation of unmodified human bone marrow CD34+ cells (hBM34+) into symptomatic ALS mice leads to restoration of capillary integrity. Three different doses (5 × 104, 5 × 105, or 1 × 106) of hBM34+ cells or media were injected via the jugular vein into male symptomatic G93A SOD1 mice at 13 weeks of age. At 17 weeks of age (4 weeks posttransplant), the mice were sacrificed, and the spinal cords were removed. Coronal cervical and lumbar spinal cord segments were cut and stored for further analyses. Immunohistochemical staining with human anti-von Willebrand factor (vWF) antibody, an endothelial cell marker, was performed to determine differentiation potential and engraftment of transplanted cells within spinal cord microvessels. Capillary rupture in the cervical and lumbar enlargements of the spinal cord (C4-C6 and L3-L5, respectively) was investigated with Perls' Prussian blue staining. The results showed that transplanted hBM34+ cells differentiated into endothelial cells in the spinal cords of all cell-treated animals. In mice receiving the high cell dose, cells engrafted into numerous microvessel walls of the cervical and lumbar spinal cord, showing significantly increased vWF immunoexpression. A substantial presence of microhemorrhages was revealed in the parenchyma of both the gray and white matter of the cervical and lumbar spinal cords of media-injected mice. A dose-dependent reduction in the average number of microhemorrhages was detected, reaching significance in the high dose-treated mice compared to the media-treated mice at both the cervical (1.00±0.63 vs. 5.17±1.19; p<0.05) and lumbar (1.17±0.79 vs. 6.50±0.92; p<0.01) levels of the spinal cord, demonstrating reduced capillary rupture. Microhemorrhages were detected in 50% of the cervical and lumbar spinal cords from the high dose-treated mice, while 100% of the media- and low dose-treated mice evidenced microhemorrhages in both regions of the spinal cord. The percentage of mid-dose-treated mice with lumbar region microhemorrhages also decreased to 66.7%, while all mid-dose-treated mice exhibited cervical microhemorrhages. In all ALS mice, some microhemorrhages were located within white matter regions associated with the ascending and descending pathways. The endothelial cell differentiation and capillary engraftment of transplanted cells might indicate reparative processes toward BSCB restoration. The significant decrease in spinal cord microhemorrhages of symptomatic ALS mice treated with the highest cell dose is also concomitant with microvascular repair. The study results provide translational outcomes supporting the utility of hBM34+ cell transplantation at optimal dose as a future therapeutic strategy leading to potential BSCB restoration in ALS patients.
This study was supported by the NIH, NINDS (1RO1 NS090962-01) grant.
L. F. Fernandes,* I. N. Soares,* T. H. Ferreira-Vieira,* S. O. A. Fernandes,† V. N. Cardoso,† F. J. G. Frezard,* and A. R. Massensini*
*Department of Physiology and Biophysics, Universidade Federal de Minas Gerais (UFMG), Belo Horizonte, Brazil
†Department of Clinical and Toxicological Analysis, UFMG, Belo Horizonte, Brazil
Stroke is one of the leading causes of death and serious long-term disability worldwide. The only therapeutic agent available is tissue-type plasminogen activator (tPA); however, the number of potential patients is limited because of its side effects and narrow therapeutic window. Angiotensin-(1-7) [Ang-(1-7)], an endogenous peptide from the renin–angiotensin system, has shown neuroprotective effects in in vitro and in vivo ischemia models. Ang-(1-7) is rapidly metabolized, but liposome nanoparticle encapsulation prevents degradation of the peptide. Liposomes are biodegradable and biocompatible and are able to increase drug efficacy in in vivo models of stroke; therefore, the use of liposomes as Ang-(1-7) carriers to the central nervous system is an asset for ischemic stroke therapy. In this study, liposomes were labeled with the fluorescent probe DiI (1,1′-dioctadecyl-3,3,3′,3′-tetramethyl-indocarbocyanine perchlorate), followed by Ang-(1-7) encapsulation. In vitro oxygen–glucose deprivation and reperfusion (OGD/R) assays were performed using CD1 mice cerebral tissue. The slices were treated with empty or loaded liposomes during reperfusion, and lactate dehydrogenase (LDH) assays were used to assess cell death. In parallel, CD1 mice underwent temporary middle cerebral artery occlusion (MCAO) surgery. Immediately after reperfusion, liposomes were intravenously injected. The brains were removed 3 or 24 h after injection. In vivo imaging and triphenyl tetrazolium chloride (TTC) staining were performed for liposome detection and confirmation of ischemia. For the in vitro OGD/R assays, we observed that Ang-(1-7) liposomes were able to protect brain cells from ischemic death. The in vivo experiments demonstrated an efficient ischemic model by TTC staining. In addition, liposomes were observed in the brain of sham animals and in the ischemic region of MCAO mice brain, showing that this type of delivery system can cross the blood–brain barrier and accumulate in the ischemic core, thereby presenting high potential for stroke treatment. Our results suggest that liposomes are an interesting perspective system to enhance the neuroprotective effects of Ang-(1-7) in ischemic stroke in vitro and in vivo.
E. Filichia,* B. Hoffer,* X. Qi,† and Y. Luo*
*Department of Neurological Surgery, Case Western Reserve University, Cleveland, OH, USA
†Department of Physiology and Biophysics, Case Western Reserve University, Cleveland, OH, USA
Accumulating evidence suggests that mitochondria-mediated pathways play an important role in dopaminergic neuronal cell death in Parkinson's disease (PD). Dynamin-related protein 1 (Drp1), a key regulator of mitochondrial fission, has been shown to be activated and translocated to mitochondria under stress, leading to excessive mitochondria fission and dopaminergic neuronal death in vitro. However, whether Drp1 inhibition can lead to long-term stable preservation of dopaminergic neurons in the PD-related mouse model remains unknown. In this study, using a classical 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP) animal PD model, we showed for the first time Drp1 activation and mitochondrial translocation in vivo after MPTP administration. Inhibition of Drp1 activation by a selective peptide inhibitor, P110, blocked MPTP-induced Drp1 mitochondrial translocation and attenuated dopaminergic neuronal loss, dopaminergic nerve terminal damage, and behavioral deficits caused by MPTP. MPTP-induced microglial activation and astrogliosis were not affected by P110 treatment. Instead, inhibition of Drp1 mitochondrial translocation diminished MPTP-induced tumor protein 53 (p53), BCL2-associated X (BAX), and p53 upregulated modulator of apoptosis (PUMA) mitochondrial translocation. This study demonstrates that inhibition of Drp1 hyperactivation by a Drp1 peptide inhibitor P110 is neuroprotective in a MPTP animal model. Our data also suggest that the protective effects of P110 treatment might be mediated by inhibiting the p53-mediated apoptotic pathways in neurons through inhibition of Drp1-dependent p53 mitochondrial translocation.
D. J Finneran, A. Pitre, C. Schuetz, D. Morgan, and K. R. Nash
Department of Molecular Pharmacology and Physiology, Byrd Alzheimer's Institute, University of South Florida Health, Tampa, FL, USA
The use of gene therapy to reduce pathology or prevent neuron loss in neurodegenerative diseases has gained significant interest in recent years. Specifically, the use of adeno-associated viral (AAV) vectors has become a popular delivery method for gene therapy due to a number of positive attributes including safety. However, one limitation has been targeting the entire brain, which would be required for neurodegenerative diseases such as Alzheimer's disease. Therefore, we aimed to determine an optimal delivery method to maximize recombinant protein distribution throughout the brain while minimizing the number of injected regions within the brain. Using different recombinant AAV (rAAV) serotypes that show distinctive transduction properties, we examined alternative routes of administration and compared green fluorescent protein (GFP) expression throughout the central nervous system (CNS). Intrathecal, intracisterna magna, and intraventricular administration of rAAV1, 5, and 9 did not result in significant transduction that would be useful for a therapeutic approach. Interestingly, a novel mutant AAV9 termed PhP.B did result in significant transduction throughout the entire CNS after tail vein administration. The use of the human synapsin promoter in combination with the PhP.B was also able to express significant levels of GFP throughout the CNS. As an alternative novel strategy, we also examined a protein replacement approach. This approach relies on targeting a select group of cells that would act as a factory to secrete a therapeutic protein that could be utilized by neighboring cells. This we approached with a natively secreted protein, fractalkine, which we have previously shown can reduce tau pathology and neuron loss in the tau mouse rTg4510, and with an engineered GFP protein, termed secGFP. secGFP has a secretion signal peptide for release and a cell-penetrating peptide sequence for reuptake into neighboring cells. To take advantage of neuronal networks within the CNS, we injected AAV2, AAV5, AAV7, AAV8, and AAV9 into the thalamus, the ventral tegmental area, and raphe nucleus (all regions that have significant projections throughout the CNS). Injection into each of these regions resulted in GFP-labeled projections into numerous other brain regions such as the hippocampus, cortex, striatum, and cerebellum. We show that the thalamic injections resulted in the greatest expression in the hippocampal formation and cortex but lacked expression in the cerebellum. Furthermore, we show the relative overexpression of soluble fractalkine after intraventricular administration of AAV4, tail vein administration of AAV-PhP.B, and parenchymal administration of AAV9.
L. Gelineau, V. A. Guedes, D. J. Eve, M. G. Liska, C. Stonesifer, M. G. Crowley, S. A. Acosta, 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
Traumatic brain injury (TBI) is a major cause of death and might result in chronic sequelae. TBI-induced emotional dysfunction is prevalent in survivors and negatively impacts on their quality of life. The mechanisms underlying the emotional dysfunctions following TBI are poorly understood, and therapeutic options are very limited. Hyperbaric oxygen therapy (HBOT) is considered a potential treatment option for TBI. In this study, we evaluated anxiety-like behavior in rats submitted to a well-established TBI model. In addition, we evaluated the effects of HBOT on the amygdala, a critical brain structure implicated as playing a central role in the processing of emotional and stressful stimuli. Sprague–Dawley rats (n = 20) were submitted to the controlled cortical impact model of TBI. Anxiety-like behavior was evaluated using the elevated plus maze (EPM). Rats were tested before the injury (baseline) and at days 7, 14, 21, and 28 and weeks 6, 8, 12, and 14. HBOT sessions started at 30 days post-TBI (90 min, 100% oxygen, 1.5 ATA). Rats were sacrificed after the HBOT treatment, and the brain tissue was processed for immunohistochemistry. We observed TBI-induced anxiety behaviors at all time points as revealed by a significant decrease in the time spent in open arms in the EPM (p < 0.0001, one-way ANOVA, followed by Bonferroni's test). The number of entries in closed arms decreased at day 1 and then returned to baseline values (p < 0.05, one-way ANOVA followed by Bonferroni's test). After the HBOT, we observed higher average time spent in open arms and number of entries in closed arms in the treatment group compared to the nontreated rats. However, the HBOT effect on time spent in open arms or number of entries was not statistically significant (p > 0.05, two-way ANOVA followed by Bonferroni's test). Histopathological results suggest alterations in amygdala morphology as mediating the observed anxiety behaviors, which appear amenable to HBOT. We observed anxiety-like behaviors after TBI in acute and chronic stages. Immunohistochemistry experiments are underway to fully evaluate neuronal loss in specific cell types of amygdala and the histological correlates of the HBOT treatment. These experiments should provide insights into the mechanisms underlying post-TBI emotional sequelae and in the potential of HBOT as a treatment option for TBI.
C.V.B. is supported by NIH NINDS 1R01NS071956-01, NIH NINDS 1 R21 NS089851-01, DOD TATRC W81XWH-11-1-0634, James and Esther King Foundation for Biomedical Research Program 1KG01-33966, SanBio Inc., Celgene Cellular Therapeutics, KMPHC, and NeuralStem Inc.
H. Ghuman,*† M. Gerwig,‡ F. J. Nicholls,*§ J. Liu,† J. Donnelly,¶ B. Wahlberg,§ S. F. Badylak,*†# and M. Modo*†§
*McGowan Institute for Regenerative Medicine, University of Pittsburgh, Pittsburgh, PA, USA
†Department of Bioengineering, University of Pittsburgh, Pittsburgh, PA, USA
‡Department of Neuroscience, University of Pittsburgh, Pittsburgh, PA, USA
§Department of Radiology, University of Pittsburgh, Pittsburgh, PA, USA
¶Department of Biological Sciences, University of Pittsburgh, Pittsburgh, PA, USA
#Department of Surgery, University of Pittsburgh, Pittsburgh, PA, USA
Stroke is the leading cause of adult disability, and a significant effort is underway to develop therapies to repair the damaged tissue. One of the key challenges in treating chronic stroke is the dramatic loss of brain tissue and the formation of a cavity filled with extracellular fluid (ECF) and cell debris. Extracellular matrix (ECM) constitutes 20% of brain tissue volume. Biomaterials composed of mammalian ECM promote constructive tissue remodeling with minimal scar formation in peripheral tissue and organs. However, the biodegradation and functional effect of injecting a large volume of ECM hydrogel into the brain are unknown. The current study therefore aimed to determine if biodegradation occurs and if ECM remodeling will affect the behavioral deficits of animals with stroke damage. At an 8 mg/ml concentration, ECM hydrogel has rheological properties similar to brain tissue. It can be formulated in a fluid phase at room temperature while forming hydrogels at body temperature. Two weeks after stroke, magnetic resonance imaging (MRI)-defined lesion volume equivalents of ECM were injected into the lesion cavity of stroke rats. A battery of behavioral tests including grip strength, bilateral asymmetry test (BAT), foot fault, and rotameter were performed at pretreatment and at 1, 4, and 12 weeks posttreatment for control (n = 14), untreated (n = 11), and ECM-treated (n = 11) groups. Retention, gelation, and biodegradation of the ECM, as well as host cell invasion and phenotype, were analyzed at 12 weeks postinjection using immunohistochemistry. Brain tissue deformation analysis using T2-weighted MRI scans indicated a 10% decrease in whole-brain tissue volume, twofold increase in ventricle size, 10% midline shift, and 30% decrease in tissue in the stroke-affected hemispheres over 12 weeks. There was no significant difference between untreated and treated groups. Behavioral tests indicated a functional impairment that was not affected by the injection of a large volume of ECM into the cavity. The ECM showed a robust gelation and retention in the lesion cavity with a 30% decrease in volume over 12 weeks. A significant host cell invasion into the ECM hydrogel was seen, with an average of 72,000 cells present within the hydrogel. Monocytes accounted for 55% of the total invading cells and expressed a neutral M1/M2 [cluster of differentiation 86/206 (CD86/206)] phenotype indicating a shift from the acute inflammatory phase to an ECM remodeling phase. Significant proportions of oligodendrocyte progenitor cells (30%) and endothelial cells (4%-5%), essential for repopulation of the neural tissue, were also present. This characterization demonstrates that an ECM hydrogel can be readily injected and retained within the lesion cavity, while promoting an acute endogenous repair response, without deleterious effects. A time course study with varying ECM concentrations is necessary to determine the optimal rate of in vivo biodegradation to further improve the endogenous repair processes.
H. Ghuman,*† B. Wahlberg,‡ J. Liu,† and M. Modo*†‡
*McGowan Institute for Regenerative Medicine, University of Pittsburgh, Pittsburgh, PA, USA
†Department of Bioengineering, University of Pittsburgh, Pittsburgh, PA, USA
‡Department of Radiology, University of Pittsburgh, Pittsburgh, PA, USA
Stroke remains the leading cause of adult disability without an effective treatment. Cell therapy is gradually emerging as a potentially viable therapy with an extended therapeutic window and requires injection of high-density cell preparations near the site of injury using small-gauge needles. Nevertheless, one persisting key challenge for intracerebral cell therapy is its poor efficiency, with only 5%-10% of injected cells being present within the brain after a few weeks of injection. While the inflammatory host microenvironment around the injured site may affect the survival after transplantation, cell damage may first occur during injection from the shear mechanical forces inside the syringe or needle. The aim of this study was to investigate the different aspects of cell injection that influence cell viability, including needle size (20, 26, and 32 gauge), syringe size (10, 50, and 250 μl), ejection rate (1, 5, and 10 μl/min), and the choice of vehicle for cell suspensions (PBS, HypoThermosol, and Pluronic). The results indicate that a combination of 10-μl syringe with a 32-gauge needle requires maximum ejection pressure in the presence of Pluronic as a vehicle for suspension. Although cells passing through a 20-gauge needle experience much lower shear stresses (<5 Dyn/cm2) compared to a 32-gauge needle (5-15 Dyn/cm2), insertion of a 20-gauge needle in brain tissue shows significantly more tissue damage and inflammatory response. Cell transplantation can only be an efficient therapeutic approach if we ensure an optimal transfer of cells from the dish to the brain, as well as keeping the transplanted cells alive and functional for extended periods. Untangling mechanisms of action might not be necessary for clinical translation, but to ensure maximal efficacy, it is essential to know and control the biomechanical stresses that may affect the therapeutic potential of cell therapy.
K. Gimse,* A. Olin,† and C. Burger*‡
*Cellular and Molecular Pathology Graduate Program, University of Wisconsin, Madison, WI, USA
†College of Letters and Sciences, University of Wisconsin, Madison, WI, USA
‡Department of Neurology, University of Wisconsin, Madison, WI, USA
The effect of aging on cognitive ability is considerably diverse. While most people will experience cognitive decline, this can range from mild memory impairments to severe dementia, and many experience no deficits even while surpassing ages of 100 years. Currently, little is known regarding the molecular mechanisms underlying these differing cognitive fates. Previous research in our lab has suggested a role for Homer1c. Long-form Homer proteins such as Homer1c belong to a family of synaptic scaffolding proteins that interact with various proteins in the postsynaptic density, including group 1 metabotropic glutamate receptors (mGluR1/5). Homer1c transcripts are downregulated in the hippocampi of aged learning-impaired (AI) rats compared to aged superior learners (SL) who have been trained in a hippocampal-dependent learning task. Additionally, recombinant adeno-associated virus (rAAV)-mediated overexpression of hippocampal Homer1c is sufficient to rescue synaptic plasticity and memory deficits in Homer1 knockout mice and improve learning in AI rats. In this study, to determine if hippocampal Homer1c expression is necessary for successful cognition, small hairpin RNA sequences targeting Homer1c were cloned into rAAV to knock down Homer1c expression in the hippocampi of adult male Sprague–Dawley rats. The impact of this knockdown on learning ability was assessed through a 1-day version of the Morris water maze (MWM) hidden platform task and probe trial, contextual fear conditioning (CFC) and cued fear conditioning, and novel object recognition (NOR). Homer1c knockdown led to significant deficits in CFC (n = 18; t-test, p = 0.03) but not in cued fear conditioning, and deficits trended toward significance in the MWM hidden platform test (n = 7; t-test, p = 0.23), MWM probe test (n = 7; t-test, p=0.12), and NOR (n = 7; t-test, p = 0.27). To determine the impact of knockdown on synaptic plasticity, the mGluR1/5 agonist (S)-3,5-dihydroxyphenylglycine (DHPG) was used to induce long-term depression (LTD). In contrast to control uninfected animals, application of DHPG (100 μM for 10 min) failed to elicit LTD in the Shaffer collateral pathway of hippocampal slices from knockdown animals [n = 10 slices (seven animals), repeated-measures ANOVA, F(1,8) = 11.29, p = 0.01]. These preliminary results provide proof of concept that Homer1c knockdown leads to deficits in learning and synaptic plasticity. Future studies will focus on the role of Homer1c in age-associated cognitive decline. We will assess the impact of knockdown on learning and synaptic plasticity in aged SL rats, comparing memory task performance before and after knockdown and characterizing the effect of knockdown on LTD. The results of this research will increase understanding of the mechanisms underlying memory consolidation and synaptic plasticity, and how these mechanisms are affected by the aging process, potentially influencing treatment and prevention of age-related neuropathies.
T. G. Golos*†
*Department of Comparative Biosciences, University of Wisconsin-Madison, Madison, WI, USA
†Wisconsin National Primate Research Center, University of Wisconsin-Madison, Madison, WI, USA
Zika virus (ZIKV) is spread by Aedes mosquitoes and sexual contact. ZIKV is now endemic in the Americas following its introduction in 2014. In utero infection with ZIKV in Oceania and the Americas has been associated with increased incidence of fetal microcephaly, and other fetal findings include placental calcifications, growth restriction, arthrogryposis, severe central nervous system (CNS) malformations, intraocular calcifications, cataracts, and skeletal and sensory disorders. The constellation of developmental abnormalities observed following ZIKV infection during pregnancy is termed “congenital Zika syndrome.” Nonhuman primates are important models for human infectious disease, and nonhuman primate pregnancy has salient similarities to human pregnancy, including hemochorial placentation with extensive trophoblast invasion, and prolonged gestation with a similar trajectory of fetal development. A rhesus macaque model has been developed demonstrating consistent susceptibility to ZIKV and the appearance of viral RNA in blood, saliva, urine, and cerebrospinal fluid. However, there is a robust innate and adaptive peripheral immune response, which drives a rapid (within 10 days) clearance in nonpregnant individuals, similar to human infection. To model fetal outcomes in nonhuman primates, we administered Asian-lineage ZIKV subcutaneously to four pregnant rhesus macaques. While nonpregnant animals clear viremia within 10-12 days, maternal viremia was prolonged (up to 70 days) in three of four pregnancies. Fetal head growth velocity in the last month of gestation determined by ultrasound assessment of head circumference was decreased in comparison with biparietal diameter and femur length within each fetus, which were both within normal range. ZIKV RNA was detected in tissues from all four fetuses at term cesarean section; thus, there was 100% vertical transmission. In all pregnancies, neutrophilic infiltration was present at the maternal–fetal interface (decidua, placenta, and fetal membranes), in various fetal tissues, and in fetal retina, choroid, and optic nerves (first-trimester infection only). Finally, ZINV was detected by immunofluorescence microscopy in several fetal tissues, as well as the maternal decidua, confirming viral RNA distribution. The confirmed vertical transmission of ZIKV in this primate model, but without severe neurodevelopmental pathology, is consistent with the sporadic appearance of microcephaly and other serious outcomes in human pregnancies in affected areas. Our results raise the question as to whether infection may be more common than is currently appreciated due to lack of the appearance of congenital Zika syndrome in these infected infants. In addition, the nonhuman primate model may provide a platform to assess risk factors and test therapeutic interventions for interruption of fetal infection.
R. Gonzalez,* I. Garitaonandia,* T. Abramihina,* G. Sherman,* A. Noskov,* A. Semechkin,* A. Shahrul,† G. Nair,† A. H. Evans,† and R. Kern*
*International Stem Cell Corporation, Carlsbad, CA, USA
†The Royal Melbourne Hospital, Parkville, VIC, Australia
Proof-of-concept studies have shown that intranigrostriatal transplantation of current good manufacturing practice (cGMP)-manufactured human parthenogenetic stem cell-derived neural stem cells (ISC-hpNSC®) are safe and functionally integrate and provide significant symptomatic relief in both rodent and primate Parkinson's disease (PD) models. Based on these results, the Australian Therapeutic Goods Administration (TGA) granted approval to conduct a first-in-human phase 1/2a study to evaluate the safety and functional activity of ISC-hpNSC® in PD patients. The phase 1/2a clinical study is designed as a dose-escalation, safety, and preliminary efficacy study of ISC-hpNSC®, intracranially transplanted into patients with moderate to severe PD. The open-label, single-center, uncontrolled clinical trial evaluates three different dose regimens (30, 50, or 70 million ISC-hpNSC®). A total of 12 participants with moderate to severe PD, divided into three cohorts of four are treated. Following transplantation, the patients are monitored for 12 months at specified intervals to evaluate the safety and biologic activity of ISC-hpNSC®. PET scans are performed at baseline, as part of the screening assessment, and at 6 and 12 months after surgical intervention. Clinical responses compared to baseline after the administration of ISC-hpNSC® are evaluated using various neurological assessments such as Unified Parkinson Disease Rating Scale (UPDRS), Hoehn and Yahr, and other rating scales. Patients will be followed up for 5 additional years. Interim safety data show that there are no test article-related adverse events 6 months after transplantation. Efficacy data for patients from the first cohort receiving 30 million ISC-hpNSC® will be presented.
K. Gresham,* T. R. Brickler,* J. Chen,* R. Gourdie,† and M. H. Theus*
*Department of Biomedical Sciences and Pathobiology, Virginia-Maryland Regional College of Veterinary Medicine, Blacksburg, VA, USA
†Center for Regenerative Medicine, VT-Carilion Research Institute, Roanoke, VA, USA
Adult neurogenesis occurs in the dentate gyrus (DG) of the hippocampus and is a highly coordinated process that participates in learning and memory throughout the life span. Neurogenesis begins when nestin+ neural stem cells (NSCs) are instructed to differentiate into polysialylated neural cell adhesion molecule-positive (PSA-NCAM+) neural progenitors (NPs) before further instructional cues to differentiate into newly born neurons. Traumatic brain injury (TBI) produces an initial loss of the NP population and an increase in NSC proliferation, causing a delay as neurogenesis starts again from the beginning. This neurogenic delay correlates with a slow progression in cognitive recovery. The mechanism(s) underlying the NP-specific death and the NSC-specific survival and proliferation remain elusive. Previous data have linked high connexin 43 (Cx43) expression to NSCs and low Cx43 expression to NPs, but no one has discovered a link between Cx43 and NSC survival or NP death in the dentate gyrus. Following TBI, it has been published that there is an increase in Cx43 expression in the hippocampus that correlates with data from another investigator showing an increase in nestin+ NSCs in the dentate gyrus. Our recent data have shown a more specific increase in the dentate gyrus after TBI, but changes in Cx43 expression on specific cell types is still under further investigation. To further elucidate the role of Cx43 in NSC/NP behavior, we used a C-terminal Cx43 mimetic peptide to influence gap junction-associated Cx43 and found a significant reduction in cultured NSC/NP proliferation and survival. Further studies will involve histological investigation of the size and number of Cx43 aggregates on NSCs and NPs as well as on terminal deoxynucleotidyl transferase dUTP nick-end labeling-positive (TUNEL+) dying cells following TBI. We will also be running Western blots to quantify protein expression in the hippocampus following TBI and looking for links between potential phosphorylation sites of Cx43 and outside pathways. Knowledge gained from these studies will improve our basic understanding of the mechanism(s) controlling the neurogenic response in the DG following TBI.
B. Grimmig,* L. Daly,† C. Hudson,*† and P. C. Bickford*†‡
*Department of Molecular Pharmacology and Physiology, 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
‡Research Service, James A. Haley Veterans Affairs Hospital, Tampa, FL, USA
Aging is a primary risk factor for the development of many diseases including neurodegenerative disorders. Aging leads to numerous physiological changes associated with loss of tissue integrity and organ function. Within the brain, the age-related anatomical and physiological changes can compromise cognitive functions including memory, attention, executive function, and perception. Although the exact molecular mechanisms through which brain aging causes significant changes in cognition are still being elucidated, it is widely accepted that increased inflammation, mitochondrial dysfunction, disrupted calcium homeostasis, and elevated oxidative stress all contribute to neurodegeneration. Astaxanthin (AXT) is a carotenoid with multiple health benefits. It is currently marketed as a health supplement and is known for its antioxidant capacity. However, recent evidence has emerged to suggest a much more broad range of biological activities. Results from the current research are beginning to come together to suggest a potential for AXT to promote or maintain neural plasticity. These emergent mechanisms of action implicate AXT as a promising therapeutic agent for cognitive decline and neurodegeneration. Therefore, we investigated the efficacy for AXT supplementation to modulate cognitive function by administering an AXT-enriched diet (delivering 30 mg/kg) to young and aged C57Bl/6N mice for 6 weeks. After 4 weeks of AXT treatment, cognitive performance was assessed using a battery of behavioral tests. We show that AXT facilitated hippocampal-dependent recall in 24 h of contextual fear conditioning as well as augmented motor learning and coordination on the rotarod. We determined that the observed improvement in hippocampal-dependent behavior was accompanied by a concomitant increase in neurogenesis in the subgranular zone of the dentate gyrus. We evaluated every sixth sagittal section for immunohistochemical markers of cell proliferation and quantified positively labeled cells using unbiased stereology (Stereo Investigator, MicroBright Field). AXT supplementation rescued the age-related decrease in the number of Ki-67+ cells in aged mice. Taken together, these results indicate that AXT supplementation may enhance certain aspects of cognitive function across age and can increase hippocampal neurogenesis, indicating a therapeutic role for AXT in attenuating age-related cognitive decline and neurological dysfunction. We are currently exploring the molecular alterations that occur in response to AXT supplementation likely mediating enhanced cognitive performance in both young and aged mice, including neurotrophins, microglial activity, and mitochondrial function.
V. A. Guedes, D. J. Eve, M. G. Liska, C. Stonesifer, L. Gelineau, M. G. Crowley, S. A. Acosta, 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
Traumatic brain injury (TBI) is a major cause of long-term disability and death worldwide, generating high personal and social costs. TBI may lead to sensory, motor, cognitive, and emotional sequelae. Therapy options for TBI are very limited, and therefore, the development of novel therapeutic alternatives represents an urgent clinical need. Hyperbaric oxygen therapy (HBOT) is a Food and Drug Administration-approved treatment for many conditions including wound healing, gangrene, and carbon monoxide poisoning. HBOT has been suggested to have relevant therapeutic indications for neurological diseases. Here we investigated the effects of HBOT in chronic TBI. Sprague–Dawley rats (n = 20) were submitted to the controlled cortical impact model of TBI. Rats received four HBOT sessions starting at 30 days after the TBI (90 min, 100% oxygen, 1.5 ATA). Behavior tests were performed before (baseline) and after the injury (days 1, 7, 14, 21, and 28 and weeks 6, 8, 12, and 14). We evaluated the animals in anxiety tests including elevated plus maze (EPM) and acoustic startle reflex (ASR), in cognitive performance utilizing the radial arm water maze (RAWM) test, and in motor function using the elevated body swing test, forelimb akinesia, and paw-grasp tests. TBI produced motor deficits across all tests and also elicited stress behaviors as evidenced by decreased amplitude in ASR and time spent in open arms in EPM (p < 0.0001, one-way ANOVA), but no significant HBOT effects were observed on these TBI-induced impairments (p > 0.05). TBI also generated cognitive deficits, which were attenuated by HBOT as revealed by a statistically significant difference between the average number of errors in the treated group (0.80 ± 0.63) compared to the control group (1.8 ± 0.42) in the RAWM (p < 0.001, unpaired t-test). Our results suggest that HBOT has a therapeutic potential to treat cognitive deficits in chronic TBI. Future studies should optimize HBOT strategies directed at ameliorating TBI-induced sensory and motor deficits. Histological and immunohistochemical studies to evaluate secondary cell loss and inflammation markers are underway.
Support: C.V.B. is supported by NIH NINDS 1R01NS071956-01, NIH NINDS 1 R21 NS089851-01, DOD TATRC W81XWH-11-1-0634, James and Esther King Foundation for Biomedical Research Program 1KG01-33966, SanBio Inc., Celgene Cellular Therapeutics, KMPHC, and NeuralStem Inc.
A. Habib,* D. Sawmiller,* Y. Xiang,* D. Rongo,* J. Tian,* H. Hou,* J. Zeng,* B. Giunta,* A. Smith,† A. Feng,* T. Mori,‡ G. Currier,* R. D. Shytle,† and J. Tan*
*Department of Psychiatry and Behavioral Neurosciences, University of South Florida Morsani College of Medicine, Tampa, FL, USA
†Center for Excellence in Aging and Brain Repair, Department of Neurosurgery and Brain Repair, University of South Florida Morsani College of Medicine, Tampa, FL, USA
‡Departments of Biomedical Sciences and Pathology, Saitama Medical Center and Saitama Medical University, Kawagoe, Saitama, Japan
Lithium has been used for a long time as a mood stabilizer for bipolar disorder as well as for the treatment of mania, depression, and suicidal thoughts. In addition, recent studies indicate that lithium can prevent the cognitive decline associated with Alzheimer's disease (AD). However, one of the main problems that exist in the currently FDA-approved lithium pharmaceutics (carbonate and citrate) is that they have a narrow therapeutic index, and lithium plasma levels change drastically, which can cause adverse side effects. Here we investigated the safety, pharmacokinetics, and therapeutic efficacies of LISPRO (ionic cocrystals of lithium salicylate with organic L-proline), lithium salicylate, and Li2CO3. We found that LISPRO reduces β-amyloid plaques and phosphorylation of tau through modulation of inflammation and glycogen synthase kinase 3β (GSK3β) inactivation in the AD mouse. Specifically, cytokine profiles in the brain, plasma, and splenocyte suggest that LISPRO (8 weeks) downregulates proinflammatory, upregulates anti-inflammatory, and suppresses renal cyclooxygenase 2 (COX2) expression in Swedish mutation of amyloid precursor protein transgenic (Tg2576) mice. Plasma and brain pharmacokinetics of lithium indicated that LISPRO showed significantly higher brain and steady plasma lithium levels in B6129F2/J (2 weeks) and Tg2576 (8 weeks) mice. Interestingly, chronic administration of LISPRO for 28 weeks produces slightly higher, but nonsignificant, brain to plasma lithium levels and reduces β-amyloid plaques, and tau phosphorylation through modulation of presynaptic (synaptophysin) and postsynaptic protein [postsynaptic density protein 95 (PSD95)] expression in mutant amyloid precursor protein, tau, and presenilin (3XTg-AD) mice.
This research was funded by R01AG050253.
A. Házy, T. R. Brickler, B. Okyere, and M. H. Theus
Department of Biomedical Sciences and Pathobiology, Virginia-Maryland Regional College of Veterinary Medicine, Blacksburg, VA, USA
Traumatic brain injury (TBI) is a leading cause of acquired central nervous system injury worldwide. The immune system is a critical factor in TBI progression, and targeting aspects of this response has been identified as a promising therapeutic approach for minimizing functional deficits and long-term disability. Brain trauma elicits peripheral-derived mononuclear cell (PDMC) migration, infiltration, and polarization leading to alterations in the microenvironmental cytokine–chemokine profile, which can affect vascular, neuronal, and glia cell health. Preliminary work from our lab has identified the ephrin receptor (Eph)/ephrin pathway as a novel regulator of the hematopoietic response to TBI. Transgenic mice lacking Eph signaling in endothelial and hematopoietic compartments display smaller lesion volumes, improved motor function, and altered cortical gene expression in the murine controlled cortical impact injury (CCI) model of TBI following injury. Using flow cytometry, we find Eph expression on numerous cell types extracted from the bone marrow, including monocytes and endothelial progenitor cells. To directly address the role of PDMCs and Eph signaling in tissue homeostasis following CCI injury, we performed adoptive transfers (ATs) using freshly isolated wild-type or Eph knockout (KO) enhanced green fluorescent protein-positive (eGFP+) bone marrow injected into irradiated wild-type mice followed by CCI injury at 30 days after AT. Analysis of the density, identity, and spatial distribution of the cells as well as lesion volume, neuronal cell death, and glial activation using nonbiased StereoInvestigator Stereology is underway. Initial findings, however, indicate that wild-type mice reconstituted with bone marrow derived from Eph KO mice display smaller lesion volumes compared to those with wild-type cells at 3 days after CCI injury. Interestingly, ex vivo isolation of M1 and M2 monocyte/macrophages from Eph KO mice display decreased levels of tumor necrosis factor (TNF) compared to wild-type cells. Based on these preliminary findings, we hypothesize that Eph signaling contributes to cortical tissue damage by mediating PDMC activities such as migration, cell polarization, and/or cytokine–chemokine expression. Findings from this study will advance our basic understanding of the mechanisms underlying the peripheral-derived inflammatory response to brain trauma.
K. Hosaka, H. Fazal, L. Lin, and B. Hoh
Department of Neurosurgery, University of Florida, Gainesville, FL, USA
We have previously demonstrated that local delivery of monocyte chemotactic protein-1 (MCP-1), via a MCP-1-releasing poly(lactic-co-glycolic acid) (PLGA)-coated coil, promotes intra-aneurysmal tissue healing. Using a murine model of carotid intra-aneurysmal healing, we observed increased expression of interleukin-6 (IL-6) in MCP-1 coil-treated aneurysms and not in control PLGA only-treated aneurysms. The roles of MCP-1 and IL-6 in aneurysm healing are confirmed by MCP-1 and IL-6 expression in coiled human cerebral aneurysms. MCP-1-mediated intra-aneurysmal healing is inhibited in mice given a blocking antibody to IL-6 receptor and only at early time points. MCP-1-mediated intra-aneurysmal healing is also inhibited by a blocking antibody to osteopontin (OPN). The role of IL-6 in intra-aneurysmal healing is in recruiting, but not proliferation of, endothelial cells and fibroblasts, and not smooth muscle cells or macrophages, as demonstrated in cell migration and cell proliferation assays. Local delivery of OPN to murine carotid aneurysms via an OPN-releasing coil significantly promotes intra-aneurysmal healing, but an IL-6-releasing coil does not, suggesting IL-6 cannot promote aneurysm healing independent of MCP-1. In the MCP-1-mediated aneurysm healing pathway, OPN expression is dependent on IL-6; inhibition of IL-6 receptor significantly inhibits OPN expression in MCP-1-mediated aneurysm healing. Our findings suggest that IL-6 and OPN are key downstream mediators of MCP-1-mediated intra-aneurysmal healing.
A. Jalloh,* A. Flowers,* C. Hudson,* S. Stevens,* 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
†Research Service, James A. Haley Veterans Affairs Hospital, Tampa, FL, USA
Aging has deleterious effects on the central nervous system (CNS) that cause an increased risk of age-related neurodegenerative disease. Systemic inflammation accumulated during the aging process plays an important role in the development and progression of Alzheimer's, Parkinson's, and other neurological disorders. The inflammatory response in the CNS is governed by microglia, immune cells “activated” by proinflammatory stimuli, which react to neuronal damage during insult or injury. Microglia are also activated by anti-inflammatory signals that resolve inflammation and initiate a reparative and neuroprotective response. Previous studies have demonstrated that chronic proinflammatory microglial activation incites excess proinflammatory/blunted anti-inflammatory responses, instigating disrupted homeostasis that furthers neurological disease pathology. This shift in microglial biology is apparent in the microglia of aged animals, but the mechanism behind this dysfunction is unknown. The goal of this study was to elucidate an underlying molecular mechanism and identify valid therapeutic targets. Our study applied label-free mass spectrometry in conjunction with the Ingenuity Pathway Analysis (IPA) software to compare differentially expressed proteins between microglia extracted from aged (20-22 months) and young (3-5 months) rats. We used IPA to generate a list of canonical pathways whose function was altered between the two groups. We then identified upstream regulators of cellular functions and knocked down elements of the identified pathways with small interfering RNA (siRNA) to evaluate the consequences on microglial behavior in vitro. In addition, we applied the same proteomics paradigm to compare microglia extracted from aged rats whose diet was supplemented with NT-020, a proprietary blend of polyphenols, and control rats fed a normal diet. Our proteomic analysis highlighted a number of altered pathways when comparing microglia from aged rats to young rats. In particular, mechanistic target of rapamycin (mTOR) signaling, a nutrient-sensing pathway that mediates cell growth, was a top canonical pathway that was significantly upregulated in aged microglia. Rapamycin-insensitive component of mTOR (RICTOR) was significantly downregulated in our data set, suggesting the involvement of mTOR complex 2 (mTORC2) as a critical element in age-altered microglia biology. Indeed, micro-glia extracted from young rats generated similar cytokine expression profiles to those from aged rats after targeted siRNA knockdown of RICTOR. Both aged and siRNA-treated young microglia also demonstrated a blunted response to anti-inflammatory interleukin-4 (IL-4) treatment compared to controls. We also identified a number of affected pathways when comparing microglia in aged rats supplemented with NT-020 and controls. Interestingly, we found that mTOR signaling was downregulated in the NT-020-supplemented microglia in addition to nerve growth factor (NGF) signaling and signaling involved in the production of nitric oxide and reactive oxygen species. These changes were marked by a significant inhibition of upstream regulators IL-3, tumor necrosis factor (TNF), transforming growth factor-1β (TGF-1β), and interferon-γ (IFN-γ) and their respective pathways. Our datasets comparing aged and young rat microglia identify pathways that become dysfunctional with age and perturb cellular homeostatic mechanisms. These pathways modulate the inflammatory response in microglia, amplifying the damage caused by age-associated inflammation, aggravating disease progression. Our analysis also offers pathways whose modulation can mitigate the biological changes microglia undergo during aging. Supplementation with NT-020 can downregulate signaling cascades that can become dysfunctional with age. This phenomenon demonstrates a role for polyphenols as therapeutics for age-related neurodegenerative diseases.
V. Joers,* B. Murray,* M. E. de Sousa Rodrigues,* F. P. Manfredsson,† B. Moore,‡ and M. Tansey*
*Department of Physiology, Emory University, Atlanta, GA, USA
†Department of Translational Science and Molecular Medicine, Michigan State University, Grand Rapids, MN, USA
‡Department Pharmaceutical Sciences, University of Tennessee Health Sciences Center, Memphis, TN, USA
There is mounting evidence that neuroinflammation accompanies and may promote progression of α-synuclein (α-syn)-induced nigral dopaminergic (DA) degeneration. Research into the disequilibrium of microglial phenotypes has become an area of intense focus in neurodegenerative disease as a potential mechanism that contributes to chronic neuroinflammation and neuronal loss in Parkinson's disease (PD). Modulating the activation state or phenotype of microglia can largely alter the local environment via cytokine signaling, which can in turn impact neuronal survival. Cannabinoid receptor 2 (CB2) is highly expressed on activated microglia and circulating monocytes (Ashton and Glass, 2007) and is upregulated in the substantia nigra (SN) of PD patients (Gomez-Galvez et al., 2015) and in mouse models of nigral degeneration (Garcia et al., 2011; Price et al., 2009). Studies conducted by our collaborator Bob Moore at the University of Tennessee with a novel CB2 inverse agonist SMM-189 demonstrated its ability to directly modulate immune cell phenotypes and protect against traumatic brain injury in mice and is potentially explained by a mechanism that suppresses microglial proinflammatory markers and increases their anti-inflammatory markers (Presley et al., 2015; Reiner et al., 2015). Here we report the preliminary findings on the neuroprotective effect of SMM-189 treatment in an adeno-associated virus-human α synuclein (AAV-hα-syn) rat model of PD and the peripheral and central neuroinflammatory profiles following CB2 modulation. We hypothesize that CB2 treatment will attenuate chronic microglial activation and alter the peripheral myeloid cell populations, resulting in dopaminergic nigrostriatal protection. Sprague–Dawley rats (2-3 months of age, male, n = 28) were unilaterally injected in the SN with 5 × 1013 vectors genomes/ml of AAV-hα-syn and followed for 8 weeks, a time point that has previously been reported to produce ~40%-60% nigral degeneration (Gombash et al., 2013). One week after surgery, animals were randomly selected to receive daily systemic injections of either SMM-189 (6 mg/kg, n = 14) or vehicle (n = 14). Motor behavior was assessed monthly with a cylinder test to determine front paw preference and every 2 weeks with an adjusted steps test. At sacrifice, peripheral blood mononuclear cells (PBMCs) were isolated from cardiac blood, animals were transcardially perfused with saline, and the midbrain was postfixated in 4% paraformaldehyde (PFA) to be processed for immunohistochemistry. Preliminary analysis of the adjusted steps test suggests a trend in contralateral motor deficits across time [F(4, 96) = 2.213, p = 0.07] but no significant differences between treatment groups. Postmortem analysis demonstrates hα-syn immunoreactivity throughout the rostrocaudal axis of the rat SN revealing a 93% successful targeting of AAV-hα-syn transduction. We are currently completing analysis of stereological nigral tyrosine hydroxylase-positive (TH+) cell counts to determine the neuroprotective effect of SMM-189, microglia activation by immunohistochemical methods of ionized calcium-binding adapter molecule 1 (IBA1; microglial marker) and major histocompatibility complex II (MHCII; antigen presentation marker) to establish the microglial activation state following SMM-189, and PBMC expression of pro- and anti-inflammatory genes to elucidate systemic changes in circulating immune populations from SMM-189 treatment.
Funding support from MJFF Foundation Validation Mechanism.
N. H. Kalin
Department of Psychiatry, University of Wisconsin School of Medicine and Public Health, Madison, WI, USA
Using our nonhuman primate model of anxious temperament (AT), we delineated the neural circuitry that underlies the expression of extreme early life anxiety. Components of this AT circuit include orbitofrontal regions (OFC), anterior insula, extended amygdala [central nucleus of the amygdala (Ce) and bed nucleus of the stria terminalis (BST)], and the periaqueductal gray (PAG). We also demonstrated that the anxiety phenotype is heritable and, importantly, is coheritable with glucose metabolism in the posterior OFC, BST, and PAG. The Ce is a core component of this circuit, so we further examined the molecular underpinnings of AT by performing transcriptome analyses with RNA sequencing (RNAseq). Within a large group of animals, we searched for relations between individual differences in AT with the expression of Ce transcripts. Findings demonstrated a number of leads that included neuroplasticity-related transcripts as well as the amyloid precursor protein (APP) and the γ-aminobutyric acid (GABA) A receptor α 5 subunit. Of particular interest, and supporting our neurodevelopmental hypothesis of anxiety disorders, is the finding that the expression of the neuroplasticity receptor, tyrosine kinase receptor 3 (Trkc) or neurotrophic receptor tyrosine kinase 3 (NTRK3), is inversely related to the expression of AT. To further test the mechanistic role of Ce NTRK3 and its viability as a treatment target, we overexpressed neurotrophin 3 (NT3; NTRK3′s endogenous ligand) in the Ce. As predicted from the RNAseq data, we found that increased expression of NT3 altered Ce metabolism and reduced monkeys' levels of AT. Other studies are ongoing to develop designer receptors exclusively activated by designer drugs (DREADDs) technology in primates to examine the effects of the prefrontal–amygdala pathway-specific manipulations on extreme anxiety. Together, these studies highlight the value of the nonhuman primate in understanding mechanisms underlying anxiety disorders, as well as in discovering and testing novel molecular targets for the treatment of human anxiety and affective disorders.
Y. Lebovitz,*† J. Brabender,‡ and M. H. Theus†§
*Ph.D. Program in Translational Biology, Medicine, and Health, Virginia Tech, Blacksburg, VA, USA
†Interdisciplinary Graduate Education Program in Regenerative Medicine, Virginia-Maryland College of Veterinary Medicine, Blacksburg, VA, USA
‡Undergraduate Program in Biological Systems Engineering, Virginia Tech, Blacksburg, VA, USA
§Department of Biomedical Sciences and Pathobiology, Virginia-Maryland College of Veterinary Medicine, Blacksburg, VA, USA
Epidemiological studies on neurodevelopmental disorders, such as autism spectrum disorders and schizophrenia, highlight antibiotic usage and disruptions to the maternal immune system during pregnancy as major correlates of subsequent diagnoses in children. These results support additional findings that antibiotic use in pregnancy can cause perturbations to the developing neuroimmune system and that defective microglial cells impair functional brain connectivity and induce aberrant social behaviors in animal models. Importantly, antibiotics create dysbiosis in the gut microbial environment (“microbiome”) that can significantly affect the production of key circulating factors, such as short-chain fatty acids (SCFAs), which are critically involved in neuroimmune regulation. As such, we hypothesized that the maternal gut microbiome plays a necessary and sufficient role in proper neurodevelopment due to regulation of the fetal neuroimmune system. Through an antibiotics-based approach, mice were used as animal models of broad gut bacterial depletion during pregnancy. Preliminary data using immunohistochemistry showed neonatal pups born to bacteria-depleted (BD) dams possessed irregular microvasculature and neurogenesis, as well as premature morphology and increased numbers of resident microglia in comparison to conventionally raised (CONV) controls. In contrast, the persistence of a single species of gut bacterium in a second set of pregnant BD dams (ABXRes) resulted in microglial characteristics more comparable to CONV than BD pup brains. ABXRes pups also performed similarly to CONV pups under behavioral assays, such as the three-chamber social test. These results suggest that specific strains of bacteria and/or their metabolites may be necessary for proper microglial development and also excludes drug-specific adverse effects. Compared to ABXRes and CONV dams, BD dams possessed atypical metabolite and inflammatory cytokine profiles. In particular, BD dams showed decreased plasma levels of key SCFAs, which are common metabolites released from the microbiome that serve multiple roles in glial metabolism, synaptic function, and cerebrovascularization. These data indicate that maternally derived gut bacteria play a vital role in proper neuroimmune development in utero.
J.-Y. Lee, P.-J. Lin, 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
The advent of induced pluripotent stem cells (iPSCs) adds an alternative stem cell source for cell therapy-based regenerative medicine. Somatic cells from individuals can be reprogrammed to pluripotency offering not only sufficient cell numbers in vitro but also autologous transplant utilization without immunorejection of the allograft. Numerous studies have demonstrated viability and engraftment of stem cells, including neural progenitor cells (NPCs) and endothelial progenitor cells (EPCs) derived from iPSCs, allowing for their therapeutic application in neurological disorders, such as ischemic stroke, traumatic brain injury, Alzheimer's disease, and Parkinson's disease. In the present study, we examined the neuroprotective effects of human iPSCs in primary rat cortical neurons pretreated with 5 ng/ml tumor necrosis factor-α (TNF-α) overnight or 1.5 h of oxygen–glucose deprivation (OGD) to simulate the cell death mechanism of stroke. This 12-h coculture paradigm entailed the use of a two-cell culture chamber system with 0.4-μm pore size inserts containing the iPSCs, thereby preventing direct cell-to-cell contact between the stem cells and the rat neurons. Following this method, human iPSCs significantly increased the cell survival rate of both TNF-α- and OGD-exposed rat neurons. Since iPSCs were not able to penetrate the insert's pore size, we eliminated the integration of stem cells with rat neurons as a key mechanism of therapeutic benefit. Instead, we demonstrated here that iPSCs might secrete cytokines or extend their filopodia across the mesh of the inserts to interact with rat neurons in attenuating the stroke-induced cell death. In particular, iPSCs significantly blocked inflammatory cytokines, indicating the capability of stem cells to exert anti-inflammatory action. Moreover, the extension of the filopodia from iPSCs to reach the damaged neurons might also explain the possibility of remote communication between stem cells and the injured rat neurons. The high risk of tumorigenicity associated with directly injecting iPSCs into the brain means that the peripheral application and use of short-term survival stem cells may offer an effective alternative approach for cell therapy in stroke and relevant disorders.
Support: C.V.B. is supported by NIH NINDS 1R01NS071956-01, NIH NINDS 1 R21 NS089851-01, DOD TATRC W81XWH-11-1-0634, James and Esther King Foundation for Biomedical Research Program 1KG01-33966, SanBio Inc., Celgene Cellular Therapeutics, KMPHC, and NeuralStem Inc.
L. Li and L. A. Cunningham
Department of Neurosciences, University of New Mexico Health Sciences Center, Albuquerque, NM, USA
Focal cerebral ischemia induced by middle cerebral artery occlusion (MCAO) stimulates a multilineage cytogenic response from neural stem cells (NSCs) within the adult subventricular zone (SVZ) in mice, which includes production of both glial and neuronal lineages (Li et al., 2010). Hypoxia-inducible factor-1α (HIF-1α) is a key mediator of the adaptive cellular response to hypoxia through direct transcriptional regulation of cellular and molecular processes involved in angiogenesis, stem cell maintenance, and differentiation. Although HIF-1α is constitutively expressed in most cell types, it normally undergoes rapid proteosomal degradation under nonhypoxic conditions. Importantly, HIF-1α remains stabilized under aerobic conditions in a number of stem cell types, including bone marrow hematopoietic stem cells (Nombela-Arrieta et al., 2013), mesenchymal stem cells (Palomaki et al., 2013), and NSCs (Roitbak et al., 2011). Prior studies by our laboratory demonstrated a critical role for NSC-encoded HIF-1α in NSC maintenance and vascular stability within the natural environment of the adult mouse SVZ in vivo under normal nonpathological conditions (Li et al., 2014). In the present study, we utilized an inducible Cre-loxP approach to selectively inactivate NSC-encoded HIF-1α and evaluate the impact of Hif-1α gene deletion on the SVZ regenerative response to subsequent stroke injury. For these studies, we utilized nestin-CreERT2:Hif1afl/fl: Rosa yellow fluorescent protein (YFP) triple transgenic mice in which tamoxifen administration resulted in concomitant induction of YFP reporter expression and Hif1a gene deletion in nestin+ SVZ-NSCs and their downstream progeny. We found that the SVZ regenerative response to stroke injury was markedly attenuated following inactivation of NSC-encoded HIF-1α. Importantly, attenuation of the SVZ regenerative response was also associated with impaired revascularization, larger stroke volumes, and impaired recovery of motor function. Furthermore, Hif1a gene deletion also led to a small but significant increase in the percentage of SVZ-derived neuroblasts versus astrocytes, and reduced expression of the notch regulator thrombospondin 4 (Tsp4), suggesting the importance of HIF-1α in SVZ astrogenesis following stroke. Taken together, these findings emphasize an essential role of NSC-encoded HIF-1α in supporting the SVZ regenerative response to stroke and the importance of the SVZ regenerative response for minimizing stroke volume and promoting functional recovery.
Supported by AHA GIA 09GRNT2290178.
M. G. Liska, V. A. Guedes, D. J. Eve, C. Stonesifer, L. Gelineau, M. G. Crowley, S. A. Acosta, 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
Traumatic brain injury (TBI) results in lifelong sequelae but lacks reliable diagnostic and prognostic metrics. Acoustic startle reflex (ASR) is a conserved defensive response to loud auditory stimulus, a useful tool to evaluate the integrity of sensorimotor processes, and has been used to assess stress-related responses. Studies have found that TBI induces ASR suppression, but contradictory results have been reported. Here we investigated the effects of TBI on ASR and its histopathological correlates with disease severity. We exposed Sprague–Dawley rats to the controlled cortical impact model of TBI. ASR was measured before the injury and at 10 time points after the TBI (from day 1 to week 14) in ventilated startle chambers where a white noise burst (110 dB, 40 ms, intertrial interval between 30 and 45 s, 30 trials) was presented. Rats were perfused, and the tissues are currently being processed for histological evaluation of the impact and peri-impact areas, secondary cell death, and inflammation. We observed suppression of the ASR after TBI as revealed by an intense decrease in the amplitude of response at all time points when compared to the baseline (p < 0.0001, one-way ANOVA and Bonferroni's posttest). We also noted an increase in the latency of response at days 1, 7, and 14 (p < 0.01, 0.05, and 0.05, respectively). We demonstrated that TBI induced suppression of ASR for at least 14 weeks postinjury. Ongoing histopathological assays are likely to reveal the levels of ASR suppression based on the TBI severity. A correlation between TBI severity and ASR suppression could facilitate the diagnostic and have prognostic value, and provide a convenient means of measuring TBI severity in the clinic. Furthermore, additional behavioral and histological analyses may reveal specifics about the effect of TBI on sensory–motor integration.
Support: C.V.B. is supported by NIH NINDS 1R01NS071956-01, NIH NINDS 1 R21 NS089851-01, DOD TATRC W81XWH-11-1-0634, James and Esther King Foundation for Biomedical Research Program 1KG01-33966, SanBio Inc., Celgene Cellular Therapeutics, KMPHC, and NeuralStem Inc.
S.-W. Liu,* T. Huang,*† N. H. Greig,‡ and J.-Y. Wang*†
*Graduate Institute of Medical Sciences, College of Medicine, Taipei Medical University, Taipei, Taiwan
†Department of Physiology, College of Medicine, Taipei Medical University, Taipei, Taiwan
‡Drug Design and Development Section, Translational Gerontology Branch, Intramural Research Program, National Institute on Aging, National Institutes of Health, Baltimore, MD, USA
Traumatic brain injury (TBI) is an acute injury that disrupts normal brain function and results in both short-term and long-term behavioral and cognitive consequences. Pomalidomide (Pom), a US FDA-approved immunomodulatory agent used in the treatment of multiple myeloma and other cancers, has been shown to be a more potent inhibitor of tumor necrosis factor-α (TNF-α) with lesser side effects than thalidomide. We have previously found that Pom (0.5 but not 0.1 mg/kg, IV) administered at 5 h after TBI reduced contusion volume and improved functional deficits by reducing neuronal apoptosis in an animal model of TBI. The purpose of this study was to determine whether postinjury administration of 3,6′-dithio pomalidomide (dt-Pom), a novel analog of pomalidomide, has therapeutic effects on TBI. We also aim to compare the therapeutic efficacy of dt-Pom with Pom. We conducted an in vivo study in which adult Sprague–Dawley rats underwent experimental TBI using controlled cortical impact (CCI) and were administered dt-Pom (0.1 or 0.5 mg/kg, IV) at 5 h after TBI. Neurobehavior was evaluated using swing test, adhesive removal, modified neurological severity scores, and beam walking tests. The degree of brain injury was evaluated by contusion volume and neuronal degeneration using cresyl violet and FluoroJade C staining. Our results indicated that postinjury administration of dt-Pom (0.1 or 0.5 mg/kg, IV) at 5 h after CCI significantly improved the behavioral impairments that resulted from TBI and decreased contusion volume at 24 h postinjury. Administration of dt-Pom also significantly reduced the number of degenerating neurons at the contusion site as well as injury-induced mRNA and protein expression of cytokines in brain tissues at 24 h after TBI in the ipsilateral hemisphere cortex. Our data suggest that postinjury treatment with dt-Pom improves histological and functional outcomes after TBI and reduces neuroinflammatory responses, and dt-Pom has the potential to be developed as a new therapy for TBI.
Supported by MOST-104-2923-B-038-001-MY3, Taiwan and RO1NS094152, NIH, USA.
O. V. Lossia,*†‡ M. O. Tree,‡ S. C. Goldthorpe,‡ B. Srinageshwar,*† G. L. Dunbar,*†§¶ M. J. Conway,‡ and J. Rossignol*†‡
*Field Neurosciences Institute for Restorative Neuroscience, Central Michigan University, Mount Pleasant, MI, USA
†Program in Neuroscience, Central Michigan University, Mount Pleasant, MI, USA
‡College of Medicine, Central Michigan University, Mount Pleasant, MI, USA
§Department of Psychology, Central Michigan University, Mount Pleasant, MI, USA
¶Field Neurosciences Institute, Saginaw, MI, USA
The emergence of the Zika virus (ZIKV) has been linked to severe nervous system abnormalities such as microcephaly in infants and Guillain–Barré syndrome in adults. Recent studies have shown that ZIKV is able to infect neural progenitor cells and affect morphology, survival, and growth of these developing cells. In our study, we show that ZIKV replicates in neural stem cells (NSCs), inhibits cell proliferation, affects protein secretion, and dysregulates gene expression. Our results demonstrate the ability of the virus to effectively infect these cells and reduce the number of neurospheres formed over time, signifying a cytopathic effect. Gene expression analysis of NSC progenitors and differentiation markers suggest that infection inhibits transcription of genes involved in developing and maintaining new neurons while increasing transcription of genes involved in forming astrocytes, providing evidence that specific neuronal lineages are explicitly targeted by the virus. In addition to data from gene expression analysis, protein secretion analysis indicates a possible neuroprotective response from the cells. Glia maturation factor-β, a protein involved in neural regeneration, was detected in the supernatant from infected cells, suggesting that the cells are compensating for infection by inducing the formation of astrocytes in an attempt to resolve infection. Additional proteins secreted from infected cells included those involved in promoting infection and proteins involved in protecting against infection. These data provide detailed knowledge on how ZIKV impacts differentiation, survival, development, and function of NSCs. In summary, our results suggest that while ZIKV infection in NSCs leads to a cytopathic effect, it is being actively mitigated through attempts that limit neuropathogenesis by the induction of astrocytes, cells known to mediate viral infection.
Support for this study was provided by the College of Medicine, Field Neurosciences Institute, and the John G. Kulhavi Professorship in Neuroscience at CMU. We also thank contributors to our Experiment.com crowdfunding campaign, “Does Zika virus replicate in neural cells?”
N. Mai,*† A. Rininger,‡ H. Bazarian,* L. Prifti,* and M. W. Halterman*†§
*Center for Neurotherapeutic Discovery, University of Rochester Medical Center, Rochester, NY, USA
†Department of Neuroscience, University of Rochester Medical Center, Rochester, NY, USA
‡Department of Biology, University of Washington, Seattle, WA, USA
§Department of Neurology, University of Rochester Medical Center, Rochester, NY, USA
Postischemic neurodegeneration remains an important therapeutic target for patients that survive the initial resuscitation period after cardiac arrest. Heightened levels of systemic inflammation portend poor neurologic recovery, but the lack of preclinical models that faithfully reproduce features of post-cardiac arrest syndrome (PCAS) remains a barrier to the development of effective neuroprotective therapies. While systemic endotoxemia caused by mesenteric ischemia is well documented after cardiac arrest, its impact on cerebral ischemia reperfusion injury (IRI) remains unknown. To test this, we induced transient global ischemia in 5- to 8-week-old C57/B6 mice using a model of three-vessel occlusion (3VO), which involves basilar artery cauterization with 15-min occlusion of the common carotid arteries. To mimic enteric endotoxemia, mice received 50 μg/kg lipopolysaccharide (LPS) by IP injection during the initial reperfusion phase. While survival was equivalent across groups, 3VO/LPS treatment was associated with heightened cortical injury, end organ vascular permeability, microglial activation, and poorer performance on behavioral tests compared to ischemic controls. Combined 3VO/LPS treatment also induced neutrophil migration to the central nervous system (CNS) and other nonischemic targets including the lung, liver, and kidney. Consistent with these findings, flow analyses on peripheral blood demonstrate that while either ischemia or low-dose LPS injection induced cluster of differentiation 11b (CD11b) on circulating neutrophils, their use in combination produced synergistic effects. Collectively, these studies indicate that low-level endotoxemia has important effects on both the CNS and peripheral tissues following reperfusion after cerebral ischemia. Our findings also suggest that physiological coupling between the lung and brain during the reperfusion period may play a major role in postischemic neurodegeneration through a mechanism involving the priming of neutrophils among other factors involved in the innate immune response.
K. Malloy,*† J. Li,† G. Choudhury,* A. Torres,* S. Gupta,‡ C. Kantorak,‡ T. Gobble,‡ P. Fox,† G. Clarke,† and M. Daadi*†
*Texas Biomedical Research Institute, Southwest National Primate Research Center, San Antonio, TX, USA
†Research Imaging Institute, Radiology, University of Texas Health Science Center at San Antonio, San Antonio, TX, USA
‡MRI-Interventions Inc., Irvine, CA, USA
Parkinson's disease is characterized by the degeneration of the dopamine-producing neurons in the substantia nigra. Neural stem cell transplantation to the putamen has the potential to improve dopaminergic function in patients afflicted with Parkinson's disease. Magnetic resonance imaging (MRI) can be used as a minimally invasive tool to guide the cell transplantation. The cells were labeled with superparamagnetic iron oxide (SPIO) nanoparticles, which can be visualized as hypointense regions on MRI. The injection parameters such as infusion speed and cannula diameter were established using the agarose gel model by injecting SPIO-labeled cells into 0.6% agarose gel phantoms and using real-time MRI to monitor the experiments. These injection parameters were translated to MRI-guided injections into the baboon cadaver brain, using the ClearPoint™ system (MRI Interventions, Irvine, CA, USA), to identify the injection target and guide the cannula insertion. A whole-brain 3D MRI series was acquired, which the ClearPoint Software used to establish a 3D coordinate system. A cannula trajectory guide (SmartFrame™) with four MRI-sensitive fiducial markers was attached to the skull. The fiducial markers allowed the software to segment the SmartFrame™ and determine the trajectory of the cannula. Iterative imaging of the fiducial markers and adjustment of the SmartFrame™ was used to align the cannula trajectory with the target until there was less than 1 mm of error between the desired target and cannula trajectory. Once the cannula trajectory was finalized, a burr hole was drilled at this location. The cannula, loaded with SPIO-labeled cells, was inserted through the guide into the brain, and the injection was initiated at 1 μml/min and recorded in real time with a TurboFlash series. A whole-brain 3D image series was acquired postinjection to visualize the graft. The phantom experiments showed that the injection speed of 1 μl/min is appropriate to prevent clogging and backflow of the cells along the cannula track. The injection recording in both the phantom and MRI-guided baboon experiments showed pulsatile flow of the cells, which exited the cannula in distinct bursts throughout the injection. The postinjection MRI images confirmed that the cells were successfully delivered to the putamen. The use of MRI guidance was determined to be an effective and minimally invasive method of cell delivery to the baboon brain.
D. J. Marmion,* R. J. Mandel,† D. Kirik,‡ Y. Chu,* T. J. McCown,§¶ S. J. Gray,§# and J. H. Kordower*,**
*Department of Neurological Sciences, Rush University Medical Center, Chicago, IL, USA
†Department of Neuroscience, University of Florida, Gainesville, FL, USA
‡Department of Experimental Medical Science, Lund University, Lund, Sweden
§Gene Therapy Center, Chapel Hill, NC, USA
¶Department of Psychiatry, University of North Carolina, Chapel Hill, NC, USA
#Department of Ophthalmology, Chapel Hill, NC, USA
**The Van Andel Institute, Grand Rapids, MI, USA
Multiple system atrophy (MSA) is a rare, progressive neurodegenerative disorder with an uncertain etiology and pathophysiology. MSA is a unique proteinopathy in which α-synuclein preferentially aggregates in the oligodendroglia rather than in the neurons. Aggregated α-synuclein is thought to elicit changes in oligodendrocyte function, such as reduced neurotrophic support and demyelination. MSA presents clinically with progressive autonomic failure, as well as parkinsonian and cerebellar features in various combinations. To date, only murine models of MSA exist for the study of this disease. Toward this end, we sought to develop novel rat and nonhuman primate (NHP) models of MSA by overexpressing α-synuclein in oligodendroglia cells using a novel oligotrophic adeno-associated virus (AAV) vector, Olig001, where expression was driven by a constitutive chicken β-actin promoter. Initially, rats received unilateral injections of the Olig001 vector expressing green fluorescent protein (GFP) stereotaxically in the striatum. After 4 weeks, rodents were sacrificed, and histological methods were used to assess the specificity of the viral vector. Using unbiased stereology, our data showed 126,574 ± 22,607.54 GFP+ cells in the striatum, with 94%-97% of the GFP+ cells colocalizing with oligodendroglial marker Olig2. There was little or no coexpression in neuronal nuclei-positive (NeuN+; 2.9%-4.7%) or glial fibrillary acidic protein-positive (GFAP+; 0.18%-0.49%) cells. We next sought to test the efficacy of this vector in nonhuman primates. Three rhesus macaques received intrastriatal injections of Olig001-expressing GFP (1 × 1013 vg/ml) unilaterally (two in the putamen and two in the caudate nucleus). The animals were sacrificed after 1 month and analyzed using histological and stereological methods. As in the rodents, stereological estimates of transfected profiles revealed a large number of striatal cells expressing GFP (monkey 1: 639,877; monkey 2: 630,520; monkey 3: 161,948) in the striatum. Additionally, 90%-94% of the GFP-expressing cells colocalized with Olig2, with sparse colocalization with either NeuN (0.23%-1.42%) or GFAP (0.09%-0.12%). Finally, we recently injected the vector expressing the α-synuclein transgene (3.75 × 1012 vg/ml) into the striatum of three monkeys as before. Histological analyses in all monkeys 3 months after injection demonstrated widespread monomeric and aggregated α-synuclein expression as determined by LB509 and serine 129 α-synuclein immunoreactivity. This expression was preferentially seen in the oligodendroglia. Unbiased stereology estimates 2,606,199.5 ± 557,864.083 pSer129+ inclusions, with a volume of transfection of 124.8 ± 3.77 mm3. Demyelination occurred in the striatum and corpus callosum, corresponding with areas expressing α-synuclein, with no loss of myelin seen in GFP-injected monkeys. These data support the establishment of a viral overexpression model of MSA.
N. M. Mercado,* D. Korol,† R. Gardner,† C. E. Sortwell,*‡ T. J. Collier,*‡ and K. Steece-Collier*‡
*Department of Translational Science and Molecular Medicine, College of Human Medicine, Michigan State University, Grand Rapids, MI, USA
†Department of Biology, Syracuse University, Syracuse, NY, USA
‡Hauenstein Neuroscience Center, Mercy Health Saint Mary's Hospital, Grand Rapids, MI, USA
There continues to be interest in the transplantation of dopamine (DA) neurons as a means of repairing the parkinsonian striatum. However, recent clinical and preclinical reports suggest that factors inherent to the aged striatum per se limit successful brain repair. To address aging as a limiting factor in successful brain repair, we have used the example of cell transplantation to interrogate the aged striatum and identify factors that may, or may not, respond to interventions aimed at improving prospects for adequate repair of the aged brain. It has traditionally been thought that the decreased therapeutic benefit in the aged host results from the decreased survival of grafted cells, but recent clinical and pre-clinical evidence is inconsistent with this notion. Indeed, despite robust survival of grafted DA neurons and extensive neurite outgrowth in the aged parkinsonian striatum, clinical and preclinical evidence suggests that unknown factors inherent to the aged striatum result in grossly suboptimal behavioral efficacy. Data from our lab demonstrate that inferior behavioral recovery in the aged parkinsonian brain is associated with inferior synaptic integration between graft and host and that this is associated with decreased dendritic spine density on striatal medium spiny neurons (MSNs). Dendritic spines are the structural element upon which descending cortical glutamate and ascending nigral DA afferent signaling is integrated, which is necessary for normal motor behavior. Aging and DA depletion result in loss of these critical sites for synaptic integration. Data from our lab and others demonstrate that spine loss related to DA depletion is reversible with calcium channel voltage-dependent, L type, α 1D subunit (CaV1.3) inhibition; however, recent data from our lab demonstrate that age-related spine loss is not reversible through this mechanism. In addition, while young animals show a robust compensatory neurotrophic response after striatal DA depletion, aged animals have been shown to lack a compensatory response, indicating a possible age-related disruption of neurotrophic activity in the striatum. As the neurotrophin brain-derived neurotrophic factor (BDNF) plays a significant role in spine dynamics, we hypothesize that age-related dendritic spine loss is due to changes in BDNF dynamics in the aged parkinsonian striatum. Striatal BDNF is primarily derived from cortical afferents that release BDNF in an activity-dependent manner. Upon release, the interaction of BDNF with tropomyosin receptor kinase B (TrkB) located on striatal MSNs initiates signaling pathways involved in dendritic spine and synapse dynamics. Existing data indicate no change in basal level of BDNF in the aged striatum, but there is rationale to suggest that BDNF release and downstream signaling may be dysfunctional in the aged brain. In a series of ongoing studies, we are examining age-related changes in basal and behavioral testing-induced BDNF release using in vivo microdialysis, and characterizing whether changes in TrkB, downstream signaling, and/or spine loss are specific to the indirect versus direct pathway MSNs. These studies will provide new insight into whether BDNF dysfunction is a risk factor that negatively impacts functional remodeling of the aged parkinsonian brain and, hence, whether it is a target for enhancing DA terminal remodeling in aged parkinsonian subjects.
A. R. Muotri*†
*Department of Pediatrics, University of California San Diego, School of Medicine, La Jolla, CA, USA
†Rady Children's Hospital San Diego, Department of Cellular and Molecular Medicine, Stem Cell Program, La Jolla, CA, USA
The recent outbreak of the Zika virus (ZIKV) has been associated with the increase in newborns with congenital malformations in Brazil. In adults, other clinical manifestations have been reported. We have previously shown that the Brazilian ZIKV (ZIKVBR) infects fetuses, causing intrauterine growth restriction (IUGR) and microcephaly in mice. Moreover, the virus infects human cortical progenitor cells leading to an increase in cell death by apoptosis. Finally, we observed that infection of human brain organoids resulted in a reduction in proliferative zones and disruption of cortical layers. Our results indicate that the ZIKVBR crosses the placenta and impairs neurodevelopment in mouse models, causing microcephaly by targeting cortical progenitor cells. Given the growing threat of the ZIKV spreading, researchers worldwide have focused on vaccine development. While immunization initiatives are important, there is a need to develop clinical strategies to treat ZIKV-infected individuals, including pregnant women for whom prevention of infection is no longer an option. Indeed, ZIKV infection during the first trimester confers the greatest risk of congenital microcephaly, thus highlighting the urgent need for treatment of infected mothers. In order to provide a potential treatment against the detrimental effects of ZIKV infection, we have tested the clinically approved antiviral inhibitors, both in vitro on ZIKV-infected human neural progenitor cells (NPCs) and cerebral organoids and in vivo using animal models. Altogether, our results showed that the potential treatment was well tolerated in vivo and able to decrease ZIKV replication. The results presented here demonstrate that repurposing the FDA-approved antiviral drugs is a timely therapeutic opportunity to counteract the harmful impact of the ZIKV in exposed individuals, including pregnant women.
E. Neal, S. A. Acosta, 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
Stroke is the second leading cause of death worldwide and the third leading cause of adult disability in adults. Ischemic stroke triggers an inflammatory response in the brain that is cytotoxic. In response to ischemic stroke, γ-Δ T cells from the gut mobilize to the brain and exacerbate cytotoxic inflammation. Regulatory T cells (Tregs) exert a neuroprotective effect after ischemic stroke by inhibiting both inflammation and effector T-cell activation. Transplantation of human bone marrow-derived stem cells (hBMSCs) after ischemic stroke has a neuroprotective effect. One way that hBMSCs protect neurons from apoptosis is by attenuating innate inflammation. Our lab has closely studied the innate immune response and found that implanted stem cells accumulate in locations with known importance to the adaptive immune system. This study will characterize the dynamic T-cell response to hBMSC treatment of ischemic stroke. It is hypothesized that hBMSCs will inhibit γ-Δ T cells and stimulate Tregs following ischemic stroke. Tregs were harvested from the spleens of healthy, wild-type mice. Tregs were isolated by magnetic sorting. Briefly, splenic tissue was dissociated manually, and a single-cell suspension was filtered out. Anti-cluster of differentiation 4 (CD4) and CD25 antibodies were used to label Tregs and were conjugated with magnetic microbeads. Magnetically labeled cells were isolated by passing the cell suspension through a column containing magnetic metal substrate. Then primary rat neuronal cells (PRNCs) were cultured in vitro. PRNCs were suspended in 400 μl of medium without antibiotic in poly-l-lysine-coated eight-well plates. After 3 days in cell culture, the cells were subjected to an oxygen–glucose deprivation and reperfusion (OGD/R) condition for 90 min. The cells were reperfused and cocultured with Tregs and BMSCs for 3 days. Cells were fixed in paraformaldehyde and immediately labeled with live-cell nuclear stain (Hoechst) and imaged under a fluorescence microscope and counted. An average of 42% of PRNCs survived the OGD/R treatment, a significant (p < 0.005) reduction from PRNCs in the normoxic condition. In comparison, 84% of PRNCs cultured with BMSCs after OGD/R survived, a significant (p < 0.005) improvement compared to the OGD/R control. Similarly, 66% of PRNCs survived OGD/R when cultured with Tregs, a significant (p < 0.05) improvement compared to the OGD/R control. PRNCs cultured with both BMSCs and regulatory cells survived OGD/R at a rate of 56%. Primary rat cortical cells were protected from ischemic conditions in coculture with Tregs. These data suggest a neuroprotective role for Tregs, which is likely due to immunomodulation mediated by astrocytes. However, the double coculture of Tregs and BMSCs did not produce an augmentation of neuroprotection. Future studies will examine in BMSCs the phenotypic expression of CD4, CD25, and forkhead protein P3 (FOXP3), which are Treg biomarkers, to reveal their specific roles in the observed stem cell-mediated neuroprotection in stroke.
Support: C.V.B. is supported by NIH NINDS 1R01NS071956-01, NIH NINDS 1 R21 NS089851-01, DOD TATRC W81XWH-11-1-0634, James and Esther King Foundation for Biomedical Research Program 1KG01-33966, SanBio Inc., Celgene Cellular Therapeutics, KMPHC, and NeuralStem Inc.
J. C. Necarsulmer,* D. B. Howard,* J. M. Pickel,† C. T. Richie,* and B. K. Harvey*
*Intramural Research Program, National Institute on Drug Abuse, Baltimore, MD, USA
†Intramural Research Program, National Institute on Mental Health, Bethesda, MD, USA
Dopaminergic signaling in the mammalian central nervous system (CNS) is involved in multiple brain functions, including movement, mood, reward circuitry, and motivation. Loss or alteration of dopamine neurons is associated with numerous neurological disorders and conditions, including Parkinson's disease (PD) and substance abuse. The ability to modulate dopaminergic signaling in the rodent CNS is a much-needed tool for biomedical researchers, but many current models of dopamine dysfunction, such as the use of 6-hydroxydopmaine (6-OHDA) in PD studies, invoke cell death rather than fine-tuned manipulation. The combination of transgenic animals expressing a “causes recombination” protein (Cre) under the control of the dopamine transporter promoter (DAT-Cre) and Cre-dependent adeno-associated viruses (AAVs) allows for precise activation of an ectopic transgene but has difficulty modulating endogenous gene expression. The development of clustered regularly interspaced short palindrome repeats (CRISPR) technology has revolutionized genome engineering and may provide the genetic models of dopamine dysfunction that are presently lacking. The use of CRISPR in vivo has been described in murine models, but studies have yet to demonstrate its action in the rat brain. Herein we describe multiple examples of CRISPR-mediated genome editing of dopaminergic neurons in the rat. As a starting proof of principle, we performed unilateral injection into the substantia nigra of two AAVs, one carrying CRISPR-associated protein-9 nuclease (Cas9) and the other a guide RNA (gRNA) targeting tyrosine hydroxylase (TH) and a green fluorescent protein (GFP) reporter. This led to an observable reduction in the amount of TH immunoreactivity in the midbrain and striatum compared to controls. To ultimately benefit from the tissue-specific activity of our lab's repertoire of Cre driver rats, we inserted a Cre-dependent [aka flanked locus of crossing (x) over, P1 (floxed)-stop] Cas9 transgene into the Rosa26 locus in a Long–Evans rat background (lsl-Cas9). We first tested these rats by injecting AAVs to deliver Cre recombinase and gRNAs into the midbrain. Two weeks following injection, a reduction in TH immunoreactivity was observed in the hemisphere that received gRNA targeting TH, but not on the contralateral side, which received a control gRNA. In the absence of Cre recombinase, no alteration of TH expression was detected. Future experiments will include injection of virally encoded gRNA into a double transgenic rat resulting from the cross of DAT-inducible Cre (iCre) and lsl-Cas9 rats, allowing selective CRISPR-mediated modification of the dopaminergic pathway. This novel technology can enhance our understanding of dopamine signaling and its role in the mammalian CNS under physiological or pathological conditions.
F. Nitzsche,*† H. Ghuman,*‡ M. Gerwig,*§ J. Moorhead,*¶ A. Poplawsky,† B. Wahlberg,† F. Ambrosio,*¶ and M. Modo*†‡
*McGowan Institute for Regenerative Medicine, University of Pittsburgh, Pittsburgh, PA, USA
†Department of Radiology, University of Pittsburgh, Pittsburgh, PA, USA
‡Department of Bioengineering, University of Pittsburgh, Pittsburgh, PA, USA
§Department of Neuroscience, University of Pittsburgh, Pittsburgh, PA, USA
¶Department of Physical Medicine and Rehabilitation, University of Pittsburgh, Pittsburgh, PA, USA
Stroke is caused by the occlusion of a cerebral artery and results in death or severe, long-lasting sequelae. Treatment options, however, are extremely limited, extensive, and cost-intensive without the guarantee to restore lost functions. Alternative approaches, such as stem cell-based therapies, are promising and can support tissue regeneration and functional recovery. Despite this, the restoration of sensory and motor deficits is still not complete and requires further enhancement. Of note, physical therapy (PT) is known to also improve behavioral functions. Thus, the aim of this study was to evaluate the efficacy of a combination therapy (CoT) consisting of human neural stem cell (NSC) transplantation and PT for improved recovery in a model of stroke. Adult male Sprague–Dawley rats underwent transient middle cerebral artery occlusion (MCAO). Success of the MCAO was determined by T2-weighted magnetic resonance imaging (MRI), and animals were randomly assigned to groups (MCAO only, MCAO + NSC, MCAO + PT, and MCAO + CoT; four to five rats/group). Sham-operated animals served as healthy control. Rats subjected to the MCAO + NSC or MCAO + CoT groups received a perilesional NSC graft 2 weeks after MCAO. Experimental groups with PT underwent daily physical training. Functional deficits and improvements were followed over a time course of 12 weeks using a battery of behavioral tests, including maximal capacity test, bilateral asymmetry test, foot fault test, rotameter, and grip strength test. Serial MRI was conducted throughout the experiment, including diffusion tensor imaging (DTI), cerebral blood volume (CBV), and functional MRI (fMRI) based on electrical forepaw stimulation in the concluding imaging session. After animal perfusion, brains and upper forelimb muscles were collected for histology. Behavioral assessment revealed functional improvements after PT, NSCs, and CoT. CoT is a promising approach to further enhance the efficacy of NSCs and to improve the restoration of lost functions.
B. Okyere, A. Hazy, M. Creasey, K. Giridhar, T. R. Brickler, X. Wang, and M. H. Theus
Department of Biomedical Sciences and Pathobiology, Virginia-Maryland Regional College of Veterinary Medicine, Blacksburg, VA, USA
Pial collaterals are arteriole-to-arteriole vascular bridges in the pia mater that connect the anterior, middle, or posterior cerebral artery branches on both dorsal hemispheres. Studies suggest that the density of native collaterals and their ability to remodel into large conduit arteries are major determinants of the severity of tissue injury after vascular occlusive disease due to faster restoration of blood flow. However, the mechanisms regulating early collateral development and their response to vascular obstruction in the adult brain remain under investigation. Although ephrin receptor tyrosine kinases have been linked to critical roles in neurological development and angiogenesis, their role in cerebral arteriole collateral formation have not been elucidated. Initially, we demonstrate that ephrin receptor A4 (EphA4) is highly expressed on pial arteriole collaterals at postnatal days (P) 1 and 7, but significantly reduced by P21. Also, EphA4 expression is reduced on blood vessels in the injured side of a stroked brain compared to the uninjured side. To address the role of EphA4 in collateral formation, we generated endothelial cell (EC)-specific EphA4 knockout (EphA4f/f/Tie2:: Cre [KO]) mice, and then analyzed collateral development using a technique called vessel painting. EphA4 ablation on ECs increased the number, but not diameter, of pial collaterals at P1 to adulthood. Using the permanent middle cerebral occlusion model to induce stroke, we show that collateral remodeling and subsequent blood flow restoration is significantly increased in KO compared to WT mice. To examine the EC-specific effects of EphA4 on proliferation of ECs as it relates to prenatal collateral development, we isolated and cultured brain ECs from our engineered mice. Interestingly, cultured brain-derived ECs isolated from KO mice displayed a threefold increase in proliferation, enhanced migration, and tube formation compared to wild-type ECs. These findings correspond to an increase in the level of phospho-Akt protein. Inhibiting phospho-Akt in KO-ECs using LY294002 attenuated the proliferative and migration effects. RNA sequencing (RNAseq) analysis also revealed altered mRNA expression patterns for genes that control cell proliferation, vascular development, extracellular matrix, and immune-mediated responses. Further analysis using real-time polymerase chain reaction showed increased expression of monocyte chemoattractant protein 1 (MCP-1), matrix metallopeptidase 2 (MMP2), and angiopoietin-1 (Ang-1) in KO compared to WT ECs. We further elucidated the cross-talk between EphA4 and the Ang-1/tunica interna endothelial cell kinase (Tie2) pathways in KO ECs. Inhibition of the Ang-1 pathway using soluble Tie2 reduced phospho-Akt protein expression and proliferation of the KO ECs. These findings demonstrate a novel role for EphA4 in the early development of the pial collateral network and in modulating postnatal vascular remodeling after cerebrovascular obstruction. EphA4 may represent an attractive new target for therapeutic intervention aimed at tissue protection and functional restoration following stroke.
A. Olin,* G. Cortese,† and C. Burger†
*College of Letters and Science, University of Wisconsin, Madison, WI, USA
†Department of Neurology, University of Wisconsin, Madison, WI, USA
Accurately testing learning and memory performance in rodents is crucial to many researchers in aging and neurodegenerative diseases. A possible issue in testing cohorts multiple times over their lifetime is that they can become trained in the task, negating the discriminating effects and goals of the testing. The eight-arm radial arm water maze (RAWM) can be used as an alternative or in addition to the Morris water maze (MWM). Performance in behavioral tests such as these shows even slight differences in phenotype in experimental cohorts. Here we have optimized a RAWM protocol to detect differences and changes in learning and memory performance in aged rats. Previously used protocols show that in young adult rats, the RAWM can be completed in 15 trials per day over 2 days. This proved to be too physically demanding for the aged (≷18-month-old) rats. We have optimized the RAWM for aged rats by performing only three trials per day over 4 days. Habituation occurs on day 1; only two of the eight arms are open, one of which contains the visible escape platform. The rat is dropped in the center of the maze and given 90 s or until it finds the platform by swimming into the correct arm. Instead of performing consecutive trials with the same rat, the next rat is used after a single trial, and so on. After each rat has its turn, the first rat performs its second trial, and this system is repeated until each has performed the maze three times. On day 2, all eight of the arms are opened, and a hidden platform is placed in a different arm from the habituation session. The platform will stay in this location for the next 3 days of testing. The starting arm will change with each trial, but the order and length of the trials remain the same. Each time the rat enters an arm that does not contain the platform, or if it stays in the same location for >30 s, an error is marked. Initial validation of this RAWM method showed that one cohort of 18-month-old Fisher rats (n = 24) can be separated into different groups by cognitive ability: superior (SL) and inferior learners (IL). On day 4, the SL group averaged significantly fewer errors than the IL group (2.03 vs. 3.12, p < 0.05, unpaired t-test). This RAWM protocol is useful in testing learning and spatial memory in aged rats, particularly if the cohort has already performed other behavioral testing such as the MWM. Tracking subtle changes in cognition due to aging and neurodegenerative diseases can be difficult; however, the challenge of the RAWM provides a method in separating a cohort based on their cognitive ability.
K. L. Paumier,* T. Cole,† J. G. Bollinger,* R. Miller,* J. Jockel-Balsarotti,* B. W. Patterson,* T. M. Miller,* H. Zhao,† E. Swayze,† R. J. Bateman,* A. Booms,‡ S. Celano,‡ E. Schulz,‡ A. Strauss,‡ B. Daley,‡ A. Weihofen,§ H. Kordasiewicz,* T. Collier,‡ and P. T. Kotzbauer*
*Department of Neurology, Washington University, Saint Louis, MO, USA
†Ionis Pharmaceuticals, Carlsbad, CA, USA
‡Michigan State University, Grand Rapids, MI, USA
§Biogen, Cambridge, MA, USA
Parkinson's disease (PD) is defined by the accumulation of α-synuclein (α-Syn) fibrils in neuronal cytoplasmic and neuritic inclusions known as Lewy bodies and Lewy neurites (Forno, 1996). Multiple therapeutic approaches targeting α-Syn accumulation are being pursued for the treatment of PD, including (1) inhibition of α-Syn protein synthesis, (2) prevention or reversal of α-Syn aggregation, and (3) enhancement of α-Syn clearance. To evaluate the first approach, we collaborated with Ionis Pharmaceuticals to test their antisense oligonucleotides (ASOs) designed to target α-Syn (Snca) RNA and reduce the production of α-Syn, thereby slowing the spread of pathology and halt disease progression. Endogenous mouse and rat α-Syn transmission preformed fibril (PFF) inoculation models were evaluated. These models are based on intrastriatal injection of α-Syn PFFs that leads to the deposition and aggregation of α-Syn, which accumulates and spreads through neural networks over time. Following characterization of timing of fibril deposition in the models, both pre (preventive) and post (therapeutic) fibril deposition paradigms were evaluated with central delivery (intracere-broventricular) of Snca-targeting ASOs. Following study completion, Snca RNA was evaluated by RT-PCR and phosphorylated α-Syn pathology was evaluated histologically. Snca-targeting ASOs in both species led to Snca RNA target reduction and slowed deposition and spread of phosphospecific forms of α-Syn. While the effect of ASOs was robust in our transmission models and RNA reduction was confirmed, there is still no reliable method to assess the success of therapeutic interventions (i.e., target engagement and pharmacodynamics) in the clinic. To address this need, we developed an immunoprecipitation tandem liquid chromatography–mass spectrometry (IP-LC/MS) method based on the stable isotope labeling kinetics (SILK) technique to quantitate α-Syn and measure its turnover kinetics in human cerebrospinal fluid (CSF). Leveraging our previous experience with the development of SILK protocols and monoclonal antibodies, we first developed an IP protocol by screening a panel of both in-house and commercial antibodies under a set of commonly applied IP conditions. We then optimized an LC/MS method based on bottom-up proteomics capable of characterizing the α-Syn proteoforms present in human CSF. Using a set of 15-nitrogen uniformly labeled proteins in addition to a set of peptides with differentially labeled individual amino acids, we designed our quantitative assay around five key proteolytic peptides containing either a leucine residue (for SILK turnover analysis) or sites of documented posttranslational modifications. With optimized assay parameters in hand, we defined a suitable labeling strategy for SILK analysis to determine the production and clearance rates of α-Syn in CSF from a set of control human participants. Using this method, we determined that the physiological half-life of α-Syn in the human CNS is approximately 8 days. Future studies will utilize the SILK method in PD patients to assess α-Syn production and clearance rates and define altered α-Syn metabolism related to PD. Further, this technique will provide a valuable measure of target engagement and pharmacodynamics for future clinical studies targeting α-Syn.
S. Pianta, H. Nguyen, X. Kaya, S. A. Acosta, N. Tajiri, J.-Y. Lee, 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
Exercise has been shown to afford therapeutic benefits in stroke patients, but the mechanism of action is still unknown. The peculiar feature of ischemic stroke is the interruption in brain blood circulation; therefore, finding strategies to maintain the potency of the cerebrovasculature may provide a therapeutic effect against stroke. The present study assessed the effects of short bouts of exercise prior to stroke induction and characterized in vivo the cerebral blood flow and concomitant motor functions. First, adult Sprague–Dawley rats were exposed once to a short bout of exercise (30- or 60-min forced running wheel), afterward they were subjected to transient intraluminal occlusion of the middle cerebral artery (MCAo). Nonexercised stroke rats served as controls and no stroke rats represent the negative control (sham). Cerebral blood flow (CBF) was evaluated by laser Doppler at baseline (prior to MCAo), during MCAo, and during reperfusion. Behavioral testing using the elevated body swing test was conducted at baseline, day 0 (day of stroke), and at days 1 and 3 after stroke. Results revealed that CBF was significantly elevated during reperfusion in 60-min exercised rats compared to controls. Moreover, the animals that received 60-min exercise displayed improved motor performance than 30-min exercise and nonexercised rats. The histological analysis revealed that exercised rats show a reduction of the infarct size area and an increased number of live cells in the peri-infarct area, specifically this trend followed the duration of the exercise. Immunofluorescence staining intensity revealed increased levels of endothelial markers/angiogenesis marker angiopoietin 2 (Ang-2) and vascular endothelial growth factor receptor 2 (VEGFR-2) and endothelial progenitor cell (EPC) marker cluster of differentiation 34 (CD34) in the exercise groups compared with control. These results showed that exercise (prior to stroke) possibly acts on improving cerebrovascular integrity and function. According to the present study, we indicate that the effect of exercise could be a prophylactic treatment against stroke, and further study is needed to understand the connection between apoptosis and angiogenesis in brain injury.
This study is supported by NIH R01 and NIH R21 grants.
S. Ping and L.-R. Zhao
Department of Neurosurgery, SUNY Upstate Medical University, Syracuse, NY, USA
Cerebral autosomal dominant arteriopathy with subcortical infarcts and leukoencephalopathy (CADASIL) is the most common form of hereditary disease leading to recurrent ischemic stroke and vascular dementia. This disease is caused by a NOTCH3 mutation, which results in vascular smooth muscle cell (VSMC) degeneration of the small arteries in the brain. So far, there is no specific treatment for this disease. Our earlier study has demonstrated that systemic administration of two hematopoietic growth factors, stem cell factor (SCF) and granulocyte colony-stimulating factor (G-CSF), restricts VSMC degeneration and improves cognitive function in a mouse model of CADASIL. The aim of this study was to determine the efficacy of SCF + G-CSF on neuronal plasticity in CADASIL mice. Five-day injections of SCF + G-CSF or an equal volume of vehicle solution were given five times at age 9, 10, 12, 15, and 20 months. CADASIL mice were euthanized at the age of 22 months, and brain sections were processed for immunohistochemistry. We found that expression of phosphorylated extracellular signal-regulated kinase (pERK) was significantly elevated in cortical neurons of SCF + G-CSF-treated CADASIL mice. In addition, SCF + G-CSF treatment also significantly increased dendritic densities and enlarged the stem neurite diameters in cortical neurons. These findings suggest that SCF+G-CSF treatment in CADASIL mice enhances cortical neural network remodeling and increases cortical neuronal activities. This study provides novel insights into the contribution of hematopoietic growth factors in brain plasticity and brain repair in the condition of CADASIL.
W. A. Ratliff,*† J. N. Chang,*† N. H. Greig,‡ and B. A. Citron*†
*Laboratory of Molecular Biology, Research and Development 151, Bay Pines VA Healthcare System, Bay Pines, FL USA
†Department of Molecular Medicine, University of South Florida Morsani College of Medicine, Tampa, FL USA
‡Translational Gerontology Branch, National Institute on Aging, NIH, Baltimore, MD, USA
In the US each year, it is estimated that 1.7 million people sustain a traumatic brain injury (TBI). In our military members and athletes, in particular, TBI remains a major cause of morbidity and mortality. The majority of TBIs are mild and these can result in deleterious cognitive effects for which there is currently no effective treatment. We have demonstrated improved outcomes in both in vitro and in vivo models of brain injury following treatment with tert-butylhydroquinone (tBHQ). tBHQ is an activator of the inflammatory responsive transcription factor, nuclear factor erythroid 2-related factor 2 (Nrf2), and has been shown to induce the production of several neuroprotective factors. Pifithrin-α (PFT-α) compounds that inhibit p53 demonstrated neuroprotective effects in an in vitro excitotoxicity model and in an in vivo head injury model. To further evaluate the effects of a repetitive physical injury on a neuronal cell line and evaluate the potential protective effects of treatment, an in vitro “injury in a dish” model was used. tBHQ plus the sulfur-containing inhibitor PFT-α produced significantly improved neuronal function as measured with an MTS assay (compared to either treatment alone), while tBHQ plus SP600125, a Jun N-terminal kinase inhibitor, was not additionally protective. We investigated treatment with tBHQ, PFT-α oxygen [PFT-α (O)], and a combination of the two to determine whether simultaneous activation of Nrf2 and inhibition of p53 would provide a synergistic neuroprotective effect in terms of neuronal survival and neurite health. Observations indicated that treatment with tBHQ plus PFT-α (O) improved overall neuronal survival and neurite health, as measured by neurite length per cell, when compared to vehicle-treated cells or those that received only one treatment. In addition, qRT-PCR array data have identified a number of differentially regulated genes in response to combination treatment. In particular, anti-inflammatory heme oxygenase 1 mRNA was significantly increased by the combination treatment, which may account for some neuroprotective effects. This suggests that simultaneous activation of Nrf2 and inhibition of p53 produce enhanced neuroprotective effects in response to neuronal injury and could represent a novel treatment in response to head injury. Overall, the goal of our group is to help identify therapeutic approaches that would benefit individuals suffering from mild TBI.
This study was supported by the Department of Veterans Affairs [Veterans Health Administration, Office of Research and Development, Rehabilitation Research and Development (I01RX001520)], the Assistant Secretary of Defense for Health Affairs through the Congressionally Directed Gulf War Illness Research Program (W81XWH-16-1-0626), the Florida Department of Health James and Esther King Biomedical Research Program (4KB14), and The Bay Pines Foundation. The contents do not represent the views of the Department of Veterans Affairs or the US Government, and the opinions, interpretations, conclusions, and recommendations are those of the authors and are not necessarily endorsed by the Department of Defense.
H. Resnikoff,* J. Shultz,*† V. Bondarenko,* A. Mejia,* H. Simmons,* and M. E. Emborg*†‡
*Wisconsin National Primate Research Center, University of Wisconsin-Madison, Madison, WI, USA
†Cellular and Molecular Pathology Graduate Program, University of Wisconsin-Madison, Madison, WI, USA
‡Department of Medical Physics, University of Wisconsin-Madison, Madison, WI, USA
α-Synuclein (α-syn) is a presynaptic protein ubiquitously present in neurons of the central and peripheral nervous system. In normal, nonpathological conditions, α-syn is found in soluble form. In contrast, in Parkinson's disease (PD) and other synucleinopathies, α-syn undergoes phosphorylation and aggregation associated with the formation of Lewy bodies, a pathological hallmark of PD. Gastrointestinal (GI) dysfunction is a common nonmotor symptom of PD. It can predate the onset of motor symptoms, and it has been seen in association with α-syn pathology in the enteric nervous system (ENS). In that regard, PD, α-syn pathology, has been hypothesized to originate in the ENS and to travel via the vagus nerve, progressing up the brainstem, eventually reaching the substantia nigra and spreading throughout the CNS. Chronic GI inflammation has been proposed as a possible trigger of α-syn pathology. Interestingly, common marmoset monkeys (Callithrix jacchus) in captivity can develop chronic colonic inflammation or colitis. The ENS anatomical organization in human and marmosets is remarkably similar, including the presence of neuronal ganglia within the myenteric plexus, which is located between the circular and longitudinal muscle layers of the colon. Therefore, marmosets can be used as an experimental platform to test the hypothesis that colitis induces changes in α-syn expression and phosphorylation. In this study, we evaluated the colonic myenteric plexus of marmoset monkeys with colitis (n=5; 5.3 ± 2.3 years old; four male) and normal control animals (n = 5; 4.1 ± 1.6 years old; one male). Colonic tissues were obtained from the Wisconsin National Primate Research Center tissue bank; subjects were selected by reviewing previous diagnoses with subsequent evaluation of hemotoxylin and eosin (H&E)-stained sections by veterinary pathologists to meet study parameters. Animals with colitis had inflammatory infiltration of the lamina propria, causing mucosal expansion, glandular displacement, glandular dysplasia, and occasional gland abscessation. Sections of colon from all animals were immunostained for protein gene product 9.5 (PGP9.5; panneuronal marker), α-syn, and phosphorylated α-syn. Nigral tissue sections from a PD patient were used as positive controls for phosphorylated α-syn. Immunoreactivity of the three markers was blindly analyzed using ImageJ software. Briefly, the myenteric plexus from each immunostained colon tissue section was image captured at 63x, the ganglia were outlined, and the percent area above threshold (%AAT) was measured. PGP9.5-ir had a similar pattern in all animals; it was uniformly present throughout all neuronal cell bodies and processes in the myenteric plexus and in nerve fibers running between muscle fibers and mucosa. Quantification of PGP9.5-ir in the myenteric ganglia showed similar %AAT values in control and colitis animals. α-Syn-ir was also identified within the myenteric ganglia area and in fibers running through the muscle and mucosal layers. In all myenteric ganglia, α-syn-ir was observed as both light, diffuse immunostaining and as small, dark, granular spots. Quantification of α-syn-ir revealed significantly less %AAT in the colitis group, as compared to the control (colitis: 76.8% AAT, control: 91.6%; p=0.038). Quantification of phosphorylated α-syn showed a statistically significant greater %AAT in animals with colitis compared to controls (colitis: 17.8% AAT, control: 1.7%; p=0.016). Our findings demonstrate that colitis induces changes in the expression of α-syn and phosphorylated α-syn in the colonic myenteric plexus of common marmoset monkeys without affecting the expression of neuronal PGP9.5-ir. Next steps include assessment of the impact of colitis on the development of proteinase K-resistant α-syn aggregates, inflammatory and oxidative stress markers, and phenotyping of the enteric neuronal populations is affected.
Supported by PDF-APDA summer fellowship (H.R.), P51OD011106, R24OD019803.
C. Richie
Genetic Engineering and Viral Vector Core, National Institute on Drug Abuse, Baltimore, MD, USA
The development of clustered regularly interspaced short palindrome repeats (CRISPR) technology has revolutionized genome engineering and lowered the time and cost commitments associated with generating genetic model organisms. Moreover, CRISPR has extended our ability to manipulate the genome of somatic cells in adult tissue to study gene function and create genetic models of disease. We have developed several tools that facilitate the use of CRISPR for knocking out gene function in the rat brain. First, we have created Crerecombinase-expressing rats to selectively modify neurons based on their expression of the dopamine transporter (DAT-Cre), dopamine D1 receptor (DRD1a-Cre), dopamine receptor 2 (DRD2-Cre), parvalbumin (Pvalb-Cre), or glutamate decarboxylase 1 (GAD1-Cre). Second, we have created a Rosa26-lsl-Cas9 rat, which contains a Cre-dependent CRISPR-associated protein-9 nuclease (Cas9) programmable endonuclease. Last, we describe several novel adeno-associated virus (AAV)-based vectors as well as previously published CRISPR-related vectors to be used in combination with our transgenic rats. As an example, we manipulated gene expression in dopaminergic neurons of the nigrostriatal pathway in the rat brain. Collectively, we describe CRISPR-based tools that can be used in various combinations to create mutations in the genome of specific neurons in an adult rat brain.
I. M. Sandoval,* R. C. Sellnow,*† B. Daley,* N. Kuhn,* N. Marckini,* A. C. Strauss,* J. W. Lipton,*†‡ T. J. Collier,*†‡ and F. P. Manfredsson*†‡
*Department of Translational Science and Molecular Medicine, College of Human Medicine, Michigan State University, Grand Rapids, MI, USA
†Neuroscience Program, Michigan State University, Grand Rapids, MI, USA
‡Hauenstein Neuroscience Center, Mercy Health Saint Mary's Hospital, Grand Rapids, MI, USA
The ability to remove a desired gene in a specific cell type enables direct evaluation of gene function. The latest class of nucleases, CRISPR (clustered regularly interspaced short palindromic repeats)/CRISPR-associated protein-9 nuclease (Cas9) system, can be used to introduce frame-shifting mutations to knock out a gene with high accuracy. CRISPR/Cas9 technology has been widely used in a variety of cells and microorganisms, but its application in mature neuronal tissue has been limited. Here we aimed to investigate whether we could achieve efficient CRISPR/Cas9-mediated gene inactivation in vivo, specifically in dopaminergic neurons of the substantia nigra, the cells affected in Parkinson's disease (PD). As a proof of concept, we selected to target tyrosine hydroxylase (TH), the rate-limiting enzyme in the production of dopamine. We also targeted α-synuclein (α-syn), vesicular transport protein 35 (VPS35), and eukaryotic translation initiation factor 4 γ 1 (EIF4G1), all of which are linked to familial forms of PD. We designed guide RNAs (gRNAs) against exon 1 and exon 2 of the TH gene and confirmed genomic DNA cleavage efficiency and reduction in protein levels in PC12 cells in vitro. We delivered the protein Cas9, together with the gRNAs and green fluorescent protein (GFP), as a transduction marker into the substantia nigra of 2-month-old Sprague–Dawley rats using adeno-associated virus (AAV). Immunostaining of brain sections revealed a robust decrease of TH protein expression in CRISPR/gRNA(TH)-treated brains as compared to “unguided” control-injected rats 6 weeks after surgery. Stereological quantification of TH immunoreactive neurons revealed a 48% reduction of TH-expressing nigral neurons in the treated animals. A closer histological examination revealed that all transduced DA neurons completely lacked TH expression. These results validate the use of CRISPR technology in vivo in the nigrostriatal dopaminergic system in mature brains. Current experiments are aimed at targeting the aforementioned PD-related genes: α-syn, VPS35, and EIF4G1 using a similar strategy. Upon project completion, we will have a useful toolbox to interrogate gene function in nigral dopamine neurons in vivo while still in their native context.
This work was supported by the office of the MSU Vice President for Research and Graduate Studies (F.P.M. and I.M.S.) and The Edwin Brophy Endowment in Central Nervous System Disorders (T.J.C.).
L. A. Sandusky, A. Kovalenko, D. S. Placides, W. J. D. Fraser, J. B. Hunt Jr., S. N. Fontaine, C. A. Dickey, M.-L. Selenica, K. R. Nash, M. N. Gordon, D. G. Morgan, and D. C. Lee
Byrd Alzheimer's Institute, University of South Florida Health, Tampa, FL, USA
In a nondiseased brain, tau stabilizes microtubules; however, in Alzheimer's disease (AD) and tauopathies, tau becomes hyperphosphorylated, aggregates, and results in neuronal death. Our group recently uncovered a unique interaction between polyamines and tau fate. Polyamines exert an array of physiological effects that support neuronal function and cognitive processing. The direct link between polyamines and cognition is postulated to involve the putative polyamine-binding site on N-methyl-D-aspartate (NMDA) receptors. Specific stimuli (physical or emotional) can elicit a polyamine stress response (PSR), resulting in altered central polyamine homeostasis. Furthermore, evidence suggests that while the elevations in polyamines following a short-term stressor are beneficial, persistent stress and subsequent PSR activation may become maladaptive and lead to polyamine dysregulation. Polyamine dysregulation occurs in numerous disease states and may contribute to neuropathology and cognitive impairment. Our group recently uncovered a unique interaction between tau and the polyamine pathway. We first identified polyamine dysregulation in rTg4510 transgenic tau mice. Specifically, rTg4510 mice showed dysregulation in ornithine decarboxylase (ODC), spermidine synthase (SRM), spermine synthase (SMS), spermidine/spermine N1-acetyltransferase (SSAT), spermine oxidase (SMOX), polyamine modulated factor 1 (PMF1), and PMF binding protein 1 (PMFBP1), relative to nontransgenic controls. The reduction in ODC and increase in SSAT are most notable, as they alone are thought to be the key regulatory enzymes controlling polyamine homeostasis in the brain. Additionally, we found that the tau phenotype increased expression of PMF1, suggesting support for SSAT-induced catabolism. Further, total polyamine mass in brain homogenates was also perturbed in rTg4510 mice, specifically an increase in both putrescine and acetylspermidine, revealing a tau-dependent polyamine signature, which we will now refer to as the tau polyamine stress response (PSR). Notably, the increased putrescine does not arise from increased metabolism of ornithine, as we did not see a corresponding increase in ODC. In fact, we observed a decrease in ODC, thus suggesting activation of the SSAT-dependent back conversion as the potential mechanism for the PSR. Furthermore, acetylspermidine can only be produced through the catabolism of spermidine, also under the control of the SSAT-dependent back conversion, signifying the impact of SSAT dysregulation. Next, to determine if polyamines regulate tau aggregation, we coincubated recombinant full-length tau with polyamines and acetylated polyamines in solution. Polyamines prevented tau fibrillization in a dose-dependent manner (spermidine being more potent than putrescine), yet acetylated forms of polyamines failed to prevent fibrillization. Similar to that of rTg4510 mice, we also observe a nearly identical tau PSR signature using the caspase-cleaved/truncated form of tau (tau D421) via recombinant adenoassociated virus (rAAV) injected in the hippocampus and anterior cortices, specifically the increase in putrescine and acetylspermidine, and reduction in ODC, confirming a unique tau-dependent PSR. Further, because of the central role of SSAT in the tau-dependent PSR, we targeted SSAT by genetic deletion and prevented the increase of putrescine and acetylspermidine and decreased the accumulation of both monomeric and high-molecular weight tau. Last, crossing P301S+/- tau mice with SSAT knockout mice (P301S+/-xSSAT-/-) prevents the tau-dependent increases in putrescine, acetylputrescine, and acetylspermidine, providing further support for the ability of SSAT deletion to prevent the tau-dependent PSR. Taken together, our central hypothesis states accumulated hyperphosphorylated tau acts as a physiological stressor that elicits a maladaptive PSR and mediates the neuropathology and cognitive impairment. We hypothesize that strategies aimed at circumventing the tau-dependent PSR may serve to reduce neuropathology and ameliorate the cognitive impairments seen in AD and tauopathies.
E. Sasaki*†
*Department of Applied Developmental Biology, Central Institute for Experimental Animals, Kawasaki, Kanagawa, Japan
†Keio Advanced Research Center, Kio University, Sinjuku, Tokyo, Japan
Nonhuman primate (NHP) laboratory animals bridge the gap between basic researches and clinics. The common marmoset (Callithrix jacchus) is a New World monkey that has several valuable biological characteristics such as fecundity and ease of handling due to its small body size, which is also beneficial in assessing the efficacy and safety of newly developing drugs in small doses. Specifically, marmoset reproductive traits are suitable for breeding and producing genetically modified (GM) models because they deliver two to three pups every half year, have a gestation period of 145-148 days, and are sexually mature at approximately one and a half years old. Furthermore, recent progress of transgenic technologies in NHPs has enabled creation of many GM human disease NHP models including marmoset. GM marmosets would offer excellent, precise preclinical study systems for assessing the safety and efficacy of new therapies and drugs. In particular, GM marmosets are expected to be good models for brain science to understand the molecular mechanisms of high cognitive function and the onset of nerve system diseases and transplant models for regenerative medicine using pluripotent stem cells in, for example, Parkinson's disease or spinal cord injury. Until a few years ago, only exogenous gene overexpression models by lenti- or retroviral vectors were available. Since NHP embryonic stem cells (ESCs) lack the ability to contribute to germ cells, which is essential for producing targeted gene knockout (KO)/knock-in (KI) animals, traditional gene-targeting KO/KI techniques cannot be applied to NHPs. The recent development of innovative genome-editing technologies such as zinc-finger nucleases (ZFNs), transcription activator-like effector nucleases (TALEN), and clustered regularly interspaced short palindromic repeat (CRISPR)/CRISPR-associated protein-9 nuclease (Cas9) make it possible to generate target-gene KO animals without using ESCs. Consequently, it should be possible to produce useful GM NHP models to uncover unknown gene function and develop new therapies for many diseases, including cognitive dysfunction.
S. Seiler, S. Di Santo, and H. R. Widmer
Department of Neurosurgery, Research Laboratory and Regenerative Neuroscience Cluster, University of Bern and University Hospital Bern, Bern, Switzerland
Transplantation of human fetal ventral mesencephalic dopaminergic neurons into the host brain is an experimental strategy for patients suffering from Parkinson's disease. The success of this approach, however, is dependent on a number of parameters in the host brain including neurotrophic factors and growth inhibitors that interact with the grafted dopaminergic neurons. While the potential of neurotrophic factors has been extensively investigated, repression of growth inhibitors has been basically neglected. This is rather surprising given the significant effects reported in various models of central nervous system (CNS) injuries. Only recently, we demonstrated that the infusion of neutralizing antibodies against Nogo-A into the lateral ventricle of hemiparkinsonian rats enhanced graft function. Since the Nogo-receptor 1 also interacts with other growth inhibitors in the brain, we investigated whether a direct antagonization of the receptor would result in even more robust effects. For that purpose hemiparkinsonian rats were grafted with ventral mesencephalic tissue in combination with infusions of the Nogo-receptor 1 antagonist NEP1-40 into the lateral ventricles. Transplanted rats receiving saline infusions served as controls. Motor behavior using the cylinder test was assessed prior to the lesion as well as prior and 1, 3, and 5 weeks after the transplantations. At the end of the experimental period, the number of graft-derived dopaminergic fibers growing into the host brain, the number of surviving dopaminergic neurons, and graft volume were analyzed. We observed that the NEP1-40 treatment marginally but significantly enhanced graft-derived dopaminergic fiber outgrowth as compared to controls, while no effects were detected for graft volume and survival of grafted dopaminergic neurons. Notably, the enhanced dopaminergic fiber outgrowth was not sufficient to induce an improved functional recovery as compared to controls. In sum, our findings demonstrate that antagonization of the Nogo-receptor 1 is inferior to neutralization of Nogo-A in supporting engraftment and functional recovery in an animal model of Parkinson's disease.
This study was supported by the Swiss National Science Foundation (Grant No. 31003A-135565) and the HANELA Foundation.
R. C. Sellnow,*† E. Flores-Barrera,‡ A. R. West,‡ K. Steece-Collier,* M. J. Benskey,* I. M. Sandoval,* N. Kuhn,* K. Y. Tseng,‡ and F. P. Manfredsson*†
*Department of Translational Science and Molecular Medicine, Michigan State University, Grand Rapids, MN, USA
†Cell and Molecular Biology Program, Michigan State University Grand Rapids, MN, USA
‡Department of Cellular and Molecular Pharmacology, Rosalind Franklin University, Chicago, IL, USA
Levodopa (L-DOPA)-induced dyskinesias (LIDs) are a debilitating side effect that occurs in a majority of Parkinson's disease (PD) patients treated with L-DOPA. LIDs are involuntary motor behaviors that include chorea, dystonias, and limb hyperkinesia that are distinct from parkinsonian motor behaviors. Despite this, L-DOPA remains the most effective pharmacological treatment for the motor symptoms in PD. It has been shown previously by our group and others that an ectopic induction of the transcription factor nuclear receptor related 1 (Nurr1) in both direct and indirect pathway striatal medium spiny neurons (MSNs) is associated with LIDs. Preliminary data from our lab show that LID severity can be affected by modulating striatal Nurr1 expression with recombinant adeno-associated virus (rAAV). These data show that Nurr1 overexpression in the striatum plays an important role in LID development. Current studies aim to elucidate the mechanism of Nurr1's contribution to LIDs by examining the effect of ectopic Nurr1 on striatal physiology. The goal of these studies is to determine if Nurr1 expression in the striatum—a region where Nurr1 is not normally expressed—can change (1) electrophysiological activity of the striatum and/or (2) morphology of striatal MSNs. 6-Hydroxydopamine (6-OHDA)-lesioned Sprague–Dawley rats received either rAAV-Nurr1 or rAAV-green fluorescent protein (GFP) injections into the striatum. Animals were not treated with L-DOPA and thus did not become dyskinetic. The local field potential (LFP) of the striatum evoked following cortical stimulation was measured following a single dose of L-DOPA. While GFP-treated animals showed a depression in response, cortical stimulation induced a potentiation in Nurr1-treated animals. Remarkably, the response recorded in rAAV-Nurr1 rats was virtually identical to that in subjects with established dyskinesias, suggesting that ectopic Nurr1 expression induces pathophysiologic changes in corticostriatal transmission similar to those observed in dyskinetic animals. Additionally, preliminary data suggest that rAAV-Nurr1-treated parkinsonian rats not treated with L-DOPA show a decrease in thin spine density, suggesting a role of Nurr1 in spine loss observed in direct pathway neurons in LID+ animals. Ongoing studies aim to investigate the role of Nurr1 specifically in the direct and indirect pathway.
Y. Sheng,* K. L. Preston,† K. A. Phillips,† Z. Lin,‡ P. Tesar,§ B. Hoffer,* and Y. Luo*
*Department of Neurological Surgery, Case Western Reserve University, Cleveland, OH, USA
†National Institute on Drug Abuse, Intramural Research Program, Baltimore, MD, USA
‡Department of Psychiatry, Mclean Hospital, Harvard University, Belmont, MA, USA
§Department of Genetics, Case Western Reserve University, Cleveland, OH, USA
The dopaminergic (DA) system plays an important role in addiction. However, human DA neurons from drug-dependent subjects were not available for study until recent development in inducible pluripotent stem cell (iPSC) technology. In this study, we produced DA neurons differentiated using iPSCs derived from opioid-dependent and control subjects carrying different 3′ VNTR (variable number tandem repeat) polymorphisms in the human dopamine transporter (DAT; SLC6A3). We successfully generated midbrain dopamine neurons that expressed forkhead box protein A2, LIM homeobox transcription factor 1 α, tyrosine hydroxylase (TH), dopamine D2 receptor, nuclear receptor-related 1 (Nurr1), vesicular monoamine transporter 2, and DAT. All iPSC lines generated dopamine neurons that released DA detectable by high-performance liquid chromatography. There was no apparent difference in dopamine differentiation efficiency between cell lines derived from control and opioid-dependent subjects with regard to the percentage of cells positive for tyrosine hydroxylase. We present the first evidence suggesting that the 3′ VNTR polymorphism in the hDAT gene affects DAT expression level in iPSC-derived human DA neurons. There were also different expression levels of D2 receptors in the control group's iPSC-derived dopamine neurons compared with the opioid-dependent group's iPSC-derived dopamine neurons. Specifically, our results showed lower expression levels of D2 receptors in both opioid-dependent iPSC lines. We also examined the expression of different classes of opioid receptors. Our data showed that μ opioid receptors were not detected in our differentiated human dopamine neurons derived from iPSCs. In contrast, κ and Δ opioid receptors were detected in our human DA neuronal cultures from all four iPSC lines. In addition, the effects of valproic acid (VPA) exposure on iPSC-derived human DA neurons were also examined. In human DA neurons, VPA treatment altered the expression of several genes important for dopaminergic neuron function including DAT, NURR1, and TH; this might partly explain its action in regulating addictive behaviors. VPA treatment also significantly increased DA D2 receptor (DRD2) expression, especially in the opioid-dependent iPSC lines. Our data suggest that human iPSC-derived DA neurons may be a useful in vitro experimental model to examine the effects of genetic variation in gene regulation, to examine the underlying mechanisms in neurological disorders including drug addiction, and to serve as a platform for therapeutic development.
J. Shultz,*† H. Resnikoff,* V. Bondarenko,* J. Holden,‡ T. Barnhart,‡ P. Lao,‡ B. Christian,‡ J. Nickles,‡ C. F. Moore,§ and M. E. Emborg*†‡
*Wisconsin National Primate Research Center, University of Wisconsin-Madison, Madison, WI, USA
†Cellular and Molecular Pathology Graduate Program, University of Wisconsin-Madison, Madison, WI, USA
‡Department of Medical Physics, University of Wisconsin-Madison, Madison, WI, USA
§Department of Psychology, University of Wisconsin-Madison, Madison, WI, USA
Loss of postganglionic sympathetic innervation to the heart is a characteristic pathology of cardiac dysautonomia in Parkinson's disease (PD); disease-modifying strategies are not available. Neuronal loss in PD is associated with inflammation and oxidative stress and can be mimicked in animals by dosing of the catecholaminergic neurotoxin 6-hydroxydopamine (6-OHDA). Interestingly, activation of the peroxisome proliferator-activated receptor γ (PPARγ) decreases inflammatory response (e.g., microglial, macrophage) and production of reactive oxygen species. Here we report our studies with the PPARγ agonist pioglitazone in a nonhuman primate model of cardiac dysautonomia induced by systemic dosing of 6-OHDA. We aimed to assess the neuroprotective efficacy of pioglitazone using positron emission tomography (PET) with radioligands specific to catecholaminergic innervation ([11C] meta-hydroxyephedrine, MHED), inflammation ([11C]peripheral benzodiazepine receptor 28, PBR28), and oxidative stress ([61Cu]diacetyl-bis(N(4))-methylthiosemicarbazone, ATSM). Radioligand uptake was evaluated in eight regions (septal through inferior) and six layers (base to apex) of the left ventricle for MHED and PBR28; ATSM data analysis was limited to the anterior region due to interference by liver radioligand uptake. Ten adult, male rhesus monkeys received intravenous 6-OHDA (50 mg/kg) and 24 h later were blindly and randomly assigned to begin daily oral dosing of placebo (n=5) or pioglitazone (5 mg/kg; n=5). At baseline, 1 and 12 weeks post-6-OHDA, all animals were PET scanned with MHED, PBR28, and ATSM; 24 h after the final PET scan, animals were euthanized by transaortic perfusion and the heart tissue was collected. One week postneurotoxin, MHED uptake was dramatically reduced throughout all regions and levels of the left ventricle in all animals compared to baseline (placebo: 86% decrease; pioglitazone: 82%). Concurrently, increased PBR28 uptake was attenuated in pioglitazone-treated animals compared to placebo, most notably in the base of the left ventricle [condition X level ANOVA, F(5, 40) = 6.443, p < 0.002]. Analysis of ATSM data at 1 week also revealed significantly less uptake in pioglitazone-treated animals compared to placebo (Mann–Whitney U = 2, p = 0.032, r = 0.693). By 12 weeks postneurotoxin, partial recovery of MHED uptake was significantly greater in the pioglitazone-treated animals compared to placebo in a manner that was dependent on left ventricle region and level [condition X region X level ANOVA, F(10.07, 80.55) = 2.098, p = 0.034]. At this same time point, PBR28 and ATSM uptake values returned to baseline levels. Ongoing immunohistochemical quantification of panneuronal marker protein gene product 9.5 (PGP9.5) and catecholaminergic marker tyrosine hydroxylase (TH) in the left ventricle tissue supports the PET findings, suggesting preservation of sympathetic nerves in the heart induced by pioglitazone. Overall, these results demonstrate PPARg activation induced cardiac sympathetic neuroprotection in association with decreased inflammation and oxidative stress.
Research was supported by NIH grants P51OD011106, UL1TR000427, and R21NS084158 and the VCRGE and the Department of Medical Physics, University of Wisconsin-Madison.
I. N. Soares,* G. M. de Abreu Gomes,* A. L. de Oliveira,* T. H. Ferreira-Vieira,* R. D. Aires,* V. S. Lemos,* R. Santos,* and A. R. Massensini*†
*Department of Physiology and Biophysics, Institute of Biological Sciences, Universidade Federal de Minas Gerais, Belo Horizonte, MG, Brazil
†Department of Pharmacology, Institute of Biological Sciences, Universidade Federal de Minas Gerais, Belo Horizonte, MG, Brazil
Angiotensin-(1-7) [Ang-(1-7)] has been described as a peptide with neuroprotective properties; however, the mechanisms by which it acts are not fully understood. We have used an in vitro oxygen–glucose deprivation and reperfusion (OGD/R) model to better understand how Ang-(1-7) promotes neuroprotection under ischemic insult. To do this, the apoptotic pathways that could be modulated by Ang-(1-7) preventing cell death were investigated. Hippocampus slices from Male Swiss mice were subjected to OGD/R and treated with Ang-(1-7), and caspases 8, 9, and 3 activities were measured by fluorimetric protease assay. Additionally, reactive oxygen species (ROS) toxicity was accessed by measuring nitric oxide release. Administration of Ang-(1-7) during ischemia protected the slices from OGD-induced cell death, while coadministration of Mas receptor antagonist A779 eliminated this protection. OGD promoted increased production of nitrite and caspases 9 and 3 activities in hippocampal CA1 region, which was prevented by Ang-(1-7) through activation of the Mas receptor. Ang-(1-7)-induced inhibition of caspases 9 and 3 activities, together with an unchanged caspase 8 activity, suggests that reduced cell death observed in slices submitted to OGD is due to diminished NO release and modulation of intrinsic apoptosis pathway.
K. L. Sowalsky, M. Terza, L. Almeida, N. McFarland, and C. J. Hass
Department of Applied Physiology and Kinesiology, Applied Neuromechanics Lab, University of Florida, Gainesville, FL, USA
Persons with Parkinson's disease (PD) walk with slow gait speed, smaller steps, reduced cadence, and altered stride to stride variability. Walking to the beat of a metronome or piece of music often increases cadence, gait speed, and stride length, yet there have been few direct comparisons of the efficacy of these techniques, and little is known about their effects on the underlying structure of walking variability. The aim of this project was to compare metronome versus music on a broad range of spatiotemporal parameters and the fractal scaling of gait. Fifteen PD (69 ± 6 years old, Hoehn and Yahr: 1-3) and 15 gender-matched older adult controls (70 ± 6 years old) volunteered. PD participants were tested in the on-medication state. The Ambulatory Parkinson's Disease Monitoring system recorded gait performance. Participants walked for 2 min and their self-selected cadence was determined. Thereafter, participants walked for 5 min at a time under three randomized auditory conditions: no cue (NC), regular-beat metronome (RM), and music (M), with the beat of RM and M set to their natural cadence. Detrended fluctuation analysis (DFA) was performed on stride time. Spatiotemporal gait variables and DFA were compared using repeated-measures analysis of variance (p < 0.05) for statistical significance. Stride length was greater during M compared to NC (p = 0.002) and RM (p = 0.001). Arm swing velocity was significantly greater during M compared to both NC (p=0.004) and RM (p = 0.001). Neither stride length nor arm swing velocity was statistically different between NC and RM. DFA observed during RM was significantly greater in PD compared to controls (p = 0.014). Within PD, DFA was not significantly different across conditions, though M was higher than RM (p = 0.058). In controls, RM significantly decreased DFA values compared to M (p < 0.001) and NC (p < 0.001). DFA during M was lower than NC but did not reach statistical significance (p = 0.076). In conclusion, walking to music, compared to a regular metronome set to the same beat frequency, improved stride length, arm swing velocity, and fractal scaling. Future research is warranted to identify musical selections optimal for improving gait performance.
B. Srinageshwar,*†‡ F. L. Shalabi,§ M. N. Fini,*†‡ A. N. Stewart,*†‡ S. Peruzzaro,*†‡ M. M. M. Andrews,*†‡ K. Johnson,¶ J. Kippe,*†# O. V. Lossia,*†§ E. D. Petersen,† A. Gharaibeh,*†‡ A. Antcliff,*†§ M. N. Florendo,§ A. M. Figacz,§ D. Swanson,¶ G. L. Dunbar,*†‡# A. Sharma,¶ and J. Rossignol*†§
*Field Neurosciences Institute Laboratory for Restorative Neurology, Central Michigan University, Mount Pleasant, MI, USA
†Program in Neuroscience, Central Michigan University, Mount Pleasant, MI, USA
‡Department of Psychology, Central Michigan University, Mount Pleasant, MI, USA
§College of Medicine, Central Michigan University, Mount Pleasant, MI, USA
¶Department of Chemistry and Biochemistry, Central Michigan University, Mount Pleasant, MI, USA
#Field Neurosciences Institute, Saginaw, MI, USA
Polyamidoamine (PAMAM) dendrimers are well-defined three-dimensional, highly branched, polymeric nanoparticles that are being widely used in biomedical applications. Previous studies have shown that the dendrimers are readily taken up by the cells; however, the conventional generation 4 amine surface dendrimers [G4-NH2 with diaminobutane (DAB) core] are highly toxic when used both in vitro and in vivo due to their positively charged surface. Therefore, to overcome the toxicity, we have modified the conventional G4-NH2 dendrimers into G4 dendrimers with DAB core, having 90% of the surface covered with hydroxyl groups (-OH) and 10% of the surface covered with amine group (~4 nm in size), thereby significantly reducing the net positive charge compared to a 100% G4-NH2 surface dendrimer. These dendrimers were labeled with FITC (fluorescein isothiocyanate) and Cy5.5 (cyanine) to track them in vitro and in vivo using an in vivo imaging system (IVIS). We explored the potential uptake of the modified dendrimers by stem cells such as mesenchymal stem cells (MSCs) and neural stem cells (NSCs) in vitro and by mature cells such as neurons and glial cells in vitro and in vivo (by injecting the dendrimers intracranially into C57BL/6J background strain mouse model). We also analyzed the possibility of labeling the MSCs with our modified dendrimer and analyzing the extent to which the MSCs can retain the dendrimers in vitro and in vivo, thereby replacing the conventional Hoechst stain that has been proven to be toxic to cells due to its ability to bind to the DNA. The MSCs were labeled with dendrimers, allowed to grow in culture, and passaged once the cells become confluent. Immunocytochemistry using various markers for MSCs such as stem cell antigen 1 (sca-1) expression was analyzed to confirm the MSC phenotype after dendrimer infection. We also exploited the anti-inflammatory properties of the dendrimers to study its effect on glioblastoma multiforme (GBM). Previous studies have shown that the dendrimers have anti-inflammatory properties; therefore, we studied the uptake and impact of our novel dendrimer on the mouse-derived glioblastoma multiforme cell line (Gl261), which is an aggressive grade IV astrocytoma shown to have an increased inflammatory response in vivo. Our research findings showed that (1) the novel dendrimers were able to “infect” the MSCs, NSCs, and the mature neurons and glial cells efficiently in vitro; (2) intracranial injections of the dendrimers into the striatum of C57BL/6J animal model showed that the neurons were able to uptake the dendrimers efficiently and retained them as evidenced by IVIS; (3) the MSCs were labeled with novel dendrimers without affecting the MSC phenotype and the cells were able to retain the dendrimer for up to four passages after uptake; and (4) the dendrimers were taken up by the Gl261 cells readily, which led to the death of tumor cells after dendrimer infection compared to untreated tumor cells and treated stem cells as previously described. Taking these results together, our novel dendrimers have various applications including its therapeutic effects on tumor cell line in vitro. Future directions involve (1) studying the extent of retaining the dendrimer-labeled MSCs in the brain and (2) studying the anti-inflammatory properties of dendrimers in the GBM mouse model by intracranial injection of dendrimers at the tumor site.
Support for this study was provided by the Neuroscience program, the College of Medicine, the Field Neurosciences Institute, and the John G. Kulhavi Professorship in Neuroscience at CMU.
K. Steece-Collier,*† T. J. Collier,*† J. A. Stancati,* C. J. Kemp,* B. F. Daley,* and C. E. Sortwell*†
*Department of Translational Science and Molecular Medicine, Michigan State University, Grand Rapids, MI, USA
†Hauenstein Neuroscience Center, Mercy Health Saint Mary's Hospital, Grand Rapids, MI, USA
Parkinson's disease (PD) is a relentlessly progressive neurodegenerative disorder impacting millions of people worldwide. There is currently no available treatment to slow disease progression, and disease-modifying therapies are urgently needed for improved management of PD. “Nuclear receptor related 1 protein” (Nurr1) is a transcription factor that has received a great deal of attention in the past several years for its essential role in the survival of substantia nigra (SN) dopamine (DA) neurons and its potential to slow the death of these neurons in PD. However, while Nurr1 agonist therapy directed at nigral neurons has received international enthusiasm as a promising new avenue of neuroprotective therapy, there is compelling evidence from our labs and others that suggests that dyskinogenic levodopa (LD) results in ectopic induction of Nurr1 within the striatum. Whether systemic administration of Nurr1 agonist drugs will induce striatal Nurr1 and exacerbate LD-induced dyskinesias (LIDs) in parkinsonian subjects treated with LD remains unanswered. To begin to understand possible off-target side effects we used the highly specific Nurr1 agonist amodiaquine (AQ) at the previously documented neuroprotective dose (20 mg/kg; Smith et al., PNAS 112:8756, 2015). In Experiment 1 (Exp 1) intact young adult male Sprague–Dawley (SD) rats received daily injections of AQ or the vehicle saline (Sal) for 28 days. In Exp 2, SD rats were lesioned with 6-hydroxydopamine (6-OHDA). Three weeks after lesion, parkinsonian rats received 1 day of AQ or Sal pretreatment (0900h and 1700h) prior to receiving a single acute injection of LD (12 mg/kg) ±AQ (20 mg/kg) on day of sacrifice. All rats were sacrificed 60-180 min after final AQ and brains were processed for Nurr1 RNAscope® in situ hybridization. In Exp 1, densitometric analyses revealed robust and widespread upregulation of Nurr1 transcript in brain regions including the dentate gyrus (3.5-fold, p = 0.014), ventral tegmental area (2-fold, p = 0.021), and anterior cingulate (4.5-fold, p = 0.046; AQ vs. Sal, n= 4/group). However, in the absence of DA depletion or LD, AQ had no effect on striatal Nurr1. In Exp 2, when rats were rendered parkinsonian, acute pretreatment with AQ and a single dose of LD resulted in a 22-fold induction of striatal Nurr1 compared to intact striatum; LD + Sal resulted in 9-fold induction (ANOVA, p = 0.0034, intact vs. LD + Sal vs. LD+AQ; LD + Sal vs. LD+AQ, p < 0.05; n = 4/group). We are currently analyzing the impact of chronic AQ treatment on LIDs in response to low (3 mg/kg), moderate (6 mg/kg), and high (12 mg/kg) doses of LD. Given that the highly specific Nurr1 agonist AQ results in robust elevation of Nurr1 in the striatum where it has been critically linked to the induction of LIDs, as well as brain regions associated with addiction and learning and memory, systemic treatment with such compounds has the potential for significant off-target side effects in PD patients.
Supported by M J Fox Foundation and MSU DFI awards.
D. Stephenson
Critical Path for Parkinson's, Critical Path Institute, Tucson, AZ, USA
The lack of success in the development of disease-modifying therapeutic candidates for neurodegenerative diseases have led to the recommendation of public–private partnerships to tackle the challenges and share costs and risks among diverse stakeholders. The Critical Path Institute (C-Path) is a nonprofit organization that is dedicated to accelerating drug development by delivering on the mission outlined by the US Food and Drug Administration's (FDA's) Critical Path Initiative. Fundamental to the mission of C-Path consortia is the sharing of patient-level data from longitudinal natural history studies and clinical trials, and transformation of those data into generalizable and applicable knowledge to advance therapies for specific diseases. C-Path consortia are composed of industry members, regulatory agencies, academic experts, government agencies, and patient advocacy organizations that collaborate to achieve regulatory milestones not achievable by any one organization. Diseases to date that have achieved positive regulatory decisions from both European Medicines Agency (EMA) and FDA enabled by C-Path's consortia include Alzheimer's disease, polycystic kidney disease, and tuberculosis. Data standardization, database development, and integration are core to the success of all C-Path consortia. The Critical Path for Parkinson's consortium (CPP) was launched in 2015 with the goal of achieving regulatory endorsement for biomarkers and disease progression modeling tools to streamline the efficiency of PD clinical trials. Despite important advances in the understanding of the pathophysiology of Parkinson's over the last decade, significant challenges exist as to how to convert these discoveries into important new treatments. The design and implementation of efficient and effective clinical testing programs for experimental treatments is a critically important element of this undertaking and is closely linked to scientific, financial, clinical, and regulatory aspects of this challenge. To achieve this, a sound foundation in relevant patient-level clinical data is needed. A recent consensus article has identified that testing in early stages of the condition (earlier than 6 years from diagnosis) is expected to become increasingly important for potential disease course-modifying therapies in Parkinson's disease. However, patient-level clinical data on early disease stages are fragmentary, collected in a small number of drug trials, and in a number of disease cohort studies conducted by academic investigators. Combining patient-level data from these studies can create a robust foundation for modeling aspects of disease progression to aid in a data-driven design of clinical testing strategies. CPP is in the process of collecting and integrating patient-level data from worldwide observational cohorts and legacy clinical trials to develop a unified PD clinical database. A key regulatory milestone achieved by CPP to date is a letter of support publically posted by both EMA and FDA for the use of dopamine transporter neuroimaging at baseline as an enrichment biomarker for PD clinical trials. Regulatory science strategies enabled by active collaboration of stakeholders around the world promises to accelerate the approval of therapies for neurodegenerative diseases.
A. N. Stewart,*† U. Hochgeschwender,†‡ M. Lu,*†§ J. Rossignol,*†‡§ and G. L. Dunbar*†§¶
*Field Neurosciences Institute Laboratory for Restorative Neurology, Central Michigan University, Mount Pleasant, MI, USA
†Program in Neuroscience, Central Michigan University, Mount Pleasant, MI, USA
‡College of Medicine, Central Michigan University, Mount Pleasant, MI, USA
§Department of Psychology, Central Michigan University, Mount Pleasant, MI, USA
¶Field Neurosciences Institute, Saginaw, MI, USA
As a treatment approach, stem cell transplantation using either mesenchymal stem cells (MSCs) or neuronal stem cells (NSCs) has demonstrated an ability to improve outcomes in animal models of spinal cord injury (SCI). Additionally, the therapeutic effects of stem cell transplantation can be enhanced with the forced overexpression of trophic molecules using genetic engineering approaches. Stromal-derived factor-1 (SDF-1) is a small secreted chemokine, which exerts influence on the nervous system by encouraging NSC survival, migration, axonal growth, and differentiation. Providing SDF-1, in combination with transplanted stem cells, may potentiate a therapeutic effect derived from the transplantation of either MSCs or NSCs. This study aims to test a combinational therapy using MSCs, NSCs, and the forced overexpression of SDF-1 to improve outcomes in a rat contusion model of SCI. A retrovirus encoding for the expression of human SDF-1α was used to establish an MSC line, which stably overexpresses SDF-1 (SDF-1-MSC). Enhanced chemotactic function of SDF-1-MSCs on NSCs was examined in vitro using a Transwell migration assay and revealed an increase in migration of NSCs in response to SDF-1 secretion. These SDF-1-MSCs, along with MSCs alone, were used in a series of transplantation experiments as further described. First, SDF-1-MSCs or MSCs alone were transplanted into a rat contusion model of SCI at 9 days postinjury and evaluated for locomotor ability using the Basso, Beattie, and Bresnahan scale of locomotor recovery (BBB) for 7 weeks prior to euthanasia. No significant behavioral improvements were found when transplanting either MSCs alone or SDF-1-MSCs. However, immunohistochemistry from this study revealed significantly increased axonal growth into the lesion site in SDF-1-MSC-treated rats, as characterized by densities of growth-associated protein 43 (GAP-43) labeling, as well as decreased cavitation within the glial fibrillary acidic protein (GFAP)-labeled boundaries. Next, a cotransplantation using either unmodified MSCs or SDF-1-MSCs along with NSCs or just NSCs alone was performed in the same manner as previously described. Prior to transplantation, NSCs were infected with a lentivirus encoding for the fluorescent reporter gene, tandem-dimerized tomato (tdTomato), and the bioluminescent reporter gene firefly luciferase. NSCs were confirmed to express the cognate receptor to SDF-1, chemokine C-X-C motif receptor 4 (CXCR4), and were transplanted as neurospheres. BBB scores at 7 weeks postinjury revealed significant improvements in motor function for animals receiving cotransplantations of SDF-1-MSCs with NSCs, but no significant effect was found between any other treatment group. Transplant viability was confirmed with in vivo imaging at 1, 7, and 21 days posttransplant, as well as at 7 weeks postinjury by the presence of tdTomato or green fluorescent protein (GFP) expression in tissue sections. Results demonstrated that bioluminescent signal decreased beyond detectable levels by 3 weeks posttransplantation, and no tdTomato or GFP expression could be detected at 7 weeks. White matter sparing and neurofilament-70 kd (NF-70) densities were analyzed surrounding the lesion center after histological/immunohistological processing and revealed no significant sparing effect on white matter, but did reveal a significant axonal sparing effect, suggested by an increased NF-70 labeling surrounding the lesion, in both NSC alone- and SDF-1-MSC/NSC-treated rats. Finally, as some tumors were found following cotransplantations, an analysis of tumor composition was performed by evaluating for tdTomato, GFP, NF-70, or GFAP expression. Tumors stained positive for both neuronal lineage markers, NF-70 and GFAP, as well as for fluorescent expression of tdTomato and GFP. Results from the presented studies suggest that providing SDF-1 into the injured spinal cord through the forced over-expression from MSCs can facilitate endogenous axonal regeneration, as well as augment the therapeutic efficacy of transplanted NSCs. Treatment with either SDF-1-MSCs/NSCs or just NSCs alone significantly enhanced NF-70 densities both within the anterior horn as well as near the rubrospinal tract, suggesting a protective effect of axons around the lesion environment produced by the transplantation of stem cells. Tumors were found only in animals receiving cotransplantations, suggesting that an interaction between the two stem cell populations may potentiate uncontrolled proliferation of either NSCs or MSCs within the in vivo environment. Because of the poor viability of NSCs post-transplantation, it is likely that the therapeutic effects found in the SDF-1-MSC/NSC-treated animals are derived from the combined trophic secretion, as opposed to the differentiation and integration of NSCs into neuronal lineage cells. Taken together, these studies suggest that SDF-1 can be used to enhance therapeutic efficacy of stem cell treatments when transplanted into an injured spinal cord.
C. Stonesifer, S. A. Acosta, V. A. Guedes, J.-Y. Lee, 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
Stroke is a leading cause of death and disability in the US. Though it has a significant impact on public health, available therapeutic options are very limited. Peanuts are considered a superfood, possessing substantial quantities of valuable nutrients including polyphenols, monosaturated fats, and flavonoids. Despite the fact that peanut butter is commonly administered to rodents to aid in postoperative recovery after the induction of experimental stroke, little is known as to the specific histopathological effects of a peanut butter diet on injured neuronal tissue. Here we investigated the neuroprotective potential of an active phenolic component of peanut butter, p-coumaric acid, in vitro using stroke-like pathological insults. Primary rat cortical neurons (E18) were treated with different concentrations of p-coumaric acid (0.1, 0.5, and 1 μM) and subsequently exposed to oxygen–glucose deprivation (OGD) or tumor necrosis factor-α (TNF-α) treatment. In the OGD condition, cortical neurons were exposed to OGD for 90 min, followed by 2 h of reperfusion (normoxic condition, regular cell culture medium). For the TNF-α condition, TNF-α (5 ng/ml) and/or p-coumaric acid was added to the cultured cortical neurons for 24 h. Cell viability was measured by using calcein AM (acetoxymethyl). Cortical neurons grown in regular cell culture conditions were used as control. A significant effect of the p-coumaric acid treatment was observed in OGD- and TNF-α-induced decreases in cell viability (p < 0.001 vs. control, one-way ANOVA). In OGD, p-coumaric acid treatment increased cell viability at the concentrations of 0.05 and 1 μM (p < 0.0001 and p < 0.05, respectively, ANOVA followed by Bonferroni test). Similarly, a significant increase in cell viability was observed when the p-coumaric acid at the concentrations of 0.01 and 1 μM was added to the cell culture medium in addition to TNF-α (p < 0.0001 and p < 0.001, respectively, ANOVA followed by Bonferroni test). Our results suggest that p-coumaric acid acts as a neuroprotective agent in in vitro models of stroke pathology. Studies about the mechanisms of action and therapeutic benefits of p-coumaric acid and peanut butter are underway.
C.V.B. is supported by NIH NINDS 1R01NS071956-01, NIH NINDS 1 R21 NS089851-01, DOD TATRC W81XWH-11-1-0634, James and Esther King Foundation for Biomedical Research Program 1KG01-33966, San Bio Inc., Celgene Cellular Therapeutics, KMPHC, and NeuralStem Inc.
M. G. Tansey, G. T. Kannarkat, D. A. Cook, L. N. Eidson, A. F. Cintron, K. P. MacPherson, C. Carr, W. M. Caudle, S. A. Factor, and J. Boss
Department of Physiology, Emory University, Atlanta, GA, USA
Gene–environment interactions determine an individual's lifelong risk for age-related neurodegeneration, including Parkinson's disease (PD). Defense against environmental exposures and invading pathogens requires immunocompetence, a process that declines as we age. Moreover, the aging process is accompanied by alterations in immune cell responses and a proinflammatory state. The research interests of our laboratory include studies aimed at gaining a deeper understanding of the physiological alterations in innate and adaptive immune system activation that contribute to neuroinflammation and risk for development of PD, with the long-term goal of informing development of mechanism-based immunomodulatory interventions to delay or prevent the development of PD. Important questions under investigation include the extent to which inflammatory markers in central or peripheral biofluids could serve as bona fide biomarkers to diagnose or monitor PD pathophysiology or responses to therapy, and whether deep immunophenotypic analysis of cells in central and peripheral compartments could reveal important clues about disease pathogenesis and progression. Recent findings from our group and others indicate that environmental exposures synergize with genetic variability in human leukocyte antigen (HLA) genes, which are involved in antigen presentation and activation of the adaptive immune system, and modify an individual's risk for late-onset PD. Specifically, pyrethroid insecticides have direct effects on immune cell profiles in vivo and in vitro, and this immune dysregulation may increase the risk for neurodegeneration. Moreover, while the field has primarily focused on studying disease mechanisms in neurons, PD-related genes such as leucine-rich repeat kinase 2 (LRRK2) are highly expressed in immune cells and may play important roles in neuroprotective or maladaptive responses that increase the risk for PD. Recent studies from our group reveal alterations in LRRK2 expression in immune cells from individuals with late-onset PD relative to that in immune cells from healthy age-matched controls. Ongoing immunophenotype analyses of individuals with sporadic and autosomal dominant forms of PD are likely to reveal opportunities for immunomodulatory intervention that could delay the onset or slow the progression of PD.
Funding from the Michael J. Fox Foundation for Parkinson's Research and the NIH/NINDS.
J. Y. Teves,*† V. Bhargava,‡ M. J. Corenblum,* A. J. Flores,§ A. Annadurai,* R. Justiniano,¶ G. T. Wondrak,¶ J. Sligh,# C. Curiel-Lewandrowski,# S. J. Sherman,* D. A. Schipper,** Z. Khalpey,** and L. Madhavan*††
*Department of Neurology, University of Arizona, Tucson, AZ, USA
†Graduate Interdisciplinary Program in Applied Biosciences, University of Arizona, Tucson, AZ, USA
‡Undergraduate Biology Research Program, University of Arizona, Tucson, AZ, USA
§Graduate Interdisciplinary Program in Physiological Sciences, University of Arizona, Tucson, AZ, USA
¶Department of Pharmacology and Toxicology, University of Arizona, Tucson, AZ, USA
#Department of Medicine, University of Arizona, Tucson, AZ, USA
**Department of Surgery, University of Arizona, Tucson, AZ, USA
††Evelyn F. McKnight Brain Institute, University of Arizona, Tucson, AZ, USA
Human dermal fibroblasts are easily accessible peripheral cells that have Parkinson's disease (PD)-relevant gene expression profiles and constitute a system that recapitulates the chronological and epigenetic aging history of patients afflicted with this disease. Here we compared primary fibroblasts obtained from individuals diagnosed with late-onset sporadic PD, and healthy age-matched controls, in terms of their growth dynamics, morphology, response to stress, as well as the mitochondrial and autophagic function. It was observed that fibroblasts from PD subjects were distinctly different in culture compared to control cells. PD fibroblasts appeared smaller, less ramified, tightly packed, and directionally aligned in streams, compared to control fibroblasts, which were larger, ramified, and more evenly spaced and distributed. Quantitative analysis determined that PD cells were indeed smaller, more round, and closely affiliated with a higher number of neighboring cells than control cells. It was also determined that PD fibroblasts proliferated more, reaching higher cell densities at faster rates, than control cells. When the fibroblast lines were subjected to stress via exposure to ultraviolet irradiation (specifically UVA), the PD cells exhibited higher levels of reactive oxygen species (ROS) and lower viability, compared to control fibroblasts. Furthermore both autophagy and mitochondrial function were dysregulated in the PD fibroblasts. Specifically, Western blotting and electron microscopic data showed a significantly increased formation of degradative autophagosomes and autolysosomes, both at baseline and after UVA stress, in cells from PD subjects. With regard to mitochondrial function, the respiratory control rate (RCR), proton leak, and coupling efficiency of PD cells was found to be impaired via a Seahorse mitostress test (Billerica, MA, USA). Overall, these studies support primary skin fibroblasts as a useful patient-relevant model to study PD molecular mechanisms and potentially develop peripheral biomarkers for the disease.
Robert G. Thorne
School of Pharmacy, University of Wisconsin-Madison, Madison, WI, USA
Successful targeted delivery of macromolecules to the brain has remained a major, largely unmet medical challenge for decades. My laboratory has focused on understanding some of the key physiologic determinants of central nervous system (CNS) delivery: (i) local transport processes governing distribution within the brain parenchyma, (ii) whole-brain distribution and clearance mechanisms following intrathecal infusion, and (iii) nasal pathways for brain targeting after intranasal administration. Our past work has explored the diffusion of a variety of macromolecules after their injection into the neocortex (Thorne et al. PNAS, 2006 and 2008; Wolak and Thorne. Mol Pharmaceutics, 2013); recent studies have focused on immunoglobulin G (IgG) antibody diffusion as well as IgG penetration into brain following intrathecal infusion, with the goal of using diffusion data to better understand complex CNS distribution patterns (Wolak et al. J. Control. Release, 2015). We have also shown that a small fraction of intranasally applied macromolecules may be noninvasively targeted to the CNS through pathways associated with the olfactory and trigeminal nerves (Lochhead and Thorne. Adv. Drug Deliv. Rev., 2012; Lochhead et al. J. Cereb. Blood Flow Metab., 2015). An emerging key mechanism underlying widespread brain delivery common to both the intrathecal and intranasal approaches is access to, and distribution within, the perivascular spaces of cerebral blood vessels. We aim to show how physiological mechanisms, specific binding interactions, and size/charge effects may combine to affect central delivery and distribution by employing a variety of differently labeled tracer macromolecules, including antibodies, antibody fragments, and oligonucleotides. Examples will be presented to reveal how diffusive transport, binding, and perivascular flow affect the central delivery and distribution of macromolecules as well as suggest new ways in which to enhance delivery.
K. A. Trychta,* X. Yan,* J. Anttila,† M. Airavaara,† K.-J. Wu,‡ Y. Wang,‡ M. J. Henderson,*§ and B. K. Harvey*
*Intramural Research Program, National Institute on Drug Abuse, Baltimore, MD, USA
†Institute of Biotechnology, University of Helsinki, Helsinki, Finland
‡Center for Neuropsychiatric Research, National Health Research Institutes, Miaoli, Taiwan
§Intramural Research Program, National Center for Advancing Translational Sciences, Rockville, MD, USA
Under normal conditions, the calcium concentration in the lumen of the endoplasmic reticulum (ER) is 1,000 to 10,000 times higher than in the cytoplasm. ER calcium is important for many cellular functions, such as protein folding, lipid metabolism, and signaling pathways. Disruption of ER calcium homeostasis is implicated in multiple neurological diseases, including stroke. We used secreted ER calcium-modulated proteins (SERCaMPs) to monitor ER calcium depletion in models of stroke. Using a previously described Gaussia luciferase (GLuc)-SERCaMP in vitro, we demonstrate here that oxygen-glucose deprivation increased ER calcium depletion and decreased cell viability. Additionally, we developed a series of GLuc-based SERCaMP reporters representing ~80 ER resident proteins and observed a widespread shift in the reporters following ischemia, which suggests changes to the ER proteome. This increase in SERCaMP response was attenuated by treatment with the ryanodine receptor (RyR) antagonist dantrolene, which also attenuated ER calcium depletion and increased cell viability. Rats injected with adeno-associated virus (AAV) vectors expressing GLuc-SERCaMP in the cortex showed evidence of ER calcium depletion following occlusion of the middle cerebral artery. Furthermore, dantrolene treatment reduced the infarct volume in rats. Collectively, our data support a model in which ischemic injury causes ER calcium depletion and the secretion of important ER resident proteins. These effects can be attenuated by stabilizing ER calcium and preventing loss of ER resident proteins.
S. C. Vermilyea,*† J. Lu,‡ M. E. Olsen,§ Y. Tao,‡ S. Guthrie,* E. M. Fekete,¶ M. K. Riedel,¶ K. Brunner,* C. Boettcher,* V. Bondarenko,* E. Brodsky,§ W. F. Block,§# A. Alexander,*‡§¶ S.-C. Zhang,†** and M. E. Emborg*†§
*Preclinical Parkinson's Research Program, Wisconsin National Primate Research Center, University of Wisconsin, Madison, WI, USA
†Neuroscience Training Program, University of Wisconsin, Madison, WI, USA
‡Waisman Center, University of Wisconsin, Madison, WI, USA
§Department of Medical Physics, University of Wisconsin, Madison, WI, USA
¶Department of Psychiatry, University of Wisconsin, Madison, WI, USA
#Department of Biomedical Engineering, University of Wisconsin, Madison, WI, USA
**Department of Neuroscience, University of Wisconsin, Madison, WI, USA
Induced pluripotent stem cell (iPSC)-derived neurons present an opportunity for cell replacement strategies for neurodegenerative disorders such as Parkinson's disease (PD). Improvement in cell graft targeting, distribution, and density can be key for disease modification. Our team has previously developed a magnetic resonance imaging (MRI)-compatible brain port system that we have demonstrated to be successful for intraoperative MRI (IMRI) delivery of viral vectors for gene therapy strategies. The aim of this study was to develop procedures suitable for real-time IMRI (RT-IMRI) targeting guidance to facilitate its application for intracerebral delivery of biologics, especially cells, as it decreases the targeting time. Dopamine neuroprogenitor neurospheres were used for these studies. We first performed a set of in vitro experiments to tailor the delivery hardware (e.g., cannula) and define a range of parameters to be applied (e.g., maximal time span allowable between cell loading in the system and intracerebral injection) to ensure cell survival. Evidence of cell death, damage to neurospheres, and obstruction of the cannula were not observed after the cells were loaded and remained in the system for up to 2 h; some cell settling occurred, observed as the expelling of cylindrical neurosphere aggregates that could be easily dissociated. To assess if the process affects cell identity, a subset of the cells were plated after passing through the system and allowed to differentiate for 2 weeks. Immunostaining against tyrosine hydroxylase and βIII-tubulin confirmed that the dopaminergic identity of the cells was unchanged. Next, we performed cell injections into a 0.6% agarose gel as a brain surrogate to analyze patterns of cell distribution and associated intraline pressure changes. We found that multiple distinct deposits could be easily delivered along the cannula tract and that the pressure reflected each bout of cell expulsion. Last, we evaluated the feasibility of applying the RT-IMRI system to deliver cells into the nonhuman primate brain by injecting neurospheres labeled with Hoechst into the putamen nucleus. Experimental procedures were approved by the IACUC of the University at Wisconsin-Madison. All efforts were made to minimize the number of animals used and to ameliorate any distress. Postmortem evaluation 2 weeks later showed abundant cell survival of Hoescht+ cells that expressed nestin. Our results demonstrate that the RT-IMRI delivery system provides valuable guidance, monitoring, and visualization during intracerebral iPSC-derived neuron delivery that is also compatible with cell survival.
Supported by R01NS076352, P51OD011106-53S2 (Wisconsin National Primate Research Center, University of Wisconsin-Madison), NINDS T32-Neuroscience Training Program (S.C.V.), Departments of Radiology and Medical Physics at UW-Madison, WARF Accelerator Program, and funding from the UW-Madison Vice Chancellor for Research and Graduate Education.
J.-Y. Wang,*† Y.-E. Liao,* J.-Y. Wang,* D. Tweedie,‡ and N. H. Greig‡
*Graduate Institute of Medical Sciences, College of Medicine, Taipei Medical University, Taipei, Taiwan
†Department of Physiology, College of Medicine, Taipei Medical University, Taipei, Taiwan
‡Drug Design and Development Section, Translational Gerontology Branch, Intramural Research Program, National Institute on Aging, National Institutes of Health, Baltimore, MD, USA
Traumatic brain injury (TBI) is a major cause of death and disability worldwide. TBI induces inflammation and autophagy in brain tissue, resulting in neurodegeneration. Pomalidomide (Pom), a US FDA-approved immunomodulatory agent used in the treatment of multiple myeloma and other cancers, has been shown to be a more potent inhibitor of tumor necrosis factor-α (TNF-α) with less side effects than thalidomide. Using an animal model of TBI by subjecting Sprague–Dawley rats to controlled cortical impact (CCI), we have previously found that posttreatment with Pom (0.5 but not 0.1 mg/kg, IV) administered at 5 h after CCI reduced contusion volume and improved functional deficits by reducing neuronal apoptosis after TBI. The aim of the present study was to investigate whether the beneficial effects of Pom are related to a reduction in neuroinflammation, oxidative stress, and autophagy after TBI. By Western blotting, we found significant increases in the protein levels of 1A/1B-light chain 3 (LC3-II/LC3-I) ratio, indicating increased autophagy. Pom attenuated TBI-induced autophagy. Protein levels of cyclooxygenase-2 (COX-2; an inflammation marker) and heme oxygenase-1 (HO-1; a cellular responder to oxidative stress) were also elevated in the cortical contusion region at 8 and 24 h after TBI. Pom also attenuated these TBI-induced protein expressions. We also found that Pom decreased 4-hydroxynonenal (4-HNE) and 3-nitrotyrosine (3-NT), which are cellular adduct products of oxidative stress. Pom also inhibited TBI-induced elevation on the level of spectrin breakdown products (SBDPs), a marker of cell death. Our data suggest that multiple mechanisms such as anti-neuroinflammation, antioxidation, and anti-autophagy may contribute to the beneficial effects of Pom on TBI.
Supported by MOST-104-2923-B-038-001-MY3, Taiwan, and RO1NS094152, NIH, USA.
B. L. Waszczak and A. E.-E. Aly
Department of Pharmaceutical Sciences, Northeastern University, Boston, MA, USA
The therapeutic potential of glial cell-line derived neurotrophic factor (GDNF) for treating Parkinson's disease (PD) has been limited by its inability to cross the blood–brain barrier (BBB). Intranasal administration offers a noninvasive approach for direct delivery to the brain of therapeutic macromolecules that do not otherwise cross the BBB. We have found that even a single 50-μg intranasal dose of GDNF given 1 h prior to a 6-hydroxydopmaine (6-OHDA) lesion protected dopamine neurons in this rat model of PD. Multiple 50-μg doses were even more effective. Nevertheless, the nasally administered protein may be too transient to arrest disease progression clinically without frequent redosing. This realization led us to attempt intranasal delivery of a GDNF gene vector as a means of providing a renewable source of the protein within the brain, thereby reducing the need for repeated dosing of a costly protein. We chose to investigate intranasal delivery of a nonviral vector, that is, 10-kDa polyethylene glycol-substituted lysine 30-mer compacted DNA nanoparticles (NPs), developed by Copernicus Therapeutics (Cleveland, OH, USA). These NPs unimolecularly compact a single molecule of expression plasmid, have a minimum size of 10 nm, and can be formulated in a rod-like shape. We have shown that intranasal administration of DNA NPs encoding hGDNF can transfect brain cells in vivo, induce transgene expression, and provide neuroprotection of substantia nigra (SN) dopamine neurons in the rat 6-OHDA model. We have also shown using double-label immunohistochemistry (IHC) that transgene expression occurs throughout the rat brain, primarily in cells located immediately abluminal to the vascular endothelium, most likely pericytes. A time course study revealed that transgene expression was highest at 1 week after nasal delivery and persisted at ~30% of maximal levels at both 3 and 6 months. The intranasal route results in widespread distribution of molecules via perivascular transport, so there is an inherent problem in targeting only specific brain regions. Focused ultrasound (FUS) with circulating microbubbles has been proposed as a means of enhancing localized delivery to target areas by transiently disrupting the BBB at the sonicated sites. We next tested whether FUS could increase delivery, improve tissue penetration, and enrich transgene expression at target sites after intranasal administration of GDNF DNA NPs. Two sites, one in the right forebrain and one in the right midbrain, were sonicated with circulating microbubbles just before and after intranasal administration of the NPs, respectively. One week later, significantly higher transgene expression was detected both by enzyme-linked immunoassay (ELISA) and IHC in the sonicated regions on the right side of the brain. In addition, FUS appeared to improve tissue penetration of the NPs in the target regions, as revealed by a higher percentage of NPs within 15 μm of a transfected cell. Collectively, these results support intranasal delivery of GDNF DNA NPs for the treatment of PD and other CNS disorders and demonstrate enhancement and relative targeting of that delivery by FUS.
M. Watanabe,* P. Bhattacharya,† W. Zhao,* A. Khan,‡ J. M. Hare,‡§ M. Perez-Pinzon,*¶ A. P. Raval,*¶ and D. R. Yavagal*‡¶
*Department of Neurology, Leonard M. Miller School of Medicine, University of Miami, Miami, FL, USA
†Department of Pharmacology and Toxicology, National Institute of Pharmaceutical Education and Research (NIPER), Ahmedabad, Gujarat, India
‡Interdisciplinary Stem Cell Institute, Leonard M. Miller School of Medicine, University of Miami, Miami, FL, USA
§Department of Medicine, Leonard M. Miller School of Medicine, University of Miami, Miami, FL, USA
¶Cerebral Vascular Disease Research Laboratories, Leonard M. Miller School of Medicine, University of Miami, Miami, FL, USA
Stroke remains a leading cause of disability worldwide and needs new therapy. Cell therapy is emerging as a promising treatment for stroke. Intra-arterial (IA) mesenchymal stem cell (MSC) delivery for the treatment of ischemic stroke has a high potential for clinical translation. However, studies using a rat model of stroke have demonstrated that IA MSC delivery can decrease middle cerebral artery flow, which may limit its clinical translation (Walczak et al., Stroke 2008). Recently, our study demonstrated that the intra-arterial delivery of mesenchymal stem cells (IA MSCs) at 24 h after a reversible middle cerebral artery occlusion (MCAo) reduces the infarct volume and improves neurological score in female rats at 1 month posttreatment (Yavagal et al., PLoS One 2014). However, an optimal time window for IA MSC therapy for stroke remains unknown. Additionally, the majority of ischemic strokes in women occur after onset of menopause; therefore, it is crucial that we test the efficacy of IA MSCs in reproductively senescent females consistent with Stroke Treatment Academic Industry Roundtable (STAIR) recommendations. Therefore, we aimed to identify an optimum time window of IA MSCs and validate the efficacy of IA MSCs in reproductively senescent female rats. Female Sprague–Dawley rats were exposed to MCAo for 90 min. Rats were treated with IA MSCs (5 × 105 cells) or phosphate-buffered saline (PBS) either at 1, 2, or 4 days after MCAo. MSC- or PBS-treated rats were sacrificed at 28-30 days for infarct volume measurement using histology. To test motor function, the rotarod test was performed. Rats were trained for 3 consecutive days for the rotarod test before undergoing the MCAo procedure. The mean duration (in seconds) on the device was recorded from three rotarod measurements 1 day before surgery. The rats were tested at 1, 7, 15, and 28-30 days after MCAo. In a subsequent experiment, retired breeder female (9-11 months) rats showing estrous acyclicity were exposed to MCAo and a day later treated with MSCs or PBS. The rats were tested for neurological and motor function as described for previous experiment, and rats were sacrificed at 28-30 days for histopathological analysis. We observed significant reduction in infarct volume and improved neurological score in rats treated at 1 and 2 days, suggesting that IA MSCs are efficacious at a 2-day time window. In case of reproductively senescent rats, we observed significantly lower mean infarct volume in the MSC-treated group (12 ± 3 mm3; n = 6) compared to the PBS-treated group (29 ± 7 mm3; mean ± SEM; n = 4, p < 0.05). Treatment at 1 day after MCAO with MSCs significantly improved functional recovery, as evidenced by improved rotarod test results and neurological scores at 7, 15, and 30 days (p < 0.05) compared with the PBS-treated group. Intra-arterial stem cell treatment reduces ischemic brain injury in reproductively senescent female rats. Validating the efficacy of IA MSC treatment using a reproductively senescent animal stroke model should have high potential for future clinical translation.
Z. Wei, M. McCrary, S. P. Yu, and L. Wei
Department of Anesthesiology, Emory University School of Medicine, Atlanta, GA, USA
Ischemic stroke is a common clinical disorder with few effective treatments. In animal models, transplantation of neural precursor cells has been shown to benefit brain repair and stimulate endogenous neurogenesis. However, whether transplanted cells can form neural networks through synaptic formation has rarely been verified. Utilizing neonatal/adult stroke models in the present study, we investigated the intracranial delivery of neural progenitor cells derived from mouse induced pluripotent stem cells (iPSCs). Before transplantation, iPSCs were genetically modified to express light-sensitive channelrhodopsin-2 (ChR2) or cation-conducting ChR (VChR1) channels that can be selectively activated by optogenetic stimulation using blue laser light or luciferin–luciferase. We hypothesized that selective activation of cells after transplantation may provide a synergistic effect to cell therapy for ischemic stroke. Barrel cortex ischemic stroke was induced in both postnatal day 7 (P7) rats and adult mice. Cell transplantation was performed at 7 days after stroke. Optogenetic stimulation (15 min/day; 15 Hz) was carried out from 14 days after stroke until sacrifice. Expression of myelin basic proteins after stroke and the number of glucose transporter 1-positive/bromodeoxyuridine-positive (Glut-1+/BrdU+) colabeled cells were further increased in optogenetics treatment groups. At 21 days after stroke, immunohistochemistry and Western blot analyses showed elevated synapsin-1 (SYN-1) levels in fresh samples and perfusion-fixed brain tissue sections. Whisker touching using the left paw was measured to confirm that there was synaptic formation between transplanted and host cells and that the whisker sensory pathway was repaired by optogenetic treatment. Furthermore, significantly better functional performances were observed in the optogenetics group by performing the whisker-touching tests, adhesive removal test, corner test, computer-based automated home cage behavioral analyses, and social behavior tests. In conclusion, optogenetic stimulation was effective to (1) promote neuronal maturation, (2) increase angiogenesis in peri-infarct regions, (3) increase synaptic and axonal growth, and (4) rebuild the thalamocorticothalamic circuits after ischemic stroke.
E. S. Wires,* M. J. Henderson,*† X. Yan,* S. Bäck,* K. A. Trychta,* M. H. Lutrey,* and B. K. Harvey*
*Intramural Research Program, National Institute on Drug Abuse, Baltimore, MD, USA
†Intramural Research Program, National Center for Advancing Translational Sciences, Rockville, MD, USA
The endoplasmic reticulum (ER) is essential to many cellular processes including protein processing, lipid metabolism, and calcium storage. Dysregulation of ER function can result in ER stress and has been associated with many neurological and neurodegenerative diseases such as Parkinson's disease, Alzheimer's disease, stroke, and amyotrophic lateral sclerosis. The ability to longitudinally monitor ER homeostasis in the same organism may provide a better understanding of the molecular and cellular adaptations to physiologic or pathologic states but has so far been challenging. We have developed two secreted luciferase-based reporters that can be used to monitor ER calcium homeostasis [Gaussia luciferase-secreted ER calcium-modulated protein (GLuc-SERCaMP)] and ER stress [unfolded protein response element-secreted nanoluciferase (UPRE-secNLuc)]. Both GLuc- and NLuc-based reporters have favorable properties for in vivo studies; however, they are not easily used in combination due to overlapping substrate activities. We report a method to measure the enzymatic activities of both reporters from a single sample and validated the approach using culture medium and rat blood samples to measure GLuc-SERCaMP and UPRE-secNLuc. Measuring GLuc and NLuc activities from the same sample allows for the robust and quantitative measurement of two cellular events or cell populations from a single biological sample. We describe the first in vivo measurement of the unfolded protein response, a response to ER stress, by sampling blood using an approach that allows concurrent interrogation of ER calcium homeostasis.
K. Xu,* J.-Y. Lee,* R. Lin,* S. Pianta,* S. A. Acosta,* V. A. Guedes,* Y. Kaneko,* F. Vale,† H. van Loveren,† 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 Neurosurgery and Brain Repair, University of South Florida Morsani College of Medicine, Tampa, FL, USA
The brain possesses a lymphatic system that allows drainage of substances from the brain to the periphery. Stem cell transplantation is a potential stroke therapy. To date, brain-to-periphery stem cell migration remains unexplored. Here we tested the hypothesis that intracerebrally transplanted human bone marrow-derived stem cells (hBMSCs) utilized the lymphatic system in migrating to the spleen in sequestering stroke inflammation. At 1 h after sham or mild stroke surgery, adult Sprague–Dawley rats were intracerebrally injected with hBMSCs (3 × 105/9 μl), and then euthanized at day 1, 3, or 7 for immunohistochemical assays. In sham and stroke brain and spleen, surviving hBMSCs were detected robustly at day 3 and there was a trend of better survival in animals with severe stroke. In sham and stroke animals, few hBMSCs were deposited inside the brain lymphatic vessels at all time points, whereas many hBMSCs were visualized inside the spleen lymphatic vessels at all time points especially at day 3 in animals with severe stroke (p < 0.05). Inflammatory signal, as evidenced by major histocompatibility complex class II (MHC-II; OX6) immunostaining, was significantly elevated in both the brain and the spleen across all time points but was most pronounced at day 3 in animals with severe stroke (p < 0.05). Experiments using hBMSCs cocultured with lymphatic endothelial cells or microglia and treated with tumor necrosis factor-α (TNF-α) revealed the key roles of the lymphatic system and inflammation in directing stem cell migration. Our findings report, for the first time, that stem cells can migrate from the brain to the periphery via the lymphatic vessels, which could be further amplified by stroke-induced peripheral inflammation.
C.V.B. is supported by NIH NINDS 1R01NS071956-01, NIH NINDS 1 R21 NS089851-01, DOD TATRC W81XWH-11-1-0634, James and Esther King Foundation for Biomedical Research Program 1KG01-33966, SanBio Inc., Celgene Cellular Therapeutics, KMPHC, and NeuralStem Inc.
F.-C. Yang,* S. M. Riordan,† J. L. Vivian,‡ S. M. Shapiro†, and J. A. Stanford*
*Department of Molecular and Integrative Physiology, University of Kansas Medical Center, Kansas City, KS, USA
†Division of Child Neurology, Department of Pediatrics, Children's Mercy Hospital and Clinics, Kansas City, MO, USA
‡Department of Pathology and Laboratory Medicine, University of Kansas Medical Center, Kansas City, KS, USA
Neurotoxicity caused by excessive neonatal hyperbilirubinemia targets specific brain nuclei, including globus pallidus (GP), inferior colliculus, and cerebellum, and can lead to kernicterus. The most debilitating symptom of kernicterus is dystonia associated with damage to the GP. Stem cell transplantation has been shown to be an effective treatment for motor deficits in basal ganglia-related diseases such as Parkinson's disease and Huntington's disease. Targeting affected brain regions with neuronal stem cells is a promising therapeutic approach for treating dystonia in kernicterus. It is unknown, however, how elevated bilirubin levels in the brain will affect neural progenitor cell survival and development. The goal of the study was to compare the survival and functional development of different subtypes of neural progenitor cells (NPCs) resembling either excitatory spinal cord interneurons (SCI-like) or inhibitory basal ganglia neurons (BGN-like) transplanted into the basal ganglia of jaundiced (jj) Gunn rats and their nonjaundiced (Nj) littermates. We injected NPCs unilaterally into 21-day-old jj and Nj rats. In the SCI group, 10,000 cells were injected into the striatum; in the BGN group, 20,000 cells were injected into the GP. Three weeks later, injected rats were tested for locomotor activity and then perfused with 4% paraformaldehyde. Brain sections (30 μm) encompassing the injection site were collected for immunohistochemical analyses (IHC). STEM121™ (human cytoplasmic specific marker), Ku-80 (human cell nucleus marker), STEM123™ (human astrocyte marker), glutamic acid decarboxylase 65 (GAD-6), choline acetyltransferase (ChAT), parvalbumin (PV), and proenkephalin (PENK) were used to identify cell survival and cell phenotypes. Our results showed that (1) both types of NPCs survived and formed abundant neurites in the basal ganglia of both jj and Nj rats. (2) SCI NPCs had higher survivability than BGN NPCs after transplantation in both jj and Nj groups. (3) Transplanted cell survival was greater in the jj brain compared to the Nj brain for both types of grafts. These results suggest that elevated bilirubin levels enhance the survivability of the grafted cells. This may be due to the antioxidant and immunosuppressant properties of bilirubin. These results support the feasibility of stem cell therapy in kernicterus.
X. Yuan,* J. T. Rosenberg,*† Y. Liu,* S. C. Grant,*† and T. Ma*
*Chemical and Biomedical Engineering, The Florida State University, Tallahassee, FL, USA
†The National High Magnetic Field Laboratory, The Florida State University, Tallahassee, FL, USA
Human mesenchymal stem cells (hMSCs) have been shown to enhance stroke lesion recovery through secretion of trophic factors that mediate inflammation and tissue repair. However, low cell survival and reduced secretory functions posttransplantation of culture-expanded hMSCs are the major barriers limiting the therapeutic efficacy of hMSCs in stroke treatment (Sart et al., Tissue Eng Part B Rev. 2014). In this study, we report the impact of in vitro preconditioning of hMSCs, especially via three-dimensional (3D) aggregation of hMSCs. The 3D aggregation culture enhanced the hMSC secretory profile, resistance to ischemic stress, in vivo survival at stroke lesion site, and stroke lesion recovery. For the aggregation process, hMSCs spontaneously formed 3D aggregates on ultralow adherent (ULA) culture plates. Aggregate-derived hMSCs were labeled with superparamagnetic iron oxide (SPIO) nanoparticles for in vivo magnetic resonance imaging (MRI) analysis, and then injected intra-arterially into a stroke rat model induced via a middle cerebral artery occlusion (MCAO). The influence of 3D aggregation culture on the secretion of anti-inflammatory, proangiogenic, and antiapoptotic cytokines was analyzed. To assess the influence of preconditioning on hMSC in vivo life span and stroke lesion recovery, serial MRI at 21.1 T was performed to acquire images of lesion progression and cell migration. The results show that aggregate-derived hMSCs are smaller in size with enhanced stromal cell-derived factor 1 α (SDF-1α)-induced migration and increased chemokine C-X-C motif receptor 4 (CXCR4) expression through a caspase-mediated mechanism (Tsai et al., Tissue Eng Part A. 2015; Liu et al., Stem Cells 2016). Aggregate-derived hMSCs also have the increased secretion profile of multiple growth and anti-inflammatory factors. The phosphoinositide 3-kinase (PI3K)/Akt signaling pathway was highly activated and contributes to the resistance of ischemia and high level of reactive oxygen species (ROS) in vitro, leading to higher posttransplanted cell survival rate at stroke lesion. The MRI of the ischemic stroke lesion showed increased 1H and 23Na signal as evidence of the influx of extracellular water and disruption of ionic homeostasis. Lesion volume analysis indicated increased recovery for the 3D cultured hMSC group with statistical significance for 23Na MRI, and after 1 week of initial MRI a decrease in SPIO contrast on T2*-weighted images was calculated for cell clearance. Neurological function recovery was evaluated by behavioral test. Together, the results demonstrated that 3D aggregation of hMSCs is an effective strategy that enhances their therapeutic performance in stroke lesion recovery compared to standard adherent culture.