Abstract
Astrocyte Regulation of Neuronal Synapses
Salk Institute for Biological Studies, La Jolla, CA, USA
Our work investigates how neuronal synapses are regulated throughout life: from the formation of synapses during development, to the remodeling of synapses in the adult in response to experience, to the loss of synapses in aging. We approach this by asking how nonneuronal glial cells, specifically astrocytes, regulate synapse number and synaptic function. This has led to identification of proteins secreted by developing astrocytes that are sufficient to induce immature synapses to form, and additional signals secreted by adult astrocytes that induce synapse maturation and limit synaptic plasticity. We have further identified altered protein secretion from astrocytes in genetic neurodevelopmental disorders, and determined which of these alterations is responsible for negatively impacting neuronal development. We are now asking if manipulation of synapse-regulating factors in astrocytes is sufficient to delay progression of synaptic dysfunction in aging and neurodegeneration.
IL-1ra and CCL5 as Targets for Treating SMA Astrocyte-Mediated Pathology
Department of Cell Biology, Neurobiology and Anatomy, Medical College of Wisconsin, Milwaukee, WI, USA
Spinal muscular atrophy (SMA) is a leading genetic cause of infant mortality and is caused by a mutation or deletion of the survival of motor neuron 1 (SMN1) gene, resulting in reduced expression of the ubiquitous SMN protein. Motor neurons (MNs) are particularly impacted by decreased SMN protein levels, and MN loss is the primary phenotypic outcome in SMA patients. SMN deficient MNs show intrinsic deficits in splicing and function, but these defects alone are not sufficient to induce overt MN loss. Our lab has found that astrocytes differentiated from SMA patient-derived induced pluripotent stem cells (iPSCs) secrete high levels of pro-inflammatory ligands into their media (ACM) compared with healthy control (HC) astrocytes and that SMA ACM is capable of inducing MN loss. We also found that SMA iPSC-derived microglia display increased reactive morphology and phagocytosis when exposed to SMA ACM compared with HC ACM. We identified CCL5 and IL-1ra as promising targets for reducing the SMA pro-inflammatory astrocytic phenotype and hypothesized that reducing CCL5 while increasing IL-1ra signaling (IL-1rainc/CCL5neut) would ameliorate astrocyte-driven glial activation and MN loss in SMA. We found an overall reduction in SMA astrocytic pro-inflammatory phenotype after IL-1rainc/CCL5neut treatment. SMA IL-1rainc/CCL5neut ACM reduced microglia priming and phagocytosis in both monocultures and co-cultures with SMA MNs. IL-1rainc/CCL5neut ACM treated SMA MNs also showed an improvement in calcium function in mono- and co-culture conditions compared with SMA ACM treated MNs. Together, these data support the idea that astrocyte-targeted IL-1rainc/CCL5neut treatments decrease astrocyte-mediated microglial activation and MN loss in SMA and may allow for an extended therapeutic window. This hypothesis is now being examined in vivo using a gene therapy approach to target IL-1ra overexpression and CCL5 knockdown specifically to astrocytes in the SMNΔ7 mouse model of SMA.
Preclinical Evaluation of iPSC-Derived Neural Progenitors Secreting GDNF for the Treatment of ALS and Retinitis Pigmentosa
Cedars-Sinai Board of Governors Regenerative Medicine Institute, Cedars-Sinai Medical Center, Los Angeles, CA, USA
Expanded populations of human fetal-derived neural progenitor cells (NPCs) transduced to produce glial-derived neurotrophic factor (GDNF) have now been shown to survive for over 3 years following injection into the human spinal cord and can provide neuroprotection in many animal models. However, low availability of starting material limits their widespread use. Human induced pluripotent stem cells (iPSCs) are an alternative, renewable cell source that can be differentiated into NPCs and transduced with GDNF (iNPC-GDNF). The goal of the current study was to characterize iNPC-GDNF cells and test their therapeutic potential and safety. Single-nuclei RNA-seq showed that fetal NPC-GDNF and iNPC-GDNF had both overlapping and unique clusters of cells, suggesting that the two products were not identical. When transplanted into the subretinal space of a rodent model of retinal degeneration, iNPC-GDNF released GDNF and preserved photoreceptors and visual function. Transplantation of iNPC-GDNF into the lumbar spinal cord of a rodent model of amyotrophic lateral sclerosis (ALS) preserved motor neurons and limb function. Finally, to evaluate long-term safety and tolerability, iNPC-GDNF were transplanted into the spinal cord of athymic nude rats for 9 months. Surviving grafts secreting GDNF were found in all animals with no signs of tumor formation or cell proliferation. iNPC-GDNF survive long-term, are safe, and provide neuroprotection in models of both retinal degeneration and ALS, indicating their potential for clinical trials as a cell and gene therapy-based treatment for various neurodegenerative diseases.
Clinical Translation of Allogenic Regenerative Cell Therapy for White Matter Stroke and Vascular Dementia
1Department of Neurology, Department of Cell and Developmental Biology, University of California, Los Angeles, Los Angeles, CA, USA
2Department of Molecular, Cell and Developmental Biology, University of California, Los Angeles, Los Angeles, CA, USA
3Neurosurgery, Stanford University, Palo Alto, CA, USA
Stroke is the leading cause of adult disability. It continues to increase, paralleling the aging population, regardless of the improved acute stroke care to battle the associated debilitation and death rate. White matter stroke (WMS) is due to the occurrence of small infarcts in deep penetrating blood vessels of the brain, affecting brain regions responsible for connectivity. The immediate and imminent consequences of WMS include death of glial cells, gait abnormalities, and challenges in executive functioning, presenting as vascular dementia (VaD). These permanent damages produce an urgent need to explore and treat this neurodegenerative disease. Here, we present a stem cell therapy designed as an allogenic off-the-shelf product to allow recovery of WMS in affected patients. Immunodeficient mice had a WMS induced via focal microinjection of a vasoconstrictor N5-(1-iminoethyl)-L-ornithine (L-NIO), leading to damage of axons, myelin, astrocytes, and oligodendrocytes. These cellular changes induce motor and cognitive deficits in mice, as seen in humans. Pluripotent stem cells from the fibroblast tissue of four distinct human donors were differentiated into glial enriched progenitor cells (hiPSC-GEPs) and transplanted independently into the stroke core 7 days post-WMS. We ascertained resultant behavioral improvement by tracking shifts in gait and measuring upper limb use tendencies, via grid-walking and cylinder behavioral tasks, respectively. Furthermore, regardless of which hiPSC-GEP donor line the cells were derived from, they all led to motor improvements after stroke. These results, alongside testing with multiple donors and multiple clones from the same donor, demonstrate that our cell differentiation process is very robust. The proposed mechanism for this functional improvement is transplant-induced remyelination and axonal regeneration from the hiPSC-GEPs, characterized as having a “pro-repair” astrocytic fate. Furthermore, we then investigated other factors that may influence the efficacy of the proposed stem cell therapy. We transplanted one of the hiPSC-GEP donor lines at a subacute and chronic time point, in high and low doses, and ipsilateral and contralateral locations in both aged and young mice. Once we ascertain the greatest behavioral recovery, we can assess optimal treatment conditions for inducing motor and cognitive recovery after WMS. The results obtained on this study evidence ideal conditions for transplantation location, dosage, and therapeutic window of our proposed stem cell-based therapy useful in future clinical applications of hiPSC-GEPs in neurodegenerative diseases such as WMS and VaD.
Straight to the Source: Microglial Homeostasis Relies on Microglia-Derived TGF-β1 Ligand That Is Highly Spatially Regulated
University of Cincinnati, Cincinnati, OH, USA
Microglia are vital for homeostatic maintenance and function of the brain and can respond to immune challenges or injury by secretion of cytokines and activating the neuroinflammatory response in astrocytes. Recently, transforming growth factor beta (TGF-β) signaling has been shown to be critical for the normal development as well as maintenance of microglial quiescence in adulthood. However, the source of central nervous system (CNS) TGF-β1 ligand production and the temporal and spatial regulation of TGF-β1 signaling in the adult brain is largely unknown. To investigate this, we generated a variety of constitutive or inducible transgenic mouse lines to selectively knockout TGF-β1 ligand in different cell types (including astrocytes, neurons, and microglia) in the adult CNS. Our data show that microglia produce the TGF-β1 ligand that maintains their quiescence in adulthood and loss of microglia-derived (MG) TGF-β1 ligand also leads to reactive astrocytes, accompanied by behavioral deficits in emotional and cognitive functions. Furthermore, we investigated the bioavailability of MG-TGF-β1 ligand spatially at single-cell resolution using a somatic mosaic knockout strategy to sparsely delete TGF-β1 gene in a small percentage of microglia in adult brain (using CX3Cr1, P2RY12 and TEME119CreER mouse lines). Results from these mosaic gene deletion studies suggest that TGF-β1 ligand bioavailability is precisely regulated in an autocrine manner but ligand loss can be compensated by nearby wild-type cells with progression of time. These results together reveal novel insights on the cellular sources of TGF-β1 ligand in the adult CNS and suggest a mechanism that allows precise spatial regulation of TGF-β1 signaling to maintain homeostasis of microglia and astrocytes and subsequent neuronal function. These findings have important implications in conditions with dysregulated neuroinflammatory responses such as aging or CNS diseases.
Combined Delivery of BDNF and VEGF From an Injectable Thermoresponsive Hydrogel Promotes Functional Recovery After a focal Ischemic Stroke in Mice
1Center for Bioengineering and Nanomedicine, Faculty of Dentistry, University of Otago, Dunedin, New Zealand
2Brain Health Research Centre and Brain Research New Zealand, Department of Anatomy, University of Otago, Dunedin, New Zealand
3Ferrier Research Institute, Victoria University of Wellington, Wellington, New Zealand
Stroke remains one of the leading causes of adult disability worldwide due to the brain’s limited ability to regenerate following injury. Unfortunately, to date, no regenerative therapies are available that can restore stroke-induced loss of functions. Here, we used a novel thermoresponsive hybrid hydrogel (F6) to deliver brain-derived neurotrophic factor (BDNF) and liposome-encapsulated vascular endothelial growth factor (VEGF) into the infarct cavity and evaluated the effect of combined treatment on post-stroke recovery following photothrombotic stroke to the motor cortex in mice. Mice were intracerebrally implanted with either F6, F6+BDNF, F6+VEGF, or F6+BDNF+VEGF 5 days following stroke. Behavioral functions were assessed for 8 weeks post-stroke by measuring limb coordination using the grid-walking task and motor asymmetry using the cylinder task. Reactive astrogliosis and microgliosis, neurogenesis, and vascularisation were assessed using immunohistochemistry 2 and 8 weeks after stroke. Our data showed that intracerebral administration of F6+BDNF and F6+VEGF either alone or in combination (F6+BDNF+VEGF) improved motor function on both the grid-walking and cylinder tasks, with the combination treatment improving motor function more rapidly and significantly than F6+BDNF or F6+VEGF alone. In addition, treatment with F6 hydrogel with or without neurotrophic factors decreased reactive astrogliosis (both 2 and 8 weeks post-stroke) and microgliosis (2 weeks post-stroke) in the peri-infarct tissue. Furthermore, administration of F6 hydrogel with neurotrophic factors induced subventricular zone neurogenesis (F6+BDNF, F6+VEGF, and F6+BDNF+VEGF) and formation of new blood vessels in the peri-infarct region (F6+VEGF and F6+BDNF+VEGF) at both 2 and 8 weeks following stroke. In conclusion, F6 hydrogel could be an ideal candidate to be used in concert with numerous other treatments as a dual targeting treatment. That is, the F6 hydrogel can be used to dampen reactive astrogliosis and microgliosis with the subsequent treatment being able to target plasticity-associated mechanisms.
Combining α7 Nicotinic Acetylcholine Receptor Allosteric Modulator and Environmental Enrichment Improves Sustained Attention, Cholinergic Neurotransmission, and Systemic Inflammation After Controlled Cortical Impact Injury
Department of Physical Medicine and Rehabilitation, Safar Center for Resuscitation Research, University of Pittsburgh, Pittsburgh, PA, USA
Traumatic brain injury (TBI) is a leading cause of disability and pharmacological strategies that enhance acetylcholine (ACh) transmission may ameliorate cognitive deficits. To mimic the clinic, combining a pharmacotherapy with noninvasive rehabilitation [ie, enriched environment (EE)] may be efficient for cognitive recovery. We predicted that chronic administration of NS-1738, a novel α7 nicotinic ACh receptor (α7-NAChR) positive allosteric modulator (PAM), will improve sustained attention post-TBI, alone and in combination with EE. Blocking α7-NAChRs with methyllycaconitine (MLA) will attenuate the beneficial effects of NS-1738, confirming its mechanism of action. Adult male rats were trained in the three-choice serial reaction time task (3-CSRT), reaching stable pre-injury baselines prior to right parietal controlled cortical impact (CCI) or sham injury. Rats were randomized to NS-1738 (3 mg/kg) or vehicle (1 ml/kg saline) starting post-injury day (PID) 1 and continued daily [subacute (7d); chronic (28d)]. The chronic paradigm co-investigated daily environmental enrichment (EE; 6 h/d), and subgroups were also subjected to daily α7-NAChRs blockade via MLA (3 mg/kg) injections. 3-CSRT retrials occurred on PID 14 to 24. Medial prefrontal cortex (mPFC) Western blots assessed cholinergic markers [acetylcholinesterase (AChE), choline acetyltransferase (ChAT), and α7-NAChR]. Microarray analysis examined serum inflammatory gene expression. Statistical analysis utilized analyses of variance with Newman-Keuls post hoc tests. TBI rats exhibited impaired sustained attention versus shams (P < 0.05), which was improved by chronic (P < 0.05) but not by subacute NS-1738 (P > 0.05). Moreover, NS-1738+EE rendered an additive effect on lowering omissions and improving inflammatory markers (P < 0.05) including TREM-1 (triggering receptor expressed on myeloid cells-1) and IL-1 RA (interleukin-1 receptor antagonist). TBI decreased mPFC ChAT and AChE (P < 0.05) with partial restoration by subacute NS-1738. TBI groups that received MLA demonstrated a reinstatement of performance deficits, as hypothesized. Our findings support benefits of α7-NAChR type-I PAM and/or EE treatment after experimental TBI on sustained attention, cholinergic neurotransmission, and systemic inflammation.
This study was supported by NIH NS110609 and Research Advisory Committee, Children’s Hospital of Pittsburgh (COB).
Toward the Use of Chemogenetics to Control Spasticity
Department of Neurosurgery, Emory University, Atlanta, GA, USA
Spasticity is a common outcome for patients with spinal cord injury, multiple sclerosis, stroke, and a variety of other disorders. This condition is characterized by muscle spasms, clonus, and hyper-excitability of the muscle stretch reflexes. The patient’s quality of life can be substantially affected because spasticity can cause additional injuries and disturb sleep and work. Chemogenetics may offer a potential treatment for spasticity when other approaches fail. This therapy combines an otherwise inert ligand with an engineered receptor that modulates neuronal activity. Viral vectors can be used to deliver the gene for the receptor to the affected levels of the spinal cord, providing spatial specificity. The ligand is given orally, and the dose can be adjusted to modulate the level of motor suppression. In the current study, a collaboration with CODA Biotherapeutics, we demonstrate that a chemogenetic channel, delivered by an AAV9 vector to the lumbar spinal cord, can modulate motor function in the hind limb of the rat. Initially, three doses of the vector were evaluated (109, 1010, and 1011 vg) to identify the maximally tolerated dose. That dose (1010 vg) was taken forward for further testing. To demonstrate that motor function could be inhibited specifically by administration of the ligand and to determine the dose-response curve, four doses of ligand (1, 10, 30, and 100 mg/kg) and one control dose of water were given by gavage. Two measures of motor function, the Basso-Beattie-Bresnahan locomotor score and a grip-strength test, were evaluated. At 1 and 10 mg/kg, no effect on motor function was detected. At 30 and 100 mg/kg, motor function was reduced in a dose-dependent fashion, demonstrating that the degree of inhibition can be modulated by adjusting ligand dose. These results suggest that chemogenetic therapy may be an effective approach to control spasticity.
Cellular Mechanisms Underlying Neuroprotective Effects of Environmental Enrichment in Aged Rats
Department of Neurology, University of Wisconsin–Madison, Madison, WI, USA
The concepts of cognitive reserve and resilience are becoming a focus in the field of aging, Alzheimer’s disease, and Alzheimer’s disease–related disorders. In humans, educational attainment, a form of environmental enrichment, provides resilience to age-related cognitive impairment in Alzheimer’s disease. While it is known that environmental enrichment preserves cognition in the senescent brain, the brain structures and molecular mechanisms participating are only beginning to emerge. To address these unresolved issues, we used a rodent model of environmental enrichment (EE) to explore the behavioral, molecular, and electrophysiological benefits of enrichment in aged animals. Twenty-month-old Fischer 344 rats were exposed to 1 month of EE, which involved housing six rats in 60 × 60-cm plastic cages equipped with objects that are changed twice a week to maintain novelty. Standard cage control (SC) cages housed two rats in standard housing cages 43 × 28 cm with no objects. We carried out several well-established behavioral tests, which showed that EE improves performance on the Morris water maze, novel object recognition, and contextual fear conditioning. The enhanced learning ability of aged rats exposed to EE correlated with increases in hippocampal long-term potentiation (LTP) dependent upon group I mGluR (mGluR1/5) activation by selective agonists. Specifically, while control rats rely on activation of mGlur1/5 to convert a subthreshold stimulus into LTP, rats exposed to EE produced LTP in the absence of mGluR1/5 agonists. We also demonstrated that this enhanced plasticity and sustained activity is dependent on p70S6K activation/phosphorylation (p-p70S6K) and occurs only in EE and not in controls. These findings are important in understanding the mechanisms by which enrichment promotes healthy cognition in the aging brain.
MultiStem® Cell Therapy for Neurological Indications
Athersys, Inc, Cleveland, OH, USA
MultiStem® (invimestrocel) is an allogeneic cellular therapy product based on multipotent adult progenitor cell technology. MultiStem is being developed for the treatment of medical conditions in areas of significant clinical need, including ischemic stroke and trauma. MultiStem cells are well characterized and distinguishable based on phenotype, size, transcriptome, secretome, miRNA profile, differentiation, and expansion capacity. The therapy represents a unique “off-the-shelf” stem cell product that can be manufactured in a scalable manner, may be stored for years in frozen form, and is administered without tissue matching or the need for immune suppression. Preclinical data suggest that MultiStem cells may provide benefit through distinct mechanisms, including reducing inflammatory damage, protecting at-risk tissue at the site of injury, and upregulating reparative aspects of the immune system. Extensive preclinical data supporting the use of MultiStem have been generated in acute neurological injury models, including traumatic brain injury, spinal cord injury, and stroke. These data along with clinical data from the Phase 2 MASTERS-1 trial and Phase 2/3 TREASURE trial support our ongoing MASTERS-2 Phase 3 pivotal clinical trial to examine the potential benefit of MultiStem in acute ischemic stroke.
NeuroD1 May Promote Neurogenesis
1Department of Biomedical Engineering, University of Minnesota, Minneapolis, MN, USA
2Department of Neurosurgery, University of Minnesota, Minneapolis, MN, USA
3Graduate Program in Neuroscience, University of Minnesota, Minneapolis, MN, USA
AAV9-mediated transduction of NeuroD1 into endogenous neurons has been used in the treatment of sciatic nerve injury. We tested this method in a mouse model of traumatic brain injury (TBI) to determine whether the treatment would lead to any behavioral, anatomical, or cellular benefits. The treatment was administered immediately after TBI was induced and the mice were then harvested at 1 and 2 weeks post TBI/treatment. The treatment consisted of an AAV9 injection with an hSYN promoter forcing expression of NeuroD1 and mRuby2 within endogenous neurons. NeuroD1 was meant to promote recovery, and mRuby2 was used to label the transduced cells. Treatment effect on behavioral differences was evaluated through rotarod and beam walk tasks. The anatomical differences measured were TBI lesion volume and ventricle volume via the use of ultrasound and microscopy. Finally, the tissue was further quantified by the number of DCX or BDNF positive cells throughout the brain. Histologically, we found a statistically significant increase in cells DCX in the treated group compared with control. We did not find a significant difference in our behavior or anatomic studies. These results suggest that our reprogramming method preferentially may be targeting neuroblasts with NeuroD1 rather than more mature neurons. However, this did not result in behavior or anatomic differences in our model.
Investigation of the Effect of Deanna Protocol on the Treatment of Alzheimer’s Disease in a Human iPSC-Derived Cortical Neuron Model
NanoScience Technology Center, University of Central Florida, Orlando, FL, USA
The Deanna Protocol (DP) is a holistic therapy that has been anecdotally reported to alleviate the symptoms of amyotrophic lateral sclerosis (ALS). The DP consists of a combination of a group of over-the-counter available supplements such as alpha-ketoglutarate and gamma-aminobutyric, intending to optimize metabolism, relieve cellular stress, and regulate neural activity, thus ultimately protect the function and viability of neurons. The ability of DP, or similar formulations to preserve motor neuron function, has been demonstrated in vitro and in vivo studies. As such, it would follow that the Deanna Protocol could represent a potential therapy for other neurodegenerative diseases such as Alzheimer’s disease (AD) in which neuroprotection could also be therapeutically valuable. This study sought to utilize human induced pluripotent stem cell (iPSC) technology in combination of organ-on-a-chip technology to evaluate the effect DP for alleviating AD pathological phenotype in an iPSC-derived cortical neuron model. Cortical neurons differentiated from iPSCs harboring AD mutations (APP and PSEN1) were treated with DP for 1 week or longer and analyzed for their functional and molecular properties with periodical sampling. The results were compared with vehicle controls and the iPSC-cortical neuron system derived from healthy subjects. The electrophysiological property of these neurons was analyzed by patch clamp, and cellular and molecular phenotypes were analyzed by immunocytochemistry, especially the differences in amyloid-β aggregation under control or DP treated conditions were analyzed. To investigate the effect of DP on mitigating the cognitive deficits in AD, these neurons were also plated on microelectrode arrays to determine their capability to reproduce long-term potentiation, a surrogate for learning and memory with and without the DP treatment. The results indicated positive effect of DP in ameliorating the AD phenotype, which suggests the therapeutic potential of DP for AD treatment.
The Human Hypothalamus at Single-Cell Resolution: A Blueprint for Neural Hypothalamic Reprogramming
Naomi Berrie Diabetes Center and Columbia Stem Cell Initiative, Columbia University Irving Medical Center, New York, NY, USA
The hypothalamus is central to the control of energy homeostasis such as the regulation of body weight. Our single-cell transcriptomic analysis of the human prenatal and adult hypothalamus revealed region-specific developmental trajectories underlying neuronal diversity. In aggregate, we identified more than 500 hypothalamic cell populations. Mapping of obesity genetic signatures to discrete human hypothalamic cell populations demonstrated the need to develop cellular reprogramming approaches for the generation of specific hypothalamic neurons involved in the regulation of body weight. Here, we report the creation of functionally mature neurons of the paraventricular nucleus of the hypothalamus (PVH) that express the melanocortin-4 receptor (MC4R). We are using overexpression of MC4R neuron cell identity-defining transcription factors—as identified by single-cell transcriptomics—to drive the differentiation of human stem cells into MC4R-expressing neurons of the PVH. Heterozygous loss-of-function mutations in MC4R are the most common cause of severe, early-onset obesity in humans. These cells will enable analysis of the cell-functional consequences of alleles of MC4R and other genes related to energy homeostasis that are expressed in the PVH.
Engineering Spinal Interneurons for Repair of the Injured Cervical Spinal Cord
1Department of Neurobiology & Anatomy, Drexel University, Philadelphia, PA, USA
2Marion Murray Spinal Cord Research Center, College of Medicine, Drexel University, Philadelphia, PA, USA
3Gladstone Institutes, San Francisco, CA, USA
Spinal cord injuries (SCIs) most commonly occur at cervical levels, compromising respiratory networks leading to life-threatening impairments in breathing. This is primarily due to extensive damage to the phrenic motor network (controlling the diaphragm) located in the cervical cord. Our ongoing experiments have identified excitatory (glutamatergic), pre-motor V2a spinal interneurons (SpINs) as key therapeutic targets for promoting plasticity and functional recovery. Building on a long history of transplanting neural precursor cells (NPCs) to promote spinal cord repair, the present work harnesses advances in stem cell engineering to enrich donor NPCs with either stem cell–derived V2a SpINs, or a mixed population of excitatory and modulatory (cholinergic) pre-motor V0 SpINs. The chemogenetic receptor hM4Di was incorporated into the engineered SpINs to allow for selective de-activation of these donor cells with clozapine during electromyography recordings to evaluate the contribution of donor cells to diaphragm activity. Adult, female Sprague-Dawley rats received lateralized, mid-cervical (C3/4) contusion injury (Infinite Horizon impactor, 200 kilodynes). One week post-injury, aggregates of NPCs enriched with stem cell–derived V2a or V0 SpINs were injected into the lesion cavity. One month later, all animals were re-anesthetised and received a laparotomy, for transneuronal tracing of the phrenic motor network with pseudorabies virus. Three days later, animals underwent terminal diaphragm electrophysiology, at which time clozapine was injected intraspinally into the transplant to silence donor SpINs. Current data revealed that clozapine-induced suppression of donor cell activity reduced diaphragm activity ipsilateral to injury, indicating functional integration between donor SpINs and the injured phrenic network. This ongoing research will offer the first in-depth assessment of how these donor SpIN populations directly contribute to phrenic recovery.
Transcriptional Resilience Mechanisms for Cognitive Reserve
Department of Neuroscience and the Genetics and Genomics Program, University of Florida, Gainesville, FL, USA
The trajectory of cognitive decline during aging and neurodegenerative disease varies across individuals due to several factors including sexual dimorphisms and the history of experience. These factors act through epigenetic regulation of transcription to maintain a youthful brain (ie, brain maintenance) or activate reserve/resilience mechanisms sometimes referred to as cognitive reserve, which preserves cognition in the face of brain aging or pathology. We employ RNA-seq data to understand the molecular basis of cognitive vulnerability and resilience. In a rodent model (declining episodic memory in aging male rats), changes in transcription that correlate with cognitive impairment are linked to a rise in inflammation and oxidative stress and a decrease in synaptic genes, consistent with failure to maintain a youthful brain. Statistical filtering based on resilient individuals (aged unimpaired animals) identified potential resilience genes, which counteract the effects of aging (eg, anti-inflammatory genes). Thus, despite increased neuroinflammation and oxidative stress, expression of resilience genes accounts for the better-than expected cognition. Using the lists of genes linked to brain maintenance and resilience, we have attempted to determine whether resilience genes play a role in cognitive differences associated with environmental factors that act as positive or negative modifiers of cognition. Previous infection due to lipopolysaccharide treatment is a negative modifier, linked to greater memory impairment later in life, and associated with an inability to engage resilience genes. Senolytic treatments to reduce systemic inflammation improved cognition and prevented transcriptional changes associated with brain aging, indicating preserved brain maintenance. The results indicate that focusing on resilient individuals can provide an understanding of the molecular basis for individual variability and identify potential resilience genes or mechanisms. Furthermore, expression of resilience genes is initiated by the stressors of aging, and resilience mechanisms are available to a subset of animals, likely due to previous history and epigenetic regulation.
Ablation of Mitochondrial RCC1L in Dopaminergic Neurons Yields a Parkinson’s Disease-Like Phenotype in Mice
1College of Letters & Science, University of Wisconsin–Madison, Madison, WI, USA
2Department of Cell and Regenerative Biology, University of Wisconsin–Madison, Madison, WI, USA
3Department of Neurology, University of Wisconsin–Madison, Madison, WI, USA
We have previously identified RCC1L as a protein of the inner mitochondrial membrane involved in mitochondrial fusion. In this study, we generated mice with selective knockout of Rcc1l in dopaminergic (DA) neurons (Rcc1lKO/KO /DATCre+) to test whether RCC1L is implicated in PD pathology. Experimental and control groups were examined at 2, 3 to 4, and 5 to 6 months of age. Animals were tested in an open field task to quantify exploratory drive, locomotion, and immobility. Following behavioral testing, animals underwent post-mortem analyses. These included tyrosine hydroxylase (TH) and NeuN immunohistochemistry, Nissl staining, confocal microscopy, and densitometry analysis of TH expression. Beginning at 3 months, both female and male Rcc1lKO/KO/DATCre+ mice show rigid muscles and resting tremor, kyphosis, and a growth deficit compared with heterozygous Rcc1lKO/+/DATCre+ or wild type Rcc1lfl/fl/DATCre- littermates. Rcc1lKO/KO/DATCre+ mice also have motor impairments at 3 months, progressing until 5 to 6 months of age, dying at 6 months of age due to severe motor deficits. The significant age-dependent reduction in locomotion and rearing in Rcc1lKO/KO/DATCre+ animals was not seen in either group of controls. The motor impairments paralleled progressive and significant reduction in TH immunoreactivity in the substantia nigra pars compacta (SNc), and loss of DA projections to striatum (ST). To characterize mitochondria structure in DA neurons, we crossed Rcc1lKO/+/DATCre+ mice with the mitochondrially targeted fluorescent reporter mice Thy1-mitoDendra TM57. Fragmented spherical mitochondria were apparent in the soma of SNc neurons in Rcc1l knockout mice as early as 3 to 4 months of age. Together, the results reveal the product of the Rcc1l gene is vital to the survivability of nigrostriatal DA neurons, and its loss in mice produces a phenotype that parallels PD in humans, including bradykinesia, postural defects, progressive movement abnormalities, and degeneration of the nigrostriatal track.
Temporal Changes in Connectivity Between Transplanted Neural Tissue and the Injured Spinal Cord
1Department of Neurobiology & Anatomy, Drexel University, Philadelphia, PA, USA
2Marion Murray Spinal Cord Research Center, Drexel University, Philadelphia, PA, USA
3Gladstone Institutes, San Francisco, CA, USA
Cervical spinal cord injury disrupts motor, sensory, and autonomic networks leading to life-threatening deficits. Among the most life-threatening is impaired breathing, resulting from compromised phrenic (diaphragm) motor networks and can lead to respiratory arrest. Spontaneous plasticity has been observed both experimentally and clinically, but the extent of associated recovery is limited. To address this, transplants of developing neural cells have been used to promote repair with the goal of further enhancing recovery. This strategy provides neuronal and glial building blocks necessary for novel pathway generation and restoration of input to denervated spinal networks. However, the temporal changes and long-term stability of donor-connectivity remain poorly defined. Using a “gold standard” in neural tissue transplantation, and a preclinical model of cervical spinal cord injury, the present work assessed the extent of synaptic integration between the transplanted neurons and the injured cervical spinal cord. Donor spinal cord tissue dissected from E13.5 GFP-Sprague-Dawley embryos, mechanically dissociated and transplanted into the injured C3/4 spinal cord 1 week following a moderate lateral contusion in adult female Sprague-Dawley rats. Either 1 or 12 months post-transplant, transneuronal retrograde tracing of the phrenic network was used to trace the synaptic integration between donor neurons and the injured phrenic network ipsilateral to injury. Preliminary results show that while donor cell survival was comparable between 1 and 12 months after delivery, there was a significant loss of donor-host connectivity to the phrenic system with time. These findings could suggest that transplanting cells alone is insufficient to retain lasting integration between transplanted neurons and injured spinal networks. Ongoing studies are exploring these changes in connectivity and assessing whether combining transplantation with other strategies can stimulate greater, consistent, and persistent donor-host connectivity.
Adropin Protects Delayed Cerebral Ischemia in Subarachnoid Hemorrhage Patients
Department of Neurosurgery, College of Medicine, University of Florida, Gainesville, FL, USA
Subarachnoid hemorrhage (SAH) is a cerebrovascular emergency with high morbidity and mortality rate. Cerebral vasospasm and delayed cerebral ischemia is a major complication associated with patient morbidity and mortality following SAH with no effective treatment. Adropin is a novel peptide hormone that is secreted mostly from the brain and liver and has regulatory effects on endothelial cells. Adropin activate a G protein-coupled receptor; which is responsible for the posttranscriptional activation of endothelial NO synthase (eNOS). NO is produced in the endothelial cell by eNOS. Increasing NO in endothelial cell protects against vasospasm and delayed cerebral ischemia. We hypothesized that Adropin would have a treatment effect after SAH and can prevent vasospasm and improve neurobehavioral function. We designed 4 groups of mice using our SAH model: Adropin or vehicle treatment 6 hours after SAH induction and, Adropin or vehicle treatment 12 hours after SAH induction. SAH was induced by autologous blood injection into subarachnoid space, followed by treatment with either Adropin or vehicle. The corner test was used to measure their neurobehavioral function, unilateral abnormalities of sensory and motor functions, at baseline before SAH and day 3 and 7 post SAH. We will measure middle cerebral artery (MCA) diameter for vasospasm assessment, and neuronal apoptosis and microthrombosis will be observed by immunofluorescence staining. Our study showed on neurobehavioral testing, that SAH caused neurologic deficit (right turns in corner test) in the vehicle group, which was significantly reduced in the Adropin treatment group (normal: turns both ways). We also will measure MCA diameter and neuronal apoptosis and microthrombosis later. Ischemia post-SAH causes the neuronal apoptosis and focal neurological deficit. Adropin treatment seems to be effective in preventing delayed cerebral ischemia post SAH. These data show that Adropin might be effective in clinical setting and may improve SAH patient’s outcome.
Exploration of the Role of Schwann Cells in an ALS Pathogenesis in an iPSC-NMJ Model
1NanoScience Technology Center, University of Central Florida, Orlando, FL, USA
2Hesperos, Inc, Orlando, FL, USA
Around 14 million people globally are affected by neuromuscular diseases such as myasthenia gravis, muscular dystrophy, and amyotrophic lateral sclerosis (ALS). These disorders have a direct effect on the neuromuscular junction (NMJ) which is essential for the motor function. This makes the NMJ a key point of interest for pathogenesis study and therapeutic development. For this purpose, a functional in vitro NMJ model was developed in a dual-chamber system consisting of human induced pluripotent stem cell (iPSC)-derived skeletal muscle cells and motoneurons. Schwann cell is an important component of the tri-partite NMJ based on in vivo study but its role for the NMJ function under physiological and pathological conditions has scarcely been investigated. The goal of this project is to develop a tri-partite NMJ model to investigate the role of Schwann cells in NMJ function and integrity under physiological and ALS pathological conditions. To do this, iPSC-derived peri-synaptic Schwann cells were introduced into the already developed bi-partite NMJ system, and the NMJ function and morphology were analyzed and compared with those from the bi-partite NMJ system. NMJ function was analyzed by activating motoneurons with field electrode stimulation and recording induced muscle contractions by phase contrast microscopy, while NMJ structure was analyzed by immunocytochemistry. The role of Schwann cells in ALS pathogenesis was then evaluated in the tri-partite NMJ system by mix-matching the Schwann cells and bi-partite NMJs of different genotypes, wild type and ALS mutant specifically. This study revealed the importance of Schwann cells for NMJ under both physiological and pathological conditions as well as presented a tri-partite functional NMJ platform for the investigation of NMJ-related diseases and drug testing.
Activity of a Novel Anti-Inflammatory Agent F-3,6’-Dithiopomalidomide as a Treatment for Traumatic Brain Injury
S-C. Hsueh1, M. T. Screba1, D. Tweedie1, D. S. Kim2,3 W. R. Selman4,5, N. H. Greig1, and
1Drug Design & Development Section, Translational Gerontology Branch, Intramural Research Program, National Institute on Aging, National Institutes of Health, Baltimore, MD, USA
2AevisBio Inc, Gaithersburg, MD, USA
3Aevis Bio Inc, Daejeon, Republic of Korea
4Department of Neurological Surgery, Case Western Reserve University, Cleveland, OH, USA
5University Hospitals Cleveland Medical Center, Cleveland, OH, USA
Traumatic brain injury (TBI) has been identified as a major risk factor for several neurodegenerative disorders, including Parkinson’s disease (PD) and Alzheimer’s disease (AD). Neuroinflammation is considered the cause of later secondary cell death following TBI, has the potential to chronically aggravate the initial impact, and provides a therapeutic target albeit that has widely failed to translate into clinical trial success. Thalidomide-like compounds have well-documented neuroinflammation reduction properties across cellular and animal models of TBI and neurodegenerative disorders. They lower the generation of proinflammatory cytokines, particularly tumor necrosis factor α (TNF-α) that is pivotal in microglial cell activation. Unfortunately, thalidomide-like drugs possess adverse effects in humans before achieving anti-inflammatory drug levels. We developed F-3,6’-dithiopomalidomide (F-3,6’-DP) as a novel thalidomide-like compound to ameliorate inflammation that binds to the key protein cereblon, but does not trigger the ubiquitination of transcription factors (SALL4, Ikaros, and Aiolos) associated with the teratogenic, anti-proliferative, and anti-angiogenic responses induced by this drug class. We utilized a phenotypic drug discovery approach that employed multiple cellular and animal models. All protocols were fully approved by the IACUC of NIA. Only male animals were used to avoid estrogen neuroprotection. Sample size was based on our previous studies. F-3,6’-DP significantly mitigated lipopolysaccharide-induced inflammation and TNF-α levels in F344 8-week-old rats. We subsequently examined immunohistochemical, biochemical, and behavioral measures following controlled cortical impact (CCI) in C57Bl6 8-week-old mice, a well-characterized model of moderate TBI. F-3,6’-DP decreased CCI-induced neuroinflammation, neuronal loss, and behavioral deficits when administered after TBI, using commercially available reagents and blinded observers. In conclusion, F-3,6’-DP represents a novel class of thalidomide-like drugs with anti-inflammatory actions that possesses promising efficacy in the treatment of TBI and potentially long-term neurodegenerative disorders.
Human Neural Stem Cell–Derived Extracellular Vesicles Attenuate Cognitive Impairment and Neuroinflammation Induced by Cranial Irradiation and Chemotherapy
Department of Anatomy & Neurobiology, School of Medicine, University of California, Irvine, Irvine, CA, USA
Brain cancer survivors face debilitating cognitive dysfunction and neuroinflammation after the combined treatment of cranial radiation therapy (CRT) and chemotherapy. With the increasing survival rate of cancer patients, therapeutic interventions for these post-treatment side effects enabling patients to return to improve their quality of life (QOL) are now a priority. Previous work from our laboratory demonstrated that human neural stem cells (hNSCs) have a neuroprotective effect following CRT. Building on this work, we utilized extracellular vesicles (EVs) isolated from hNSCs. EVs are nano-scale and lipid-bound vesicles that contain bioactive cargo such as miRNA and can easily cross the blood-brain barrier. We identified miRNA-123-3p within the EV cargo as one of the contributing factors to ameliorating cognitive impairment and neuroinflammation. Our research aims to study the regenerative effects of hNSC-EVs on the immunocompetent wild-type mice after receiving clinically relevant CRT and chemotherapy regime, including CRT (8.67Gy, 3 doses) in combination with chemotherapy [temozolomide (TMZ), 25 mg/kg, 6 doses]. EVs were administered intravenously (retro-orbital vein injection) 2 days after the last TMZ treatment. The effect of EVs isolated from two different good manufacturing practice GMP-derived hNSC lines (Shef6.133.hNSC and UCI-191) on cognitive function, neuroinflammation, and synaptic integrity was tested. CRT-TMZ-exposed mice that received four doses of EVs compared with vehicle-treated mice showed substantial improvement in learning and memory, and memory consolidation tasks (object recognition and fear extinction memory). Immunohistochemical data of specific markers in the brain also showed a significant improvement in synaptic integrity, microglial activation, and astrogliosis in EVs-treated mice. Currently, we are testing the cognitive improvement and neuroprotective efficacy of hNSC-EVs in a clinically relevant brain cancer mouse model (astrocytoma) receiving CRT-TMZ. Future research on different GMP-grade hNSC lines may significantly increase the translational relevance of this regenerative therapy for radiation and chemotherapy-exposed brains that would allow to improve the QOL for thousands of cancer survivors.
Location, Location, Location: Utilizing Regional Specificity in Cell Transplantation Therapeutics
1Department of Neuroscience, University of Minnesota, Minneapolis, MN, USA
2Department of Neurosurgery, University of Minnesota, Minneapolis, MN, USA
3Stem Cell Institute, University of Minnesota, Minneapolis, MN, USA
Spinal cord injury (SCI) is associated with profound changes in sensory and motor performance that severely impact quality of life, often leaving patients paralyzed and with little chance of complete recovery. With the development and application of both cell transplantation and human induced pluripotent stem cells (hiPSCs), the potential for targeted neural regeneration and repair in sensorimotor systems after SCI has greatly improved. However, while preclinical models utilizing cell transplantation have yielded promising outcomes for recovery after injury, clinical translation has yet to be fully achieved. This can be attributed in part to a lack of host to graft integration limiting the capacity for survival, regeneration, and connectivity. Growing evidence has demonstrated that regional specification, whereby cell grafts are patterned to the phenotypic identity at the level of the injury prior to transplantation, greatly improves these outcomes. This has been shown extensively through patterning of ventral spinal and motor neurons, though additional spinal cell types, such as those involved in dorsal sensory circuitry, have yet to be explored. We report here the results of our work generating a rapid and reproducible differentiation protocol for production of region specific dorsal and ventral spinal neural progenitor cells from a shared population of hiPSCs. These cell types have been characterized using histology, reverse transcription quantitative real-time RT-qPCR, RNA sequencing, calcium imaging, and electrophysiology. Transplantation studies examining the effects of both cell types in isolation are underway, and in the future, we will transplant both dorsal and ventral populations in tandem to evaluate their capacity to attain correct repair and reconnection at the site of injury and beyond. These discoveries will push the field of spinal regeneration forward and advance the translation of cell transplantation therapies.
Targeted Circuit Manipulation for Ameliorating Huntington’s Disease Pathogenesis
Central Michigan University, Mount Pleasant, MI, USA
Almost 30 years after identifying the genetic mutation underlying Huntington’s disease (HD), treatments remain limited to managing late-stage symptoms of motoric, psychiatric, and cognitive deficits. Findings from patients and mouse models of HD point to pre-symptomatic imbalances in neuronal circuit activity, well before any overt symptoms are observed. Our central hypothesis is that manipulating the firing activity within selected microcircuits before the onset of symptoms by chemogenetic inhibition and/or excitation of key target populations will slow HD disease progression. A crucial early event in HD is the pathological increase in the overall excitatory output from cortex onto striatum. The enhanced excitability of cortical pyramidal neurons (CPNs) in pre-symptomatic HD is one key target for correctional intervention. The window before the onset of symptoms presents an opportunity to inhibit the firing rate of CPNs projecting to the striatum with the prospect of preventing or slowing disease progression. For manipulation of neuronal activity, we utilized bioluminescent optogenetics (BL-OG) that employs light-emitting luciferases to activate light-sensing opsins. We are testing the effects of circuit manipulation on preventing or delaying behavioral deficits in the R6/2 transgenic mouse model of HD. To selectively target CPNs projecting to the striatum, an AAV vector carrying a Cre-inducible inhibitory LMO (AAV-CamKIIa-DIO-NCS3-hGtACR1) was injected into the cortex of 3-week-old mice, while a retrogradely transported Cre-recombinase (AAVrg-hSyn-Cre-P2A-dTomato) was injected into the striatum. Two weeks later, luciferin or vehicle was administered once every other day for 2 weeks to decrease CPN firing. Rotarod, open field, and CatWalk were used to assess motor coordination, exploratory behavior, and gait function. We assessed cognitive behavior through water T-maze, novel object recognition test, and passive avoidance test. Our studies will contribute to understanding how microcircuit manipulation influences motor and cognitive behavior in HD and will drive translational progress toward novel therapeutic purposes.
Recombinant Human GABAergic Cells in the Therapy of Spinal Cord Injury–Induced Chronic Pain
Miami Project, Miller School of Medicine, University of Miami, Miami, FL, USA
Chronic pain following spinal cord injury (SCI) is a challenging clinical target with a need for the identification of new and potent therapeutic strategies. Our previous studies showed that spinal transplantation of recombinant rat GABAergic neuronal progenitor cells (NPCs) releasing an Food and Drug Administration–approved calcium channel blocker conotoxin MVIIA can attenuate injury-induced hypersensitivity in rats. To bring this approach closer to clinical application, human induced pluripotent stem cells (hiPSCs) were used to generate recombinant GABAergic cells. Using hiPSCs techniques, cells of various phenotypes can be autologously derived from the patient’s own cells readily obtained via blood draw or skin fibroblasts. In this study, we have engineered and evaluated analgesic effects of recombinant GABAergic hiPSCs releasing MVIIA in a model of SCI-induced chronic pain in rats. Recombinant hiPSCs derived from fibroblasts and bone marrow were differentiated into GABAergic phenotype using established protocols and transduced with AAV2/8 vector encoding analgesic conopeptide MVIIA. Sprague-Dawley rats (180–220 g) were used in SCI-induced chronic pain by spinal clip injury model. Hypersensitivity to tactile and thermal stimuli was evaluated weekly. Animals were grafted with GABAergic NPCs (historical control), GABAergic hiPSCs and GABAergic/MVIIA hiPSCs or vehicle at 4 weeks post SCI to target ongoing neuropathic pain. Results showed attenuation of hypersensitivity on all grafted animals with significantly better outcome in groups with recombinant grafts. Intrathecal injection of MVIIA antibody partially reversed the effects in animals with recombinant grafts. Reduced levels of proinflammatory cytokines interleukin-1 beta and tumor necrosis factor α were more significantly reduced in animals with recombinant grafts. Our data suggest that recombinant GABAergic hiPSCs are capable to reinstate a balance in pain signaling in the spinal cord and might be considered as a new strategy to manage chronic SCI neuropathic pain.
This study was supported by DoD Discovery Award PR182408 and The Florida Department of Health COPBC.
Attenuation of CB1 Activity by Novel Conopeptide in a Model of Chronic Pain in Rats
1Miami Project, Miller School of Medicine, University of Miami, Miami, FL, USA
2School of Biological Sciences, The University of Utah, Salt Lake City, UT, USA
Marine cone snails produce a wealth of selective peptides with analgesic activities. We have previously screened and identified possible Conus venom fractions possessing cannabinoid 1 (CB1) receptor activities. The current project aimed to obtain a purified fraction with CB1 activity and to identify its gene therapy potential. The goal is to develop a novel strategy for management of chronic pain by clinically acceptable cannabinoid peptides without detrimental psychoactive effects. Previously identified CB1 active high-performance liquid chromatography fractions from Conus Textile, CTex-185 and CTex-195 were used to engineer AAV2/8 particles encoding their sequences. Male Sprague-Dawley rats (220–250 g) underwent spinal cord clip compression injury (SCI). AAV2/8_CTex-185/CTex-195/ or control GFP were injected at 4 weeks post SCI using intraspinal, intrathecal, or intra-DRG routes. Changes in tactile, cold, and heat hypersensitivity were monitored weekly up to 10 weeks. CB1 antagonist AM251 was injected in some rats to evaluate CB mechanisms. Cerebrospinal fluid (CSF) levels of inflammatory cytokines were evaluated at the end of the experiment. Results showed analgesic effects of the CTex AAVs via all three injection routes. The CTex-195 produced more robust antinociceptive effects overall, which was partially reversed by AM251. Spinal CSF samples taken from treated animals retained CB1 internalization capacity, with CTex-195 significantly more potent than CTex-185, and this activity was trypsin sensitive, supporting CB1 peptidergic activity. The level of tumor necrosis factor α in CSF and spinal cord homogenates was reduced in animals treated with CTex-195 in contrast to control SCI animals. Interleukin-1 beta was reduced in both groups of CB1 conopeptide treated animals. These finding indicate that the identified C Tex fractions have the capacity to be used in gene therapy to manage chronic SCI pain. This study provides a first step in the identification of novel cannabinoid receptor-active substances suitable for gene therapy of chronic pain.
This study was supported by Department of Defense SC170295.
Removal of Cannabinoid Receptor 2 Reduces Alpha-Synuclein Aggregation
1Department of Neuroscience, University of Florida, Gainesville, FL, USA
2Faculty of Experimental Sciences, Universidad Francisco de Vitoria, Madrid, Spain
3Department Pharmacology and Toxicology, Medical College of Wisconsin, Milwaukee WI, USA
Parkinson’s disease (PD) is characterized by alpha-synuclein protein aggregates that contribute to neuroinflammation. Cannabinoid receptor 2 (CB2) is upregulated in the brains of PD patients and plays a role in modulating immune cell functions likely due to its expression on immune cells. Modifying CB2 pharmacologically has been shown to suppress inflammation, protect against neurotoxin models of PD, and induce the removal of Amyloid-B, a protein that aggregates in Alzheimer’s disease. Yet, it is still not clear the role of immune cell CB2 on alpha-synuclein aggregation. Previously in our lab, we demonstrate that a CB2 inverse agonist, SMM-189, reduced phosphorylated species of alpha-synuclein in a viral mediated alpha-synuclein model of PD. We extended these studies to evaluate the functional effects of CB2 deficiency in peripheral macrophages (pMacs) challenged with human alpha-synuclein pre-formed fibrils in vitro and in organotypic brain slices transduced with AAV-human-alpha-synuclein ex vivo, and evaluated both cultures for biochemical fractions of alpha-synuclein using Western blots. Preliminary results demonstrate reduced insoluble alpha-synuclein in CB2-deficient brain slice cultures (P = 0.0343) and a trend in CB2 deficient pMacs (P = 0.078). These experiments will outline whether the role of immune-CB2 is different in peripheral compared with central compartments. Future studies will investigate the autophagy-lysosomal pathway, as lysosomal dysfunction is associated with PD and the direct involvement of CB2 on immune lysosomal function is unknown.
Iron Chelator Mitigates Neurodegenerative Effects of Excess Iron in Subarachnoid Hemorrhage
Department of Neurosurgery, College of Medicine, University of Florida, Gainesville, FL, USA
Subarachnoid hemorrhage (SAH) is a debilitating event that results from trauma to the brain, such as an intracranial aneurysm. Initial injury of an SAH has a fatality rate of up to 50%, with the only medical treatment being surgical intervention and supportive care. Of the patients who survive, up to 40% will experience secondary injuries such as cerebral vasospasm, diminished cerebral blood flow, neuronal apoptosis, neurobehavioral deficits, or even death. The severity of the disease illustrates the need for better understanding of the pathways of SAH, and subsequently better treatments to prevent these potential outcomes. SAH results in excess blood degrading in the area, leading to a buildup of toxic iron by-products that causes irreversible neuronal damage by instigating an inflammatory response via ferroptosis. Microglia aggregate to the injured region to remove the iron but cannot reverse damage. Our hypothesis is that excess iron buildup becomes toxic and leads to ferroptosis of cells in the brain, resulting in neurodegenerative effects. Female C57BL/6 mice were used in an autologous blood injection SAH model and groups received either deferoxamine (DFO) treatment or vehicle. Vasospasm measurements suggested that DFO treated mice have less vasospasm than both sham and vehicle groups on day 7. Corner test data suggest that the DFO group had slightly improved neurobehavioral outcomes compared with vehicle SAH mice at both early and late time points. These results suggest iron is a major contributor to the neurodegenerative effects of SAH. This means that removing excess iron after SAH could be protective to the neurons. With further study, this could potentially lead to use of an iron chelator in the clinic to remove excess iron after SAH to prevent ferroptosis and neurodegeneration before irreversible damage occurs, thus limiting the mortality and long-term disability of the disease.
Novel, Thalidomide-Like, Non-Cereblon Binding Drug Tetrafluorobornylphthalimide Mitigates Inflammation and Brain Injury
D. Lecca1, S-C. Hsueh1, W. Luo1, D. Tweedie1, D. S. Kim2,3, A. M. Baig4, N. Vargesson5, B. J. Hoffer6,
1Drug Design & Development Section, Translational Gerontology Branch, Intramural Research Program, National Institute on Aging, National Institutes of Health, Baltimore, MD, USA
2Aevisbio Inc, Gaithersburg, MD, USA
3Aevis Bio Inc, Daejeon, Republic of Korea
4Department of Biological and Biomedical Sciences, The Aga Khan University, Karachi, Pakistan
5School of Medicine, Medical Sciences and Nutrition, Institute of Medical Sciences, University of Aberdeen, Aberdeen, UK
6Department of Neurological Surgery, School of Medicine, Case Western Reserve University, Cleveland, OH, USA
7Neuroscience Research Center, Taipei Medical University, Taipei
8Department of Neurosurgery, Taipei Medical University Hospital, Taipei
9Department of Surgery, School of Medicine, Taipei Medical University, Taipei
Quelling microglial-induced excessive neuroinflammation is a potential treatment strategy across neurodegenerative disorders, including traumatic brain injury (TBI), and can be achieved by thalidomide-like drugs albeit this approved drug class is compromised by potential teratogenicity. Tetrafluorobornylphthalimide (TFBP) and tetrafluoronorbornylphthalimide (TFNBP) were generated to retain the core phthalimide structure of the thalidomide immunomodulatory imide drug (IMiD) class. However, the classical glutarimide ring was replaced by a bridged ring structure. TFBP/TFNBP were hence designed to retain beneficial anti-inflammatory properties of IMiDs but, importantly, hinder cereblon binding that underlies the adverse action of thalidomide-like drugs. In this study, we evaluated the effect of TFBP/TFNBP in a controlled cortical impact (CCI) mouse model of TBI. Results showed that TFBP/TFNBP reduced markers of inflammation in mouse macrophage-like RAW264.7 cell cultures and in rodents challenged with lipopolysaccharide, lowering proinflammatory cytokines. Binding studies demonstrated minimal interaction with cereblon, with no resulting degradation of teratogenicity-associated transcription factor SALL4 or of teratogenicity in chicken embryo assays. To evaluate the biological relevance of its anti-inflammatory actions, two doses of TFBP were administered to mice at 1 and 24 hours post-injury following CCI TBI. Compared with vehicle treatment, TFBP reduced TBI lesion size together with TBI-induction of an activated microglial phenotype, as evaluated by immunohistochemistry 2 weeks post-injury. Behavioral evaluations at 1 and 2 weeks post-injury demonstrated TFBP provided more rapid recovery of TBI-induced motor coordination and balance impairments, versus vehicle treated mice. In conclusion, TFBP and TFNBP represent a new class of thalidomide-like IMiDs that lower proinflammatory cytokine generation but lack binding to cereblon, the main teratogenicity-associated mechanism. This aspect makes TFBP and TFNBP potentially safer than classic IMiDs for clinical use. TFBP provides a strategy to mitigate excessive neuroinflammation associated with moderate severity TBI to, thereby, improve behavioral outcome measures and warrants further investigation in neurodegenerative disorders involving a neuroinflammatory component.
Effects of Whole-Body Resistance Exercise in Young and Middle-Aged Rats
A. K. Lee1, F-C. Yang1, P. Morefield1, P. Kueck2, O. J. Veatch3, J. K. Morris2, and
1Department of Cell Biology and Physiology, The University of Kansas Medical Center, Kansas City, KS, USA
2Department of Neurology, The University of Kansas Medical Center, Kansas City, KS, USA
3Department of Psychiatry and Behavioral Sciences
Preclinical studies can reveal mechanisms underlying the effects of exercise. However, most animal studies have focused on aerobic exercise. Our goal was to compare effects of a whole-body, progressive overload resistance exercise protocol in young adult and middle-aged rats. We trained 10 male rats (3–5 months old) to climb a ladder with weights attached to their tails. The protocol then determined maximum weight loads (Mondays) and repetitions with increasing percentages of maximum weight (Wednesdays and Fridays) for 8 weeks. One year later, we repeated the protocol with four of the previously trained rats, leaving five as sedentary controls. Although the maximum load carried was similar for the rats when they were young vs older, the ratio of maximum load to body weight was greater when they were young (1.2–1.5) vs older (0.88–1.4). The rats reached the goal box quicker when they were older (~6s) vs younger (~8s). Lean mass increased with resistance exercise more when the rats were young (28%) than older (10%). Fat mass increased slightly in the young rats (12%) but decreased with exercise when they were older (39%). Blood levels of neurofilament light (NFL; neurodegeneration maker) and glial fibrillary acidic protein (GFAP; inflammation marker) increased 8-fold and 3-fold, respectively, from young adult to middle-age. Resistance exercise had no effect on NFL and GFAP in the older rats. Our results reveal age-related differences in the effects of resistance exercise on body composition in rats. Preliminary proteomic data from hippocampus and striatum tissue samples in the older exercised vs sedentary rats will also be presented.
Probing Multiple Transplantation Delivery Routes of CD+34 Stem Cells for Promoting Behavioral and Histological Benefits in Experimental Ischemic Stroke
1Center of Excellence for Aging & Brain Repair, Department of Neurosurgery and Brain Repair, University of South Florida Health, Tampa, FL, USA
2CellProthera, Mulhouse, France
Stroke is a leading cause of death in the United States and around the world but with limited treatment options. Survivors often present with long-term cognitive and neurological deficits. Stem cell–based therapy has emerged as a potential treatment for stroke. While stem cell transplantation in stroke has reached clinical trials, mostly safety outcomes have been reported with efficacy readouts warranting more studies. In an effort to optimize the stem cell regimen for stroke, here we conducted vis-à-vis comparison of different routes of transplantation, namely intracerebral, intraarterial, and intranasal delivery of expanded human CD34+ stem cells, called ProtheraCytes®, in the established stroke model of transient middle cerebral artery occlusion using adult Sprague-Dawley rats. After adjusting for the dose and subacute timing of cell delivery, animals were randomly assigned to receive either ProtheraCytes® or vehicle. Motor and neurological assays from day 7 to day 28 post-stroke revealed significant functional recovery across all three delivery routes of ProtheraCytes® compared with vehicle-treated stroke rats. In addition, ProtheraCytes®-transplanted stroke rats displayed significantly reduced infarct size and cell loss in the peri-infarct area compared with vehicle-treated stroke rats. These results highlight the safety and efficacy of transplanting ProtheraCytes®, including via the minimally invasive intranasal route, in conferring robust and stable behavioral and histological positive outcomes in experimental stroke.
Choroid Plexus–Targeted Gene Therapy to Treat Neurologic Disease
Department of Pathology, Boston Children’s Hospital, Boston, MA, USA
The choroid plexus (ChP) is an epithelial barrier located in each ventricle in the brain that produces cerebrospinal fluid (CSF) and secretes important health- and growth-promoting factors into the CSF. Here, we leveraged AAV technology to target ChP epithelial cells to treat neurologic conditions in mouse models. In our first example, we focused on treating the life-long neurologic effects termed chemotherapy-related cognitive impairment, reported in up to 75% cancer survivors. We discovered that the chemotherapy methotrexate (MTX) adversely affects oxidative metabolism of noncancerous ChP cells and the CSF. We used a ChP-targeted AAV approach in mice to augment CSF levels of the secreted antioxidant SOD3. AAV-SOD3 gene therapy increased oxidative defense capacity of the CSF and prevented MTX-induced damage to the hippocampus, including anxiety and short-term learning and memory deficits. In our second example, we focused on treating hydrocephalus, a life-threatening accumulation of CSF in the brain. We focused on post-hemorrhagic hydrocephalus (PHH), which can occur following intraventricular hemorrhage (IVH) particularly in premature infants. We found a role for the bi-directional Na-K-Cl cotransporter, NKCC1, in the ChP to mitigate PHH. We modeled IVH in mice, which led to increased CSF [K+] and NKCC1 activation. Because endogenous NKCC1 expression is low during this developmental stage, it was hypothesized to be rate limiting for removing K+ from the CSF. We therefore augmented ChP-NKCC1 expression by AAV technology, which lowered CSF-K+, prevented blood-induced ventriculomegaly, and was accompanied by persistently increased CSF clearance capacity. These data indicate that intraventricular blood triggered a trans-choroidal, NKCC1-dependent CSF clearance. Inactive, phosphodeficient AAV-NKCC1-NT51 failed to mitigate ventriculomegaly. Together, our findings support advancing ChP-targeted gene therapies as eventual treatments for select neurologic conditions.
Re-Engineered Chondroitinase ABC as a Potential Neuroregenerative Therapy for the Stroke-Injured Brain
1Department of Chemical Engineering & Applied Chemistry, University of Toronto, Toronto, ON, Canada
2Institute of Biomaterials and Biomedical Engineering, University of Toronto, Toronto, ON, Canada
3Department of Chemistry, University of Toronto, Toronto, ON, Canada
Following injuries to the central nervous system, such as stroke and spinal cord injuries, astrocytes become reactive and deposit extensive amounts of chondroitin sulfate proteoglycans (CSPGs). This astrocytic border surrounds the injury site and prevents injury expansion, yet is also considered to be inhibitory to cell migration and axonal regrowth at a later time point. The bacterial enzyme chondroitinase ABC (ChASE) is known to degrade CSPGs, thus potentially enabling tissue regeneration, yet its use is limited due to inherent thermal instability, characterized by a rapid loss of activity at physiological temperatures and pH. Moreover, continuous delivery is needed to obtain significant improvements after stroke. Therefore, we have used computational tools to redesign ChASE into a more thermostable version of the enzyme (termed “ChASE37”) by incorporating multiple point mutations within its sequence, and have shown its thermal superiority over the native enzyme. We differentiated human induced pluripotent stem cells into mature neurons and showed the ability of ChASE37 to rapidly degrade the inhibitory CSPGs in vitro and enable neuronal attachment and neurites outgrowth. Furthermore, we optimized a carboxymethylcellulose-based hydrogel to deliver ChASE37 using affinity-controlled release, based on the reversible interactions between a Src homology 3 (SH3) domain that we fused to ChASE37, and its binding peptide that we covalently crosslinked to the hydrogel. Finally, we delivered ChASE37 in the modified hydrogel to an endothelin-1 rat stroke model and showed that it penetrated deep into the tissue, retained its catalytic activity, and degraded CSPGs. Together, our data show that ChASE37 degrades CSPGs of the glial scar and thereby enhances cell adhesion and neurite outgrowth, laying the foundation for endogenous tissue regeneration. In future studies, we will investigate the co-delivery of sustained release ChASE37 with neural progenitor cells for tissue and functional repair.
CXCL1: A Novel Therapeutic Target for Aneurysm Healing
1Department of Neurosurgery, College of Medicine, University of Florida, Gainesville, FL, USA
2Department of Neurosurgery, Jacobs School of Medicine and Biomedical Sciences, Buffalo, NY, USA
Intracranial aneurysm formation is caused by an impairment of the arterial elastic lamina from endothelial dysfunction or injury and leads to an outward bulge that is prone to rupture. If left untreated, an aneurysm could rupture and lead to a lethal condition referred to as a subarachnoid hemorrhage. Currently, an endovascular technique used to prevent rupture is called coil embolization. However, this technique has a high recanalization rate for unruptured and ruptured aneurysms. Therefore, efforts should be focused on developing treatments that could be used concurrently with this endovascular technique to completely occlude the aneurysm. At inflammatory sites, neutrophils and macrophages are recruited by chemotactic factors. Macrophages polarize to a pro-inflammatory or anti-inflammatory state that enhances the inflammatory response or initiates the healing process. A pro-inflammatory chemokine called CXCL1 has been identified as an activator, chemoattractant, and a priming agonist of neutrophils at inflammatory sites. Once neutrophils aggregate to the site through CXCL1’s chemoattractant signaling, inflammatory responses become enhanced via the release of pro-inflammatory cytokines. Our studies focus on mechanisms involved in aneurysm healing and the inflammatory mediators responsible for promoting healing. Carotid aneurysms were created in female and male C57BL/6 mice. Using the murine carotid artery coil embolization model, a platinum coil was inserted and coated with neutralizing CXCL1 antibody or an IgG control. Immunohistochemistry data showed an increase in aneurysm healing in both male and female mice with locally blocked CXCL1 compared with IgG. At the 7-day, 14-day, and 21-day post coiling time points, a decrease of pro-inflammatory macrophages and an increase of anti-inflammatory macrophages were seen in the experimental group; neutrophils infiltrating the tissue also decreased. Inhibiting CXCL1 could be a therapeutic route to help improve aneurysm healing and reduce the rate of incomplete aneurysm occlusion, which in turn can be highly efficient in preventing aneurysm rupture.
A New Genetic Model for Cholinergic Functional Impairment in Septo-Hippocampal Interaction
Department of Neurology, Nagoya City University, Nagoya, Japan
The cholinergic efferent network from the medial septal nucleus to the hippocampus plays an important role in learning and memory processes. Cholinergic activation can enhance glutamatergic activity in the hippocampus under pathologic conditions, such as Alzheimer’s disease and Lewy body disease. To analyze the relationship between glutamatergic neural suppression and cholinergic neural dysfunction, we aimed to generate a new model for cholinergic functional impairment in septo-hippocampal interaction. Hippocampal cholinergic neurostimulating peptide (HCNP), which induces acetylcholine (Ach) synthesis in the medial septal nuclei of an explant culture system, was purified from the soluble fraction of postnatal rat hippocampus. Here, we confirmed direct reduction of Ach release in the hippocampus of freely moving HCNP-precursor protein (HCNP-pp) knockout (KO) mice under an arousal state by the microdialysis method. The levels of vesicular acetylcholine transporter (VAchT) were also decreased in the hippocampus of these mice in comparison with those in control mice, suggesting there was decreased incorporation of Ach into the synaptic vesicle. HCNP-pp KO electrophysiologically also presented cholinergic dysfunction, theta oscillation power, with glutamatergic dysfunction in the hippocampus with age. Interestingly, in those mice the impairment of cholinergic dysfunction with VAchT decrease in the pre-synapse may induce the reactive upregulation of the muscarinic M1 receptor in post-synapse, suggesting partly involvement of cholinergic impairment in glutamatergic dysfunction in the hippocampus of HCNP-pp KO mice. These results may support that HCNP-pp KO mice are an adequate genetic model for cholinergic functional impairment in septo-hippocampal interactions. Thus, according to cholinergic hypothesis, this mouse model might have a potential as a partial pathological animal model for Alzheimer’s disease.
SARS-CoV-2 Infection Increases the Gene Expression Profile for Alzheimer’s Disease Risk
1Department of Molecular Medicine, Morsani College of Medicine, University of South Florida, Tampa, FL, USA
2James A. Haley Veterans Hospital, Tampa, FL, USA
3Department of Internal Medicine, Morsani College of Medicine, University of South Florida, Tampa, FL, USA
4Byrd Alzheimer’s Center and Research Institute, University of South Florida, Tampa, FL, USA
5Center of Excellence for Aging & Brain Repair, Departments of Neurosurgery and Brain Repair, and Molecular Pharmacology and Physiology, Morsani College of Medicine, University of South Florida, Tampa, FL, USA
The COVID-19 pandemic is a global outbreak of coronavirus, an infectious disease caused by the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). One in five adults who have had COVID-19 in the past was still experiencing any one of the symptoms of long COVID like headache, brain fog, fatigue, and shortness of breath. Up to 30% of individuals with mild to severe infection show diverse neurological symptoms, including dementias. Hence, it is very much important to characterize the neurotropism and neurovirulence of the SARS-CoV-2 virus. This helps us understand the mechanisms involved in initiating inflammation in the brain, further leading to the development of early-onset Alzheimer’s disease and related dementias (ADRDs). In our brain gene expression analysis, we found that severe COVID-19 patients showed increased expression of innate immune response genes and genes that are implicated in AD pathogenesis. To study the infection-induced ADRDs, we used a mouse-adapted strain of the SARS-CoV-2 (MA10) virus to infect mice of different age groups (3, 6, and 20 Months). In this study, we found that aged mice showed evidence of viral neurotropism, prolonged viral infection, increased expression of tau aggregator FKBP51, interferon-inducible gene Ifi204, and complement genes like C4 and C5AR1. Brain histopathology also showed the AD signature including tau-phosphorylation, tau-oligomerization, and alpha-synuclein expression in aged MA10-infected mice. The results from gene expression profiling of SARS-CoV-2 infected and AD brains and studies with MA10 aged mice show that COVID-19 infection increases the risk of AD in the aged population. Furthermore, this study helps us to understand the crucial molecular markers that are regulated during COVID infection that could act as major players in developing ADRDs. Future studies will be involved in understanding the molecular mechanisms of ADRD in response to COVID infection and developing novel therapies targeting AD.
iPSC-Derived Mononuclear Phagocytes Have Regenerative Effects on Cognition and Neural Health in Mouse Models of Aging and Alzheimer’s Disease
1Cedars-Sinai Board of Governors Regenerative Medicine Institute, Cedars-Sinai Medical Center, Los Angeles, CA, USA
2Department of Veterinary Medicine, University of Maryland, College Park, MD, USA
Several studies have demonstrated that treatment with young blood or plasma has restorative effects on cognition and neural health in mouse models of aging and Alzheimer’s disease (AD), and our lab has previously shown that bone marrow transplants from young to aged mice have similar benefits. These approaches have practical disadvantages, as they may result in graft vs host disease, immune system rejection, or the transfer of communicable diseases from donor to recipient. Thus, the aim of the current work is to identify the cell type responsible for the beneficial effects of young blood and bone marrow, and to develop a cell-based therapy using human induced pluripotent stem cells (iPSCs). We generated iPSC-derived mononuclear phagocytes (iMPs) and administered them to aging, genetically immunocompromised NOD-scid-gamma (NSG) mice, as well as to the 5xFAD mouse model of AD. Treatment with iMPs significantly improved performance in tasks relying on spatial working memory and on hippocampus-dependent short-term memory in both aging and AD mice. Moreover, a number of neural health markers were improved by iMPs, including hippocampal expression of the synaptic transporter VGLUT1, which was reduced in aging vehicle-treated mice, but restored in aging mice treated with iMPs. iMPs also modulated neuroinflammation, as both astrocyte and microglia numbers were increased, while microglial branching was decreased, in aging and AD mice; changes that were reversed by iMP treatment. Single nucleus RNA sequencing of hippocampus and proteomic analysis of plasma revealed several genes and proteins that were significantly different between young and aging mice, but restored in iMP-treated mice, pointing to potential pathways that may mediate the regenerative effects of iMPs. These findings demonstrate the potential of using an autologous iPSC-based product as a new therapeutic strategy for aging and AD-associated declines in cognition and neural health.
An In Vitro Model to Assess the Consequences of Tau Aggregation in Neurons
1Department of Translational Neuroscience, College of Human Medicine, Michigan State University, Grand Rapids, MI, USA
2Neuroscience Program, Michigan State University, East Lansing, MI, USA
3Hauenstein Neuroscience Center, Mercy Health Saint Mary’s, Grand Rapids, MI, USA
Millions of Americans are living with Alzheimer’s disease (AD), a relentless age-related neurodegenerative disease that has no cure. As such, there is a critical need for disease-modifying therapies. One of the defining features of AD is the pathological accumulation of tau protein which spreads throughout the brain in a stereotypical pattern. Pathological tau species can be propagated from cell to cell in vitro and in vivo through a process called tau seeding. The functional consequences of these pathological species are not fully understood. To address this gap, we developed a tau seeding model in human tau knock-in (hTau-KI) mouse primary hippocampal neurons. Treatment of hTau-KI neurons at DIV5 with AD brain-derived pathological tau (AD-tau) resulted in robust and progressive formation of pathogenic tau species in the hTau-KI neurons from DIV9 to DIV31. The accumulation of pathogenic tau was dose and time dependent and occurred primarily in neuronal processes, with infrequent accumulation in the soma of neurons. Using immunological methods, we identified known disease-associated tau species in the AD-tau seed material that are propagated in the hTau-KI neurons. This included tau phosphorylated at the PHF1 and pS422 sites, as well as oligomeric tau and tau species with the phosphatase-activating domain exposed. To assess the effect of tau aggregation on cell viability, we utilized CellTiter-Glo and ApoTox-Glo assays, as well as performed neuron and astrocyte counts using immunocytofluorescence. No significant effect of tau seeding on cell viability was observed using these approaches. The lack of overt neurodegeneration in this model is consistent with the vast majority of tau seeding models that currently exist. We hypothesize that the effects of tau aggregation on neuronal function are more subtle (eg, deficient axonal transport and synaptic dysfunction). Identifying functional consequences of tau seeding will help guide the development of disease-modifying therapies for AD.
This project was supported by the NIA (R01 AG067762, F31 AG074521), NINDS (R01 NS082730), the Integrative Pharmacological Sciences Training Program (NIH 5T32GM092715), the Michigan Alzheimer’s Disease Center (P30AG053760 and P30AG072931), and Secchia Family Foundation Research Fund.
Cellular Reprogramming for Spinal Cord Repair
1Department of Neurobiology & Anatomy, College of Medicine, Drexel University, Philadelphia, PA, USA
2Spinal Cord Research Center, College of Medicine, Drexel University, Philadelphia, PA, USA
3Gladstone Institutes, San Francisco, CA, USA
Traumatic spinal cord injury (SCI) destroys nerve cells and tissue, resulting in permanent sensory and motor deficits. While there is evidence for spontaneous plasticity, it is limited, and life-threatening deficits persist. Injury at cervical levels is particularly devastating; it can compromise the phrenic motor network controlling diaphragm function. The present work takes a novel approach to promoting repair, first building on prior success transplanting pro-reparative glial precursor cells to promote tissue repair, then taking advantage of advances in cellular engineering and direct reprogramming to provide new populations of neurons to the injured spinal cord. The first phase of this research has been to establish methods for reprogramming transplantable glial cells into functionally viable neurons. Primary astrocyte cultures derived from neonatal rat cortex or postnatal spinal cord (postnatal days 2–3) were targeted with lentivirus promoting expression of neurogenic transcription factors Ascl1 or a combination of microRNAs (miRs) miR124, miR9/9*. Both brain and spinal cord astrocytes initially exhibited the typical star-shaped astrocytic morphology. Fifteen days following doxycycline administration, neurons from brain-derived glia took on a typical neuronal morphology, were positive for the neuronal markers NeuN (neuronal nuclear protein) and β3-tubulin (neuronal cytoskeletal marker) and negative for the astrocytic marker GFAP (glial fibrillary acidic protein), supporting successful conversion. Multielectrode array (MEA) recordings of these cells revealed spontaneous firing consistent with neuronal activity. In contrast, most of the spinal cord astrocytes continued to show astrocytic morphology even after 15 days of reprogramming, were positive for GFAP, and showed little to no neuronal activity during MEA recording. These results show that brain astrocytes display a higher efficiency for reprogramming to neurons (via either Ascl1 or miRs), compared with spinal cord astrocytes. Accordingly, there is a need to develop alternative strategies that may more efficiently promote conversation of spinal glia to spinal neuron populations.
Building a New Spinal Cord: Considerations for Chronic SCI Patients
1Department of Neurosurgery, University of Minnesota, Minneapolis, MN, USA
2Stem Cell Institute, University of Minnesota, Minneapolis, MN, USA
3Department of Neuroscience, University of Minnesota, Minneapolis, MN, USA
4Department of Mechanical Engineering, University of Minnesota, Minneapolis, MN, USA
Chronic spinal cord injury (SCI) devastates patients and families with few clinical options. Little research has been focused on this area as regeneration after SCI has met with little success over the past few decades of research. However, recently the possibility of regeneration via cellular transplantation, specifically by creating a relay mechanism through the site of injury, has become more realistic. Some key factors/questions contributing to this new potential therapy include the following. The use of autologous induced pluripotent stem cells to avoid immune rejection provides a mechanism to avoid the use of potentially toxic long-term immunosuppressants. This further brings to light our potential past mistakes in interpreting glial differentiation after cell transplantation after SCI as being driven by microenvironmental cues rather than basic immune rejection of neurons. The increased recognition of regional specificity with neural transplantation (ie, brain versus spinal neurons) highlights the fact that all stem cells are not the same, even within the “neural stem cell domain.” Furthermore, the surprising innate ability of regionally specified neural stem cells to form spinal organoids with three-dimensional bioprinting techniques provides further hope for patient-specific implants. Finally, the addition of electrical stimulation and/or focused physical therapy could also help reinforce appropriate neuronal connections to significantly impact the lives of patients with chronic SCI.
Propentofylline and IL-4 Alleviates Central Neuropathic Pain in Male Spinal Cord Injured Rats by Suppressing P38 MAP Kinase Activation
The Miami Project to Cure Paralysis, Department of Neurological Surgery, Miller School of Medicine, University of Miami, Miami, FL, USA
The development of central neuropathic pain (CNP) occurs in 40% to 50% of patients with spinal cord injury (SCI). Microglial cell activation plays a central role in neuroinflammation associated with CNP development and persistence after SCI. The current study builds upon prior work from the laboratory that has demonstrated the therapeutic effects of acute xanthine derivative propentofylline (PPF) administration, independently or with the anti-inflammatory cytokine interleukin-4 (IL-4), in promoting the conversion of activated microglia from a pro- to anti-inflammatory phenotype. This conversion was accompanied by a reduction in the development and severity of CNP across genders in rodent SCI that was more pronounced in males. Here the gender disparity in the therapeutic response to PPF+IL-4 was further explored by immunohistopathological assessment. For this work, the dorsal horn of the L4-L5 spinal cord from injured animals across the different treatment and control cohorts was probed to determine whether a correlation existed between specific proteins implicated in CNP and gender-dependent behavioral pain outcomes. It was identified that the phosphorylation of the P38 MAP kinase [pP38 MAPK (Thr180/Tyr182)], a known pain-potentiating kinase, was reduced in microglia of spinal cord–injured male rats following PPF+IL-4 treatment. A significant correlation was found between the treatment-induced reduction in microglial P38 activation and improved sensory outcomes. These findings identify PPF+IL-4 as a potential combined therapeutic approach to perturb SCI-induced CNP that acts putatively through reducing the phosphorylation of the pain-inducing MAPK kinase P38.
Age-Specific Imprinting Through Direct Reprogramming Reveals a Developmental Loss of Intrinsic Neurite Growth Ability in Human Neurons
1Department of Neuroscience, University of Wisconsin–Madison, Madison, WI, USA
2Department of Biostatistics and Medical Informatics and Department of Computer Science, University of Wisconsin–Madison, Madison, WI, USA
3Leonard Davis School of Gerontology, University of Southern California, Los Angeles, CA, USA
*Indicates equal contribution.
Spinal cord injury (SCI) leads to life-long disability, with limited treatment options. After injury, central nervous system (CNS) axons fail to regenerate due both to extrinsic and intrinsic factors. Rodent studies have revealed a developmental regulation of axon growth ability, such that embryonic CNS neurons extend long axons, whereas postnatal CNS neurons cannot. Yet, whether this is similar in human CNS neurons is unknown. Recently, our lab generated an in vitro, age-relevant, human model to identify novel intrinsic factors which regulate axon growth. This direct reprogramming protocol transdifferentiates human fibroblasts directly into neurons (Fib-iNs), skipping pluripotency which restores cells to an embryonic state. Using human fibroblast samples from 8 gestational weeks to 72 years old, we confirmed that Fib-iNs maintained the original cell’s age. Furthermore, we found that early fetal Fib-iNs grew longer neurites relative to late fetal and adult ages, mirroring the age-dependent decrease in regenerative ability during development in rodents. Interestingly, these neurons are environmentally naive, suggesting an intrinsic aging clock may drive changes in neurite growth ability. Using RNA sequencing on all ages of Fib-iNs, we identified dramatic transcriptional shifts between ages with high versus low intrinsic growth ability. We performed a small screen to identify regulators of neurite growth based on these differentially expressed genes, and identified ARID1A, a subunit of the BAF nucleosome remodeling complex, to be a key developmentally regulated gene that drives neurite outgrowth. These results suggest that age-specific imprinting at the chromatin level may be the driver for the intrinsic loss of neurite growth ability in human CNS neurons during development. Taken together, we characterized neurite outgrowth in human neurons, providing a new resource for future studies in axon growth and regeneration that can be used to identify novel therapeutic candidates for SCI.
Role of PTPRS in Axonal Sprouting of Midbrain Dopaminergic Neuron in Parkinson’s Disease
University of Cincinnati, Cincinnati, OH, USA
Parkinson’s disease (PD) is the second most prevalent neurodegenerative disease and is characterized by the loss of dopaminergic (DA) neuron bodies and fibers in the substantia nigra pars compacta and striatum, respectively, which cause various motor deficits in these patients. Current treatments only target the symptoms of PD. However, axonal repair has been proposed as a therapeutic strategy for PD treatment. Our recent paper shows that inhibiting the chondroitin sulfate proteoglycans (CSPGs)-receptor PTPsigma (PTPRS) signaling stimulates axonal regrowth of survival cortical neurons and improve the functional recovery after stroke. Analysis of published single-cell RNA-seq data on rodent and human DA neurons show that PTPRS can be used for all per author, they are the same protein is also abundantly expressed in DA neurons during development and adulthood. In this study, we investigate whether inhibition of this critical inhibitory signaling pathway is able to stimulate the axonal regrowth of remaining DA neuron fibers in the striatum and improve motor function in PD animals. To inhibit CSPGs-PTPRS signaling, we utilized a combined pharmacological (via the small peptide, Intracellular sigma peptide ISP, which mimics PTPRS’s dimerization state and inhibit the catalytic activity and downstream signaling) and dopaminergic neuron-specific PTPRS cKO genetic approach. Utilizing a human in vitro Embryonic stem cell ES-cell derived DA neuron PD model, we show that ISP treatment increases the length of the axons in human DA neurons with and without the addition of 6-OHDA and 1-methyl-4-phenylpyridinium MPP+. We are currently in the process of testing the efficacy of ISP peptide in a 6-hydroxydopamine 6-OHDA unilateral lesion in vivo PD model. Moreover, we have generated the DA-specific RPTPS cKO mice, which will be used to examine whether genetic deletion of RPTPS gene in DA neurons confers better regrowth of DA fibers after lesion.
CRISPR/Cas9 Mediated Gene Knockout in Adult Rat Astrocytes to Inhibit Notch, GSK-3 β, and BMP Cell Signaling Pathways
1Field Neurosciences Institute Laboratory for Restorative Neurology, Central Michigan University, Mount Pleasant, MI, USA
2Program in Neuroscience, Central Michigan University, Mount Pleasant, MI, USA
3College of Medicine, Central Michigan University, Mount Pleasant, MI, USA
4Department of Chemistry and Biochemistry, Central Michigan University, Mount Pleasant, MI, USA
5Department of Psychology, Central Michigan University, Mount Pleasant, MI, USA
6Field Neuroscience Institute, Saginaw, MI, USA
Ischemic stroke is caused by the cessation of blood flow in the brain leading to hypoxic damage to the brain tissue eventually killing the neurons and causing neuroinflammation. This activates astrocytes into reactive astrocytes for defensive functions. Previous studies have shown that inhibition of Notch, GSK-3 β, and BMP pathways can convert/reprogram reactive astrocytes into functional neurons. In this study, target genes for the above-mentioned pathways were identified through an extensive review of the literature: Hes5, NFkB1, and Blc2 for Notch; NLRP3 inflammasome for GSK-3β; and Smad1, Smad5, and Smad8/9 for BMP. We inhibited the pathways through gene knockout with CRISPR/Cas9 editing in cultured adult primary rat astrocytes. The goal was to reprogram astrocytes into neurons by knocking out the genes in the specified pathways which could be a potential treatment for ischemic stroke. We transfected CRISPR ribonucleoprotein specific to target genes in the identified pathways in astrocytes that were extracted from adult rat brains. We used HEK T293 to transfect with Trac CRISPR/Cas9 to measure editing efficiency of different method of transfection. HEK T293 underwent 3- and 6- days of forward and reverse transfection. Six days reverse transfection showed maximum transfection efficiency in HEKT292. This transfection timeline was used for further experiment. HEK T293 underwent 3 and 6 days of forward and reverse transfection. Sanger sequencing showed successful editing of Hes5, Nfkb1, Bcl2, Nlrp3, Smad1/5, and Smad9. In addition, Western blot analysis confirmed a significant reduction in the expression of Hes5, Nfkb1, Bcl2, Smad1, and Smad5 protein, confirming the inhibition of Notch and BMP pathways. Here, in this study, we confirmed the knockout of six genes in three pathways. This study represents the first step to confirm the feasibility of the reprogramming of astrocytes into neuroblasts.
Support for this study was provided by the American Heart Association (AIREA grant #957277), the College of Medicine, John G Kulhavi Professorship, Neuroscience Program, E. Malcolm Field and Gary Leo Dunbar Endowed Chair in Neuroscience at Central Michigan University, and partially by the Graduate Research Grant from the Office of Sponsored Programs at Central Michigan University.
Contributions of PSEN1 and APP Genetic Mutations Toward Alzheimer’s Disease Pathology
1Nanoscience Technology Center, University of Central Florida, Orlando, FL, USA
2Hesperos, Inc, Orlando, FL, USA
Alzheimer’s disease (AD) is a neurodegenerative disorder hypothesized to occur as a result of the accumulation or impaired clearance of amyloid-β (Aβ) and hyperphosphorylated Tau (p-Tau) in the brain, as well as aberrant inflammatory response by glial cells. Genetic mutations of the presenilin 1 (PSEN1) presenilin 2 (PSEN2) and amyloid precursor protein (APP) genes have been causally associated with early-onset AD (EOAD). Integration of human-on-a-chip technologies with cortical neurons differentiated from human induced pluripotent stem cells (hiPSC-CNs) allows for the study of functional deficits and pathological mechanisms caused by the aforementioned mutations. In this study, we focus on characterizing how the PSEN1 and APP mutations modulate molecular and electrophysiological functions of hiPSC-CNs. CRISPR-Cas9 technology has been used to introduce a single point mutation of the APP gene—creating an ideal system for AD modeling with an isogenic wild-type (WT) control. Action potential characteristics of these iPSC-CNs were observed and analyzed by whole-cell patch clamp electrophysiology. Long-term potentiation (LTP) capabilities of populations of neurons were measured using microelectrode array (MEA) technology. Biomarkers associated with AD pathology, including Aβ and p-Tau, were detected through immunocytochemical staining, allowing for the comparison of their accumulation and localization in cultures of WT and AD mutation carrying iPSC-CNs. A variety of pro- and anti-inflammatory cytokines secreted by WT and AD iPSC-CNs were detected through a membrane-based sandwich immunoassay and visualized via chemiluminescence. This provides the means for a greater understanding of the effects of AD mutations on the inflammatory characteristic of iPSC-CNs. AD modeling with hiPSC-CNs harboring AD mutations is a powerful technique that aids in uncovering the mechanisms of this neurodegenerative disorder and has the potential to be used as a platform to test and develop therapeutics for the treatment of AD.
Peripheral Neuron Transplantation Overexpressing NaChBac as a Therapeutic Intervention for Spinal Cord Injury
S. Hingorani Jai Prakash1, C. Sánchez Huertas1,2, E. M. Villalba Riquelme3, A. Ferrer Montiel3, and
1Neuronal and Tissue Regeneration Laboratory, Centro de Investigación Príncipe Felipe, Valencia, Spain
2Laboratory of Bilateral Neural Circuits, Instituto de Neurociencias (CSIC-UMH), Alicante, Spain
3Instituto de Investigación, Desarrollo e Innovación en Biotecnología Sanitaria de Elche (IDiBE), Universidad Miguel Hernández, Elche, Spain
Spinal cord injury (SCI) is a devastating, debilitating, and life-altering injury that hampers the life of a patient and is characterized by partial or complete loss of muscle function. Current consensus defends two major impediments limiting the neuronal regeneration, first, the inhibitory extrinsic signal generated by the hostile microenvironment after injury and the limited intrinsic capacity of adult central neurons to regenerate. Then, we propose a combinatory cell transplantation approach to overcome the above-mentioned limitations. Since peripheral nerve grafts transplantation creates a permissive substrate and has been reported to allow central axons to regenerate, we propose the use of dissociated dorsal root ganglia neurons as a cell transplantation approach. The genetic modification of the DRGs by overexpression of NaChBac (NC), a bacterial sodium channel, known to increase neuronal survival and integration in brain circuits, was intended to overcome the ephemeral life cell engraftment and increase the axonal growth. We found that the NC expressing neurons grafts survived and integrated significantly better in the injured tissue, inducing a significant improvement in the locomotor outcomes 2 months after severe SCI. The modified peripheral neurons expressing NC would be able to modulate the lesion environment, provide neuronal rewiring postulating as a strategy for SCI cell therapy.
SCF+G-CSF Treatment Enhances Remyelination in the Chronic Phase of Severe Traumatic Brain Injury
X. Qiu, S. Ping, M. Kyle, L. Chin, and
Department of Neurosurgery, State University of New York Upstate Medical University, Syracuse, NY, USA
Severe traumatic brain injury (TBI) causes long-term disability and death in young adults. White matter is vulnerable to TBI damage. Demyelination is a major pathological change of white matter injury after TBI. Demyelination which is characterized by myelin sheath disruption and oligodendrocyte cell death leads to long-term neurological function deficits. Stem cell factor (SCF) and granulocyte colony-stimulating factor (G-CSF) treatments have shown neuroprotective and neurorestorative effects in the subacute and chronic phases of experimental TBI. Our previous study has revealed that combined SCF and G-CSF treatment (SCF+G-CSF) enhances myelin repair in the chronic phase of TBI. However, the long-term effect and mechanism of SCF+G-CSF-enhanced myelin repair remain unclear. In this study, we uncovered persistent and progressive myelin loss in the chronic phase of severe TBI. SCF+G-CSF treatment in the chronic phase of severe TBI enhanced remyelination in the ipsilateral external capsule and striatum. The SCF+G-CSF-enhanced myelin repair is positively correlated with the proliferation of oligodendrocyte progenitor cells in the subventricular zone. These findings reveal the therapeutic potential of SCF+G-CSF in myelin repair in the chronic phase of severe TBI and shed light on the mechanism underlying SCF+G-CSF-enhanced remyelination in chronic TBI.
This study was supported by the National Institutes of Health/National Institute of Neurological Disorders and Stroke in the United States (R01NS118166).
Feasibility and Safety of Implanting Autologous Regenerative Peripheral Nerve Tissue Into the Basal Ganglia of Animal Models of DBS-Plus
1Department of Neurosurgery, College of Medicine, University of Kentucky, Lexington, KY, USA
2Department of Neuroscience, College of Medicine, University of Kentucky, Lexington, KY, USA
3Department of Neuroscience Medical University of South Carolina, Charleston, SC, USA
One strategy to aid sick or dying cells in Parkinson’s disease (PD) is to provide cell-survival factors. In clinical trials we have termed DBS-Plus, we combine the implantation of reparative, autologous peripheral nerve tissue, at the time of deep brain stimulation (DBS) surgery, to provide anti-inflammatory, anti-apoptotic, and other cell-survival factors. Our objective was to assess the feasibility and safety of implanting peripheral nerve tissue into the basal ganglia in nonhuman primates and canines to match the surgical approach of ongoing clinical trials. All animal procedures were first approved by the UK IACUC. In two macaques, we implanted autologous sural nerve grafts into the substantia nigra using magnetic resonance imaging–guided surgeries. For sural nerve preparation, 5 days before the nerve tissue graft implant, the sural nerve was exposed in the leg. The distal extremity was cut and sutured in place to stimulate production of pro-regenerative factors from the cells within the nerve. On the day of implantation, a 5-mm section of the sural nerve was harvested immediately before unilateral implantation into the midbrain and cut into 1 mm pieces then loaded into a guide tube for delivery. Meanwhile in four research naive, purpose-bred male beagles, a similar procedure was carried out except (1) acutely injured, rather than chronically degenerating, surae caudalis was used, and (2) the tissue was deployed to the caudate. Both groups of animals successfully received the peripheral nerve tissue delivery. Macaques and dogs were followed for 2 months without any severe or serious adverse events. Post-mortem histopathology did not identify any major tissue disruption or abnormal growth. We used p75-NTR- and S100β-immunoreactivity to identify the peripheral nerve graft and tyrosine hydroxylase staining in the caudate and substantia nigra regions to identify dopaminergic neurons. The deployment of peripheral nerve tissue to the basal ganglia was feasible with a safe profile.
A Novel Target Essential to Axonal Regeneration After Spinal Cord Injury
Department of Neurobiology & Anatomy, College of Medicine, Drexel University, Philadelphia, PA, USA
SPAST, also called SPG4, encodes spastin, a multi-functional protein. Vertebrate SPAST has two start codons that produce a longer isoform called M1-spastin and a slightly shorter isoform called M87-spastin (M85 in rodents). Lines of evidence demonstrated that M87-spastin is a potent microtubule-severing protein that cuts long microtubules into short pieces, while M1-spastin is associated with the morphogenesis and homeostasis of endoplasmic reticulum. Previous studies on Drosophila showed a dose-dependent detriment in the ability of amputated axons to regenerate when spastin is reduced. Here, we sought to determine whether those observations extend to vertebrate axons and whether such functional impairment is isoform specific. Indeed, when spastin is either functionally inhibited or depleted in cultured cortical neurons, the regenerative capacity of severed axons was significantly reduced accompanied with decreased neuronal activities. Such defects were rescued in the spastin-depleted neurons in dose-dependent fashion by re-expression of M87-spastin, but not M1-spastin. Interestingly, enhancing neuronal activities via a DREADD-based (designer receptors exclusively activated by designer drugs) chemogenetic tool rescued the impaired axonal regeneration in the spastin-deficient neurons. Thus, we conclude that axonal regeneration, including the capacity of injured neurons to re-establish connections, depends on sufficient levels of M87-spastin. Furthermore, our findings suggest controlled overexpression of spastin may serve as a novel therapeutic strategy for augmenting axonal regeneration after spinal cord injury.
NeuroD1-Mediated Cell Reprogramming for Functional Recovery in Spinal Cord–Injured Rats
1Graduate Program in Neuroscience, University of Minnesota Twin Cities, Minneapolis, MN, USA
2Department of Neurosurgery, University of Minnesota Twin Cities, Minneapolis, MN, USA
Spinal cord injury (SCI) is a globally prevalent issue with no currently available treatments to restore nervous system function outside of physical therapy rehabilitation regimens that allow for limited functional recovery. Viral vector–mediated delivery of proneural factors is an emerging experimental approach that makes use of cellular reprogramming technology—glia-to-neuron reprogramming—to address the need for therapies that promote nervous system repair. Current literature shows that NeuroD1, a significant proneural developmental factor, is sufficient to convert astrocytes into neurons in vitro and in animal models of neurological disorders. However, this has been met with recent controversy with some studies highlighting a lack of consistency and ambiguity in the cellular origin of “reprogrammed cells.” In response, the viral titer and timing of treatment intervention have been identified as significant factors capable of influencing efficacy and reproducibility. In addition, the capability of this platform to improve functional deficits within the context of SCI has yet to be explored. We hypothesize that the AAV9-NeuroD1 reprogramming platform reprograms astrocytes into neurons within the spinal cord of a spinal cord–injured rat in a dose-dependent manner, ultimately restoring nervous system function. Here we use a two-vector AAV9 DIO/FLEx-based delivery platform for the select expression of NeuroD1-mRuby2 (reprogramming) or mRuby2 alone (control) in spinal cord astrocytes after SCI. We used immunofluorescence and microscopy-based analyses to examine cellular characteristics and extent of transduced cells throughout the spinal cord in response to the viral platform at one of two titers (1013 or 1011 GC/ml) and time points (acute or subacute stage of injury). Contrary to our hypothesis, trends in our preliminary motor functional analysis do not indicate viral platform–mediated functional recovery. Future work will aim to continue motor and sensory functional analysis with additional numbers of rats and exploring the transduction pattern of NeuroD1 through spatial transcriptomics and neuronal lineage tracing.
A Comparison of Pathologies in Those With Down Syndrome, Early-Onset Alzheimer’s, and Late-Onset Alzheimer’s in the Locus Coeruleus
Department of Neurosurgery, University of Colorado Anschutz Medical Campus, Aurora, CO, USA
Down syndrome (DS) or trisomy 21 is the most common chromosomal disorder worldwide and among the most complex genetic conditions resulting in numerous comorbidities and neurological phenotypes. While advancements in medical care have increased the life expectancy of individuals with DS to 60 years, incidences of age-related disorders like Alzheimer’s disease (AD) have risen. The hallmarks of AD are amyloid-β plaques, neurofibrillary tangles, and glial activation. These pathologies are associated with neurodegeneration in various brain areas, specifically the hippocampus, manifesting clinically as memory loss. However, it is believed that neuron degeneration in the locus coeruleus (LC) precedes all other neuronal loss. As the LC degenerates, there is a significant decline in norepinephrine levels in other brain regions including the hippocampus, which may contribute to initiating neurodegeneration in such areas. The main purpose of this study was to examine the pathological hallmarks in the LC and identify the relationship between AD and DS-AD in human tissue. We utilized immunofluorescent techniques on 39 postmortem human brain sections from individuals with AD, DS-AD, and controls to examine amyloid, tau, and glial cell pathologies in the LC. We found sex-dependent differences in amyloid deposits as well as various tau markers in the LC in DS-AD and AD cases. Morphological differences in glial activation states were observed in the LC between DS-AD, early-onset AD, and late-onset AD. Most notably, the glia response correlated to a mid-stage tau tangle marker. DS-AD is now considered a genetic form of AD; however, the genetic complexity and comorbidities associated with DS means that DS-AD has similarities to both early-onset AD as well as late-onset AD. This provides unique opportunities to further our understanding of the mechanisms governing AD which can lead to better diagnostic and therapeutic strategies for both those with DS and the general population.
Identifying Spinal Interneurons That Contribute to Respiratory Plasticity After Cervical Spinal Cord Injury
1Department of Neurobiology & Anatomy, College of Medicine, Drexel University, Philadelphia, PA, USA
2Marion Murray Spinal Cord Research Center, College of Medicine, Drexel University, Philadelphia, PA, USA
3Universitat Autònoma de Barcelona, Barcelona, Spain
4University of St Andrews, St Andrews, UK
5Gladstone Institutes, San Francisco, CA, USA
Spinal cord injury (SCI) is a devastating and irreversible event that affects almost 20,000 people each year. Most injuries occur at the cervical level, compromising the phrenic motor network, resulting in life-threatening respiratory deficits. Despite the devastating outcomes, there is significant evidence for spontaneous neuroplasticity after cervical SCI. Spinal interneurons are now widely recognized as a key component of this plasticity, which can alter their function and connectivity to provide new neuronal pathways that facilitate partial functional recovery. While many spinal interneuron subtypes have been identified, their characterization and involvement in respiratory plasticity after injury has remained limited. Building upon our previous studies on the contribution of excitatory, pre-motor V2a interneurons in neuroplasticity after SCI, the present study uses a battery of outcome measures to explore how inhibitory and modulatory pre-motor interneurons, V1 and V0c, respectively, contribute to plasticity after a high cervical SCI. Transgenic mice underwent a lateral left hemisection at the second cervical segment (C2) which denervates the ipsilateral spinal phrenic motor circuit (C3-C5/6) that controls the diaphragm—a primary muscle of breathing. Weeks to months post-injury, following modest neuroplastic changes and partial recovery, a transsynaptic retrograde tracer—pseudorabies virus—was applied to the left hemidiaphragm to label phrenic motoneurons and interneurons ipsilateral to injury. Respiratory function was examined 4 weeks post-injury through terminal diaphragm electromyography under anesthesia. Tissues were perfuse-fixed, and immunohistochemistry on cervical spinal cord sections was used to map and quantify the distribution of pseudorabies virus PRV-labeled spinal interneurons connected to the injured phrenic network. Preliminary data suggest increased interneuronal connectivity after SCI. The long-term goal of this research study is to understand the neuroplastic potential of these spinal interneurons in contributing to motor recovery after traumatic spinal cord injury.
Novel, Interesting, Surprising, and Useful Properties of a Panel of NF-L Monoclonal Antibodies
1EnCor Biotechnology Inc, Gainesville, FL, USA
2Department of Neuroscience, College of Medicine, University of Florida, Gainesville, FL, USA
3McKnight Brain Institute, University of Florida, Gainesville, FL, USA
4Department of Physical Therapy, University of Florida, Gainesville, FL, USA
5Breathing Research and Therapeutics Center, University of Florida, Gainesville, FL, USA
Neurofilament light protein (NF-L) can be detected at informative levels in blood, cerebrospinal fluid, and other fluids in studies of a variety of neurodegenerative states using excellent commercial assays from Uman Diagnostics, Quanterix, and others. We have fully characterized the two key monoclonal antibody reagents used in these assays. Surprisingly, neither antibody recognizes typical neurofilament-rich profiles in healthy neurons and their processes in sectioned material or in neural cultures, instead recognizing a minority of processes which have the appearance of neurodegeneration. In stark contrast, following a mid-cervical spinal cord contusion injury in rats, both antibodies reveal numerous strongly stained nerve fibers in regions expected to contain compromised processes. Many of these processes appear beaded, sinusoidal, or discontinuous as expected for degenerating axons. We localized the epitopes for both Uman antibodies to a short NF-L peptide and made novel monoclonal and polyclonal antibodies to this region, which share these interesting selective staining properties. The unmasking of the degeneration specific epitopes appears to be due to proteolysis and can be mimicked by treating sections of healthy tissue with proteases, which results in previously unreactive NF-L containing profiles becoming strongly reactive with this specific type of NF-L antibody. We have shown that NF-L contains many more epitopes which are hidden in healthy neurofilaments but revealed on degeneration and that the homologous regions of NF-M and NF-H also contain epitopes only revealed on degeneration. We have also characterized antibodies which recognize a proteolytically labile site on NF-L in healthy axons which disappears in degenerating processes, allowing positive identification of both healthy and degenerated processes. We propose that the hidden epitopes are part of functionally important binding sites important for filament assembly. These reagents are robust, excellent, novel and specific markers of neurodegeneration of wide utility in future studies of central nervous system disease and injury.
Optimization of Single Cell-Repliseq for Studying Replication Timing in Early-Stage Wild-Type and Chimeric Mouse Embryos for Enhancing Interspecies Chimerism
University of Minnesota, Minneapolis, MN, USA
The field of interspecies chimerism holds the potential to develop functional human organs and cells in host animals like pigs for organ transplantation in the clinic. However, there are multiple hurdles that need to be overcome before this becomes a reality. One of the major hurdles is the low efficiency of chimerism between evolutionarily distant donor–host species such as human–mouse when compared with more closely related species such as rat–mouse. The current hypothesis is that the differences in developmental speeds between cells of the donor and host in the chimeric embryos lead to low chimerism. To understand and overcome this hurdle, there is a need to define “developmental speed.” Here, we propose to optimize single cell-Repliseq (sc-Repliseq) for the purpose of defining developmental speed in cells of wild-type and chimeric mouse embryos. Through sc-Repliseq, we are able to analyze cell cycle phases (G1, S, or G2) and the genes that are replicated early versus late during the S phase of the cell cycle, at the single-cell level. By analyzing sc-Repliseq of cells in wild-type mouse embryos from the zygote (E0.5) to the early gastrulating embryo (E5.5), we develop the baseline developmental speed in these host embryos. Since replication timing of cells in early mouse embryos is still not fully established, this analysis will also provide insight into how cells in the early embryo replicate and what genes are expressed early and late in the cell cycle. Subsequently, analysis of marmoset-mouse interspecies chimeric embryos will allow us to observe any differences in developmental speed between the donor and host cells during different stages of the chimeric mouse embryo. Observation of developmental speed differences between donor and host cells will allow us to address these differences and overcome a major hurdle toward optimizing interspecies chimerism for organ and cell development.
Determining the Impact of CRISPR/Cas9 Mediated AVIL Gene Knockout on Human Glioblastoma In Vitro
1Field Neurosciences Institute Laboratory for Restorative Neurology, Central Michigan University, Mount Pleasant, MI, USA
2Program in Neuroscience, Central Michigan University, Mount Pleasant, MI, USA
3Department of Chemistry and Biology, Central Michigan University; College of Medicine, Central Michigan University, Mount Pleasant, MI, USA
4Department of Psychology, Central Michigan University, Mount Pleasant, MI, USA
5College of Medicine, Central Michigan University, Mount Pleasant, MI, USA
6Field Neuroscience Institute, Saginaw, MI, USA
Glioblastoma (GB) is the deadliest and most common form of brain cancer. The standard treatment protocol for GB is maximum safe surgical resection and adjuvant chemotherapy; however, this only extends median overall survival to 12 to 14 months subsequent diagnosis. Despite a wealth of novel research being pursued, the last major advancement in the treatment of GB occurred in 2005 with the emergence of the Stupp Protocol. Clearly, there is a need to identify and pursue new therapeutic targets for the treatment of GB. One promising target is the AVIL gene, which produces the cytoskeleton-regulating protein p92. Recent research has elucidated that p92 is overexpressed in GB cells and is crucial for GB tumorigenesis, migration, invasion, and the function of GB stem cells. To our knowledge, no prior studies have used CRISPR/Cas9 to knockout the AVIL gene in GB cells. Ergo, in this study we delivered CRISPR/Cas9 ribonucleoprotein (RNP) to U87 and HEK293 cells using Lipofectamine CRISPRMAX Cas9 Transfection reagent. We designed three independent guide RNA for AVIL knockout and determined the optimal concentrations of CRISPR/Cas9 RNP and Lipofectamine CRISPRMAX Cas9 Transfection reagent in working conditions. The CRISPR-Cas9-mediated knockout of AVIL resulted in the death of nearly all U87 cells, while untreated cells were unaffected. Further analysis will include Western blot for protein expression, Sanger Sequencing to confirm gene knockout, and BRDU assay to quantify cell proliferation.
Support for this study was provided by the College of Medicine, John G Kulhavi Professorship, Neuroscience Program, E. Malcolm Field and Gary Leo Dunbar endowed Chair in Neuroscience at Central Michigan University.
Montelukast Treatment Attenuates Cognitive Dysfunction in a Model of Chronic Gulf War Illness With Modulation of Leukotriene Signaling and NLRP3-Inflammasome Activation
Institute for Regenerative Medicine, Department of Cell Biology and Genetics, School of Medicine, Texas A&M University, College Station, TX, USA
Chronic Gulf war illness (GWI), typified by persistent cognitive and mood impairments, is associated with chronic neuroinflammation. Montelukast (MLK), a Food and Drug Administration–approved drug for treating asthma, could improve brain function due to its ability to reduce neuroinflammation by inhibiting cysteinyl leukotriene (CysLT) signaling. This study investigated the effects of long-term MLK treatment to improve brain function in rats afflicted with GWI-like symptoms for prolonged periods. Young adult rats were exposed to GWI-related chemicals (pyridostigmine bromide, oral; DEET N,N-Diethyl m-Toluamide, dermal; and permethrin, dermal) and 15 minutes of restraint stress for 28 days. Nine months post-exposure, the GWI rats received MLK (10 mg/kg, 5 days/week) or vehicle for 6 months. Neurobehavioral tests performed during the last 2 months of MLK treatment (ie, at 15–16 months post-exposure to GWI-related chemicals and stress) revealed improvements in hippocampus-dependent object location and spatial recognition memories and pattern separation ability. Anhedonia was also reversed in MLK-treated GWI rats. Analysis of hippocampal tissues showed elevated CysLT and LTB4 levels in GWI rats receiving vehicle treatment. However, GWI rats receiving MLK treatment displayed CysLT and LTB4 levels that were similar to age-matched naive control rats. Such changes in LT signaling in MLK-treated GWI rats were also associated with reduced astrocyte hypertrophy and decreased incidence of activated microglia displaying NOD-, LRR-, and pyrin domain-containing protein 3 (NLRP3) inflammasomes. The biochemical analysis also revealed suppression of NLRP3 inflammasome activation in MLK-treated GWI rats, evident from reduced concentrations of its mediators, nuclear factor kappa B, NLRP3, adapter protein apoptosis-associated speck-like protein containing a CARD (ASC), and cleaved caspase-1 as well as end products, interleukin-1 beta (IL-1b) and IL-18. Thus, oral MLK treatment improves cognitive and mood function in rats afflicted with chronic GWI. Modulation of LT signaling leading to reduced neuroinflammation likely underlies the MLK-mediated improved brain function in chronic GWI.
This study was supported by Department of Defense (DOD) to A.K.S.
PAMAM Dendrimer Delivered Nocodazole as a Potential Treatment for Human Glioblastoma in SCID Mice
1Field Neurosciences Institute Laboratory for Restorative Neurology, Central Michigan University, Mount Pleasant, MI, USA
2Program in Neuroscience, Central Michigan University, Mount Pleasant, MI, USA
3College of Medicine, Central Michigan University, Mount Pleasant, MI, USA
4Department of Chemistry & Biochemistry, Central Michigan University, Mount Pleasant, MI, USA
5Department of Psychology, Central Michigan University, Mount Pleasant, MI, USA
6Field Neurosciences Institute, Saginaw, MI, USA
Glioblastoma (GB) is a grade IV astrocytoma, which is an aggressive form of brain cancer. There is no cure for GB. Current treatments include use of temozolomide along with chemotherapy and radiation therapy. The survival rate of GB patients is 12 to 15 months. There is a need for more effective treatments for GB, including the use of better delivery systems, to maximize therapeutic efficacy. To this end, we propose Nocodazole, an anti-neoplastic agent that halts proliferation and migration of tumor cells. However, Nocodazole is not water soluble and delivering water-insoluble compounds to the brain is a challenge. Herein, we sought to demonstrate whether encapsulation of Nocodazole within cavities of polyamidoamine (PAMAM) dendrimers (D-Noco) increases its packaging efficiency, aqueous transport, and eventual release into in vitro U87 glioblastoma cell lines and in vivo U87-induced tumors in severe combined immunodeficiency disease (SCID) mice. Our in vitro results showed that U87 cells treated with D-Noco survived less than cells treated with Nocodazole and vehicle controls, confirming increased solubility of Nocodazole when encapsulated in the dendrimer as compared with its native form. This also confirmed the treatment effects of D-Noco compared with free form Nocodazole in U87 cell lines. As a next step, we injected D-Noco directly into U87-induced brain tumors in SCID mice. Following treatment, in vivo imaging was performed to analyze the tumor growth twice a week until the end point of the mice. Kaplan–Meier survival analysis of the mice following tumor formation showed that mice which received treatment with D-Noco survived significantly longer than those which did not. Overall, our results suggest that PAMAM dendrimers can improve the solubility and drug delivering efficacy of water insoluble cancer drugs, such as Nocodazole, both in vitro and in vivo.
Support for this study was provided by the Neuroscience program, the College of Medicine, the Field Neurosciences Institute, department of chemistry and biochemistry and the John G. Kulhavi Professorship in Neuroscience at CMU.
Regeneration of the Sensory Tract After Spinal Cord Injury Using AAV Gene Therapy
K. Stepankova1,2, B. Smejkalova1,2, L. M. Urdzikova1, J. CF. Kwok1,3,
1Institute of Experimental Medicine, Czech Academy of Sciences, Prague, Czech Republic
2Department of Neuroscience, Second Faculty of Medicine, Charles University, Prague, Czech Republic
3Faculty of Biological Sciences, University of Leeds, Leeds, UK
4John van Geest Centre for Brain Repair, Department of Clinical Neurosciences, University of Cambridge, Cambridge, UK
Regeneration of sensory axons in the injured spinal cord (SCI) has been a research focus in many labs. The regenerating axons must overcome nonpermissive extracellular matrix of the glial scar, in which tenascin-C is upregulated after SCI and contributes to the inhibitory environment around the lesion. The migration-inducing tenascin-binding integrin is alpha9beta1, which is downregulated in adults and not upregulated after injury. Integrin α9 needs an activator kindlin 1 which prevents an integrin inactivation by inhibitory chondroitin sulfate proteoglycans (CSPGs). The aim of this study was to achieve sensory axon regeneration in animals with SCI using viral vector delivery of the relevant genes (integrin α9 and kindlin 1, kindlin 1 alone or GFP) to the DRGs. We addressed two levels of SCI, C4 lesion with DRG C6 and C7 injections for forelimb sensory restoration and T10 lesion with DRG L4 and L5 injections for hind limb sensory restoration. The animals underwent dorsal column crush injury with concurrent DRG injections followed by 12 weeks of behavioral testing. Significant improvement was observed in Von Frey test for mechanical perception and Hargreaves test for thermal sensation in treated animals with both, cervical and thoracic lesions when compared with controls. Functional improvement was confirmed by cFos staining. Immunohistochemistry shows regenerating axons from the integrin α9 and kindlin 1 group overcoming the lesion growing on tenascin-C substrate. GFP and V5 staining confirmed that transport of integrin and kindlin occurred over the full regeneration distance in the re-growing axons in the spinal cord, from L4, L5 up to cervical cord. About 40% of axons below the lesion regenerated their axons to at least 4 cm above the lesion. In conclusion, the AAV-mediated gene therapy leads to robust sensory regeneration after SCI at C4 and T10 level, proved by behavioral tests and immunohistochemical staining.
This study was supported by NEURORECON .02.1.01/0.0./0.0/15_003/0000419.
The Neuroprotective Effect of Caffeic Acid Phenethyl Ester on Fibrinogen-Induced Damage of Primary Neurons
Department of Surgery, Morsani College of Medicine, University of South Florida, Tampa, FL, USA
Caffeic acid phenethyl ester (CAPE), an active component of honeybee produced propolis, has been used for many years in traditional medicine for its anti-inflammatory and antioxidant properties. It is a specific inhibitor of nuclear factor-κB (NF-κB) activity. Elevated levels of fibrinogen are found during inflammatory conditions, including traumatic brain injury, resulting in its extravasation and deposition in the brain parenchyma. We have shown that an elevated level of fibrinogen caused the upregulation of pro-inflammatory cytokine C-C motif chemokine ligand-2 (CCL2), interleukin 6 (IL-6), and the activation of NF-κB through specific interactions with its neuronal receptors intercellular adhesion molecule 1 and cellular prion protein. It was also shown that fibrinogen enhanced the production of nitric oxide and reactive oxygen species in astrocytes and neurons, which contributed to neurodegeneration and increased cell death. The anti-inflammatory and antioxidant effects of CAPE in fibrinogen-induced neurodegeneration were tested using primary mouse brain cortex neurons from C57BL/6 mice. Cells were plated at 7×105 cells/ml in poly-d-lysine- and laminin-coated cell culture plates and treated for 1 hour with either media alone (control) or Fg (0.5 or 1 mg/ml) in the presence or absence of CAPE. To investigate the expressions of mRNAs for CCL2, IL-6, and neuronal nitric oxide synthase (nNOS), real-time polymerase chain reaction was performed. NF-κB activity was assessed by TransAM™ NF-κB p65 assay, Western blot, and immunocytochemistry. Cell viability was assessed with a Live/Dead assay using calcein and ethidium homodimer. Data showed that CAPE attenuated fibrinogen-induced increases in mRNA levels of CCL2 and IL-6 as well as NF-κB activation in neurons. Also, it reduced the fibrinogen-induced upregulation of nNOS and neuronal death. Thus, our results suggest that CAPE offers protection against fibrinogen-induced oxidative damage and neuronal death despite the propensity of the interaction between fibrinogen and neurons.
A Combined Gene and Stem Cell Therapy for ALS Targeting Both Lower and Upper Motor Neurons
Cedars-Sinai Board of Governors Regenerative Medicine Institute, Los Angeles, CA, USA
We have been working for over a decade to generate a cell product that can replace damaged astrocytes in neurological diseases and at the same time deliver vital growth factors to dying neurons. Amyotrophic lateral sclerosis (ALS) has been our lead indication, and we have shown that fetal-derived neural progenitors engineered to release GDNF (CNS10-NPC-GDNF) can survive transplantation, generate astrocytes, and release GDNF that protects motor neurons for up to a year in animal models. Recently, we completed a unique Phase IIa safety clinical trial where the cells were transplanted unilaterally into the lumbar spinal cord of 18 ALS patients. The cells were safe, generated astrocytes that released GDNF, and survived for up to 3 years. We are currently planning to follow up with more spinal cord patients in a small efficacy trial. However, as the upper motor neuron also degenerates in ALS, we went on to show CNS10-NPC-GDNF can also protect these motor neurons in animal models and have now started another trial in ALS patients transplanting into the hand knob region of the motor cortex strip. Both these trials will be discussed along with how astrocyte replacement and growth factor delivery may also be valuable for other neurodegenerative diseases.
Delivery of Curcumin Using Mixed-Surface Generation 4 Poly-Amido(Amine) Dendrimers Prevents the Development of Motoric Deficits in the GFAP-IL 6 Mouse Model
1Field Neurosciences Institute Laboratory for Restorative Neurology, Central Michigan University, Mount Pleasant, MI, USA
2Program in Neuroscience, Central Michigan University, Mount Pleasant, MI, USA
3College of Medicine, Central Michigan University, Mount Pleasant, MI, USA
4Department of Chemistry & Biochemistry, Central Michigan University, Mount Pleasant, MI, USA
5Department of Psychology, Central Michigan University, Mount Pleasant, MI, USA
6Field Neurosciences Institute, Saginaw, MI, USA
*Contributed equally.
The recently established GFAP-IL 6 mouse model congenitally expresses hippocampal and cerebellar neuroinflammation via upregulation of interleukin 6 (IL-6). Neuroinflammation is an essential component of numerous neuropathologies. Previous studies have identified curcumin as a promising treatment for neuroinflammation; however, the efficacy of this treatment is hindered by its low bioavailability and solubility. One method to increase the treatment efficacy is to deliver curcumin using poly-amido(amine) (PAMAM) dendrimers. Generation 4 (G4) 70/30 PAMAM dendrimers contain hydroxyl and amine surface functional groups at a 70:30 ratio. In this study, we used G4 70/30-cystamine core PAMAM dendrimer-encapsulated curcumin (D-Curc) for intracranial administration into the hippocampus and cerebellum of the GFAP-IL 6 mouse model. The efficacy of D-Curc was measured by testing motoric and cognitive functioning via accelerated rotarod (accelerod) and water T-maze (WTM). Prior to treatment administration, a preoperative sex-dependent genotype difference was observed between heterozygous GFAP-IL 6 mice (HET) and wild-type (WT) mice. The female HETs exhibited significantly reduced latency to fall when compared with female WT mice (P = 0.028), whereas male HETs exhibited significantly greater latency to fall than WT mice (P = 0.021). After treatment, we observed that control (HETs) injected with Hank’s Balanced Salt Solution (HBSS) and the dendrimer alone developed significant motoric deficits, whereas HETs injected with D-Curc did not. Finally, no difference was observed between HET and WT WTM performance. To our knowledge, this is the first time that WTM and accelerod tests have been used to characterize the GFAP-IL 6 mouse model of neuroinflammation following treatment with curcumin.
This GFAP-IL6 model was a generous gift from Emeritus Professor Iain L. Campbell, University of Sydney, Australia. Support for this study was provided by the College of Medicine, John G Kulhavi Professorship, Neuroscience Program, E. Malcolm Field and Gary Leo Dunbar endowed Chair in Neuroscience at Central Michigan University.
Rehabilitation Accompanied With Kampo Medicine Improves Motor Deficits Score After Intracerebral Hemorrhage in Rats
N. Tajiri, S. Ueno, D. Mustika, T. Shimizu, and
Department of Neurophysiology and Brain Science, Graduate School of Medical Sciences, Nagoya City University, Nagoya, Japan
We reported that rehabilitation by forced limb use (FLU) after intracerebral hemorrhage near the internal capsule (ICH) shows a causal relationship between the functional recovery and the cortico-rubral tract in rats (J. Neurosci 36(2),455–67:2016). Although the importance of Kampo medicine on rehabilitation is recently increasing, the precise mechanism of Kampo medicine on rehabilitation is still unknown. We investigated whether combination of rehabilitation and Kampo medicine can promote disturbed motor function after ICH, Ninjin’yoeito (NYT), which effects on the muscle (sarcopenia) and the brain (cognitive dysfunction), was given to the rats with FLU after ICH. ICH model was made by the injection of type IV collagenase (15 units/ml, 1.4 µl) near the internal capsule of male rats. FLU was given from 1 day after the region (D1) for 7 days with chows containing 1% NYT until D56. Five groups (sham-operated group, ICH-only group, ICH+FLU group, ICH+NYT group, ICH + FLU+NYT group) were prepared, and motor deficit scores (MDS: beam walk ability test, forepaw grasp test, hind limb retraction test) and open field test were used for behavioral assessment until D56. Significant increase of MDS was shown in ICH-only group (8.3 ± 1.9, n = 10) at D28, which is comparable to ICH+NYT group (6.8 ± 2.8, n = 11) and ICH+FLU group (6.6 ± 1.7, n = 14). Significantly better functional recovery was shown in ICH + FLU+NYT group (5.3 ± 2.0, n = 13). In open field test, total distance of the walk was the longest, and the max speed in locomotion was significantly different from ICH+NYT group in ICH + FLU+NYT group that was comparable to sham control. Data suggest that NYT is additive to the effect of FLU. However, the mechanism of NYT seems to be different from FLU, probably effecting on the muscle rather than promoting the plasticity by FLU.
Novel Choroid Plexus Ablation Model Unveils the Role of Cerebrospinal Fluid in Adult Neurogenesis in the SVZ
1Department of Molecular Genetics, College of Medicine, University of Cincinnati, Cincinnati, OH, USA
2Cincinnati Children’s Hospital Medical Center, Cincinnati, OH, USA
Despite their prominent role in multiple developmental and pathological processes, the choroid plexus (ChP) and cerebrospinal fluid (CSF) remain the most understudied compartments of the central nervous system, largely due to a lack of noninvasive tools to directly and selectively manipulate ChP function, CSF production, and flow. In this study, we present a new model (the ROSA26iDTR mouse line) allowing for noninvasive, specific, and temporally controllable ablation of CSF-producing ChP epithelial cells, leading to profound and sustained loss of ventricular volume due to apoptosis in the ChP epithelium upon administration of diphtheria toxin. Using a novel three-dimensional fluid-sensitive magnetic resonance imaging sequence, we show that in this mouse line, ablation of choroid plexus and loss of CSF volume is irreversible in the adult brain. Surprisingly, ablation of ChP does not lead to an overt neurological phenotype, nor does it result in motor differences or changes in cognitive function acutely at 1 month after ablation. However, longer-term CSF loss (at 3 and 6 months post-ablation) results in a pronounced decrease in adult neurogenesis in the subventricular zone (SVZ). Using SVZ whole-mount tissue immunohistochemistry analysis, we further show that ChP-ablated mice display a reduced number of newly born neuroblasts (DCX+) in the SVZ, without any significant differences in the overall number of proliferating (ki67+) cells, suggesting that the CSF is critical for the differentiation or survival of adult-born neuroblasts. In addition, we observed a diffused and disorganized pattern of ependymal cilia bundles in the ventricular wall of ChP-ablated animals, which may contribute to the disrupted migratory network of DCX+ neuroblasts we observed in the SVZ whole mounts. Thus, our study characterizes a first-of-its-kind noninvasive in vivo tool to selectively ablate the ChP and establishes an important and direct role of CSF in maintaining the regenerative capacity of the adult brain.
Neuronal BIN1 Modulation of Tau Pathogenesis Reveals Potential Resistance Mechanisms
Byrd Alzheimer’s Center and Research Institute, Department of Molecular Medicine, University of South Florida, Tampa, FL, USA
Genetic factors and lifestyle play essential roles in age-associated cognitive decline and an individual’s susceptibility to developing Alzheimer’s disease (AD) and related dementia. Bridging integrator 1 (BIN1) is the second most prevalent genetic risk factor identified by genome-wide association studies for late-onset AD. As an adaptor protein, BIN1 regulates membrane dynamics in the context of endocytosis and neurotransmitter vesicle release. BIN1 also limits extracellular tau seed uptake by endocytosis and binds to tau in the cytosol. We have characterized the in vivo roles of BIN1 in Alzheimer’s tau pathophysiology using conditional Bin1 knockout mice generated in human tau transgenic background. PS19 mice develop age-dependent tau neuropathology and motor deficits and are commonly used to study AD tau pathophysiology. The loss of BIN1 expression in forebrain excitatory neurons of PS19 mice exacerbated tau pathology in the somatosensory cortex, thalamus, spinal cord, and sciatic nerve. Intriguingly, the loss of BIN1 also mitigated neuropathology in select regions, including the hippocampus, entorhinal/piriform cortex, and amygdala, thus attenuating hippocampal synapse loss, neuronal death, neuroinflammation, and brain atrophy. Digital spatial profiling revealed that the loss of forebrain BIN1 expression elicited complex neuronal and nonneuronal transcriptomic changes across the brain regions, including impaired microglial and astrocyte transition toward the disease-associated phenotypes in the hippocampus. Moreover, we observed increased expression of genes regulating the ATP metabolic process and response to stress. These results provide crucial new information on in vivo BIN1 function in the context of tau pathogenesis. We conclude that forebrain neuronal BIN1 expression promotes hippocampal tau pathogenesis and neuroinflammation, and loss of this function protects hippocampal synapses. Our findings highlight an exciting region specificity in neuronal BIN1 regulation of tau pathogenesis and reveal resistance mechanisms involved in the modulation of tau neuropathology.
A Repair-Cell Therapy Approach for Parkinson’s Disease: A Summary of Safety, Feasibility, and Clinical Data for 68 Participants
University of Kentucky, Lexington, KY, USA
The lack of any treatment that alters the course of Parkinson’s disease (PD) represents a major therapeutic gap. Our objective is to investigate a disease-modifying therapy utilizing the direct delivery of autologous repair cells to the substantia nigra (SN). We developed a clinical trial platform, DBS Plus, that provides the opportunity for direct delivery of investigational therapeutics for patients undergoing deep brain stimulation (DBS) surgery. Sixty-eight participants with idiopathic PD participated in the clinical trials (NCT01833364, NCT02369003). Participants received autologous, peripheral nerve tissue (PNT) grafts to the SN. Graft dosing was explored in four cohorts by depositing either single or double grafts unilaterally or bilaterally. Primary endpoints were safety and feasibility. Secondary endpoints captured clinical data, including MDS-UPDRS Movement Disorder Society-Unified Parkinson’s Disease Rating Scale measures. Studies were institutional review board approved and reviewed every 4 months by a data and safety monitoring board. All 68 participants underwent successful DBS placement and received PNT grafts to the SN: single-unilateral (33), dual-unilateral (9), single-bilateral (11), and dual-bilateral (15). All adverse events related to the grafting procedure occurred at the PNT harvest site. All were transient and included paresthesias (16) and superficial infections (3). End-of-study neurocognitive evaluations revealed no significant cognitive decline for all groups. UPDRS III scores in the practical OFF state (Off-Med, Off-stim for 12 hours) showed a significant decrease at the end of study, −8.8 points (−11.8 to −5.9, 95% CI, P < .0001). Historical controls, DBS patients without graft (n = 16), showed no change, + 0.4 (−7.7 to +8.4, 95% CI, P = 0.93). Dual-bilateral grafts trended toward greater improvement compared with single-unilateral grafts. Our results provide evidence for safety and feasibility of the DBS Plus approach for the direct delivery of PNT to the SN. All participants received their intended DBS therapy, and the grafting procedure was well tolerated. Despite being open-label without placebo controls, the clinical outcomes are encouraging.
Inducible Ablation of TGF-β Signaling in Adult Microglia Leads to Activation of Microglia and Stimulation of Adult Neurogenesis in Hippocampus
University of Cincinnati, Cincinnati, OH, USA
Microglia are the brain-resident immune cells that remain in a homeostatic state that allows them to survey their microenvironment and maintain healthy brain function under physiological condition. Dysregulation of microglia in the brain has been implicated in neurodegenerative diseases and aging. Previous studies have implicated transforming growth factor beta (TGF-β) signaling as a key modulator for microglia homeostasis, and microglia has been speculated to modulate adult hippocampal neurogenesis. However, the exact molecular and cellular mechanisms are not well understood. Based on previous work and our preliminary data, we hypothesize that TGF-β signaling regulates adult neurogenesis through an indirect mechanism via microglia–neuronal stem cell (NSC) crosstalk. To test this hypothesis, we generated novel inducible microglia-specific TGF-β receptor TβRI(ALK5) knockout (iKO) mice in which adult inducible specific silencing of TGF-β signaling in microglia allows us to dissect out the role of TGF-β signaling on the crosstalk of microglia and adult NSCs in vivo. Our data show that microglia morphology shifts from a quiescent, ramified state to an activated phenotype in the absence of TGF-β signaling which is accompanied by loss of homeostatic microglia signature genes (such as TMEM119 and P2RY12). Interestingly, we also observed increase in number of adult-born immature neuroblasts (doublecortin, DCX+) in the subgranular zone SGZ in microglia-ALK5 iKO mice at 3 weeks after ALK5 gene deletion. Utilizing BrdU labeling and birth dating of adult-born neuroblast, currently we are investigating whether the increase in adult-born immature neurons is due to increased proliferation or decreased apoptosis. In addition, we will carry out single-cell RNA-seq to delineate the microglia-neuron crosstalk/interaction in wild-type and microglia-specific ALK5 KO hippocampus.
Ablation of Microglia Alters Reactive Astrocytes Gene Expression and Alleviates Dopaminergic Neuronal Loss in the Subacute MPTP Model of Parkinson’s Disease
1Department of Molecular and Cellular Biosciences, University of Cincinnati, Cincinnati, OH, USA
2Neuroscience Graduate Program, University of Cincinnati, Cincinnati, OH, USA
Parkinson’s disease (PD) is the second most common neurodegenerative disorder characterized by degeneration of dopaminergic (DA) neurons in the substantia nigra (SN) and loss of dopamine in the striatum (STR). Current therapies attempt to relieve the motor symptoms of PD without addressing the underlying neurodegeneration. Activation of microglia and astrocytes and elevated proinflammatory cytokines have been reported in PD; however, it is unknown whether this neuroinflammation plays a causal role in DA neurodegeneration. To investigate the role of neuroinflammation and glial activation as drivers of neuron death in PD, we ablated microglia using the CSF1R inhibitor PLX5622 (>90% ablation versus control mice) in the subacute neurotoxin 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine MPTP mouse model. We evaluated its effect on DA neuron survival, astrocyte activation, and cytokine production. MPTP treatment resulted in initial degeneration of DA neuron fibers in the STR, followed by prolonged degeneration of DA neurons in the SN. We observed acute (24 hours post) and robust activation of microglia in the STR and SN, followed by prolonged activation of striatal astrocytes. Unbiased stereological immunohistochemistry analysis showed that ablation of microglia was protective against loss of striatal DA fiber density and DA neurons in the SN. Interestingly, a multiplex cytokine assay also showed that MPTP treatment increases CXCL10 levels in the STR, and microglia ablation abolishes this increase in MPTP treated mice. This novel finding aligns well with human data showing plasma CXCL10 levels negatively correlate with cognitive function in PD patients. Moreover, our data show that ablation of microglia even after the initial 5 days of MPTP treatment also showed protection of DA neurons evaluated at 30 days post MPTP, suggesting microglia play an important role in the delayed progression of DA neuronal loss after the initial insults and support the clinical relevance of modulating microglia response as a potential treatment strategy after symptoms onset.
Neuronal Tau Pathology Cell Culture Model for Alzheimer’s Disease Therapeutic Discovery
Cambridge Research Center, AbbVie, Cambridge, MA, USA
Alzheimer’s disease (AD) involves misfolded tau protein hijacking synaptic transmission between neurons to spread pathology among brain regions resulting in cognitive impairment. To model the tau protein pathology, we built a highly sensitive, physiologically relevant in vitro murine hippocampal and cortical culture system that (1) accumulates tau aggregates upon seeding with AD brain-derived tau seeds and (2), synaptically spreads from neuron to neuron in microfluidic devices. The readout assay employs high content analysis (HCA) and automated image analysis to generate quantitative, fluorescence-based multi-parametric data for measuring tau pathology. This model is applicable for tau-antibody immunotherapy and inhibitors of tau aggregation small molecule screen.
Author is employees of AbbVie. The design, study conduct, and financial support for this research were provided by AbbVie. AbbVie participated in the interpretation of data, review, and approval of the publication.
Microglia Sense and Regulate Neuronal Activity Through Adrenergic Mechanisms
Department of Neurology, Mayo Clinic, Rochester, MN, USA
Microglia are resident immune cells of the central nervous system (CNS) and play key roles in brain homeostasis. Microglia processes dynamically survey the brain parenchyma and interact with neuronal elements. However, how microglia are engaged in the neuronal network activity is not entirely clear. Using in vivo two-photon imaging, here we demonstrate that general anesthesia increases microglial process dynamics and territory surveillance. Reduced norepinephrine signaling is necessary for anesthesia-induced increase in microglial process surveillance. Interestingly, beta 2 adrenergic receptors in microglia suppress the microglia dynamics in awake mice. Electron microscopy–based synaptic reconstruction after two-photon imaging reveals that microglial processes enter into the synaptic cleft to shield GABAergic inputs. Microglial ablation or loss of microglial β2-adrenergic receptors prevents post-anesthesia neuronal hyperactivity. Thus, under general anesthesia, the disinhibition of adrenergic regulation increases microglial dynamics and interaction with neuronal dendrites and cell bodies. During anesthesia recovery, the increased microglia-neuron interaction remains and promotes neuronal activity. These findings indicate that microglia sense and regulate neuronal network activity via adrenergic signaling during anesthesia to maintain brain homeostasis.
Transplanted Human Stem Cell–Derived Interneurons Functionally Integrate With the Injured Cervical Spinal Cord
1Gladstone Institutes, San Francisco, CA, USA
2Department of Neurobiology & Anatomy, Drexel University, Philadelphia, PA, USA
Advances in cell-based strategies offer new promise for some of the most devastating neural injuries like spinal cord injury (SCI). Engineering stem and neural precursor cells toward specific neural cell phenotypes that are optimal for repair is offering renewed enthusiasm for cell therapies. To harness the full therapeutic potential of stem cells, it is necessary to understand how to direct their differentiation to appropriate cell phenotypes and ensure that their phenotype and function persist after transplantation into a pathologic environment. Using a preclinical cervical contusion SCI in adult rats, we transplanted human induced pluripotent stem cell (hiPSC)-derived pre-motor spinal interneurons (SpINs). We hypothesized that donor spinal interneurons, known to contribute to plasticity post-injury, will become synaptically integrated with damaged spinal networks to promote novel neuronal relay formation and improve functional outcome. Quantitative polymerase chain reaction, immunocytochemistry, multielectrode array (MEA) analysis, and single cell and nuclei RNA sequencing were used to characterize engineered human SpINs prior to transplantation to confirm identity and neuronal function. Neuroanatomical tracing (transynaptic pseudorabies virus) and immunohistochemistry were used to assess anatomical integration between donor neurons and injured spinal networks. Optogenetic activation of either bulbospinal axons at the level of the transplant, or transplanted hiPSC-SpINs, was used to assess synaptic host-donor and donor-host connectivity, respectively. These stimulations were also performed during bilateral terminal diaphragm electromyography, which was used to quantitatively assess functional contribution of transplanted human SpINs to the recovery of phrenic function after injury and transplantation. These studies demonstrated that transplanted human SpINs survived and integrated with injured cervical spinal cord circuits, and displayed anatomical and functional evidence of connectivity. Having rigorously established improvement in diaphragm muscle activity with objective metrics, this strategy holds great promise to establish motor recovery post-SCI.
Role of aging in modulating the response to human Adipose Stem Cell derived exosomes following Traumatic Brain Injury
S. S. Abdelmaboud, L. D. Moss, C. Hudson, M. Avlas, R. Patel, N. A. Patel and P. C. Bickford
Department of Molecular Pharmacology and Physiology, Morsani College of Medicine, University of South Florida, Tampa, FL
Traumatic brain injury (TBI) is a leading cause of neurological complications including chronic memory deficits, dementia, and tau pathology. It is well known that aging is the primary risk factor for neurodegenerative diseases and is associated with increased morbidity and mortality from acute and chronic brain injuries. Older TBI patients are at a higher risk for cognitive and motor decline after TBI exposure than younger individuals. Additional data suggests that older subjects may show less responsiveness to therapeutic interventions, making treatment options for this vulnerable population more difficult. Aging and TBI are independently associated with increases in inflammation and cognitive function, The aim of the current study is to explore how these factors interact to worsen prognosis in older TBI patients and to develop targeted strategies for this vulnerable population.
Exosomes are a critical part of the human adipose stem cells (hASC) secretome, containing cargo that limits the secondary cell death following TBI by modulating neuroinflammation. Recently, our lab has showed efficacy of exosomes through intranasal delivery at 48 h post TBI in young mice. We aim to find the most effective dose in older TBI mice and characterize how exosomes interact with the immune system.
We conducted dose response experiments on young (3 months) and aged (15,20 month) mice treated with intranasal hASC exosomes at three different dose 10, 20 and 50ug. The end point was motor (Elevated body swing test EBST) and cognitive (Y maze and novel object and novel place test). We also did lesion volume analysis to assess how exosomes decrease the lesion volume in TBI mice vs sham. Moreover, we performed IHC and stained for microglia marker (MHCII) and Astrocytes (GFAP) to assess the neuroinflammation associated with TBI in aged vs young mice.
From that we concluded that hASC exosomes significantly improved motor dysfunction and significantly decrease cognitive deficit in aged vs young TBI mice. In addition, a major action of hASC exosomes is to modulate the secondary immune response to injury by interacting with the immune system cells mainly Microglia and astrocytes.
In this study we aim to understand how different ages impact response to hASC exo. treatment and to move this promising therapy forward and developing it for clinical practice.
Investigating the Therapeutic Utility of Adipose stem cell derived Exosomes on a-synuclein induce neurodegeneration in Rats
C. Logan, C. Hudson, R. Patel, K. Nash, N. A. Patel and P. C. Bickford
Center of Excellence for Aging and Brain Repair, Departments of Neurosurgery and Brain Repair and Molecular Pharmacology & Physiology, USF, James A Haley VA Hospital
Parkinson’s Disease (PD) is the second most prevalent neurodegenerative disease with over 90,000 people diagnosed each year. Treatment options are limited but include medications, surgery, and lifestyle changes. Previous data has shown that exosomes can migrate to injury sites to slow down the progression of a disease or injury. Our goal is to use exosomes derived from human adipose stem cells to treat Parkinson’s Disease (PD).
To model PD, we are using recombinant adeno-associated virus (AAV) expressing a-synuclein (Syn) injected into the substantia nigra (SN) of male Fisher rats. Injection unilaterally into the SN of rats results in dopaminergic neurodegeneration and impairment of front paw usage on the contralateral side to injection. We tested the rats for paw bias at baseline to ensure there was not a paw preference difference between groups prior to initiation of the study. Rats received 2 mL of either rAAV9-Syn or the control vector rAAV9-mKate (0.5x1013 vector genomes/µL). Three weeks after injection of the rAAV9-Syn rats, they were split into different groups which received intranasal administration of either: 1) 100 mg hASC exosomes in PBS; 2) 60 mg hASC exosomes in PBS; or 3) PBS (control). mKate injected rats received intranasal PBS administration. Once complete the rats were euthanized and we will use immunohistochemistry to determine the extent of cell loss in the SN and examine for changes in microglia, astrocytes and T cells.
We expect to see preservation of dopaminergic neurons in rats that received 100μg of exosomes more than animals that received 60μg. We also hope to observe a similar number of neurons between the 100μg and control group. If our hypothesis is correct, exosomes are a potential treatment to slow the progression of PD.
Engineering of glial-targeted AAVs for neurodegenerative disease
I. M. Sandoval1,2, J. D. Elsworth, and
1Department of Translational Neuroscience, Barrow Neurological Institute, Phoenix AZ
2nVector Inc, Phoenix AZ
3Yale University School of Medicine, New Haven CT
4Virscio, St. Kitts Biomedical Research Foundation, St. Kitts, West Indies
It is becoming increasingly appreciated that glial cells play an active role in neurodegenerative disease processes, with, for instance, inflammation being a key histopathological feature in diseases such as Parkinson’s disease, Alzheimer’s disease, and ALS, amongst others. Gene Therapy is emerging as a strong therapeutic modality for such disorders, negating the risk for systemic off-targets effects and affording anatomical precision within the CNS, with Adeno-associated virus being a leading viral vector candidate. However, wildtype AAVs exhibit largely a neuronal tropism and engineering efforts have been unsuccessful in generating AAV capsids that target specific populations of glia, specifically microglia. To that end, we engineered an AAV library where portions of every known glial ligand was inserted into receptor binding sites across the capsid. We thereafter delivered the viral library using intraparenchymal delivery and performed selection in clinically relevant subjects, including young, aged, and diseased (6-OHDA or MPTP) rats and non-human primates, as well as in acutely collected postmortem brain and spinal cord material from ALS patients. Four weeks after library delivery specimens were collected and either processed for bulk RNA seq, or we enriched the microglial population using magnetic assisted cell sorting. In addition, library targeting was confirmed using immunohistochemistry against the AAV reporter (GFP) and relevant cell populations such as oligodendrocytes, astrocytes, and importantly, microglia. Sequencing of collected tissue is currently ongoing which will allow us to identify specific ligands that target microglia and other subsets of glia. In parallel, we have devised a computational workflow to facilitate additional enhancement of binding pockets.
Neuromodulation using bioluminescent optogenetics in sympathetic neurons
T. Tian1,2,3, J. Owyoung3,4, H. SiMa3, and
1Medical Scientist Training Program, Emory University School of Medicine, Atlanta, GA
2Neuroscience Graduate Program, Laney Graduate School, Emory University, Atlanta, GA
3Department of Cell Biology, Emory University School of Medicine, Atlanta, GA
4Genetics and Molecular Biology Graduate Program, Atlanta, GA
The concept of dual innervation of the neuromuscular junction (NMJ) was hotly debated and eventually rejected in the 1930s. However, advances in tools and techniques have recently returned scientific interest to the dual innervation of NMJs by both sympathetic neurons and motoneurons. To begin to understand the physiological function of this innervation, we used bioluminescent optogenetic, electrophysiology, electromyography, pharmacologic, and RNA sequencing approaches. Traditional electrical and optogenetic stimulation were insufficient to target lumbar post-ganglionic axons/neurons. Therefore, we used a transgenic mouse line in which a luminopsin fusion protein — a light-sensing opsin fused to a light-emitting luciferase — was targeted to sympathetic neurons, enabling them to produce biological (internal) light in the presence of substrate. By using these photosensory proteins and luciferase, this technique (called bioluminescent optogenetics) permits spatiotemporal neuromodulation via fiber optic and/or by administration of luciferin. Furthermore, bioluminescent optogenetics advantageously allows for targeting of these difficult to reach lumbar sympathetic neurons via chemogenetics but independent of GPCR coupled pathways. We found that sympathetic denervation to skeletal muscle profoundly impacted muscle mitochondrial gene expression; sympathetic activity modulates muscle function; sympathetic innervation to skeletal muscle is required for optimal muscle health; and post-ganglionic sympathetic axons fail to regenerate following injury even when stimulated with bioluminescent optogenetics or traditional electrical stimulation paradigms.