Abstract
Introduction
The annual American Society for Neural Therapy and Repair (ASNTR) conference provides for the highest exchange of scientific information related to the field of cell therapies and brain repair for the treatment of neurological disorders. This Society consists of leading scientists and physicians, as well as postdoctoral fellows and students, who are all actively engaged in research and education in the field of neural therapy and repair.
This annual conference is at the forefront of translational medicine, and as the abstracts reflect, there will be topics that range from very basic development of stem cell therapies to clinical trials using existing technologies. With the emphasis on translation, many innovative presentations will be discussed as well as new technologies being developed for translation into the clinic.
The ASNTR conference provides for the highest exchange of scientific information related to the field of cell therapies and brain repair for the treatment of neurological disorders. The passionate scientific discussions during our annual conferences have developed into so many fruitful collaborations over the years, resulting in truly pioneering discoveries in the field of neural therapy.
The 2024 annual conference is being held at the Sheraton Sand Key Resort, Clearwater Beach, Florida. Without the continued support of NIH/NINDS, our corporate sponsors and members, none of this would be possible. Special thanks to our current President, Michael Lane, President-Elect, John Stanford, the 2024 Program Committee, Education Committee, Fundraising Committee and our staff for all their efforts in planning what will, without a doubt, be an exciting meeting.
Transcriptional analysis reveals new mechanisms of SCF+G-CSF-reduced neuropathology in APP/PS1 mice
SUNY Upstate Medical University, Syracuse, New York, USA
Alzheimer’s Disease (AD) is a neurodegenerative disease characterized by amyloid plaque deposition, tau hyperphosphorylation and neurofibrillary tangles, neuroinflammation, and cognitive decline. Our previous studies have demonstrated that the combination treatment of stem cell factor (SCF) and granulocyte colony-stimulating factor (G-CSF) reduces AD neuropathology in APP/PS1 mice. The purpose of this study is to explore the mechanism of SCF+G-CSF-ameliorated AD neuropathology. Six 17–18-month-old male APP/PS1 mice received subcutaneous injections of SCF+G-CSF or saline for 12 days. On day 13, RNA was extracted from brain tissue for gene chips. Reactome, ShinyGo 0.77, DAVID, and STRING 12.0 software were used for analysis of gene chip data. Gene chip data showed 315 differentially expressed genes (DEGs) after SCF+G-CSF treatment. Seventy-nine genes met the 2-fold expressional change threshold, including both upregulated DEGs (UDEGs) and down-regulated DEGs (DDEGs). Functional and tissue enrichment analysis unveiled that the majority of the 79 DEGs were associated with immune system function. Reactome pathway analysis revealed that many DEGs had immune system-related functions and/or were highly enriched for metal sequestration which promotes inflammation site targeting. Other identified functions were innate immune cell migration in injury, antimicrobial peptides and macrophage migration, and myeloid differentiation gene regulation. DAVID analysis revealed that the UDEGS were highly enriched in immune stimulation, cytoplasmic to extracellular ion transport, and cytoplasmic-cell membrane trafficking. Additionally, the DDEGs were highly enriched in metal ion binding, cytoplasmic-nuclear transcriptional regulation, and ubiquitination. STRING 12.0 analysis identified one cluster of seven interacting UDEGs (S100a9, Lnc2, S100a8, Ngp, Chil3, Ltf, and Ifitm6). PPI enrichment score indicates the significant value of the interactive relationship among these genes. The findings of this study suggest that SCF+G-CSF treatment in old APP/PS1 mice promotes and modulates a suitable inflammatory environment in the brain to optimize immune cell functions necessary to reduce AD neuropathology.
This study was supported by The Franks Foundation.
Modifying behavior with cortical layer specific neuromodulation
Central Michigan University, Mount Pleasant, MI, USA
The recent evolution of genetic tools to target neural circuits allows an unprecedented precision in neuromodulation. Genetically targeted circuit manipulation allows to probe nervous system function in the healthy brain, explore pathophysiology of neurodevelopmental and neurodegenerative diseases, and might be used to manipulate neural circuits for therapy. For Parkinson’s disease, the goal has been to modulate the basal ganglia circuits in a way that is achieved with deep brain stimulation but with better spatial and temporal control. For Huntington’s disease, circuit specific modulation to correct aberrant firing has shown promising rescue of motor deficits. For stroke, excitatory optogenetics has been used to strengthen the function of intact circuits so that they can restore lost motor function. After spinal cord injury, control of spinal neurons distal to the lesion established control of circuits that have been disconnected from brain control. We previously showed that systematically enhancing activity levels of pan-neocortical Emx1-positive pyramidal neurons during postnatal days 4 – 14 using non-invasive BioLuminescent-OptoGenetic (BL-OG)-mediated activation of luminopsin 3 (LMO3) led to decreased social interaction and increased grooming activity in adult animals. In vivo, both prefrontal neural activity and functional markers of cortico-striatal connectivity were impaired in developmentally hyperexcited adult Emx1-LMO3-positive mice. We wanted to further dissect the neural populations and their specific target areas mediating the observed behavioral and electrophysiological changes. Neurons in layer 5 integrate information between cortical areas but also project to subcortical structures involved in the generation of behavior. We carried out developmental hyperexcitation in layer 5-specific Rbp4-LMO3 mice, thus restricting LMO3 expression to L5 projection neurons. The behavioral consequences were compared to those of pan-laminar neocortical developmental hyperexcitation. We hypothesized that narrowing down the population of cells that are hyperexcited in early development will similarly narrow down the set of phenotypes that are altered in adulthood.
Developing ECM bioscaffolds to regenerate brain tissue after a stroke
N. Didwischus*, A. Kisel*, and
*contributed equally
Department of Radiology & McGowan Institute for Regenerative Medicine, University of Pittsburgh, Pittsburgh, PA, USA
The stroke-damaged brain does not spontaneously regenerate lost tissue. Inductive bioscaffolds derived from extracellular matrix (ECM) are widely used in peripheral organs to regenerate soft tissues. Formulation of these ECM bioscaffolds as a hydrogel affords the injection of these through a thin needle into a tissue cavity caused by a stroke. We developed an injection-drainage approach of ECM hydrogel to ensure a robust coverage of the cavity. ECM concentration emerged as a major factor determining the rheological properties of the bioscaffold that affect its degradation, as well as cell invasion. A compact interface between bioscaffold and host tissue is required to induce and support the invasion of host cells into the hydrogel. Immune cells invade and degrade the scaffold in a first phase, followed by brain and endothelial cell invasion. Gradually the scaffold is degraded and populated with host cells, which deposit a de novo ECM. Post-stroke timing of implantation affects the efficiency of hydrogel distribution and degradation. Hydrolysis and matrix metalloproteinases contribute to ECM hydrogel degradation. Implantation between 14 and 28 days post-stroke achieves the most efficient degradation and tissue replacement, whereas implantation at 90 days post-stroke has a more limited success. To enhance the neuronal content of de novo tissue, we investigate the stimulation of endogenous neurogenesis using growth factor to increase the number of neural progenitor that can invade the bioscaffold. Improving our understanding of how bioscaffolds interact with damaged host brain tissue will be essential to determine how the materials can be implemented in a clinical setting.
Preclinical evaluation of transaxial intraputaminal trajectory for enhanced distribution of grafted cells in Parkinson’s disease
M.E. Emborg1,2,3, A. Mancinelli1, J.C. Colwell1,3, A. Zinnen1, B. Pape1, K. Brunner1, V. Bondarenko1, C. Fitz1, J. Coonen1, V. Menna1, K. Fuchs1, Schultz-Darken1, H. Simmons1, H. Tran4, P. Larson5, M. Olsen6, S. Hurley7, A. Bratt-Leal4, E. Wirth4, and J.M. Metzger1
1Wisconsin National Primate Research Center; Madison, WI, USA
2Department of Medical Physics, University of Wisconsin-Madison; Madison, WI, USA
3Cellular and Molecular Pathology Graduate Program; Madison, WI, USA
4Aspen Neuroscience Inc., San Diego, CA, USA
5Neurosurgery, Department of Medicine, University of Arizona, Tucson, AZ, USA
6Department of Psychiatry, University of Wisconsin-Madison; Madison, WI, USA
7Department of Radiology, University of Wisconsin – Madison; Madison, WI, USA
This project aimed to develop and evaluate the feasibility and safety of a novel transaxial surgical approach for delivery of human iPSC-derived dopaminergic neuron progenitor cells (DANPCs) into the putamen nucleus of nonhuman primates, with surgical techniques and tools relevant to clinical translation. Nine immunosuppressed, unlesioned adult cynomolgus macaques received intraputaminal injections of vehicle or DANPCs (~1x105 cells/µL; 25µL and 50 µL, right and left putamen, respectively) under real-time, intraoperative-MRI guidance. The infusates were combined with 1mM gadoteridol (for intraoperative-MRI visualization) and delivered via two tracks per hemisphere (ventral and dorsal) using a transaxial approach. Animals were evaluated with a battery of clinical and behavioral tests and euthanized 7- or 30-days post-surgery; full necropsies were performed by a board-certified veterinary pathologist. Brain tissues were collected and processed for immunohistochemistry, including against the human-specific marker STEM121. The optimized surgical technique and tools produced successful targeting of the putamen via transaxial approach. Intraoperative-MRI scans confirmed on-target intraputaminal injections in all animals. All animals survived to scheduled termination without clinical evidence of neurological deficits. The first four animals to undergo surgery had mild brain swelling noted at the end of surgery, of which 3 had transient reduced vision; inclusion of mannitol therapy and reduced intravenous fluid administration during the surgical procedure addressed these complications. STEM121-immunoreactivity confirmed the presence of grafted cells along the injection track within the targeted putamen area of DANPC-treated animals. All adverse histological findings were limited in scope and consistent with surgical manipulation, injection procedure, and post-surgical inflammatory response to the mechanical disruption caused by cannula insertion. The delivery system, injection procedure, and DANPCs were well tolerated in all animals. Our results establish that this novel transaxial approach can be used to safely target DANPCs injections with precision to the post-commissural putamen and support its clinical investigation for Parkinson’s disease.
Molecular Insights into Fkbp5 Gene Deletion: Circadian Modulation and Brain Proteomics in Aged Mice
1Byrd Alzheimer's Center and Research Institute, Tampa, Florida, USA
2Department of Molecular Medicine, University of South Florida, Tampa, Florida, USA
3Department of Molecular Biosciences, University of South Florida, Tampa, Florida, USA
4Research Service, James A. Haley Veterans Hospital, Tampa, FL, USA
The FK506-binding protein 5 (Fkbp5) gene is a chaperone protein that regulates endocrine stress response in mammals by mediating glucocorticoid release within the hypothalamic-pituitary-adrenal axis. Several single nucleotide polymorphisms within Fkbp5 have been associated with increased susceptibility to psychiatric disorders. Because of this and the increased expression of Fkbp5 observed with age and stress, it offers potential therapeutic avenues for psychiatric disorders. This study explores the impact of Fkbp5 deletion on homeostasis in aged mice, using circadian activity assessments and brain proteomics to unravel molecular changes. We hypothesize that Fkbp5 ablation will be protective from age-associated deficits. To test this, we used 17-month-old Fkbp5 knockout (KO) mice and control (WT) littermates housed in circadian phenotyping chambers. Wheel running activity served as a circadian rhythm proxy during a 12-hr light/dark (to establish basal rhythm), 7-hr phase advance and 24-hr dark periods (measure endogenous rhythm). Acute stress was induced 48-hrs before sacrifice. At 18-months of age, corticosterone levels were assessed by ELISA, and brain tissues were collected. Hippocampal tissues were processed by 4D proteomics in aged KO and WT mice, along with 6-month-old WT mice. Basal circadian rhythm patterns were comparable in aged KO and WT groups. Exposure to acute stress revealed sex-specific differences in circadian period and intra-daily variability. Immunohistochemical analysis revealed increased BMAL1 levels in aged KO mice, while other clock proteins remained unchanged. Proteomic analysis uncovered distinct protein expression patterns in aged KO and young WT compared to aged WT. Enrichment of pathways related to endocytosis, GPCR and serotonin signaling, and phagosome formation was observed. In conclusion, Fkbp5 gene deletion in aged mice exerts modest protection against stress-induced circadian rhythm changes, likely through alterations in key signaling pathways. Understanding these molecular intricacies involving Fkbp5 and the aging brain may have implications in developing targeted therapeutic interventions for psychiatric disorders.
New technology for neurological disorders using machine learning, AI, and spatial transcriptomics
1Department of Neurosurgery, University of Colorado Anschutz Medical Campus, Aurora, CO, USA
2Department of Cell and Developmental Biology, University of Colorado Anschutz Medical Campus, Aurora, CO, USA
3Department of Pathology, University of Colorado Anschutz Medical Campus, Aurora, CO, USA
4Bioinformatic Solutions LLC, Sheridan, WY, USA
New omics approaches involve a spatial component, allowing assessment of gene expression as a function of cytoarchitectural organization which is critical for validating drug targets and identifying specific novel pathways involved in disease processes in discrete areas of the brain. Defining the spatial distribution of mRNA molecules will allow us to uncover cellular heterogeneity in specific cell types in the brain as well as determining subcellular distribution of transcripts during disease conditions. Identifying disease-specific areas of change can be used to develop novel algorithms for clinical trial success and/or lead to novel treatments associated with specific clinical symptoms and outcomes. We have implemented a spatial transcriptomics platform at CU Anschutz, focused on both intracranial tumors and neurodegenerative disorders. Tissue blocks from patients are examined for RNA quality and subsequently sequenced using the 10X Visium Spatial Gene Expression platform. The spatially derived expression profiles from individual patients are then sent for downstream AI-driven meta-analysis across all patients. To date, brain tissue from patients with epilepsy, Alzheimer’s disease, and Down syndrome-related Alzheimer’s disease have been analyzed and already revealed novel pathological pathways in discrete populations of cells within the hippocampus. The findings can generate important clinical data for future novel drug targets, as well as function as a training tool for ascending pathologists or neurologists, when we create a “library” of transcriptomic changes in these conditions. Deep analytics using AI-developed algorithms can also identify traits in subgroups of patients that determine prognosis for success in clinical trials. Data obtained with spatial transcriptomics can be validated by performing immunohistochemical analysis of specific targets in adjacent sections.
Atypical Neurogenesis, Astrogliosis, and Excessive Hilar Interneuron Loss Are Associated with the Development of Post-Traumatic Epilepsy
1Department of Biomedical Sciences and Pathobiology, Virginia Tech, Blacksburg, VA, USA (E.A.H.)
2Department of Cell, Developmental and Integrative Biology, University of Alabama at Birmingham, Birmingham, AL, USA
3Department of Biomedical Engineering, Florida International University, Miami, FL, USA
4School of Neuroscience, Virginia Tech, Blacksburg, VA, USA
5Translational Biology Medicine and Health Graduate Program, Blacksburg, VA, USA
6Department of Biomedical Engineering and Mechanics, Blacksburg, VA, USA
7Department of Neurology and Rehabilitation, University of Illinois at Chicago, Chicago, IL, USA
8Center for Engineered Health, Virginia Tech, Blacksburg, VA, USA
9Department of Physiology and Aging, College of Medicine, University of Florida, Gainesville, FL, USA
Traumatic brain injury (TBI) is a significant risk factor for post-traumatic epilepsy (PTE). The pathophysiological mechanisms underlying TBI-induced epileptogenesis are an active area of research, particularly focusing on the dentate gyrus, known for its susceptibility to injury and seizure development and progression. In this study, we utilized the murine unilateral focal control cortical impact (CCI) injury model and conducted continuous EEG video monitoring to detect seizure onset at 2-4 months post-injury. We used unbiased stereology and Imaris image analysis to quantify cellular changes in the dentate gyrus and hilus of the hippocampus, including Prox1-positive cell migration, astrocyte branching, and morphology, as well as neuronal loss at four months post-injury. To identify potential genetic factors involved in epileptogenesis, RNA-Seq analysis on astrocytes from specific brain regions was performed to profile differentially expressed genes between PTE-positive and negative animals. We showed that CCI injury resulted in 37% PTE incidence, with a strong correlation with injury severity and hippocampal damage. Notably, we observed a significant loss of hilar interneurons that coincided with aberrant migration of Prox1-positive granule cells and reduced astroglial branching in PTE-positive +compared to PTE-mice. Furthermore, we uniquely identified Cst3 as a PTE+-specific gene signature in astrocytes across all examined brain regions, and showed Cst3 increased expression in astrocytes from the hilus of PTE-positive mice. Together, these results suggest that epileptogenesis may arise following TBI due to distinct aberrant cellular remodeling and key molecular changes within the dentate gyrus of the hippocampus.
Adropin Protects Delayed Cerebral Ischemia in Subarachnoid Hemorrhage Patients
1University of Florida College of Medicine, Department of Neurosurgery, Gainesville, Florida, USA
Characterization of CMT2s iPSC-Human Motoneurons for Drug Application
1NanoScience Technology Center, University of Central Florida, Orlando, FL
2Hesperos, Inc., Orlando, FL
Charcot-Marie-Tooth (CMT) disease is a hereditary motor and sensory neuropathy affecting roughly 2.6 million people worldwide, making it the most common inherited peripheral neuropathy. This inherited disorder has a direct effect on the neuromuscular junction (NMJ), which is essential for motor function, causing gradual paralysis in most cases. CMT2s, a subtype of CMT affecting the IGHMBP2 gene associated with helicase binding, leads to loss of motor and sensory function in the lower extremities. To study the pathology, patient-derived induced pluripotent stem cells (iPSC) were differentiated into motoneurons for phenotypic analysis. Axonal development was analyzed by immunocytochemistry with a focus on axonal branching and axonal varicosity formation, electrophysiological function by patch clamp, and mitochondrial integrity by tetramethylrhodamine ethyl ester (TMRE). Significant deficits were identified in CMT2s motoneurons at both the early and late stages of the culture. The therapeutic effect of a potential drug is evaluated, with an emphasis on cultivating and maintaining normal axonal development. The motoneurons were treated with the drug in both acute and chronic dosing schemes, utilizing clinically relevant dosages. This study uncovers essential pathological mechanisms for motor dysfunction in CMT2s and provides a model for therapeutic development.
Ergogenic effects of invasive and non-invasive spinal cord stimulation strategies following spinal cord injury: a case series
1School of Sport, Exercise and Rehabilitation Sciences, University of Birmingham, Birmingham, UK
2International Collaboration on Repair Discoveries (ICORD), Vancouver, BC, Canada
3School of Kinesiology, University of British Columbia, Vancouver, BC, Canada
4Department of Medicine, Division of Physical Medicine and Rehabilitation, University of British Columbia, Vancouver, BC, Canada
5Experimental Medicine Program, University of British Columbia & Department of Cardiology, Vancouver Coastal Health, Vancouver, BC, Canada
6GF Strong Rehabilitation Center, Vancouver Coastal Health, Vancouver, BC, Canada
Disrupted sympathetic cardiovascular (CV) control lowers blood pressure (BP) and stroke volume (SV), thus impairing exercise performance in individuals with cervical and upper-thoracic spinal cord injury (SCI, ≥T6). Epidural spinal cord stimulation (ESCS) has previously demonstrated an effective modulation of CV control to augment aerobic capacity yet is an expensive and invasive strategy. This case-series compared the effects of ESCS and transcutaneous spinal cord stimulation (TSCS) (an affordable, non-invasive strategy) on modulating CV control and submaximal time-to-fatigue upper-body exercise performance in individuals with SCI. Participants (aged 24-59yrs) had a chronic (>1yr), motor-complete SCI. Two had an implanted epidural stimulator (C6 and T4 injuries), and two without an implant received TSCS (both T4 injuries). A mapping session identified the specific stimulation parameters (frequency, intensity, epidural electrode configuration, transcutaneous electrode locations) that optimally elevated BP (CV-SCS). A sham condition (SHAM-SCS) was included as a comparison. A graded arm-crank exercise test identified individual’s ventilatory thresholds (VT). Following a familiarisation trial, participants exercised to fatigue at a fixed workload above their VT on separate days, with CV-SCS or SHAM-SCS. CV-SCS increased time-to-fatigue with ESCS (Δ15min 29s) and TSCS (Δ17min 9s), relative to SHAM-SCS. Relative to baseline, change in systolic BP at rest was greater with CV-SCS versus SHAM-SCS with ESCS (Δ12 vs 1mmHg) and TSCS (Δ21 vs 9mmHg). Peak oxygen pulse, a reasonable surrogate for SV, was greater with CV-SCS in ESCS (Δ2.1mL/beat) and TSCS (Δ1.7mL/beat), relative to SHAM-SCS. Rating of perceived exertion also tended to be lower with CV-SCS than SHAM-SCS. Seemingly, modulating BP and peak oxygen pulse with CV-SCS increased supraspinal sympathetic output to the vasculature to improve upper-body exercise performance. While further research is necessary, these data suggest TSCS is as effective as ESCS as an ergogenic aid and may potentially be used to support exercise rehabilitation in this population.
Expression of alpha synuclein in the amygdala and midbrain nuclei of hemiparkinsonian rhesus monkeys
1Preclinical Parkinson’s Research Program, Wisconsin National Primate Research Center, University of Wisconsin-Madison, Madison, WI, USA
2Neuroscience Training Program, University of Wisconsin-Madison, Madison, WI, USA
3Department of Medical Physics, University of Wisconsin-Madison, Madison, WI, USA
Amygdala dysregulation has been linked to anxiety and depression, which are highly prevalent in patients with Parkinson’s disease (PD). Interestingly, the central amygdala nuclei complex (CeA) which is composed of the central medial (CeM) and the central lateral (CeL) nuclei, receives projections from midbrain dopaminergic neurons which are lost in PD. Moreover, PD patients present alpha-synuclein (aSyn)+ Lewy bodies in the CeA. This study aimed to characterize aSyn expression in the amygdala and its relationship with midbrain dopaminergic loss in hemiparkinsonian monkeys. Six adult rhesus macaques received a single right intracarotid artery injection of MPTP. Twenty-eight months later the subjects were euthanized and their brains processed for immunohistochemistry. In the midbrain, tyrosine hydroxylase (TH) immunoreactivity (-ir) was present in the soma and fibers of neurons within the substantia nigra pars compacta (SNpc) and ventral tegmental area (VTA). Percentage area above threshold (%AAT) quantification detected less TH-ir in the MPTP-treated SNpc (22.65%±1.79) compared to the contralateral side (69.77%±2.76) (p<0.0001). In the rostral VTA, TH-ir was very similar between ipsilateral (59.29±2.91) and contralateral (61.38%±1.80) hemispheres (p=0.47). In the amygdala, TH-ir was limited to neuronal fibers and was more abundant in the CeL and CeM nuclei. The MPTP-treated amygdala had less TH-ir (1.72% ±0.34) compared to the intact side (4.54%±0.44) (p=0.0005); a significant difference in TH-ir was also detected in the CeM (p=0.04). aSyn-ir was abundantly present in the amygdalar nuclei of both hemispheres (MPTP 79.36±3.30; intact 80.65%±1.76); no significant differences in expression were detected (p=0.64). These results demonstrate that dopaminergic innervation in the amygdala declines after MPTP-induced nigral dopaminergic neurodegeneration, but this loss does not significantly affect amygdalar aSyn expression. Next steps will include to analyze the effects of MPTP in other midbrain dopaminergic neuronal populations and how they correlate with amygdalar TH, aSyn, and phosphorylated aSyn expression.
Neuroprotective efficacy of human neural stem cell-derived exosomes for breast cancer chemobrain
Department of Anatomy and Neurobiology, School of Medicine, University of California Irvine, California, USA
As of 2023, the US had more than four million women with a history of breast cancer, and 60-75% of these survivors are affected with cancer therapy-related cognitive impairments (CRCI) or chemobrain. This condition is defined as thinking, recalling, and memory problems during or post-treatment. Previously we showed the regenerative efficiency of human neural stem cell (hNSC)-derived extracellular vesicles (EV) to reverse CRCI in irradiated and brain cancer mouse models. The current study is focused on a breast cancer mouse model to test the effectiveness of GMP-ready hNSC line (UCI-191)-derived EV treatment for chemobrain. EVs are lipid-bound nano-sized vesicles, can cross the blood-brain barrier, and contain bioactive cargo including lipids, proteins, nucleic acids, and mitochondrial components. We have identified miRNA-124-3p as a potential contributor to the neuroprotective effect following EV treatment. The current study used a syngeneic, immunocompetent WT mouse breast cancer model. Murine Py230 cancer cells with an epithelial-like morphology form adenocarcinoma tumors in the mammary fat pads. The mice received a clinically relevant adjuvant chemotherapy schedule including Adriamycin (ADR, doxorubicin, 2 mg/kg) and cyclophosphamide (CYP, 50 mg/kg) an hour apart, once weekly for four weeks. The EVs were delivered intravenously (retro-orbital vein injection) three days post-chemotherapy. One-month post-EV mice were administered cognitive function tests (object or place recognition, fear extinction, puzzle box), and brains were evaluated for neuroinflammation, gliosis, and synaptic integrity. ADR-CYP-treated mice showed significantly declined learning and memory, increased anxiety, and reduced memory consolidation compared to ADR-CYP-treated mice receiving EVs. Immunofluorescence analyses showed significant improvements in synaptic integrity and reductions in astroglial and microglial activation in the EV-treated mice. These data show the neuroprotective impact of stem cell-derived EV in ameliorating breast cancer chemobrain.
Neuroprotective impact of human neural stem cell-derived exosomes following cranial irradiation and chemotherapy for brain cancer
C. Hudson, R. P. Krattli, S. M. El-Khatib, A. H. Do, A. R. Vagadia, M. T. Usmani, S. Madan, A. J. Anderson, B. J. Cummings, and M. M. Acharya
Department of Anatomy & Neurobiology, School of Medicine, University of California Irvine, California, USA
CNS (central nervous system) cancers, with 85-90% brain tumors, are debilitating with survival rates around <30%. Brain cancer survivors face debilitating cognitive dysfunction and neuroinflammation after the combined treatment with cranial radiation therapy (CRT) and chemotherapy (temozolomide, TMZ). As survivorship is increasing, therapeutic interventions for these irreversible side-effects are becoming a priority to increase patients’ quality of life (QOL). Past work from our laboratory demonstrated that human neural stem cell (hNSC)-derived extracellular vesicles (EVs) have a neuroprotective and regenerative effect following acute CRT. EVs are nano-scale lipid-bound vesicles that contain bioactive cargo and can cross the blood-brain barrier. We identified miRNA-124-3p within the EV cargo as one of the contributing factors to ameliorating acute, single-dose CRT-induced cognitive impairment in mice without cancer. The current study used a syngeneic, immunocompetent WT mouse brain cancer model to expand its clinical relevance. Murine CT2A astrocytoma line was used to induce brain cancer, and the mice received a clinically relevant CRT-TMZ regime, including CRT (8.67 Gy x3 fractionated doses) in combination with TMZ (6 doses) each 48h apart. EVs were administered intravenously (retro-orbital vein injection) two days post-ultimate TMZ treatment. The effect of EVs isolated from two GMP-derived hNSC lines (Shef6.133.hNSC and UCI-191) on cognitive function, neuroinflammation, and synaptic integrity was tested. CRT-TMZ-exposed mice receiving 4 doses of EVs compared to vehicle-treated showed substantial improvement in learning and memory, and memory consolidation tasks (object recognition and fear extinction memory). Immunofluorescence analyses also showed significant improvements in synaptic integrity, reduced microglial activation, and astrogliosis in EVs-treated mice. Currently, RNAseq are being analyzed to characterize the miRNA content of the EV cargo. Future research aims to elucidate the mechanism of action, increasing the translational relevance of this regenerative therapy for radiation and chemotherapy-exposed brains.
Generation of 3D Printed Dorsal Spinal Neural Progenitor Cell Scaffolds for Spinal Cord Injury
1Department of Neuroscience, University of Minnesota, Minneapolis, MN, USA
2Department of Mechanical Engineering, University of Minnesota, Minneapolis, MN, USA
3Department of Neurosurgery, University of Minnesota, Minneapolis, MN, USA
4Stem 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. Cell transplantation is among the most promising treatments for SCI given its potential for targeted neural regeneration and repair in sensorimotor systems. However, while preclinical models utilizing cell transplantation have yielded promising outcomes, 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. The use of spinal scaffolds following SCI is known to provide a support structure that promotes regeneration, though this is not always sufficient to translate to behavioral recovery. Combinatorial approaches that utilize both scaffolds and cell transplantation may provide the necessary balance of cellular and structural support needed to promote meaningful improvement. For the injured sensory system where incoming signals are lost, relay formation is a critical step for restoring sensory function, though limited work has been done to produce the sensory spinal cell types needed to achieve this goal. Our lab has previously utilized a multi-channel silicone scaffold containing ventral spinal interneurons in a rodent SCI model demonstrating moderate functional motor recovery. We report here preliminary results generating and characterizing 3D-printed, stem cell derived dorsal spinal neural progenitor cell (dsNPC) scaffolds to target repair in sensory circuits. These cells form 3D assembloids within the scaffold and produce a diverse array of dorsal neuronal subtypes. In the future, dsNPC scaffolds will be transplanted to evaluate their capacity to attain correct repair and reconnection at the site of injury and beyond. This technology will push the field of spinal regeneration forward and advance the translation of cell transplantation therapies.
Optogenetic Enhancement of Neuronal Networks for Spinal Cord Injury Repair
Gladstone Institutes, San Francisco, CA, USA
Spinal interneurons are pivotal in modulating spinal neural network function, playing a vital role in both normal motor, sensory and autonomic function and post-injury plasticity. Transplantation of spinal interneuron progenitors after spinal cord injury (SCI) has demonstrated their ability to spontaneously survive and integrate with injured networks, contributing to improved functional outcome. The extent of this connectivity, however, can be variable. We propose that one way to address this is by increasing activity in neural networks (e.g., neural stimulation) which can enhance more consistent connectivity. Building on these Hebbian principles, we hypothesize that optogenetic activation of spinal interneuronal networks will increase neurotrophic factor production and neuronal connectivity, thereby fostering consistent integration that could be harnessed post-transplantation to improve repair and recovery. To test our hypothesis, we used optogenetic human induced pluripotent stem cells (hiPSC) to engineer pre-motor spinal interneurons previously transplanted into a model of cervical SCI. These cells were characterized through immunocytochemistry, single-cell RNA sequencing, and multi-electrode array (MEA) recordings. Optogenetic spinal interneurons were cultured on MEAs and stimulated with blue light every four days (20Hz for five-minute intervals) over four weeks. The electrophysiological activity was compared between stimulated an unstimulated cells, and the supernatant from each well was collected post-stimulation to assess neurotrophin (BDNF) synthesis/secretion via ELISA. Preliminary results revealed that stimulation enhanced neural network activity (increased neuronal spiking and synchronous bursting), and BDNF levels, suggesting improved neuronal support, maturation and connectivity. Accordingly, targeted neuronal stimulation via optogenetic stimulation can be used to improve network connectivity between engineered spinal interneurons. Future studies will focus on implementing these tools to enhance connectivity of these cells post-transplantation into the injured nervous system.
Defining the Role of Viral and Cellular Insulators in Promoting Durable HSV-1 Vector Mediated Transgene Expression to the Central Nervous System
1Department of Microbiology and Molecular Genetics, University of Pittsburgh School of Medicine, Pittsburgh, PA, USA
Herpes simplex virus 1 (HSV-1) replication defective vectors are promising gene therapy vectors for expression of large or multiple genes. We have developed safe HSV-1 gene transfer vectors by functional deletion of all immediate-early (IE) genes and the reiterated JOINT region flanking the unique long (UL) and unique short (US) genomic components, together providing a payload capacity of ~30kb. Deletion of the gene encoding the highly cytotoxic infected cell protein 0 (ICP0) completely abrogated viral gene expression and eliminated vector cytotoxicity in vivo. While this enhanced vector safety, the transgene payload was also rapidly silenced. Transgene expression was rescued by replacing either the latency associated transcript (LAT) or the ICP4 lytic gene with the transgene cassette close to naturally occurring viral insulator elements that help maintain euchromatin. The viral insulators are enriched in binding sites for the CTCF protein, a master regulator of chromatin structure. Surprisingly, the ICP4 locus was more permissive for transgene expression in differentiated SH-SY5Y neuroblastoma cells suggesting that the two loci are resistant to transgene silencing in a cell-specific manner. Following stereotactic injection of mouse hippocampus transgene expression from both loci lasted up to 5-months and was essentially restricted to neurons. However, the level of expression in brain neurons tended to decline over time. To enhance transgene expression, we designed a novel insulator construct by combining cellular and viral endogenous insulator elements. The new design included mouse tRNA genes and scaffold/matrix attachment regions (S/MARs) known to function as chromatin barrier elements. This novel combination of insulator elements provided significantly higher levels of transgene expression in the brain and eliminated the difference in transgene activity observed between the two loci. This new cassette design will ensure high and durable transgene activity in multiple tissue types and should prove beneficial for treating a wide range of brain disorders.
Role of Protein-R in cognition and pathology in a mouse model of amyloidosis
1Morsani College of Medicine, Molecular Pharmacology and Physiology Department; University of South Florida, Tampa FL, USA
Alzheimer’s disease is a progressive neurodegenerative disease characterized by cognitive impairments as well as deficits in brain metabolism, oxidative damage induced mitochondria dysfunction, and increased inflammation, which correlates with memory deficits and AD lesions. Protein-R is a scavenger protein for reactive oxygen species currently studied in metabolic diseases. Recently, Protein-R has been shown to decrease inflammation and have neuroprotective effects in models of traumatic brain injury. Hence, the objective of this study was to test if increased levels of Protein-R in the brain of a mouse model of amyloidosis would have beneficial effects in cognition and pathology. APP/PS1 mice aged 6 months old were injected intracranially in the hippocampus and anterior cortex, with either an empty Adeno-Associated Virus 9 (AAV9) vector or AAV9 vector expressing the Protein-R gene. Non-transgenic littermates were used as baseline for behavior and brain biomarkers. After 6 months of treatment, behavior testing was performed, followed by tissue collection. We show cognitive benefits in particular increased learning and memory during radial arm water maze in APP/PS1 mice treated with AAV9-Protein-R when compared to control. Tissue analysis revealed that these cognitive improvements are associated with an increase of antioxidant enzymes in the hippocampus but with no change in amyloid levels.
Repopulation of Microglia Following Partial Ablation Improves Cognitive Performance and Diminishes Neuroinflammation in a Mouse Model of Chronic Gulf War Illness
Institute for Regenerative Medicine, Department of Cell Biology and Genetics, Texas A&M University College of Medicine, College Station, Texas, USA
Over a third of the 700,000 United States military personnel who served in the first Gulf War display a range of unexplained persistent symptoms referred to as Gulf War Illness (GWI). Epidemiological studies have implied that GWI has likely resulted from exposure to pesticides like permethrin and the nerve gas prophylactic drug pyridostigmine bromide. Cognitive dysfunction observed in GWI has been linked to chronic neuroinflammation in the CNS. Previous studies in animal models displaying chronic neuroinflammation have shown that short-term pharmacological ablation of activated microglia using a small molecule PLX5622 results in the repopulation of microglia with homeostatic function, leading to improved cognitive function associated with reduced neuroinflammation. We investigated whether transient ablation of microglia in chronic GWI could also alleviate cognitive impairments by reducing neuroinflammation. C57BL6 mice received permethrin and pyridostigmine bromide in DMSO (i.p.) for ten days, which led to cognitive dysfunction in an object location test (OLT) at ten months post-exposure. A cohort of GWI mice displaying object location memory (OLM) dysfunction next received PLX5622 via diet for 28 days, which resulted in a 70% depletion of microglia in the cortex and 64% in the hippocampus. When examined immediately after partial microglial ablation, GWI mice demonstrated the persistence of OLM impairment. However, PLX5622-treated GWI mice showed improved OLM thirty days after PLX5622 withdrawal, compared to age-matched GWI mice receiving no treatment displaying incessant OLM impairment. Analyses of microglia and astrocytes in the cerebral cortex and the hippocampus revealed signs of microgliosis and astrocyte hypertrophy in untreated GWI mice, in contrast to PLX5622-treated GWI mice exhibiting reduced microgliosis and astrocyte hypertrophy. The results underscore that persistent cognitive dysfunction and neuroinflammation in chronic GWI could be alleviated by spontaneous repopulation of microglia following their partial ablation.
Iron Chelator Mitigates Neurodegenerative Effects of Excess Iron After Subarachnoid Hemorrhage
1University of Florida College of Medicine, Department of Neurosurgery, Gainesville, Florida, 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. Up to 40% of survivors will experience secondary injuries such as cerebral vasospasm, neurobehavioral deficits, or even death. The severity of the disease illustrates the need for better understanding of the pathways of SAH and better treatments to prevent these outcomes. SAH results in excess blood degrading in the area, leading to a buildup of toxic iron byproducts that causes irreversible neuronal damage by instigating an inflammatory response via intracellular iron-dependent cell death, ferroptosis. Microglia aggregate to the injured region to remove the iron but cannot reverse damage. Our hypothesis is that excess iron buildup is toxic to neuronal and microglial cells and leads to ferroptosis of these cells, resulting in neurodegenerative effects. Female C57BL/6 mice were used in an autologous blood injection SAH model and groups received either an iron chelator treatment with deferoxamine (DFO) or vehicle. Artery measurements suggested that DFO treated mice have less vasospasm than both sham and vehicle groups on days 1 and 5 post-SAH. Immunofluorescence staining indicated greater microglial activation and ferritin presence in SAH tissues without DFO treatment. Rotarod testing also suggested DFO tissues had slightly improved outcomes compared to vehicle SAH mice. These results suggest that iron is potentially a major contributor to cellular death and neurodegenerative effects of SAH, and that removing excess iron quickly after SAH could be protective of neurons and microglia. With further study, this could lead to use of an iron chelator in the clinic to prevent ferroptosis before irreversible damage occurs, thus limiting the mortality and long-term disability of the disease.
Improved Brain Function Mediated by Extracellular Vesicles from hiPSC-NSCs in 5xFAD Mice is Linked with Enhanced Hippocampal Neurogenesis and Reduced mTOR Signaling
Institute for Regenerative Medicine, Department of Cell Biology and Genetics, Texas A&M University School of Medicine, Bryan/College Station, Texas, USA
Alzheimer's disease (AD) is a type of late-onset dementia that is characterized by the accumulation of amyloid plaques and formation of neurofibrillary tangles which lead to neuronal loss and cognitive impairments. Early changes in AD include reduced hippocampal neurogenesis and hyperactivation of the mechanistic target of rapamycin (mTOR) signaling in the brain. Disruption of hippocampal neurogenesis in AD contributes to cognitive and memory impairments. Increased mTOR signaling can lead to neuroinflammation, impair autophagy, and cause phagocytic dysfunction in microglia, causing the accumulation of amyloid plaques. This study examined the effectiveness of extracellular vesicles (EVs) released by human-induced pluripotent stem cell (hiPSC)-derived neural stem cells (hNSCs) for alleviating neurogenesis decline and hyperactivation of mTOR signaling in 5x familial AD (5xFAD) mice, a model of early-onset AD. Three-month-old 5xFAD mice received intranasal administrations of either hiPSC-NSC-EVs (~30 billion/week for two weeks, AD-EVs group) or the vehicle (AD-Veh group). Two months later, the effects of treatment on hippocampus-dependent cognitive function and anhedonia were measured through object location and sucrose preference tests. Brain tissues were processed for doublecortin immunostaining to quantify hippocampal neurogenesis. Accumulation of phosphorylated ribosomal protein S6 (pS6) accumulation within neurons was also assessed, as increased pS6 accumulation indicates activated mTOR signaling. The animals in the AD-EVs group exhibited no cognitive or mood impairments. In contrast, animals in the AD-Veh group displayed object location memory dysfunction and anhedonia. Maintenance of a higher level of hippocampal neurogenesis was observed in the AD-EVs group compared to the AD-Veh group. A reduced pS6 accumulation, implying diminished mTOR signaling, was also observed within neurons in the AD-EVs group. The results suggest that intranasal administrations of hiPSC-NSC-EVs maintain better cognitive and mood function in AD mice for extended periods. Notably, such improvements in the AD-EVs group were associated with higher levels of hippocampal neurogenesis and attenuated mTOR signaling. This study was supported by a grant from the National Institute for Aging (1RF1AG074256-01A1 to A.K.S).
A Rat-Based Progressive Overload Resistance Exercise Task for Research in Aging and Age-Related Neurodegenerative Disease
1Department of Cell Biology and Physiology, University of Kansas Medical Center
2Department of Neurology, University of Kansas Medical Center
3Department of Psychiatry and Behavioral Sciences, University of Kansas Medical Center
Preclinical studies can reveal mechanisms underlying the effects of exercise. Most animal studies have focused on aerobic exercise protocols. Our goal was to determine effects of a progressive overload resistance exercise (RE) task in different groups of rats. We trained young and middle-aged male and female rats to climb a ladder with weights attached to their tails. Each rat’s maximum load (MAX) was determined on Mondays and then rats climbed eight times with increasing percentages of MAX on Wednesdays and Fridays. In young rats, MAX was initially greater in the males, but females exceeded males by week 6. The ratio of MAX to body weight was greater in the females across all testing sessions. An aging comparison revealed that MAX was similar for young and middle-aged males but was greater in young than older females. The ratio of MAX to body weight was greater in young rats of both sexes. We compared blood plasma levels of neurofilament light (NFL) and glial fibrillary acidic protein (GFAP) in male rats as young adults and at middle-aged (the same rats). NFL increased with age but did not differ between RE and sedentary (SED) males. Striatum and hippocampus were harvested from the RE and SED middle-aged male rats and processed for proteomic analysis. Neurofilament proteins and GFAP were significantly greater in RE than SED striatum, but not hippocampus. This suggests a disconnect between these peripheral markers and their CNS activity. Our results reveal sex- and age-related differences in RE performance and effects of RE in rats. Proteomic data provide targets for future studies to determine the functional significance of up- and downregulated genes and how RE can prevent sarcopenia and improve brain health.
Synergistic Delivery of Thermostabilized Enzyme and Human Neural Progenitor Cells via Tailored Hydrogels Enhances Recovery after Stroke
1Department of Chemical Engineering and Applied Chemistry, University of Toronto, Toronto, ON, Canada
2Institute of Biomedical Engineering, University of Toronto, Toronto, ON, Canada
3Department of Surgery, University of Toronto, Toronto, ON, Canada
4Department of Chemistry, University of Toronto, Toronto, ON, Canada
Stroke stands as a leading global contributor to morbidity and long-term disability, lacking clinical interventions for promoting tissue regeneration. While transplanting neural cells is a promising approach, cell survival poses a significant challenge. To overcome these obstacles, we designed an injectable hyaluronan-based hydrogel that mimics the brain’s extracellular matrix and supports the survival and neuronal differentiation of human induced pluripotent stem cell (iPSC)-derived neural progenitor cells (iPSC-NPCs). We encapsulated iPSC-NPCs in the hydrogel, injected them through a fine needle, and showed high cell viability and differentiation for at least 14 days in vitro. To further optimize cell survival and promote neuroplasticity, we co-delivered chondroitinase ABC (ChASE), which we re-engineered to be more thermally stable with 37-point mutations (ChASE37). Similar to its native counterpart, we showed that ChASE37 effectively degrades inhibitory chondroitin sulfate proteoglycans (CSPGs) that are secreted post-injury to the central nervous system. We expressed the enzyme as a fusion protein with a Src homology 3 (SH3) domain (SH3-ChASE37) and modified an injectable crosslinked methylcellulose hydrogel with SH3 binding peptides, enabling affinity-controlled release. This hydrogel attenuated the release of bioactive SH3-ChASE37, and demonstrated efficacy by CSPG degradation both in vitro and in vivo. We co-delivered iPSC-NPCs and SH3-ChASE37 directly to the brain of endothelin-1-induced stroke-injured rodents, and observed improved motor function relative to vehicle controls. We are currently examining the fate of the transplanted cells in terms of promoting tissue repair and the impact of SH3-ChASE37 release on CSPG degradation and tissue plasticity. This multifaceted approach holds immense potential for advancing stroke therapeutics by addressing key challenges in cell survival and promoting neuroplasticity.
This work was supported by the Stem Cell Network, Heart & Stroke Foundation, CFREF through Mend the Gap, and PRiME clinical catalyst fellowship.
Investigating the molecular mechanisms of adipose stem cell derived exosomes for prevention of neurodegeneration in a-synuclein model of Parkinson’s Disease
1University of South Florida, Tampa FL
2James A Haley VA Hospital, Tampa FL
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 research 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 (hASC) to treat PD. To model PD, we injected recombinant adeno-associated virus (rAAV) expressing a-synuclein (Syn) into the substantia nigra (SN) of male Fisher rats. Unilateral injection into the SN of rat’s 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, then divided animals into groups to receive 2uL of either rAAV9-Syn or the control vector rAAV9-mKate (0.5 x 1013 vector genomes/mL). Three weeks post-injection animals were split to received intranasal administration of either: 100ug hASC exosomes or PBS (control). Weeks four and eight behavior was conducted again. Once complete rats were euthanized and immunohistochemistry was conducted to determine the extent of cell loss in the SN, and we conducted single cell RNA sequencing (scRNA seq) on the SN to observe any changes in microglia, astrocytes, and T cells. We found dopaminergic neurons were preserved in animals that received 100ug of exosomes and found exosomes potentially play a role in reducing microglia activation in the SN. For scRNA seq, we expect to observe differences in microglia, astrocytes, and T cells between Syn+exosomes and Syn+PBS treated animals. In exosome treated animals, we expect to observe increased homeostatic microglia compared to Syn+PBS animals. If our hypothesis is correct, exosomes are a potential treatment to slow the progression of PD.
The Effects of a Supportive Enriched Environment on Neurological Function Recovery of Chronic Severe TBI in a Murine Model
Department of Neurosurgery, SUNY Upstate Medical University, Syracuse, NY 13210, United States of America
Severe traumatic brain injury (sTBI) causes permanent disability in adults worldwide. Although enriched environments (EE) have been shown to improve TBI recovery, it remains unknown about the efficacy of EE in the chronic settings of sTBI. The objective of this study is to evaluate neurological function recovery in chronic sTBI mice housed in EE or a supportive enriched environment. Adult male C57BL mice were subject to sTBI by controlled cortical impact. After sTBI, mice returned to standard environments (SE) for 7 months. Thereafter, sTBI mice were randomly assigned to standard (TBI-SE), enriched (TBI-EE-1), or supportive enriched environments (TBI-EE-2). Sham mice were housed in either the standard environment (Sham-SE) or supportive enriched environment (Sham-EE-2). TBI-EE-1 mice were housed in a large 1m-diameter tank with toys changed 3 times a week (EE tank), and the EE-2 combined TBI-EE-2 mice with Sham-EE-2 mice in another EE tank. Mice remained in these environments for 10 weeks, with a battery of neurobehavioral tests beginning in week 6. In RotaRod test, motor impairments were observed in TBI-SE mice, while TBI-EE-2 mice showed robust motor functional recovery. The motor function in TBI-EE-1 mice was improved, but it was not as robust as TBI-EE-2 mice. Among the TBI mice, only the TBI-EE-2 mice showed improvements in motor learning. In Morris water maze test, both TBI-EE-2 and TBI-EE-1 mice showed better performance in spatial learning and memory than the TBI-SE mice. In Y-maze test, decreased short-memory was seen in TBI-EE-1 mice, while TBI-EE-2 mice showed short-memory improvements. Anxiety-like behavior observed in open field and light-dark box tests was also reduced in the TBI-EE-2 mice. These findings suggest that supportive enriched environments, compare to EE alone, are more effective in reducing anxiety and improving motor function and cognitive function in the chronic phase of severe TBI.
High-resolution spatial mapping of neuroimmune interactions after mouse spinal cord injury
1Division of Neuroscience, Department of Brain Sciences, Imperial College London, London, UK
Axonal regenerative failure restricts neurological recovery after traumatic spinal cord injury (SCI). The intricate interactions between surviving neurons and the surrounding cells are determinant for the success of axonal growth and reconnection, but there is limited understanding of their spatiotemporal orchestration. Glial reactivity and immune cell infiltration following injury lead to complex and dynamic microdomains that could affect axons differently throughout the tissue. Mapping these cellular interactions is pivotal for the development of targeted therapies for SCI. We performed high-resolution spatial transcriptomics, using deterministic barcoding in tissue (DBiTseq) to map and characterize microdomains of interest in the SCI site. We integrated the results with single-cell RNA sequencing (scRNAseq) data from the injury site to identify specific cell types contributing to axonal regenerative failure. Our findings offer insights into SCI pathophysiology and pave the way for novel therapeutic interventions favoring axonal regeneration.
The Impact of Age on Stem Cell Transplantation and Gene Therapy
Texas A&M, Bryan, TX, USA
Spinal cord injury (SCI) is a lifelong disease that results in countless negative life changes, the most visible of which is paralysis and sensory loss below the site of injury. One of the most promising strategies for recovering motor and sensory ability after SCI is grafting stem cells into the site of injury. These grafts can potentially serve as relay stations between axons rostral of the injury site and their targets caudal and may also enhance regrowth of the host axons. However, such regrowth is limited with very few host axons existing in the graft. Here, we first interrogate if combining stem cell transplantation in the spinal cord with gene therapy targeting the cortico-spinal neurons can further enhance axon growth of the cortico spinal tract (CST) observed with each individual strategy. In addition, while it is known that aging reduces axon growth potential even after gene therapy, the impact of age on stem cell grafts has not been explored. Therefore, in this study we also examine how age modulates stem cell graft survival and integration, and whether combining stem cell transplantation and gene therapy can reduce the age-dependent effect on CST regeneration. Specifically, we determine how age impacts stem cell graft survival (size and spread), how the graft impacts CST axon growth after gene targeting (within the graft and exiting the graft caudal of the injury site), and how age influences CST axon growth in the presence of both the graft and gene targeting. This work demonstrates that age can impact both stem cells transplantation and gene therapy and its impact should be better characterized to increase the potential of combining these strategies in the future.
The Impact of Neutrophil Depletion on Aneurysm Healing
1Department of Neuroscience, University of Florida, Gainesville, Florida, USA
2Department of Microbiology and Cell Science, University of Florida, Gainesville, Florida, USA
3Department of Neurosurgery, University of Florida, Gainesville, Florida, USA
Intracranial aneurysm formation arises from endothelial dysfunction or injury, causing thinning of the intracranial arteries and an outward bulge that can lead to rupture. If untreated, this condition may result in a subarachnoid hemorrhage. Coil embolization is an endovascular technique used to prevent aneurysm rupture; however, this technique has a high recanalization rate for ruptured and unruptured aneurysms, possibly due to incomplete aneurysm occlusion leading to recurrence. Therefore, efforts should be concentrated on developing treatments that could be used concurrently with this endovascular technique to enhance the aneurysm healing process. This study focuses on the cellular-mediated wound healing in aneurysms, particularly the role of neutrophils, first responder cells that are recruited to injury or inflammatory sites. Neutrophils initiate the healing process through immune response activation and inflammatory cytokine release. However, exacerbation of this response may impede aneurysm healing. We hypothesize that neutrophil depletion can enhance aneurysm healing by reducing neutrophil infiltration and promoting tissue ingrowth. Using female C57BL/6 mice, carotid aneurysms were induced, and a PLGA-coated coil was inserted using the murine carotid artery embolization model. Neutrophil depletion was achieved through anti-Ly6G antibody injections 7 days before the coil embolization procedure and every other day thereafter for 21 days. The control group received rabbit serum injections and followed the same timeline. Tissues were analyzed at 21 days post-coiling using immunohistochemistry to assess tissue ingrowth and neutrophil infiltrates. The results indicated enhanced tissue ingrowth and reduced neutrophil infiltration in the neutrophil-depleted group compared to controls. While suggesting a positive impact on aneurysm healing, it’s essential to note the impracticality of clinical neutrophil depletion. Further studies should explore the downstream pathways and inflammatory effector functions of neutrophils to develop strategies that modulate inflammation and enhance aneurysm healing without directly depleting neutrophils.
Novel Nano-gene therapeutic approach for treating SARS-CoV-2 induced tauopathy
1Department of Molecular Medicine, Morsani College of Medicine, University of South Florida, Tampa, FL, USA
2Department of Internal Medicine, Morsani College of Medicine, University of South Florida, Tampa, FL, USA
3Byrd Alzheimer’s Research Institute, University of South Florida, Tampa, FL, USA
4Center of Excellence for Aging and Brain Repair, Departments of Neurosurgery and Brain Repair, and Molecular Pharmacology and Physiology, Morsani College of Medicine, Tampa, FL, USA
5Taneja College of Pharmacy Graduate Programs, University of South Florida, Tampa, FL
6James A Haley VA Hospital, Tampa, FL, USA
The SARS-CoV-2 (CoV2) pandemic has left large numbers (>100 million) of Americans suffering from post-acute sequelae of CoV2 (PASC, or long-COVID). A majority of PASC patients report ongoing neurological sequelae known as neuro-COVID. Specifically, the role of CoV2 infection-triggered changes in the brain that increase the risk of neurologic disorders, such as tauopathy remains unclear. We hypothesize that heightened inflammatory response in the brain due to severe SARS-CoV-2 infection drives the early onset of tauopathy leading to dementia. Our gene expression studies with the SARS- CoV-2 infected brains led to the identification of a hub gene Fkbp5 that is linked to microtubule-associated protein tau (MAPT) expression, which is key to tauopathy. The Fkbp5 gene encodes for FK506-binding protein 51 (FKBP51) protein. FKBP51 and Heat shock protein 90 (Hsp90) synergize to induce tau hyperphosphorylation and tau oligomerization promoting tau accumulation in the brain. Gene silencing by shRNA is a powerful RNAi strategy that offers a safe, target-specific attenuation of protein expression. However, the efficacy of the gene therapy depends on the successful delivery of the gene to the target site. Hence we developed a novel nano-gene-delivery platform using dendrimers complexed with plasmids encoded with shRNA. We treated (intranasally) SARS-CoV-2 Mouse Adapted 10 infected C57BL6 mice with dendriplexes (DPX) comprising PAMAM dendrimers (polyamindoamines) and plasmids encoding 4 different shRNAs targeting Fkbp5 (pshFkbp5) as the payload. The downregulation of Fkbp5 expression significantly reduced GFAP expression and IBA1 expression in the olfactory bulb, cortex, and hippocampus of pshFkbp5 DPX treated mice. Pathological evidence also revealed that pshFkbp5 DPX treatment significantly reduced tau phosphorylation (pTauT231) and oligomerization (Tau T22) in the olfactory bulb, cortex, and hippocampus when compared to the infection-only or pshScr DPX group. In summary, our approach has the potential to ameliorate the SARS-CoV-2 infection-induced abnormal tau accumulation using the novel nano-gene-delivery of pshFkbp5 nanodendriplexes to the brain.
Investigating the Neuroprotective Effects of ACMSD in a Synergistic Model of α-syn/LPS
1Parkinson’s Disease Research Unit, Barrow Neurological Institute, Phoenix, AZ, USA
2Barrett, the Honors College, Arizona State University, Tempe, AZ, USA
3Van Andel Research Institute, Grand Rapids, MI, USA
Alpha-synuclein (α-syn) plays an important role in synaptic function, but its aggregated form(s) are heavily implicated in the pathology of Parkinson’s disease (PD). Moreover, environmental exposures including inflammation are thought to influence α-syn aggregation. We therefore examined the synergistic effects of α-syn overexpression in a neuroinflammatory environment. Intrastriatal delivery of lipopolysaccharide (LPS) activates microglia and results in nigrostriatal lesions, thereby impairing motor function and recapitulating PD pathology. Moreover, LPS induces indoleamine 2,3-dioxygenase (IDO1/2), the rate-limiting enzyme of the kynurenine pathway (KP), which is dysregulated in PD. We performed intrastriatal injections (4 sites) of 30 micrograms (ug) of lipopolysaccharide (LPS) in both naïve and experimental adult Sprague Dawley rats which four months prior, had received unilateral injections of a “sub-toxic” dose of adeno-associated virus (AAV) expressing human α-syn together with either FLEX-GFP as control or with amino-3-carboxymuconate 6-semialdehyde decarboxylase (ACMSD), to the substantia nigra. As our previous studies demonstrated that ACMSD provides neuroprotective effects in the AAV-α-syn PD animal model, an enzyme that resides within the KP, we hypothesized that ACMSD would prevent the hypothesized deleterious synergistic effects of LPS and AAV-α-syn. One week following intrastriatal injections of LPS, motor function was assessed, and animals were thereafter euthanized. The striatum was dissected for biochemical analyses of KP metabolites, CSF was collected for measurement of proinflammatory cytokines, and the substantia nigra was processed for histological analyses. We found that LPS induces profound motor dysfunction, which was preserved in animals previously treated with AAV-α-syn/ACMSD suggesting neuroprotective effects by ACMSD in the LPS intrastriatal model of neurodegeneration. Paradoxically, animals injected with AAV-α-syn/FLEX-GFP likewise showed preservation of motor function in the test, a finding suggesting that α-syn at “sub-toxic” levels acts as a neuromodulator to influence the nigrostriatal pathway, a finding congruent with the role of α-syn in synaptic function. Quantitative histological analyses are undergoing.
Harnessing Histone Deacetylase 6 Inhibition: A Breakthrough Therapy for Lower Spinal Cord Spasticity
1Department of Neurobiology and Anatomy, College of Medicine, Drexel University, Philadelphia, PA, USA
Hereditary Spastic Paraplegia (HSP) is an inherited neurodegenerative disorder marked by the progressive weakening of lower limb muscles, spasticity, and profound gait impairments, primarily attributed to the degeneration of the corticospinal tract—a bundle of axons for upper motor neurons. SPAST that encodes spastin, a microtubule severing enzyme, is the most common gene mutated in HSP. SPAST-based HSP, also known as SPG4, is driven by gain-of-toxicity mechanisms and currently lacks effective treatment options. To tackle this challenge, we have developed a double-heterozygous transgenic mouse model that closely mimics SPG4 genetics, expressing a human spastin variant with the C448Y mutation alongside just one functional mouse spastin allele. Concurrently, we have cultivated human forebrain organoids derived from isogenic human induced pluripotent stem cell (hiPSC) lines, each carrying a distinct SPAST mutation. These hiPSC-derived organoids are enriched with upper motor neurons, the cell type most vulnerable in HSP. In light of emerging evidence implicating hyperactive histone deacetylase 6 (HDAC6) in the gain-of-toxicity mechanisms, we are actively investigating HDAC6 as a promising therapeutic target. Our approach encompasses the administration of a highly selective HDAC6 inhibitor, tubastatin A, in both the transgenic mouse and hiPSC-organoid models. Encouragingly, systemic administration of tubastatin A in both models did not elicit any overt toxicity. Moreover, our experimental findings demonstrated its potential to ameliorate functional deficits and effectively rescue anatomical axon degeneration features in these models. Our research extended its impact to postmortem brain tissues from HSP patients, revealing a consistent hyperactivity of HDAC6, thereby underscoring its relevance in HSP pathogenesis. In all, these findings robustly endorse the prospect of HDAC6 inhibitors as a viable avenue for rectifying the therapeutic shortfall in treating SPG4, HSP, lower spinal cord injury, and promoting neural repair.
Strain-Specific Variations in Motor Recovery Following Spinal Cord Injury: A Comparative Study in Sprague-Dawley and Wistar Rats
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 Psychology, Central Michigan University, Mount Pleasant, MI, USA
Rodent models of spinal cord injury (SCI) are critical for testing potential treatments. Reports of strain-dependent differences in spontaneous motor recovery from SCI in rats often lack comprehensive assessments of locomotive function, gait, step precision, inter-limb coordination, and pathophysiology following severe and moderate SCI. This study compared the hindlimb movement aspects and related pathophysiology of both male and female SD and Wistar rats following moderate and severe SCI. The clip-compression model of SCI was used to induce moderate (15-gram clip for 180 seconds) or severe (50-gram clip for 60 seconds) SCI in SD (males, n=6; females, n=6) and Wistar (males, n=6; females, n=6) rats. A battery of behavioral tests (including spontaneous locomotor evaluations, CatWalk, and horizontal ladder) were conducted preoperatively and weekly for seven weeks. Fresh samples were extracted from half of the rats for Western blotting with Iba1 antibody, while the others underwent perfusion for either transverse cavity size measurement (eosin hematoxylin staining) or longitudinal demyelination assessment (Luxol fast-blue staining) using ImageJ. SD rats showed greater spontaneous motor recovery assessed by Basso, Breattie, and Bresnahan (BBB) assessments than Wistar rats after moderate SCI (p=0.008), but not after severe SCI. Additionally, no significant differences were observed between the strains in horizontal ladder testing and Iba1 antibodies on Western blotting for either moderate or severe SCI. Results from CatWalk showed that SD rats had significantly greater hindlimb print width (p=0.022) and swing speed (p=0.028) relative to Wistar rats after moderate SCI. Moreover, histology indicated that both severe and moderate Wistar rats had larger cavity sizes than the SD rats with the same models (p<0.05). In conclusion, our study revealed that SD rats exhibited more spontaneous motor recovery following moderate SCI, while Wistar rats had larger cavity sizes, emphasizing the relevance of strain-specific considerations in assessing SCI outcomes.
Support for this study was provided by the Neuroscience program, the College of Medicine, Office of Research and Sponsored Program, the E. Malcolm and Gary Leo Dunbar Endowed Chair, and the John G. Kulhavi Professorship in Neuroscience at CMU.
UBE3A and tauopathy
A. Joly-Amado1, J. Pena1, A. Zitnyar2, H. Choudhary1, E. Stewart1, U. Jinwal2, and
1Morsani College of Medicine, Molecular Pharmacology and Physiology Department; University of South Florida, Tampa FL
2Taneja College of Pharmacy, Department of Pharmaceutical Sciences; University of South Florida, Tampa FL
Post translational modifications (PTMs) play a significant role in normal protein function, but can also be instrumental in disease pathology. Hyperphosphorylation of the Tau protein has been extensively studied and tied to its aggregation in tauopathies. However, other PTMs of Tau (methylation, ubiquitination, acetylation) which likely contribute to Tau pathology are less studied. Ubiquitination of proteins is critical for signaling a protein for degradation with the ubiquitin-proteasome system (UPS). It is well known that the UPS is disrupted in neurodegenerative diseases such as Alzheimer’s disease (AD). While most ubiquitin chains can target proteins for proteasomal degradation, ubiquitination can contribute to other functions within the cell, including protein localization, protein activity, endocytosis, transcription and autophagy. Ubiquitin ligase 3A (UBE3A) an E3 ligase is well known for its critical involvement in normal synaptic plasticity and has garnered much attention in autism spectrum disorders, which may have intersectionality with diseases like AD. A genome wide association neural networks study has linked UBE3A with AD, and transcriptomics has shown reduced UBE3A in human AD and mouse models. It has been proposed that loss of UBE3A may be a contributor to dendritic spine loss in AD. We examined long term overexpression of UBE3A through gene therapy in a mouse model of tauopathy, Tg4510 mice. Three-month-old Tg4510 and non-transgenic mice were injected in the hippocampus and cortex with AAV9-GFP or AAV9-UBE3A. Behavior testing was performed after 2 months of viral expression, followed by brain tissue collection. We observed improvement in cognitive function and a decrease in phospho-tau. We also show in vitro that Tau can be ubiquitinated by UBE3A. This suggests that increasing UBE3A activity in AD could be a potential therapeutic avenue, maybe by increasing the ubiquitination and degradation of tau.
CRISPR inactivation strategies for ALS/FTD and other dominant neurogenetic diseases
1Gladstone Institutes, San Francisco, CA, USA
2University of California San Francisco, San Francisco, CA, USA
3University of California, Berkeley, Berkeley, CA, USA
4Innovative Genomics Institute, Berkeley, CA, USA
Inactivation of a mutant allele could be therapeutic for many dominant genetic diseases. CRISPR/Cas systems have successfully been deployed to knockout disease alleles by targeting the causative mutation directly. However, for most diseases, this mutation-dependent approach is not scalable due to a diversity of rare mutations, absence of PAMs, or poor gRNA discrimination between mutant and wildtype alleles. Instead, we developed a CRISPR platform that takes advantage of single nucleotide polymorphisms (SNPs) to inactivate any mutant allele that occurs in cis with common SNPs. Targeting a single coding SNP creates indels that knockout expression of the mutant allele, while targeting pairs of non-coding SNPs can excise the mutant allele entirely. >60 dominant mutations in fused in sarcoma (FUS) have been linked to amyotrophic lateral sclerosis (ALS) and frontotemporal dementia (FTD). We identified two coding SNPs whose alleles can each be targeted by CRISPR/Cas9 gRNAs. Based on the prevalence of these SNPs across human populations, optimization of just four gRNAs could be therapeutic in up to 64% of patients. To compare editing strategies, we engineered isogenic FUS mutations into a human iPSC line that is heterozygous at both SNPs, then tagged the endogenous FUS genes with either GFP or HaloTag in order to track the wildtype and mutant proteins independently. CRISPR/Cas9 gRNAs targeting each of the four SNP alleles demonstrate efficient and specific generation of indels, functional knockdown of mutant protein, and prevention of disease phenotypes. We are now optimizing lipid nanoparticle delivery of Cas9 and gRNA directly to in vitro iPSC-neurons and in vivo rodent models in order to asses editing and rescue of disease phenotypes in a neuron-specific context. Together with new bioinformatics algorithms capable of mapping all SNP-gRNA around a given gene, these studies demonstrate new strategies for CRISPR gene inactivation with broad applications to dominant disease.
Engineered Spinal Cord Organoids: A Novel Cell Therapy Approach to Repair the Injured Spinal Cord
1Department of Neurobiology and Anatomy, College of Medicine, Drexel University, Philadelphia, PA, USA
2Gladstone Institutes, University of California San Francisco, San Francisco, CA, USA
Spinal cord injuries (SCIs) inflict profound damage and loss of local neurons and their associated circuitry, yielding severe functional impairments. The majority of SCIs occur at the cervical level, often compromising the phrenic circuit responsible for diaphragm function, culminating in impaired breathing. Evidence suggests that neuroplasticity reliant on preserved tissue following cervical SCIs supports only limited functional recovery. To address this limitation, the present work uses cell-based therapies to promote neural repair. Prior work has shown that transplanting excitatory V2a spinal interneurons can contribute to novel neural relays within the phrenic circuit that improve function. Moreover, recent advancements in self-organizing spinal cord organoids have positioned themselves as an even more sophisticated engineered tissue source for SCI cell replacement therapies. These organoids recapitulate the histological architecture of the developing spinal cord and feature a diverse array of interneurons, including V2a phenotypes. In this study, we investigated the transplantation of human induced pluripotent stem cell-derived, ventralized, cervical spinal cord organoids, consisting of a diverse interneuron population, as a prospective therapy for cervical SCI. We characterized the organoid composition and regional cell identities prior to transplantation into the epicenter of a cervical 3/4 lateral contusion injury in adult Sprague Dawley rats. We used immunohistochemistry, retrograde pseudorabies virus tracing, and terminal electromyography to characterize transplant survival and composition, elucidate donor-to-host connectivity with the host phrenic circuit, and assess functional outcomes. This pilot project serves as a proof-of-concept, innovative therapeutic avenue with the potential to significantly advance SCI treatment.
iPSCs-based Regenerative Therapy for Spinal Cord Injury
H. Okano
Keio University, Japan
Since the birth of human induced pluripotent stem cell (iPSC) technology in 2007, its application in regenerative medicine has significantly advanced. Our research focuses on iPSC-based cell therapy for spinal cord injury (SCI), a condition that leads to motor and sensory dysfunctions due to axonal degeneration and the death of neurons and glial cells following ischemia-induced secondary injury. We have developed a method involving the transplantation of allogeneic iPSC-derived neural stem/progenitor cells (NS/PCs) into patients with complete SCI in the subacute phase. Preclinical studies have demonstrated that these transplanted NS/PCs can induce long-term functional recovery in SCI model animals without causing tumor formation.
Utilizing a chemical genetic approach, specifically DREADD technology, we have shown that the activity of graft-derived neurons and synaptic formation between host and graft neurons are crucial for the functional recovery. This underlines the importance of cell replacement therapy in contributing to the restoration of SCI functions.
In December 2021, Keio University initiated the first-in-human (FIH) clinical research, transplanting human iPSC-NS/PCs into patients with complete SCI in the subacute phase. This research is conducted under the Safety of Regenerative Medicine Act, using clinical-grade, integration-free hiPSC lines prepared in a Good Manufacturing Practice facility. The cells undergo rigorous quality control, including genome and safety analysis, before transplantation. Approximately 2 million iPSC-NS/PCs are transplanted into the injury's epicenter after being processed through a series of steps to promote differentiation.
The recruitment for this FIH study has been completed, with patients showing good postoperative conditions. Efforts are ongoing to prepare for clinical trials targeting patients in the chronic phase of SCI. These efforts include enhancing the therapeutic potential of iPSC-NS/PCs through ex vivo gene therapy, improving the spinal cord's microenvironment, and integrating rehabilitation techniques.
Characterizing White Matter Stroke Behavioral Phenotype Through Machine Learning
1Department of Neurosurgery, Stanford School of Medicine, Stanford University. Palo alto, California
White matter strokes (WMS) occur in the deep penetrating blood vessels of the brain and makeup 30% of stroke subtypes. Previous research has shown that WMS leads to impairments in forelimb usage, motor coordination, learning, memory, and other cognitive domains with limited recovery over time. However, most studies have focused primarily on individual behavioral measurements rather than characterizing overall behavioral differences across multiple domains. They have also typically examined mouse behavior in constrained environments rather than observing behaviors expressed naturally. To address these gaps, we used Motion Sequencing (MoSeq) to automatically identify a broad array of mouse behavioral motifs through analysis of 3D depth videos of young adult mice moving freely in an open arena. We used MoSeq to comprehensively track alterations in these behavioral motifs before and after white matter stroke. Our results showed that MoSeq could identify distinct differences in behaviors such as vertical rearing, jumping, darting, grooming, and pausing. Comparative analysis between pre-WMS and post-WMS mice revealed detectable changes in the expression of these behavioral syllables indicative of an exclusive WMS-associated phenotype, including changes in velocity and duration. Our findings were validated through the use of a machine learning classifier which, given a summary of MoSeq syllables expressed in a video, classified them as WMS-affected or unaffected with a high rate of accuracy. This demonstrates that automated behavioral analysis with MoSeq enables the characterization of overall behavioral alterations following WMS with high granularity.
Electrical stimulation affects the differentiation of transplanted regionally specific human spinal neural progenitor cells (sNPCs) after chronic spinal cord injury
1Department of Neurosurgery, Stem Cell Institute, University of Minnesota, Minneapolis, MN, USA
2Massachusetts Institute of Technology, 77 Massachusetts Ave, Cambridge, MA, USA
3Department of Neuroscience, University of Minnesota, Minneapolis, MN, USA
4Department of Mechanical Engineering, University of Minnesota, Minneapolis, MN, USA
There are currently no effective clinical therapies to ameliorate the loss of function that occurs after spinal cord injury. Electrical stimulation of the rat spinal cord through the rat tail has previously been described by our laboratory. We propose combinatorial treatment with human induced pluripotent stem cell-derived spinal neural progenitor cells (sNPCs) along with tail nerve electrical stimulation (TANES). The purpose of this study was to examine the influence of TANES on the differentiation of sNPCs with the hypothesis that the addition of TANES would affect incorporation of sNPCs into the chronically injured spinal cord, which is our ultimate goal. We found that sNPCs were multi-potent and retained the ability to differentiate into mainly neurons or oligodendrocytes after this transplantation paradigm. The addition of TANES resulted in more transplanted cells differentiating into oligodendrocytes compared with no TANES treatment, and more myelin was found. TANES not only promoted significantly higher numbers of sNPCs migrating away from the site of injection but also influenced long-distance axonal/dendritic projections especially in the rostral direction. Further, we observed localization of synaptophysin on STEM121 (human cytoplasmic marker) positive cells, suggesting integration with host or surrounding neurons, and this finding was enhanced when TANES was applied. Also, rats that were transplanted with sNPCs in combination with TANES resulted in an increase in serotonergic fibers in the lumbar region. This suggests that TANES contributes to integration of sNPCs, as well as activity-dependent oligodendrocyte and myelin remodeling of the chronically injured spinal cord. Together, the data suggest that the added electrical stimulation promoted cellular integration and influenced the fate of human induced pluripotent stem cell-derived sNPCs transplanted into the injured spinal cord.
Elevated Expression of Chitinase-3-like Protein 1 in Parkinson’s Disease
1Department of Translational Neuroscience, Barrow Neurological Institute, Phoenix, AZ, USA
2ASU-Banner Neurodegenerative Disease Research Center, Biodesign Institute, Tempe, AZ, USA
Chitinase-3-like protein-1 (CHI3L1) belongs to the chitinase and chitinase-like protein (C/CLP) family, is associated with neuroinflammatory microglia cells, and is elevated in various neurodegenerative diseases, including Alzheimer’s disease (AD) and amyotrophic lateral sclerosis (ALS). On the other hand, whether C/CLPs are altered in Parkinson’s disease (PD) remains controversial. Here we assessed and characterized astrocytic CHI3L1 expression in human PD postmortem brain tissue and in an adeno-associated virus (AAV)-induced α-synuclein (α-syn) overexpression rodent model of PD. Preliminary histological analysis shows that levels of CHI3L1 expression are elevated in the α-syn model prior to neuronal loss. Quantitative immunohistochemistry revealed that CHI3L1 protein was significantly increased 1.8-fold in the substantia nigra compared to non-diseased controls. In situ hybridization (RNAscope) analysis is underway to further characterize astrocytes expressing CHI3L1. Currently, we are engineering an AAV to manipulate CHI3L1 expression specifically in astrocytes. Preliminary findings indicate that AAV5 combined with the astrocytic-specific promoter GFAP can increase astrocytic-specific expression in the rat striatum. Our findings indicate increased astrocytic expression of CHI3L1 PD as reported in ALS and AD. Moreover, ongoing work in using an AAV-α-syn animal model will aid in determining the role that CHI3L1 plays in the pathological progression of PD.
Comparative Analysis of Dopamine Neuron Activity in Pavlovian Versus Non-Contingent Methamphetamine Exposure
University of Florida, Evelyn F. and William L. McKnight Brain Institute, Department of Neuroscience Gainesville, FL, USA
There is a notable increase in methamphetamine abuse. Despite this increase, the distinct impacts of environmentally associated (Pavlovian) and non-contingent (uncoupled) methamphetamine exposure on the activity of midbrain dopamine neurons and the dynamics of neuronal networks are not well understood. Our data suggests that acute non-contingent exposure to methamphetamine increases firing rate of dopaminergic neurons and alters neuronal network dynamics, evidenced by increased synchrony, modularity, and assortativity (N=5, p<0.05). However, the effects of exposure to methamphetamine in an environmentally conditioned versus an unconditioned context on dopamine neurons remain unknown. Our data, and the literature, support the hypothesis that Pavlovian versus uncoupled methamphetamine exposure influences dopamine neuron activity and neuronal network connectivity in distinct ways and differential mechanisms. In the current study, male and female mice were administered methamphetamine (2mg/kg, intraperitoneally) or saline, either through a conditioned place preference paradigm or in their home-cage environment. Forty-eight hours after the last drug administration, we employed ex vivo patch-clamp electrophysiology to measure the baseline firing activity of dopamine neurons in the ventral tegmental area (VTA). Our data indicates that forty-eight hours after the last drug administration non-contingent administration of methamphetamine increases the baseline firing activity of VTA dopamine neurons and leads to sensitization upon exogenous methamphetamine application. This effect could potentially be attributed to the desensitization of inhibitory D2 receptors. Current studies are exploring whether environmentally conditioned drug exposure similarly or differently influences the baseline activity of dopamine neurons and examining potential sex-dependent responses in each drug exposure model. This research offers important insights into the neurobiological effects of various methamphetamine exposure models in the context of developing pharmacological treatments for patients.
Exploring Hemispheric Lateralization: Implications for Parkinson's Disease and Cell Transplantation Therapies
*
1Department of Neurology, University of Toledo, College of Medicine and Life Sciences, Toledo, OH, USA
2Department of Neurology and Neurosciences, University of Toledo, College of Medicine and Life Sciences, Toledo, OH, USA
3Department of Neurology & Neurosciences, and Bioengineering, University of Toledo, College of Medicine and Life Sciences, Toledo, OH, USA
Hemispheric lateralization refers to the distinct information-processing abilities exhibited by the cerebral hemispheres of the human brain [1]. There is a well-established specialization of function in the two hemispheres, with the left hemisphere primarily specialized for language function in right-handed individuals. In contrast, the right hemisphere is mainly specialized for visuospatial function in right-handed individuals and ambidextrous individuals with no hand preference [2]. Evidence also suggests that paw preference in rats is similar to human handedness [3]. Despite the evolutionary development of hemispheric specializations in humans, their role in cell transplantation for Parkinson’s disease (PD) remains poorly studied. Previous studies have indicated that cell transplantation in the striatum of the dominant hemisphere, as opposed to the non-dominant hemisphere in 6-hydroxydopamine lesioned rats, resulted in improved motor behavior [4]. However, the potential underlying factors for the improvement in motor behavior have not been explored. This experiment aims to investigate whether lateralization exists in the case of the substantia nigra pars compacta (SNpc) and striatum between the dominant and non-dominant hemisphere animal groups. We hypothesize that animals within the dominant hemisphere will exhibit a significantly higher population of dopaminergic neurons, as well as variation in volume in SNpc and striatum compared to the non-dominant hemisphere animal group. (N=15) Sprague Dawley rats will be assigned to a paw preference test to determine the degree of handedness (right, left, or ambidextrous) as a measure of hemispheric dominance. Subsequently, a rodent behavioral battery test (RBBT) will be performed, followed by euthanasia, Cresyl violet (CV) staining and Tyrosine hydroxylase (TH)-immunohistochemistry. Stereological quantification of TH expression will be done on SNpc and striatum in each hemisphere using Stereo Investigator (MBF Bio.) software. The potential finding of variation in intrinsic factors between dominant hemisphere and non-dominant hemisphere animals is crucial for understanding successful cell transplantation in PD patients.
References
1. Josse, G., & Tzourio-Mazoyer, N. (2004). Hemispheric specialization for language. Brain research. Brain research reviews, 44(1), 1–12.
2. Gotts S. J., Jo H. J., Wallace G. L., Saad Z. S., Cox R. W., Martin A. (2013). Two distinct forms of functional lateralization in the human brain. Proc. Natl. Acad. Sci. U.S.A. 110, E3435–E3444
3. Pençe S. (2002). Paw preference in rats. Journal of basic and clinical physiology and pharmacology, 13(1), 41–49.
4. Nikkhah, G., Falkenstein, G., & Rosenthal, C. (2001). Restorative plasticity of dopamine neuronal transplants depends on the degree of hemispheric dominance. The Journal of neuroscience: the official journal of the Society for Neuroscience, 21(16), 6252–6263.
Investigation of Alzheimer’s disease-related neuromuscular dysfunction using hiPSC-derived cells in a compartmentalized bioMEMs platform
1Nanoscience Technology Center, University of Central Florida, Orlando, FL
2Hesperos, Inc. 3459 Progress Dr. Orlando, FL
Understanding the peripheral manifestations of Alzheimer’s disease (AD) is essential as motor deficits, particularly gait impairment, have been increasingly recognized as potential indicators of AD progression, with some studies suggesting their presence prior to amyloid deposition in the brain. Despite the considerable research on the central nervous system (CNS) aspects of AD, there is a critical gap in knowledge regarding its peripheral nervous system (PNS) symptoms. This study addresses this gap by employing an innovate in vitro model that focuses on the neuromuscular junction (NMJ), a crucial interface between the nervous system and skeletal muscles. Administration of amyloid beta (Aβ) oligomers, a recognized hallmark of AD, to this model allows for an investigation of PNS-related motor dysfunction in AD. The use of human induced pluripotent stem cell (hiPSC)-derived motoneurons (MNs) and skeletal muscle (SkM) in a compartmentalized BioMEMs device provides a controlled environment for assessing Aβ -induced NMJ dysfunction. Fluidic and electrical isolation of the two cell types is achieved by a barrier composed of polydimethylsiloxane (PDMS) microtunnels, only allowing for the passage of MN axons to form NMJs with SkM (Badu-Mensah et al., 2022; Guo, et al., 2020). NMJ function (fidelity, stability, fatigue index) was evaluated after acute and chronic treatment of Aβ in MN and/or SkM chambers, and significant NMJ functional deficits were identified. Additionally, MN and SkM monocultures treated with Aβ allowed for the identification of the cellular and molecular deficits associated with the two cell types independent of NMJ synapses. The findings from this study contribute valuable insights into the peripheral manifestations of AD and guides the identification of functional markers and biomarkers for preclinical diagnostics. This work underscores the utility of hiPSC-derived models in advancing our understanding of the intricate functional aspects of AD pathology and its implications for early diagnosis and intervention.
References
Badu-Mensah A, Guo X, Nimbalkar S, Cai Y, Hickman JJ. ALS mutations in both human skeletal muscle and motoneurons differentially affects neuromuscular junction integrity and function. Biomaterials. 2022 Oct;289:121752. doi: 10.1016/j.biomaterials.2022.121752. Epub 2022 Aug 19. PMID: 36084484.
Guo X, Smith V, Jackson M, Tran M, Thomas M, Patel A, Lorusso E, Nimbalkar S, Cai Y, McAleer CW, Wang Y, Long CJ, Hickman JJ. A Human-Based Functional NMJ System for Personalized ALS Modeling and Drug Testing. Adv Ther (Weinh). 2020 Nov;3(11):2000133. doi: 10.1002/adtp.202000133. Epub 2020 Aug 11. PMID: 33709015; PMCID: PMC7942691.
Long-term clinical outcome of a participant with Parkinson’s disease who received autologous cell-based investigational therapy at the time of deep brain stimulation surgery
1Department of Neurosurgery, University of Kentucky College of Medicine
2Department of Neurology, University of Kentucky College of Medicine
3Department of Neuroscience, University of Kentucky College of Medicine
4Department of Neurosurgery, University of Colorado Anschutz Campus
The peripheral nervous system can readily regenerate after injury through a transformation of cells that support repair mechanisms including the production of neurotrophic, anti-apoptotic, and anti-inflammatory factors. Over the last ten years, we have been conducting open-label clinical trials exploring the safety, feasibility, and preliminary clinical outcomes of implanting peripheral nerve tissue (PNT) into the substantia nigra of patients with Parkinson’s disease. Here we report on one study participant who completed a seven year post-implantation visit and who, after 10-years post implantation, recently came to autopsy of unrelated causes. A 48 year-old male with a reported 12-year history of Parkinson’s disease consented to participating in a clinical trial where he was unilaterally implanted with autologous PNT into the substantia nigra at the time of standard-of-care deep brain stimulation (DBS) surgery. There were no serious adverse events. Exam raters were blinded to the side of implant. Before surgery, in the practically-defined OFF-state (>12 hours off antiparkinsonian medications) his Unified Parkinson’s Disease Rating Scale (UPDRS) Part III (motor) score was 34 points (5 points in the ON-state). Meanwhile OFF-state lateralized Part III scores were 12 points on the side contralateral to the PNT graft/10 points ipsilateral to the graft. At 7 years after surgery, the OFF-state (>12 hours off medication/>12 hours off stimulation) UPDRS score was 34 points (6 points in the ON-state). The lateralized scores were 8 points on the side contralateral to the graft/14 points on the side ipsilateral to the graft. Histopathological studies examining the graft/host interaction are ongoing and will be reported.
Novel interaction partners of spastin reveal potential therapeutic targets for axonal regeneration
1Department of Neurobiology and Anatomy, College of Medicine, Drexel University, Philadelphia, PA, USA
SPAST, also known as SPG4, encodes for spastin, a multifunctional protein. Spastin has been known for its ability to sever microtubules (MT) with the help of its ATPase associated with diverse cellular activities (AAATPase) domain. Lines of evidence have shown spastin couples severing with protein interactions. Interestingly, our studies with SPAST -/- mice and pharmacologically inhibited SPAST showed defective axonal regeneration, decreased calcium signaling and reduced neuronal activity. To further delve into the underlying mechanisms, we performed co-immunoprecipitation (co-IP) coupled with mass spectrometry using wild type (WT) and mutant spastin (SPAST-C448Y) as baits. Strikingly, the mutant spastin, not the WT, co-IPed microtubule associated protein 6 (MAP6). MAP6 is known to stabilize MTs in neurons, which may act as a regulator for MT dynamics. The localization of MAP6 on MTs is known to be regulated by Ca2+/calmodulin and/or through phosphorylation by calcium calmodulin kinase II (CaMKII). So far, we confirmed that WT spastin and MAP6 have distinct subcellular distribution, and the majority of MAP6 does not associate with the MT lattice. Whereas mutant spastin binds to MAP6 and retains it to the MT lattice. Such aberrantly potentiated MAP6 and MT interaction may lead to hyper-stabilized MTs, thereby damaging the axonal integrity and impairing their capacity to regenerate. Interestingly, we also identified that T-complex containing Protein 1 (TCP1) is a strong binding partner for both WT and mutant forms of spastin. TCP1 is a chaperonin protein known for its essential role in ensuring the proper folding of tubulin subunits. Overall, we posit that spastin regulates axon regeneration through regulating the localization of MAP6 through Ca++ signaling pathways as well as tubulin availability through TCP1, and MT dynamics to ensure optimal growth conditions for the regenerating axon. Understanding these mechanisms allows us to utilize spastin and its interacting partners as novel therapeutic targets for axon regeneration.
NeuroD1-Mediated Effects on Motor Function for Subacute Spinal Cord Injury
1Graduate Program in Neuroscience, University of Minnesota Twin Cities, Minneapolis, MN
2Department of Neurosurgery, University of Minnesota Twin Cities, Minneapolis, MN
3College of Biological Sciences, University of Minnesota Twin Cities, Minneapolis, MN
Spinal cord injury (SCI) remains a significant global concern with no available treatments that restore function after injury. As such, a major goal for SCI research is the regeneration of the spinal cord to promote functional recovery after injury. To address this, viral-mediated delivery of proneural factors for in vivo glia-to-neuron reprogramming has emerged as a potential experimental approach for central nervous system (CNS) restoration. Current literature indicates that NeuroD1, a developmental proneural factor, is sufficient to convert astrocytes into neurons in vitro and in several animal models of CNS injury. Significantly, however, there is only one published study using NeuroD1 for SCI, and this study did not assess motor or sensory recovery. Further, other studies have highlighted an inconsistency in reprogramming efficiency and ambiguity in the cellular origin of “reprogrammed” cells. In response, researchers have identified viral dosage and intervention timing as significant factors influencing reprogramming efficacy and reproducibility. We hypothesize that the AAV9-NeuroD1-based reprogramming platform is capable of reprogramming spinal cord astrocytes into neurons following SCI in a dose-dependent manner to restore nervous system function. Here we employed a two-vector, AAV9 DIO/FLEx-based delivery platform for selective expression of NeuroD1-mRuby2 (reprogramming) or mRuby2 alone (control) in astrocytes after moderate SCI in female rats. Histological analysis was used to examine viral transduction throughout the spinal cord at one of two viral titers (1013 or 1011 GC/mL) and timepoints (acute or subacute stage of injury). Motor and sensory functional analysis of subacute NeuroD1-treated rats did not reveal any functional recovery up to 6 weeks post-injury. This is in contrast to rats treated in the acute stage of injury where there was a trend toward a deleterious effect on motor functional recovery in response to NeuroD1 treatment. Future work will explore the spatial resolution and extent of reprogramming throughout the spinal cord.
Small neuron-derived extracellular vesicles from individuals with Down syndrome propagate AD pathology and affect behavior of trisomic Ts65Dn mice
H. Saternos, J. Banh, A-C. Granholm, and
Department of Neurosurgery, University of Colorado Anschutz Medical Campus, Aurora CO, USA
Down Syndrome (DS) is the most common chromosomal disorder worldwide. Individuals with DS develop Alzheimer’s disease (AD) decades earlier than the general population, with accumulation of amyloid-β plaques and neurofibrillary tangles as well as glial activation. Research studies suggest that neurodegeneration in the locus coeruleus (LC) precedes all other neuronal loss, and that the it results in a significant decline in norepinephrine levels, which may contribute to initiating neuroinflammation. However, the consequences of early AD pathology in the LC to the spread of pathology has not been examined in DS. The main purpose of this study was to determine whether neuron-derived extracellular vesicles (NDEVs) from individuals with DS-AD can exacerbate AD pathology in the LC neurons and/or seed AD pathology into other areas. NDEVs carry pathological proteins such as pTau and Aβ that reflect the cells they originate from, and evidence suggest that they can spread toxic misfolded proteins from cell to cell, contributing to the propagation of the pathology to nearby brain areas. We injected NDEVs isolated from plasma of individuals with DS-AD into the LC of Ts65Dn mice and assayed for pathological and cognitive hallmarks 3 months after injection. Using the novel object recognition task along with the double-H maze, we found that Ts65Dn mice injected with DS-AD NDEV performed on these 2 tasks compared to those injected with healthy control NDEVs. Using immunofluorescent techniques, we found alterations in glial morphology as well as indicators of neuronal distress and early AD pathology. These data not only suggest that EVs contribute to the spread of AD pathology but that pathology in the LC-NE is sufficient to trigger pathology in other brain regions, affecting behavior. This furthers our understanding of the biological mechanisms for AD in DS which can lead to better diagnostic and therapeutic strategies for both those with DS and the general population.
Intranasal Administration of Extracellular Vesicles from hiPSC-Derived Neural Stem Cells as an Anti-Aging Treatment to Prevent Age-Related Cognitive and Mood Dysfunction
Institute for Regenerative Medicine, Department of Cell Biology and Genetics, Texas A&M University School of Medicine, College Station, Texas, USA.
Brain aging is the primary risk factor for developing neurodegenerative diseases. Unfortunately, there are currently no effective therapies to prevent cognitive and mood impairments associated with brain aging, which are often linked to neuroinflammation and decreased hippocampal neurogenesis. However, recent studies have shown that extracellular vesicles (EVs) released by neural stem cells (NSCs) derived from human induced pluripotent stem cells (hiPSCs) carry therapeutic miRNAs and proteins that have potent antiinflammatory, pro-cognitive, and neurogenic effects. Therefore, we employed C57BL6/J mice to investigate whether intranasal administration of hiPSC-NSC-EVs during early middle age (12 months) or early and late middle age (12 and 16 months) could improve cognitive and mood function in old age. The mice received two doses of hiPSC-NSC-EVs or vehicle at 12 months or at 12 and 16 months of age. Each dose of hiPSC-NSC-EVs contained approximately 11 billion EVs. After receiving the treatments, mice underwent behavioral tests to assess cognitive and mood function at 20 months of age. The cognitive tests examining the integrity and function of various brain regions included novel object recognition (perirhinal cortex), object location memory (dorsal hippocampus), object-in-place (medial prefrontal and perirhinal cortices, and the hippocampus), temporal pattern processing (hippocampal CA1 subfield), pattern separation (dentate gyrus and hippocampal neurogenesis), and delayed alternation Y-maze (medial prefrontal cortex) tasks. Mood function was assessed via a sucrose preference test measuring anhedonia, novelty-suppressed feeding, and elevated plus maze tests examining anxiety. The results showed that vehicle-treated animals displayed impairments in all cognitive and mood function tests. In contrast, animals receiving hiPSC-NSC-EVs displayed improved cognitive and mood functions in all tasks. Immunohistochemical analysis of brain tissues is underway to ascertain the antiinflammatory and neurogenic effects of hiPSC-NSC-EVs treatment. Overall, the findings suggest that hiPSC-NSC-EVs have the potential to be an effective anti-aging treatment to prevent age-related cognitive and mood impairments.
Supported by a National Institutes for Aging grant (R01AG075440-01 to A.K.S.).
An FDA-approved Blood Test for Concussion: Anatomy of a Discovery
1Walter Reed Army Institute of Research, Silver Spring, MD, USA
2U.S. Army Medical Materiel Development Activity, Fort Detrick, MD
3Combat Casualty Care Research Program, Fort Detrick, MD
4U.S. Army Medical Research and Materiel Command, Fort Detrick, MD
5U.S. Army Headquarters, Dept of the Army, Arlington, VA
GFAP (glial fibrillary acidic acid protein) and UCH-L1 (ubiquitin C-terminal hydrolase-L1) were recently approved by the United States Federal Drug Administration (FDA) as blood-based biomarkers for traumatic brain injury (TBI). The inception of the TBI biomarker field began in 2001 with a partnership formed between scientists at the University of Florida (UF) and Walter Reed Army Institute of Research (WRAIR). The discovery, development, and validation process that led to FDA approval 2 decades later was immense, involving multiple stakeholders, and recently culminated in Abbott Diagnostics obtaining FDA clearance in 2021 for marketing of a point-of-care i-STAT Alinity™ plasma blood test using GFAP and UCH-L1 as an aid to diagnosing concussion. Abbott’s i-STAT TBI plasma test also received CE Mark from the European Medical Authority, paving its way for commercial launch in Europe. The most recent published results from TRACK TBI NET have further indicated that GFAP and UCH-L1 levels in blood are predictive of patient outcome at 6 months post injury. The FDA approval of a blood test for GFAP and UCH-L1 to aid in diagnosing concussion represents a significant advance for the field. The importance of having a capability to be provide objective/quantitative assessment of the effects of exposure to blast or impact concussion at the point of injury cannot be over-emphasized. The i-STAT TBI plasma test rapidly provides a quantitative analysis from just a few droplets of blood. This capability is important for guiding triage decisions and tracking the progression of brain injury, but even more importantly, will become part of the TBI patient’s permanent medical record. There still remains a critical need for research on TBI-specific biomarkers capable of tracking chronic TBI pathology and that can serve as diagnostic, prognostic, and perhaps most importantly, theragnostic indicators.
Delivery of PAMAM dendrimers across natural barriers (blood-brain barrier and placental barriers) in healthy pregnant 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 Psychology, Central Michigan University, Mount Pleasant, MI, USA
5Department of Chemistry & Biochemistry, Central Michigan University, Mount Pleasant, MI, USA
Polyamidoamine (PAMAM) dendrimers are a new modality to deliver molecular payloads across physiological barriers such as cell membranes, the blood brain barrier (BBB), and potentially the placenta. Dendrimers have advantages over traditional vectors, such as viral or plasmids, due to the ability to tune the size and the chemistry of the dendrimer and to target specific tissues. We have previously shown that PAMAM dendrimers can cross the blood brain barrier following different routes of systemic injections. However, their ability to cross the placental barrier is unknown. In this study, cyanine 5.5 tagged PAMAM dendrimers (D-Cy5.5) were intraperitoneally (IP) injected into pregnant mice (E14). In vivo imaging (IVIS) was performed 3 days post-injection with the animals positioned both supine and prone. On the last day of gestation (day 21), the maternal and fetal brains were extracted, sliced, and imaged to evaluate the presence of D-Cy5.5 in the brain. We found that the surface modified D-Cy5.5 had crossed the BBB in pregnant mothers following IP injection. The D-Cy5.5 was found in the neurons and glial cells in the maternal brain. No D-Cy5.5 was observed in the fetal brains; however, it was present in the placental membrane showing that the dendrimers did not cross the placenta to the fetus. To conclude, PAMAM dendrimers can be used to deliver treatments to the maternal brain during pregnancy. This is an important finding, suggesting that dendrimers may be a viable vector to deliver drugs or other molecules intended specifically for pregnant mothers, while minimizing potential risk to the developing fetus.
Support for this study was provided by the Neuroscience program, the College of Medicine, department of chemistry and biochemistry and the John G. Kulhavi Professorship in Neuroscience at CMU
Orally administered inhibitor of perineuronal nets leads to functional recovery, structural changes and modulation of the immune response after chronic spinal cord injury
K. Štepánková1, L. Machová Urdzíková1, J. C. F. Kwok1,2, and
1Institute of Experimental Medicine, Czech Academy of Sciences, Prague, Czech Republic
2Faculty of Biological Sciences, University of Leeds, Leeds, UK
Spinal cord injury repair is partially inhibited by chondroitin sulfate proteoglycans (CSPGs). CSPGs are also part of perineuronal nets (PNNs), a compact and specialized form of extracellular matrix (ECM) that is unique to the central nervous system (CNS). PNNs limit the plasticity of adult neural tissue, which is particularly evident after injury. We studied the effect of PNN downregulation on spinal cord repair in the chronic phase of spinal cord injury (SCI) using 4-methylumbelliferone (4-MU), an inhibitor of hyaluronan (HA) synthesis. Rats underwent spinal cord contusion injury (using Horizon impactor) and were orally administered 4-MU 2 g/kg/day for 8 weeks along with rehabilitation on treadmill. After the end of treatment, the rats were rehabilitated for additional 2 weeks. 4-MU downregulated PNNs in spinal cord including those around motoneurons. In SCI rats treated with 4-MU, astrogliosis decreased, axon sprouting increased and cavity was filled with immune cells when compared with placebo-treated animals. The increase in the M2 macrophage marker CD206 suggests that systemic reduction of CSPG may alter the macrophage phenotype in favor of alternatively activated M2 macrophages. 4-MU also promoted remodeling of the specific ECM molecules filling the cystic cavity and increased vascularity at the lesion epicenter. These effects of 4-MU treatment were reflected by improvements in sensorimotor function as assessed by behavioral tests (BBB, ladder rung walking test, maximum speed test). The improvement in function corresponded to axonal tracing (DiD) increased serotonergic innervation, and a higher number of excitatory synapses in ventral horn above and, bellow the lesion. Thus, we demonstrated that oral treatment with 4-MU at a dose of 2 g/kg/day can modulate neuroplasticity and secondary damage processes, leading to long-term improvement in functional outcomes after spinal cord injury even in the chronic stage.
Supported by EXREGMED CZ.02.01.01/00/22_008/0004562 and Czech Science Agency 19-10365S.
Downregulation of the expressions of brain noradrenergic receptors during traumatic brain injury is alleviated in mice with a reduced blood level of fibrinogen
Department of Surgery, Morsani College of Medicine, University of South Florida, Tampa, FL USA
The noradrenergic system in the brain plays an important role in cognition. Dysfunction of the noradrenergic system has been observed during neuroinflammatory pathologies, e.g. Alzheimer’s disease. Inhibition of the adrenergic-α1 receptor has been associated with impaired learning while the activation of the receptor with its improvement. Here we tested a vascular effect, particularly the effect of the blood level of fibrinogen (Fg), on the noradrenergic system during neuroinflammation. As a model of neuroinflammation, we generated mild-to-moderate traumatic brain injury (m-mTBI) in transgenic heterozygous mice with a Fg gamma-chain knockout (FgKO), that had reduced blood levels of Fg, and in control C57BL6 wild-type (WT) mice. The effects of the reduction of Fg on the expression of adrenoceptors α-1A (ADRA-1A), α-1D (ADRA-1D), and β-3 (ADR-B3) were tested in brain samples collected 14 days after trauma or sham surgery. The expression of ADRA-1A, ADRA-1D, and ADRB-3 mRNAs was assessed with real-time PCR. The short-term memory (STM) of mice was assessed by a novel object recognition test. We found that m-mTBI caused the downregulation of all three adrenoceptors in mice during TBI and that the reduction of Fg was able to alleviate this effect. There was a reduction of STM in mice with m-mTBI. However, FgKO mice demonstrated better STM than the WT mice. Here, we are the first to show that a reduction in the blood level of Fg was associated with the alleviation of the downregulation of brain noradrenergic receptors during m-mTBI and the accompanied reduction in STM compared to that in WT mice. These results suggest a possible connection between the blood level of Fg and the brain noradrenergic system in the formation of STM during neuroinflammation, such as m-mTBI.
Vascularized Brain Assembloids with Enhanced Cellular Complexity Provide Insights into the Cellular Deficits of Tauopathy
1Drexel University College of Medicine, Department of Neurobiology and Anatomy, Philadelphia, PA, USA
2Washington University in St. Louis, Department of Psychiatry, Missouri, MO, USA
Advanced technologies have enabled the engineering of self-organized 3-dimensional (3D) cellular structures from human induced pluripotent stem cells (hiPSCs), namely organoids, which recapitulate some key features of tissue development and functions of the human central nervous system (CNS). While hiPSC-derived 3D CNS organoids hold promise in providing a human-specific platform for studying CNS development and diseases, most of them do not incorporate the full range of implicated cell types, including vascular cell components and microglia, limiting their ability to accurately recreate the CNS environment and their utility in the study of certain aspects of the disease. Here we have developed a novel approach, called vascularized brain assembloids, for constructing hiPSC-derived 3D CNS structures with a higher level of cellular complexity. This is achieved by integrating forebrain organoids with common myeloid progenitors and phenotypically stabilized human umbilical vein endothelial cells (VeraVecs), which can be cultured and expanded in serum-free conditions. Compared with organoids, these assembloids exhibited enhanced neuroepithelial proliferation, advanced astrocytic maturation, and increased synapse numbers. Strikingly, the assembloids derived from hiPSCs harboring the tauP301S mutation exhibited increased levels of total tau and phosphorylated tau, along with a higher proportion of rod-like microglia-like cells and enhanced astrocytic activation, when compared to the assembloids derived from isogenic hiPSCs. Additionally, the tauP301S assembloids showed an altered profile of neuroinflammatory cytokines. This innovative assembloid technology serves as a compelling proof-of-concept model, opening new avenues for unraveling the intricate complexities of the human brain and accelerating progress in the development of effective treatments for neurological disorders.
Characterization of nonmotor aspects of the paraquat and lectin rat model of parkinsonism
1Department of Neurology, University of Toledo College of Medicine and Life Sciences, Toledo, OH, USA
2Department of Neurology and Neurosciences, University of Toledo College of Medicine and Life Sciences, Toledo, OH, USA
3Department of Neurology, Neurosciences, and Bioengineering, University of Toledo College of Medicine and Life Sciences, Toledo, OH, USA
Parkinson’s disease (PD) is defined by its cardinal motor deficits, although increasing attention has been given to the many nonmotor symptoms present(1). REM behavior disorder (RBD) is one well-recognized nonmotor symptom of PD(1). Cognitive deficits are also seen, particularly when PD progresses to Parkinson’s Disease Dementia (PDD), in which visuospatial and executive function deficits are common(2). One model of PD that aims to recapitulate natural etiology involves administering paraquat and lectins (P+L) to rats via oral gavage(3). In this model, pathology begins in the gut and spreads retrogradely to the brain via the nigrovagal pathway(3). We have previously shown that oral administration of the biosalt squalamine protects from motor deficits in this model(4). The aim of this study was to evaluate nonmotor aspects of the P+L model including sleep and cognitive abnormalities and determine if squalamine protects from nonmotor deficits. Sprague dawley rats were oral gavaged for 7 days with paraquat and lectin (n=10, P+L). A separate group were given squalamine in their drinking water for 30 days following treatment (n=9, P+L+S). A control group were oral gavaged with sucrose solution alone (n=9). Cognitive deficits were assessed with the Y-maze and sleep abnormalities were assessed with 24-hour video analysis four weeks post-P+L treatment. Histological analysis was done to assess for cholinergic degeneration in basal forebrain regions. P+L rats show significant deficits in Y-maze performance compared to controls, while P+L+S rats show no significant difference in Y-maze performance compared to controls. P+L rats show significant sleep abnormalities compared to controls. Choline acetyltransferase immunohistochemistry suggests a qualitative difference in cholinergic neuron density in basal forebrain regions in P+L rats compared to controls. Studies are ongoing to further characterize sleep with polysomnography including EEG and EMG, quantitate neuronal loss via stereology, and determine the potential neuroprotective role of squalamine.
Sources
1. Pfeiffer, R.F., Non-motor symptoms in Parkinson's disease. Parkinsonism & related disorders, 2016. 22: p. S119-S122.
2. Gomperts, S.N., Lewy body dementias: dementia with Lewy bodies and Parkinson disease dementia. Continuum: Lifelong Learning in Neurology, 2016. 22(2 Dementia): p. 435.
3. Anselmi, L., et al., Ingestion of subthreshold doses of environmental toxins induces ascending Parkinsonism in the rat. npj Parkinson's Disease, 2018. 4(1): p. 30.
4. Caroline C. Swain, Khoi Le, Julia Arnold, Karen Sauter, Thyagarajan Subramanian and Kala Venkiteswaran. “Squalamine is protective against paraquat and lectin model of Parkinson’s disease” Society for Neuroscience 2022. Virtual poster.
Effects of Mitochondrial Rcc1-like Gene on Hippocampal Learning and Memory
1College of Letters and Science, University of Wisconsin-Madison, Madison, WI, USA
2Department of Neurology, University of Wisconsin-Madison, Madison, WI, USA
The Rcc1l gene encodes for the inner mitochondrial membrane protein RCC1L, important for mitoribosome assembly and mitochondrial fusion. Disruption of these processes has been linked to neurodegenerative disorders such as Alzheimer’s Disease (AD). To understand the effect Rcc1l has on hippocampal learning, memory, and neurodegeneration, we used the Cre-loxP system to ablate Rcc1l in the hippocampus and forebrain of mice (Rcc1l/Camk2a-cre+). Rcc1l knockout (Rcc1lKO/KO/Cre+, n=3), heterozygous (Rcc1lKO/+/Cre+, n=8), and wild-type mice (Rcc1lfl/fl/Cre-, n=5) were evaluated at 3- and 6-months of age in the open field task, radial arm water maze (RAWM), and novel object recognition (NOR) behavioral tasks. At 3 months of age, the experimental and control groups showed no significant difference in behavioral performance. However, at 6 months of age, KO mice, but not heterozygous or wild-type mice, began to show deficits in the NOR task (Rcc1lKO/KO/Cre+vs. Rcc1lKO/+/Cre+, unpaired t-test t=2.53, df=9, p .032; Rcc1lKO/KO/Cre+vs. Rcc1lfl/fl/Cre-, t-test t=2.46, df=6, p=.047). These results are encouraging considering the low number of animals available at the time of analysis, and they likely reflect the progressive nature of neurodegeneration. Surprisingly, KO mice displayed exercise-induced seizures during RAWM training, so we could not test them in this task. Future experiments will explore this phenotype. Additionally, we crossed Rcc1l/Camk2a-cre+mice with PhAMFloxed mice. This latter strain contains the fluorescent protein Dendra that permits the expression of green fluorescence in mitochondria. We are currently examining mitochondrial morphology using the ImageJ/FIJI plugin Mitochondrial Analyzer. Future studies will include more animals and later time points since aging is a risk factor for neurodegenerative diseases. In summary, this is the first investigation on the role of the mitochondrial RCC1L protein on hippocampal learning and memory and provides insight into the influence of mitochondrial dysfunction on memory-associated phenotypes that may be linked to neurodegeneration in AD.
The Development of a Novel Bilateral Rodent Model of Parkinsonian Alpha-Synuclein Pathology
1Department of Translational Neuroscience, Barrow Neurological Institute, Phoenix, AZ, USA
2Behavioral Neuroscience Program, Department of Psychology, Binghamton University, Binghamton, NY, USA
Although there is a wealth of different rodent models aimed at modeling Parkinson’s disease (PD), these often lack face validity, or as is the case of acute toxin models, can only be readily achievable unilaterally. The neuronal protein alpha-synuclein (α-syn) is recognized as a key etiopathological agent in (PD) neurodegeneration and intranigral overexpression of this protein is often used to emulate the synucleinopathy, neuronal dysfunction, and neuronal death seen in PD. However, published reports to date using this model have largely utilized an unilateral approach and focused on motor outcomes. Herein, to create a new model of PD with improved face validity and taking non-motor functions into account, adult Sprague-Dawley rats received bilateral injections of adeno-associated virus (AAV) expressing human α-syn, or AAV carrying an empty transgene cassette as control. Moreover, in contrast to prior work, we injected a larger volume of the AAV in order to facilitate spread of the virus, beyond the substantia nigra to additional areas important to non-motor symptoms such as the amygdala, to expand our understanding of non-motor deficiencies. Animals were thereafter subject to a panel of behavioral analyses aimed to capture both motor and affective phenotypes, including gait analysis, rotarod testing, open field, elevated plus maze, amongst others. Moreover, at the same time we are developing an AI platform intended to phenotype subtle behaviors in this novel model during home cage activity. Ongoing behavioral testing shows that AAV-hα-syn animals exhibit motor impairment and an anxiogenic phenotype. Postmortem, we will perform quantitative histological measures to validate vector transduction, formation of pathological α-syn, and nigral neurodegeneration.
With the conclusion of this study, we aim to have phenotyped changes in motor and non-motor features of this model that emulate features of human disease to be able to confidently utilize this novel rat model in studies going forward.
The APOE4 allele is involved in extracellular vesicle-dependent neurodegeneration in Alzheimer’s disease and Down syndrome
1Department of Neurosurgery, University of Colorado Anschutz Medical Campus, Aurora CO, USA
2University of Colorado Alzheimer’s and Cognition Center, Department of Neurology, University of Colorado Anschutz Medical Campus, Aurora CO, USA
3Linda Crnic Institute for Down Syndrome, University of Colorado Anschutz Medical Campus, Aurora CO, USA
People with Down syndrome (DS) develop Alzheimer’s disease (AD) decades earlier than the general population and make up the largest AD patient group under the age of 65. Among genetic factors known to increase the risk to develop AD, individuals with two copies of the apolipoprotein Eε4 allele (APOE4) are up to 12-fold more susceptible to develop cognitive impairment. Strong evidence shows that the apoE4 protein impacts the endosomal-lysosomal pathway leading to a downregulation of extracellular vesicle (EV) biosynthesis and release by the cells. In typical AD and DS-associated AD (DS-AD), EVs are implicated in neuronal degeneration by spreading toxic misfolded proteins from cell to cell, including amyloid-beta and the microtubule-associated protein tau. While it is known that the APOE4 allele accelerates and enhances the development of AD in individuals with DS, it is unclear how APOE4 exacerbates the AD pathology in the brain.
We hypothesize that EV biogenesis and function are impacted by APOE allelic variations existing in the human population. Using human isogenic DS and euploid pluripotent stem cell-derived cerebral organoids (CO), we investigate how different APOE genotypes impact EV biogenesis, surface markers and cargo in the context of DS.
We found that APOE variants affect the number of EVs, as well as membrane and cargo composition. Interestingly, EV populations evolve as the CO mature and change in cell type composition. Finally, we found that amyloid-beta and hyperphosphorylated tau proteins dramatically accumulate in EVs released from the DS APOE4 CO models, suggesting a significant default of clearance.
Overall, our results suggest that apoE is an important modifier of EV release and trafficking in the brain. In the context of DS, our data point to a significant role of EVs in the spread of AD neuropathology. Our studies also shed light on the usefulness of EV-based biomarkers for identifying disease-modifying markers of AD and DS-AD.
Combining Neural Progenitor Cell Transplantation with Respiratory Training after Cervical Spinal Cord Injury
1Gladstone Institutes, University of California San Francisco, San Francisco, CA, USA
2Department of Neurobiology and Anatomy, College of Medicine, Drexel University, Philadelphia, PA, USA
There is a growing interest in the use of neural progenitor cells (NPCs) to repair the injured spinal cord. Despite extensive preclinical research, it remains unclear as to how donor cells differentiate and integrate with host injured circuitry, and if integration can be enhanced and/or guided using non-invasive means such as activity-based therapy. With a focus on the phrenic circuit and respiratory dysfunction after cervical spinal cord injury (SCI), the present work tests the hypothesis that pairing cellular transplantation with respiratory training - daily acute intermittent hypoxia (dAIH) - will enhance neuroplasticity and promote connectivity. NPCs (developing neuronal and glial restricted progenitors) were transplanted into a cervical contusion SCI in adult rats, one week after injury. Animals then received 4 weeks of dAIH, beginning one-week post-transplantation. Donor cells survived, differentiated, and spontaneously integrated with the host spinal cord as assessed with anatomical tracing and immunohistochemistry. Respiratory training enhanced donor-host connectivity to ipsi- and contralateral-to-injury phrenic circuit. At least a subset of these newly integrated donor spinal interneurons are cholinergic, and the number of donor cholinergic neurons connected to the injured circuit correlated with motor recovery. Interestingly, transplant and dAIH training recipients demonstrated a greater ability to respond to hypoxic but not hypercapnic respiratory challenge. Ongoing experiments are focused on assessing host-to-donor axonal integration and whether respiratory training is selective in the subsets of donor neuron phenotypes and targets. These experiments suggest that rehabilitative strategies may be an effective way for enhancing donor cell outgrowth and connectivity.