ASNTR 2025 Meeting Abstracts

The annual conference held by the American Society for Neural Therapy and Repair (ASNTR) is a premier venue for exchanging scientific findings and ideas for cell therapies and brain repair following injury and disease. The Society consists of leading scientists and physicians, as well as postdoctoral fellows and students, 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 the abstracts included in this volume reflect the diverse and innovative research being conducted in the field of neural therapy and repair. Topics range from disease mechanisms and basic development of stem cell therapies to clinical trials using cutting-edge technologies. With an emphasis on translation, presentations will discuss innovations in discovery as new techniques in data science and -omics emerge and lead to clinical application. The collegial exchange of ideas during our annual conferences have led to many fruitful collaborations over the years, resulting in truly pioneering discoveries in the field of neural therapy.

The 2025 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 our members, none of this would be possible. Special thanks to our current President John Stanford, the 2025 Program Committee, Education Committee, Fundraising Committee, Donna Morrison, Inger Mills, and our staff for all their efforts in planning what will, without a doubt, be an exciting meeting.

Comprehensive Examination of Nose-to-Brain Administration using G4 70/30 Poly (Amidoamine) (PAMAM) dendrimers in the C57BL/6J Mouse Brain for Non-Invasive Drug Delivery

N.A. Khiabani^1,2,3, O. Dubey^1,3,4, D. Doyle^1,2,3, B. Srinageshwar^1,2,3,5, D. Story⁶, A. Sharma⁷, D. Swanson⁷, G. L Dunbar^1,2,5, and J. Rossignol^1,2,3,4

¹Field Neurosciences Institute Laboratory for Restorative Neurology, Central Michigan University, Mount Pleasant, MI 48859 USA

²Program in Neuroscience, Central Michigan University, Mount Pleasant, MI 48859 USA

³College of Medicine, Central Michigan University, Mount Pleasant, MI 48859 USA

⁴Program in Biochemistry, Cell and Molecular Biology, Central Michigan University, Mount Pleasant, MI 48859 USA

⁵Department of Psychology, Central Michigan University, Mount Pleasant, MI 48859 USA

⁶Department of Psychology, Arts and Behavioral Sciences, Saginaw Valley State University, Saginaw, MI 48710 USA

⁷Department of Chemistry & Biochemistry, Central Michigan University, Mount Pleasant, MI 48859 USA

Various strategies for delivering drugs to the brain have been investigated, yet the blood-brain barrier (BBB) remains a formidable obstacle in achieving effective treatment for neurological disorders. Over the years, nanocarriers have appeared as a promising approach to overcome this barrier. Nose-to-brain (intranasal) drug delivery is emerging as a promising non-invasive approach for enhancing therapeutic efficacy circumventing the BBB. Dendrimers are spherical nanomaterials with extensive branching that are manufactured for diagnostic and therapeutic purposes showing potential as carriers for CNS and offering reduced systemic exposure and limited side effects after in vivo administration. The current study focuses on investigating in vivo effects and safety of our PAMAM dendrimers (G4 70/30) following intranasal exposure in the C57BL/6J mouse brain. This research seeks to elucidate their biodistribution, safety, and therapeutic implications, paving the way for advancements in CNS drug delivery strategies.

Our study was designed with male (n=9) and female (n=9) C57BL/6J mice. The treatment group received daily intranasal delivery of CY5.5-labeled G4 70/30 PAMAM dendrimers, while the control group received HBSS.

Biodistribution was measured using the In Vivo Imaging System on a weekly basis for a period of three weeks. Following a three-week period, organs including the brain, lung, liver, and kidney were extracted from all experimental mice. Utilizing fluorescence microscopy, the sections were imaged to assess the distribution of our dendrimers. Notably, the results of fluorescence evaluation revealed a notable accumulation of dendrimers in the brain of the intranasally inoculated mice, but no toxic effects were observed in any organs. Results showed that male brains showed higher fluorescent intensity compared to female brains and confirm that ability to deliver dendrimers intranasally opens avenues for the delivery of therapeutic agents such as genes and drugs for therapeutic applications in neurological disorders and offering opportunities for the development of innovative treatment strategies.

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.

An integrated proteome and transcriptome approach to promoting stem cell proliferation, regeneration, and cognition in diabetic mice brain

S. Behera¹, A. Kesharwani¹, S.K. Singh², K. Pandey², C.L. Limoli³, V. Ravichandiran¹, and V.K. Parihar¹

¹Department of Pharmacology and Toxicology, National Institute of Pharmaceutical Education and Research (NIPER), Hajipur, Bihar, 844102, India

²Department of Clinical Medicine, ICMR-Rajendra Memorial Research Institute of Medical Sciences, Patna - 800007, India

³Department of Radiation Oncology, University of California, Irvine, CA 92697-2695

Patients with poorly managed diabetes are more likely to develop dementia and have accelerated brain aging as a result of high blood glucose levels. Furthermore, diabetic individuals usually experience difficulties with memory, recollection, and focus when doing regular activities. This study examined the transcriptome and proteome alterations in diabetic mouse brains and their putative link to brain dysfunction. A well-established mouse model of type 2 diabetes generated by a high-fat diet and low streptozotocin dosages was used to study how hyperglycemia affects stem cell proliferation, regeneration, and cognition. Our findings indicate that hyperglycemia impairs hippocampus, prefrontal, and entorhinal cortex-dependent spatial, temporal, and episodic memory. Additionally, mice with diabetes show impaired extinction memory strengthened fear-related memory, and reduced cognitive adaptability. An integrated transcriptome and proteome analysis revealed that 26 genes encoding mitochondrial energetics (Cox6), insulin resistance (Etnppl), lipid metabolism (Apod, Plin4), accelerated-brain aging (Gm11639), and inflammation (Ighg2c), stem cell (SOX2) are differentially expressed in the diabetic mouse brain at both mRNA and protein level. The bioinformatic study reveals the level of FOXO3 significantly increases in the diabetic brain, whereas dehydrozingerone promotes SOX2 expression. Glutamate-glutamine/GABA cycle abnormalities may worsen diabetes-related cognitive decline. Quantification of mature neurons (NeuN+ neurons), dividing neurons (PCNA+), and newly born cells (Ki67+ cells) in the hippocampus showed that hyperglycemia significantly inhibits neurogenesis and cell proliferation in diabetics. In diabetic mice, HMGB1 and CD200 expression in the medial frontal cortex increased, indicating cellular and neurovascular inflammation. Our data also show that diabetes lowers GABA and dopamine. Further, dehydrozingerone, a half analog of curcumin, had a variety of advantages, including decreased neuroinflammation and cell death, as well as the promotion of critical genes and proteins necessary to promote cognitive performance. As a consequence, dehydrozingerone may be a potential treatment option for diabetics with persistent neuroinflammation and cognitive impairments.

Exploring the Impact of rAAV-based Gene Delivery on the Terminal Differentiation of hESC-Derived Dopaminergic Progenitors

D.H. Beligala, D. Pokharel, T. Subramanian, and K. Venkiteswaran

Department of Neurology, University of Toledo, Toledo, OH, 43614, United States.

Disclosures

D. H. Beligala: None. D. Pokharel: None. T. Subramanian: Speaker’s bureau -Supernus, Neurocrine, Acadia; Royalty CRC Press; and Founder of StereoRx. K. Venkiteswaran: None.

In our previous study by Gilmour et al. (2011) we showed that the fetal ventral mesencephalic (FVM) cell transplants in parkinsonian rats can repair basal ganglia neuronal circuitry that is not achieved via pharmacotherapy. However, whether transplants of mesencephalic dopaminergic (mesDA) progenitor cells derived from human embryonic stem cells (hESCs), that are now in clinical trials, can achieve similar outcomes remains unexplored. Therefore, we examined if recombinant adeno-associated virus (rAAV) mediated ex-vivo gene therapy can be used in hESC-derived mesDA cells to provide the ability to “turn on/off” graft function as we have successfully shown with FVM transplants. Thus, the aim of this study was to examine the effects of rAAV-based gene delivery on terminal differentiation of hESC-derived mesDA progenitors in vitro and in vivo. We optimized the transduction protocol using rAAV8/Ef1a-mcherry-IRES-WGA-Cre and rAAV9/TR-eGFP viral vectors. The percentage cell survival after 24 hrs was 12.9%, 71.3%, 88.2% and 97.1% when plated at 155,000, 232,500, 310,000 and 387, 500 cells/cm² densities, respectively. Furthermore, the highest transgene expression was observed with the lowest viral titer used in this study (10,000 MOI). Immunoblot analysis showed that the expression of intrinsic dopaminergic factors: tyrosine hydroxylase (TH), dopamine transporter (DAT), dopa-decarboxylase (DDC) and vesicular monoamine transporter-2 (VMAT2), in hESC-derived neurons was not affected by the viral transduction. Moreover, immunohistochemistry analysis confirmed that rAAV-transduced mesDA progenitors transplanted into rat striatum produced TH positive neurons in vivo within 5 weeks. Post transplantation evaluation of these ex-vivo transduced hESC-derived mesDA cells in parkinsonian rats and their ability to restore basal ganglia neural circuitry are ongoing. Our results suggest that rAAV-based ex-vivo gene delivery does not compromise the differentiation capacity of mesDA progenitors despite needing a higher cell density and that rAAV vectors can be used in place of the high-risk lentiviral vectors to achieve durable ex-vivo gene transduction.

Research Funding from DoD NETP 13204752, NIDDK R01 DK124098 and NINDS R01 NS104565 to Thyagarajan Subramanian and from the Anne M. and Phillip H. Glatfelter III Family Foundation to Thyagarajan Subramanian and Kala Venkiteswaran

Using the Optical Synapse for Corrective Circuit Manipulation in Huntington's Disease Mice

C.L. Bermúdez, M.O. Tree, G.L. Dunbar, J. Rossignol, and U. Hochgeschwender

Central Michigan University, Mount Pleasant, MI 48859, USA

Huntington’s disease (HD) is characterized by a cytosine-adenine-guanine (CAG) triplet expansion in exon 1 of the huntingtin gene, resulting in the late-stage symptoms of motoric, psychiatric, and cognitive deficits. In presymptomatic HD patients and mouse models, excitatory overdrive of cortical output onto striatal neurons has been observed. Our central hypothesis is that manipulating the firing activity within selected microcircuits before the onset of symptoms will slow HD disease progression. We previously utilized bioluminescent optogenetics (BL-OG) to inhibit the firing rate of cortical pyramidal neurons (CPNs) projecting to the striatum. Dampening excitatory drive of CPNs during weeks 5 – 8 in R6/2 HD mice using inhibitory luminopsins, fusion proteins of a light-emitting luciferase and a light-sensing inhibitory channelrhodopsin, significantly slowed progression of motor deficits as seen in improved motor coordination and gait parameters. We now want to apply the Interluminescence strategy to convert the excitatory overdrive of CPNs into dampening the activity of striatal target neurons. This is achieved through the optical synapse where a vesicle-targeted luciferase is released into the synaptic cleft when the presynaptic CPN neuron fires; in the presence of luciferin the bioluminescence emitted will activate an inhibitory optogenetic channel expressed in postsynaptic striatal neurons. AAVs of a Cre-dependent luciferase (AAV2/9 EfIa DIO hPOMC1-26 sbGluc dTomato) will be used to transduce the M1 motor cortex of 3-week-old Emx1-Cre::R6/2 mice and AAVs of an inhibitory opsin (AAV2/9 hSyn hGtACR1 EYFP) to transduce neurons in the striatum. Intraperitoneal application of luciferin once daily during weeks 5 – 8 is expected to convert the overexcitation of CPNs into dampening of striatal neuronal activity and HD motor recovery in the mice. Mice will be assessed through various motor-based tests such as balance beam and ladder tests, as well as rotarod and grip strength to assess recovery in motor function.

Automated scoring system using deep learning for abnormal involuntary movement assessment in a rat model of levodopa-induced dyskinesia

C. Budrow¹, A. K. Janapareddi², A. Velazquez², F. Manfredsson², C. Kelley², and C. Bishop¹

¹Department of Psychology, Behavioral Neuroscience Program, Binghamton University, Binghamton, NY, 13902

²Barrow Neurological Institute, Department of Neurobiology, Phoenix, Arizona, 85013

Parkinson’s Disease (PD) is a neurodegenerative movement disorder, arising in part through dopamine (DA) neuronal loss within the substantia nigra pars compacta (SNc). While levodopa (L-DOPA) is a widely accepted gold-standard DA replacement therapy, chronic use results in the development of levodopa-induced dyskinesia (LID), characterized by debilitating choreic and dystonic movements that plague upwards of 70% of the clinical population. Behaviorally, abnormal involuntary movements (AIMs), characterized by axial, limb and orolingual features, are pivotal in estimating disease progression and treatment efficacy. While reliable, AIMs scoring requires manual, qualitative observations that are subject to reduced temporal specificity and inter-rater variability. Alternatively, deep learning models (DLM), when integrated with animal pose estimation (APE), can automate accurate and objective behavioral assessments. However, current models demonstrate variable accuracy and flexibility from time-constraints and visual fatigue. To overcome these barriers, we first compiled a diverse and comprehensive dataset for training DLM reliant on APE. TPH2-CRE+ Long Evans and Sprague Dawley rats (N=29) were rendered hemiparkinsonian via 6-hydroxydopamine (6-OHDA) lesioning to the left medial forebrain bundle, followed by chronic L-DOPA (6-12 mg/kg, s.c.) treatment, where AIMs were captured by 3 GoPro Hero9 Black cameras positioned 120° around AIMs cylinders at 2 min time-bins, every 10 min, for 120-180 min. Second, frames were extracted from video recordings of dyskinetic rats in varying lighting and resolution conditions, where frames were segmented into discrete dimensions repositioned from the DeepLabCut library. Key-feature points were manually annotated for model training. Results demonstrated increasingly promising likelihood scores across subsequent generations in pinpointing AIMs, and with continued training and additional key-feature point extractions, posits clear utility in purposing this model for AIMs scoring in preclinical and clinical assessments of LID.

Bioluminescent Kinase Sensors for detection of growth factor and inflammation signaling

M. Chatterton^1,2, B. St. Ogne ^1,2, J. Rossignol ^1,2, J.L. Bakke¹, and E.D. Petersen^1,2

¹College of Medicine, Central Michigan University, Mount Pleasant, MI 48859, USA

²Program in Neuroscience, Central Michigan University, Mount Pleasant, MI 48859, USA

Growth factor signaling is an important component of a large variety of cellular processes including metabolism, differentiation, proliferation, and migration. When growth factor signaling is altered it can lead to pathologies like cancer cells forming and proliferating within the body such as glioblastoma multiforme (GB). In this study, we investigate and propose novel therapeutic approaches utilizing genetically encoded Bioluminescent Kinase Sensors (BlinKS) to respond to growth factor signaling via kinases in the epidermal growth factor receptor (EGFR) signaling pathway. Specifically, this study targets the kinases within the MEK, RAS, and RAF signaling pathways. We developed a rational library of BlinKS variants with altered phospho-amino acid binding domains (PAABD) and varying kinase substrate peptides and permutations of the linker regions, either flexible or rigid at the interfaces of the protein fusion sites. We tested our BlinKS constructs in U87 glioblastoma cells expressing our candidate sensor variants, treated the cells with epidermal growth factor (EGF), and measured the response of the BlinKS sensors for light emitted by the sensor by measuring an optogenetic transcriptional readout via a fluorescent reporter protein. Bioluminescence readings were conducted on a plate reader, and it was found that the cells treated with EGF produced more luminescence than those not treated with EGF. We also found our sensors targeting this signaling cascade to be able to control an optogenetic transcription system, reporting EGFR activation with GFP expression. We have also developed versions of BlinKS that respond to inflammatory signaling via the JAK/STAT pathway downstream of the IL-6 receptor, reporting the presence of an inflammatory compound (LPS) with an increase in light emission. We aim to test these sensors in a variety of neuroscience applications. We will also be testing the inflammation-sensing BlinKS to report and control neuroinflammation with the ultimate goal of limiting neurodegeneration.

Development of a Bioluminescent Probe for Real-Time Detection of Serotonin in Migraines

J. Covell¹, K. Taylor^1,2, and E.D. Petersen^1,2

¹College of Medicine, Central Michigan University, Mount Pleasant, MI 48859, USA

²Program in Neuroscience, Central Michigan University, Mount Pleasant, MI 48859, USA

Migraines are a common neurological disorder characterized by moderate to severe headaches, nausea, and photosensitivity. The serotonergic system has been previously linked to the pathophysiology of migraine, but there is still much to be discovered about the exact nature of this connection. Existing methods of measuring serotonin do not enable non-invasive real-time detection in deep brain regions. In addition, current clinical techniques can only quantify serotonin in the periphery or measure metabolites of serotonin in the CNS. Therefore, there are no definitive answers about serotonin levels in the migraine brain. To resolve the current limitations of recent research, this project aims to engineer and optimize a bioluminescent indicator for serotonin that will eventually allow measurements of deep brain processes in vivo related to migraine onset. The sensors were developed and enhanced with manual cloning of various domains and linker screening, then analyzed for a large change of bioluminescent light emission in response to serotonin. The current variants exhibit a 20-25% change in luminescence in response to 10 μM serotonin. The results are sensors that can continue to be optimized for future real-time, in vivo testing of serotonin levels in the brain before, during, and after a migraine attack. Once we have optimized sensors that light in response to rising serotonin levels, we plan to adapt them as optogenetic neuromodulators to prevent migraines and as neuromodulators in neurodegenerative disease.

Single Nuclei RNA-seq and Motion Sequencing Analyses Reveal Distinct Pathophysiology Signatures between White Matter and Cortical Stroke

Á.M. Cruz-Lociel^1,2, A. Panditrao^1,2, K.G. Vlassopoulos^1,2, E. González-Cubero^1,2, and I.L. Llorente^1,2

¹Department of Neurosurgery, Stanford School of Medicine, Stanford University, Palo Alto, California, 94304, USA

²Palo Alto Veterans Institute for Research, Palo Alto Veterans Affairs Medical Center, Palo Alto, California, 94304, USA

Ischemic strokes affect >795,000 patients per year in the United States and account for ~87% of all stroke types. Cerebral ischemia can occur in large blood vessels, such as those affected by cortical stroke (CS), or in deep penetrating vessels, such as those affected by subcortical white matter stroke (WMS). While infarction in the cortex typically produces an acute injury, white matter infarcts can progress over time and develop into vascular dementia. Despite current treatment modalities, there exists a lack of disease-modifying therapeutics in the clinic partly because there are still gaps in knowledge regarding the specific responses across brain regions after injury. To this aim, we have utilized single nuclei RNA-sequencing (snRNA-seq) and an unsupervised behavioral approach through Motion Sequencing (MoSeq) analysis to characterize tissue-specific and motion-based changes during the subacute and chronic phases of ischemia. MoSeq analysis reveals a distinct injured phenotype characterized by stationary behavioral motifs (i.e., supported rears) after both stroke types in young male mice. Interestingly, this data also shows statistically significant motifs between WMS and CS with the latter being associated with more frequent paused behaviors at the subacute time point. In addition to our behavioral analysis, snRNA-seq data from young males indicates that inflammatory responses are similarly enhanced during the subacute phase in both models. However, these results also suggest a possible dysfunctional oligodendrocyte population marked by the downregulation of myelination markers (i.e., Plp1) exclusively after WMS. In contrast, our chronic phase data shows increased subcellular responses such as vesicular transport and translation. Current analysis of spatial transcriptomics datasets from both models should enhance our understanding of the transcriptional responses after injury with in situ anatomical context. This approach should provide insight into cell-specific and behavioral profiles of brain ischemia and help us generate precise hypotheses about the underlying pathophysiology.

This research is supported by the California Institute of Regenerative Medicine TRAN1-12891 & DISC2-15137 grants.

Stem cell-based therapies to repair the spinal cord

M. D’Ambra¹, S. Azarapetian², L. Lydaki¹, E. Gonzalez-Cubero¹, and I.L. Llorente¹

¹Department of Neurosurgery, Stanford School of Medicine, Stanford University. Palo alto, California, 94304, U.S.

²Department of Neurology, David Geffen School of Medicine, UCLA. Los Angeles, California, 90095, U.S.

More than 300,000 people are living with SCI in the U.S., with approximately 18,000 new cases per year. Over 60% of SCIs occur at the cervical spinal cord levels resulting in the most severe functional deficits, financial cost, and a reduction in life expectancy. Injury to the spinal cord disrupts axonal tracts and damages glial cells and regeneration is limited by inhibitory signals and fibrotic barrier formation, causing permanent disability. Previous work has demonstrated the potential benefit that axonal growth induction or the modulation of the astrocytic scar has on the treatment of SCI using stem cell-based therapies or biopolymers. Therefore, a cell-based therapy that can replace lost glia and induce structural repair after SCI is of great promise. In this study, we have characterized the cellular properties and in vivo tissue repair activity of glial enriched progenitor cells differentiated from human induced pluripotent stem cells, termed hiPSCs-GEPs. To date, we have demonstrated reproducible production of the hiPSC- GEP, having produced them from at least five independent donors. We have also qualified the entire manufacturing process for the intended therapeutic candidate, hiPSC-GEPs, through safety, identity, purity, activity, and stability qualification assays. To demonstrate the scale-up manufacturing capabilities of the potential therapeutic product, we have developed a new cGMP manufacturing protocol and produced well above the necessary number of cells for phases 1 and 2 of a future clinical application. This study demonstrates the reliable and safe generation of patient derived hiPSC-GEPs that are clinically ready as a cell-based therapeutic approach for SCI. Future work will establish the safety and efficacy of hiPSC-GEPs transplantation and determined the appropriate therapeutic time window, location, and dose of hiPS-GEPs needed to induce motor improvement after SCI.

This research was supported by the Paralyzed Veterans for America endowment fund.

CNS-wide engraftment of Human Monocytes, but not Microglia, induces a chronic proinflammatory state associated with demyelination, astrogliosis, synapse loss, and behavioral dysfunction

H. Davtyan^1,2, J.P. Chadarevian^1,2, J. Hasselmann^1,2, G. Eskandari-Sedighi¹, S. Lin-Koch⁴, A.L. Chadarevian^2,3, J.K. Capocchi¹, D. Duong⁵, J. Nguyen¹, C. Tu^1,2, S.K. Shabestari³, F. Wu⁵, A. Shantaraman⁵, K. Mansour², W. Carlen-Jones¹, M. Mgerian³, K. Deynega¹, T.E. Lim¹, A.K. Mai¹, L. Le¹, A. Agababian³, D.A. Hume⁶, C. Pridans⁷, E. Head¹, S.R. Datta⁴, N.T. Seyfried⁵, and M. Blurton-Jones^1,2,3

¹Institute for Memory Impairments & Neurological Disorders, University of California, Irvine, CA, USA

²Stem Cell Research Center, University of California, Irvine, CA, USA

³Department of Neurobiology & Behavior, University of California, Irvine, CA, USA

⁴Harvard Medical School, Boston, MA, USA

⁵Department of Biochemistry, Emory University School of Medicine, Atlanta, GA, USA

⁶Mater Research Institute, University of Queensland, Brisbane, Australia

⁷Centre for Inflammation Research, University of Edinburgh, Edinburgh, UK

Hematopoietic stem cell transplantation (HSCT) in combination with microglia depletion is increasingly being examined as a potential therapy for neurological disorders. The premise of this approach is that HSCT-derived monocytes (MN) may infiltrate the brain and differentiate into “microglia-like” cells. However, induced pluripotent stem cells derived microglia (iMG) provides a potential alternative source of therapeutic cells. As many questions remain regarding the similarities and differences between microglia and monocytes, we utilized a xenotransplantation-compatible model that lacks endogenous microglia (hFIRE mice) to examine the transcriptional and functional properties of human iMG and MN. iMG and MN from four patients were transplanted into adult hFIRE brains and four months later behavioral testing was performed. Brains were then examined via spatial RNA sequencing, proteomics, histological, and biochemical approaches. Despite four months of brain residence and near complete chimerism, human iMG and MN continued to exhibit many important differences. In particular, we found that MN, but not iMG, induced a chronic proinflammatory state associated with significant levels of astrogliosis, demyelination, synaptic loss, and behavioral impairment. Taken together, these results demonstrate the critical role of ontogeny on myeloid cell function within the brain and provide important implications for the development of CNS-wide microglial replacement therapies.

Enhancing PAMAM dendrimers for targeted delivery of YWHAB siRNA as a promising therapeutic approach for Glioblastoma treatment

O. Dubey^1,2,3,4, N.A. Khiabani^1,2,3, A. Poudel^1,2,3, B. Srinageshwar^1,2,3,5, A. Sharma⁶, D. Swanson⁶, G.L. Dunbar^1,2,5, J. Rossignol ^1,2,3,4, and J. Bakke ^2,3,4

¹Field Neurosciences Institute Laboratory for Restorative Neurology, Central Michigan University, Mount Pleasant, MI 48859 USA

²Program in Neuroscience, Central Michigan University, Mount Pleasant, MI 48859 USA

³College of Medicine, Central Michigan University, Mount Pleasant, MI 48859 USA

⁴Program in Biochemistry, Cell and Molecular Biology, Central Michigan University, Mount Pleasant, MI 48859 USA

⁵Department of Psychology, Central Michigan University, Mount Pleasant, MI 48859 USA

⁶Department of Chemistry & Biochemistry, Central Michigan University, Mount Pleasant, MI 48859 USA

Glioblastoma (GB) is an aggressive brain tumor with limited treatment options and high recurrence rates. Current therapies, including surgery, radiotherapy, and chemotherapy, provide poor outcomes due to lack of precision medicine and tumor resistance. Small interfering RNAs (siRNAs) are emerging as a promising therapeutic approach due to their specificity and potency. The YWHAB gene encodes the 14-3-3β protein, which is overexpressed in glioblastoma and contributes to malignancy. Previous study from our lab highlighted that the knockdown of YWHAB significantly reduced spheroid formation and proliferation in glioblastoma cells. Our study demonstrates the delivery of YWHAB siRNA using generation 4 (G4) 70/30 PAMAM dendrimers (containing 70% hydroxyl and 30% surface amine groups), which offer advantages such as biocompatibility, stability, and blood-brain barrier penetration when using in vivo. Using G4 PAMAM dendrimers complexed with siRNA, we achieved a 70% knockdown efficiency of YWHAB in HEK293 cells. Optimal knockdown results were obtained after delivery of dendrimer-siRNA complex to the cells, with significant efficacy observed over seven-day post transfection. MTT assays confirmed that the dendrimer-siRNA complex was not toxic to cells indicating its safety. Wound healing assays showed a significant reduction in the migratory capacity of cells treated with the dendrimer-siRNA complex, suggesting potential to inhibit tumor invasiveness. These findings highlight the potential of PAMAM dendrimers as a safe and efficient siRNA delivery system for glioblastoma treatment, providing a robust foundation for further preclinical development of targeted therapies.

Support for this study was provided by 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.

Roles of microglial heparan sulfate in brain homeostasis and Alzheimer’s disease

J. Faulkner¹, D. Gonzalez¹, K. Drago¹, S. Soleimani¹, X. Song¹, H. Yang¹, C. Alvarez¹, L. Zoungrana¹, and L. Wang¹

¹Department of Molecular Pharmacology and Physiology, Morsani College of Medicine, University of South Florida, Tampa, FL, USA

Microglia, specialized immune cells in the brain, are crucial in maintaining normal brain function. They shape neuronal circuits, eliminate cellular debris, and constantly monitor the brain for signs of infection, injury, or abnormalities. However, in Alzheimer's disease (AD), microglia become dysregulated, responding to abnormal protein aggregates, such as beta-amyloid (Aβ) plaques and neurofibrillary tangles (NFTs). This dysregulation leads to chronic inflammation, further impairing neuronal survival and function. Therapeutic strategies aiming to modulate microglial function are being pursued to slow AD progression and preserve brain function. The success of these strategies relies essentially on a comprehensive understanding of the molecular mechanisms that govern microglial function. Heparan sulfate (HS) is a structurally variable polysaccharide that is expressed on various cell surfaces, including microglia, and in the extracellular matrix. It plays critical roles in multiple cellular processes, including cell-cell interactions and cell signaling. Moreover, HS has been implicated in various mechanisms central to inflammatory events and is involved in AD pathogenesis. Studies have demonstrated that microglial HS plays a role in facilitating the CD14/TLR4-dependent inflammatory response in culture, presuming through direct binding to CD14. Additionally, microglial HS co-deposits with Aβ plaques in both AD patients and the AD Tg2576 mouse model. However, the exact roles of microglial HS in brain homeostasis and AD are currently unknown. Building upon our unexpected findings that reducing microglial HS expression enhances LPS-induced microglia activation and ameliorates Aβ deposition in an AD mouse model, we have hypothesized that microglial HS is required for brain homeostasis and exacerbates amyloidopathy in AD.

AAV-mediated delivery of APOE4-Christchurch mitigates amyloid deposition, neuronal dystrophy, and alters microglia responses in APP/PS1 mice

K. Fredriksen¹, M. Kim², H. Korthas², and L. Li²

¹Graduate Program in Neuroscience, University of Minnesota, Minneapolis, MN 55455, USA

²Department of Experimental and Clinical Pharmacology, University of Minnesota, Minneapolis, MN 55455, USA

Alzheimer’s disease (AD) is the leading cause of dementia worldwide. It is characterized pathologically by the presence of Aβ plaques and tau neurofibrillary tangles, causing brain atrophy and neurodegeneration. Apolipoprotein E (APOE) is the greatest genetic risk factor for developing AD and in humans exists as 3 isoforms-APOE2, APOE3 (E3), and APOE4 (E4). E4 confers an increased risk of developing AD and causes widespread dysfunction including disrupted synapse formation, altered neuroinflammatory responses, and enhanced Aβ and tau aggregation. A recent case study revealed that the presence of a rare APOE mutation R136S, known as the Christchurch mutation (Ch), was associated with a significant delay in the onset of symptoms in an individual harboring a familial AD mutation which causes early-onset AD. Recent studies have shown the protective effect of E3-Ch on Aβ and tau pathology and related toxicity in multiple in vitro and in vivo AD mouse models. However, the effects of Ch on Aβ pathology in E4 backgrounds remain poorly understood. Here, we utilized a translational gene therapy approach by injecting an adeno-associated virus (AAV) vector expressing E4-Ch or E4 into 6–8-month-old APP/PS1 mice, an amyloid mouse model. We used a combination of immunofluorescence and biochemical techniques to determine the effect of E4-Ch 3 months after AAV delivery. Remarkably, AAV-mediated E4-Ch overexpression significantly reduced amyloid plaques and Aβ levels compared to E4-injected mice. Furthermore, we observed a reduction in plaque-associated neuronal dystrophy and an increased microglial response to plaques in E4-Ch injected mice. Studies are underway to further characterize microglia morphology and activation state in mice expressing E4-Ch. Overall, these findings provide evidence that exogenous Ch expression in the context of E4 reduces amyloid-related pathology and neurotoxicity in APP/PS1 mice and highlights the potential of gene therapy approaches that enhance Ch expression as a therapeutic strategy for AD.

Preclinical evidence supporting therapeutic application of apolipoprotein A1 in post-syptomatic ALS mice

S. Garbuzova-Davis, L. Manora, L. Gefen, and C.V. Borlongan

Department of Neurosurgery and Brain Repair, Center of Excellence for Aging and Brain Repair, University of South Florida, Morsani College of Medicine, Tampa, USA.

Amyotrophic Lateral Sclerosis (ALS) is a fatal neurodegenerative multifactorial disease, and dyslipidemia is considered an essential component of the pathologic disease process. Dysregulation of lipoprotein metabolism was noted in ALS patients and a mouse model of ALS. Lipid metabolism in plasma is mainly regulated by apolipoprotein constituents. Apolipoprotein A1 (ApoA1), the major protein component of high density lipoprotein, provides anti-oxidative and anti-inflammatory actions, preventing vascular damage. ApoA1 has shown beneficial effects in treatment of several diseases. Our recent in vitro study demonstrated therapeutic potential of ApoA1 for endothelial cell repair under pathologic conditions reminiscent of ALS. The aim of the current study was to determine the effect(s) of ApoA1 administration into symptomatic G93A SOD1 mutant mice of both genders. A single 100 µg dose of ApoA1 from human plasma or media was injected intravenously into G93A SOD1 male and female mice at 13 weeks of age and behavioral disease outcomes were evaluated for 4 weeks post-injection. The astrocyte and microglial cell statuses were determined via immunohistochemistry in cervical and lumbar spinal cords of both ALS male and female mice euthanized at 17 weeks of age. Spinal cord capillary permeability was evaluated for 2% Evans Blue (EB) fluorescent dye injected 30 min prior to perfusion of mice. Histological staining with 0.1% cresyl violet of motor neurons in the ventral horn of the cervical and lumbar spinal cords was performed in randomly selected ApoA1- and media-treated G93A SOD1 male and female mice. Results demonstrated that ApoA1 administration into symptomatic G93A SOD1 mice provided significant benefits in both males and females by: 1) retarded behavioral disease progression; 2) decreased EB extravasation into spinal cord parenchyma; 3) reduced astrogliosis and microgliosis; and 4) increased survival of spinal cord motor neurons. These novel data demonstrated that intravenous administration of ApoA1 into symptomatic ALS mice delayed disease progression and enhanced motor neuron survival, likely due to reparative effects of this protein on damaged microvasculature, potentially leading to blood-spinal cord-barrier repair. Also, the anti-inflammatory effect of ApoA1 was demonstrated by reductions of astrogliosis and activated microglia, resulting in possible mitigation of central nervous system vascular damage in ALS mice of both genders. The present study provides key preclinical evidence suggesting ApoA1 as a promising protein-mediated therapeutic for vascular restoration. Our innovative strategy to probe the role of ApoA1 in CNS barrier restoration via protein-facilitated endothelium homeostasis will prompt future clinical trials towards providing safe and effective treatment for ALS patients.

This study was supported by the NIH, NINDS (1R21NS132579-01) grant.

Generation and Characterization of hiPSC-Inhibitory Interneurons to treat cortical strokes

E. Gonzalez-Cubero¹, and I.L. Llorente¹.

¹Department of Neurosurgery, Stanford School of Medicine, Stanford University, Palo Alto, California.

The persistent incidence of stroke, despite declining mortality rates, has transformed this condition into a long-term, highly disabling health problem. In this study, we aim to address the limitations of current treatments, which are restricted by a critical time window and have not been possible to bring to the clinic. Aiming to overcome these challenges, our research focuses on the innovative application of inhibitory interneurons derived from human induced pluripotent stem cells (hiPSCs) as a novel therapeutic strategy to treat cortical stroke. Previous work has shown that inhibitory interneurons can modulate neuronal activity and promote synaptic plasticity, representing a promising regenerative solution. In this study, we have developed and optimized a differentiation protocol to generate hiPSCs- inhibitory interneurons from three independent donors. Comprehensive in vitro characterization has confirmed the identity and functionality of these cells. Through qPCR and immunostaining, we have demonstrated the expression of key markers, including LHX6, indicative of MGE-derived interneurons, as well as somatostatin (SST) and gamma-aminobutyric acid (GABA). Functional assays, including ELISA, demonstrated active GABA secretion, while migratory assays showed the cells’ ability to respond to chemotactic cues. Single-cell RNA sequencing further validated the expression of key interneuronal markers, providing a detailed transcriptomic profile. To assess electrophysiological properties, microelectrode array (MEA) recordings confirmed the cells’ ability to generate inhibitory synaptic activity. These preclinical findings were extended in vivo using a mouse model of cortical stroke. Following transplantation of the hiPSC-derived inhibitory interneurons encapsulated in biocompatible hydrogels, we observed enhanced tissue recovery. Immunohistochemical analysis revealed enhanced integration of transplanted cells and signs of synaptic connectivity in the peri-infarct regions. These results highlight the therapeutic potential of interneurons for modulating post-stroke neuroplasticity and promoting functional recovery. Our study demonstrates the feasibility of generating functionally mature interneurons from hiPSCs and their potential utility in stroke therapy.

"Long-Term Impact of Traumatic Brain Injury and Alcohol Misuse on Sleep-Wake Disturbances, Circadian Rhythms, and Neuroinflammation in Mice"

E.K. Gudenschwager-Basso¹, A.R. Morris¹, M.A. Gutierrez-Monreal¹, R.D. Arja², O. Kenyon¹, A. Garg¹, F.H. Kobeissy², C.G. Janus³, J. Zhu², K.K.W. Wang², and A.C. Liu¹

¹Department of Physiology and Aging, College of Medicine, University of Florida, 1345 Center Drive, Gainesville, FL 32610-0274, USA

²Center for Neurotrauma, Multiomics & Biomarkers, Department of Neurobiology and Neuroscience Institute, Morehouse School of Medicine, 720 Westview Drive, Atlanta, GA 30310, USA

³Center for Translational Research in Neurodegenerative Disease (CTRND), Department of Neuroscience, College of Medicine, University of Florida, 1275 Center Drive, Gainesville, FL 32610, USA

Traumatic brain injury (TBI) is a leading cause of mortality and diminished healthspan^1–3. TBI induces, poor sleep quality, and chronic morbidity^4–8. Sleep-wake disturbances (SWD) after TBI are one of the most common long-term consequences of injury^9,10. Sleep supports brain health and TBI recovery, in contrast, poor sleep is associated with neurodegeneration and health decline^8,11. Both TBI sequelae and SWD are exacerbated by prolonged alcohol misuse¹². At the same time, TBI raises the risk of alcohol misuse ¹³, although the underlying mechanisms remain poorly understood. In this study, we utilized a repetitive midline fluid percussion injury (rmFPI) mouse model to examine the long-term effects of TBI and alcohol on sleep, circadian rhythms, and neuroinflammation. Using the non-invasive PiezoSleep system, we observed that adult TBI mice exhibited reduced sleep duration starting 3 months post-injury. TBI mice also demonstrated altered circadian locomotor activity and decreased lean mass and body water content. Histological analysis revealed brain tissue loss, and microglial hyperactivity in the hypothalamus, indicating chronic neuropathology. We extended these findings by exploring the interaction between TBI and binge alcohol use (BAU) in female mice. While TBI alone induced sleep deficits at 4 months post-injury, the combination of rmFPI and BAU resulted in reduced sleep during specific phases (ZT 18–23) and increased sleep fragmentation during the dark-to-light transition at 18 months post-injury. RNA sequencing revealed changes in inflammation, immunity, and circadian rhythm-related genes. Histopathological analysis revealed increased microglial activation and neurodegeneration. These findings demonstrate that TBI induces SWD and circadian rhythm dysfunction, exacerbated by alcohol misuse. This work highlights the critical role of neuroinflammation and circadian regulation in TBI pathology. Further studies will identify molecular mechanisms and potential therapeutic targets.

References

1. Thompson, H. J., McCormick, W. C. & Kagan, S. H. Traumatic brain injury in older adults: epidemiology, outcomes, and future implications. J. Am. Geriatr. Soc. 54, 1590–1595 (2006).

2. Mattingly, E. & Roth, C. R. Traumatic Brain Injury in Older Adults: Epidemiology, Etiology, Rehabilitation, and Outcomes. Perspect. ASHA Spec. Interest Groups 7, 1166–1181 (2022).

3. Wood, R. L. Accelerated cognitive aging following severe traumatic brain injury: A review. Brain Inj. 31, 1270–1278 (2017).

4. Utomo, W. K., Gabbe, B. J., Simpson, P. M. & Cameron, P. A. Predictors of in-hospital mortality and 6-month functional outcomes in older adults after moderate to severe traumatic brain injury. Injury 40, 973–977 (2009).

5. Susman, M. et al. Traumatic brain injury in the elderly: increased mortality and worse functional outcome at discharge despite lower injury severity. J. Trauma Acute Care Surg. 53, 219–224 (2002).

6. Verkaik, F., Ford, M. E., Geurtsen, G. J. & Van Someren, E. J. W. Are sleep-related beliefs and behaviours dysfunctional in people with insomnia after acquired brain injury? A cross-sectional study. J. Sleep Res. e13998 (2023).

7. Gardner, R. C. et al. Effects of age and time since injury on traumatic brain injury blood biomarkers: a TRACK-TBI study. Brain Commun. 5, fcac316 (2023).

8. Fleming, M. K. et al. Sleep Disruption After Brain Injury Is Associated With Worse Motor Outcomes and Slower Functional Recovery. Neurorehabil. Neural Repair 34, 661–671 (2020).

9. Malinowska, K. B. et al. Effect of self-reported quality of sleep on mobility in older adults. Geriatr. Gerontol. Int. 16, 266–271 (2016).

10. Stenholm, S. et al. Sleep-related factors and mobility in older men and women. J. Gerontol. A Biol. Sci. Med. Sci. 65, 649–657 (2010).

11. Shen, Y. et al. Circadian disruption and sleep disorders in neurodegeneration. Transl. Neurodegener. 12, 8 (2023).

12. Jorge, R. E. et al. Alcohol misuse and mood disorders following traumatic brain injury. Arch. Gen. Psychiatry 62, 742–749 (2005).

13. Weil, Z. M., Corrigan, J. D. & Karelina, K. Alcohol use disorder and traumatic brain injury. Alcohol Res. 39, 171–180 (2018).

Adropin Protects Neuronal Injury in Subarachnoid Hemorrhage Patients

Z. Hasanpour Segherlou¹, H. Xu¹, H. Hutchinson¹, E. Klaas¹, M. Martinez¹, K. Hosaka¹, and B. Hoh¹

¹University of Florida College of Medicine, Department of Neurosurgery, Gainesville, Florida, 32611, USA

Background: Subarachnoid hemorrhage (SAH) is a cerebrovascular emergency with high morbidity and mortality rate. Cerebral vasospasm and delayed cerebral ischemia (DCI) are a major complication associated with patient morbidity and mortality following SAH with no effective treatment. Adropin, encoded by the Enho gene, is a novel peptide hormone that regulates endothelial function and is highly expressed in the brain. There is strong evidence of the protective effects of adropin in stroke, heart disease, aging, and other diseases. Adropin regulates endothelial cells and maintains blood-brain barrier integrity through an endothelial nitric oxide synthase (eNOS)-dependent mechanism. We aim to investigate adropin's therapeutic benefits under clinically relevant delayed treatments. Methods: SAH was induced in mice via autologous blood injection into the subarachnoid space under a stereotactic microscope. Mice received adropin treatment 6 or 12 hours post-SAH, with additional doses on days 1, 3, and 5, and were compared to a vehicle-treated group. Results: Adropin treatment reduced delayed cerebral vasospasm in SAH models, indicated by larger middle cerebral artery diameters in treated mice. Microthrombi formation decreased with adropin, restoring cerebral blood flow, as shown by fibrinogen immunostaining. Adropin also increased eNOS phosphorylation one day post-SAH, preserving nitric oxide bioavailability and attenuating SAH pathology. Functionally, adropin enhanced cognitive function in Y maze and novel object recognition test, suggesting improved recovery. It preserved ZO-1 expression, reduced cerebral edema, and activated microglial cells, reducing neuronal apoptosis. These results suggest adropin may mitigate vascular and neural damage, enhancing recovery in SAH models. Conclusion: DCI post-SAH causes neuronal apoptosis and neurological deficits. Adropin treatment improved cognitive tests performance and maintained cerebral perfusion on day 3, likely through eNOS activation, increasing NO bioavailability. Administering adropin at 6 and 12 hours post-SAH supports its potential for clinical use. Further studies are needed to confirm these promising results.

Transplantation of 3D printed dorsal spinal neural progenitor cell scaffolds for spinal cord injury

A. Huntemer-Silveira¹, A. Frie¹, M. Madhaparia², M. McAlpine², and A. Parr^3,4

¹Department of Neuroscience, University of Minnesota, Minneapolis, MN, 55455, USA

²Department of Mechanical Engineering, University of Minnesota, Minneapolis, MN, 55455, USA

³Department of Neurosurgery, University of Minnesota, Minneapolis, MN, 55455, USA

⁴Stem Cell Institute, University of Minnesota, Minneapolis, MN, 55455, USA

Spinal cord injury (SCI) is associated with profound changes in sensory and motor performance that severely impact quality of life. Cell transplantation is among the most promising treatments for SCI, however, while preclinical models utilizing cell transplantation have yielded promising outcomes, clinical translation has yet to be 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, therefore combinatorial approaches that utilize both scaffolds and cell transplantation may provide the necessary balance of cellular and structural support needed to promote 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 our results transplanting 3D-printed, stem cell derived dorsal spinal neural progenitor cell (dsNPC) scaffolds to target repair in sensory circuits. Briefly, differentiated dsNPCs were 3D-printed into the microchannels of silicone scaffolds utilizing previously published protocols. These cell-laden scaffolds were matured for three weeks and transplanted into an acute transection in athymic nude rats. Functional sensorimotor testing and neuroanatomical approaches were performed to evaluate the capacity of these cells in supporting repair of sensory systems. Future work will utilize both dorsal and ventral sNPC scaffolds in order to support both sensory and motor systems and maximize cellular targeting for neural therapy and repair after injury. This technology will push the field of spinal regeneration forward and advance the translation of cell transplantation therapies.

Therapeutic potential of novel construct rAAV9-R36 in heterozygous Reeler mice

N. Hurst-Calle¹, E. Stewart¹, M. Levis-Rabi¹, A. Joly-Amado¹, and K. Nash¹

¹Department of Molecular Pharmacology and Physiology, Morsani College of Medicine, University of South Florida, 12901 Bruce B Downs Blvd, Tampa FL-33612, USA

Reelin is a large extracellular matrix glycoprotein that plays a crucial role in neuronal migration, positioning, and synaptic plasticity in brain development and adulthood. Reelin signaling is found to be dysregulated in neuropsychiatric conditions such as autism and Alzheimer’s disease. We previously identified that the central fragment of Reelin, R36 can initiate reelin signaling and R36 single protein injection in the brain could rescue behavior deficits in heterozygous reeler mice (HRM). However, sustaining increased levels of R36 requires repeat injections in the brain which are invasive and non-practical. Therefore, this study aims to examine the use of rAAV9-R36, a novel construct deriving from the previously identified R36 protein as a gene therapy for sustained delivery of R36. We used HRM, which carry one copy of the reeler mutation (rl/+) resulting in a 50% decrease of Reelin levels and mild cognitive impairments. Wild type (WT) littermates and HRM aged 18-20 weeks were injected intracerebroventricularly with either rAAV9-R36 (n=12 [6F, 6M]), or rAAV9-GFP (n=11 [5F, 6M]). Two months after surgery, mice underwent behavioral evaluation (open field maze, hidden platform water maze, and fear conditioning). Following behavior testing, brain tissue was collected for western blot analysis of hippocampal tissue. Western blot analysis of brain tissue is underway to assess the levels of R36, as well as Reelin signaling pathway, such as phosphorylated Disabled-1 (Dab-1), Src and Fyn kinases, Reelin receptors (Apolipoprotein E receptor 2 and very low density lipoprotein receptor) and changes in synaptic markers. Behavior is currently being analyzed and will indicate if enhancing Reelin pathway through AAV-R36 will rescue cognitive impairments in HRM. Positive outcomes of these findings may identify new targets to be considered in diseases with dysregulated Reelin signaling in the brain such as Alzheimer’s disease and neuropsychiatric disorders alike.

Establishing a novel clinically relevant rodent model to understand pathophysiological consequences following Sepsis after Spinal Cord Injury

K. Iyer¹, K. Zamiar¹, S. Rippy², J. Patel¹, D. Patel¹, T.P. Garg¹, D. Winchester¹, A.M. Galvan Lara², T.A. Butterfield³, H. Saito², and S.P. Patel¹

¹Spinal Cord & Brain Injury Research Center, Department of Physiology, University of Kentucky, Lexington, KY, 40536, US

²Departments of Surgery and Physiology, University of Kentucky, Lexington, KY, 40506, US

³Athletic Training and Clinical Nutrition, University of Kentucky, Lexington, KY, 40506, US

Motor paralysis and post-injury complications severely impacts quality of life of individuals with Spinal cord injury (SCI). Post-SCI sepsis is a predominant secondary complication, impairing motor recovery and increasing mortality. The focus of this study is to develop a clinically relevant rodent model to mimic long-term complications in sepsis survivors following SCI.

Animals were divided in 4 groups – Sham, Sepsis, SCI and SCI+Sepsis. SCI and SCI+Sepsis groups underwent T10-laminectomy and contusion (200kDyn) using Infinite Horizon impactor. Sepsis was induced by intraperitoneal injection of cecal slurry (3ml) immediately post-SCI. Fluid resuscitation and antibiotics were initiated at 8-hours post-injury/sepsis induction, then twice daily for 5 days. Outcome measures include bacteremia, survival, body weight, cytokine levels (blood and tissues), locomotor function (BBB-LRS, horizontal ladder), in vivo muscle strength test. At 12 weeks-post SCI, spinal cords were collected for quantitative histology to corelate with behavior.

Animals in SCI+Sepsis group showed significant bacteremia at 6 hours post-sepsis induction compared to Sepsis or SCI alone that was associated with lowest survival in SCI+Sepsis. In addition, SCI+Sepsis also resulted in further impaired hindlimb locomotor recovery compared to SCI. No locomotor deficits were seen in Sham or Sepsis alone groups. In vivo muscle-strength test showed significant muscle weakness in SCI+Sepsis vs SCI. At acute observations, Splenomegaly, reduced leg skeletal muscles weights and elevated levels of cytokines in blood and spinal cord was evident in SCI+Sepsis group.

This study provides the first clinically relevant model to investigate post-SCI sepsis-induced complications at acute phase, laying the groundwork for understanding mechanisms and developing therapeutic strategies. Ongoing studies investigate pathophysiological outcomes of sepsis at chronic phases of SCI.

Acknowledgement: NIH/NINDS 1R21NS128749-01A1 (SP/HS), P20 M148326/GM/NIGMS NIH HHS/United States.

The Interplay Between Alzheimer's Disease and Glucose Metabolism via Reg3A Mediation

M. Levis Rabi¹, N. Hurst-Calle¹, K. Nash¹, C. Brechot¹, and A. Joly-Amado¹

¹Morsani College of Medicine, University of South Florida, 12901 Bruce B Downs Blvd, Tampa, FL 33612, USA

The main hallmarks of Alzheimer's Disease (AD) are the accumulation of extracellular amyloid beta plaques and intracellular tau neurofibrillary tangles. Additionally, AD is characterized by glucose metabolism dysregulation in the brain, contributing to neurodegeneration. In AD mice, inducement of diabetes and insulin resistance amplified the accumulation of amyloid beta plaques, oxidative stress, inflammation of nervous tissue, and impairment of mitochondrial function. Regenerating islet-derived protein 3A (REG3A), a secreted protein with antioxidant properties, has been shown to improve sensitivity to insulin and partly prevent cognitive deficits attributed to diabetes. Our previous data revealed that REG3A improved cognition and increased antioxidant levels in an amyloid mouse model. This study aims to investigate the effects of REG3A supplementation in AD mice with diet induced diabetes and will provide understanding into the intersection of AD and glucose metabolism, and as to how REG3A rescues cognitive deficits. APP/PS1 mice and non-transgenic littermates aged 7 months old were bilaterally injected with a recombinant adeno-associated virus serotype 9 (rAAV9) vector overexpressing REG3A or with an empty rAAV9 vector into the anterior cortex and hippocampus (n=8/9). Mice were then either left on their standard rodent chow diet or placed on a high-fat diet. Glucose metabolism was assessed through glucose and insulin tolerance tests and behavior testing was performed at 12 and 16 months. Brain tissue, blood, fat, and organs were collected at 17 months. We successfully induced diabetes after 3 months of high fat diet. Mice under high fat diet had significantly higher glucose levels during glucose tolerance test and insulin tolerance test, indicative of glucose intolerance and insulin resistance. Behavior analysis is ongoing and will indicate if treatment with REG3A prevented the worsening of cognitive impairments in AD mice.

Regenerative Effects of Dedifferentiated Peripheral Nerve Tissue Transplanted into the Substantia Nigra of Patients with Parkinson’s Disease

A. Marlonsson¹, J. Phares⁴, J.E. Quintero^1,2,3, C.G. van Horne^1,2,3, L. Granholm⁴, and G.A. Gerhardt^1,2,3

¹Department of Neuroscience, University of Kentucky, Lexington, KY 40536, USA

²Department of Neurosurgery, University of Kentucky, Lexington, KY 40536, USA

³Neurorestoration Center, University of Kentucky, Lexington, KY 40536, USA

⁴Department of Neurosurgery, University of Colorado Anschutz Medical Campus, Aurora, CO 80045, USA

ABSTRACT: We have investigated the long-term effects of autologous peripheral nerve tissue (PNT) grafts transplanted into the substantia nigra (SN) of participants with Parkinson’s disease (PD) receiving deep brain stimulation (DBS). This grafting procedure aims to leverage PNT’s regenerative potential against PD's neurodegenerative effects. A primary objective has been to characterize the PNT graft after extended survival in the central nervous system (CNS). We've begun analyzing brain donations from multiple participants who died of unrelated causes and were post-op up to 126 months. Immunohistochemistry (IHC) revealed a mild glial fibrillary acidic protein (GFAP) border forming around the PNT graft, indicative of a permissive form of astrogliosis allowing for restorative properties provided by PNT to influence surrounding host brain tissue. We observed diffuse IHC staining of P75 NTR and tyrosine hydroxylase (TH) within the PNT graft. P75’s presence in the graft suggests ongoing Schwann cell activity, while TH indicates dopaminergic activity, crucial for any potential PD treatment. The PNT grafts also stained positively for angiogenic factors including VEGF, CD31, and GAP43, indicating the graft could still be in a repair state after more than 10 years in the participant, actively producing new axons and blood vessels. Spatial transcriptomic analysis has supported the notion that PNT grafts have restorative effects by revealing a diffuse increase in the expression of BCL2L2, PRMT8, and ATG14 in the brain tissue immediately surrounding the graft compared to regions further away. These changes suggest enhanced cell survival and autophagy in the vicinity of the graft, contributing to a supportive environment for tissue repair and regeneration. These findings indicate a favorable interaction between the graft and host tissue and highlight the potential restorative effects of PNT grafts. The long-term survival and activity of these grafts underscore the importance of further research into the mechanisms underlying PNT-mediated neuroprotection and regeneration.

(Supported in part by NIA AG081356)

Optimizing 660nm Photobiomodulation for Motor Recovery in Wistar Rats with Spinal Cord Injury

N. Mojarad^1,2, B. Stewart^1,2, S. Wright^1,2, K. Reed^1,2, A. Uprety^1,2, J. Rossignol^1,2,3, and G.L. Dunbar^1,2,3,4

¹Field Neurosciences Institute Laboratory for Restorative Neurology, Central Michigan University, Mount Pleasant, MI 48859 USA

²Program in Neuroscience, Central Michigan University, Mount Pleasant, MI 48859 USA

³College of Medicine Central Michigan University, Mount Pleasant, MI 48859 USA

⁴Department of Psychology, Central Michigan University, Mount Pleasant, MI 48859 USA

Spinal cord injury (SCI) is the leading cause of disabilities worldwide, resulting in temporary or permanent impairment of function. Photobiomodulation therapy (PBMT) has emerged as a promising avenue due to its multifaceted benefits, including suppressing inflammation and repairing damage tissue. While initial preclinical studies have shown potential of PBMT for axonal regeneration and inflammation reduction, there is currently no standardized protocol or timeline for its application in SCI treatment. We conducted three studies aimed at optimizing PBMT as therapy for SCI. Firstly, we compared the effects of one-week PBMT, two-weeks PBMT with treatments using methylprednisolone sodium succinate (MPSS) on motor function and inflammation in male rats with moderate compression SCI. We found that two weeks of PBMT treatment was as efficacious as MPSS in motor recovery and inflammation reduction, without causing weight loss or mortality, suggesting long-term PBMT as a preferable option due to fewer side effects. Our second study investigated the efficacy of two PBMT protocols administered over two and four weeks in male rats with moderate compression SCI. The four-week PBMT showed greater effectiveness in alleviating motor dysfunction compared to the shorter duration protocols, emphasizing the importance of extended PBMT durations for optimal outcomes in motor function recovery. Our third study extended the PBMT timeline to seven weeks in both male and female rats with severe compression SCI. Daily PBMT administration throughout the experiment demonstrated enhanced motor recovery compared to untreated groups, highlighting the potential of extended PBMT durations in improving outcomes post-SCI. Our studies indicate that prolonged PBMT therapy can significantly enhance motor recovery following SCI. Our results indicate that PBMT is an effective therapy, even following severe SCI, suggesting that it will enhance therapeutic outcomes when used in combination with other therapies.

3D Printed Scaffolds Promote Enhanced Spinal Organoid Formation for Use in Spinal Cord Injury

N. Patil¹, G. Han², N.S. Lavoie¹, O.G. Korenfeld¹, H. Kim², M. Esguerra³, D. Joung⁴, M.C. McAlpine², and A.M. Parr¹

¹Department of Neurosurgery, University of Minnesota, Minneapolis, MN, 55455, USA

²Department of Mechanical Engineering, University of Minnesota, Minneapolis, MN, 55455, USA

³Department of Neuroscience, University of Minnesota, Minneapolis, MN, 55455, USA

⁴Department of Physics, Virginia Commonwealth University, Richmond, VA, 23284, USA

The transplantation of regionally specific spinal neural progenitor cells (sNPCs) has shown promise for functional restoration after spinal cord injury (SCI) by forming connections with host neural circuits. Here, we developed 3D printed organoid scaffolds for transplantation using clinically relevant human induced pluripotent stem cell-derived regionally specific sNPCs. Scaffolds with microscale channels were printed, and sNPCs were subsequently printed within these channels. The scaffolds directed axonal projections along the channels and guided the cells to simulate in vivo-like conditions, leading to more effective cell maturation and the development of neuronal networks crucial for restoring function after SCI. The scaffolds, with organoids assembled along their lengths, were transplanted into the transected spinal cords of rats. This significantly promoted the functional recovery of the rats. At 12 weeks post-transplantation, the majority of the cells in the scaffolds differentiated into neurons and integrated into the host spinal cord tissue. These results demonstrate the potential to create a relay system along the spinal cord and form synapses in both the rostral and caudal directions relative to the scaffold. We envision that combining sNPCs, organoid assembly, and 3D printing strategies can ultimately lead to a transformative treatment approach for SCI.

The Influence of Limb Dominance on Potential Vulnerability to Parkinsonian Neurodegeneration in Rats

D. Pokharel², D. Beligala¹, C. Swain², V. Peshattiwar¹, K. Le¹, I. Nester¹, T. Subramanian³, and K. Venkiteswaran²

¹Department of Neurology, ²Department of Neurology and Neurosciences, ³Department of Neurology & Neurosciences, and Bioengineering, University of Toledo, College of Medicine and Life Sciences, Toledo, OH, 43614, USA.

Limb dominance has been studied in the context of Parkinson’s disease (PD), but its role in susceptibility to neurotoxin-induced parkinsonism or alpha-synucleinopathy remains unclear.[1] PD is characterized by the progressive, asymmetric loss of dopaminergic neurons in the nigrostriatal pathway and the unilateral onset of symptoms (Stage 1 disease), which results in persistent asymmetry in disease manifestation throughout its course.[2] Factors such as intrinsic brain differences, localized vulnerability, compensatory mechanisms, and alpha-synuclein pathology progression may contribute to this asymmetry, with the initially affected side remaining more impaired as the disease progresses.[3] Our lab previously demonstrated that oral administration of paraquat (P) combined with lectin (L) for seven days induces bilateral parkinsonism in rats.[4] Here, we tested the hypothesis of whether limb dominance influences the side of onset of parkinsonism following P+L administration. Limb dominance was assessed using the Collins paw preference test in N=123 Sprague-Dawley rats (55 right-paw dominant, 48 left-paw dominant, and 20 ambidextrous). Parkinsonian symptoms were evaluated using a rodent behavioral battery of tests (RBBT). Following P+L exposure (N=16; 8 right-paw dominant, 8 left-paw dominant), 13 rats initially manifested right hemiparkinsonism (HP) 8 right-paw dominant and 5 left-paw dominant. In contrast, 3 animals manifested left HP, all left-paw dominant. HP status was statistically confirmed using RBBT in right HP rats (p<0.03) and left HP rats (p<0.02). Statistical analysis of hemispheric risk for parkinsonism (expected 8:8 versus observed 13:3) using Fisher’s exact test revealed significantly higher left hemispheric vulnerability for parkinsonism (p=0.01). Histological analyses, including design-based stereology and bulk proteomics; and the investigations into differential effects of left and right vagus nerve effects on gut-brain alpha-synuclein transmission, aim to elucidate mechanisms underlying this increased left hemispheric vulnerability to parkinsonism. Our findings provide novel insights into the neurobiology of PD and may identify important targets for disease prevention or mitigating progression.

References

1. van der Hoorn, A et al., (2011). Handedness and dominant side of symptoms in Parkinson's disease. Parkinsonism & related disorders, 17(1), 58–60. https://doi.org/10.1016/j.parkreldis.2010.10.002

2. Barrett, M. J., et al., (2011). Handedness and motor symptom asymmetry in Parkinson's disease. Journal of neurology, neurosurgery, and psychiatry, 82(10), 1122–1124. https://doi.org/10.1136/jnnp.2010.209783

3. Shi, J., et al., (2014). Handedness and dominant side of symptoms in Parkinson's disease. Medicina clinica, 142(4), 141–144. https://doi.org/10.1016/j.medcli.2012.11.028

4. Anselmi, L., et al., (2018) Ingestion of subthreshold doses of environmental toxins induces ascending Parkinsonism in the rat. NPJ Parkinson's disease, 4, 30. https://doi.org/10.1038/s41531-018-0066-0

Knockout of Psen1, Gsk3b, and Bmpr1a in primary rat astrocytes using CRISPR/Cas9 converted them into neuron-like cells: applications for stroke therapy

A. Poudel^1,2,4, B. Srinageshwar^1,4, E.A. Nisper⁴, S.A. Schwind^1,2,3, N.J. Day^1,5, L.K. Bolen^1,2,3, A.M. Uprety^1,2,3, A. Sharma⁵, G.L. Dunbar^1,2,3, J.L. Bakke⁴, and J. Rossignol^1,2,4.

¹Field Neurosciences Institute Laboratory for Restorative Neurology

²Program in Neuroscience

³Department of Psychology

⁴College of Medicine

⁵Department of Chemistry & Biochemistry Central Michigan University, Mount Pleasant, MI, USA

Hypoxic injury, notably observed after ischemic stroke leads to neuroinflammation, causing neuronal death, which triggers an immune response that activates the protective functions of astrocytes, also known as reactive astrocytes. Previous research has shown that it is possible to convert these astrocytes into functional neurons by inhibiting specific pathways, including the Notch, GSK-3 β, and BMP pathways. The goal was to reprogram astrocytes into neurons by knocking out the genes in the specified pathways which could be a potential treatment for ischemic stroke. In this study, we used the CRISPR/Cas9 gene-editing tool to knock out the target receptor-associated genes Psen1 for Notch pathways, Bmpr1a for BMP pathway, and Gsk3b for GSK-3β in adult rat brain-derived astrocytes. Sanger sequencing showed knockout of the above-mentioned genes. Western blot analysis showed an alteration in the expression of the target proteins of each pathway in HEK T293 cells, which were used as control, suggesting the successful knockout of the above-mentioned target genes. We performed immunocytochemistry in Psen1, Gsk3b, and Bmpr1a CRISPR/Cas9 treated primary rat astrocytes and observed the expression of neuronal marker Tuj1 after 28 days of transfection.

This study aimed to confirm the delivery of CRISPR/Cas9 to reprogram astrocytes in primary rat cell culture using G4 PAMAM dendrimers, as well as in an MCAo rat model, to edit the genes involved in the pathways described above to convert reactive astrocytes into neuroblasts. This in vitro study suggests that using CRISPR/Cas9 to edit genes and pathways to convert astrocytes into neuron-like cells in stroke could be a promising strategy for these therapeutics in the brain. Applications of this delivery tool are underway in MCAo rat model.

Support for this study was provided by the American Heart Association (AIREA grant #957277), the College of Medicine, John G Kulhavi Professorship, Neuroscience Program, E. Malcolm Field and Gary Leo Dunbar Endowed Chair in Neuroscience at Central Michigan University, and partially by the Graduate Research Grant from the Office of Sponsored Programs at Central Michigan University.

Administrations of Human Astrocyte-derived EVs in Adulthood Can Promote the Maintenance of Enhanced Cognitive Ability in Early and Late Middle Age

S. Rao, M. Kodali, G. Shankar, A. Morton, Y. Somayaji, S. Attaluri, L.N. Madhu, B. Shuai, and A.K. Shetty

Institute for Regenerative Medicine, Department of Cell Biology and Genetics, Texas A&M University School of Medicine, College Station, Texas, USA-77845

Astrocytes are vital for maintaining the blood-brain barrier, neuronal function, neurotransmitter recycling, regulating immune response, and promoting repair after injury or disease. Hence, it is believed that extracellular vehicles (EVs) shed by astrocytes have therapeutic properties. This study examined the proficiency of EVs from human induced pluripotent stem cell (hiPSC)-derived astrocytes (iAstrocytes), to enhance cognitive and memory function in aging. The media from mature iAstrocyte cultures were used to isolate astrocyte-derived EVs (ADEVs). Small-RNA sequencing revealed the presence of multiple miRNAs capable of mediating antiinflammatory and neuroprotective effects in ADEVs cargo. A biodistribution study in 6-month-old C57BL6/J mice confirmed the ability of intranasally (IN) administered ADEVs to target neurons, microglia, astrocytes, and oligodendrocytes in the entire brain. We next administered ADEVs to a larger cohort of 6-month-old mice (weekly IN doses for two weeks, 30-billion/dose). Analyses of cognitive function 6 months after EVs administration revealed improved ability in mice receiving ADEVs for perceiving minor changes in their environment in an object location test (OLT) and pattern separation function in a pattern separation test (PST). Such improvements in hippocampus-dependent tasks were evident from ADEVs or VEH-treated mice showing proficiency with shorter inter-trial intervals (ITIs; 3-hours in OLT and 30-minutes in PST), but only ADEVs-treated mice exhibiting competence with longer ITIs (7-hours in OLT and 4-hours in PST). Analyses of cognitive function 12-months after EVs administration also revealed improved cognitive ability in mice receiving ADEVs, apparent from ADEVs or VEH-treated mice showing proficiency in OLT and PST tasks with shorter ITIs (30-min in OLT and 15-min in PST) but only EVs-treated mice exhibiting competence with longer ITI’s (3-hours in OLT and 1-hour in PST). The results suggest that IN administrations of ADEVs in adulthood can promote enhanced cognitive performance in early and later middle age.

Support: Supported by a grant from the National Institute for Aging (R01AG075440 to A.K.S.).

Acute and subacute administration of AAV9-NeuroD1 for functional restoration after spinal cord injury

A. Roman^1,2, M. Sorensen³, A. Parr^1,2, A. Grande^1,2, and W. Low^1,2

¹Graduate Program in Neuroscience

²Department of Neurosurgery

³College of Biological Sciences, University of Minnesota Twin Cities, Minneapolis, MN 55455

The viral delivery of proneural factors for in vivo glia-to-neuron reprogramming, has emerged as a promising—and controversial—strategy for central nervous system (CNS) restoration. Published studies have characterized NeuroD1, a developmental proneural factor, as sufficient to convert astrocytes into neurons in vitro and in animal models of CNS injury and disease. Of these published studies, only four examined the platform within the context of spinal cord injury (SCI). Notably, these studies report varying extents of functional recovery. Other studies have separately challenged the premise of single-factor-mediated reprogramming, citing inconsistencies in reprogramming efficiency and questioning the cellular origin of “reprogrammed” cells. In response, viral dosage and intervention timing have been identified as factors capable of influencing reprogramming efficacy and reproducibility. We hypothesize that the AAV9-NeuroD1 platform is capable of restoring nervous system function following SCI through reprogramming and neuroprotection. Here we employed a two-vector, AAV9 DIO/FLEx-based delivery platform for selective expression of NeuroD1-mRuby2 (reprogramming) or mRuby2 alone (control) in GFAP-expressing reactive astrocytes after moderate SCI in female rats. The viral treatment was administered either immediately after injury (i.e. acute stage of injury) or 1 week post-contusion (i.e. subacute stage of injury) at one of two viral titers (10¹³ or 10¹¹ GC/mL); these rats were assessed for motor and sensory functional recovery up to 6 weeks post-contusion. Acute viral intervention revealed a dose-dependent deleterious effect on motor function of the viral platform, where the use of AAV9 at the high titer was associated with lower BBB scores, irrespective of NeuroD1 involvement. Interestingly, this did not translate to subacute viral intervention, where the motor functional recovery of NeuroD1-treated rats did not significantly differ from injury-only controls. However, preliminary analysis of spinal cord anatomical restoration revealed dose-dependent, NeuroD1-mediated tissue preservation. Future work will continue to focus on the neuroprotective capabilities of this platform.

Unveiling the pioneering potential of phytoestrogens in promoting stem cell functions and neural regeneration in post-menopausal brain

Ambika¹, R.L. Gajbhiye², P. Ramalingam², D. Prasad³, V. Ravichandiran¹, and V.K. Parihar¹

¹Department of Pharmacology and Toxicology, National Institute of Pharmaceutical Education and Research, Hajipur, Bihar, 844102, India

²Department of Pharmaceutical Analysis, National Institute of Pharmaceutical Education and Research, Hajipur, Bihar, 844102, India

³Department of Obstrectics and Gynaecology, Indira Gandhi Institute of Medical Sciences, Patna, Bihar, 800014, India

Chronic estrogen deprivation increases the risk of post-menopausal females developing cognitive impairment, dementia, and accelerated brain aging. Menopause may impede cognition by disrupting the hippocampal neurogenesis, persistently increasing neuroinflammation, and disrupting glial-neuron bioenergetics. Nevertheless, the biochemical mechanisms by which estrogen deficiency during menopause contributes to cognitive deterioration remain inadequately elucidated. This study investigated the integrated proteomic and transcriptomic changes in the blood of post-menopausal women and in ovariectomized mice model, both showing chronically estrogen depletion states. Our findings confirmed that menopause significantly alters the genes and proteins regulating olfactory system, at both mRNA and protein levels. Further these olfactory genes strongly influence neuroinflammation, brain immunity, formation of new stem cells, and promotes neural tissue repair and regeneration. In addition, integrated omics approach determined that women who show maximum anxiety, and depressive behavior have parallel dysregulation in the genes regulating neurogenesis, neuroinflammation, neuronal senescence, synaptic plasticity and cognition. Likewise, our animal study has confirmed that depleted estrogen in ovariectomized mice exhibited impairment of hippocampal, and pre-frontal cortex-dependent episodic, spatial and temporal order memory. Moreover, this decrease of estrogen has been directly associated with the reduced expression of genes and proteins regulating mitochondrial bioenergetics (NDUFB8), neurodegeneration (GFAP), epigenetics (5-HMC), and neuroinflammation (HMGB1). Moreover, estrogen depletion also showed reduced expression of markers associated with stem cell proliferation (Ki67), neuronal differentiation (Sox2, Tuj1), and regeneration (MAP2). Furthermore, immunohistochemical analysis revealed significant loss of mature neurons (NeuN+), immature neurons (DCX+), and impaired synaptic plasticity (PSD-95). The neurocognitive benefits following phytoestrogen diet treatment were concomitant with multiple favorable, cellular and molecular alterations in post-menopausal mice, which comprised diminished neuroinflammation, enhanced neurogenesis, and normalized expression of multiple genes and proteins maintaining normal mood and memory functions. Thus, administration of diet is an efficacious approach to reducing chronic neuroinflammation and cognitive impairments in chronically estrogen deprived mice.

The effect of fibrinogen on brain adrenergic receptors and the resultant changes in memory during traumatic brain injury

N. Sulimai, J. Brown, and D. Lominadze

Department of Surgery

Morsani College of Medicine

University of South Florida, Tampa, FL 33612 USA

Traumatic brain injury (TBI) often leads to memory impairment. Currently, TBI has been shown to cause significant changes in adrenergic receptor (AR) signaling. ARs (categorized into alpha (α) and beta (β) subtypes), which are cell surface proteins that bind catecholamines and mediate sympathetic nervous system responses, play crucial roles in memory consolidation and synaptic plasticity in the brain. ARα1, especially the α1A subtype, regulates synaptic efficacy, short- and long-term synaptic plasticity, and various types of memory. Understanding the molecular signaling of AR during TBI may provide insights into potential strategies for improving cognitive deficiency during TBI. We have previously shown that TBI, as an inflammatory disease, was associated with increased extravascular deposition of fibrinogen (Fg) and an elevation in systemic and brain expression of pro-inflammatory cytokines, resulting in neurodegeneration. However, an association of extracellularly deposited Fg with the expression of ARs in the brain and the effect of this possible association on cognition has not been explored. Mouse brain endothelial cells were treated with 2mg/ml or 4mg/ml of Fg. Fg dose-dependently reduced the expression of ARα1A-(ADRA-1A). A cortical contusion injury (CCI)-induced model of mild-to-moderate (m-m) TBI was generated in C57BL/6 mice. Novel object recognition and fear conditioning tests were used for short-term memory assessment and brains were collected 14 days after CCI for immunohistochemistry and PCR analyses. M-mTBI resulted in reduced expression of ADRA-1A, AR-α1B (ADRA-1B), and AR-B3 (ADRB3), increased extravascular deposition of Fg, and a short-term memory deficit. Mice with a reduced Fg concentration in their blood demonstrated improvement in short-term memory. Our findings suggest that Fg is in part responsible for the reduced expression of AR during m-mTBI and cognitive deficits. Future studies targeting AR may have therapeutic potential in managing TBI-associated cognitive deficits.

Effects of chronic levodopa on cognitive deficits in paraquat and lectin rat model of Parkinson’s disease

C. Swain², D. Pokharel², K. Le¹, V. Peshattiwar¹, K. Venkiteswaran², and T. Subramanian³

¹Department of Neurology

²Department of Neurology and Neurosciences

³Department of Neurology, Neurosciences, and Bioengineering, University of Toledo College of Medicine and Life Sciences, Toledo, OH, 43614 USA

Parkinson’s disease dementia (PDD) is diagnosed in the setting of a pre-existing Parkinson’s disease (PD) diagnosis, when symptoms of cognitive decline occur at least one year after the onset of motor deficits (1). It is currently unknown whether chronic dopamine replacement therapy in the setting of PD contributes to the development of PDD (2). The paraquat and lectin (P+L) rat model of parkinsonism involves administering subthreshold doses of herbicide paraquat (1mg/kg) and dietary lectins (0.05%) via oral gavage following an i.p. injection of cholecystokinin (CCK, 4µg/kg) to rats for 7 days (3). In this model, parkinsonian motor deficits are observed as early as two weeks post-P+L treatment, and visuospatial cognitive deficits are observed four weeks post-P+L treatment. The aim of this study was to evaluate whether chronic levodopa treatment following the onset of motor deficits in the P+L rat model would accelerate cognitive decline. Sprague-dawley rats (n=6) were orally gavaged for 7 days with paraquat and lectin. After the onset of bilateral motor deficits, rats were administered 30 days of twice daily i.p. levodopa (4mg/kg) + benserazide (15mg/kg) injections, followed by a 30 day washout period. Rats were tested for cognitive deficits via the Y-maze at baseline, at four weeks (prior to levodopa), and after levodopa washout. Rats show a gradual decline in percent spontaneous alternations from baseline (81%), 4 weeks (59%), and post-levodopa washout (56%), but not significant with one way ANOVA (p=0.052). While preliminary results show that chronic levodopa does not appear to impact cognitive decline, studies are ongoing to repeat with a higher sample size and compare against a behavioral control group administered 30 days of twice daily i.p. saline following motor deficits. Histological analysis is also ongoing to determine any changes in pathology, including changes in dopaminergic neuron loss, cholinergic neuron loss, or phospho-S¹²⁹-α-Synuclein pathology.

Sources

(1) Gomperts SN. Lewy body dementias: dementia with Lewy bodies and Parkinson disease dementia. Continuum: Lifelong Learning in Neurology. 2016;22(2 Dementia):435.

(2) Molloy SA, Rowan EN, O'Brien JT, McKeith IG, Wesnes K, Burn DJ. Effect of levodopa on cognitive function in Parkinson's disease with and without dementia and dementia with Lewy bodies. J Neurol Neurosurg Psychiatry. Dec 2006;77(12):1323-8. doi:10.1136/jnnp.2006.098079

(3) Anselmi L, Bove C, Coleman F, et al. Ingestion of subthreshold doses of environmental toxins induces ascending Parkinsonism in the rat. npj Parkinson's Disease. 2018;4(1):30.

Developing and optimizing bioluminescent neurotransmitter sensors and neurotransmitter dependent neuromodulators

K. Taylor^1,2, H. Galvin¹, Z. Pinderi¹, and E. Petersen^1,2,3

¹CMU Biochemistry, Molecular and Cell Biology Program

²CMU College of Medicine

³CMU Neuroscience Program

Mt. Pleasant, MI 48859, United States

Many neurological diseases such as Alzheimer’s Disease, Parkinson’s Disease, and autism spectrum disorder have been shown to be associated with neurotransmitter imbalance. Expanding on the types of neurotransmitters and methods to study them is important for revealing disease mechanisms and creating new treatments. In this study, we focus on gamma-aminobutyric acid (GABA) and acetylcholine (ACh), which are neurotransmitters that are involved in many neurological disorders. We developed a variety of genetically encoded bioluminescent GABA and ACh sensors that are an attractive alternative to using fluorescent sensors because they do not require an excitation light source, allowing deeper areas of the brain to be recorded without autofluorescence and damaging tissue. We created a library of GABA and ACh sensor variants based on our prior glutamate sensors and tested their neurotransmitter response. Taking bioluminescence readings on a plate reader, we found that the sensors with an optimized transmembrane domain have a signal-to-noise ratio (SNR) of 1.7 and up to 300% higher response to saturating amounts of neurotransmitter than variants with the traditionally used domain. To further improve the neurotransmitter response of the sensors with the goal of using them to image brain activity in rodents, we are using rational design with the goal of improving response amplitude and SNR. We also paired these light emitting sensors with light sensitive optogenetic channels to excite or inhibit neurons based on the presence of a specific neurotransmitter. For example, the ACh sensor was paired with an excitatory channel to depolarize cells in the presence of ACh and luciferin. This showed a 50 pA response to ACh and luciferin compared to a 10 pA response to luciferin alone, giving a SNR of 3.8. Our sensors will allow scientists to modulate and correct over- or underactive neural circuits to treat neurodegenerative disease.

Constraints on gene delivery to stem/progenitor cell populations for cellular engineering or reprogramming

M. Thaqi, M. Schlyer, E. Reisenbigler, J. Lee, C. Speil, W. Parsons, L. Monroy, and D.A. Peterson

Center for Stem Cell and Regenerative Medicine, The Chicago Medical School, Rosalind Franklin University of Medicine and Science, North Chicago, IL 60064, USA

Gene delivery has become a powerful tool for engineering cell expression for scientific investigation and, increasingly, as an accepted therapeutic tool. From the perspective of therapeutic applications, the field has invested heavily in the use of adeno-associated viruses (AAV) as a preferred viral vector. Recently, lineage respecification of somatic or progenitor cell populations has been demonstrated in vitro and in vivo. As neurons are not replaced in most of the brain once damaged or killed, lineage respecification by reprogramming could lead to the recruitment of non-neuronal populations into replacement neurons. However, the lack of cellular selectivity with AAV-mediated gene delivery, its potential toxicity, and the potential leakiness of supposedly cell-specific promoters has mitigated progress. We found that most natural AAV serotypes infect a variety of neural cell types, even if predominantly infecting one specific cell type. We are interested in targeting a particular glial progenitor cell population, the oligodendrocyte progenitor cells (OPCs) in our reprogramming studies, but found that the cell specific promoter, NG2, did not limit reporter gene expression following AAV delivery. In addition, we noticed that 21 days following gene delivery, virtually no NG2-positive cells were expressing the fluorescent reporter. This is in contrast to a sustained expression of GFP following retroviral gene delivery. Using a dual thymidine labeling approach, we demonstrate that OPC populations are mitotically active and postulate that with non-integrating vectors, like AAV, there is a dilution effect with continued cell proliferation. AAV has also been reported to be toxic to dividing cells. We suggest that proliferating stem/progenitor cell population should be targeted with integrating viral vectors like retrovirus and lentivirus. As initiation of lineage reprogramming in fully differentiated somatic cells can result in their initial cell cycle reentry, integrating viral vectors may be the best choice for all reprogramming approaches.

Early rehabilitation attenuates late-onset symptoms and restores circuit function in a preclinical traumatic brain injury model

T.C. Thomas^1,2,3,4, C. Bromberg^1,2,5, T. Curry-Koski^1,2,3, Z. Sabetta^1,2, L. Curtin^1,2,3, P.D. Adelson, and G. Krishna^1,2

¹University of Arizona College of Medicine-Phoenix, Department of Child Health, Phoenix, AZ

²Barrow Neurological Institute at Phoenix Children’s Hospital, Phoenix, AZ

³Midwestern University, Glendale, AZ

⁴Phoenix VA Health Care System, Phoenix, AZ

⁵Arizona State University, Tempe AZ

Up to 50% of people who have experienced a traumatic brain injury (TBI) report late-onset and/or persistent sensory hypersensitivity, somatic, cognitive, and affective symptoms that prevent return to pre-injury health and well-being. Our preclinical studies demonstrate that diffuse axonal injury (DAI)—the most common neuropathology associated with TBI—decreases neuronal circuit integrity, promotes maladaptive circuit reorganization, which precedes the development of late-onset and persistent sensory hypersensitivity correlating with abnormal thalamocortical glutamate signaling by 28 days post-injury (DPI; 28DPI=2 clinical years post-injury). Early rehabilitation is well-established in driving adaptive neuroplasticity and reducing chronic symptoms after severe neurological injuries. However, more evidence is needed to justify its use as standard practice to prevent chronic symptoms in the more prevalent mild-to-moderate cases. We hypothesize that early rehabilitation can promote adaptive neuroplasticity and attenuate persisting symptoms and associated neuronal dysfunction. Midline fluid percussion injury (FPI) induced DAI in young adult male and female rats (n=10-12/group). Rats received thalamocortical circuit-directed rehabilitation (Early Rehab) for two weeks (15min/day, 3times/week, between days 7 and 18) or no rehabilitation (No Rehab). Immediately Post-Rehab, brain-derived neurotrophic factor (BDNF) mRNA levels were measured using rtPCR. At 28DPI, sensory hypersensitivity was assessed, followed by in vivo amperometry with glutamate-selective microelectrodes to measure glutamate signaling. Statistical analysis employed one- or two-way ANOVAs with Tukey’s post-hoc comparisons. Rehab upregulated BDNF for 2-5 hours post-treatment (p<0.01), indicating enhanced adaptive neuroplasticity. The No Rehab group exhibited significantly greater sensory hypersensitivity than controls (p<0.001). At 28DPI, early Rehab reduced sensory hypersensitivity in both males (43%) and females (34%) and restored abnormal glutamate signaling to control levels (p<0.05). Data support the efficacy of early rehabilitation to attenuate a common late-onset symptom and restore circuit function, reinforcing early rehabilitation strategies as a potential non-invasive approach to improve outcomes and enhance the long-term quality of life for TBI patients.

Funding: NIH R01NS100793, Phoenix Children’s Mission Support

Bioluminescent Kinase Sensors for detection of insulin-stimulated site-specific phosphorylation of Glucose Transporter 4 regulatory proteins

A. VanDusen¹, M. Chatterton^1,2, E.D. Petersen^1,2, and J.L. Bakke¹

¹College of Medicine, Central Michigan University, Mount Pleasant, MI 48859, USA

²Program in Neuroscience, Central Michigan University, Mount Pleasant, MI 48859, USA

Type 2 Diabetes, insulin resistance, and obesity are significant risk factors for developing neurodegenerative diseases. Those with poorly managed type 2 diabetes are at significantly higher risk for neurovascular damage, amyloid beta overaccumulation, Tau phosphorylation, chronic neuroinflammation, blood brain barrier damage, developing dementia or Alzheimer’s disease, and stroke. Blood glucose homeostasis is a highly regulated process essential for cellular metabolism, particularly in the brain, which has a high energy demand and limited energy storage capacity. This biological process is prone to dysregulation leading to failure to properly maintain glucose levels, leading to metabolic dysregulation, oxidative damage, weight gain, inflammation, and other factors that all contribute to the development of diabetes-related neurodegeneration. Our project focuses on designing, developing, and optimizing a Bioluminescent Kinase Sensor (BlinKS) to detect insulin receptor signaling in glucose transport pathways in muscle and adipose tissue. Specifically, the sensor will measure site-specific phosphorylation of mimic regulatory proteins AS160 and TBC1D1. In our experiments, we tested nine different phosphorylation-site-specific substrate variants in HEK293 and C2C12 cells. Once variant achieved an average maximal bioluminescence of 45.2% higher in insulin-treated C2C12 cells compared to serum-starved control cells. By enabling precise monitoring of insulin-stimulated regulation of GLUT4 activity, this tool could significantly advance basic research and be applied for targeted modulation of insulin signaling for self-regulated enhancement of cellular responses to insulin which we plan to test in a diabetic Alzheimer’s mouse model. Ultimately, insulin-responsive BlinKS may improve our understanding and treatment of type 2 diabetes and insulin resistance, paving the way for enhanced clinical interventions, and preventing neurodegenerative diseases caused by type 2 diabetes.

Utilizing the Barnes Maze to Assess Cognitive Flexibility after Early Intervention with Gabapentin in Male Rats with Traumatic Brain Injury

J. Venegas^1,2, C. Bromberg^2,3, and T.C. Thomas ^1,2,3

¹Midwestern University, Glendale AZ, 85308, United States ²University of Arizona-College of Medicine-PHX AZ, 85004, United States ³Arizona State University-Tempe AZ, 85281, United States

Traumatic brain injury (TBI) often leads to long-term cognitive impairments, including deficits in spatial learning and cognitive flexibility, which profoundly affect quality of life. To date there are no early pharmacological intervention to prevent long-term consequences of TBI. Early injury-induced activation of astrocyte-secreted thrombospondins is hypothesized to drive maladaptive plasticity, contributing to the development of cognitive deficits. Gabapentin (GBP), by blocking thrombospondin-mediated synaptogenesis, is proposed as a therapeutic strategy to prevent these deficits. The Barnes Maze (BM), a validated tool in preclinical research, was used to evaluate these cognitive domains after TBI. Adult, male Sprague-Dawley rats underwent TBI using midline fluid percussion. Rats were randomly assigned to groups (n=10-12/group; sham+vehicle, injury+vehicle, sham+GBP, injury+GBP) where GBP was administered at 30, 100, or 300mg/kg/day in both sham and injured rats for 10 days post-injury (DPI), and BM was conducted at 30 days post-injury. While spatial learning was similar across all groups, reverse learning was impaired in injury+vehicle rats (p<0.05). Early treatment with GBP at 100 mg/kg/day mitigated these deficits, reducing their severity by 30% (p<0.05) compared to injury+vehicle rats. In a separate cohort, pretraining was implemented prior to injury to evaluate whether it enhances the sensitivity of the BM in detecting cognitive deficits and improves the reproducibility of the outcomes; analysis of these data is currently ongoing. These findings indicate GBP as a promising early intervention to mitigate chronic cognitive deficits after TBI and support the need for further investigation sex differences and specific mechanisms of action.

The Therapeutic Potential of ADSC-secreted LEFTY2 in treating Alzheimer’s Disease

W. WuLi^1.2, H-J Harn³, C. Tjandra⁴, I. Wijaya⁴, and S-Z Lin^5*

¹Bioinnovation Center, Buddhist Tzu Chi Medical Foundation, Hualien, 97002, Taiwan,ROC

²Department of Medical Research, Hualien Tzu Chi Hospital, Buddhist Tzu Chi Medical Foundation, Hualien, 97002, Taiwan,ROC

³Department of Pathology, Hualien Tzu Chi Hospital, Hualien 97002, Taiwan. Taiwan,ROC

⁴Department of Molecular Biology and Human Genetics, Tzu Chi University, Hualien 97004, Taiwan,ROC

⁵Department of Neurology, Hualien Tzu Chi Hospital, Hualien 97002, Taiwan,ROC

Adipose-derived mesenchymal stem cells (ADSCs) have exhibited promising therapeutic potential in Alzheimer's disease (AD), although the underlying mechanisms remain poorly understood. Previously established Alzheimer's disease neuron models derived from Ts21-induced pluripotent stem cells (Ts21-iPSCs) have been shown to exhibit progressive amyloid beta accumulation during neuronal differentiation. In this study, we employed a Transwell co-culture system to investigate the interaction between neurons derived from Ts21-iPSCs and ADSCs. Our findings revealed that co-culture with ADSCs significantly enhanced the survival rate of AD neurons. Proteomics analysis identified significant upregulation of left-right determination factor 2 (LEFTY2) protein in the co-culture medium. Supplementation with 2 nmol LEFTY2 markedly improved the survival and growth of AD neurons. Furthermore, LEFTY2 effectively downregulates the expression of apolipoprotein E4 and amyloid beta 1-42, along with attenuating phosphorylated tau231 levels in AD neurons. These results suggest the potential of LEFTY2 as a promising therapeutic candidate for Alzheimer's disease.

Epigenetic Regulation of Axon Regeneration by H3K27me3

C. Zhang^{1, 2}, M. Xiao³, C. Wang³, and X. Wang^{1, 2}

¹Department of Molecular Medicine, University of South Florida Morsani College of Medicine, Tampa, FL 33612

²Byrd Alzheimer's Center and Research Institute, University of South Florida Morsani College of Medicine, Tampa, FL 33613

³Center for Global Health and Infectious Diseases Research, College of Public Health, University of South Florida, Tampa, FL 33612

Axon degeneration and loss are common consequences of neural injuries and neurodegenerative disorders, leading to nervous system disconnection and functional impairment. Although neurons in the peripheral nervous system (PNS) of mammals have the ability to regenerate axons at the speed of ~1 mm/day, in humans, it is often incomplete, resulting in unsatisfactory functional recovery and oftentimes permanent disabilities, especially in cases with proximal damage where long-distance regeneration of axons is required. In addition, the critical time window for successful reinnervation is relatively short due to atrophy of denervated targets. Therefore, accelerating PNS axon regeneration can greatly enhance the possibility of functional restoration. Dorsal root ganglion (DRG) neurons, which are sensory neurons in the PNS, can regenerate axons upon peripheral nerve injury in a transcription-dependent manner. However, it remains unclear how global transcriptomic changes in DRG neurons are epigenetically coordinated upon peripheral nerve injury to support axon regeneration. Trimethylation of histone H3 at lysine 27 (H3K27me3) is a histone modification that silences transcription of nearby genes. Our prior study demonstrated that levels of H3K27me3 and its methyltransferase Ezh2 are elevated in mouse DRG neurons after peripheral nerve injury, and that such elevation contributes to maintaining the 1-mm/day spontaneous axon regeneration of DRG neurons. Specifically, depleting H3K27me3 by Ezh2 loss-of-function in DRG neurons impairs axon regeneration in vitro and in vivo. Here, to explore the molecular mechanisms through which this transcriptionally repressive histone mark facilitates axon regeneration of DRG neurons, we first establish a method to purify DRG neurons from mice with or without peripheral nerve transection, and then combine multiomics (RNA-seq and H3K27me3 ChIP-seq) of purified DRG neurons with functional screening to identify key axon regeneration suppressing genes that are epigenetically silenced by H3K27me3.

Secondary dystonia associated with focal thalamic lesions in cynomolgus macaques

Z. Zhang¹, F. Pomerleau¹, T. Mims², A. Hall², G.A. Gerhardt¹, W.F. Kaemmerer³, and R. Grondin¹

¹Department of Neuroscience, University of Kentucky, Lexington, KY 40536 USA

²Division of Laboratory Animal Resources, University of Kentucky, Lexington, KY 40536 USA

³CGTA Research Group, Minneapolis, MN, USA

Dystonia is a movement disorder causing involuntary muscle spasms categorized as either primary or secondary when identifiable causes, such as brain lesions, are present. Dystonia may affect one body part (focal) or two or more areas of the body next to each other (segmental). Here, we used the nonhuman primate due to its close anatomical and functional similarities to the human motor systems, particularly the basal ganglia and thalamus, to examine the role various thalamic nuclei play in the presentation of dystonic and parkinsonian symptoms. Eighteen adult cynomolgus monkeys received unilateral thalamic injections of AAV6 viral vectors expressing shRNA constructs. Of these 18 monkeys, 14 developed dystonia or co-occurring dystonia and parkinsonian signs on the contralateral side that became more noticeable over time. Four of these 14 animals exhibited hemidystonia and parkinsonism, characterized by involuntary twisting or turning of the neck, abnormal posture, muscle spasms on the contralateral limbs, and bradykinesia. Two monkeys displayed segmental dystonia involving the contralateral arm and leg. Eight animals exhibited focal dystonia, with muscle spasms localized to the contralateral arm/hand or leg. Post-operative MRI and gross anatomical examinations of the brains recovered 9 months post AAV6-shRNA administration revealed the presence of tissue damage in the thalamus, primarily located in lateral regions of the ventral posterolateral nucleus (VPL) and the ventral posteromedial nucleus (VPM) in monkeys with hemidystonia and parkinsonism. In contrast, tissue damage associated with segmental dystonia was localized in medial regions of the VPL and VPM and parts of the lateral posterior (LP) and lateral dorsal (LD) nuclei. Monkeys with focal dystonia exhibited tissue damage in the medial regions of the LD and VPM. These findings support causality between alterations within specific thalamic areas and the expression of specific dystonia symptoms and lay the foundation for developing novel therapies to treat dystonia.

The Role of Mitochondrial Rcc1-like Gene on Hippocampal Cognitive Impairment

J. Zhu¹, P. Ung¹, S. Osting², and C. Burger²

¹College of Letters and Science, University of Wisconsin-Madison, Madison, WI 53706, USA

²Department of Neurology, University of Wisconsin-Madison, Madison, WI 53706, USA

Inner mitochondrial membrane protein RCC1L is important for mitoribosome assembly and mitochondrial fusion. Disruption of Mitochondrial dysfunction has been linked to neurodegenerative disorders To elucidate the role of Rcc1l on cognition, and neurodegeneration, we selectively ablated Rcc1l in the hippocampus and forebrain of mice. Rcc1l knockout, heterozygous, and wild-type mice were evaluated at 3- and 6-months of age in the open field (OF), 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 differences in behavioral performance. However, at 6 months of age knockout mice- but not heterozygous or wild-type littermates-, began to show higher distance and time spent in the periphery of the OF arena (Rcc1l^KO/KO/Cre⁺ vs. Rcc1l^fl/fl/Cre^-, p= 0.03; Rcc1l^KO/KO/Cre⁺ vs. Rcc1l^KO/+/Cre⁺, p= 0.008; one way ANOVA with multiple comparisons)and deficits in the NOR task. No significant differences were found in the RAWM task at 3 months, but knockout mice displayed seizures during RAWM training at 6 months of age, so we could not test them in this task. Preliminary data (n=2) indicates apoptotic cell death was observed in 6 month old Rcc1l^KO/KO/Cre⁺CA1 hippocampal neurons using the TUNEL system. These results are encouraging and suggest progressive neurodegeneration. Ongoing studies include more animals to examine seizure-like behavior that we have observed in Rcc1l^KO/KO/Cre⁺ mice . In summary, mitochondrial RCC1L protein on hippocampal learning and memory may provide insight into the influence of mitochondrial dysfunction on memory-associated phenotypes that may be linked to neurodegenerative disorders.

Spatially Mapping Insulin/PI3K/AKT/mTOR Dysregulations in Down Syndrome with Alzheimer’s Disease Human Hippocampi

A. Wohlfert¹, K. Jones², S.J. Guzman³, and A-C Granholm¹

¹Department of Neurosurgery, University of Colorado Anschutz Medical Campus, Aurora, CO.

²Bioinformatic solutions, LLC, Sheridan, WY

³Department of Pathology, University of Colorado Anschutz Medical Campus, Aurora, CO.

Down syndrome (DS) is a chromosomal disorder characterized by widespread systemic dysregulations, which increase the risk of various age-related diseases, including Alzheimer’s disease (AD). This heightened susceptibility to AD pathology may stem from the triplication of the Amyloid precursor protein (APP) gene or disruptions in key mechanistic pathways, such as the mTOR signaling pathway. mTOR signaling influences many metabolic processes, including Tau protein homeostasis, cytoskeletal organization, autophagy, and cell survival. Dysregulation of upstream mTOR regulators, including insulin and PI3K/AKT, has been associated with increased accumulation of amyloid-beta (Aβ) plaques and neurofibrillary tangles (NFT), hallmark pathologies of AD. Despite these associations, mTOR dysregulations that potentially drive disease progression in DS-associated AD (DS-AD) remain poorly understood. In the current study, we used a novel spatial transcriptomic analysis method coupled with immunohistochemical validation methods to investigate possible aberrant expression of genes involved in insulin, PI3K/AKT, and mTOR signaling in postmortem human hippocampi of individuals with DS-AD and healthy controls (HC). The expression of IGF1, PI3K, AKT, and mTOR was upregulated in DS-AD cases compared to HC, especially in the dentate gyrus, CA 3 neurons, CA 4 neurons, and subiculum. In contrast, insulin and tuberous sclerosis complex 1/2 (TSC1/2) expression was downregulated, particularly in these same hippocampal regions. The density of phospho-mTOR immunostaining was elevated in the hippocampus of DS-AD cases, compared to age-matched controls and AD cases, especially in the dentate gyrus, CA 3 neurons, CA 4 neurons, and subiculum.

These studies were supported by grants to ACG from the NIH (R01AG071228-02, R01AG061566, and 1U24AG092191-01) as well as funding from the BrightFocus Foundation (grant no. CA2018010) and the Lejeune Foundation (grant no. GRT-2023b/2277).

Corona virus in the brain: Long-term consequences for nuclear function

A-C Granholm^1*, E. Englund², H. Olofsson², and A. Wohlfert¹

¹Department of Neurosurgery, University of Colorado Anschutz Medical Campus, Aurora, CO, 80045, USA

²Division of Pathology, Department of Clinical Sciences, Lund University, Lund, Sweden

Background: The SARS-CoV-2 virus that led to COVID-19 is associated with significant and long-lasting neurological symptoms in many patients, with an increased mortality risk for people with Alzheimer’s disease (AD) and/or Down syndrome (DS). We have previously demonstrated that SARS-CoV-2 nucleoproteins remain in the brain for at least one year following severe COVID-19. Further, glial cells in SARS-CoV-2-infected individuals with DS exhibited significant abnormalities. Methods: Fixed 5-micron postmortem hippocampal, frontal cortex, and brainstem sections from DS-AD, AD, or healthy controls (HC) with or without COVID-19 were stained with phosphorylated mTOR and phospho-Tau(S396) antibodies. Results: There were alterations in the cytoplasmic vs. nuclear distribution of p-Tau and p-mTOR immunostaining. Severe COVID-19 cases from all three groups showed that p-mTOR staining was transposed from granular cytoplasmic immunostaining to nuclear staining. Since the PI3K/Akt signaling pathway plays a critical regulatory role in RNA processing, translation, autophagy and apoptosis, viruses may hijack the mTOR pathway to survive. We also found nuclear immunostaining of p-Tau in locus coeruleus noradrenergic neurons in severe COVID-19 cases. Discussion: Nuclear presence of phosphorylated mTOR indicates that mTOR is active and signaling within the nuclear compartment. A similar transposition of p-mTOR to the nucleus has been observed in cells infected with cytomegalovirus and it has been suggested that the virus can hijack nuclear proteins to benefit survival and replication of the virus. Others have shown that herpes simplex virus 1 (HSV-1) and SARS-CoV-2 can cause p-Tau(S396) to accumulate in the nucleus of neurons. It has been suggested from tissue culture studies that neurons may increase p-Tau production and accumulation in the nucleus to combat viral infection, but this could also be caused by viral hijacking of normal cell functions.

This work was supported by grants from the NIH (R01AG070153 and R01AG061566), and grants from BrightFocus foundation (CA2018010) and Lejeune Foundation (GRT-2023b/2277).