Abstract
Characterization of IP3R as a Potential New Target for p70 Ribosomal S6 Kinase Following Exposure to Environmental Enrichment in Aged Rats
1 College of Engineering, University of Wisconsin–Madison, Madison, WI, USA
2 Department of Biology, University of Northern Michigan, Marquette, MI, USA
3 College of Letters & Sciences, University of Wisconsin–Madison, Madison, WI, USA
4 Department of Neurology, Medical Sciences Center, University of Wisconsin–Madison, Madison, WI, USA
Our lab has previously shown that exposure of aged rats to environmental enrichment (EE) triggers sustained phosphorylation of p70 ribosomal S6 kinase (p70S6K), a protein involved in dendritic protein translation of genes central to learning and memory formation and synaptic plasticity. To further explore the role p70S6K activation plays in EE, we used stimulated emission depletion (STED) microscopy to obtain and analyze super-resolution images from stimulated hippocampal slices to identify the localization of this protein following synaptic stimulation. We found that animals exposed to EE displayed increased co-localization of activated p70S6K (p-p70S6K) with endoplasmic reticulum inositol triphosphate receptor isoform1 (IP3R1) when compared with controls, 30 min after half train of theta burst stimulation. IP3R is involved in the regulation of Ca2+ release from the ER, and dysregulation of Ca2+ release has been associated with age-related cognitive impairment and neurodegeneration. Thus, we hypothesize IP3R1 is a phosphorylation target for P70S6K. To test our hypothesis, we first identified a p70S6K consensus substrate motif [R/K]XRXX[pS/pT][M/L/V/I] in amino acid position 2642 of rat IP3R1. Next, we cloned a rat IP3R1 lacking the P70S6K phosphorylation site (S2642A). We will use wild-type IP3R1or IP3R1 mutant S2642A expression vectors to transfect an HEK 293 cell line with the three isoforms of IP3R knocked out (HEK 293Δ IP3R). Extracts will be co-immunoprecipitated and samples will be incubated with recombinant active p70S6K in the presence of [33P] ATP to determine whether IP3R is phosphorylated by p-p70S6K. We predict that if p-p70S6K interacts with IP3R1, we will observe an increased phosphorylation of IP3R1 at S2642 not present in IP3R1 mutant S2642A. These results will inform future studies to investigate the functional consequences of this newly identified target of IP3R phosphorylation on internal calcium ([Ca2+]I) signaling. Dysregulation of [Ca2+]I is associated with brain aging and changes in synaptic plasticity.
Developing a Method to Identify the Protein Interactome of Tau Using the BioID2 Approach
1 Department of Translational Neuroscience, College of Human Medicine, Michigan State University, Grand Rapids, MI, USA
2 Neuroscience Program, Michigan State University, East Lansing, MI, USA
3 Hauenstein Neuroscience Center, Mercy Health Saint Mary’s, Grand Rapids, MI, USA
Aberrant aggregation of tau protein is a hallmark of neurodegenerative diseases collectively known as tauopathies, of which the most common is Alzheimer’s disease. Although tau protein is most commonly known as a microtubule-associated protein involved in regulating microtubule dynamics, accumulating evidence suggests tau is likely involved in many other biological functions. For instance, several studies implicate a scaffolding protein role for tau where it can act to regulate cellular pathways through direct or indirect interactions with multiple other proteins. Deciphering the tau protein–protein interactome is a critical step toward better understating the physiological and pathological roles of tau. The goal of this work is to identify tau-interacting partners using a method that allows in situ identification of protein–protein interactions. We are adapting a technique based on biotin ligase (BioID2) that allows in situ protein labeling with biotin followed by biotin-targeted pulldown and mass spectrometry. Here, we describe the methodological approach used to adapt this technology for studying tau interactions. To map the tau protein interactome, we began by creating fusion proteins between tau and BioID2, with BioID2 on the N-terminus (BioID2-ht40) or C-terminus (ht40-BioID2) of full-length human tau. To further dissect the role of each tau domain in protein interactions, we created BioID2 fusion proteins with the following tau domains: (1) 1–224 amino acids (Nterm-BioID2), (2) 225–380 amino acids (MTBR-BioID2), (3) 381–441 amino acids (Cterm-BioID2), (4) 1–380 amino acids (Nterm-MTBR-BioID2), and (5) 225–441 amino acids (MTBR-Cterm-BioID2). The constructs are then produced in lentiviruses. Ongoing studies will use validated lentiviruses to stably express the tau-BioID2 proteins in E18 primary cortical neurons from tau knockout mice. Studies will isolate biotinylated proteins from neurons expressing the various tau-BioID2 proteins through affinity purification using streptavidin-coated magnetic beads and identified by mass spectrometry. We expect this approach will provide a method to identify novel tau-interacting partners that could help shed light on tau’s growing functional roles in neurons.
This work was supported by NIH/NIA R01AG067762, R01AG044372, and R01NS082730.
Development of a Functional Long-Term Potential Model Utilizing Human iPSC-Cortical Neurons for Alzheimer’s Drug Testing
1 Nanoscience Technology Center, University of Central Florida, Orlando, FL, USA
2 Hesperos, Inc., Orlando, FL, USA
Alzheimer’s disease (AD) is commonly associated with a characteristic loss of cognitive functions associated with memory and learning due to synaptic decline. One of the hallmarks for AD is the synaptic deterioration following the accumulation of amyloid-β42 (Aβ42) oligomers, formed from improper cleavage of amyloid precursor protein to amyloid-beta40 (Aβ40). Our study aimed to develop a human-based AD model for the evaluation of cognitive integrity by reproducing this pathology on a chip. The use of multi-electrode arrays (MEAs) with the surface patterned using photolithographic chemistry enabled synaptic development between paired populations of neurons. By integrating a previously published human iPSC-cortical neuron culture onto these MEAs, a synapse-driven model of long-term potentiation (LTP) was validated for biologically relevant evaluation of synaptic function. We were able to assess synaptic plasticity in each neuronal pair by stimulating one neuronal population and analyzing the connected population for synapse-mediated response and the induction of LTP. LTP induction was confirmed by AMPA (α-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid) and NMDA (N-methyl-
Can hESC-Derived Dopamine Grafts Improve Non-Motor Impairments?
1 Brain Repair Group, School of Biosciences, Cardiff University, Cardiff, UK
2 Neuroscience and Mental Health Research Institute, School of Medicine, School of Bioscience, Cardiff University, Cardiff, UK
3 Developmental and Regenerative Neurobiology, Department of Experimental Medical Science, Wallenberg Neuroscience Center, Lund University, Lund, Sweden
4 Lund Stem Cell Center, Lund University, Lund, Sweden
5 School of Pharmacy and Pharmaceutical Sciences, Cardiff University, Cardiff, UK
Non-motor (NM) symptoms in Parkinson’s disease have a significant impact on the quality of life for patients. Cell therapies, such as human fetal ventral mesencephalic tissue (hfVM), can replace lost dopamine (DA) cells and alleviate motor symptoms. Preclinical studies have also demonstrated the amelioration of some dopamine-dependent neuroleptic malignant syndrome (NMS), such as visuospatial dysfunctions. It was unknown whether recovery of NM impairments could be achieved with human embryonic stem cell–derived (hESC) ventral midbrain DA (vmDA) grafts. To understand this, female lister hooded rats underwent operant training on the lateralized choice reaction time (LCRT) task before receiving unilateral 6-OHDA medial forebrain bundle (MFB) lesions. Lesion-induced deficits were measured prior to receiving transplants of either hfVM or hESC-derived DA cells differentiated according to one of two different in vitro protocols. Animals were kept for 21 weeks post-transplantation and tested on amphetamine-induced rotations and the LCRT task before brain tissue was taken for immunohistochemical analysis. Grafts from all three cell types alleviated amphetamine-induced rotational bias. However, only rats with hfVM grafts demonstrated improvements in visuospatial function on the LCRT task. Correlational analysis revealed an association between more accurate visuospatial performance and deeper medial neostriatal innervation (P = 0.006), lower TH+ neuron content (P = 0.008), and a higher percentage of A10 neurons present within the graft (P = 0.011). Poorer visuospatial performance was associated with greater lateral neostriatal innervation (P = 0.007). These data suggest that the cellular composition of the graft and extent of medial neostriatal innervation may be important for the recovery of visuospatial deficits, thus highlighting that consideration of these factors may ultimately yield a cell therapy product capable of targeting dopamine-dependent NM deficits.
Olfactory Bulb Mitral Cells in Synucleinopathy Pathogenesis
1 Graduate College, Rush University Medical Center, Chicago, IL, USA
2 Parkinson’s Disease Center, Department of Neurodegenerative Science, Van Andel Institute, Grand Rapids, MI, USA
3 ASU-Banner Neurodegenerative Disease Research Center, Arizona State University, Tempe, AZ, USA
Lewy pathology (LP) is a central pathological feature of synucleinopathies, a group of neurodegenerative disorders, including Parkinson’s disease (PD) and dementia with Lewy bodies. LP consists of intercellular aggregates of a protein called α-synuclein, which are predominantly phosphorylated at serine 129 (PSER129). LP is consistently detected in the brain region responsible for smell called the olfactory bulb (OB), and anosmia (loss of smell) is an early prodromal symptom of several synucleinopathies. Together these observations suggest that the synucleinopathy disease process begins in the OB. In the current study, we examined the distribution of PSER129 in nondiseased brains of mammals using a sensitive tyramide signal amplification (TSA) technique to detect low levels of endogenous PSER129 under ideal antibody-binding conditions. In wild-type nondiseased mice, PSER129 was detected in the OB and several regions of the olfactory cortex across the neuroaxis. In contrast, we did not observe PSER129 immunoreactivity in any of the α-synuclein knockout mice tested in any brain region. In the nondiseased wild-type mice, PSER129 was contained in the mitral cell layer and the external plexiform of the OB. Dual labeling for the PSER129 and the mitral cell marker TBX21 confirmed PSER129-positive structures localized to mitral cells of the OB. In addition, evidence of the same PSER129-positive structures in the OB was found in nondiseased rats, nonhuman primates, and healthy humans. However, PK-sensitive PSER129 was not observed in individuals diagnosed with PD. These findings suggest that phosphorylation of the α-synuclein mechanism is constitutively active in OB mitral cells and may be predisposed to pathological aggregation that can spread throughout the brain via fibers of the olfactory tract. Future studies should focus on understanding the cellular function of alpha-synuclein in OB mitral cells.
Potential Benefits of Reprogramming Astrocytes Into Neurons in Canine Stroke Model
1 Department of Biomedical Engineering, University of Minnesota, Minneapolis, MN, USA
2 Department of Neurosurgery, University of Minnesota, Minneapolis, MN, USA
3 Graduate Program in Neuroscience, University of Minnesota, Minneapolis, MN, USA
AAV9-mediated delivery of NeuroD1 has been used in the treatment of mouse stroke for the purpose of reprogramming astrocytes into neurons. The translation of this technology to the clinical field will require the evaluation of cell reprogramming in the brains of large animals. We therefore developed a canine stroke model to determine whether astrocytes could in fact be reprogrammed into neurons in a canine model of ischemic brain injury. Three dogs were used for this experiment, each of which had a stroke induced via the use of a temporary aneurysm clip applied to the middle cerebral artery for 1 h. One week after the stroke, two dogs were given the treatment vector mGfap::Cre+CAG::DIO-NeuroD1-mRuby2 and one was given a control vector mGfap::Cre+CAG::DIO-mRuby2. The vector system was designed with the hGFAP promoter to transduce astrocytes, while the NeuroD1 gene is meant to promote the reprogramming of astrocytes to become neurons. The inclusion of the mRuby2 gene is meant to allow us to track which cells are transduced. Three weeks after the treatment, the models were harvested. The data that have been collected thus far show evidence of reprogramming as well as a potential anatomical benefit for the treated models. Immunohistochemistry analysis has shown many of the transduced cells express neuronal biomarkers, suggesting they are either astrocytes that have been reprogrammed into neurons or endogenous neurons that were transduced despite the GFAP promoter. In addition, analysis of the ventricular volume has shown that the control canine model had significantly more atrophy than the treated models. These results provide very promising evidence that this treatment has some anatomical benefit.
Characterization of Rat iPSC-Cortical Neurons and Its Comparison With Human iPSC-Cortical Neurons
1 NanoScience Technology Center, University of Central Florida, Orlando, FL, USA
2 Hesperos, Inc., Orlando, FL, USA
For the last several decades, one of the most predominant models for preclinical research has been the rodent model. However, as previous clinical trials have shown, efficacy in murine and rat models can be poor predictors of drug efficacy in humans. Combining induced pluripotent stem cell (iPSC) technology with human-on-a-chip technology presents a human-based platform for more sophisticated electrophysiological characterization of neuronal networking, which could facilitate the development of potential therapies of central nervous system (CNS) diseases linked to synaptic dysfunction and neurodegeneration, such as Alzheimer’s disease and other cognitive diseases. iPSC-based modeling also provides an achievable bridge to translate rodent-based preclinical findings to human systems. This study sought to compare a rat iPSC–derived (riPSC) cortical neuron model with a human iPSC–derived (hiPSC) cortical neuron model for their functional characteristics and drug sensitivity. riPSC-derived cortical neurons were differentiated, characterized by immunocytochemistry, and plated on microelectrode arrays (MEAs) to determine their capability to reproduce long-term potentiation (LTP), a surrogate for learning and memory. Phase microscopy was first used to monitor the morphology of riPSC-cortical neurons throughout differentiation and maturation. Cortical neuron differentiation and network formation were characterized and confirmed through the expression of a group of markers (Nestin, Ctip, MAP2, and Synaptophysin, etc) using immunocytochemistry. Then, the electrophysiological characteristics of the riPSC-cortical neurons was analyzed by whole-cell patch clamp at multiple time points. There neurons were further analyzed using MEAs for their electrical activity and LTP induction capability, in a system which has been previously validated using hiPSC-cortical neurons. Their sensitivity to multiple drugs was then characterized with the LTP assay and compared with those from the hiPSC-cortical neuron system. Comparison between rat and human iPSC-derived cortical neuron systems will shed light on the species gap between rat and human, which would facilitate the translation of preclinical data collected from rat to clinic applications.
Surface EMG of the Biceps–Triceps Pair in Tetraplegic Patients
N. Datta and J. D. Guest
The Miami Project to Cure Paralysis, Department of Neurological Surgery, University of Miami, Miami, FL, USA
The commonest level of cervical spinal cord injury (SCI) is C4/C5, with usual recovery of biceps function but frequent weakness in the triceps. The triceps is a critical muscle for activities of daily living and its recovery is a target of several interventions. Surface electromyography (EMG) is a tool to quantitatively assess muscle activation in real time. The biceps/triceps (B/T) muscle pair task efficacy is affected by post-SCI neuroplasticity that can dysregulate motor control through co-contraction and spasticity. Furthermore, the proximity of B/T and chronic SCI changes such as atrophy lead to potential crosstalk in the surface EMG signals. Studies to improve motor control require methods to interpret the voluntariness in the face of these events. Prior methods applied to lower extremity muscle pairs after SCI include intermuscular coherence in the lower frequency range. Methods to detect crosstalk use the detection of differences in high-frequency content between muscle pairs. In one suitable subject with motor complete injury, we recorded from a bilateral EMG upper extremity montage during attempted maximal and submaximal isolated contraction of biceps and triceps. EMG signals were analyzed offline with suitable digital processing and determination of the frequency content contributing to signal power and also for intermuscular coherence to detect spasms and coactivation. The subject had difficulty to inhibit co-contraction in the isometric tasks. The frequency analysis of the B/T pair indicated that probable crosstalk signal lacked higher frequencies due to the effects of volume conduction and distance on frequency content. Strong coherence in the biceps and triceps indicated lack of independent motor control. Frequency and coherence analysis of real-time EMG is a valuable tool to track motor control after C4/C5 SCI.
Engineering Spinal V2a Populations for Developmental Modeling and Targeted Cellular Therapies
1 Developmental and Stem Cell Biology Graduate Program, University of California, San Francisco, San Francisco, CA, USA
2 Gladstone Institutes for Cardiovascular Disease, San Francisco, CA, USA
3 Department of Cellular and Molecular Pharmacology, University of California San Francisco, San Francisco, CA, USA
4 Sana Biotechnology, Inc., South San Francisco, CA, USA
Advances in cellular neurobiology have begun to elucidate the heterogeneity of neuronal phenotypes within the spinal cord, improving our understanding of how these cells form motor networks, how they can be affected by injury and disease, and how we can develop treatments to repair these networks. Cellular engineering presents us with the tools to generate specific populations of spinal neurons to model neural network formation and develop tailored cellular therapies. One example is the V2a class of interneurons, a population of excitatory neurons throughout the hindbrain and spinal cord, which have been identified as integral components of the central pattern generator and a promising neural population for cell transplantation strategies. However, in vitro differentiation protocols produce heterogeneous populations and V2a neurons can be transiently marked by the canonical transcription factor CHX10, limiting V2a purification. To improve the generation and characterization of V2a neurons in vitro, we have generated a lineage-traced human pluripotent stem cell line that allows live readout of differentiation of V2a neurons and purification of V2a populations for downstream characterization. Briefly, CRISPR/Cas9 was used to knock in a CRE sequence downstream of FOXN4, a unique marker of the V2 progenitors, in a cell line containing a floxed GFP reporter. Using GFP as a readout, we were able to screen 16 differentiation conditions in parallel to improve V2a differentiation by 20%. In contrast with other mature neuronal populations, committed V2a neurons survive fluorescent-activated cell sorting. Electrophysiological characterization of sorted V2a neurons demonstrated rhythmic neuronal activity. Finally, by including axial patterning steps in the V2a differentiation, we propose to characterize the phenotypic diversity that has been recently described within the developing spinal cord. The results from this ongoing work will enable refinement of cellular therapies for repairing the injured nervous system, including spinal cord.
Engineering Spinal Interneurons for Repair of the Injured Cervical Spinal Cord
1 Department of Neurobiology and Anatomy, Drexel University, Philadelphia, PA, USA
2 Marrion Murray Spinal Cord Research Center, College of Medicine, Drexel University, Philadelphia, PA, USA
3 Gladstone Institutes, San Francisco, CA, USA
Cervical spinal cord injuries (SCIs), the most common SCI, compromise respiratory networks, leading to life-threatening impairments in breathing. One primary reason is the extensive damage that occurs to the phrenic motor network (controlling the diaphragm) located in the cervical cord. Our ongoing experiments have identified spinal interneurons (SpINs) as key therapeutic targets for promoting plasticity and functional recovery. Building on a long history of transplanting spinal neural precursor cells (NPCs) to promote spinal cord repair, the present work harnesses advances in stem cell engineering to enrich donor NPCs with specific subsets of stem cell–derived SpINs, which are known to reside in the phrenic network—excitatory V2a and cholinergic (modulatory) V0 SpINs. The chemogenetic receptor hM4Di was incorporated into the engineered SpINs to allow for selective de-activation of these donor cells with clozapine during electromyography recordings.
Adult female Sprague-Dawley rats received mid-cervical contusion injury (Infinite Horizon impactor, intended impact force 200 kilodyne). One week after injury, aggregates of NPCs with stem cell–derived V2a SpINs were injected into the lesion cavity. One month later, all animals were re-anesthetized and received a laparotomy for transneuronal tracing of the phrenic motor network with pseudorabies virus. Three days later, animals underwent terminal diaphragm electrophysiology, at which time clozapine was injected intraspinally into the transplant to silence donor SpINs. Preliminary data revealed that clozapine-induced suppression of donor cell activity reduced diaphragm activity ipsilateral to injury, with no measurable effect on the contralateral side. This ongoing research will offer the first in-depth assessment of how these donor SpIN populations contribute to phrenic recovery.
Adeno-Associated Viral Gene Delivery of Wild-Type Human Tau Induces Progressive Neurodegeneration and Formation of Insoluble Tau Species in the Hippocampi of Middle-Aged Rats
1 University of Wisconsin–Madison, Madison, WI, USA
2 University of California, Los Angeles, Los Angeles, CA, USA
3 University of South Florida, Tampa, FL, USA
Tauopathies are a broad class of diseases, including Alzheimer’s disease (AD), characterized by hyperphosphorylation and aggregation of the microtubule-associated protein tau. To generate a rat tauopathy model, we used bilateral AAV9 injections of wild-type human tau (AAV-htau) into the hippocampus of 6-month-old adult male Fischer 344 rats. Controls included AAV-green fluorescent protein and uninjected rats. Compared with controls, AAV-htau rats showed neuronal loss in hippocampal areas CA1 and CA2 at 3, 8. and 12 weeks after injection, with significant loss beginning at 8 weeks measured by Nissl stain. Immunohistochemical analysis revealed human tau expression by 3 weeks that continued at 8 and 12 weeks. Western blot analysis of total tau revealed a significant 1.5-fold increase at 8 weeks and 12 weeks. By 3 weeks, AAV-htau animals exhibit tau hyperphosphorylation at residues S202-T205, characteristic of early/mid tauopathy, but no significant change in the late tauopathy phosphorylation site S396 was found at any time point. Hippocampal extracts analyzed via sarkosyl fractionation show aggregation of monomeric and oligomeric tau species by 3 weeks after injection. Data suggest aggregation in monomeric, oligomeric, and polymeric fractions at 12 weeks. No significant behavioral deficits in fear conditioning or Morris water maze were observed at any time point, but a trend toward significance was found in cued fear conditioning at 12 weeks after injection. Investigation of behavioral and cellular phenotypes at later time points is currently in progress. This includes coupling mass spectrometry with aggregate fractionation techniques to define site-specific phosphorylation in the AAV-tau model to understand its relevance to human tauopathy. In conclusion, AAV-mediated gene delivery of human tau offers a fast and reliable approach to generate progressive tauopathy models, which have the advantage of administration at any age to study age as a variable in disease progression, which currently is not possible in tau transgenic mouse lines.
Investigation of the Functional Integration and Efficacy of hESC-Derived Grafts in a Model of Huntington’s Disease
1 Brain Repair Group, Sir Martin Evans Building, School of Biosciences, Cardiff University, Cardiff, UK
2 Stem Cell Neurogenesis Group, Neuroscience and Mental Health Research Institute, School of Medicine and School of Bioscience, Hadyn Ellis Building, Cardiff University, Cardiff, UK
Huntington’s disease (HD) is a neurodegenerative and autosomal dominant genetic disorder that affects the medium spiny neurons (MSNs) of the striatum. Degeneration of the striatum is associated with progressive deterioration of movement and cognition in gene-positive individuals, and currently available treatments are limited to symptom relief. Cell replacement therapy (CRT) is in development as a potential therapeutic intervention for HD patients. Promising preclinical and clinical data suggest that intracerebral transplantation of live striatal progenitors can result in grafts of mature MSNs capable of alleviating motor and cognitive deficits. It is hypothesized that functional recovery may rely largely on the reconstruction of the basal ganglia circuitry, which is disrupted in the degenerated brain. The aim of this project is to determine whether grafts of human embryonic stem cell (hESC)-derived striatal cells can form functional synapses with the host brain. We address this by mapping efferent and afferent graft-brain projections using the monosynaptic rabies technology. We have created an hESC line expressing the TVA receptor and used this cell line to demonstrate maturation of grafts harboring MSNs in the brains of rats with quinolinic acid lesions of the striatum. Preliminary data suggest that host-to-graft connectivity may be established, and further investigation of graft-to-host connectivity will be undertaken. Although further investigation is required in genetic models of HD, these data will be important to establish the ability of hESC-derived MSN grafts to form functional synapses and to develop our understanding of the mechanism of recovery conferred by striatal transplant.
Single-Cell RNA Sequencing Reveals Immunomodulatory Effects of Stem Cell Factor and Granulocyte Colony-Stimulating Factor in Aged APP/PS1 Mice
Department of Neurosurgery, State University of New York Upstate Medical University, Syracuse, NY, USA
Alzheimer’s disease (AD) is a progressive neurodegenerative disease that most commonly occurs in advanced age. Neuropathological hallmarks of AD include accumulation of beta-amyloid (Aβ) plaques that lead to disrupted cell signaling and reduced neurites. Aβ can be cleared by activation of microglia and monocytes/macrophages which phagocytose Aβ. Our previous studies have shown that combined treatment with stem cell factor and granulocyte colony-stimulating factor (SCF+G-CSF) increases Aβ uptake by microglia/macrophages and restores neurite morphology in aged APPswe/PS1dE9 (APP/PS1) mice. The current study uses a transcriptomic approach to explore molecular mechanisms underlying SCF+G-CSF-enhanced Aβ uptake by microglia/macrophages in age-related AD. APP/PS1 mice (male, ~28 months) were injected with SCF+G-CSF or vehicle solution for 5 days. Following injections, single-cell RNA sequencing was performed on isolated CD11b-positive cells from the brain, thought to reflect monocytes/macrophages and microglia. In the isolated CD11b-positive cells, SCF+G-CSF treatment resulted in upregulated gene-sets linked to bone marrow–derived monocytes/macrophages (Hp, Emilin2, C3, Sell, F10, Gda) and to tissue infiltration of leukocytes (CCR2, s100a8/9). Findings from a flow cytometry experiment further confirmed that the levels of peripheral macrophages (CD11b+/ CD45high) were significantly increased by SCF+G-CSF treatment in the brains of aged APP/PS1 mice. In addition, SCF+G-CSF treatment upregulated both pro- and anti-inflammatory genes related to macrophage activation as well as Aβ load and clearance (s100a6, CCR2, Anxa1, IL-1β, Trem1, s100a8/9) in the isolated CD11b-positive cells. Collectively, the findings of this study reveal that SCF+G-CSF treatment in aged APP/PS1 mice increases the recruitment of peripheral macrophages into the brain and promotes multifaceted regulations of Aβ-macrophage interactions. This transcriptomic study provides deep insight into how SCF+G-CSF treatment regulates peripheral macrophages in the removal of pathological Aβ from the aged brain.
This study was supported by NIH/NIA (1R01AG051674).
Modeling of the Human Cortical-Spinal Neuraxis Using Organoids
1 John van Geest Centre for Brain Repair and Division of Stem Cell Neurobiology, Department of Clinical Neurosciences, University of Cambridge, Cambridge, UK
2 MRC Laboratory of Molecular Biology, Cambridge, UK
3 Department of Physiology, Development and Neuroscience, University of Cambridge, Cambridge, UK
Restoring functional connectivity after spinal cord injury (SCI) is still a major challenge. While there has been great progress in stimulating axon regeneration and removing inhibitory cues at the lesion site, this work has been conducted in rodent models and its translatability is yet to be judged. Issues with translatability are to be expected as rodents and humans differ anatomically, in immune response, and with regard directly to cell phenotypes. For instance, human astrocytes are larger and more morphologically complex than their rodent counterparts and have vastly different transcriptomes. To overcome these issues, we have developed an all-human model of the cortical-spinal neuraxis in a dish. Using stem cell–derived cerebral and spinal organoids, we can mimic corticospinal connections and circuitry in an in vitro setting. This model allows us to be able to observe human axon dynamics in real time in a three-dimensional system and to look at the effects of potentially proregenerative agents in a human environment.
In Drug-Naïve Parkinson’s Disease Patients, Dopamine Transporter and Tyrosine Hydroxylase Expression Are Increased in Peripheral Immune Cells
1 Department of Neuroscience, College of Medicine, University of Florida, Gainesville, FL, USA
2 Carnegie Mellon University, Pittsburgh, PA, USA
3 University of South Carolina, Columbia, SC, USA
4 Drexel University, Philadelphia, PA, USA
5 ICBR Flow Cytometry, University of Florida, Gainesville, FL, USA
6 Department of Neurology, College of Medicine, University of Florida, Gainesville, FL, USA
While the scientific debate about the origin of sporadic Parkinson’s disease (PD), that is, body-first versus brain-first hypothesis, has not been settled, PD pathology affects both central nervous system (CNS) and peripheral dopamine transmission. PD research has largely focused on neuronal and glial dysfunction, but there is strong evidence demonstrating the dysregulation of innate immunity in PD at the systemic level. In PD, there is a loss of dopamine neurons indicated by decreased neuronal dopamine transporter (DAT) and tyrosine hydroxylase (TH). As peripheral immune cells express DAT and TH, we tested the hypothesis that in drug-naïve PD patients, there is a reduction in DAT+/TH+ peripheral blood mononuclear cells (PBMCs). We assessed expression in PBMCs of 12 drug-naïve PD patients (newly diagnosed), 84 PD patients receiving treatments, and 67 age-/sex-matched healthy subjects. We found there is no change in total PBMCs, but we found that percentages of DAT+/TH+, but not NET+, PBMCs are significantly higher in drug-naïve PD patients (n = 12) compared with healthy controls (n = 67). In PD patients receiving treatments (n = 84), irrespective of the treatment modality, the %DAT+/TH+ PBMCs declined significantly compared with drug-naïve patients, but significantly higher than controls, suggesting that treatment for PD modulates DAT/TH+ PBMCs. The percentages of DAT+/TH+ myocytes are increased in two animal models of PD and reduced following L-DOPA+Carbidopa. To test whether changes in DAT+/TH+ PBMCs are increased in other neurodegenerative diseases, we recruited a patient cohort with Alzheimer’s disease and found increased DAT+/TH+ PBMCs are specific for PD. We also discovered PD patients exhibit increased DAT+/TH+ myeloid-derived suppressor cells (MDSCs), suggesting a compensatory immune response. Ongoing studies are investigating the mechanism underlying increased DAT+/TH+ PBMCs in PD. Collectively, these data suggest a system-wide dysregulation of the dopamine signaling on peripheral immune cells in PD, providing a novel diagnostic and therapeutic perspective for PD.
Efficacy of Intra-Arterial Mesenchymal Stem Cell–Derived Exosome Treatment Following Acute Ischemic Stroke
1 Department of Neurology, Miller School of Medicine, University of Miami, Miami, FL, USA
2 Department of Neurosurgery, Jikei University of Medicine, Tokyo, Japan
3 Interdisciplinary Stem Cell Institute, Miller School of Medicine, University of Miami, Miami, FL, USA
4 Department of Medicine, Miller School of Medicine, University of Miami, Miami, FL, USA
Stroke is the fifth leading cause of death and a leading cause of disability in the United States. While current clinical management offers an increased survival rate, more than 50% of stroke survivors are chronically disabled. It is imperative for the field to develop more effective therapies for stroke. Research in the past two decades has shown promise in cell and cell-based therapies for stroke. Our group has shown the safety and efficacy of intra-arterial (IA) mesenchymal stem cells (MSCs) for stroke. It is known that MSCs exert their effects by secretion of extracellular vesicles called exosomes. While IA-MSC therapy is still in optimization, it is of crucial importance to also determine the efficacy of IA exosomes. The reduced size (40–100nm) of exosomes, easy availability, and notable benefits make them a significant candidate for preclinical testing. To address this gap, we determined the efficacy of multiple doses of IA exosomes following ischemic stroke in rats. This was achieved by using female Sprague-Dawley rats that were subjected to middle cerebral artery occlusion (MCAO). Rats were injected with phosphate-buffered saline (PBS), MSCs, and 10 µg, 50 µg, or 100 µg of exosomes intra-arterially at 24 h after stroke. The efficacy of different doses of exosomes compared with other groups was determined using behavior analysis like neurodeficit scoring (NDS), rotarod test (RTD), and stroke volume (SV) determination via histology. The NDS and the SV improved the most in the IA 50 µg group compared with the IA-MSC and 10 µg/100 µg groups. Both 50 µg and 100 µg exosome treatment showed improvements in motor function using RTD. Preliminarily, our data indicate that 50 µg of IA exosome treatment appears to be more effective. Furthermore, the exosome treatment was more effective than MSCs alone. In conclusion, our study provides strong preclinical evidence for efficacy of IA exosome treatment for stroke.
Glial Reactions to Cortical and Spinal Electrical Stimulation in a Rat Model of Cervical Spinal Cord Injury
P. J. Horner
Houston Methodist Research Institute, Houston, TX, USA
Electrical stimulation of the corticospinal tract as well as stimulation of the spared sensorimotor circuitry below a spinal cord injury leads to improved forelimb recovery after cervical spinal cord injury. The mechanisms of activity-dependent plasticity are largely unknown, particularly in the context of the chronically injured nervous system. In these studies, we applied transcriptomic and anatomical approaches to explore how glial populations respond to electrical stimulation. We discovered that mature glial cells undergo significant structural plasticity as a result of sub-threshold, patterned electrical activity applied to the motor cortex. Furthermore, we show subpopulations of glia, including microglia, have significant acute changes in master regulatory gene networks related to inflammation that may have secondary impact on neural plasticity. Altogether, these results point to the potentially important role glial cells play in the functional changes manifest in activity-dependent plasticity in the injured spinal cord.
This research is supported by The Craig H. Neilsen Foundation.
Development of Dorsal Spinal Neural Progenitor Cells for Application in Spinal Cord Injury Therapeutics
1 Department of Neuroscience, University of Minnesota, Minneapolis, MN, USA
2 Department of Neurosurgery, University of Minnesota, Minneapolis, MN, USA
3 Stem Cell Institute, University of Minnesota, Minneapolis, MN, USA
In the United States, there are approximately 18,000 new cases of spinal cord injury (SCI) each year, with individual economic costs in the millions. SCI can diminish or abrogate both motor and sensory systems, although most research to date has emphasized motor impairments and recovery. With the application of human-induced pluripotent stem cells (hiPSCs), the potential for targeted neural regeneration and repair in sensorimotor systems after SCI has greatly improved. In the past decade, the use of hiPSC-derived motor neurons has increased our understanding of degenerative spinal motor diseases, as well as shown successful transplantation in rat models. However, comparatively little has been published applying the same strategies to dorsal sensory neurons and promotion of sensory recovery, despite the prevalence of sensory dysfunction and chronic pain in SCI patients. An imperative next step is to develop protocols to generate the necessary sensory and interneuron populations. We report here the results of our work generating a rapid differentiation protocol for region-specific dorsal spinal neural progenitor cells (dsNPCs) from hiPSCs. This novel protocol allows us to generate both dorsal and ventral spinal populations (ventral protocol previously published) from a shared lineage. These cell populations are being used to study injury and sensorimotor recovery in vitro and in vivo. In the future, we will use our dorsal and ventral cells in tandem in a rodent transplantation model to evaluate their capacity to attain correct repair and reconnection at the site of injury, restoring both motor and sensory systems. These discoveries will push the field of spinal regeneration forward and advance the translation of cell transplantation therapies.
Noninvasive Optogenetic Stimulation in a Rat Model of Spinal Cord Injury
Central Michigan University, Mount Pleasant, MI, USA
The ability to manipulate specific neuronal populations of the spinal cord following spinal cord injury (SCI) could potentially prove highly beneficial for rehabilitation in patients through maintaining and strengthening still existing neuronal connections and/or facilitating the formation of new connections. A noninvasive and highly specific approach to neuronal stimulation is bioluminescent-optogenetics, where genetically expressed light-emitting luciferases are tethered to light-sensitive channelrhodopsins (luminopsins, LMO); neurons are activated by the addition of the luciferase substrate coelenterazine (CTZ). This approach takes advantage of using ion channels for current conduction while activating the channels through application of a small chemical compound, thus allowing noninvasive stimulation and recruitment of all targeted neurons. We previously showed the efficacy of this approach in improving locomotor recovery following severe spinal cord contusion injury in rats expressing the excitatory LMO3 under control of a pan-neuronal and motor neuron–specific promoter; CTZ was applied through a lateral ventricle cannula (Petersen et al., Front Neurol 2022). Here, we transduced spinal cord neurons with a synapsin promoter–driven LMO that contains an opsin with higher light sensitivity. In this construct, the Gaussia luciferase variant sbGLuc is fused to CheRiff, a channelrhodopsin from Scherffelia dubia. Taking advantage of the high light sensitivity of CheRiff, we stimulated transduced lumbar neurons after thoracic SCI by intraperitoneal application of CTZ, allowing for a less invasive treatment. The efficacy of this noninvasive bioluminescent optogenetic approach was confirmed by improved open-field locomotion [Basso, Beattie and Bresnahan (BBB)] scores, BBB subscores, and catwalk subscores. This study demonstrates that peripheral and intraperitoneal injection of the luciferin CTZ can be used to activate LMOs expressed in spinal cord neurons that employ an opsin with increased light sensitivity, CheRiff. Activation of sbGLuc-CheRiff after a severe thoracic level contusion injury resulted in ameliorated motor deficits.
Paraoxonase-2: A Druggable Mitochondrial Enzyme With Differential Age, Sex, and Brain Region Expression That Demonstrates Neuroprotective Potential for Parkinson’s Disease
1 Department of Psychiatry, Yale University School of Medicine, New Haven, CT, USA
Mitochondrial dysfunction and oxidative stress are key pathogenic events that underlie Parkinson’s disease (PD); however, there is no clinically available drug that targets these events. PON2 is a mitochondrial enzyme with significant antioxidant and anti-inflammatory characteristics. Earlier, our lab identified an age-associated decline in PON2 expression in nonhuman primates (NHPs) that correlates with the sensitivity of dopamine neurons to oxidative stress induced by parkinsonian toxins. We also discovered a 2- to 2.5-fold greater PON2 expression in NHP striatum compared with other brain regions and that females exhibit 2- to 3-fold higher expression compared with males. These findings on age-, sex- and region-dependent PON2 expression stimulated our interest in activating PON2 in vivo as a strategy to halt PD progression, particularly as PD is 2–2.5 times more prevalent in men than women. Preclinical studies indicate that the PPARγ activator, pioglitazone, is neuroprotective in PD models by reducing oxidative stress and/or inflammation, and clinical studies suggest it may reduce the risk of PD; however, its precise mechanism has not been elucidated. We hypothesized that pioglitazone’s action involves stimulation of PON2. Indeed, we documented that subchronic pioglitazone treatment significantly augmented striatal expression of PON2 in mice. This led to us determining the effect of pioglitazone on PON2 expression in a more translatable species, male African green monkeys, which were treated with oral pioglitazone (5 mg/kg/day) or vehicle for 1 or 3 weeks. Pioglitazone significantly increased PON2 expression in substantia nigra, striatum, dorsolateral prefrontal cortex, and hippocampus, but after 3 weeks of treatment the effect on PON2 had subsided. An explanation for the short-term effect might be compensatory mechanisms that are activated in the nondiseased brain. These studies establish the therapeutic potential of pharmacologically stimulating PON2 and reveal a novel mechanism for pioglitazone-induced neuroprotection. We anticipate that these outcomes will contribute to the development of neuroprotective treatments for PD.
Ovine-Sourced GM1 Ganglioside Reduces Motor Deficits and Elevates BDNF Levels in the R6/2 Mouse Model of Huntington’s Disease
1 Field Neurosciences Institute Laboratory for Restorative Neurology, Central Michigan University, Mount Pleasant, MI, USA
2 Program in Neuroscience, Central Michigan University, Mount Pleasant, MI, USA
3 Department of Psychology, Central Michigan University, Mount Pleasant, MI, USA
4 College of Medicine, Central Michigan University, Mount Pleasant, MI, USA
5 Field Neurosciences Institute, Saginaw, MI, USA
Huntington’s disease (HD) is an autosomal dominant neurodegenerative disease caused by mutations producing elongated polyglutamine repeats. These mutations lead to excessive production and mutant huntingtin protein, which cause motor and cognitive impairments. Although no effective treatment has been approved for HD, a promising therapy involves the use of GM1 ganglioside, as reduced levels of this glycophospholipid in HD patients correlate to disease progression. Research using bovine- and synthetic-sourced GM1 has shown remarkable reduction in behavioral and neuropathological deficits in rodent models of HD, but these sources of GM1 are very expensive. Recently, a novel procedure for procuring GM1 has been developed whereby a ganglioside storage disease can be induced in sheep to yield 40 times the amount of GM1 obtained from bovine brains. However, the efficacy of treatment using ovine-sourced GM1 has yet to be tested. The goal of this study was to compare the efficacy of ovine- and bovine-sourced GM1 on behavioral deficits and survivability in the R6/2 mouse model of HD. Assessments of motor abilities, using the rotarod, open-field, and catwalk tasks, as well as cognitive abilities, using the Barnes maze and novel object recognition tasks, were made in GM1-treated and vehicle-treated R6/2 mice, which carry a partial transgene for HD. Results indicated that ovine-sourced GM1 gangliosides significantly reduced deficits on the motor tasks (ie, rotarod, open-field, catwalk), but not on cognitive tasks (on the Barnes maze and novel object recognition tasks) at levels matching or exceeding that of bovine-sourced GM1. Both sources of GM1 tended to extend the lifespan of R6/2 mice, but only ovine-sourced GM1 significantly reduced weight loss in the R6/2 mice. Importantly, brain-derived neurotrophic factor (BDNF) levels were increased dramatically in the cortex and striatum of HD mice treated with only ovine-sourced GM1. Our results indicate that ovine-sourced GM1 ganglioside provides a cost-effective and highly efficacious treatment strategy for HD.
Support for this study was provided by the Shepherd’s Gift, the John G. Kulhavi Professorship in Neuroscience, and the E. Malcolm Field. and Gary Leo Dunbar Endowed Chair in Neuroscience at CMU.
Enabling Neuroregeneration After Stroke With an Engineered Thermostabilized Chondroitinase ABC
1 Department of Chemical Engineering and Applied Chemistry, University of Toronto, Toronto, ON, Canada
2 Institute of Biomaterials and Biomedical Engineering, University of Toronto, Toronto, ON, Canada
3 Department of Chemistry, University of Toronto, Toronto, ON, Canada
Following injuries to the central nervous system (CNS), such as spinal cord injury and stroke, astrocytes become reactive and deposit large quantities of proteoglycans that form a glial scar. The scar is essential to prevent the spread of the injury to healthy tissue at the acute phase after stroke, while at the chronic phase, it limits the regenerative capacity of the tissue. The degradation of the glial scar was therefore suggested as a therapeutic strategy after CNS injuries to improve local plasticity and promote axonal regrowth. The bacterial enzyme chondroitinase ABC (ChABC) can degrade chondroitin sulfate proteoglycans, a major component of the glial scar, and has been proposed as a therapeutic strategy for multiple disorders. Yet, its inherent thermal instability, characterized by a rapid loss of activity at physiological temperatures and pH, limits its therapeutic potential. Moreover, continuous delivery is needed to obtain significant improvements after stroke. To address the need for a sustained local release of the enzyme, we developed an affinity-controlled delivery platform composed of an injectable cross-linked methylcellulose hydrogel modified with Src homology 3 (SH3) binding peptides. The enzyme was expressed as a fusion protein with an SH3 domain. To address the intrinsic instability of ChABC, we designed a thermostable mutant of ChABC (ChABC-37-SH3) using computational remodeling. ChABC-37-SH3 demonstrated a 6.5 times higher half-life than the native enzyme, with a higher melting temperature and increased activity for its substrate. Affinity-controlled release from the hydrogel was achieved for at least 7 days in vitro, and the released enzyme retained its catalytic activity. We demonstrated the efficacy of our minimally invasive delivery system in vivo, in an endothelin-1-induced stroke injury model in rats, where we focused on the effect of ChABC-37-SH3 on the glial scar and the ability of the tissue to regenerate.
Iduronidase Gene Therapy Improves the Functional Brain Connectome in Mice With Mucopolysaccharidosis Type I as Determined by Resting-State fMRI
1 Deptarment of Neurosurgery, University of Minnesota, Minneapolis, MN, USA
2 Graduate Program in Neuroscience, University of Minnesota, Minneapolis, MN, USA
3 Deptarment of Biomedical Engineering, University of Minnesota, Minneapolis, MN, USA
4 Graduate Program in Molecular, Cellular, Developmental Biology, and Genetics, University of Minnesota, Minneapolis, MN, USA
5 Center for Magnetic Resonance Research, University of Minnesota, Minneapolis, MN, USA
6 Division of Biostatistics, University of Minnesota, Minneapolis, MN, USA
7 Minnesota Supercomputing Institute, University of Minnesota, Minneapolis, MN, USA
8 Deptarment of Pediatrics, Cell Biology and Development, University of Minnesota, Minneapolis, MN, USA
9 Deptarment of Genetics, Cell Biology and Development, University of Minnesota, Minneapolis, MN, USA
Mucopolysaccharidosis type I (MPS I) is a genetic disorder resulting from mutations of the gene that encodes the enzyme alpha-
Advances in Epidural Spinal Cord Stimulation to Restore Function After Spinal Cord Injury: History and Systematic Review
1 Department of Neurosurgery, University of Minnesota, Minneapolis, MN, USA
2 Division of Neurosurgery, Hennepin County Medical Center, Minneapolis, MN, USA
3 Department of Bioinformatics and Computational Biology, University of Minnesota, Minneapolis, MN, USA
4 Division of Neurosurgery, VA Healthcare System, Minneapolis, MN, USA
Epidural spinal cord stimulation (eSCS) has been recently recognized as a potential therapy for chronic spinal cord injury (SCI). eSCS has been shown to uncover residual pathways within the damaged spinal cord. The purpose of this review is to summarize the key findings to date regarding the use of eSCS in SCI. Searches were carried out using Medline, EMBASE, and Web of Science database and reference lists of the included articles. A combination of medical subject heading terms and keywords was used to find studies investigating the use of eSCS in SCI patients to facilitate volitional movement and to restore autonomic function. The risk of bias was assessed using Robins-I tool for nonrandomized studies. We were able to include 30 articles that met our eligibility criteria. The studies included a total of 105 patient experiences with incomplete or complete SCI. The majority of the studies used the Medtronic 16 paddle lead. Half of the studies reported lead placement between T11 and L1. We included studies that assessed motor (n = 22), autonomic (n = 9), and other outcomes (n = 9). The majority of the studies reported improvement in outcomes assessed. The wide range of included outcomes demonstrates the effectiveness of eSCS in treating a diverse SCI population. However, the current studies cannot definitively conclude which patients benefit the most from this intervention. Further study in this area is needed to allow improvement of the eSCS technology and allow it to be more widely available for chronic SCI patients.
Engineered Pre-Vascularized Microporous Implants for Brain Repair After Stroke
1 The Lundquist Institute for Biomedical Innovation at Harbor-UCLA Medical Center, Torrance, CA, USA
2 Department of Neurology, David Geffen School of Medicine, University of California, Los Angeles, Los Angeles, CA, USA
Stroke is the leading cause of disability in the United States. To date, no clinical trials have succeeded in alleviating stroke survivors’ neurological impairment. The clinical and economic burden of this disease urges the need for a medical solution outside the confines of conventional practices in neurology. While the overwhelming majority of research on brain repair is centered on neurons, an increasing body of evidence suggests that post-stroke angiogenesis plays a fundamental role in guiding repair and functional recovery through the activation of endogenous repair mechanisms. Recent advances in tissue engineering have led to the development of hydrogel materials that can be injected directly into the stroke lesion site to form cell-instructive scaffolds for tissue repair. However, these materials have not been actively designed to promote the regeneration of a functional and mature vasculature within the lesion site. We have recently developed a new class of injectable biomaterials made of annealable micro-scale hydrogels and showed that their in vivo immunomodulating effect is far superior to their bulk hydrogel counterpart. Our preliminary in vivo data show that the injection of these microgels into the stroke cavity reduced local inflammation by changing perilesional astrocyte polarization and reducing microglia activity. In addition, we showed that these injectable microgels can be pre-vascularized by encapsulating endothelial progenitor cells (EPCs) to form vascular capillaries prior to transplantation in vivo. Here, we propose to use a systematic approach to develop an injectable proangiogenic and immunomodulating material that combines pre-vascularized microgels and immunomodulation. We hypothesize that these injectable microgels specifically designed to simultaneously promote vascular regeneration and immunomodulation will promote tissue regeneration and functional recovery after stroke.
A Single-Cell View of Degeneration and Regeneration in the Mammalian Spinal Cord
K. Matson1,2, A. Yadav3, L. Li1, I. Hua1, G. Courtine4,5, E. Bourinet6, P.-F. Mery6, M. Hildebrand7,8, E. Tsai8, V. Menon3, and
1 Spinal Circuits and Plasticity Unit, National Institute of Neurological Disorders and Stroke, Bethesda, MD, USA
2 Department of Biology, Johns Hopkins University, Baltimore, MD, USA
3 Department of Neurology, Center for Translational and Computational Neuroimmunology, Columbia University, New York, NY, USA
4 Center for Neuroprosthetics and Brain Mind Institute, Faculty of Life Sciences, École Polytechnique Fédérale de Lausanne, Lausanne, Switzerland
5 NeuroRestore, Department of Clinical Neuroscience, Lausanne University Hospital and University of Lausanne, Lausanne, Switzerland
6 Institute of Functional Genomics, Montpellier University, CNRS, INSERM, Montpellier, France
7 Neuroscience Program, Ottawa Hospital Research Institute, Ottawa, ON, Canada
8 Department of Neuroscience, Carleton University, Ottawa, ON, Canada
The mammalian spinal cord and peripheral nervous system forge the links between the brain and the body to enable sensorimotor control. These functions are mediated by a diverse array of spinal cord cell types, each with their own molecular repertoires, morphology, activity, and contribution to behavior. Spinal neurons can also display differential vulnerabilities to disease or specific potential for plasticity, suggesting that cell type is a critical factor for understanding spinal cord pathophysiology. Key examples include that neurodegenerative diseases that affect the spinal cord such as amyotrophic lateral sclerosis (ALS) and spinal muscular atrophy (SMA) are characterized by the loss of spinal motoneurons, while injured neurons display widely different regeneration depending on the cell type. To address the molecular basis of neuronal diversity in the spinal cord, we used single-nucleus RNA sequencing to profile the mouse and human adult spinal cord. Within human motoneurons, we observed a unique gene expression signature that provides insight into the selective degeneration of motoneurons. Within the mouse spinal cord, we profiled the spared tissue distal to an injury and discovered rare populations of spinal neurons that express regeneration-associated genes and undergo sprouting outgrowth and remodeling after injury. Ultimately, we hope that our work will provide fundamental knowledge and enhanced therapeutic targets in spinal cord injury and disease.
Neurological Consequences Associated With SARS-CoV-2 Infection in Repeated Traumatic Brain-Injured Mice
1 Department of Molecular Medicine, University of South Florida, Tampa, FL, USA
2 James A. Haley VA Hospital, Tampa, FL, USA
3 Department of Neurosurgery and Brain Repair, University of South Florida, Tampa, FL, USA
4 Department of Internal Medicine, University of South Florida, Tampa, FL, USA
Traumatic brain injury (TBI) is a neurological disorder that affects ~10 million people globally every year. The underlying pathology of repeated TBI (rTBI) is poorly understood, which makes the development of therapeutics more challenging. Coronavirus disease 2019 (Covid 19) caused by SARS-CoV-2 is declared a pandemic by the World Health Organization since March 2020. Although Covid-19 is considered a disease that primarily affects the lungs, it can also cause damage to multiple organs including the brain. Specifically, CoV-2 infection in TBI patients can exacerbate the neuroinflammation, leading to long-term neurological sequelae of viral infection. In our studies, we found that SARS-CoV-2 infection in TBI mice showed significantly increased viral load in the brain when compared with infection-only mice. And we also detected there is a significant elevation of inflammatory markers in these TBI-CoV-2-infected mice. CCL20 is one such inflammatory mediator that is elevated during this double-barreled insult. It specifically interacts with CCR6 receptors, attracting B cells, T cells, and dendritic cells to the site of injury, thereby promoting neurodegeneration. Our study shows that blocking CCL20–CCR6 axis by delivering dendrimer complexed with plasmids encoding shRNA to knock down CCL20 and CCR6 decreases the viral load and also ameliorates the inflammation in the brain. Thus, blockade of chemokine pathways, including CCR6/CCL20, may be a possible avenue for therapeutic intervention to prevent dissemination of the virus to key immunological sites.
Reprogramming by Sequential Gene Expression for Survival and Specification of Induced Neurons From Oligodendrocyte Progenitor Cells
M. Thaqi, E. Reisenbigler, R. Greene, and
Center for Stem Cell and Regenerative Medicine, The Chicago Medical School, Rosalind Franklin University of Medicine and Science, North Chicago, IL, USA
Direct reprogramming of cell identity from glial to neuronal phenotype has been demonstrated both in vitro and in vivo. The induction of phenotypic neurons from glial cell populations follows viral delivery and forced expression of pioneering transcription factors, such as Ngn2, NeuroD1, and Ascl1, which are normally expressed during development to specify neuronal fate. As induction of astrocytes may remove an important functional glial population from the targeted region, we have chosen to target oligodendrocyte progenitor cells (OPCs), also known as NG2 glia. NG2 glia, understood to represent a reserve cell population to replace oligodendrocytes, are actively dividing in the mature central nervous system (CNS), perform no known critical neural activity, and respond to injury by proliferation and subsequent population homeostasis. One challenge facing the reprogramming field, for which a solution is needed to advance therapeutic advancement of this approach, is the need to target a defined glial population with specificity. Although the use of adeno-associated viral (AAV) vectors for gene delivery is favored for their potential therapeutic advantages, we show that many serotypes of AAV are promiscuous regarding the cell types infected in the rodent brain. Furthermore, the use of cell-specific promoters in wild-type animals, a more suitable model for therapeutic delivery, does not accomplish selective expression of OPCs. We show that retroviral vectors offer a way to deliver reprogramming genes to OPCs with high probability. Another challenge of significant therapeutic relevance facing the field is directing lineage respecification to the desired neuronal subtype following overall neuronal induction. The expression of only one initiating reprogramming gene does not facilitate control over subsequent survival and subtype specification. We describe the development of a retrovirally delivered, regulatable gene expression system to control temporal expression of sequential genes instructive for survival and subtype specification of neurons induced from OPCs.
Overexpression of ACMSD Prevents Neuroinflammation in the P301S Tauopathy Alzheimer’s Mouse Model and Improves Learning and Memory in Hippocampal-Dependent Spatial Navigation
K. T. Meyers1, D. J. Marmion1, I. M. Sandoval1, J. Alvarez-Rucoba1, L. Brundin2, and F. P. Manfredsson1
1 Parkinson’s Disease Research Unit, Barrow Neurological Institute, Phoenix, AZ, USA
2 Van Andel Research Institute, Grand Rapids, MI, USA
Alterations to the kynurenine metabolic pathway measured by disease-related changes in metabolites from cerebrospinal fluid (CSF) and plasma are observed in numerous neurodegenerative diseases including Alzheimer’s disease (AD). Interestingly, metabolites from this pathway can be either directly neurotoxic, quinolinic acid (QA), or promote neuroinflammation. Our overarching hypothesis thus posits that shifting the kynurenine pathway away from the production of neurotoxic compounds to that of neuroprotective compounds, picolinic acid (PA), will provide neuroprotection in the face of varying neurodegenerative insults. We recently showed that overexpression of 2-amino 3-carboxymuconate 6-semialdehyde decarboxylase (ACMSD), an enzyme that shifts the kynurenine pathway away from production of QA toward PA, provides neuroprotection in the adeno-associated virus-mediated (AAV) alpha-synuclein (α-syn) model of Parkinson’s disease. We therefore hypothesized that AAV-mediated overexpression of ACMSD would limit neurodegenerative and neuroinflammatory processes and prevent cognitive decline in P301S mutant human tau transgenic mice (Tau P301S mice), a model of early-onset frontotemporal dementia. Tau P301S mice (or wild-type littermates) received AAV-ACMSD or AAV-FLEX-GFP (control) at two months of age prior to pathology onset, delivered to the dorsal hippocampus. The Barnes Maze spatial navigation test was administered to animals 5–7 months of age. ACMSD overexpression correlated with increased learning and memory performance. Interestingly, improvement was also observed in wild-type mice, suggesting that ACMSD can modulate neuronal function regardless of the disease state. Animals were sacrificed at 8 months of age, a time point where significant losses of neurons and synapses are observed with concomitant increases in gliosis. Whereas significant increases in astrogliosis and microgliosis were observed in the hippocampus of AAV-FLEX-GFP P301S mice, AAV-ACMSD P301S mice show an observable reduction in markers of these neuroinflammatory processes. Ongoing work aims to quantify additional histopathological indices including tau phosphorylation and neurodegeneration. Nonetheless, these results provide further support for the use of ACMSD in alleviating neuroinflammatory and neurodegenerative processes.
Erodible Thermogelling Hydrogels for Subdural Transplantation of Exogenous Mitochondria Following Spinal Cord Injury
1 Department of Physiology, University of Kentucky, Lexington, KY, USA
2 Spinal Cord & Brain Injury Research Center, University of Kentucky, Lexington, KY, USA
3 Department of Chemical and Materials Engineering, University of Kentucky, Lexington, KY, USA
4 Department of Chemistry Lexington, University of Kentucky, Lexington, KY, USA
Mitochondrial dysfunction leads to secondary damage following spinal cord injury (SCI). Acute mitochondrial transplantation via intraspinal injections improves mitochondrial bioenergetics of injured tissues a week after injury, but associated functional improvement was not observed long-term. This study tested, first, whether supplementing exogenous mitochondria into cell lines increases metabolism associated with documented cellular uptake. In experiments using human neuroblastoma cell lines (SH-SY5Y) and rat pheochromocytoma cell lines (PC-12), we found that mitochondrial transplantation augmented the metabolic activity of recipient cells in dose-dependent manners, although efficiency varied depending on recipient cells (syngeneic vs xenogeneic) and mitochondrial source (ie, tissue-derived). To document mitochondrial uptake, we used the presence of human mitochondrial DNA (mtDNA) as a surrogate marker within rat PC12 cells co-incubated with human mitochondria isolated from SH-SY5Y cells and noted dose-dependent difference in uptake. Due to technical limitations such as needle tract injury, accumulation at injection sites, and rapidly reduced mitochondrial integrity ex vivo, we secondly sought to test a delivery system using a hydrogel designed to erode over the course of 1–3 h in situ to facilitate mitochondrial release for cellular uptake. Experiments using hydrogels composed of (1%) methylcellulose and (1%) hyaluronic acid show that it remarkably maintained integrity of isolated mitochondria for up to 1 h at 37°C. Based on preliminary studies injecting the same hydrogel composition labeled with dye subdurally into rats with and without SCI, we determined that the spread and extent of mitochondrial distribution were improved following subdural delivery compared with focal injection. While the presence of mtDNA extended to regions distal to the injection sites, mitochondrial uptake in the injured cord was lower compared with naïve cord. Thus, we are repeating our studies and modulating the gel composition to further test components that improve mitochondrial health, such as sodium oxide dismutase, catalase, and glutathione.
Unique Gut Microbiome Signature Changes in Stroke Pathology and Treatment
1 USF Center for Microbiome Research, University of South Florida, Tampa, FL, USA
2 Department of Neurosurgery and Brain Repair, University of South Florida, Tampa, FL, USA
3 Center for Excellence of Aging and Brain Repair, University of South Florida Morsani College of Medicine, Tampa, FL, USA
Stroke is a debilitating neurological problem that causes long-term disability affecting 7 million adults in the United States each year. It is one of the major causes of death in this population. Emerging evidence indicates that stroke alters the gut microbiome in stroke patients; however, the role of the microbiome in stroke etiology and its importance in the stoke treatments is not known. Adult (8 weeks old) male Sprague-Dawley rats underwent unilateral middle cerebral artery occlusion (MCAO) for 1 h with one group (n = 8) also subjected to the therapeutic conditioning of transient jugular vein clamping (JVC) over the first 15 min of MCAO, while another group of MCAO animals (n = 8) only received jugular vein isolation without clamping. Here, we observed that MCAO produced a unique gut microbiome signature characterized by significantly reduced microbiome diversity—an indicator of microbiome health and increased the ratio of major bacterial phyla like Firmicutes/Bacteroidetes. In particular, MCAO significantly enhanced the abundance of pathogenic commensals like Clostridium, Turicibacter, and Romboutsia and reduced the abundance of beneficial bacteria like Lactobacillus, Barnesiella, and Bacteroides. Interestingly, JVC when combined with MCAO protected against the gut dysbiosis and was linked with improved stroke measures like Neuroscore, infarct volume, and peri-infarct cell loss. Overall, the results demonstrated that the stroke-induced behavioral and histological deficits coincided with significant gut microbiome alterations as evidenced by enhanced pathogenic and suppressed beneficial bacteria, while JVC prevented these gut microbiome abnormalities, along with improved stroke outcomes, suggesting that the gut microbiome serves as a sensitive pathological biomarker and therapeutic index in stroke.
Extracellular Matrix–Based Hydrogel for the Treatment of Stroke
M. Modo
McGowan Institute for Regeneration Medicine, Departments of Radiology & Bioengineering, University of Pittsburgh, Pittsburgh, PA, USA
Stroke remains a devasting neurological condition with a lack of treatment opportunities. One major challenge posed by stroke damage is the evolution of the core of the lesion into brain tissue cavity. Inductive bioscaffold, such as extracellular matrix (ECM)-based hydrogel, in other tissues can invoke a regenerative response that restores site-appropriate lost tissue. To achieve a tissue restoration in a stroke-induced cavity, we have established that bioscaffold needs to (1) completely fill the tissue cavity, (2) gel within the cavity, (3) invoke an immune response, (4) produce angiogenesis, (5) attract neural progenitors, and (6) develop neural circuitry that is integrated with the vertebrate brain. We have further established an advantageous time window that defines minimum delivery conditions (>10 days), as well as a decreased regenerative capacity with a delayed implantation (90 days). Defining these key aspects of bioscaffold administration is essential to evaluate the potential of these materials for the treatment of stroke.
Nicotinic Receptor Antagonist and Pioglitazone Combination Therapy Decreases Neuroinflammation and SARS-CoV-2 Replication
S. Mohapatra1, S. S. Mohapatra2, K. Mayilsami1, M. Das1, A. R. McGill1,2, C. Brechot1, and P. R. Sanberg3
1 Molecular Medicine, University of South Florida, Tampa, FL, USA
2 Internal Medicine, University of South Florida, Tampa, FL, USA
3 Neurosurgery and Brain Repair, Center of Excellence for Aging and Brain Repair, University of South Florida Morsani College of Medicine, Tampa, FL, USA
The emergence of SARS-CoV-2 coronavirus has caused the ongoing global pandemic COVID-19 with millions of deaths thus far. A large proportion of the infected population exhibits multi-organ inflammatory syndrome causing lung acute respiratory distress syndrome (ARDS) and neurologic deficits in the brain. There is a dire need to develop therapies that target both viral infection and ensuing inflammation in the lung and brain. To address this, we conducted a molecular modeling–based combination therapy approach involving two Food and Drug Administration (FDA)-approved drugs, that is, mecamylamine (MM), a nicotinic receptor antagonist that can traverse through the blood–brain barrier easily, and pioglitazone (PG), a PPAR-γ agonist with anti-inflammatory, hypoglycemic, and antiviral properties. We found that MM and PG combination treatment each at 5 µM synergistically reduces replication of mouse coronavirus MHV4 in infected IMG mouse glial cells and decreases gene expression of inflammatory cytokines such as interleukin (IL)-6, IL-β, and tumor necrosis factor-α (TNFα). Furthermore, the effect of these drugs alone or in combination on the viral infection was examined in a mouse coronavirus infection model. The results showed that C57BL6 mice treated with MM (or its isomer) and PG (each at 2 mg/kg) exhibit a greater reduction in the expression of MHV-N gene in the lung and brain and the inflammatory cytokine genes such as IL-6, IL-β, and TNFα versus MM or PG used alone. Also, we examined the efficacy of MM plus PG treatment on SARS-CoV-2 replication in infected human Calu3 lung cells, and the results show that MM plus PG treatment significantly decreases viral N gene expression and the expression of genes encoding inflammatory cytokines such as IL-6, IL-β, and TNFα compared with single drugs. Taken together, these results indicate the MM plus PG combination therapy can synergistically decrease viral replication and cytokine storm in both the lung and the brain. This therapy is expected to decrease the severity of the infection and decrease long-COVID, which involves continued neurologic manifestations following acute infection.
Disclosure: SM, SSM, CB, and PRS are inventors on a pending patent filed by USF.
Countering Fragile X Syndrome With Reelin
1 Morsani College of Medicine, Department of Molecular Pharmacology and Physiology, University of South Florida, Tampa, FL, USA
2 PTC Therapeutics Inc., Plainfield, NJ, USA
Fragile X Syndrome (FXS) is the most common inherited form of intellectual disability and is characterized by autistic behaviors, childhood seizures, and abnormal dendritic spines. The loss of function of the FMR1 gene leads to a lack of Fragile X Mental Retardation Protein (FMRP) expression. FMRP has been shown to play a critical role in synaptic plasticity and leads to significant behavioral deficits in spatial learning and memory tests. Currently, there are no therapeutic treatments for FXS. Here, we propose that increased Reelin activity could offer a novel therapeutic intervention for FXS. Reelin is an extracellular signaling protein important for normal hippocampal synaptic plasticity, and reductions in Reelin have been implicated as a contributing factor to several neurological disorders including schizophrenia, autism, and temporal lobe epilepsy. We demonstrate a reduction in Reelin, as shown in Western blot analysis, in FMR1 KO mice, thus implicating a loss of Reelin activity as a contributing factor for FXS. It has previously been shown that a single small increase in Reelin can enhance long-term potentiation (LTP), increase dendritic spine density, and improve cognition in wild-type mice. Reelin supplementation has also shown beneficial effects in a model of Angelman syndrome, suggesting a benefit in neurodevelopmental disorders. We demonstrate here that Reelin protein supplementation in FMR1 KO mice can profoundly rescue cognitive deficits in Hidden Platform Water Maze, Open Field, and Fear Conditioning. More importantly, we have generated a truncated Reelin peptide that can be expressed using recombinant adeno-associated virus, and this virus was able to rescue the cognitive deficits in the FMR1 KO mice. These data suggest that increasing Reelin signaling with our new gene therapy approach could offer a novel therapeutic for FXS.
Astrocyte-to-Neuron Reprogramming for Spinal Cord Repair
1 Department of Neurobiology & Anatomy, Drexel University College of Medicine, Philadelphia, PA, USA
2 Spinal Cord Research Center, Drexel University College of Medicine, Philadelphia, PA, USA
3 Gladstone Institutes, San Francisco, CA, USA
Traumatic spinal cord injury (SCI) disrupts neural pathways and results in devastating tissue damage and loss of function, with subsequent reactive gliosis and tissue scarring that limits further loss, but also limits endogenous repair. One therapeutic strategy gaining some attention in recent years is cellular reprogramming, which harnesses advances in cellular engineering to directly convert cells from one phenotype to another. The present work explores strategies for converting either mature or developing astrocytes into neurons. Our initial experiments have begun exploring viral targeting of glial cells to promote doxycycline-inducible overexpression of neurogenic transcription factors (TFs) Ascl1 or a combination of microRNAs (miRs) miR124 and miR9/9* for neuronal conversion. Our primary goal was to establish optimal conditions for consistent and efficient reprogramming of developing spinal glia and mature reactive spinal astrocytes. Primary astrocyte cultures were generated from postnatal days 2–3 (P2–3) rat cortex and spinal cord, and then were activated with TGFβ (transforming growth factor-beta) 1 day prior to viral transduction. Fifteen days following doxycycline administration, astrocytes took on a typical neuronal morphology. Immunocytochemistry confirmed that these cells were positive for neuronal markers MAP2, NeuN, and TAUR1 and negative for GFAP. Multielectrode array recordings of converted cells revealed spontaneous firing consistent with neuronal activity. These preliminary results confirm that cortical and spinal astrocytes can be efficiently reprogrammed into neurons by either Ascl1 or miRs. Advances in cellular reprogramming are providing novel ways to manipulate the cytoarchitecture of the injured cervical spinal cord. Our ongoing work is now using this same approach to modify developing glial precursors that can be harness for their proreparative properties for spinal cord repair.
Neuroinflammatory Changes in the Brain Following Intra-Arterial Mesenchymal Stem Cell Therapy in a Canine Endovascular Model of Ischemic Stroke
1 Department of Neurology, University of Miami Miller School of Medicine, Miami, FL, USA
2 Department of Pathology, University of Georgia, Athens, GA, USA
3 Department of Neurosurgery, Jikei University School of Medicine, Tokyo, Japan
Stroke is the fifth leading cause of death in the United States. With the current standard of care for stroke, more than 50% of stroke survivors are chronically disabled, leading to more than $46 billion in annual economic burden. It is therefore imperative to develop novel therapies for stroke. Preclinical studies in the past two decades have shown promise in cell therapies for stroke. Intra-arterial (IA) delivery of mesenchymal stem cells (MSCs) is minimally invasive, making it attractive for clinical translation. Studies from our lab have shown the safety and efficacy of IA-MSC treatment in rodent and canine stroke models. However, the mechanism of action of the IA MSCs and the underlying inflammatory changes remain to be understood, specifically in the large animal model which is crucial for effective translation. In this study, we determined the expression pattern of microglia, astrocytes, and the Nod-like receptor 3 (NLRP3) inflammasome pathway markers in the large animal canine brain following IA MSC therapy after ischemic stroke. Immunohistochemistry staining and fluorescence imaging of the canine brain cortex and hippocampi were performed to determine the expression of inflammatory markers. Sequence identity and match were predetermined using BLAST software to confirm antigenic specificity for the rare canine research model. Numerical quantification identified the expression patterns of GFAP, Iba1, NLRP3, apoptosis-associated speck-like protein containing a CARD domain (ASC), caspase 1, and interluekin-1beta at different doses of IA-MSCs following stroke in the canine brain at 30 days after stroke. These novel findings help us understand inflammation in the canine brain after stroke. Our data further reveal how IA-MSC therapy mediates its action via inflammatory changes. Overall, this approach contributes to developing more robust and engineered IA-MSCs therapies for stroke patients, leading to more effective translation.
Host-to-Graft Propagation of Alpha-Synuclein in Parkinson’s Disease: Intra-Nigral Versus Intra-Striatal Transplantation
1 Laboratoire de Neurosciences Expérimentales et Cliniques, Inserm U1084, Université de Poitiers, Poitiers, France
2 Hôpital de la Milétrie, CHU Poitiers, Poitiers, France
Parkinson’s disease (PD) is a neurodegenerative disorder characterized by the degeneration of nigro-striatal dopaminergic (DA) neurons and by an abnormal accumulation of aggregated alpha-synuclein (a-syn), the main protein component of Lewy bodies. Intrastriatal transplantation of DA neurons from fetal ventral mesencephalic (fVM) tissue in PD patients has provided proof-of-principle for the cell replacement strategy in this disorder. However, 10–22 years after transplantation, some of the grafted neurons contained Lewy bodies similar to those observed in the host brain. We hypothesize that the striatum represents an unfavorable environment for the development of transplanted neurons, making DA neurons more vulnerable to the disease process. Our project aims to investigate the host-to-graft propagation of a-syn by comparing two transplantation sites, the substantia nigra (SN) and the striatum. For this purpose, we set up two mouse models of PD by injecting AAV2-human a-synuclein A53T viral vector into either the SN or the striatum of C57BL6 or RAG2−/− mice. In these two models, DA progenitors originating from mouse fVM or human-induced pluripotent stem cells (iPSC) were transplanted into either the SN or the striatum. The host-to-graft propagation of human and phosphorylated a-syn was quantified from 1 month up to 12 months after transplantation. The polarization of the inflammatory response (pro vs anti) was assessed in astrocytes and microglia, into the graft and in the surrounding host tissue, to determine whether the inflammatory state is correlated with the level of a-syn propagation. Our preliminary results show that the accumulation of human a-syn gradually increases over time in both nigral and striatal fVM-derived grafts. After normalization with host surrounding tissue, our first data show 5 months after transplantation that the amount of human a-syn is higher in striatal grafts of striatal viral injected animals compared with other conditions, confirming our hypothesis. Analyses are underway in fVM and iPSC-derived grafts to see whether similar results are obtained with phosphorylated a-syn and whether this accumulation is linked to tissue inflammation.
Hydrogel-Assisted Cannabidiol Brain Delivery for the Treatment of Ischemic Stroke
1 The Lundquist Institute for Biomedical Innovation at Harbor-UCLA Medical Center, Torrance, CA, USA
2 Department of Neurology, David Geffen School of Medicine, University of California, Los Angeles, Los Angeles, CA, USA
Stroke is a leading cause of adult disability and the fifth cause of death in the United States. Currently, the only Food and Drug Administration (FDA)-approved therapeutics for ischemic stroke target reperfusion of the occluded blood vessel by administration of an intravenous thrombolytic agent or mechanical retrieval of the clot via an endovascular stent, however the utilization of these therapies, is limited by a brief therapeutic window, making the majority of stroke patients ineligible for treatment and subject to further neurological damage. The immune response to ischemic stroke induces acute and chronic inflammation, resulting in a dynamic lesion that expands over time and exacerbates the neurological insult. In addition, the stroke lesion forms a compartmentalized cavity devoid of extracellular matrix and encapsulated by an astrocytic scar, both challenges to cellular infiltration and tissue regeneration. Cannabidiol (CBD), a cannabinoid with nonpsychotropic action, has received great scientific interest due to its neuroprotective, anti-inflammatory, and antioxidant properties. This compound extracted from Cannabis sativa has shown efficacy in neuropathologies such as Alzheimer’s disease and epilepsy. In mouse models of cerebral ischemia, the bolus intraperitoneal or intracerebral injection of CBD has shown immunomodulatory benefits with decreased stroke volume and blood–brain barrier permeability. However, the benefits of CBD bolus administration are not sustained long-term and do not alleviate mice after stroke neurological deficit. We recently developed an injectable hydrogel-based engineered system to deliver drugs in a controlled-release manner while simultaneously providing structural support in the lesion site. In this study, we use our engineered system to encapsulate, deliver, and slowly release CBD directly into the infarct cavity to supply the local environment with sustained immunomodulation with the hypothesis that localized controlled release of CBD within the lesion site will promote long-term immunomodulation, activation of the endogenous repair machinery, and functional recovery after stroke.
Engineered Proangiogenic and Immunomodulating Hydrogel for Neural Stem Cell Transplantation After Stroke
1 The Lundquist Institute for Biomedical Innovation at Harbor-UCLA Medical Center, Torrance, CA, USA
2 Department of Neurology, David Geffen School of Medicine, University of California, Los Angeles, Los Angeles, CA, USA
Stroke is the leading cause of disability in the United States. To date, no clinical trials have succeeded in alleviating stroke survivors’ neurological impairment. Stem cell transplantation after stroke has shown promise in enhancing brain repair in pre-clinical models but has not advanced to a clinically relevant treatment due to poor cell survival rates and poor control over these cells’ differentiation. Recent advances in tissue engineering have led to the development of hydrogel materials that can be injected directly into the stroke lesion to form cell-instructive scaffolds that can serve as a cell transplantation vehicle. We have recently developed an injectable extracellular matrix (ECM)-mimetic hydrogel platform for neural progenitor cell (NPC) transplantation in the stroke brain using a systematic approach to optimize the material’s capacity to control stem cell survival and differentiation into specific neural lineages in vivo. These findings successfully led to a series of hydrogel “recipes” with distinct mechanical, structural, and biological properties that can precisely predict the distinct cell fate of transplanted cells. Although this technology significantly improved cell viability and controlled differentiation, encapsulated cells did not survive long enough to enhance functional recovery due to the immunological attack on the transplant and a lack of vascularization of the injected hydrogel. In this study, we propose to further develop our proneuronal hydrogel platform to incorporate immunomodulating and proangiogenic capabilities. We hypothesize that this novel material platform can enhance long-term survival and tissue integration of the transplanted graft and promote brain repair and recovery after stroke.
Therapeutic Neural Stem Cell Candidate Line for Chronic Cervical Spinal Cord Injury
1 Sue & Bill Gross Stem Cell Research Center, University of California, Irvine, Irvine, CA, USA
2 Institute for Memory Impairments & Neurological Disorders, University of California, Irvine, Irvine, CA, USA
3 Anatomy & Neurobiology, University of California, Irvine, Irvine, CA, USA
4 Physical & Medical Rehabilitation, University of California, Irvine, Irvine, CA, USA
Spinal cord injury (SCI) is a devastating neurological condition with no established treatment to improve the quality of life of those affected, making development of therapeutic intervention targeting chronic SCI a high priority. Multipotent fetal brain–derived human neural stem cells (hF-NSCs) have shown reproducible efficacy in preclinical therapeutic transplantation studies in subacute and early chronic thoracic and cervical SCI models. Small Phase I/II clinical trials with hF-NSC in thoracic and cervical SCI support both safety and tolerability of tissue-derived neural stem cells and the potential for functional improvement. Multiple studies suggest that the intrinsic properties of stem cells influenced by the cell source are a critical variable for clinical success. The therapeutic bioactivity may be affected by numerous factors including in vitro culture conditions, scale-up/manufacturing, and cell banking. Consequently, the lack of in vitro assays that can successfully discriminate between cell lots/lines that yield efficacy, versus ones that do not, has become a significant bottleneck for clinical translation. To better understand the intrinsic differences between neural stem cells, we have derived a series of new CD133-enriched hF-NSC lines, including cGMP-compliant cell lots, and developed an in vitro gene expression classifier that can a priori correctly predict post-transplantation outcome in a chronic cervical SCI model. Based on these data, we are testing an RNA array for use in monitoring therapeutic candidate line stability during cGMP process development and scale-up from seed bank to final product bank.
This work has been funded by CIRM DISC2-10753 and TRAN1-13059.
Investigation of AD Pathology in iPSC-Cortical Neurons Carrying PSEN1 and APP Mutations
1 Nanoscience Technology Center, University of Central Florida, Orlando, FL, USA
2 Hesperos, Inc., Orlando, FL, USA
Alzheimer’s disease (AD) can onset as a result of familial AD (FAD) mutations of the presenilin 1 (PSEN1) or amyloid precursor protein (APP) genes. PSEN1 is a proteolytic component of the gamma-secretase complex, cleaving APP into amyloid β 40 (Aβ40). Mutations in the PSEN1 gene result in a defective presenilin 1, leading to abnormal cleavage of APP. Under normal conditions, APP is cleaved by PSEN1 and released outside of the cell as soluble APP and Aβ40. However, this abnormal cleavage gives rise to a longer version of amyloidβ peptide 42 (Aβ42), forming destructive amyloid oligomers, fibrils, and plaques which cause synaptic dysfunction of neural networks. Differentiating cortical neurons (CNs) from human-induced pluripotent stem cells (hiPSCs) carrying these mutations offers the ability to reproduce and investigate the AD pathology in human cortical neurons, thus establishing efficient Alzheimer’s disease models. An iPSC-CN model capable of generating functional long-term potentiation (LTP) in an organ-on-a-chip system has already been established and validated. For this study, we attempt to characterize FAD PSEN1 and APP mutant hiPSC-CNs to identify AD-relevant pathologies by comparing with their isogenic controls. Phase microscopy was used to monitor morphological differences throughout 40 days of differentiation. Immunocytochemistry using markers of ctip2 and MAP2 confirmed successful differentiation of cortical neurons, while staining of synaptic puncta indicated synaptic deterioration over time. Patch-clamp electrophysiology was used to characterize the electrophysiological properties of the differentiated neurons. Neural network activity formed by the iPSC-CNs was evaluated by their culture on multi-electrode arrays to analyze their basic electrical activity and the induction and maintenance of LTP. The ability to construct these AD models and effectively compare their functional and characteristic behavior allows for further applications such as screening of current and novel treatments for AD.
Recommendations for Research Guidelines That Involve Stem/Progenitor Cells and Human–Animal Chimeras
1 University of Minnesota, Minneapolis, MN, USA
2 Central Michigan University, Mount Pleasant, MI, USA
3 University of California at Davis, Davis, CA, USA
4 Upstate Medical Center, Syracuse, NY, USA
5 University of California, San Francisco, San Francisco, CA, USA
Advances in stem cell biology and the generation of interspecies chimeras for producing exogenic organs and cells have reached the limit of current guidelines for research that involve stem/progenitor cells and human–animal chimeras. The International Society for Stem Cell Research (ISSCR) has recommended new guidelines for their membership to address these issues. Many of these new guidelines are also relevant for members of the American Society for Neural Therapy and Repair (ASNTR). ISSCR guidelines relevant to ASNTR include (1) studies transplanting human stem cells or their derivatives into the central nervous system of animals, (2) human–animal chimera embryo research, (3) culture of human embryos beyond formation of the primitive streak or 14 days, (4) mitochondrial replacement techniques, (5) emerging categories of embryo research that merit close review—heritable gene editing, and (6) classifying stem cell–based, cell-based, and tissue-based interventions. To address these issues, an ASNTR Task Force was assembled to discuss each of these topics with the objective of providing recommendations to the ASNTR membership for their consideration as to whether to adopt the ISSCR guidelines as part of ASNTR’s own guidelines. The recommendations of the ASNTR Task Force will be presented at the ASNTR 2022 annual meeting.
Reduction of Behavior Deficits Following Delivery of hSOX2 Gene to Stroke Brain Using PAMAM Dendrimer Nanomolecules
1 Field Neurosciences Institute Laboratory for Restorative Neurology, Central Michigan University, Mount Pleasant, MI, USA
2 Program in Neuroscience, Central Michigan University, Mount Pleasant, MI, USA
3 College of Medicine, Central Michigan University, Mount Pleasant, MI, USA
4 Department of Chemistry & Biochemistry, Central Michigan University, Mount Pleasant, MI, USA
5 Department of Psychology, Central Michigan University, Mount Pleasant, MI, USA
6 Field Neurosciences Institute, Saginaw, MI, USA
PAMAM dendrimers are three-dimensional nanomolecules that can deliver a large cargo of drugs and biomolecules to the cells in vitro and in vivo. In the past, we used the generation-4 surface-modified PAMAM dendrimers having 90% hydroxyl groups and 10% amine groups (G4-90/10) which can cross the blood–brain barrier and deliver the cargo into the cells. Unlike some of the viral vectors, these dendrimers can carry plasmids that are more than 10 kb in size. In the current study, we used G4-90/10 PAMAM dendrimers to deliver a therapeutic gene to the infarct in a stroke rat brain. Following a stroke event, many astrocytes are recruited at the stroke region to perform their normal maintenance functions, creating severe inflammation. The goal of this study was to reprogram a fraction of these glia into neuronal lineage and repopulate the infarct region. We have used a transcription factor (hSOX2 gene), which was then delivered to the stroke brain in rats using the G4-90/10 dendrimers. Our initial behavior analysis indicated that, following PAMAM dendrimer–based delivery of hSOX2, there was recovery of behavioral functioning in stroked rats compared with vehicle-treated stroked rats and sham-operated controls. Furthermore, as an effort to optimize and fine-tune our in-house synthesized PAMAM dendrimer–based delivery, our collaborators have synthesized another type of surface-modified PAMAM dendrimers (G4-70/30). In a parallel study, we complexed these G4-70/30 dendrimers (in activated and nonactivated states) with plasmids having reporter genes and tested their delivery efficiency in rat-derived primary cortical neurons. Our results showed that the G4-70/30 dendrimers in an activated state are able to deliver and express twice the number of plasmids compared with the nonactivated dendrimers. Overall, these results show that PAMAM dendrimers have significant potential delivering therapeutic molecules for treating stroke and other neurological conditions.
Support for this study was provided by the American Heart Association (18AIREA 33990094), National Institute of Heath NIH R21 (1R21EY030012), Neuroscience program, the College of Medicine, the Field Neurosciences Institute, Department of Chemistry and Biochemistry, and the John G. Kulhavi Professorship in Neuroscience at Central Michigan University.
Fibrinogen Modulates NF-kB Signaling in Neurons
Department of Surgery, Morsani College of Medicine, University of South Florida, Tampa, FL, USA
Activity of nuclear factor-κB (NF-kB) has been implicated in the pathogenesis and early mechanisms of neuronal viability during traumatic brain injury (TBI). We have previously shown that TBI-induced hyperfibrinogenemia was associated with upregulation of inflammatory cytokines in astrocytes and neurons, resulting in oxidative stress and increased neuronal death through activation of intercellular adhesion molecule-1 (ICAM-1). It has been shown that expression of ICAM-1 is regulated by NF-kB. However, the signaling pathway for the Fg-induced neurodegeneration was never characterized. Here, we tested whether Fg’s interaction with its neuronal receptors, cellular prion protein (PrPC) and ICAM-1, induced activation of NF-kB. Primary mouse brain cortex neurons from C57BL/6 mice plated at 7 × 105 cells/ml in poly-
Connectivity Mapping of Neural Progenitor Cells for Restoring Locomotor Circuits After Spinal Cord Injury
1 Department of Biology, Texas A&M University, College Station, TX, USA
2 Texas A&M Institute for Neuroscience, Texas A&M University, College Station, TX, USA
Keywords: neural progenitor cells, spinal cord injury, transsynaptic tracing
Spinal cord injury (SCI) is a traumatic and life-altering event that frequently results in the loss of voluntary motor function after injury. Currently, there are no clinically effective treatments to restore severed locomotor circuitry after injury. Neural progenitor cells (NPCs) are a promising potential therapeutic intervention for SCI due to their ability to mature into all neuronal subtypes of the spinal cord and make functional synaptic connections with surviving host neurons. To reconstruct locomotor circuitry following traumatic spinal cord injury using NPCs, it is first critical to determine the number and phenotypes of transplanted neural progenitor cell–derived neurons that establish direct synaptic connections onto spinal cord motor neurons after spinal cord injury. In this study, we used monosynaptic rabies and pseudorabies tracing to characterize the presynaptic inputs onto spinal cord lumbar motor neurons in mice that received a T12 spinal cord injury and NPCs. Using immunohistochemistry, we first identified rabies+ neurons outside and within the NPC transplantation site. Rabies+ neurons found within the NPC transplantation site were marked for their location within the graft. We are currently characterizing rabies+ NPCs through the usage of ventral spinal cord interneuron markers Chat, CAMKII, and GAD67 based on our tracing studies in the uninjured spinal cord. Future work will include the usage of Designer Receptors Exclusively Activated by Designer Drugs (DREADDs) to further elucidate the potential role of these rabies+ interneuron populations on motor recovery. Ultimately, these findings will inform future work to elucidate the transplanted neuron populations that will be most critical for restoring locomotor function after spinal cord injury.
Stem Cells Derived From Human Umbilical Cord Blood for Treating Ischemic Brain Injury
1 Department of Neurosurgery, University of Minnesota, Minneapolis, Minneapolis, MN, USA
2 Deptarment of Veterinary Population Medicine, University of Minnesota, Minneapolis, MN, USA
3 Stem Cell Institute, University of Minnesota, Minneapolis, MN, USA
4 Saneron CCEL, Tampa Bay, FL, USA
Human umbilical cord blood (hUCB) is a rich source of stem cells that has been evaluated for the treatment of various disorders and diseases. We have isolated a CD34-negative population of stem cells from hUCB that exhibits neuroprotective properties in experimental animal models of ischemic brain injury. To translate these cells to the clinical setting, we have conducted investigational new drug (IND)-enabling studies in consultation with panel members at the US Food and Drug Administration. We have carried out toxicity studies, tumorigenicity studies, and efficacy studies on our nonhematopoietic umbilical cord blood stem cells (nh-UCBSCs). For the toxicity studies, adverse events such as seizures, respiratory distress, decreased weight, and death were monitored. To determine whether the administration of the cells led to systemic inflammation addition, serum cytokines were monitored. For the tumorigenicity studies, karyotyping of nh-UCBSCs was conducted at different passages to assess chromosomal translocation. Telomerase activity was evaluated in nh-UCBSCs. The presence of possible tumors in the various organs throughout the body of each animal was evaluated. Biodistribution of nh-UCBSCs after intravenous administration was assessed and resulted in trafficking to bone marrow and spleen. For the therapeutic efficacy studies, rats were subjected to middle cerebral artery occlusion and administered nh-UCBSC 2 days following ischemic brain injury. To determine functional improvement, animals treated with nh-UCBSCs and vehicle controls were tested on a battery of behavioral tests, which included modified neurological severity score and performances on staircase task and vertical pole to determine. The results of these IND-enabling studies for using nh-UCBSCs to treat ischemic stroke will be presented.
Highly Enriched Populations of V1 Interneurons From Mouse Embryonic Stem Cells
Department of Biomedical Engineering, The University of Texas at Austin, Austin, TX, USA
Neurological disease and injury models in vitro frequently use neuronal populations derived from embryonic stem cells that are isolated using fluorescent sorting techniques. However, there are several ventral progenitor pools that yield numerous cell populations within a single class of ventral interneuron (IN) often defined by the same marker; therefore, fluorescent sorting methods might inadvertently be involving numerous subtypes of a given ventral IN. The progenitor pool for the V1 IN class is known to produce Renshaw Cells (RCs) prior to producing V1-derived IaINs(V1-IaINs), both with distinct functional roles; however, with fluorescently sorted systems, both RCs and V1-IaINs could be isolated, as they both express En1 prior to specification. Here, we employed a genetic strategy to enrich for mouse embryonic stem cell–derived V1 INs by inserting the puromycin n-acetyltransferase into the En1 locus. As En1 is expressed prior to V1 IN subtype specification, antibiotic selection would permit temporal isolation of En1+ cells through the course of induction. We found that puromycin selection enriched the population for En1+ V1 INs; notably, they were found to be immature and exclusively expressing vesicular inhibitory ammino acid transporters. Furthermore, the selected V1 INs also exhibited functional activity when cocultured to maturity in a microelectrode array with V2a INs. These data demonstrate that the En1-PAC line provides a method to generate V1 INs for use in disease modeling, cell transplantation, or in vitro network analysis.
Plasticity and Functional Recovery Can Be Promoted by Oral Administration of 4-Methylumbelliferone in Chronic Model of Spinal Cord Injury in Rats
P. Jendelova1,2, K. Stepankova1,2, M. Chudickova1, S. Kubinova1, L. M. Urdzikova1,2, J. W. Fawcett1,3 and J. CF. Kwok1,4
1 Institute of Experimental Medicine, Czech Academy of Sciences, Prague, Czech Republic
2 Second Faculty of Medicine, Charles University, Prague, Czech Republic
3 John van Geest Centre for Brain Repair, Department of Clinical Neurosciences, University of Cambridge, Cambridge, UK
4 Faculty of Biological Sciences, University of Leeds, Leeds, UK
Perineuronal nets (PNNs) are specialized extracellular matrix (ECM) structures on the surface of neurons that act as key plasticity regulators to the central nervous system (CNS). Previous experiments demonstrated that the removal of PNNs is able to reactivate neuroplasticity for functional recovery after spinal cord injury (SCI). PNNs might also be removed by the medicinal drug 4-methylumbelliferone (4MU), which inhibits the synthesis of hyaluronan and chondroitin sulphate proteoglycans (CSPGs) and regulates PNN formation in the nervous system.
We investigated the pharmacological, biochemical, and biomechanical effects of long-term 4MU (2.5% w/w) treatment in noninjured rats. Results of hematological and serological parameters showed nonsignificant changes between groups. Tissue biochemistry showed significant downregulation of glycosaminoglycans (GAGs) in 4MU-treated group, suggesting attenuated PNNs, which was also confirmed by immunohistochemistry of Wisteria floribunda agglutinin (WFA) and hyaluronan binding protein (HABP).
We therefore tested 2.5% (w/w) 4MU to promote axonal plasticity and functional recovery in the rat chronic contusion model of SCI. Animals were fed daily with 4MU for 8 weeks, starting 6 weeks after SCI, followed by daily treadmill training. Our results showed that oral administration of 2.5% (w/w) 4MU reduced glial scar and promoted anatomical plasticity, but not functional recovery in the chronic stage of SCI.
We thus increased the dose of 4MU to 5% (w/w) to test in the chronic SCI paradigm. We observed the significant locomotor improvement of rats with SCI. Our results indicate that 5% (w/w) 4MU reopened a window of plasticity in chronic SCI, allowing rehabilitation to promote functional recovery.
This work was supported by Czech Science Foundation GACR 19-10365S, by Operational Programme Research, Development and Education in the framework of the project “Center of Reconstructive Neuroscience,” registration number CZ.02.1.01/0.0./0.0/15_003/0000419.
Safety and Feasibility of Regenerative Peripheral Nerve Tissue Delivery to the Nucleus Basalis of Meynert for Cognitive Decline in Patients With Parkinson’s Disease
J. E. Quintero1, L. M. Koehl2, A. Ledreux3, A. Gilmore3, J. T. Slevin2, A.-C. Granholm3, G. A. Gerhardt1,2, and C. G. van Horne1
1 Department of Neurosurgery, University of Kentucky College of Medicine, Lexington, KY, USA
2 Department of Neurology, University of Kentucky College of Medicine, Lexington, KY, USA
3 Department of Neurosurgery, University of Colorado Anschutz Campus, Aurora, CO, USA
Previously, we showed safety evidence when autologous peripheral nerve tissue (PNT) is deposited in the substantia nigra in conjunction with standard deep brain stimulation surgery (DBS). In the current pilot study, we examined cognitive and motor outcomes in a Parkinson’s disease (PD) group following bilateral globus pallidus interna (GPi) DBS plus nucleus basalis of Meynert (NBM) PNT. We hypothesized that regenerative PNT delivery to the NBM would support cholinergic cell survival, resulting in stable cognitive outcomes. Outcome data were analyzed for seven participants with bilateral GPi DBS plus unilateral NBM PNT. Post-DBS neurocognitive evaluations were on average 12.40 months post-surgery (SD = 3.05). Pre- to post-DBS cognitive scores were compared via paired-sample t tests. On-state and off-state Unified Parkinson’s Disease Rating Scale (UPDRS) scores were obtained 12 months after surgery. Two participants died of unrelated causes >12 months after grafting followed by pathological examination. No significant changes were noted in verbal memory, working memory, processing speed, or executive functions; phonemic fluency neared significance (FAS t = 2.295; P = 0.061). Twelve-month UPDRS Part III OFF-state scores indicated no change from enrollment (mean difference = 1.9; 95% confidence interval: −4.6 to +8.3); ON-state scores remained stable (M = 22.0; SD = 2.7). Pathology demonstrated cholinergic fibers extending toward the PNT. DBS plus NBM participants showed stable cognition. There was no change in motor function, while pathology supported accurate deposition of NBM grafts. Overall, the procedure appears safe with potential for graft-induced preservation of cholinergic neurons coupled with potential cognitive benefit.
This work was supported by gifts to the Brain Restoration Center; Ann Hanley Parkinson’s Research Fund; Pro’s Players Fore Parkinson’s; the UK College of Medicine BRAIN Alliance; and through NIA grant 1RF1AG070952 and NCATS, through NIH grants UL1TR000117 and UL1TR001998.