William Aisenberg, UPenn, Postdoc (PDS)

Krabbe disease, also named Globoid Cell Leukodystrophy (GLD) for its distinct lipid-laden macrophages, is a severe leukodystrophy caused by galactosylceramidase (GALC) mutations. Hematopoietic stem cell transplant (HSCT) ameliorates disease and is associated with central nervous system (CNS) engraftment of GALC+ donor macrophages. Yet, the role of macrophages in GLD pathophysiology and HSCT remains unclear. Single-cell sequencing revealed early interferon response signatures, followed by progressively severe macrophage dyshomeostasis, including a molecular signature of globoid cells. Genetic depletion and direct microglia replacement by CNS monocyte injection rapidly replaced >80% of endogenous microglia with healthy monocytes in the twitcher (GalcW355*) mouse, a faithful model of GLD. Perinatal microglia replacement completely normalized transcriptional signatures, rescued histopathology, and doubled average survival. Overall, we uncovered distinct forms of microglia dysfunction, and evidence that direct, CNS-limited microglia replacement improves a monogenic neurodegenerative disease, a promising therapeutic target.

Erin Anderson, UPenn, Grad Student (SGS)

Repetitive head trauma is a significant concern in contact sports, contributing to heterogeneous outcomes following concussion. A major challenge is identifying biomarkers that can predict cognitive outcomes while accounting for differences in impact history. Extracellular vesicles (EVs), which are passively released by all cells, reflect the molecular state of their parent cell. In a mouse model of repetitive head trauma, we previously found that rapid subconcussive impacts precondition the brain against concussion, protecting the brain and preventing memory deficits. We isolated neuron-derived EVs (NDEVs) from the bloodstream and identified miRNA biomarkers linked to injury and outcomes, and found those miRNAs were primarily involved in inflammation and angiogenesis. Histological analysis revealed that subconcussive impacts triggered an acutely reactive microglia morphology, while preconditioned concussions did not, suggesting that subconcussive impacts may serve as a desensitizing immune stimulus. We hypothesized that microglia play a key role in determining cognitive outcomes. Depleting microglia at the time of injury improved memory in unconditioned concussion but negated the cognitive benefits in preconditioned concussion, indicating that microglia are sensitive to impact history and influence cognitive outcomes. We further investigated how microglial depletion affected NDEV contents and discovered miRNAs dependent on both injury and microglial presence, suggesting that NDEVs can be used to monitor neuro-immune crosstalk. Future studies will employ snRNAseq to more precisely define the microglia/macrophage subpopulations responding to injury and their effects on neuronal molecular states.

Sudha Anilkumar, Nemours, Undergrad (UGT)

Occurring in 1.5 per 1000 live births, hypoxic ischemic encephalopathy (HIE) is a leading cause of death and long-term disability in children born at term. HIE is caused by insufficient oxygen delivery to the brain at birth. This injury triggers an inflammatory response mediated by microglia, the primary immune cells of the central nervous system. We developed a novel two-hit mouse model of HIE that exposes the fetus to maternal immune activation (MIA) via lipopolysaccharide in late gestation followed by hypoxia at postnatal (P) day 6 to investigate the pathophysiology of the interaction between MIA and subsequent hypoxic exposure. We hypothesized that microglia from the brains of mice exposed to this dual-hit HIE model will have amplified inflammatory responses that contribute to overall injury and worsen functional outcomes.

In this study, we characterized the transcriptional response in microglia exposed to HIE or control conditions. Bulk RNA sequencing of microglia isolated at P7 and P14 was performed on HIE and saline/normoxia controls. Gene set enrichment analysis revealed upregulation of TNF signaling via NF-kB in HIE microglia, particularly at the P7 timepoint, suggesting a pathway for inflammation. Single cell RNA sequencing was also conducted on brain cells from HIE and saline/normoxia controls during the early postnatal period (P8 & P10) to identify a microglial subpopulation unique to our HIE model. This analysis yielded increased expression of chemotaxis genes and lysosomal degradation pathways. These results will be utilized to further understand the molecular mechanisms of injury involved in neonatal HIE.

Sanjana Bandi (Undergrad) & Sophie Lake (Grad Student), Drexel, (JGS)

Astrocytes, a primary class of glial cells in the CNS, are essential for synapse formation and function, yet their morphological and transcriptional development is poorly understood. Their development coincides with neural circuit formation and refinement, and understanding astrocyte-circuit interactions can reveal how their dysfunction contributes to neurodevelopmental disorders. We aim to investigate astrocyte-like glia (ALG) morphology and transcription across Drosophila melanogaster development; Drosophila provides a valuable model with developmental stages comparable to vertebrates. We utilized multicolor stochastic labeling to mark ALG in the optic lobe, selected for its conserved visual system and well-characterized neuronal wiring. We labeled ALG at key timepoints: 72 hours after pupal formation (APF), marking peak synaptogenesis and the start of refinement, 84h APF, 96h APF, and in adults. Analysis via 3D reconstructions revealed that astrocytes in different optic lobe regions exhibit distinct structural characteristics. Our filament data shows a trend of increasing branching complexity from 72h APF to Adult, suggesting ongoing maturation across development. This progression can provide insights into how astrocyte morphology supports neural circuit maturation and function. In parallel, single-cell RNA sequencing identified 19 glial clusters in the developing Drosophila optic lobe, with clusters G49 and G51 showing strong astrocyte marker expression and low expression of other glial markers. From this data, we were able to analyze transcriptional patterns of ALG. Many genes show interesting patterns that coincide with key features in development, suggesting potential roles in ALG-synapse interactions. Overall, our work provides a foundation for further research into astrocyte roles in neural development.

Dimitris Boufidis, UPenn, Grad Student (JGS)

Astrocytes, the most abundant cells in the brain, play a crucial role in maintaining neural health and function, with newer evidence suggesting potential therapeutic value upon injury and neurological disorders [1]. Various nanomaterials, including 2D MXenes, are promising candidates for enabling targeted neural and astrocyte stimulation [2, 3]. While previous studies have indicated biocompatibility of 2D MXenes with neuronal cells [4-6], limited research exists on the interaction between MXenes and astrocytes. Our preliminary data suggest minimal cytotoxicity of Ti3C2Tx MXene flakes with astrocytes [7], but the fate of MXene flakes upon interfacing with astrocytes remains unclear. This study aims to investigate astrocyte viability, changes in metabolic activity and morphology, and cellular uptake of MXenes. Understanding MXene/astrocyte interactions could enable novel mechanisms for regulating astrocyte function as potential therapeutic targets for neurological diseases.

References:
1. Lee, H.-G., M.A. Wheeler, and F.J. Quintana, Function and therapeutic value of astrocytes in neurological diseases. Nature Reviews Drug Discovery, 2022. 21(5): p. 339-358.
2. Wang, Y., et al., Neural modulation with photothermally active nanomaterials. Nature Reviews Bioengineering, 2023. 1(3): p. 193-207.
3. Fabbri, R., et al., Graphene oxide electrodes enable electrical stimulation of distinct calcium signalling in brain astrocytes. Nature Nanotechnology, 2024.
4. Driscoll, N., et al., Two-Dimensional Ti3C2 MXene for High-Resolution Neural Interfaces. ACS Nano, 2018. 12(10): p. 10419-10429.
5. Wu, W., et al., Evaluating the Cytotoxicity of Ti3C2 MXene to Neural Stem Cells. Chemical Research in Toxicology, 2020. 33(12): p. 2953-2962.
6. Wang, Y., et al., Photothermal Excitation of Neurons Using MXene: Cellular Stress and Phototoxicity Evaluation. Advanced Healthcare Materials, 2023. p. 2302330.
7. Boufidis, D., D.K. Cullen, and F. Vitale, Biocompatibility and Distribution of 2D MΧene Flakes with Neural Cells In Vitro [Poster Abstract]. in 2023 Biomedical Engineering Society (BMES) Annual Meeting. 2023: Seattle, WA, United States.

Bridget Boyle, Jefferson, Grad Student (SGS)

Chordin-like 1 (Chrdl1) is an astrocytic protein that stabilizes GluA2-containing AMPA receptors (GluA2-AMPARs) at synapses and is expressed heterogeneously across different brain regions. GluA2-AMPARs play crucial roles in calcium homeostasis, which is vital for learning and memory processes. After excitotoxic events like ischemic stroke, GluA2-AMPARs decrease at hippocampal synapses, heightening neuronal vulnerability to excitotoxicity. We hypothesize that increasing astrocytic Chrdl1 will stabilize GluA2-AMPARs at hippocampal synapses, protecting neurons from excitotoxicity-induced cell death. To test this, we used in vitro (oxygen/glucose deprivation, OGD) and in vivo (photothrombosis, PT) models of ischemic stroke. Immunocytochemistry in primary hippocampal neuronal cultures was employed to assess surface and internalized GluA2 levels, comparing neurons treated with or without Chrdl1 prior to OGD and those under normoxic conditions. In vivo, we transduced Chrdl1 in astrocytes via AAV.PHP-eB, induced focal ischemic stroke using PT in the mouse hippocampus, and analyzed GluA2 levels post-PT by immunohistochemistry around the injury site. To evaluate cell death in vitro, we used a lactate dehydrogenase release assay for cytotoxicity levels and assessed cleaved-caspase3 in hippocampal neurons immediately after OGD and 72 hours later for delayed cell death. In vivo, we co-stained NeuN with TUNEL using immunohistochemistry. Our findings demonstrate that Chrdl1 treatment or overexpression before OGD or hippocampal PT prevents GluA2-AMPAR internalization, enhancing hippocampal neuron viability. This data highlights Chrdl1's role in stabilizing GluA2-AMPARs in vulnerable hippocampal neurons after excitotoxicity induced by ischemic conditions, suggesting that astrocytic Chrdl1 could serve as an innovative ‘resilience factor’, preventing neuronal death in early phases after ischemia.

Viet Bui, Temple, Grad Student (SGS)

Glial cells in the central nervous system (CNS) display characteristic reactive responses to inflammation induced by disease or injury. Understanding the nature and roles of disease-associated glial responses in specific disease contexts is critical, as they can significantly impact on disease progression and injury outcomes. While the reactive responses of OPCs, astrocytes, and microglia to CNS injury are well-documented, including enhanced cell proliferation, changes in cell morphology and/or gene expression, little is known about disease-associated oligodendroglial reactive responses. Oligodendrocytes (OLs) form myelin sheaths along the neuronal axons and do not divide or exhibit obvious morphological changes, except for ultrastructural changes in the myelin sheath during diseases.
In this study, we used RNA sequencing data specific to OLs in a mouse model of Alzheimer’s disease (AD) and further explored genes that are commonly upregulated in OLs under various disease conditions. Our analysis revealed that STAT3 signaling is activated in OLs located near amyloid plaques in APP/PS1 mice, in the spinal cord of SOD1 (G93A) mice, and in brain injury after glutamatergic excitotoxicity. We also observed significant increases in p-cJUN, a well-known neuronal injury marker, in OLs in SOD1 (G93A) mice, which is, however, specific to the disease. The p-STAT-3 immunoreactivities disappeared after crossing with OL-specific cKO mice for glycoprotein 130 (gp130), a subunit of the IL-6 receptor family. Our results reveal at least two molecular pathways defining OLs' reactive responses to ALS: GP130/STAT3 signaling and cJUN signaling. The significance of those transcription factor-mediated signaling and upstream signaling mechanisms is under investigation.

Sean Callahan, UPenn, Postdoc (PDS)

Microglia are resident immune cells of the central nervous system, but the role of metabolism-driven epigenetic modifications in their function is not fully understood. In the present study, we discovered acetyl-CoA synthetase 2 (ACSS2), which generates acetyl-CoA from acetate, dictates microglia development and regulates activation potential. Interestingly, ACSS2 engages in numerous neurological processes and diseases, including addiction, alcohol metabolism, Alzheimer’s disease, glioblastoma, and memory formation. While ACSS2 localizes in the cytoplasm or nucleus of cells, I observe nuclear ACSS2 in microglia. Yet, there is a significant decrease in microglia abundance in ACSS2KO mice compared to wildtype mice. However, the microglia present within the ACSS2KO brain possess an activated morphology. Through transcriptomics of wildtype and ACSS2KO microglia, I observe ACSS2 regulating expression of numerous immune regulation genes, whereby numerous anti-inflammatory polarization regulators are downregulated. As such, there is increased expression of inflammatory genes in ACSS2KO microglia. Fascinatingly, we see numerous inflammasome components increase in ACSS2KO microglia. Analogously, we observe increased apoptotic cells accumulate in the brains of ACSS2KO mice. Therefore, we hypothesize ACSS2 utilizes acetate to generate local pools of acetyl-CoA directly in the nucleus, allowing for histone acetylation, and resultant upregulation of key microglia maturation and anti-inflammatory genes to prevent pyroptotic cell death. Since ACSS2 expression is lost in aged microglia and ACSS2 overexpression ameliorates neurodegeneration, these findings could give us crucial insight into the role of ACSS2 in microglia physiology and disease.

Serena Chen, UPenn, Grad Student (JGS)

Acetyl-CoA Synthetase 2 (ACSS2) is an important metabolic-epigenetic enzyme that uses acetate to produce metabolite acetyl-CoA, which fuels histone acetylation in the brain, and ultimately contributes to long-term memory formation. Our group has shown that knocking out ACSS2 attenuates long-term learning and memory by disrupting histone acetylation. However, the specific cells and pathways that ACSS2 acts in remains unknown. Here, we investigate the function of ACSS2 in astrocytes, central regulators of metabolism and the most abundant glial cells in the brain. Astrocytes undergo many transcriptional changes during memory formation, but the mechanisms underlying this are poorly understood. Our preliminary data shows ACSS2 is highly expressed and nuclear localized in astrocytes, suggesting its potential role in regulating astrocyte gene expression. This is further supported by our finding that ACSS2 is required for the transcription of immediate early genes (IEGs) in astrocytes: IEG expression occurs after memory stimulation via fear conditioning in vivo, and knocking out ACSS2 attenuates this upregulation in astrocytes. These results are the first to suggest the impact of ACSS2 on gene expression in astrocytes. However, the function and mechanism of ACSS2 in astrocytes has not yet been fully determined. Given the importance of ACSS2 in memory-associated histone acetylation, we hypothesize astrocytes utilize a metabolic-epigenetic axis, driven by ACSS2, to establish gene expression programs that allow for proper astrocyte-neuron cooperation and long-term memory formation. This project is one of the first to investigate the direct connection and contribution of metabolism and epigenetics in astrocytes to learning and memory formation.

Grace Clark, Drexel, Grad Student (JGS)

Astrocytes are the most abundant glial cells in the CNS and serve many functions. Specifically, astrocytes are involved in supporting synapse function and synaptogenesis. Glutamatergic activity during synaptogenesis is linked to increased arborization of astrocytic processes. Throughout these processes, as well as in the body of the cell, mitochondria are present and provide energetic support for astrocytic activities. Mitochondrial networks, and therefore metabolism, in astrocytes are known to be regulated by PGC1a. Additionally, it is known that the transcription factor CREB is activated during early astrocyte maturation. It is unknown if CREB activation can directly drive PGC1a activation in astrocytes, though evidence suggests that it might. The aim of this project is to investigate the impact of CREB on mitochondrial networks and metabolism in astrocytes, using astrocyte-targeted AAVs for both constitutive active (Y134F) and dominant negative (S133A) CREB, then investigating their mitochondrial networks with IHC and their metabolic changes with seahorse assays.

Bailey Collins, Nemours, Grad Student (JGS)

Hypoxic ischemic encephalopathy (HIE) is a brain injury that occurs in 1-3 per 1000 live births and is one of the leading causes of long-term disability or death in infants. Maternal immune activation (MIA) has been implicated in the pathophysiology of HIE, with epidemiological studies indicating that infants exposed to systemic inflammation in utero are 8 times more likely to be diagnosed. We used a novel model of HIE, which includes maternal immune activation on gestational day 18 followed by deep hypoxia to better capitulate the injury as seen in human infants. Microglia and infiltrating regulatory T-cells are the primary responders to this injury, suggesting that modulating this response using cytokines can provide mechanisms for treatment. We hypothesize that the delivery of immunomodulatory biomaterials can modulate the brain’s immune response to induce more favorable outcomes. Interleukin 33 (IL-33) has been previously shown to have a neuroprotective effect after HIE when delivered systemically. We administered a sustained release IL-33 loaded alginate hydrogel to neonatal mice treated with HIE or control. The impact on the brain’s inflammatory response was measured using RT-qPCR. Microglia and T-cell response to treatment was determined by microglia density (IBA-1+ cells) and flow cytometry. Overall, we seek to develop a novel immunomodulatory delivery system that will reduce inflammation to improve outcomes in human infants.

Andrew Ebenezer, Nemours, Research Tech (UGT)

Hypoxic Ischemic Encephalopathy (HIE) is the leading cause of several severe neuronal disabilities in children born at term and affects up to 4 million children per year globally. Many risk factors contribute to HIE; however, this disease is ultimately due to insufficient oxygen delivery to the fetal brain during birth. Brain inflammation begins soon after injury and likely contributes to the extent of neurologic impairment after HIE. Prior research has demonstrated that microglia subpopulations arise during development and disease with unique functions related to the environmental niche in which they arise. We hypothesize that Pseudobulk analysis will reveal specific biomarkers that arise in microglia and macrophages after HIE that the regulation of gene function and pathways. To identify differentially expressed genes in the neonatal brain after HIE we performed Pseudobulk sequencing using single-cell RNAseq data. After which, a REVIGO analysis from the data sets were performed to identify pathways and functions affected.
This study will allow us to determine whether there are microglial populations that can be specifically targeted to influence the outcomes of neonatal HIE. Our Pseudobulk analysis identifies increased microglial motility gene expression and upregulation of epigenetic machinery and neurodevelopmental genes in macrophages following HIE.
Future directions will confirm the presence and localizations of transcriptional markers in different regions of the brain affected by HIE. This will be performed by Immunohistochemistry staining and utilizing certain biomarkers discovered after Pseudobulk analysis

Lindsay Festa, UPenn/CHOP, Postdoc (PDS)

The transient receptor potential mucolipin 1 (TRPML1) is the main Ca2+ exporter on the lysosome and interacts with the Rho GTPase, Rac1, a key regulator of the actin cytoskeleton via activation of numerous downstream mediators, including PAK. Intriguingly, inactivating mutations of TRPML1 result in the rare neurodevelopmental disorder mucolipidosis type IV, which is characterized by oligodendrocyte deficits and hypomyelination. These data implicate TRPML1 in playing a critical role in oligodendrocyte maturation and myelination, though the precise mechanisms underlying these observations are unknown. We hypothesized that activation of TRPML1 is required for the actin cytoskeleton changes that underlie process extension. Utilizing our well-established in vitro OPC differentiation and artificial nanofiber myelination paradigms, we found that stimulation of TRPML1 resulted in striking morphological alterations, including increased OL process number, process length, and process complexity. Our data suggests that this results from a shift in actin polymerization. Lastly, we demonstrated that stimulation of TRPML1 rapidly activates Rac1 and its downstream effectors PAK1, LIMK1, and cofilin, revealing a potential mechanism by which TRPML1 controls the OL actin cytoskeleton; indeed, our data demonstrates that blockade of Rac1 activation prevents TRPML1-induced actin alterations and process outgrowth. Ongoing work is currently investigating the relationship between lysosomal Ca2+ efflux and actin cytoskeleton dynamics, as well as the role of TRPML1 during remyelination. Taken together, our work highlights a previously unknown function of TRPML1 in modulating OL function and has implications not only for homeostatic regulation but also disease states where lysosomal function is known to be dysregulated.

Madison Fuller, UPenn, Research Tech (UGT)

Astrocytes play a critical role in the formation and maintenance of neuronal synapses. Astrocytes form a tripartite synapse with neurons via protrusions known as peripheral astrocytic processes (PAPs). PAPs are actin enriched and are highly plastic to allow for extension and retraction around neuronal synapses. Ezrin-radixin-moesin (ERM) are actin associated proteins that facilitate the plasticity of PAPs. Phosphorylated ERM links actin to the plasma membrane, promoting the outgrowth of astrocytic processes. The actin cytoskeleton and PAP proteome of astrocytes is disrupted in the lysosomal storage disorder Mucolipidosis Type IV (MLIV). MLIV is caused by loss of function mutations in the lysosomal cation channel TRPML1. TRPML1 is a major source of calcium efflux from the lysosome. TRPML1’s calcium activity has been shown to regulate the organization of the actin cytoskeleton in cancer cells and immune cells . Thus, we sought out to investigate if TRPML1 regulates PAP plasticity in astrocytes. With our neuron-astrocyte coculture system, we are able to form stellated astrocytic branches to view PAP-like structures. When activating TRPML1 pharmacologically, we observed rapid calcium efflux from the lysosome and extension of PAPs by live-cell imaging. Surprisingly, this activation corresponded with dephosphorylation of ERM shown by immunofluorescence and immunoblotting. Conversely, when TRPML1 was knocked down, we observed an increase in phosphorylated ERM. Together, these data suggest that lysosomes may influence PAP activity through TRPML1 mediated calcium release. Current work aims to connect ezrin dephosphorylation by TRPML1 to PAP extension, as well as identify potential kinases and phosphatases involved in this pathway.

Taylor Gardner, Drexel, Undergrad (UGT)

Stroke is the third leading cause of death and disability worldwide. Ischemic stroke occurs due to the blockage or narrowing of the arteries that supply oxygen and glucose-rich blood to the brain. The only approved treatment for ischemic stroke has a narrow therapeutic window, thus, there is a critical need for novel drug targets. Stroke research has heavily focused on targeting neuronal cell death mechanisms, but recently, non-neuronal cells have been
demonstrated to have a significant role in stroke pathobiology. One of these non-neuronal cells are astrocytes; the major glial cells in the CNS. After ischemia, astrocytes become reactive,
which indicates changes in morphology, signaling, and metabolism. Additionally, calcium levels within the cell are elevated and prolonged. These changes in calcium signaling may increase local brain inflammation and inhibit functional brain recovery. A gene of particular interest to the Jackson Lab is camp-response-element-binding-protein (CREB). CREB is implicated in
regulatory functions in astrocytes, but its role post-stroke is unknown. In neurons, the CAMKK/CAMKIV signaling pathway activates CREB. Preliminary analyses suggest a time-dependent activation of CREB and downstream signaling pathways. We aim to explore the mechanism associated with CREB activation in astrocytes after ischemic insult by pharmacologically targeting the CAMKK signaling pathway, as well as determine if CREB
activation is calcium dependent. Additionally, we will investigate the downstream effects of CREB activation after stroke in-vivo utilizing AAVs to manipulate its expression. This study will provide a greater understanding of astrocytic response to ischemia and potential avenues for future therapeutic development.

Simran Gill, Drexel, Grad Student (SGS)

Glutamate transporters play a pivotal role in maintaining glutamate homeostasis throughout the central nervous system (CNS), working to clear glutamate from the synaptic space following neurotransmission. These transporters are implicated in a wide variety of neurological disorders that are associated with glutamatergic dysregulation and excitotoxicity, such as ischemic stroke. This has led to an increasing interest in the targeting of these transporters as a therapeutic strategy to mitigate ischemic injury. However, the regulatory response of these transporters following ischemic insult is not well defined. In this study, we report a prolonged reduction in glutamate clearance following oxygen glucose deprivation (OGD) that is dependent on the severity of ischemic insult. Additionally, we observed a marked reduction in surface expression levels of GLT-1, a glutamate transporter subtype responsible for the majority of glutamate clearance in the CNS. However, we did not observe changes in total cellular levels of the GLT-1 protein, suggesting aberrations in protein trafficking following ischemic insult. Administration of ceftriaxone (CEF), a beta-lactam antibiotic previously found to increase GLT-1 expression, was only able to increase uptake and surface expression of the transporter in baseline conditions, with no effect following OGD. We therefore sought to determine possible regulatory mechanisms that could lead to a reduction in GLT-1 surface expression, finding an increase in the sumoylation of GLT-1 following severe OGD insult, possibly pointing to a mechanism leading GLT-1 internalization after ischemic injury. Findings from these studies suggest that intricate mechanisms regulating GLT-1 expression and activity after OGD may hinder ischemic recovery.

Michael Grovola, Upenn, Postdoc (PDS)

Traumatic brain injury (TBI) is a public health concern with an estimated 42 million cases globally every year. The majority of TBIs are mild TBI, also known as concussion, and result from the application of mechanical forces on the head. Most patients make a complete recovery and mortality is rare, therefore studies investigating cellular changes after mild TBI in a clinical setting are limited. To address this constraint, our group utilizes a pig model of closed-head rotational acceleration-induced TBI, which recreates the biomechanical loading parameters associated with concussion on a large gyrencephalic brain similar to humans. To further characterize single mild TBI pathology, we evaluated axonal degeneration, blood-brain barrier disruptions, astrogliosis, and microglial reactivity out to 1 year post-injury (YPI). Immunohistochemical staining revealed the presence of a hyper-ramified microglial phenotype – more branches, junctions, endpoints, and longer process lengths – out to 1 YPI in distinct gray and white matter structures, and in regions with axonal pathology. Interestingly, we did not find neuronal loss or astroglial reactivity paired with these chronic microglia changes. Additionally, we utilized transcriptomic assays to evaluate an array of TBI-induced neurodegenerative analytes. Differential expression analysis revealed significant genes at 3 days post injury (DPI) including IRF8, a regulator of microglia activity, but no significant genes at 30 DPI or 1 YPI compared to sham. This study highlights potential therapeutic targets following mild TBI. Further understanding of the brain’s inflammatory activity after mild TBI will hopefully provide understanding of pathophysiology that translates to clinical treatment for TBI.

Michael Grovola, Upenn, Postdoc (NA) *Presented by John O'Donnell

Neural precursor cells (NPCs) are generated in the subventricular zone (SVZ) and travel through the rostral migratory stream (RMS) to replace interneurons in the olfactory bulb of most adult mammals. Following brain injury, SVZ-derived NPCs divert from the RMS and migrate toward injured brain regions but arrive in numbers too low to promote functional recovery. Our lab has biofabricated a tissue-engineered rostral migratory stream (TE-RMS) that replicates the structural and functional features of the glial tube of the endogenous RMS. This regenerative medicine strategy is designed to facilitate stable and sustained NPC delivery into neuron-deficient brain regions following injury or neurodegeneration and serves as an in vitro tool to investigate the mechanisms of neuronal migration and cell-cell communication. We have previously shown that TE-RMS astrocytes exhibit elongated nuclei and longitudinally aligned cytoarchitecture, mimicking the unique morphology of endogenous RMS astrocytes. Here, we perform RNAseq on TE-RMS constructs to identify gene expression changes that may underlie these morphological changes and compare these expression levels to planar astrocytes in vitro. Differential expression analysis revealed 4008 significant genes in TE-RMS constructs, with 2076 downregulated genes and 1932 upregulated genes compared to planar astrocytes. Moreover, there were 256 downregulated genes and 91 upregulated genes that had greater than a 3-fold change. Finally, we conducted gene set analysis on cellular component sets related to cytoskeleton and nuclear structure, revealing significant enrichment of cytoskeletal components. The TE-RMS offers a unique opportunity to study the interplay between cytoskeleton, nuclear shape, and gene expression regulation.

David Harary, Jefferson, Grad student (JGS)

Hypoxic Ischemic Encephalopathy (HIE) affects 1-3 per 1000 births in developed countries, with a 40% mortality rate. Despite therapeutic hypothermia, 40% of affected infants suffer permanent disabilities. Notably, 50% of neonates with HIE are born to mothers with chorioamnionitis or infection, significantly increasing the risk of HIE and associated neurological conditions such as cerebral palsy, autism, and schizophrenia. The mechanisms underlying these associations remain unclear, though neuroinflammatory responses involving glial cells, cytokines, and chemokines are implicated.

This study aimed to explore the associations of parent factors, particularly maternal infection, with neurologic and developmental outcomes in an HIE cohort from Nemours. Medical records of 325 patients diagnosed with HIE (ICD-10 codes) from January 2019 to May 2024 were analyzed. Inclusion criteria included patients with EEGs performed within the first 10 days of life. Diagnostic codes were available for 177 patients and provided information on HIE severity and all documented diagnoses.

Results indicated a significant increase in the prevalence of seizures and sepsis in patients with severe HIE. These findings support the role of neuroinflammatory processes in the severity and outcome of HIE, though the specific involvement of glial cells remains to be clarified. Limitations include a loss of chronology of medical diagnoses in analysis.

Future work aims to correlate maternal health conditions with neonatal outcomes and collect cord blood samples to study immune cell development. These efforts will help identify biomarkers and underlying mechanisms of HIE, potentially guiding targeted therapies to mitigate the impact of HIE-related disabilities.

Zhongqi Hou, CHOP, Research Tech (UGT)

Hypomyelination with Atrophy of Basal Ganglia and Cerebellum (H-ABC) is a rare leukodystrophy with heterozygous p.Asp249Asn (D249N) [P.745G>A] variant in the tubulin beta class IVA (TUBB4A). This leukodystrophy is defined by MRI with characteristic hypomyelination and atrophy in basal ganglia. The affected cell types include oligodendrocytes (OLs), cerebellar granule neurons (CGNs), and medium spiny neurons (MSNs). However, the mechanisms through which TUBB4A mutations impact these cell types are unknown, and there is no cure for this debilitating disorder. Herein, we generated TUBB4AD249N human induced pluripotent stem cells (hiPSCs) from H-ABC-affected individuals to model and understand the disease pathophysiology. We differentiated the TUBB4AD249N hiPSCs into MSN- and OL-like cells. TUBB4AD249N MSNs exhibited decreased maturation and increased apoptosis compared to wild-type control neurons. TUBB4AD249N OLs fail to mature and show impaired complexity and myelination. CRISPR-mediated knockout (KO) of TUBB4A in TUBB4AD249N hiPSC-derived neurons and OLs rescued cellular deficits without affecting wild-type TUBB4AKO hiPSC neurons and OLs. This suggests that TUBB4A suppression could be a viable treatment approach for H-ABC. We also screened antisense oligonucleotides (ASO) targeted against TUBB4A in hiPSC-derived neurons and OLs, finding that the selected ASO candidate effectively reduced TUBB4A expression with no toxicity. ASO treatment in TUBB4AD249N OLs partially improved OL maturation and complexity; however, it failed to improve the cellular phenotypes in MSNs. Ongoing studies are focused on treatment timing and developing a potent ASO candidate. Overall, this study provides proof-of-concept for ASO treatment and advances pre-clinical therapeutics for H-ABC disease.

Marisa Jeffries, CHOP, Postdoc (PDS)

Human immunodeficiency virus (HIV)-associated neurocognitive disorders (HAND) affect 30-50% of people with HIV (PWH) and are associated with white matter pathologies. New HIV infection rates are climbing among adolescents, who may be vulnerable to white matter disruption during the critical window of adolescent myelination. The HIV-1 transgenic (Tg) rat model is a noninfectious model of HIV neuropathology which exhibits an altered transcriptome suggestive of deficient myelination, but no studies have directly examined oligodendroglial myelination and potential mechanisms of white matter disruption in this model. Transcriptome analyses in PWH indicate reduced lipid metabolism and myelin proteins; disrupted brain lipid metabolism results in myelin abnormalities and is predictive of cognitive decline in HIV. We hypothesized that glial lipid metabolism is disrupted by HIV-1 and impairs adolescent myelination. Western blot analyses of myelin protein expression in control and HIV-1 Tg rat microdissected caudate, cortex, hippocampus, and callosum indicate no significant changes in myelin basic protein (MBP), 2’,3’-cyclic-nucleotide 3’-phosphodiesterase (CNPase), myelin oligodendrocyte glycoprotein (MOG), or myelin associated glycoprotein (MAG). However, there are brain-region specific changes in expression of fatty acid synthase (FASN) and acetyl-CoA carboxylase (ACC) at 3 and 9 weeks. To determine whether myelin lipid composition is affected, we performed assays for total cholesterol and free fatty acids on purified whole brain myelin and discovered significantly increased total cholesterol at 9 weeks. If changes in myelin lipids impair adolescent myelination, lipid metabolism may be a promising therapeutic target to improve white matter integrity and ameliorate associated HAND pathology in people living with HIV.

Geena John, Drexel, Grad Student (SGS)

Stroke is the third leading cause of death and disability worldwide. In response to ischemic stroke, astrocytes, which are considered homeostatic regulators of the CNS, undergo prolonged and enhanced Ca2+ elevations, associated with reactivity. Although many studies have explored the mechanisms by which Ca2+ levels increase after stroke, there is a significant knowledge gap in Ca2+ extrusion mechanisms from astrocyte processes. Here, we used organotypic hippocampal and primary astrocyte cultures to investigate the kinetics of Ca2+ clearance in astrocyte processes, to evaluate expression levels of identified Ca2+ clearance proteins after oxygen-glucose deprivation (OGD; an in-vitro stroke model), and to explore changes in downstream gene expression. Using live cell calcium imaging, we identified plasma membrane calcium ATPase (PMCA) as the primary mediator of Ca2+ extrusion. PMCA inhibition resulted in significant changes in the frequency of Ca2+ events, spatial distribution, duration, and clearance rate, as compared to other probed clearance related pathways. After OGD, we observed a loss of PMCA isoforms, specifically PMCA2. We re-established baseline astrocytic Ca2+clearance rates after OGD through PMCA2 overexpression; thus, uncovering an avenue to mitigate aberrant Ca2+signaling in astrocytes after ischemia. Using immunoblots, we discovered CREB activation in astrocytes 1 hour after OGD. By reducing Ca2+ signals either through PMCA2 overexpression or use of a calcium chelator (BAPTA-AM), CREB activation was suppressed, suggesting a direct link between Ca2+ signaling and early gene activation after stroke. Our data demonstrates OGD impairs Ca2+ clearance in astrocytes through the loss of PMCA2, resulting in downstream expression changes of CREB.

Kaleb Kelley, CHOP, Research Tech (UGT)

Pelizaeus-Merzbacher Disease (PMD) is a rare X-linked hypomyelinating leukodystrophy associated with severe neurologic impairment. It is caused by pathogenic variants in the PLP1 gene leading to oligodendrocyte dysfunction. PMD-related impact of Quality-of-Life (QoL) and health-related priorities remain poorly understood. In this study, we aim to characterize PMD-specific QoL impacts, and QoL priorities using Patient Reported Outcomes (PRO) for clinical trial readiness.
Caregivers of subjects with genetically confirmed PMD were administered the Vineland Adaptive Behavior Scale (VABS-3). In addition, the following Health-Related Quality of Life (HRQOL)-parent version questionnaires were completed electronically: Caregiver Priorities & Child health Index of Life with Disabilities (CPCHILD), the Pediatric Quality of Life InventoryTM-Generic Core (PedsQL-GC) and Family Impact (PedsQL-FI) Module, and the Traumatic Brain Injury -Care Quality of Life (TBI-CareQoL). Data was summarized using medians, interquartile ranges [IQR], and mixed effects analysis with Bonferroni correction.
Caregivers [N=47, median(IQR) age=7.25(18.3) years] reported a severe impairment of motor skills compared to social abilities on the VABS-3; (p<0.0001) and Motor Skills Domain vs. Communication Domain (p=0.0143). Also, daily living skills were severely impaired compared to social abilities (Daily Living Skills Domain versus Socialization Domain, p<0.0001). On HRQOL questionnaires caregivers reported severe impairment of motor abilities and daily living skills with significant dependency for most motor tasks assessed.
ObsRO permit improved understanding of disease-related impact on QoL and identification of caregiver priorities. We characterized the impact on QoL of impairments in motor and daily living skills associated with PMD, as well as the priorities of affected individuals and their caregivers.

Elizabeth Krizman, UPenn, Center/Lab Manager (PDS)

Brain injury can result in long-term neuronal loss that is exacerbated by the limited regenerative capacity of the central nervous system. The rostral migratory stream (RMS) facilitates neuroblast migration from the subventricular zone to the olfactory bulb throughout adulthood. Brain lesions attract neuroblast migration out of the RMS, but resultant regeneration is insufficient without intervention. Our lab has biofabricated a “living scaffold” that is implanted to enhance and redirect endogenous neuroblast migration from the subventricular zone to neuron-deficient brain regions. This approach utilizes the first implantable, biomimetic tissue-engineered RMS (TE-RMS), designed to leverage the brain’s natural mechanism for sustained neuronal replacement by replicating the native RMS to direct neuroblasts to distal sites of injury. Our previous work has characterized the structure of the TE-RMS fabricated from primary rat postnatal cortical astrocytes (rat TE-RMS). We have recently demonstrated that astrocyte-like cells can be non-genetically derived from adult human gingiva mesenchymal stem cells and used for TE-RMS fabrication (human TE-RMS), and that key proteins (ezrin, robo2, and glial fibrillary acidic protein) enriched in the endogenous adult rat RMS are also enriched in the rat and human TE-RMS, but not in planar astrocyte sister cultures. Additionally, we demonstrate that astrocytes in the endogenous RMS possess an elongated nuclear shape and distinct cytoskeletal arrangement compared to surrounding protoplasmic astrocytes, and that astrocytes in the rat TE-RMS mimic this distinct astrocytic nuclear shape and intermediate filament arrangement whereas planar astrocyte sister cultures do not. Furthermore, the human TE-RMS facilitates directed migration of immature rat cortical neurons in vitro at an average migration rate of at least 56 microns/hour which is comparable to neuroblast migration through the endogenous RMS, whereas acellular collagen and acellular collagen and laminin control columns do not direct immature neuronal migration in vitro. Finally, human TE-RMSs implanted in athymic rat brains redirect migration of neuroblasts out of the endogenous RMS. The rat and human TE-RMS mimic the basic structure and function of the endogenous rat RMS. By emulating the brain’s most efficient means for directing neuroblast migration, the TE-RMS offers a promising new regenerative medicine therapy for endogenous neuronal replacement following brain injury.

Elise Lemanski, Nemours, Grad Student (SGS)

Hypoxic ischemic encephalopathy (HIE) is one of the most serious causes of neurological deficits in children born at term, and is the leading cause of cerebral palsy. Few animal models of HIE incorporate maternal immune activation (MIA) despite the significant risk MIA poses to HIE incidence and diagnosis. Our model pairs maternal immune activation via the administration of lipopolysaccharide (LPS) on gestational day 18 with a progressive hypoxia to 0% oxygen for 8 minutes on postnatal day 6 (P6). This two-hit model produces a significant delay in the neonatal acquisition of motor, reflexive, and sensory behaviors. In adulthood, HIE animals exhibited long-term motor deficits including reduced forelimb strength and gait disturbances. Bulk RNA-seq on microglia one day following hypoxia showed an upregulation of pro-inflammatory, proliferation-related, and apoptotic gene sets. At this same timepoint, anatomical MRI showed that HIE animals trended towards a decrease in overall brain volume. Single cell RNAseq revealed two microglia subclusters of interest following HIE. Pseudobulk analysis revealed increased microglia motility gene expression and upregulation of epigenetic machinery and neurodevelopmental genes in macrophages following HIE. This model results in a milder phenotype compared to established HIE models; however, HIE is a clinically heterogeneous injury resulting in a variety of outcomes in humans. These findings support our two-hit model as a valid model of neonatal HIE in which to explore interactive effects between MIA and hypoxia.

Kathryn Markey, Drexel, Grad Student (SGS)

Establishment of proper cell numbers is vital for the proper development of the central nervous system(CNS). Here we focus on astrocytes, a major class of glial cells in the CNS, which have roles in synapse formation and function. How the brain achieves the correct number of astrocytes is poorly understood. Elimination of neurons and oligodendrocytes is well established and accomplished by apoptosis and non-apoptotic microglial engulfment during their respective developmental period. Whether astrocytes experience elimination to sculpt the adult population, and mechanism by which this is achieved, is unknown. In this study, we focus on cortical astrocytes during postnatal development, during this time astrocytes are generated and undergo maturation. We investigated whether astrocytes are eliminated during the early postnatal period. Immunostaining with the microglia marker Iba1 and astrocyte marker Sox9 revealed that microglia engulf astrocytes. We examined tissues between postnatal day (P)3 and P10 and found that microglial engulfment is most pronounced at P7. Further evaluation with the apoptosis marker, cleaved caspase 3(CC3), indicates that astrocytes at P7 are not undergoing apoptosis, suggesting that engulfed astrocytes are otherwise viable cells. Colocalization analysis with Iba1, Sox9 and proliferation marker, Ki67, demonstrated that engulfed astrocytes are postmitotic. We next investigated whether astrocytes undergo elimination during late postnatal development. Stereological analysis shows that astrocyte numbers decline between P14 to P28 and stabilize into adulthood. Colocalization analysis with Sox9 and CC3 suggests that at P28 astrocytes are undergoing apoptosis. Taken together, my data suggest astrocytes undergo stage-specific elimination, achieved through two different mechanisms.

Silva Markovic-Plese, Jefferson, PI (NA)

Background:
IL-11+ cells were enriched in the CSF and in active brain lesions in RRMS patients. Using scRNAseq, we characterized the IL-11-induced transcriptome changes
in immune cells from MS patients. Following reports on the NLRP3 expression in neurons and caspase 1-induced pyroptotic neuronal cell death, which is reversed by NLRP3 gene KO or silencing, and by its inhibitor MCC950, we examined the
molecular mechanisms of neurotoxicity of the MS CSF, and of IL-11 in human inducible pluripotent stem cell (iPS)-derived neurons.
Objective:
To study the role of IL-11-mediated NLRP3 inflammasome activation in neuronal cell death in patients with relapsing remitting multiple sclerosis (RRMS).



Design/Methods:
iPS-derived human neuronal cultures were used to detect neurotoxic effect of MS CSF and IL-11. In order to test if the effect was mediated via NLRP3 inflammasome activation, the the cultures were treated with NLRP3 inflammasome inhibitor MCC950.

Results:
We report that IL-11 induced neuronal death in dose- dependent manner in human cortical neuronal
cultures. The effect is inhibited by NLRP3 inhibitor MCC950, supporting IL-11-NLRP3 inflammasome-induced neuronal death. CSF samples from MS patients induced a similar degree of neuronal cell death and the studies of inflammasome-dependant signaling pathways involved in neuronal death revealed IL-11R, NLPR3 and caspase-1 protein expression in iPNSC human neurons.

Conclusions:
IL-11 induces NLRP3 inflammasome priming, whose therapeutic targeting may prevent neuronal loss in RRMS.

Ivette Martorell Serra, Jefferson, Grad Student (SGS)

ALS and FTD, two neurodegenerative disorders on a shared continuum, display progressive pathology that spreads throughout the CNS and is potentially associated with gliosis, indicating that neuroinflammation is a contributing factor. A hexanucleotide repeat expansion in the C9ORF72 gene, which causes a reduction of C9orf72 protein (C9) levels, is the most common genetic cause of ALS/FTD. Impaired C9 function induces altered trans-Golgi vesicle trafficking and aberrant extracellular vesicles (EVs) secretion. EVs, bilayer membrane vesicles released by cells, play critical roles in intercellular communication and inflammation. Our hypothesis posits that C9-haploinsufficiency affects this pathway, leading to abnormal EV production and composition, potentially contributing to neuroinflammation. To test this, we generated cerebral organoids (COs) from iPSC-derived controls (C9+/+), C9-linked ALS/FTD patients (C9+/-), and engineered C9 KOs (C9-/-). We observed an increased EVs production at 6 months in vitro in C9+/- and C9-/- COs compared to control. This increase was not caused by higher basal neural activity, as determined by calcium imaging experiments, nor by a difference in cell composition, as indicated by protein measurement of SOX9 (astrocytes) and CTIP2 (mature neurons) via WB and immunofluorescence. Our immediate objective is to study EV composition/origin in our model using RNA-seq, lipidomics, and proteomics analysis. We will also assess the capacity of C9+/- and C9-/- COs-derived EVs to induce an inflammatory response in cultured microglia. Overall, this will help us investigate the potential involvement of EVs in the initiation and progression of neuroinflammation. Our ultimate goal is to gain a deeper understanding of the role played by C9 in disease progression.

Jordan McKinney, UPenn/CHOP, Grad Student (JGS)

Aicardi-Goutières Syndrome (AGS) is a severe, progressive pediatric neurodegenerative disorder caused by pathogenic mutations in genes that regulate antiviral and innate immune responses. Among these mutations, over-expression of IFIH1 and under-expression of ADAR lead to neurological symptoms, progressive encephalopathy, and systemic interferonopathy due to errors in sensing endogenous double-stranded RNA. Current treatments for AGS primarily focus on reducing the inflammatory response through the systemic use of JAK-STAT inhibitors; however, these approaches do not address the underlying mechanisms of the disease and are associated with significant side effects. There is a pressing need to develop targeted therapies that can more precisely modulate these pathological processes at the cellular level. In this context, we hypothesized that adeno-associated viruses (AAVs) engineered with cell-type specific promoters could enable targeted gene therapy in the central nervous system, thereby reducing disease pathology by selectively infecting astrocytes and endothelial cells, which are critically involved in AGS pathology. These AAVs were designed to increase ADAR expression through the delivery of the ADAR gene and to suppress MDA5 (encoded by IFIH1) expression using short hairpin RNA (shRNA) within a miR30 cassette. This dual strategy aims to correct the underlying genetic defects and modulate the immune response, thereby reducing systemic inflammation and preventing the progression of neurodegeneration in AGS. By precisely targeting the molecular drivers of the disease within specific cellular compartments, this approach holds the potential to alleviate the debilitating symptoms of AGS while minimizing the adverse effects associated with current systemic therapies.

Prabhat Napit, CHOP, Postdoc (PDS)

Hypomyelination and Atrophy of the Basal Ganglia and Cerebellum (H-ABC) is a rare leukodystrophy resulting in progressive loss of neurological skills in childhood. It is caused by heterozygous mutations in TUBB4A, which encodes tubulin beta class IVA (TUBB4A). Representing 10% of leukodystrophies, the p.Asp249Asn (D249N), a recurring variant in TUBB4A, results in cell-autonomous deficits in oligodendrocytes (OLs), medium spiny neurons, and cerebellar granule neurons. TUBB4A, a subtype of -tubulins, heterodimerizes with -tubulin to form microtubules (MT). MTs are essential for neuronal and OL structure, function, and cellular transport of critical proteins. In this study, we sought to determine how Tubb4a mutation causes OL deficits. Oligodendrocyte precursor cells (OPCs) generated from the Tubb4aD249N mice failed to differentiate into OLs. The few differentiated mutant OLs exhibited impaired morphology and reduced myelin proteins (proteolipid (PLP) and myelin basic protein) as compared to the controls. Furthermore, isolated Tubb4a mutant OLs demonstrated reduced lysosomal puncta and aberrant mitochondrial puncta on OPCs and PLP+ OLs. In vivo electron microscopy in these mice supported the disrupted mitochondrial morphology and abnormal accumulation of lysosomes in axons and OLs. Ongoing studies are focused on understanding if Tubb4a mutation impacts the transport of these key organelles. This study highlights how the Tubb4a variant alters the fundamental cellular functions.

Carleigh O'Brien, Penn, Postdoc (PDS)

Originating in the embryonic yolk sac, microglia are long-lived brain resident macrophages capable of responding rapidly to brain injury. Circulating monocyte-derived macrophages are typically short-lived cells that originate from hematopoietic stem cells (HSCs) in the bone marrow. Despite differing origins, HSC-derived macrophages are programmed by the brain environment to become “microglia-like cells” (MLCs), though they maintain some transcriptional hallmarks of their HSC origin. The goal of this project is to explore how macrophage origin impacts microglial and MLC responses to excitotoxic injury in adulthood. We developed an in vivo neonatal transplantation model to deplete and replace endogenous microglia with either MLCs or yolk sac-derived microglia. Using the glutamate receptor agonist kainic acid (KA) to induce seizures following engraftment of donor macrophages, we found that mice with transplanted microglia displayed more severe seizure behaviors, hyper-activated microglial morphology, increased GFAP expression, and increased neurodegeneration compared to both mice with unmanipulated endogenous microglia or transplanted MLCs. Interestingly, bulk RNA-sequencing studies revealed approximately 500 differentially expressed genes between transplanted microglia and transplanted MLC following seizures, but revealed very few differentially expressed genes between transplanted microglia and unmanipulated endogenous microglia. Ongoing studies aim to characterize the differentially regulated pathways in transplanted microglia, endogenous microglia, and MLCs in response to seizure activity in the brain, as well as uncover the effects of neonatal microglia depletion and repopulation on the developing brain environment. This work will provide a better understanding of how origin, transplantation, and repopulation influence brain development and macrophage responses to excitotoxic injury.

John O'Donnell, Penn, PI (NA)

Traumatic brain injury (TBI) precipitates a cascade of pathophysiological events that significantly influence patient prognoses and survival outcomes. The primary focus of acute neurocritical care is evaluating and minimizing the risk of cerebral ischemia caused by edema and intracranial hypertension (ICH). Studies have shown that ATP-binding cassette protein receptor, sulfonylurea receptor-1 (SUR1), upregulates in response to TBI, initiating a rapid increase in oncotic pressure through its interaction with aquaporin-4 (AQP4) in astrocytic end feet, resulting in cytotoxic edema. Using the porcine closed-head rotational acceleration TBI model, we hypothesized that increases in ICH and vascular SUR1/AQP4 expression will be detected post-TBI. Following multimodal neuromonitoring in neurocritical care, immunohistochemistry was conducted to assess changes in GFAP, SUR1, and AQP4. TBI resulted in ICH and increases in perivascular SUR1 and AQP4 throughout the brain, and we also observed SUR1 signal in cortical grey that was lost after injury. Identifying a highly translational animal model for human TBI is crucial for advancing therapeutic intervention and clinical outcomes. Our study highlights the relevance of using a large animal closed-head rotational acceleration TBI model that recreates the mechanisms and manifestations (e.g. ICH, loss of consciousness) of human TBI, while integrating comprehensive neuromonitoring and histology. Our approach provides unique insights that are not possible in small animal TBI models for studying the complex mechanisms involved in humans, narrowing the translational gap between research and clinical application.

Oluwatofunmi Oteju, Drexel, Grad student (SGS)

Stimulant use is highly prevalent in HIV-infected populations, but the mechanism(s) by which stimulants impact HIV infection are undefined. Our data show that CNS dopamine, which is released following stimulant use, increases HIV replication in CNS myeloid cells. However, it is not clear if stimulants act only via changes in dopamine or if they have other discrete effects on HIV replication. To test this, we inoculated human induced pluripotent stem cell-derived microglia (iMg) with HIV in the presence of dopamine (DA) or cocaine (Coc). High-content image analysis and p24 AlphaLISA were used to define infection dynamics, evaluating % infected cells and level of secreted p24 protein. DA and Coc increased p24 secretion and % infected cells in iMg, and inhibition of dopaminergic signaling did not attenuate the impact of Coc. However, inhibition of sigma-1 (S1R), a chaperone protein targeted by Coc, did block the effect of Coc, and a S1R agonist independently increased p24 secretion. This indicates that Coc acts, at least in part, via S1R and not through dopaminergic mechanisms. Immunofluorescence staining showed increased S1R in p24 negative cells in HIV-infected, Coc-treated cultures, suggesting that Coc-induced changes in sigma-1 prior to infection could be increasing susceptibility to HIV. To identify mediators downstream of S1R driving cocaine-mediated changes in HIV-infection, we examined transcriptomic (single-cell RNAseq) and inflammatory (multiplex cytokine panel) changes in HIV-infected iMg +/- Coc. Future studies involve using mixed-culture systems (syngeneic iMg with iPSC-derived neurons/astrocytes) to examine changes in inflammation, neuron/glial health, and function in response to HIV+cocaine.

Gregory Perrin, Penn, Postdoc (PDS)

In progressive MS, cortical demyelinating lesions contribute to cognitive and motor decline, with remyelination failure driving disease progression. In healthy cortex, astrocytes and oligodendrocytes express specific connexins, allowing them to form long-term connections with each other via gap junctions, forming the A:O Network. During demyelination, oligodendrocytes and their connexins are lost, leading to astrocyte reactivity and dysregulated connexin expression. Prior studies suggest that simply making new myelin is insufficient for recovery; replacement oligodendrocytes must also re-form gap junctions with cortical astrocytes to rebuild the A:O Network. We propose that dysregulated astrocyte-astrocyte (A⇄A) coupling hinders replacement oligodendrocytes from re-establishing astrocyte-oligodendrocyte (A⇄O) gap junctions, thus impairing recovery from cortical demyelination.
We test this hypothesis at three scales throughout cuprizone-induced demyelination and recovery. At the cellular level, connexin immunofluorescence is used to determine if dysregulated astrocyte connexin expression persists and if the required connexins for A⇄O gap junctions reform. In local microcircuits, patch clamp physiology and dye transfer methods assess functional changes in A⇄A and A⇄O gap junctions. Additionally, we developed a network theory-based framework for longitudinal in vivo imaging to track changes in gap junction function and A:O Network structure at a larger scale.
These ongoing experiments aim to structurally and functionally map the A:O Network during demyelination and remyelination. Our results will offer insights into disease mechanisms and identify new therapeutic targets to promote cortical remyelination in progressive MS.

Katelyn Reeb, Drexel, Grad Student (SGS)

Excitatory amino acid transporters (EAATs) are critical proteins in the CNS that regulate synaptic glutamate levels, preventing excitotoxicity. The astrocytic transporter EAAT2 is responsible for the majority of glutamate clearance in the CNS. Aberrant EAAT2 activity and glutamatergic signaling occurs in many neuropsychiatric disorders. Our work focused on ischemic stroke, a condition that urgently needs new treatments. In ischemic stroke, excessive levels of released glutamate cause excitotoxicity, leading to secondary damage which ultimately results in cognitive deficits. We have developed novel allosteric modulators (AMs) of EAATs, including selective-EAAT2 positive allosteric modulators (PAMs), and non-specific and broad-acting analog AMs. We hypothesize the pharmacological activity of AMs is determined by differential interactions with critical amino acid residues. We have two main goals: to further understand the mechanism of these AMs, and to study their effects in an in vitro stroke model. Computational modeling predictions suggest specific amino acid residues on EAAT2 that are important for mediating the action of NA-014, an EAAT2-specific PAM. Dose-response assays evaluating the effect of NA-014 and other AMs offered further insights on which residues mediate their action. Additionally, we evaluated potential translatability of EAATs PAMs in a model of ischemic stroke. We hypothesize that these compounds can restore glutamatergic balance by augmenting glutamate clearance. We evaluated NA-014 using oxygen glucose deprivation in primary neuron-glia cultures, and found that it demonstrated neuroprotective properties. Collectively, these studies expand our mechanistic understanding of the EAAT AMs and demonstrate their clinical utility for ischemic stroke.

Julia Riley, Penn, Grad Student (SGS)

Astrocytes are the most abundant cells in the brain, and their ability to promote neuroinflammation is an aspect of Parkinson’s disease (PD) pathology. Accumulating data suggest a link between neuroinflammation and mitochondrial dysfunction, another hallmark of PD. Mutations in the enzymes PTEN-Induced Kinase 1 (PINK1) and Parkin, which facilitate clearance of damaged mitochondria via mitophagy, are sufficient to cause PD. However, the clearance mechanism for damaged mitochondria in astrocytes and the inflammatory signaling that is initiated by mitochondrial damage require further investigation. Here, we used OXPHOS inhibitors Antimycin A and Oligomycin A to induce mitochondrial damage in murine cortical astrocytes. Damaged mitochondria accumulate phospho-ubiquitin and Parkin, targeting them for clearance via PINK1/Parkin mitophagy. Parkin recruitment to mitochondria and subsequent mitochondrial degradation are both PINK1-dependent. We then sought to identify autophagy receptors that participate in this process. We observed upregulation of p62 expression, and colocalization of p62 with phospho-ubiquitin on damaged mitochondria, suggesting involvement of this receptor in astrocytic mitophagy. Next, we examined a link between mitophagy initiation in astrocytes and inflammatory signaling. Previous work from our group has shown that mitochondria targeted for mitophagy recruit the NF-κB effector molecule (NEMO), promoting activation of the NF-κB pathway for innate immunity. Using the same damage paradigm that induces mitophagy, we observed NEMO recruitment to damaged mitochondria in astrocytes. Using qPCR, we determined that levels of NF-κB-associated cytokines TNF-α and IL6 are elevated following mitochondrial damage; this response does not occur in PINK1-/- astrocytes. Further, inhibition of NF-κB ameliorates mitochondrial damage-induced upregulation of TNF-α expression. Ongoing experiments using RNA-sequencing will more fully define the inflammatory pathways induced by mitochondrial damage in astrocytes, which may be neuro-protective, neuroinflammatory, or both. These results provide new insights into cell non-autonmous mechanisms linking mitochondrial stress to neuroinflammation and neurodegeneration.

Steve Rutledge, Drexel, Grad Student (JGS)

The ability for melanoma to metastasize to the brain results in poor clinical outcomes, with over 60% of patients suffering from stage IV melanoma developing a brain metastasis during the course of their disease. Tumor progression is impacted by the tumor microenvironment comprised of a unique extracellular matrix, neurons, and glial cells (oligodendrocytes, microglia, and astrocytes). Following injury or disease within the central nervous system, astrocytes can become reactive in a process called astrogliosis. Reactive astrocytes display altered morphologies, often undergoing hypertrophy and increases in length and number of processes, as well as numerous translational changes. Importantly, reactive astrocytes surround the tumor in the setting of brain metastases.

Unlike neurons, astrocytes are not electrically excitably and instead utilize both intracellular and intercellular calcium signaling to control functions including neurotransmitter regulation, blood-brain barrier permeability, and ion homeostasis. Additionally, in many pathologies, reactive astrocytes are known to show altered calcium signaling, but the influence of melanoma brain metastases on calcium signaling remains understudied. Therefore, we are developing a model of calcium imaging in peri-lesional astrocytes utilizing a membrane tethered, genetically encoded calcium indicator (LckGCaMP6F) under an astrocyte promoter (GfaABC1D) to quantify progression of fluorescently labeled melanoma cells (D4m.3a labeled with LSSmOrange or Td-Tomato). Alterations to calcium signaling in peri-tumor and tumor free sections of organotypic slice cultures are observed using 2-photon microscopy and compared via use of Astrocyte Quantitative Analysis (AQuA) software.

Gregory Salimando, CHOP, Staff Scientist (PDS)

Microglia are an attractive emerging target for gene & cell-based therapies due to their immune effector functions, crosstalk with other neural cell types, and transplantability. Despite this promise, a major hurdle in their utility is their high resistance to viral transduction by two of the most common vectors used for gene and cell therapy applications: the adeno-associated viruses (AAV) and lentiviruses (LV). To understand the molecular basis of microglial antiviral immunity, we are: (1) investigating the role of cGAS/STING in restricting viral infection in microglia, and (2) establishing a broadly applicable discovery platform to interrogate the role of other signaling pathways in driving this viral restriction via the creation of a Microglial Lentivirus CRISPR Knockout (MVrik) library. Preliminarily, we find that inhibition of cGAS/STING with small molecule inhibitors increases LV-transduction in primary rodent-derived microglia in vitro. Selective STING deletion in microglia also globally enhances in vivo LV-transduction, suggesting that the targeting of select pathway components may improve viral transduction of microglia for downstream applications. To investigate such additional elements that confer viral resistance, we have developed a custom ~1,200 gene sgRNA library to knock out the function of these potential anti-viral genes of interest via pooled CRISPR screens and in vitro testing for changes in viral transduction efficacy. Successful hits will be escalated to in vivo applications to test if either reduction or inhibition of these targets boosts viral transduction in rodent models. Taken together, this work aims to expand upon our current understanding of the viral resistance mechanisms inherent to microglia and create versatile screening platforms that may lead to more efficient and effective gene and cell therapy development for intractable neurological disorders.

Sunetra Sase, CHOP, Staff Scientist (PDS)

Hypomyelination and atrophy of basal ganglia and cerebellum (H-ABC) is a rare leukodystrophy associated with causal variants in -tubulin 4A (TUBB4A). The recurring variant p.Asp249Asn (D249N) presents in infancy with dystonia, communication deficits, and loss of ambulation during the first decade of life. In this study, we characterized a genetic murine series (Tubb4aKO/KO, Tubb4aD249N/+, Tubb4aD249N/KO and Tubb4aD249N/D249N) to demonstrate that disease severity correlates with the expression of mutant Tubb4a and relative preservation of WT tubulin. To further evaluate the translational potential of Tubb4a suppression as a therapy in H-ABC, we identified aTubb4a-targeted antisense oligonucleotide (ASO) candidate that selectively reduces Tubb4a in vitro and in vivo with no toxicity. Notably, single intracerebroventricular (i.c.v.) administration of ASO into postnatal Tubb4aD249N/KO mice drastically extends its lifespan, improves motor phenotypes, and reduces seizures. ASO treatment in Tubb4aD249N/KO mice restored myelin and oligodendrocyte survival. Furthermore, in vivo visual evoked potential latencies recovered in ASO-treated Tubb4aD249N/KO mice. This is the first preclinical proof-of-concept for Tubb4a suppression via ASO as a disease-modifying therapy for H-ABC.

Erin Smith, Penn, Grad Student (SGS)

The lysosome is an essential degradative organelle for proteome maintenance. Damage to lysosomes aid protein aggregation in many neurodegenerative diseases (NDDs). In some NDDs, astrocytes are less prone to aggregate accumulation than neurons. Further, emerging evidence shows cell-type-specific differences in lysosomal biology. How neuronal and astrocytic lysosomes differ is unknown. In response to lysosomal damage, lysosomal repair pathways such as the ESCRT, PITT and the autophagic lysosomal reformation (ALR) pathways are activated. Gaps remain in our understanding of differences in lysosomal repair in astrocytes versus neurons and how these differences impact lysosomal resilience. To address these gaps, we used a neuron-astrocyte coculture system that recapitulates interactions observed in vivo. To model lysosomal damage, we used the lysomotropic agent LLOMe. LLOMe induces lysosomal damage in both neurons and astrocytes as measured by 1. Accumulation of lysosomal damage markers (galectin-3, lysophagy receptors) and 2. Reduced Lysotracker signal. Despite inducing lysosomal damage in both cell-types, ESCRT (CHMP2B, ALIX) and ALR (TBC1D15) machinery were more robustly recruited to lysosomes in astrocytes versus neurons. Interestingly, PITT machinery (PI4K2A and ORP-9) were recruited to LLOMe damaged lysosomes in both cell-types. Together, our data suggest that in response to lysosomal damage, there is differential recruitment of lysosomal repair machinery between astrocytes and neurons. Differences in lysosomal repair pathways could confer cell-type-specific lysosomal vulnerabilities to pathophysiological lysosomal damage.

Sidney Smith, Penn, Grad Student (JGS)

Adult-onset leukoencephalopathy with axonal spheroids and pigmented glia (ALSP) is a rare, autosomal-dominant white matter disorder that causes debilitating neurological symptoms including neuropsychiatric impairment, motor dysfunction, and dementia. Disease pathology is characterized by patchy white matter abnormalities in the brain that grow progressively confluent over time. ALSP is caused by mutations in the colony stimulating factor type 1 receptor gene (CSF1R) which is necessary for microglial and myeloid-lineage cell survival. It is not well understood how expression of mutant CSF1R in circulating myeloid cells may lead to damage of the central nervous system. Due to its variation in symptoms and age of onset, ALSP is often misdiagnosed as other disorders such as multiple sclerosis, Alzheimer’s disease or frontotemporal dementia. There is an unmet need for an early diagnostic biomarker for ALSP.
Using flow cytometry, we measured expression of CSF1R and molecules associated with quiescence, activation/inflammation, and phagocytosis in ALSP patient and healthy control blood samples with the goal of identifying unique ALSP sample signatures that can be used diagnostically. It is expected that ALSP peripheral monocytes will have a distinct phenotypic and functional profile that exhibits reduced expression of CSF1R, altered expression of chemokine receptors and phagocytic propensity, and increased levels of activation and pro-inflammatory cytokines. It is expected that immuno-stained brain tissue from mutant CSF1R mice will demonstrate changes in glia populations. The results of these experiments will provide a framework for identifying ALSP in patient blood and tissue samples and may serve as future disease biomarkers.

Bailey Spangler, Penn, Research tech (UGT)

Multiple Sclerosis (MS), Neurosarcoidosis, Neuromyelitis Optica Spectrum Disorder (NMOSD) and Myelin Oligodendrocyte Glycoprotein Associated Disorder (MOGAD) are all acquired immune-mediated disorders affecting the central nervous system white matter. Inherited leukodystrophies are a group of rare and highly diverse disorders defined by the genetic mutation associated with them, while leukoencephalopathies have extensive white matter disease without a known genetic cause identified. Despite great heterogeneity in pathophysiology and clinical courses, misdiagnosis amongst these disorders is common due to overlapping clinical presentations and imaging findings. MRI is a powerful tool for diagnosis; however, clinical imaging biomarkers rely on recognizing patterns which can be slow to develop in many diseases. Additionally, clinical MRI sequences lack specificity to most pathological processes. In advanced contrast MRI sequences, can have improved biological specificity, and ultra-high field MRI (7-tesla) enables improved spatial resolution and tissue contrast over conventional clinical MRI. In this study, we are utilizing advanced 7T MRI to measure tissue injury across the spectrum of white matter disorders. We are applying established MRI biomarkers with putative disease specificity to MS including the central vein sign and paramagnetic rim lesion to cohorts of patients with different diseases to determine similarities and differences between these disorders. In this pilot study, we have imaged 14 patients that include 7 with genetically defined leukodystrophies, 3 with genetically undefined leukoencephalopathy, 2 with progressive MS, 1 with neurosarcoidosis, and 1 with NMOSD. These participants have undergone 7T MRI as well as measures of cognitive function, motor function, and mood.

Joseph Vithayathil, CHOP, Instructor (PDS)

Iron deposition has previously been identified in areas of perinatal focal ischemic brain injury, while recent data has shown that lipid peroxidation may play a role in neonatal hypoxic-ischemic (HI) brain injury. However, the timing and cell specific changes to iron and lipid peroxidation continue to be important questions that will provide insight into the development of targeted therapies and treatment windows. In order to evaluate this question, we used a mouse model of HI in post-natal day 9 mouse pups and focused on injury in the hippocampus. Dissociated cells from HI hippocampi had increased intracellular labile iron at 24h (2.3 fold increase compared to sham, p<0.001) that decreased at 72h (1.8 fold increase compared to sham, p<0.001). Lipid peroxidation was then assessed by measuring isoprostanes and neuroprostanes in hippocampal tissue. F4-neuroprostanes were elevated at 24h (97% increase compared to sham, p<0.001), but then decreased below sham levels by 72h (62% decrease compared to sham, p<0.001). To assess which cells accumulate iron, L-ferritin immunoreactivity in HI tissue peaked at 72h post-injury and co-localized with Iba1+ cells (microglia/macrophages). Isolated CD11b+ cells (microglia/macrophage enriched) from HI hippocampi showed gene expression changes in L-ferritin (38% increase, p<0.05), transferrin receptor (78% decrease, p<0.05) and Ferroportin1 (44% decrease, p<0.05) when compared to microglia/macrophages from sham hippocampus, which is consistent with a cellular iron sequestration response (CISR) in microglia/macrophages. In conclusion, Iba1+ microglia/macrophages activate a CISR response that peaks at 72h post-HI and correlates with a decrease in labile iron and lipid peroxidation.

Eliana von Krusenstiern, Penn, Grad student (GS)

HIV-associated neurocognitive disorder (HAND), a spectrum of neurocognitive impairments, occurs in up to 50% of people with HIV (PWH), even if their viral load is undetectable due to antiretroviral therapy (ART). Pathological features include white matter abnormalities, which worsen with increased time on ART. Work in our lab has demonstrated that select ART drugs prevent the maturation of oligodendrocytes (OLs) and remyelination, at least partially through the activation of the Integrated Stress Response (ISR). Co-treatment with the ISR inhibitor ISRIB rescues differentiation. We also show that OLs treated with select ART drugs form cytoplasmic stress granules (SGs), thought to be downstream of the ISR. SGs sequester proteins and mRNAs critical for cell survival. We demonstrate that when OLs are co-treated with ART drugs and ISRIB, SGs do not form. We further show that co-treatment with an inhibitor of PERK, one of 4 ISR kinases, also decreases SG-positive OLs. Ex vivo analysis of both mice treated with ART, and PWH revealed the presence of SGs within oligodendrocytes of the corpus callosum and cortical white matter, respectively. PWH with HAND had an increased percentage of OLs with stress granules and an increase in the number of stress granules/OL compared with neurocognitively normal individuals. These findings suggest that formation of SGs in OLs may contribute to persistent white matter pathology in PWH with HAND. These studies are among the first to study stress granules in OLs, and have the potential to contribute to the improvement of patient outcomes for PWH on ART.

Johnathan Wong, Penn, Grad student (SGS)

In the central nervous system, oligodendrocytes (OLs) synthesize myelin, a lipid-enriched membrane crucial for facilitating transmission of action potentials and trophic support to axons. The loss of myelin contributes to motor and neurocognitive dysfunction in demyelinating diseases such as multiple sclerosis (MS). Diseases involving an excess or deficiency in cytoplasmic copper (Wilson’s and Menkes disease, respectively) also show impaired myelination in the CNS, suggesting oligodendrocytes may be keenly sensitive to copper deprivation. Consistent with this, feeding rodents the copper chelator cuprizone causes demyelination, primarily in the corpus callosum, and is commonly used as a model in demyelination/remyelination studies. The exact mechanism by which cuprizone induces demyelination is not clear. We treated cells in culture with cuprizone at 3 specific stages of differentiation: as oligodendrocyte progenitor cells (OPCs), differentiating OLs (DOLs), and mature OLs (MOLs). We found that only cultures allowed to differentiate for 3 days before being treated with cuprizone (MOLs) showed significant levels of oligodendrocyte death. These data indicate that cuprizone exclusively kills mature OLs, suggesting that copper chelation may be specifically impactful to mature OLs and their stage-specific proteins such as MBP and PLP. Additionally, we found that activation of the endolysosomal cation channel TRPML1 via Mucolipin synthetic agonist 1 (MLSA1) rescued the copper-chelating deficits in oligodendrocytes, possibly implicating lysosomal function in the actions of cuprizone. The results of this study will provide valuable insights into the effects of copper storage and transport on oligodendrocytes maturation and function.

Feiyi Xiong, Johns Hopkins, Research tech (UGT)

Postoperative delirium is a common and significant cognitive complication after major surgery in older people. Nonetheless, the molecular and cellular mechanisms underlying the pathophysiology of postoperative delirium remain poorly understood, delaying advances in its prevention and treatment. Systemic inflammation induces neuroinflammation – largely regulated by brain resident immune cells, resulting in acute cognitive dysfunction. Recent studies suggest that deterioration of glymphatic waste clearance system may contribute to cognitive decline in the elderly. Interestingly, glutamate induces astrocyte swelling via altered expression of aquaporin-4 water channel (AQP4) in astrocytes, which is a critical regulator of the glymphatic system. In the current study, using an aged mouse model of abdominal surgery, we are investigating whether abdominal surgery-induced systemic inflammation results in excess glutamate production in the CNS, which may affect AQP4 signaling in perivascular astrocytes, leading to aberrant glymphatic waste clearance system and cognitive impairments. We are also exploring whether pharmacological inhibition of specific enzymes for regulation of glutamate production could normalize glial cell and glymphatic phenotypes, as well as cognitive impairments. We hope our studies determine the potential of the glymphatic system not only in addressing the astrocyte-mediated pathological mechanisms of postoperative delirium but also in providing novel molecular targets for its prevention and treatment in the elderly.

Yixuan Erica Zhu, Penn, Research tech (UGT)

Several genetic risk factors for Alzheimer’s disease implicate genes involved in lipid metabolism and many of these lipid genes are highly expressed in glial cells. However, the relationship between lipid metabolism in glia and Alzheimer’s disease pathology remains poorly understood. Through single-nucleus RNA sequencing of brain tissue in Alzheimer’s disease, we have identified a microglial state defined by the expression of the lipid droplet-associated enzyme ACSL1 with ACSL1-positive microglia being most abundant in patients with Alzheimer’s disease having the APOE4/4 genotype. In human induced pluripotent stem cell-derived microglia, fibrillar Aβ induces ACSL1 expression, triglyceride synthesis and lipid droplet accumulation in an APOE-dependent manner. Additionally, conditioned media from lipid droplet-containing microglia lead to Tau phosphorylation and neurotoxicity in an APOE-dependent manner. Our findings suggest a link between genetic risk factors for Alzheimer’s disease with microglial lipid droplet accumulation and neurotoxic microglia-derived factors, potentially providing therapeutic strategies for Alzheimer’s disease. The Haney lab is continuing to investigate the mechanisms by which lipid droplet containing microglia secrete damaging factors to neurons through conditioned media experiments with primary microglia and neurons and through CRISPR KO screens. The Haney lab is also continuing this research by combining spatial transcriptomics and oil red O staining of post-mortem human brain tissue.

Joseph Gallegos, UPenn, Graduate Student

Multiple sclerosis (MS) is a disease characterized by loss of oligodendrocytes and myelin ensheathment of axons (demyelination). Demyelination in the cortex is a prominent feature of MS pathogenesis, as cortical demyelination and atrophy predicts physical and cognitive decline. Despite this, the mechanisms underlying cortical demyelination and remyelination are poorly understood.

By pairing longitudinal in vivo imaging with the cuprizone model of demyelination, our lab recently characterized the loss and recovery dynamics of cortical oligodendrocytes (Orthmann-Murphy et al. 2020). We found that recovery of cortical oligodendrocytes is inefficient in deeper cortical regions, and this impaired recovery is correlated with persistent astrocyte reactivity. I hypothesized that cortical astrocytes acquire divergent reactive states, where astrocytes in deep cortical regions maintain a persistent pro-inflammatory profile that impairs oligodendrocyte recovery.

To test this, I performed single-cell mRNA sequencing on purified cortical astrocytes from adult mice. Tissue was collected from untreated mice, mice treated for 4 weeks with cuprizone (or sham), and mice treated with cuprizone and then allowed to recover for 2 or 5 weeks (remyelination). Collectively, I have assembled a dataset containing > 450,000 cells, chronicling the molecular landscape of cortical astrocytes throughout demyelination and remyelination. I have found distinct subpopulations of astrocytes that are produced during remyelination, and identified putative gene networks employed by these cells which may contribute to impaired recovery of cortical oligodendrocyte. This dataset represents a new tool for understanding astrocyte responses to demyelination, providing a framework to base new hypothesis driven questions, towards the goal of developing remyelination therapies.

Sonia Lombroso, UPenn, Graduate Student

Achieving microglia replacement, which entails depleting endogenous microglia and substituting them with surrogate macrophages, has been a challenging and elusive goal in glial biology for the treatment of neurological diseases. Despite the significant therapeutic potential of this approach, its application has been hindered by the lack of safe and specific methods. Our recent work demonstrated that microglial depletion using colony-stimulating factor 1 receptor inhibitors (CSF1Ri), combined with intracranial cell delivery of inhibitor resistant CSF1R (IR-CSF1R) donor cells, is feasible for direct replacement of microglia. However, there is a pressing need for less invasive methods to target macrophages in both the central nervous system (CNS) and peripheral tissues.
Here, we introduce a novel approach: CSF1Ri-dependent microglia replacement through the peripheral delivery of IR-CSF1R hematopoietic stem cell-derived myeloid cells in both neonates and adults. This method offers a promising alternative for microglia replacement therapy.
Combining an IR-CSF1R transgenic mouse with host CSF1Ri treatment, we tested the hypothesis that IR-CSF1R-expressing donor cells could successfully infiltrate the CNS following peripheral delivery. Notably, peripheral transplantation of bone marrow expressing this variant resulted in CNS penetration and significant donor cell engraftment without the need for chemotherapy or irradiation. This CNS engraftment was long-lasting and persisted even after CSF1Ri cessation. Furthermore, combining the IR-CSF1R approach with ex vivo expansion of hematopoietic stem cells, we demonstrate delivery of potentially therapeutic payloads to the CNS from the periphery. This suggests a method for delivering engineered, effector immune cells as living drugs for the treatment of neurological diseases.
In summary, by engineering an IR-CSF1R variant resistant to pharmacological inhibitors, we have developed a microglia replacement strategy that allows for peripheral delivery of therapeutic payloads to the CNS without chemotherapy or irradiation.