Lindsay Festa, CHOP, Assistant Professor, Gliadelphia New Investigator Speaker

In rodents and humans, the oligodendrocyte lineage is an active player in shaping neuronal circuitry and function. Moreover, oligodendrocytes have an endogenous capacity to restore their cell population via remyelination, a process driven primarily by the migration and differentiation of oligodendrocyte precursor cells (OPCs). The progression of OPCs and subsequent remyelination is closely regulated by intrinsic mechanisms; however, in conditions of chronic neuroinflammation and aging, spontaneous remyelination is often inefficient and eventually fails, leading to permanent neuronal degradation and clinical disability. There is a current unmet clinical need to identify regulators of oligodendrocyte homeostasis that are disrupted during disease states and can be utilized to promote functional recovery in patients. In particular, the role of intracellular organelles, including the (endo)lysosome and mitochondria, in modulating oligodendrocyte function has not been well described. My postdoctoral work identified the (endo)lysosome as a novel regulator of actin polymerization, a process necessary for appropriate oligodendrocyte differentiation and initial ensheathment of axons. Now, as an independent investigator, my group is utilizing an innovative and intersectional approach to identify and manipulate novel functions of organelles within the oligodendrocyte lineage to uncover intrinsic mechanisms underlying developmental myelination and demonstrate its therapeutic potential to alleviate cellular and functional white matter abnormalities. Currently, we are investigating the organelle “contactome” within the oligodendrocyte lineage and how disruption of these contact sites leads to white matter loss and impairment of remyelination.

Sara Bernardez Noya, UPenn, Postdoctoral Fellow, Sehgal Lab, Gliadelphia Trainee Selected Speaker

Sickness behavior is a coordinated set of adaptive responses observed across species to promote recovery and survival following peripheral infection or injury. Recent studies in mammals have begun to uncover a distributed array of neural circuits underlying sickness behaviors, suggesting the existence of coordinating mechanisms that operate across cell types and circuits. Here, we combined single-cell RNA sequencing with functional analyses in Drosophila to uncover a central role for glia in orchestrating sickness-associated brain states. Specifically, ensheathing glia, exhibits the most pronounced gene expression changes following sterile injury. Targeted perturbations demonstrated that adrenergic signaling onto these cell type is necessary for the induction of sickness-induced sleep. In vitro calcium imaging further confirmed that octopamine, the invertebrate analog of norepinephrine, stimulates glial calcium influx and glutamate release. In addition, genetic disruption of glial glutamate signaling impaired both neuronal activation and the behavioral expression of sleep during sickness. Together, these findings identify glia as critical integrators of neuromodulatory and immune inputs, advancing a framework in which glia, rather than neurons alone, orchestrate state transitions that underlie sickness behavior.

Natasha O'Brown, Rutgers & HHMI, Assistant Professor, Invited External Speaker

The blood-brain barrier (BBB) is formed by brain endothelial cells that develop restrictive barrier properties through signals from neurons, pericytes, and astrocytes. Our prior work detailed the timeline of zebrafish BBB maturation, noting a posterior-anterior pattern of functionalization by 5 days post-fertilization (dpf) due to the suppression of endothelial transcytosis. We recently identified the neuronal signal Spock1 as a conserved factor in both mouse and zebrafish for inducing BBB properties during development. While both mutants show embryonic leakage, zebrafish spock1 mutants remain leaky in adulthood, whereas Spock1 knockout mice fully recover BBB function, suggesting species-specific mechanisms in maintaining BBB integrity. We hypothesized that postnatally born astrocytes are responsible for the recovery in Spock1 knockout mice. To explore this, we used transgenic zebrafish and electron microscopy to first characterize glial-vascular interactions throughout zebrafish life. We observed a significant increase in glial-vascular contacts with age, and some glial ensheathment of vessels. However, these glial-vascular interactions never reached the near full coverage observed in mice. Using cross-species xenografts where we transplanted mouse astrocytes and neural stem cells into zebrafish spock1 mutant brains, we observed a complete restoration of BBB function throughout the mutant brain with the addition of astrocytes, similar to what is seen in adult Spock1 knockout mice. These results suggest that specialized mammalian glial mechanisms, absent in zebrafish, play a crucial role in BBB maintenance.

Michael Haney, UPenn, Assistant Professor, Gliadelphia New Investigator Speaker

While much of the visible pathology in neurodegenerative diseases is neuronal, microglia play a fundamental role through dynamic transitions into various states that can drive pathology or confer protection. Recent studies from human genetics and experimental models have revealed that microglia-expressed genes can mitigate or delay neurodegeneration, highlighting an underexplored therapeutic landscape. In this talk, I will outline why the present moment is uniquely exciting for advancing our understanding of the role microglia play in neurodegenerative disease, with a case study focusing on APOE and lipid-accumulating microglia in Alzheimer’s disease. I will also share emerging projects and methodological developments from the Haney lab aimed at dissecting microglial biology and identifying pathways with therapeutic potential.

Melissa Cooper, NYU, Postdoctoral Fellow, Liddelow Lab, Invited External Speaker

Traditionally, neuronal axons have been considered the primary mediators of functional connectivity among brain regions. However, the role of astrocyte-mediated communication has been largely underappreciated. While astrocytes communicate with one another through gap junctions, the extent and specificity of this communication remain poorly understood. Astrocyte gap junctions are necessary for memory formation , synaptic plasticity, coordination of neuronal signaling, and closing the visual and motor critical periods. These findings indicate that this form of communication is essential for proper central nervous system development and function. Despite their significance, studying astrocyte gap junctional networks has been challenging. Current methods like slice electrophysiology disrupt network connectivity and introduce artifacts due to tissue damage. To overcome these limitations, we developed a vector-based approach that labels molecules as they are fluxed by astrocyte gap junctions in awake, behaving animals. We then used whole-brain tissue clearing to image these intact, three-dimensional astrocyte networks. We show that multiple astrocyte networks traverse the mouse brain. These networks selectively connect specific regions, rather than diffusing indiscriminately, and vary in size and organization. We observe local networks are confined to single brain regions and long-range networks robustly interconnecting multiple regions across hemispheres, often exhibiting patterns distinct from known neuronal networks. Further, we demonstrate that astrocyte networks undergo structural reorganization in adult brain following sensory deprivation. These discoveries reveal a previously unrecognized mode of communication between distant brain regions, mediated by plastic networks of gap junction-coupled astrocytes.

Alex Hulegaard, UPenn, Postdoctoral Fellow, Granato Lab, Gliadelphia Trainee Selected Speaker

Insults to the central nervous system cause tissue-wide changes involving various cell types to neutralize toxic debris. Among these are astrocytes, changing their morphology and expression profiles. Despite their prominence, the complex role of astrocytes in injury and regeneration is poorly understood. Namely, what are the dynamic behaviors of astrocytes in vivo triggered directly by neuron injury and what molecular signals regulate them? These outstanding questions remain elusive in part due to the challenges precluding visualizing astrocytes in real time using most model systems. Furthermore, while many genes have been identified as differentially expressed in astrocytes upon injury, their functional roles regarding astrocyte dynamics remain largely unknown. Using zebrafish, we interrogate these questions by examining astrocytes in the spinal cord following injury. We use a laser to transect the Mauthner axon in the spinal cord and record its regeneration over the course of a few days. Using live-imaging, we monitor astrocytes with single-cell resolution following axotomy and during regeneration. Astrocytes near the transection site exhibit dynamic behavior within 1-2 hours of injury and send new processes towards the injury site. Using a genetically encoded calcium sensor, we reveal increased astrocytic calcium activity immediately upon axon transection. From a candidate screen to target genes upregulated in astrocytes in neurodegeneration models using CRISPR/Cas9 we identified several genes required for axon regeneration. Ongoing work will clarify how these genes regulate astrocyte dynamics in vivo and elucidate molecular pathways in astrocytes that promote CNS axon regeneration.

Joshua Jackson, Drexel, Assistant Professor, Gliadelphia Faculty Speaker

TBD

Alan Gu, UPenn, Grad Student, Yuanquan Song Lab (JGS)

Central nervous system neurons exhibit limited regenerative capacity following injury, contributing to permanent functional deficits in patients with brain and spinal cord injury (SCI). While mechanical forces inhibit axon regeneration through the Piezo-Atr-Chek1 pathway, the downstream effectors mediating this inhibition remain unknown. Here, we investigated whether Drep-1 and Drep-4, two DNA fragmentation factor-related proteins traditionally associated with apoptosis and lipid metabolism, function as a critical mediator of mechanosensory-induced regeneration failure. Using the Drosophila dendritic arborization (da) neuron injury model, we performed laser axotomy followed by confocal imaging to quantify regeneration capabilities. Sample sizes were determined through power analysis and previous literature, establishing statistical requirements for detecting meaningful differences in regeneration capabilities. We first established baseline regenerative capacities, demonstrating that Class IV da neurons in the PNS exhibit robust regeneration while Class IV da neurons in the CNS shows limited regenerative potential. Importantly, RNAi-mediated knockdown of Drep-1 and Drep-4 significantly enhanced axon regeneration in Class IV neurons within the ventral nerve cord, an environment typically non-permissive for regeneration (p<0.05 and p<0.0001 respectively). Transcriptome analysis revealed that Drep-1 expression is upregulated following Chek1 overexpression and downregulated in Atr knockout conditions, suggesting its function as a downstream effector of the Piezo-Atr-Chek1 pathway. These findings identify Drep-1 as a potential novel inhibitor of axon regeneration and may reveal an unexpected role for metabolic machinery in neural repair.

Alison Salinas, Mount Sinai, Grad Student, Allison Bond Lab (SGS) +Flash

Alzheimer’s Disease (AD) is a neurodegenerative disorder that leads to degeneration of multiple brain regions, including the hippocampus, and precedes onset of symptoms. Apolipoprotein E4 (APOE4) is one of the strongest genetic risk factors for late-onset AD, but the mechanism of increased risk is not well understood. APOE is expressed predominantly by astrocytes and, to a lesser extent, by microglia and neural stem cells (NSCs) within the dentate gyrus across development and adulthood. APOE4 is known to disrupt glial function, however most APOE-related studies are limited to the adult brain. I hypothesize that the APOE4 risk allele will lead to altered glial development and dysfunction that contributes to AD susceptibility in adulthood. Using human APOE variant knock-in mice I aim to investigate how the APOE4 variant alters the development of NSCs and their differentiation into neurons and glia through birth dating methods and immunohistochemistry. APOE4 mice exhibited altered glial cell numbers including reduced oligodendrocyte precursor cells (OPCs) and increased number of astrocytes compared to APOE3. Future work is aimed at determining whether these effects persist across different developmental stages and define mechanisms underlying altered glial crosstalk, such as disrupted astrocyte lipid support and altered microglia phagocytic activity resulting in fewer OPCs. This research furthers our understanding of how the APOE4 risk variant alters development and will be crucial in identifying future therapeutic targets that prevent cognitive symptoms in APOE4 carriers.

Andrea Berghella, Jefferson, Grad Student, Blanco-Suarez Lab (JGS)

Chordin-like 1 (Chrdl1) is an astrocyte-secreted protein that induces synapse maturation in the cortex, limiting plasticity. Chrdl1 is aberrantly upregulated in peri-infarct astrocytes after ischemic stroke, and Chrdl1 KO mice show better stroke outcomes, including preserved dendritic spines, smaller injury volumes, and improved motor function. Ketamine is an intriguing candidate compound to target Chrdl1 based on its plasticity-promoting properties and ability to modulate astrocyte biology, including protein secretion. We hypothesize that subanesthetic ketamine treatment limits post-stroke astrocytic Chrdl1 upregulation and stimulates recovery. Methods: In vitro, we cultured primary mouse cortical astrocytes and treated them with low-dose ketamine (10µM) or vehicle. We measured Chrdl1 mRNA and protein secretion via RT-qPCR and ELISA, respectively. In vivo, we generated ischemic stroke by photothrombotic occlusion of the distal middle cerebral artery in adult male WT mice. We administered intranasal subanesthetic ketamine (10mg/kg) or vehicle daily on days 2-8 after the insult. We quantified Chrdl1 levels adjacent to the injury by single-molecule fluorescent in situ hybridization; we determined stroke volume using small animal MRI and evaluated sensorimotor behavior on the adhesive removal test. Results: We found that ketamine decreases astrocytic Chrdl1 levels in vitro and dampens post-stroke upregulation of Chrdl1 in vivo late after administration. Ketamine does not impact stroke volume, but preliminary data indicate that ketamine treatment may improve sensorimotor recovery after stroke. Conclusion: Our results suggest that ketamine could be a pharmacological approach to regulate astrocytic Chrdl1 for potential therapeutic benefit following stroke.

Andrew Nguyen, UPenn, Grad Student, Sehgal Lab (SGS)

Glia–neuron metabolic coupling is essential for maintaining brain homeostasis. A key component of this interaction is the glia–neuron lactate shuttle, which supports neuronal activity by supplying lactate, a glycolytic byproduct, during periods of high energy demand. Because neuronal activity fluctuates across the day, glial lactate shuttling must also be dynamically regulated. Further, glial lactate contributes to neuronal lipid synthesis and peroxidation. Peroxidized lipids generated in neurons are transferred to glia, where they accumulate in lipid droplets (LDs) and are degraded in a sleep–wake–dependent manner. Here, we demonstrate time-of-day–dependent changes in the glia–neuron lactate shuttle and show how perturbations in lactate transport, lipid transfer, and mitochondrial fission–fusion factors collectively regulate glia–neuron metabolic coupling.

Anna Foltz, CHOP, Predoc, Mariko Bennett Lab (UGT)

Aicardi-Goutières Syndrome (AGS) is a type I interferonopathy characterized by systemic inflammation and progressive leukodystrophy. To reveal the immune mechanisms underlying disease pathogenesis, we have characterized the ADAR1 D1113H mutant mouse model, which recapitulates AGS neuropathology. By 8 weeks of age, D1113H mice exhibited astrocytic and microglial activation, as well as significant demyelination. Peripheral immune system analysis revealed an expansion of activated CD8+ T cells expressing Ly6C and LFA-1, as well as Ly6C⁺ monocytes.
To determine whether CD8+ T cells or monocytes drive disease, we crossed D1113H mice onto a Rag1-/- background to address the role of T cells in neuropathology. While neuropathology persisted in the absence of mature B and T cells, Ly6C⁺ monocytes remained abundant, implicating monocytes as early drivers of pathology. Antibody-mediated blockade of Ly6C and IFNAR reduced type I interferon (IFN-I) signaling both in the periphery and CNS. We also show that full genetic deletion of IFNAR in D1113H mice significantly reduced pathology, confirming aberrant interferon signaling as major driver of the pathology. Together, our findings suggest the potential role of inflammatory monocytes as key effectors in AGS. Currently, we are manipulating these monocytes to better understand their role as mediators of interferonopathy.

Ashley Carper, Drexel, Grad Student, Denise Garcia Lab (JGS)

Astrocytes are a major type of glial cell in the brain that perform diverse functions, including maintaining the blood-brain barrier, promoting synapse formation and elimination, and maintaining neurotransmitter homeostasis. Recent studies have found that astrocytes exhibit activity-dependent gene expression to regulate formation, elimination, and plasticity of their synaptic partners. Furthermore, astrocytes and synapses exhibit bidirectional interactions, with synaptic activity influencing astrocyte function and vice versa. RNA sequencing data from our lab and others identified that cortical astrocytes express AMPA receptors (AMPAR). The role of neuronal-AMPAR is well established, however their role and functionality on cortical astrocytes is not understood. In the cerebellum, Bergmann glia express functional AMPAR that are vital for forming and maintaining proper astrocyte-synapse interactions. In preliminary experiments, my data show that cortical astrocytes express AMPAR subunits GluA1 and 2 under baseline conditions. GluA2 is expressed at much higher levels than GluA1. Interestingly, mice trained on a motor learning task show differential upregulation of GluA1, but not GluA2, subunits. This same pattern is seen in neuronal-AMPAR following activity, suggesting that astrocytic AMPARs may also play a role in learning-induced synaptic plasticity. In this study, I am investigating the role of AMPARs in cortical astrocytes following motor learning.

Bailey Collins, Nemours, Grad Student, Elizabeth Wright-Jin Lab (SGS)

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. To better capitulate the injury seen in human infants, we used the Rice Vannucci (RV) model of injury on C57/B6 mice that included maternal immune activation on gestational day 18. The primary responders to this injury are microglia and infiltrating regulatory T-cells, suggesting that modulating this response using cytokines can provide mechanisms for treatment. Interleukin 33 (IL-33), a cytokine that recruits regulatory T cells, has been previously shown to have a neuroprotective effect after HIE when delivered systemically. We administered a sustained release IL-33 loaded alginate hydrogel via intracerebroventricular injection to neonatal mice treated with the full Rice Vannucci model or a control carotid ligation immediately post injury. Functional outcomes such as gait analysis, grip strength, and rotarod were assessed. Histological outcomes were assessed using hematoxylin and eosin. Staining for microglia density and regulatory T cells will be conducted. Overall, males had worse outcomes than females. Male mice treated with RV and a blank hydrogel displayed the lowest normalized grip strength and the most abnormal gait, as well as the largest ventricular area increase. Males treated with RV and the IL-33 loaded gel showed similar functional and histological outcomes as the controls. The results indicate that IL-33-loaded alginate hydrogels are a promising technique for the treatment of HIE.

Brennan Lewis, UPenn, Grad Student, Haney Lab (JGS)

Alzheimer’s disease (AD) is the most prevalent age-related dementia in the world. The hallmarks of Alzheimer’s disease (AD) include extracellular Aβ plaques and intracellular tau neurofibrillary tangles. APOE genotypes dramatically modify AD pathology, with the APOE4 genotype exacerbating Aβ and tau pathology and APOE3ch genotype protecting against Tau pathology. However, the mechanisms of this genotype influence remain unknown. Our goal is to characterize the cellular pathways that govern how ApoE modifies AD pathology in microglia and neurons. We utilize isogeneic APOE varient iPSC lines to derive microglia (iMG) and neurons (iNeurons) to address these questions. In this system we evaluate the transcriptomic, metabolic, and cellular phenotypes resulting from challenging microglia with pathological protein aggregates such as Aβ, tau, and alpha-synuclein. We will take the conditioned media from these challenged iMG to test effects in iNeurons of APOE transfer with Halo-tagged APOE iMGs and Tau propagation using Tau-FRET reporters.

Briana Lisi, Jefferson, Grad Student, Pasinelli/Weinberg ALS Center (SGS) +FLASH

One of the most aggressive and often juvenile-onset forms of ALS is associated with mutations in the DNA/RNA binding protein Fused in Sarcoma (FUS), causing it to mis-localize to the cytoplasm. FUS mis-localization disrupts its phase separation dynamics and promotes the formation of toxic aggregates, contributing to disease through both LOF and GOF mechanisms. Using purified protein assays, we identified a small RNA oligonucleotide, RNAS1, capable of preventing and reversing FUS aggregation, and utilized it in vivo to test our working hypothesis that preventing FUS mis-localization and aggregation can restore its nuclear function, reduce toxicity, and mitigate motor dysfunction and motor neuron degeneration. Using an adult model of acute astrocyte-restricted mutant FUS (mutFUS) expression, in which astrocytic cytoplasmic aggregation of mutFUS is sufficient to cause motor neuron dysfunction and degeneration (Jensen BK, Glia 2022), we found that RNAS1 treatment reduced cytoplasmic FUS aggregation. One week post-treatment, RNAS1-treated mice exhibited diffuse, punctate mutFUS in GFAP+ astrocytes, compared to saline-treated animals with persistent fibrillar cytoplasmic mutFUS. Nuclear localization of mutFUS also increased in RNAS1-treated mice, starting three days after treatment. These findings suggest that RNAS1 can enter cells, reverse mutFUS aggregation, and partially restore its nuclear localization in vivo.

Despite these improvements in FUS localization, RNAS1 treatment did not prevent motor neuron loss or preserve motor function. This lack of efficacy may be due to suboptimal timing, dosing, or delivery of RNAS1. In vitro studies using primary mouse spinal cord astrocytes expressing human mutFUS (R521G) are ongoing to optimize RNAS1 treatment.

Cassandra Mikkelson, Jefferson, Grad Student, Juliet Goldsmith (JGS)

Neuroinflammation is a natural defense mechanism against pathogens, disease, and injury. However, when prolonged or dysregulated, chronic neuroinflammation caused by hyperactivation of microglia can lead to neuronal damage and death, contributing to the progression of neurodegenerative diseases. Macroautophagy (autophagy) is a recycling system that regulates the clearance of misfolded/aggregated proteins and damaged organelles. Critically, autophagy degrades inflammasomes and damaged cell components to help terminate a pro-inflammatory response in microglia. Our goal is to therapeutically induce microglial autophagy to reduce neuroinflammation associated with disease or injury. However, the current in vitro models of microglia have artificially activated autophagy, so our understanding of the regulatory pathways of microglial autophagy is lacking. To address this critical gap in knowledge, we developed human iPSC-derived microglia (iMicroglia) that express inducible transcription factors (MAFB, CEBPα, IRF8, PU.1, CEBPβ, and IRF5) inserted via a piggyBac-mediated transfection. This model allows for rapid differentiation in under 10 days with over 95% efficiency. The iMicroglia express canonical microglial markers (Iba1, TMEM119, and P2RY12) and display an increase in Iba1 expression upon stimulation with lipopolysaccharide (LPS) via immunofluorescence. The iMicroglia perform phagocytosis and respond to autophagy manipulation as expected: Bafilomycin A1 causes a stall in autolysosome degradation while Rapamycin increases autophagosome biogenesis. We observed an increase in the number of autophagosomes following LPS, which we are further confirming and investigating. Our next steps will continue to validate our microglia model by monitoring cytokine production and calcium activity following LPS stimulation, understanding how this model compares to autophagy in vivo, and identifying the signaling pathways regulating autophagy following immune stimuli in microglia.

Diana Renteria, UPenn, Grad Student, Vanderver Lab (JGS)

Aicardi-Goutières Syndrome (AGS) is a progressive neurodevelopmental disorder caused by overexpression of interferon-alpha (IFNα), a type I interferon, in the blood and cerebrospinal fluid. The neurovascular unit, which includes astrocytes and brain microvascular endothelial cells (BMECs), is implicated in AGS neuropathology by the microvascular phenotype seen in patients and astrocyte production of IFNα. In a mouse model of astrocyte-derived IFNα overexpression, endothelial cells mediate IFNα-induced neuropathology. However, the extent to which the IFNα response in endothelial cells contributes to AGS neuropathology is unclear. I hypothesize that IFNα induces endothelial dysfunction in AGS, and that inhibiting IFNα signaling in endothelial cells will rescue neuropathology in an AGS mouse model. To test this hypothesis, I will use AGS patient-derived induced pluripotent stem cells (iPSCs) to determine whether AGS iPSC-derived BMECs (iBMECs) exhibit endothelial dysfunction intrinsically or when co-cultured with AGS iPSC-derived astrocytes (iAstrocytes). I will evaluate whether iBMEC dysfunction induced by AGS iAstrocytes is due to IFNα by inhibiting IFNα signaling with antibodies against IFNα or IFNAR, the type I interferon receptor, to rescue AGS astrocyte-induced endothelial dysfunction (Aim 1). Next, to determine whether inhibiting type I interferon signaling in endothelial cells is sufficient to rescue AGS neuropathology, I will delete Ifnar1 in an AGS mouse model using Ifnar1 floxed alleles with an endothelial-specific Cre (Aim 2). I expect to uncover a novel role of IFNα in endothelial dysfunction in AGS and highlight endothelial cells as a potential cellular target for future AGS therapies.

Eli Levitt, UPenn, Pre-doc, Chris Bennett Lab (UGT)

Krabbe disease is a pediatric neurodegenerative condition caused by loss of function mutations in the gene encoding galactosylceramidase (Galc), an enzyme needed to metabolize lipids enriched in myelin. Defective lipid catabolism leads to an accumulation of toxic lipid species, which results in demyelination and the formation of pathologic macrophage aggregates called globoid cells. Introducing GALC+ macrophages via hematopoietic stem cell transplant is the current standard of care for Krabbe disease but this requires toxic immune conditioning and has limited engraftment of donor cells in the brain. Using a genetic model of microglia replacement, we previously showed that high engraftment of GALC+ donor monocytes ameliorated disease in a mouse model of Krabbe disease. We therefore set out to identify a method of microglia transplant that i) avoids toxic chemotherapy pretreatment and ii) allows for higher engraftment of healthy donor cells. One potential translationally relevant solution involves pre-conditioning with the CSF1R inhibitor PLX-3397 and then transplantation of inhibitor-resistant engineered donor cells. We have shown that PLX-3397 allows for high donor cell engraftment and low-toxicity, however it has known off-target effects that may negatively impact disease course. Therefore, we compared PLX-3397 to a different CSF1R inhibitor (PLX-5622). Preliminary data suggests that PLX-5622 enables less efficient brain engraftment. Future studies will test extending PLX-5622 treatment and assessing engraftment outcomes in Twitcher mice.

Eliana von Krusenstiern, UPenn, Grad Student, Grinspan & Jordan-Sciutto Lab (SGS)

Approximately half of people with HIV (PWH) experience HIV-Associated Neurocognitive Disorder (HAND). While the majority of PWH in the United States are virally suppressed, the overall proportion of PWH with HAND symptoms remains unchanged. A persistent pathologic feature of HAND is white matter abnormalities, with the duration of antiretroviral (ART) treatment in patients correlating with observed thinning of the corpus callosum, suggesting that ART drugs may contribute to this pathology. We have found that the ART drug elvitegravir (EVG), prevents the maturation of oligodendrocytes (OLs) and remyelination via activation of the Integrated Stress Response (ISR). During ISR activation, stress granules (SGs) often form, sequestering proteins, mRNAs and translation machinery. Here we show that treating differentiating OLs with the ART drugs bictegravir (BIC) and EVG leads to formation of cytoplasmic SGs. Treatment washouts lead to rapid decrease in SG presence, demonstrating their dynamic nature. Co-treatment of these ART drugs with the ISR inhibitor ISRIB and PERK inhibitor PERKi prevents SG formation, indicating the SGs form canonically via the PERK activated ISR. Ex vivo analysis of mice treated with BIC revealed the presence of SGs within OLs of the corpus callosum, and in post-mortem cortical white matter of PWH with and without HAND, we observed increased SG formation in PWH with HAND compared with neurocognitively normal individuals. These findings suggest that SG formation in OLs may contribute to persistent white matter pathology in PWH with HAND, and implicate SGs as a potential therapeutic target for improving outcomes for PWH on ART.

Erin Smith, UPenn, Grad Student, Maday Lab (SGS)

Lysosomal damage impairs proteostasis and contributes to neurodegenerative diseases. In response to lysosomal membrane damage, cells can employ several mechanisms to restore lysosomal integrity. However, cell-type-specific usage of lysosomal repair pathways in neurons versus astrocytes is poorly understood. Here, we compare the response of neurons versus astrocytes to damage induced by LLOMe, a lysosomotropic methyl ester, with a focus on three key pathways: ESCRT-mediated membrane repair, TBC1D15-mediated membrane reformation, and PITT pathway-mediated lipid shuttling. To elucidate cell-type-specific responses, we used a neuron-astrocyte coculture system. Both neurons and astrocytes showed lysosomal damage, marked by galectin-3 recruitment to lumenal lysosomal beta-galactosides, elevated lysosomal pH, and engagement of lysophagy receptors. Despite lysosomal damage occurring in both cell types, astrocytes showed a preferential recruitment of ESCRT repair machinery to damaged lysosomes, as compared to neurons. This enhanced ESCRT recruitment in astrocytes was observed with multiple components of the ESCRT pathway (e.g., CHMP2B, CHMP2A, ALIX and IST1). Moreover, enhanced ESCRT recruitment was conserved in astrocytes isolated from different brain regions (e.g., the hippocampus or cortex). Additionally, the lysosomal membrane reformation pathway regulated by the RAB7-GAP, TBC1D15, was more robustly activated in astrocytes. By contrast, PITT pathway components (e.g., PI4K2A and ORP9) were activated in both cell types. Our data reveal a divergence in how neurons and astrocytes mobilize repair pathways to manage lysosomal damage. These data may reflect differences in lysosomal resilience between astrocytes and neurons and inform therapeutic strategies to correct lysosomal dysfunction in neurodegenerative diseases.

Gabrielle Spagnuolo, Jefferson, Postdoc, Lorraine Iacovitti Lab (PDS) +FLASH

Current ischemic stroke treatments largely focus on exogenous means of neural repair, with endogenous mechanisms being less understood. Here, we examine the cellular and molecular foundation of an endogenous neuroprotective mechanism using in vitro and in vivo models of stroke. We demonstrate that after oxygen-glucose deprivation (OGD) in vitro, dying cortical neurons release ATP to activate microglia. There is a simultaneous increase in released B-NGF and TrkA receptor expression on astrocytes and a consequent downregulation in astrocyte P2Y1 receptors. These critical interactions, mimicked by P2Y1R knockout astrocytes, were further accompanied by a decline in neuronal intracellular calcium levels and enhanced neuronal survival. Similarly, in vivo there is decreased P2Y1R expression in the peri-infarct region compared to the core injury after middle cerebral artery occlusion (MCAO). Additionally, P2Y1R knockout mice have reduced infarct size and cell death compared to wildtype mice after MCAO. Inhibiting microglial activation with minocycline treatment or CSF1R inhibition reverses these rescue mechanisms, increasing infarct size and P2Y1R expression in the peri-infarct region. Together, these results suggest the downregulation of P2Y1R in astrocytes by activated microglia is a critical endogenous neuroprotective mechanism after ischemic injury. By understanding these inherent non-cell autonomous mechanisms and their molecular mediators, it may be possible to improve intrinsic neuroprotection and recovery from stroke.

Geena John, Drexel, Grad Student, Jackson Lab (SGS)

Stroke remains a leading cause of death and disability worldwide. Following ischemic injury, astrocytes exhibit prolonged calcium elevations associated with reactivity. Yet there is a significant knowledge gap regarding the mechanisms regulating calcium termination in astrocyte processes.
Organotypic hippocampal and primary astrocyte cultures were used to investigate the kinetics of calcium clearance, assess calcium clearance protein levels after oxygen-glucose deprivation (OGD; in-vitro stroke model), and examine downstream gene expression and metabolic changes. Plasma membrane calcium ATPase (PMCA) was identified as the primary mediator of calcium extrusion using live-cell calcium imaging. PMCA inhibition significantly altered calcium event frequency, spatial distribution, duration, and clearance compared to other clearance-related pathways.
Following OGD, we observed reduced expression of PMCA isoforms, specifically PMCA2. Overexpressing PMCA2 restored basal astrocytic calcium clearance rates after OGD, suggesting a potential therapeutic avenue to mitigate aberrant astrocytic calcium signaling. Additionally, we identified CREB, a regulator of mitochondrial morphology and network dynamics in astrocytes, as a downstream transcription factor differentially expressed after OGD. Reduction of calcium signals through PMCA2 overexpression suppressed CREB activation, suggesting a direct link between calcium signaling and transcriptional regulation. Furthermore, reducing astrocytic calcium signaling increased infarct volume in an in-vivo photothrombotic stroke model, indicating a functional role for astrocytic calcium in stroke recovery.
Taken together, our findings demonstrate that ischemic stroke impairs calcium clearance in astrocytes through the loss of PMCA2. Restoring PMCA2 attenuates calcium dynamics, alters CREB activation, and unexpectedly, increases infarct size, highlighting a complex role for astrocytic calcium in post-stroke outcomes.

Gregory Perrin, UPenn, Postdoc, Orthmann-Murphy Lab (PDS)

Cortical demyelinating lesions in multiple sclerosis 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 full recovery; we hypothesize that 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 are testing this hypothesis at three scales throughout cuprizone-induced demyelination and recovery. At the cellular level, scRNAseq data show a transient decrease in astrocyte connexin expression after demyelination, while gap junction puncta appear reduced in demyelination and early recovery. In local microcircuits, we will use patch clamp physiology and dye transfer methods to 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.

Hannah Gong, UPenn, Undergraduate, Mariko Bennett Lab (UGT)

Microglia are dynamic resident macrophages of the central nervous system, which exert critical roles in neuroimmune regulation and development. Due to their integral role in neuroinflammation, microglia are implicated in many neurological diseases and represent a promising therapeutic target. However, distinguishing microglia from other myeloid cells remains a major challenge. Genetic tools such as inducible-Cre systems enable controlled, cell-specific labeling and manipulation of genes in vivo. Current “microglia-specific” knock-in models, driven by microglia-associated promoters, often exhibit non-specific recombination in other cells such as macrophages. Here, we validate a novel knock-in/knock-out (KI/KO) inducible-Cre mouse line driven by the microglia-specific Tmem119 promoter. Coupled with a floxed Ai9-tdTomato reporter, tamoxifen-induced activation of Cre in Tmem119+ cells enables identification of recombined microglia. Using these Tmem119CreERT2;Ai9tdTomato mice, we assessed recombination specificity and efficiency in microglia compared to other cell types via immunohistochemistry and flow cytometry. Our KI/KO line exhibits high specificity for microglia with minimal recombination in other cells. Flow cytometry of peripheral organs revealed reporter expression in the bone marrow, suggesting the presence of a Tmem119+ progenitor population. In contrast, recombination in the blood, spleen, and thymus was minimal. Collectively, these findings establish this model as a reliable tool for microglia-specific genetic manipulation. Future experiments in our group will leverage this system to selectively replace diseased microglia with engineered cells and advance microglia-targeted immunotherapies.

Jordan McKinney, UPenn, Grad Student, Mariko Bennett Lab (JGS)

Aicardi-Goutieres syndrome (AGS) is a severe pediatric neurodegenerative disorder characterized by systemic interferonopathy and elevated type I interferon (IFN) signaling, leading to profound white matter degeneration and early childhood morbidity. Current therapeutic approaches have poor central nervous system (CNS) penetration, cause significant side effects, and fail to address the underlying disease mechanisms, highlighting the need for targeted cellular therapies. Mutations in adenosine deaminase acting on RNA 1 (ADAR1) cause accumulation of immunogenic double-stranded RNA (dsRNA), driving excessive interferon (IFN) production. Astrocytes have emerged as key drivers of interferonopathies and neurodegeneration, supported by post-mortem analyses identifying them as primary IFNa producers in AGS and by mouse models overexpressing astrocytic IFNa that recapitulate AGS pathology. Our preliminary findings also demonstrate that astrocyte-specific loss of ADAR1 induces AGS-like pathology, suggesting a critical role for astrocytes in disease progression. Leveraging recent advancements in astrocyte-specific viral targeting and a patient mutation-derived (D1113H) mouse model that recapitulates human AGS pathology, we hypothesize that restoring astrocytic ADAR1 function or reducing aberrant dsRNA and IFN sensing in astrocytes will ameliorate interferonopathy and improve clinical outcomes. Overall, this project, by precisely targeting the molecular drivers of disease within a specific cellular compartment, has the potential to alleviate the debilitating symptoms of AGS while minimizing adverse effects associated with current systemic therapies, as well as provide insights into the biology of astrocyte-driven RNA metabolic dysfunction and innate immune activation.

Joseph Gallegos, UPenn, Grad Student, Orthmann-Murphy Lab (SGS)

Cortical demyelination is a prominent feature of multiple sclerosis (MS) pathology, but the mechanisms underlying cortical demyelination and remyelination are not well understood. Using the cuprizone model, we recently showed that oligodendrocyte regeneration is impaired in the deep cortex. This inefficient replacement correlates with persistent upregulation of Gfap only by deep cortical astrocytes. Based on these observations, I hypothesized that cortical astrocytes undergo spatially and temporally divergent reactive responses following demyelination. To test this, I isolated cortical astrocytes and performed single-cell mRNA sequencing to comprehensively profile their transcriptional state at key time-points: adult mice treated with sham or 4 weeks of cuprizone, or following 2 (early) or 5 (late) weeks of recovery post-cuprizone.

Julia Coakley, Temple, Assistant Scientist, Kang Lab (UGT)

The expressions of Cspg4, Pdgfra, or Matn4 serve as markers of oligodendrocyte precursor cells (OPCs), and their genetic loci have been utilized for OPC-specific mouse genetic engineering. However, the sole use of each locus leads to the unintended additional sampling of other brain cell types, such as pericytes, perivascular fibroblasts, and neurons. To enhance the target cell specificity while expanding the scope of OPC-targeted studies to more complex biological contexts, we generated a novel mouse line that expresses FlpO recombinase in OPCs in a Cre-dependent manner, named NG2-cFlp mice. In NG2-cFlp mice, the transgene also contains sequences of HA-tagged RPL22, placed upstream of FlpO with T2A, by which translating ribosomes can be immunoprecipitated for the subsequent RNA sequencing.

When crossed with tdTomato-expressing Flp reporter (Ai65F) and with Olig2-Cre or Sox10-CreER, the resulting triple transgenic mice expressed tdTomato and HA-RPL22 exclusively in OPCs, without sampling other types of cells. The tdTomato-labeled OPCs differentiated into tdTomato+ pre-mature and mature oligodendrocytes with time, enabling OL lineage tracing. On the other hand, crossing these with Pdgfrb-CreER resulted in tdTomato and HA expression confined to NG2+PDGFRβ+ pericytes. Importantly, the expression of HA-tagged RPL22 remained in NG2+ OPCs and pericytes, respectively, even after a long period of time. This demonstrated that NG2-cFlp mice specifically sample various subsets of NG2-expressing cells, depending on Cre lines utilized. The NG2-cFlpO mice will provide a powerful tool for more advanced OPC fate tracing, morphological analysis, and OPC-specific bulk expression profiling in experimental settings where reliable OPC analyses are challenging.

Julia Kovalski, Drexel, Grad Student, Denise Garcia Lab (SGS)

In response to an enriched environment (EE), synapse number increases and synaptic communication becomes stronger. Astrocytes, glial cells closely associated with synapses, support synaptic function and even participate in synaptic communication. Recently, RNA sequencing studies have identified drastic changes in astrocyte gene expression following novel experience, suggesting astrocytes respond to changes in neuronal activity through alterations in gene expression. We found that following exposure to EE, astrocytes upregulate synapse modifying cues, SPARC and Hevin, demonstrating astrocytes alter their gene expression following experience to promote synaptic plasticity. How these gene expression changes are mediated in response to experience is not well understood. We identified a novel astrocyte-specific gene, Luzp2, that encodes a leucine zipper protein, a class of proteins known to be involved in regulating gene expression. Although GWAS studies suggest this gene is associated with cognitive function, its precise role in cellular function is completely unknown. We found that Luzp2 is expressed specifically in cortical astrocytes. Mice exposed to EE for two hours show a significant increase in Luzp2 expression in cortical astrocytes, suggesting rapid upregulation in response to experience. Interestingly, the regulatory network prediction tool, SCENIC, identified Hevin and the glutamate transporter, GLAST, as regulated by Luzp2. This, in combination with our finding that Hevin and SPARC expression increase following sensory stimulation, suggests LUZP2 may regulate expression of experience-dependent genes that promote synaptic plasticity. Ongoing work using viral-mediated knock down of Luzp2 in cortical astrocytes aims to investigate the role of Luzp2 in regulating activity-dependent gene expression in astrocytes.

Junyoung Shin, UPenn, Grad Student, Grinspan and Jordan-Sciutto Labs (SGS) + FLASH

Approximately 30-50% of people with HIV (PWH) are affected by HIV-associated neurocognitive disorders (HAND) that are strongly linked to white matter abnormalities. In this study, primary mixed glial cultures from neonatal HIV-1 transgenic (Tg) rats showed reduced oligodendrocyte precursor cell (OPC) numbers and potentially impaired differentiation into mature OLs compared to wild-type (WT) cultures. While MBP expression remained unchanged, CNP—an essential myelin enzyme—was significantly decreased. We also observed reduced expression of low-density lipoprotein receptor (LDLR), a key mediator of lipid uptake for myelination. Similarly, WT OPCs treated with an antiretroviral therapy (ART) drug combination (tenofovir disoproxil fumarate and emtricitabine) showed decreased LDLR fluorescence intensity. These findings suggest that HIV and ART disrupt OL maturation and myelin lipid homeostasis, potentially contributing to white matter deficits in HAND. Ongoing studies are examining how HIV and ART impair astrocyte-to-OL lipid transport, a critical pathway for myelin synthesis. Understanding these mechanisms could inform therapeutic strategies to improve cognitive outcomes in PWH.

Kei Katsura, CHOP, Postdoc, Mariko Bennett & Elizabeth Bhoj Labs (PDS)

BANDDOS (Brain Abnormalities, Neurodegeneration, and Dysosteosclerosis) is a rare autosomal recessive syndrome caused by biallelic mutations in CSF1R and is characterized by severe neurodevelopmental, skeletal, and craniofacial abnormalities. While the role of CSF1R in brain development and bone remodeling is well-established, its function in tooth morphogenesis and epithelial organization remains unknown.
Here, we summarize the dental phenotypes in BANDDOS patients and pair these findings with deep phenotyping of the Csf1r-/- mouse model, uncovering a previously unrecognized requirement for CSF1R signaling in dental development. Children with BANDDOS can present with hypomineralized enamel, shortened or missing roots, and delayed tooth eruption. Parallel analysis in the mouse revealed that although tooth formation occurs, these teeth fail to erupt, in addition to exhibiting delayed mineralization and abnormal morphogenesis.
Using a GFP-labeled bone marrow transplant model, micro-computed tomography, and histology, we show that macrophage engraftment supports tooth eruption, possibly in organizing key structures. Furthermore, our findings suggest that macrophages provide key signaling molecules implicated in epithelial-mesenchymal interactions. Notably, we demonstrate that neonatal bone marrow transplant partially rescues both eruption and morphogenesis, indicating that tissue-resident macrophages are indispensable for normal dental architecture.
This work reveals a novel role for CSF1R in coordinating immune-epithelial crosstalk during tooth development and highlights new aspect of the BANDDOS phenotype, underscoring the importance of macrophage-driven regulation in craniofacial organogenesis.

Khalil Rust, Jefferson, Grad Student, Juliet Goldsmith (JGS)

Alzheimer’s Disease (AD) is the leading cause of dementia worldwide, characterized by progressive memory loss due to neuron death. The most common genetic risk factor for AD is the Apolipoprotein E 4 (APOE4) variant. APOE is responsible for cholesterol transfer between cell types. Although cholesterol is necessary for neuronal homeostasis, neurons do not synthesize their own cholesterol. Instead, astrocytes supply neurons with cholesterol. APOE is expressed highly in astrocytes and not expressed in neurons. In astrocytes, APOE4 leads to impairments in autophagy, the multi-step cellular degradation process resulting in the degradation of proteins and organelles in the lysosome. We hypothesize that the cholesterol accumulated in APOE4 astrocyte lysosomes contributes to the impaired autophagy. To further investigate the changes to lysosomal degradation and signaling and its impact on autophagic regulation downstream of cholesterol accumulation, we are developing an in vitro iPSC-derived system of APOE4 astrocytes and glutamatergic neurons. Our astrocytes express GFAP, S100b, GLAST and ALDHL1 by day 21. This model allows us to monitor the function and survival of the isogenic cell types individually and in co-culture. We will assess (1) whether APOE4 leads to changes in the lysosomal processing of cholesterol in astrocytes, (2) how this alters the biogenesis and degradation of autophagy cargo in astrocytes, and (3) the downstream effects on astrocyte metabolic function and neuronal viability. We will use immunocytochemistry and western blot to determine interactions between APOE4 and lysosomes, as well as the fluorescent cholesterol analog Bodipy to visualize cholesterol in astrocytes. Astrocyte conditioned media experiments will be used to determine variant impact on neuronal health.

Leanne Holt, Mount Sinai, Instructor, Eric Nestler Lab (incoming Temple Faculty) +FLASH

Drug addiction is characterized by neurobiological adaptations that support a shift from goal-directed behaviors to habitual, compulsive drug-seeking with persistent effects on cognition and decision-making. To better understand its biological underpinnings, investigations of the transcriptional response to drugs of abuse have demonstrated lasting changes in gene expression throughout the brain’s reward circuitry. Despite increasing evidence of the role of astrocytes in neuropsychiatric disorders, including addiction, the astrocyte-specific transcriptome and its regulation following exposure to drugs of abuse have not yet been investigated. We performed RNA-sequencing on whole-cell sorted astrocytes in the nucleus accumbens (NAc), a key brain region involved in reward-processing, following cocaine self-administration, withdrawal, and “relapse” in mice and determined that astrocytes exhibit a robust and context-specific transcriptional response, including context-specific transcriptional signatures. Bioinformatic analysis revealed CREB as a highly ranked predicted upstream regulator and CUT&RUN-sequencing identified increased astrocytic CREB DNA binding in response to cocaine. Viral-mediated manipulation of CREB activity selectively in NAc astrocytes in combination with addiction related behaviors reveals that astrocytic CREB increases the rewarding and reinforcing properties of cocaine. This effect is sex-specific, with no change in preference found in females. Finally, we identified potential molecular mechanisms of astrocytic CREB’s influence through modulating astrocytic calcium dynamics and selectively increasing D1-type medium spiny neuronal activity. Together, these data demonstrate that the astrocyte transcriptome responds robustly to cocaine administration and indicates, for the first time, that CREB is a cocaine-induced transcriptional regulator in astrocytes, with implications on neuronal activity and the rewarding properties of cocaine.

Lucy Maxim, University of Delaware, Predoc, Jaclyn Schwarz (UGT)

Hypoxic-ischemic encephalopathy (HIE) is one of the leading causes of neonatal fatalities worldwide. The only current treatment, therapeutic hypothermia (TH), has many limitations. First, it must be performed within 6 hours of birth, and even then, it's not effective at preventing disabilities and all fatalities. Even with TH, 30% of severe cases still end in fatalities, with male neonates having an increased risk of severe HIE than their female counterparts. Another risk factor for HIE is maternal immune activation (MIA), with nearly 40% of HIE events being associated with MIA. Due to the ineffectiveness of TH for treating severe HIE and its limited availability, there have been many attempts to find an alternative treatment using various animal models and molecules that target immune activation. Among such attempts, amphiregulin has been proposed as an alternative to TH. To better understand the potential impacts of amphiregulin on the neonatal brain, BV2 cell culture experiments were performed.
Our experimental model used two main groups: normoxia and hypoxia. Out of those two groups, half were exposed to lipopolysaccharide (LPS) to stimulate the immune system, simulating MIA. Subsequently, the hypoxia group went through a 6-hour-long hypoxic event approximately 24 hours after LPS exposure. Amphireuglin was administered at varying timepoints for both groups, with some not receiving the drug, some receiving it at the start of the hypoxic event, some receiving it 4 hours into the event, some receiving it 6 hours into the event, and some receiving it 4 hours after the end of the event. These time points aim to explore the anti-inflammatory effects of amphiregulin for the potential treatment of HIE with associated immune activation and to determine the optimal treatment window.

Madison Sangster, UPenn, Grad Student, Mariko Bennett Lab (JGS)

Microglia are resident macrophages of the central nervous system and are the only replaceable cells in the brain, making them prime targets for treating neurological disorders that are not fully responsive to pharmacologic interventions. Recent research has demonstrated the feasibility of transplanting macrophages into the brain to replace microglia. These cells can be engineered to express synthetic receptors, but the extent to which these receptors can alter the transcriptional and functional states of macrophages is not well understood. Different intracellular domains of engineered receptors affect how these receptors are expressed and function within the cell, which can be shown using the domains of known transmembrane receptors. Exploring the differing effects of various receptors in macrophages will further our understanding of how we can harness and engineer these cells to replace microglia.

Marisa Jeffries, CHOP, Postdoc, Grinspan Lab (PDS)

Human immunodeficiency virus (HIV)-associated neurocognitive disorders affect 30-50% of people with HIV (PWH) and associate with white matter pathologies. The HIV-1 transgenic (Tg) rat is a noninfectious model of HIV neuropathology which exhibits an altered transcriptome suggestive of disrupted myelination. Myelin is highly enriched in lipids, which are critical for appropriate myelin structure and function. Interestingly, disrupted brain lipid metabolism results in myelin abnormalities and is predictive of cognitive decline in HIV. We hypothesized that HIV-1 disrupts glial lipid metabolism, impairing myelin integrity and function. Immunoblotting for myelin proteins in 3 or 9 week-old control and HIV-1 Tg striatum, cortex, hippocampus, and callosa indicated no changes in expression of major myelin proteins. However, expression of fatty acid synthase was significantly decreased in HIV-1 Tg cortex and striatum at 3 and 9 weeks. To assess myelin lipid composition, we purified whole brain myelin at 3 and 9 weeks. While lipidomics on whole brain myelin extracted at 3 weeks indicated no significant changes in the myelin lipidome, myelin extracted at 9 weeks of age showed significant increases across a range of lipid classes in the HIV-1 Tg, particularly in diacylglycerols with specific increases in ceramides, phospholipids, and lysophosphatidylcholines. Likewise, total cholesterol content was unchanged at 3 weeks, but significantly elevated at 9 weeks, suggestive of an acquired dysregulation in myelin lipid metabolism. If changes in myelin lipids result in impaired myelin structure and function, lipid metabolism may be a promising therapeutic target to improve white matter integrity and cognitive function in PWH.

Maya Bassuk, Jefferson, Medical Student, Elizabeth Jin-Wright Lab (JGS)

Neonatal hypoxic-ischemic encephalopathy (HIE) is a leading cause of brain injury in newborns, often resulting in permanent disability or death. Microglia, the brain’s resident immune cells, are key mediators of the neuroinflammatory response in HIE. While global microglial ablation worsens outcomes in animal models, suggesting protective functions, emerging evidence indicates that distinct microglial subpopulations may play divergent roles in injury, regeneration, and neurodevelopment.
Using single-cell RNA sequencing in a novel mouse model of HIE combined with maternal immune activation (HIE/MIA), we identified three microglial subclusters significantly associated with hypoxic injury. Two of these subclusters showed enriched expression of genes involved in nervous system development, including axonogenesis and neuronal motility.
Among the differentially expressed genes, SOX11 emerged as a highly expressed marker of these subpopulations. We validated SOX11 expression using RNAscope and immunohistochemistry, supporting its reliability as a marker for HIE-associated microglia in the neonatal mouse brain. Preliminary analyses suggest that SOX11+ microglia may display regional and morphological differences in P8 MIA/HIE brains compared to controls, pointing to functional heterogeneity within the microglial response to injury.
These findings support the existence of specialized microglial populations in neonatal HIE and highlight SOX11 as a promising marker for further investigation. Ongoing studies aim to define the roles of these subpopulations in neuroinflammation and repair, with the goal of identifying novel therapeutic targets for neonatal brain injury.

Nehalee Surve, UPenn, Grad Student, Vanderver Lab (JGS)

Microtubules (MTs), composed of α-β tubulin heterodimers, are essential for neuronal function, intracellular trafficking, and myelination. Mutations in tubulin genes cause a range of neurological disorders, including leukodystrophies—rare genetic diseases involving abnormalities in brain white matter. Heterozygous missense mutations in the TUBB4A gene result in TUBB4A-related leukodystrophy (TUBB4A-LD), with a clinical spectrum from isolated hypomyelination to severe Hypomyelination with Atrophy of the Basal Ganglia and Cerebellum (H-ABC). Milder TUBB4A-LD variants result in isolated hypomyelination with no cerebellar atrophy. Conversely, the recurrent TUBB4AD249N variant causes H-ABC, resulting in severe dystonia, loss of previously acquired motor skills, and early childhood mortality. H-ABC impacts oligodendrocytes, striatal neurons, and cerebellar granule neurons (CGNs). While CGN loss is a hallmark feature of H-ABC, the impact of the toxic gain-of-function Tubb4aD249N variant on CGN death remains unclear. Our studies employ novel mouse models to examine the cellular impact of specific TUBB4A-LD variants, revealing differences in neuronal vulnerability. Using high-resolution imaging and biochemical approaches, we assess alterations in MT dynamics and trafficking. Although a previously developed antisense oligonucleotide therapy effectively mitigates certain pathological features, it fails to prevent CGN loss—highlighting the need for more targeted interventions. To address this, we will test whether CGN–specific gene suppression can mitigate CGN loss and improve functional outcomes. By defining cell-type–specific mechanisms and testing targeted therapies, my work aims to advance treatment strategies for TUBB4A-LD and related tubulinopathies.

Nikolas Kinney, Jefferson, Grad Student, Hristelina Ilieva Lab/Weinberg ALS Center (SGS)

Amyotrophic Lateral Sclerosis (ALS) is a devastating, uniformly lethal progressive neuromuscular disease. Current pharmacological management of ALS aims to slow disease progression, extending life by ~2-6 months. However definitive identification of ALS is an imperfect diagnosis of exclusion that causes on average a 10-16 month delay from first symptom to final diagnosis.

Our study aims to identify an early stage ALS biomarker based upon TDP-43 regulated RNA splicing. Cytoplasmic aggregation and dysfunction of TDP-43, a hallmark of ALS pathology, can cause non-canonical splicing of various RNA transcripts. Current effort to utilize these transcripts, termed cryptic exons (CEs), as a biomarker have been extensive in cerebrospinal fluid, with less success in peripheral blood. Cryptic splicing is a rare event even in a disease state. Thus, we hypothesized that RNA targets of TDP-43 that are highly and ubiquitously expressed will have a greater abundance of CEs generated in disease, and therefore have more utility as a blood biomarker. We compiled previously identified RNA targets of TDP-43 and stratified them by basal expression level. We identified CEs in the Vimentin gene that can differentiate between ALS and controls in peripheral blood. Post-mortem imaging shows a subset of astrocytes in both genetic and non-genetic forms of ALS that upregulate the Vimentin protein. This suggests that astrocytes in disease could potentially be producing CEs in Vimentin which are usable as an ALS blood biomarker. We are actively working to confirm this hypothesis.

Nikole Fandino Pachon, CHOP, Lindsay Festa Lab, Research Assistant (UGT)

Myelin, in the CNS, is generated by oligodendrocytes and is crucial for proper and efficient neuronal signaling. Oligodendrocytes mature from oligodendrocyte precursor cells (OPCs) in a highly regulated process that begins at birth. However, the intrinsic regulation of these events, especially from an organelle standpoint, remain inefficiently understood and require further study. Previous studies have demonstrated an essential role of lysosomes in oligodendrocyte differentiation. The lysosome is a significant source of ions for the cell, and mutations to many of its non-selective cation channels result in several lysosomal disorders characterized by hypomyelination. Additionally, lysosomes participate in the newly described contact sites with mitochondria that have shown to aid in the maintenance and promotion of cellular events such as metabolic signaling and mitochondrial fission. Moreover, mitochondria also play a role in driving the differentiation of the oligodendrocyte lineage. Therefore, we hypothesized that mitochondria and lysosomes promote oligodendrocyte maturation through calcium exchange via these contact sites. To test this, we used primary rat oligodendrocytes to first investigate mitochondrial and lysosomal spatial and temporal localization across oligodendrocyte differentiation. We observed mitochondrial expansion occurring primarily in the processes of oligodendrocytes and increased mitochondrial and lysosomal velocity during day 1 of differentiation. However, further experiments such as the immunohistochemical identification of contact sites, and myelin sheath formation assessment during inhibition or activation of ion channels, are needed to better clarify the spatial and temporal activity of the mitochondrial and lysosomal crosstalk.

Nishell Savory, Jefferson, Grad Student, Ramesh Raghupathi Lab (SGS)

Abusive head trauma (AHT), characterized by mild, repetitive brain injury, accounts for 40% of TBIs in infants and is often associated with severe long-term deficits or even death. One major risk factor for AHT is lower socio-economic status, which is associated with early life stress (ELS). ELS exposure during childhood has been shown to alter brain development and may contribute to chronic behavioral problems such as psychiatric and cognitive deficits. This study aimed to develop a clinically relevant model of AHT in infant rats that incorporated both repeated mild TBI and ELS. Forty-one neonate male and female Sprague Dawley rats, along with their dams, were exposed to limited bedding and nesting (LBN) from post-natal days 2-14 and underwent either repeated mild TBI (n=22) or sham injury (n=19) on post-natal days 11, 12, 13. We found that animals exposed to both mild repetitive TBI and LBN showed a robust increase in microglia levels in the cortex and corpus callosum. Traumatic axonal injury was evident in the cortex, white matter, and thalamus, and persisted for up to 8 days post-injury. Additionally, in the hippocampus, we found that animals exposed to both mild repetitive TBI and LBN showed increased levels of IL1β expression, whereas animals exposed to either TBI or LBN independently showed decreased expression, though not significant. These data provide evidence of a working model of AHT in the neonatal rat, which will allow us to investigate further the behavioral deficits as these rats age into adolescence and adulthood.

Ryan Anderson, UPenn, Grad Student, Chris Bennett and Michael Haney Labs (JGS)

Alzheimer’s Disease (AD) affects more than 55 million people worldwide and AD risk is heavily influenced by variants of the gene apolipoprotein E (APOE). A recent case study highlighted a homozygous carrier of a rare APOE-R136S Christchurch (APOE3ch) variant that was protected against AD. APOE3ch has since been shown to alter microglia response to AD pathology. Whether the mechanism of APOE3ch protection occurs through the APOE expressed solely by microglia has yet to be elucidated. In this work, I employ a novel method of murine microglia replacement to establish APOE3ch expressing microglia-like-cells in the murine brain and assess whether the protective effects of APOE3ch can be phenocopied in a microglia specific manner. Using previously established methods of hematopoietic stem cell (HSC) editing and clonal expansion, we have the ability to establish APOE-modified cells in the brain of 5XFAD mice to assess the effect of rare variants on AD related phenotypes. The combination of microglia replacement and the recent innovations of HSC gene editing, permits the generation of microglia that carry compound variants containing multiple protective alleles, as well as tagged constructs to enhance clarity of downstream readouts. If successful, this work will discern the role of microglial APOE in AD, and lead to advances in microglia replacement as a tool for studying genetic variants in microglia.

Ryan Rahman, UPenn, Grad Student, Mariko Bennett Lab (JGS) +FLASH

Brain diseases are the second leading cause of death and the number one cause of disability worldwide. Inflammation of the central nervous system (CNS) underlies most neurological diseases and is mediated in large part by the innate immune cells that call the CNS home: microglia. As the brain’s friendly (and sometimes not so friendly) neighborhood cleanup crew, microglia mold the extracellular environment and can respond to (or propagate) disease in many ways. A striking example of this is the development of microglial aggregates. In lysosomal storage disorders, Rasmussen’s encephalitis, viral encephalitides, and neurodegenerative diseases, microglia clump together in regions of high pathology, forming a wide variety of multicellular masses whose roles are not well understood. Leveraging the advent of spatial transcriptomics RNA sequencing with subcellular 2m x 2m resolution using 10X Visium HD technology, we characterize the composition of multinucleate, lipid-laden “globoid cells”, the pathological hallmark of globoid cell leukodystrophy. We describe components of pathologic microglial aggregates, identify transition markers from healthy to diseased tissue, isolate ligand-receptor pairs, and establish subcellular spatial transcriptomics as a broader tool for deciphering complex multicellular biological phenomena.

Salma Mami, Temple, Medical Student, Shin Kang Lab (JGS)

The deposition of amyloid-β (Aβ) plaques and accompanying neuroinflammation are central features of Alzheimer’s disease (AD). While interleukin-33 (IL-33), a glial cytokine in the CNS, is thought to influence microglial phagocytic activity, the role of IL-33 from distinct glial sources in AD pathology is not well understood. Oligodendrocyte (OL)-specific RNA sequencing revealed that OLs produce increasing levels of IL-33, as they age. OL-specific IL-33 deletion in Mog-Cre mice confirmed that OLs are the predominant source of IL-33 in the aged brain.

To explore the impact of OL-derived IL-33 on microglial phenotype and Aβ accumulation, we introduced OL-specific IL-33 cKO to APP/PS1 mice. We found that IL33 cKO significantly increased Aβ plaque deposition in the cerebral cortex and hippocampus of 12-month-old mice. Meso scale detection analysis of Aβ1-40 and Aβ1-42 showed that the levels of both Aβ species were markedly elevated in the cKO mice. Importantly, microglial occupancy, migration, phagocytic engulfment of Aβ, and plaque compaction were not altered by OL IL-33 cKO, indicating that the increased Aβ levels did not result from impaired microglial Aβ clearance. However, OL-specific IL-33 deletion led to a more inflammatory microglial phenotype, characterized by elevated intracellular ASC specks, and exacerbated neurite dystrophy. Notably, when IL-33 was deleted from astrocytes using ALDH1L1-CreER mice, there were no significant changes in Aβ levels.

Our results suggest that OL-derived IL-33 suppresses Aβ plaque accumulation by negatively regulating microglial inflammatory signaling, rather than by enhancing microglial phagocytic clearance. In contrast, astrocyte-derived IL-33 seems to have minimal effects on AD pathology.

Samar Oubarri, Temple, Predoc, Shin Kang Lab (UGT) +FLASH

Apolipoprotein E (APOE) regulates lipid transport and exists in three common human isoforms: ε2, ε3, and ε4, with ε4 (APOE4) conferring the greatest genetic risk for Alzheimer’s disease (AD). Although astrocytes are considered the principal source of APOE in the brain, oligodendrocytes (OLs) increase APOE expression with age. The functional role of OL-derived APOE, however, remains unclear. To study APOE trafficking from OLs, we generated adeno-associated viruses (AAVs) encoding HA-tagged human APOE3 or APOE4 and mCherry under Cre control, linked via either IRES or T2A elements. AAVs were delivered to the cortex and hippocampus of MOBP-iCre;EGFP mice for OL-specific expression. At two weeks post-injection, mCherry localized exclusively to EGFP+ OLs, whereas HA-APOE3 and HA-APOE4 signals from IRES constructs were also detected in neurons, suggesting that the APOE initially expressed by OLs was being secreted and transported to other cell types. T2A constructs, which produced APOE–mCherry fusion products, showed diffuse labeling across OLs, extracellular space, and neurons, further supporting intercellular transfer. For both APOE3 and APOE4, transfer increased between 2 and 4 weeks post-injection. No transfer was detected when constructs were expressed in astrocytes, demonstrating that rapid glia-to-neuron trafficking is unique to OLs. Notably, at 2 weeks post-injection, APOE4 exhibited greater neuronal transfer than APOE3 in the cingulate cortex, whereas both variants showed comparable transfer in the somatosensory cortex. These results identify a previously unrecognized mechanism of OL-to-neuron communication mediated by APOE. The variant- and region-specific trafficking patterns may provide new insight into how OLs contribute to AD pathogenesis.

Serena Chen, UPenn, Grad Student, Berger Lab (SGS)

Diet and alcohol consumption are major sources of blood acetate, which can pass through the blood brain barrier (BBB) and induce various changes in the brain. Even under normal homeostatic conditions, exogenous acetate exposure alters the epigenetic landscape of the brain. Specifically, acetate is rapidly metabolized and incorporated into histone acetylation to drive gene expression. This acetate-induced gene expression enhances long-term memory formation across models of alcohol use disorder and Alzheimer’s disease. Importantly, this process depends on a metabolic-epigenetic enzyme, acetyl-CoA synthetase 2 (ACSS2), which converts acetate into acetyl-CoA for histone acetylation in the nucleus. However, whether ACSS2 acts in specific cell types to influence transcription and behavior is unknown. Astrocytes are central regulators of metabolism and are poised at the BBB as first responders to metabolites such as acetate entering the brain. Acetate is also preferentially taken up by astrocytes. Here, we show high expression and strict nuclear localization of ACSS2 in astrocytes. We hypothesize that astrocytes utilize exogenous acetate to drive ACSS2-dependent histone acetylation, which induces transcriptional programs related to learning and memory. Indeed, we have observed through RNA-sequencing that acetate exposure induces over 800 differentially expressed genes in primary cultured astrocytes, including astrocyte activity and signaling genes. This effect is attenuated in the presence of an ACSS2 inhibitor, suggesting ACSS2 is a positive regulator of acetate-induced transcriptional programs in astrocytes. Our work highlights a previously unknown mechanism for how astrocytes modulate gene expression in response to changes in their external environment, possibly contributing to ACSS2-dependent behavioral phenotypes.

Sidney Smith, UPenn, Grad Student, Orthmann-Murphy (SGS) +FLASH

Adult-onset leukoencephalopathy with axonal spheroids and pigmented glia (ALSP) is a rare, autosomal-dominant neurodegenerative disorder characterized by progressive psychiatric, motor, and cognitive impairment. White matter pathology has patchy regions of demyelination, loss of microglia and axonal spheroids. ALSP is caused by pathogenic variants in CSF1R, which is necessary for myeloid-lineage cell survival. It is not well understood how mutant CSF1R expression in circulating monocytes and microglia leads to CNS damage. Due to heterogeneous symptoms and age of onset, ALSP is often misdiagnosed, can be progressively fatal, and there is no FDA-approved disease modifying therapy. There is an unmet need for early diagnosis and development of treatments.
To define a distinct immune signature in ALSP monocytes, we use flow cytometry to quantify monocyte phenotype, CSF1R cell surface expression and response to ligands, and compare to healthy control monocytes. To date, we have defined a unique signature in a rare patient with biallelic CSF1R variants, and I will compare this to our ALSP samples. In parallel, to determine whether reduced Csf1r signaling in microglia alters recovery from demyelination, I treated adult Csf1r-haploinsufficient mice with cuprizone to induce demyelination, and then allowed recovery for two or five weeks. After two weeks of recovery, there were fewer Iba1+ microglia and more ASPA+ oligodendrocytes compared to wild type. Based on this preliminary data, Csf1r-haploinsufficient microglia alters oligodendrocyte recovery from demyelination. In ongoing experiments, I am defining the reactive state of Csf1r-haploinsufficient mice exposed to cuprizone.

Simran Gill, Drexel, Grad Student, Mortensen Lab (SGS) +FLASH

Ischemic stroke is one of the leading causes of death in the United States, however, effective treatments to improve patient outcomes are still lacking. After stroke, cessation of blood flow to the brain prevents oxygen and glucose delivery, leading to cell death, primarily through glutamate-induced excitotoxicity, which involves excess release of extracellular glutamate, over-activation of glutamate receptors and calcium-mediated cell death pathways. 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, implicating them in a wide variety of neurological disorders associated with glutamatergic dysregulation, such as ischemic stroke. However, the regulatory response of these transporters following ischemic insult is not well defined. Therefore, an understanding of transporter response to and trafficking after ischemia can provide a novel strategy for therapeutic targeting. In this work, we report aberrant trafficking of GLT-1 that is due to an increase of transporter internalization and degradation via the proteasome that coincides with a decrease in glutamate uptake capacity. With a focus on post-translational modifications (PTMs), we find that the ubiquitination and sumoylation of the transporter is increased after ischemic insult and may play a role in transporter internalization as we are able to rescue transporter surface expression and restore glutamate uptake by preventing these PTM interactions. Future studies will determine if the inhibition of these PTMs in an ex-vivo model of ischemic injury is able to confer neuroprotection, paving the way for potential therapeutic strategies.

Steve Rutledge, Drexel, Grad Student, Jackson Lab (SGS)

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 (MBM) 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, astrocytes can become reactive in a process called astrogliosis, displaying altered morphologies and translational changes. Importantly, reactive astrocytes surround the tumor in the setting of brain metastases.

In many pathologies, reactive astrocytes show altered calcium signaling, but the influence on MBM remains understudied. Using an ex-vivo MBM model, we demonstrate that peri-lesional astrocytes exhibit increased frequency, duration, and amplitude of calcium events. Additionally, our preliminary analyses suggest: 1) Attenuating calcium activity in-vivo results in increased tumor volumes 2) Intercellular calcium events occur between astrocytes and melanoma.

Given that changes in calcium signaling can influence astrocyte secretion, we profiled astrocyte conditioned media (ACM) from reactive astrocytes (A1 and A2) via mass spectrometry. Analysis of the differentially abundant proteins revealed an increase in mitochondrial proteins in A2-ACMs. Two-photon microscopy confirmed the presence of active, polarized mitochondria in our A2-ACM and revealed mitochondrial transfer from astrocytes to melanoma in-vitro and ex-vivo.

Sydney Mason, UPenn, Grad Student, Haney Lab (SGS)

As the resident immune cells of the brain, microglia play an essential role in neurodevelopment as well as CNS disease progression and degeneration. Microglia, similar to peripheral macrophages, are reliant on the secreted cytokine macrophage colony-stimulating factor (mCSF) for differentiation and survival. This reliance on mCSF, and its receptor CSF-1R, has been exploited in recent years to explore microglia replacement therapy in mouse models of disease. This method uses PLX-3397 (PLX), a CSF-1R antagonist, which can be administered to mice orally in chow (Lombroso et al 2025). PLX depletes endogenous microglia leaving an available niche for peripherally injected hematopoietic stem cells (HSCs) to fill and become microglia-like in the brain environment. However, if mice are taken off PLX, endogenous microglia will repopulate and out-compete donor cells. To further explore potential methods for microglia replacement, we performed an in vitro CRISPR knock-out screen to identify genes which, when knocked out, confer survival of macrophages in the absence of mCSF. The results from this screen provide potential new targets for the generation of inhibitor resistant cells to be used in microglia-replacement. This platform will also be utilized to understand mechanisms of microglial phenotypes in neurodegenerative disease models such as phagocytosis and lipid accumulation.

Taylor Phillips-Jones, Upenn, Grad Student, Cristancho and Chris Bennett Labs (JGS)

Prenatal hypoxia is characterized by a loss of oxygen or nutrients during the perinatal period. This can lead to severe brain dysfunction known as hypoxic-ischemic encephalopathy (HIE), a leading cause of lifelong neurological disability. How mild prenatal hypoxia leads to persistent neurological dysfunction remains unclear, posing a barrier to developing effective therapies. Our lab addresses this gap using a model of mild, transient late gestation hypoxia in which pregnant dams are exposed to 5% inspired oxygen for 8 hours. This insult decreases juvenile dendritic spine density in corticothalamic neurons and reduces seizure threshold in adulthood despite the absence of neuronal death. Microglia, the brain’s resident immune cells, are key regulators of neurodevelopment and neural activity. They also retain an epigenetic memory of early life insults, leading to maladaptive responses to later challenges. I hypothesize that prenatal hypoxia primes microglia by triggering a proinflammatory response that disrupts neurodevelopment and causes persistent epigenetic reprogramming that impairs microglial regulation of neural hyperactivity. Preliminary data show no change in Iba1+ cell number one hour after prenatal hypoxia exposure, but an increase in microglia with an amoeboid morphology in the hippocampus and basal ganglia compared to normoxic controls. Future studies will investigate changes in microglial gene expression and chromatin accessibility in response to prenatal hypoxia using single-nuclei RNA and ATAC sequencing and determine the effects of depleting microglia during the exposure. This work is foundational to understanding the pathophysiology of mild and preterm HIE and evaluating microglial targeting as a potential therapeutic.

Teshawn Johnson, UPenn, Grad Student, Jordan-Sciutto and Grinspan Labs (SGS)

Oligodendrocytes are vital for myelinating axons for neuronal conductance and stability in the central nervous system (CNS). During early development, oligodendrocyte progenitors (OPCs) undergo differentiation to become mature oligodendrocytes and make myelin. This differentiation requires coordination from other glial cell types such as the microglia. As the immune cells of the CNS, microglia aid in the process of oligodendrocyte differentiation by phagocytosing excess oligodendroglia and secreting factors that aid in differentiation. Recently, a population of microglia that expresses CD11c has been identified and implicated in various neurodevelopmental processes including oligodendrocyte development. CD11c microglia mainly populate white-matter regions during early development and highly express oligodendrocyte-supportive genes like insulin-growth factor 1 (IGF1) and osteopontin. Preliminary data from our lab has shown that co-treatment of OPCs with IGF1 and osteopontin have an additive effect to enhance oligodendrocyte maturation, but the downstream mechanisms remain unknown. These findings suggest that IGF1 and osteopontin work together to enhance oligodendrocyte differentiation. To investigate the mechanisms of CD11c-derived IGF1 and osteopontin, we have generated novel methods of isolating and generating CD11c microglia from postnatal rats and human-induced pluripotent stem cells (hiPSCs). From the hiPSC model, we have confirmed that the CD11c microglia overexpress and secrete higher concentrations of osteopontin than non-CD11c hiPSC microglia. Furthermore, we are the first to demonstrate that we can isolate CD11c-positive microglia from postnatal rat pups. Using these models, we will be able to determine how CD11c microglia promote oligodendrocyte development through IGF1 and osteopontin, as well as compare the effects between species.

Viet Bui, Temple, Grad Student, Shin Kang Lab (SGS)

In response to injuries or diseases , glial cells in the central nervous system (CNS) undergo significant changes in morphology, gene expression, and cellular activities. These responses, called glial reactivity, significantly affect the course of cellular degeneration or post-injury repair of the neuronal network. While the reactive responses of astrocytes and microglia are well documented, the responses of oligodendrocytes (OLs), the myelin-forming CNS glia, have been largely underexplored.
In a preliminary study, OL-specific RNA-Seq with 14-month-old APP/PS1 mice and age-matched control revealed signal transducer and activator of transcription 3 (STAT3) was among the most strikingly upregulated genes in OLs. Anti-phospho-STAT3 (p-STAT3) immunoreactivities also increased in OLs near Aβ plaques. In this study, we showed an increase in STAT3 activation in OLs within the spinal cords of SOD1 (G93A) mice, in the injured spinal cords of rats, and in brains exposed to glutamate excitotoxicity. Thus, STAT3 activation is associated with OL changes across different types of injuries and diseases. To identify the upstream receptor for STAT3, we crossed SOD1 (G93A) and APP/PS1 mice with OL-specific cKO mice of GP130. The disappearance of p-STAT3 signal in crossed mice suggested that GP130 is a key upstream signal transducer for OLs’ STAT3 activation in both models. In SOD1 (G93A) mice with OL-specific STAT3 cKO, we observed a significant increase of p-STAT3 in astrocytes and reduction of cleaved Caspase3 immunoreactivity in OLs, hinting at a signaling network between OLs and astrocytes; and OLs-STAT3 signaling might negatively regulate OL survival in ALS-like conditions.

Wesley Evans, Rutgers, Postdoc, Huda Lab (PDS) +FLASH

Parkinson’s disease (PD) is characterized by the degeneration of dopaminergic nigrostriatal inputs, which causes striatal network dysfunction and leads to pronounced motor deficits. Recent evidence highlights astrocytes as a potential local source for striatal neuromodulation. It is currently unknown how dopamine loss affects striatal astrocyte activity and whether astrocyte activity regulates behavioral deficits in PD. We addressed these questions by performing astrocyte-specific calcium recordings and manipulations using in vivo fiber photometry and chemogenetics. Unilateral dopamine depletion significantly reduced astrocyte calcium responses, which were facilitated by activating astrocyte specific Gi-DREADDs. Further, astrocyte Gi-DREADD activation rescued asymmetric paw usage and moderately improved open field exploratory behavior in dopamine lesioned mice. Although long-term dopamine depletion severely curtailed astrocyte calcium activity, administration of the broad spectrum dopamine antagonist flupenthixol showed only a minor effect of acute dopamine receptor antagonism. To further explore neurotransmitters mediating astrocyte locomotion responses, we co-expressed an astrocyte-specific calcium indicator alongside a neuronal optogenetic activator (hSyn-ChrimsonR). Neuronal optogenetic stimulation evoked robust astrocyte calcium responses, which were reduced by scopolamine, an acetylcholine muscarinic receptor antagonist. Further, restricting ChrimsonR expression to ChAT+ neurons using a Cre-dependent strategy also elicited astrocyte calcium responses during optogenetic stimulation. These results suggest that cholinergic signaling influences neuronal-astrocyte communication in the striatum. In current work, we are exploring how this system may be affected in parkinsonian conditions.

Yixuan Erica Zhu, UPenn, Grad Student, Haney Lab (JGS)

Microglia-derived cytokines IL-1α, TNF, and C1q have been shown to induce A1 astrocytes, a subtype of reactive astrocytes, which lose neuroprotective functions and become neurotoxic. Recently, Lombroso et al. engineered an inhibitor-resistant murine CSF1R variant G793A that enables efficient, nontoxic, and brain-specific replacement of microglia. Harnessing this platform, we propose to engineer hematopoietic stem cells (HSCs) using CRISPR/Cas9 to simultaneously knock out IL-1α, TNF, and C1q. Microglia differentiated from these CRISPR-edited G793A HSCs would engraft robustly, and remain incapable of inducing A1 astrocytes. This dual strategy offers a targeted, safer therapeutic approach: microglia replacement with engineered cells that engraft robustly but will not mediate A1 astrocyte induction. If successful, this platform could hold great potential to treat a variety of neurological diseases.