Faculty Bibliography

Studies of the choroid plexuses lag behind those of, the more widely known, blood brain barrier in spite of a much longer history. This review has two overall aims. The first is to outline longstanding areas of research where there are unanswered questions, such as control of cerebrospinal fluid (CSF) secretion and blood flow. The second part reviews research over the past ten years where the focus has shifted to the idea that the choroid plexuses make specific contributions to brain development and function through molecules they generate and circulate throughout the CSF; these appear to be particularly important for aspects of normal brain growth. Most research in the 20^th Century dealt with the choroid plexuses as one of the brain barrier interfaces that make an important contribution to the composition and stability of the internal environment of the brain in the adult and during its development. More recent research has shown the importance of choroid plexus generated CSF in neurogenesis, influence of sex and other hormones on plexus function, and their role in circadian rhythms and sleep. Of clinical importance are attempts to develop methods to deliver brain-specific drugs via the CSF and understanding the implications of drug entry into developing brain when administered to pregnant women.

Microglia and astrocytes play an important role in the neuroinflammatory response and contribute to both the destruction of neighboring tissue as well as the resolution of inflammation following stroke. These reactive glial cells are highly heterogeneous at both the transcriptomic and functional level. Depending upon the stimulus, microglia and astrocytes mount a complex, and specific response composed of distinct microglial and astrocyte substates. These substates ultimately drive the landscape of the initiation and recovery from the adverse stimulus. In one state, inflammation- and damage-induced microglia release tumor necrosis factor (TNF), interleukin 1α (IL1α), and complement component 1q (C1q), together "TIC." This cocktail of cytokines drives astrocytes into a neurotoxic reactive astrocyte (nRA) substate. This nRA substate is associated with loss of many physiological astrocyte functions (e.g., synapse formation and maturation, phagocytosis, among others), as well as a gain-of-function release of neurotoxic long-chain fatty acids which kill neighboring cells. Here we report that transgenic removal of TIC led to reduction of gliosis, infarct expansion, and worsened functional deficits in the acute and delayed stages following stroke. Our results suggest that TIC cytokines, and likely nRAs play an important role that may maintain neuroinflammation and inhibit functional motor recovery after ischemic stroke. This is the first report that this paradigm is relevant in stroke and that therapies against nRAs may be a novel means to treat patients. Since nRAs are evolutionarily conserved from rodents to humans and present in multiple neurodegenerative diseases and injuries, further identification of mechanistic role of nRAs will lead to a better understanding of the neuroinflammatory response and the development of new therapies.

Background: Astrocytes can have helpful or harmful effects on neuron health and brain function in disease. While they normally provide trophic support to neurons during development and normal functioning, in response to many stimuli their heterogeneous "˜reactive"™ responses can alter these functions drastically. Changes in astrocyte function depends on their "˜reactive"™ sub-state. Understanding when and where sub-states of reactive astrocytes occur, and how these altered functions contribute to disease will pave the way for novel strategies to protect neurons. Method: We performed combined 10x genomics single cell and spatial transcriptomics in wildtype and Alzheimer"™s disease (AD) model mice, combined with single nuclei RNA sequencing of human postmortem non-symptomatic and AD patient brains. Results: With improved capture rates and subsequent powering of astrocyte sequencing we highlight lowly abundant, biologically important, reactive astrocyte sub-states that are positioned in strategic locations throughout the brain "“ namely at sites of entry for peripheral immune cells (e.g. adjacent to penetrating vessels in layer I of the cortex, and around the ventricles). Further, we integrate our datasets with previously published scRNAseq and snRNAseq datasets to confirm these small populations exist in other patient populations. Most surprising was that interferon-responsive reactive astrocytes were present early progression of pathology in the 5xFAD mouse AD model, but not at later time points "“ suggesting important early (possibly protective) roles for astrocytes early in AD. Additionally, when comparing mouse and human datasets we find most disease pathology-associated reactive astrocytes are located around strategic points of entry to the brain, and express many inflammation-responsive transcripts. Probing for "˜modules"™ of genes associated with inflammation-response and reactive sub-states of microglia and other immune cells highlights putative interactions likely integral for feedback between these two cell types. Conclusion: Optimization of astrocyte capture for single cell/nuclei sequencing combined with integration of previously published datasets increased the size of datasets for analysis and power of our analysis. Our data highlight several novel reactive astrocyte sub-states that warrant additional functional characterization and further investigation.

Multiple sclerosis (MS) is a central nervous system (CNS) autoimmune disease characterized by inflammation, demyelination, and neurodegeneration. The ideal MS therapy would both specifically inhibit the underlying autoimmune response and promote repair/regeneration of myelin as well as maintenance of axonal integrity. Currently approved MS therapies consist of non-specific immunosuppressive molecules/antibodies which block activation or CNS homing of autoreactive T cells, but there are no approved therapies for stimulation of remyelination nor maintenance of axonal integrity. In an effort to repurpose an FDA-approved medication for myelin repair, we chose to examine the effectiveness of digoxin, a cardiac glycoside (Na⁺ /K⁺ ATPase inhibitor), originally identified as pro-myelinating in an in vitro screen. We found that digoxin regulated multiple genes in oligodendrocyte progenitor cells (OPCs) essential for oligodendrocyte (OL) differentiation in vitro, promoted OL differentiation both in vitro and in vivo in female naïve C57BL/6J (B6) mice, and stimulated recovery of myelinated axons in B6 mice following demyelination in the corpus callosum induced by cuprizone and spinal cord demyelination induced by lysophosphatidylcholine (LPC), respectively. More relevant to treatment of MS, we show that digoxin treatment of mice with established MOG_35-55 -induced Th1/Th17-mediated chronic EAE combined with tolerance induced by the i.v. infusion of biodegradable poly(lactide-co-glycolide) nanoparticles coupled with MOG_35-55 (PLG-MOG_35-55 ) completely ameliorated clinical disease symptoms and stimulated recovery of OL lineage cell numbers. These findings provide critical pre-clinical evidence supporting future clinical trials of myelin-specific tolerance with myelin repair/regeneration drugs, such as digoxin, in MS patients.

COVID survivors frequently experience lingering neurological symptoms that resemble cancer-therapy-related cognitive impairment, a syndrome for which white matter microglial reactivity and consequent neural dysregulation is central. Here, we explored the neurobiological effects of respiratory SARS-CoV-2 infection and found white-matter-selective microglial reactivity in mice and humans. Following mild respiratory COVID in mice, persistently impaired hippocampal neurogenesis, decreased oligodendrocytes, and myelin loss were evident together with elevated CSF cytokines/chemokines including CCL11. Systemic CCL11 administration specifically caused hippocampal microglial reactivity and impaired neurogenesis. Concordantly, humans with lasting cognitive symptoms post-COVID exhibit elevated CCL11 levels. Compared with SARS-CoV-2, mild respiratory influenza in mice caused similar patterns of white-matter-selective microglial reactivity, oligodendrocyte loss, impaired neurogenesis, and elevated CCL11 at early time points, but after influenza, only elevated CCL11 and hippocampal pathology persisted. These findings illustrate similar neuropathophysiology after cancer therapy and respiratory SARS-CoV-2 infection which may contribute to cognitive impairment following even mild COVID.

Resolving glial contributions to Alzheimer's disease (AD) is necessary because changes in neuronal function, such as reduced synaptic density, altered electrophysiological properties, and degeneration, are not entirely cell autonomous. To improve understanding of transcriptomic heterogeneity in glia during AD, we used single-nuclei RNA sequencing (snRNA-seq) to characterize astrocytes and oligodendrocytes from apolipoprotein (APOE) Ɛ2/3 human AD and age- and genotype-matched non-symptomatic (NS) brains. We enriched astrocytes before sequencing and characterized pathology from the same location as the sequenced material. We characterized baseline heterogeneity in both astrocytes and oligodendrocytes and identified global and subtype-specific transcriptomic changes between AD and NS astrocytes and oligodendrocytes. We also took advantage of recent human and mouse spatial transcriptomics resources to localize heterogeneous astrocyte subtypes to specific regions in the healthy and inflamed brain. Finally, we integrated our data with published AD snRNA-seq datasets, highlighting the power of combining datasets to resolve previously unidentifiable astrocyte subpopulations.

Brain metastasis is a significant cause of morbidity and mortality in multiple cancer types and represents an unmet clinical need. The mechanisms that mediate metastatic cancer growth in the brain parenchyma are largely unknown. Melanoma, which has the highest rate of brain metastasis among common cancer types, is an ideal model to study how cancer cells adapt to the brain parenchyma. Our unbiased proteomics analysis of melanoma short-term cultures revealed that proteins implicated in neurodegenerative pathologies are differentially expressed in melanoma cells explanted from brain metastases compared to those derived from extracranial metastases. We showed that melanoma cells require amyloid beta (AB) for growth and survival in the brain parenchyma. Melanoma-secreted AB activates surrounding astrocytes to a pro-metastatic, anti-inflammatory phenotype and prevents phagocytosis of melanoma by microglia. Finally, we demonstrate that pharmacological inhibition of AB decreases brain metastatic burden.

Across neurodegenerative diseases, common mechanisms may reveal novel therapeutic targets based on neuronal protection, repair, or regeneration, independent of etiology or site of disease pathology. To address these mechanisms and discuss emerging treatments, in April, 2021, Glaucoma Research Foundation, BrightFocus Foundation, and the Melza M. and Frank Theodore Barr Foundation collaborated to bring together key opinion leaders and experts in the field of neurodegenerative disease for a virtual meeting titled "Solving Neurodegeneration". This "think-tank" style meeting focused on uncovering common mechanistic roots of neurodegenerative disease and promising targets for new treatments, catalyzed by the goal of finding new treatments for glaucoma, the world's leading cause of irreversible blindness and the common interest of the three hosting foundations. Glaucoma, which causes vision loss through degeneration of the optic nerve, likely shares early cellular and molecular events with other neurodegenerative diseases of the central nervous system. Here we discuss major areas of mechanistic overlap between neurodegenerative diseases of the central nervous system: neuroinflammation, bioenergetics and metabolism, genetic contributions, and neurovascular interactions. We summarize important discussion points with emphasis on the research areas that are most innovative and promising in the treatment of neurodegeneration yet require further development. The research that is highlighted provides unique opportunities for collaboration that will lead to efforts in preventing neurodegeneration and ultimately vision loss.

During neurogenesis, mitotic progenitor cells lining the ventricles of the embryonic mouse brain undergo their final rounds of cell division, giving rise to a wide spectrum of postmitotic neurons and glia^1,2. The link between developmental lineage and cell-type diversity remains an open question. Here we used massively parallel tagging of progenitors to track clonal relationships and transcriptomic signatures during mouse forebrain development. We quantified clonal divergence and convergence across all major cell classes postnatally, and found diverse types of GABAergic neuron that share a common lineage. Divergence of GABAergic clones occurred during embryogenesis upon cell-cycle exit, suggesting that differentiation into subtypes is initiated as a lineage-dependent process at the progenitor cell level.

Astrocytes respond to injury, infection, and inflammation in the central nervous system by acquiring reactive states in which they may become dysfunctional and contribute to disease pathology. A sub-state of reactive astrocytes induced by proinflammatory factors TNF, IL-1α, and C1q ("TIC") has been implicated in many neurodegenerative diseases as a source of neurotoxicity. Here, we used an established human induced pluripotent stem cell (hiPSC) model to investigate the surface marker profile and proteome of TIC-induced reactive astrocytes. We propose VCAM1, BST2, ICOSL, HLA-E, PD-L1, and PDPN as putative, novel markers of this reactive sub-state. We found that several of these markers colocalize with GFAP⁺ cells in post-mortem samples from people with Alzheimer's disease. Moreover, our whole-cells proteomic analysis of TIC-induced reactive astrocytes identified proteins and related pathways primarily linked to potential engagement with peripheral immune cells. Taken together, our findings will serve as new tools to purify reactive astrocyte subtypes and to further explore their involvement in immune responses associated with injury and disease.