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Isolation of Pure Astrocytes and Microglia from the Adult Mouse Spinal Cord For <em>In Vitro</em> Assays and Transcriptomic Studies

Astrocytes and microglia play pivotal roles in central nervous system development, injury responses, and neurodegenerative diseases. These highly dynamic ...

The George Washington University School of Medicine

Astrocytes and microglia are essential cellular elements of the CNS that are critical for normal development, function, and injury responses. Both cell types are highly pleiotropic and respond rapidly to environmental changes, making them challenging to characterize. One approach is to develop efficient isolation paradigms of distinct cell populations, allowing for characterization of their roles in distinct CNS regions and in pathological states.

Cell type specific isolation of primary astrocytes and microglia from adult mouse spinal cord.

Multiple sclerosis (MS) is characterized by a compromised blood-brain barrier (BBB) resulting in central nervous system (CNS) entry of peripheral lymphocytes, including T cells and B cells. While T cells have largely been considered the main contributors to neuroinflammation in MS, the success of B cell depletion therapies suggests an important role for B cells in MS pathology. Glial cells in the CNS are essential components in both disease progression and recovery, raising the possibility that they represent targets for B cell functions. Here, we examine astrocyte and microglia responses to B cell depleting treatments in an animal model of MS, experimental autoimmune encephalomyelitis (EAE). B cell depleted EAE animals had markedly reduced disease severity and myelin damage accompanied by reduced microglia and astrocyte reactivity 20 days after symptom onset. To identify potential initial mechanisms mediating functional changes following B cell depletion, astrocyte and microglia transcriptomes were analyzed 3 days following B cell depletion. In control EAE animals, transcriptomic analysis revealed astrocytic inflammatory pathways were activated and microglial influence on neuronal function were inhibited. Following B cell depletion, initial functional recovery was associated with an activation of astrocytic pathways linked with restoration of neurovascular integrity and of microglial pathways associated with neuronal function. These studies reveal an important role for B cell depletion in influencing glial function and CNS vasculature in an animal model of MS.

B cell depletion modulates glial responses and enhances blood vessel integrity in a model of multiple sclerosis.

Ammonia, which is toxic to the brain, is converted into non-toxic urea, through a pathway of six enzymatically catalyzed steps known as the urea cycle. In this pathway, N-acetylglutamate synthase (NAGS, EC 2.3.1.1) catalyzes the formation of N-acetylglutamate (NAG) from glutamate and acetyl coenzyme A. NAGS deficiency (NAGSD) is the rarest of the urea cycle disorders, yet is unique in that ureagenesis can be restored with the drug N-carbamylglutamate (NCG). We investigated whether the rarity of NAGSD could be due to low sequence variation in the NAGS genomic region, high NAGS tolerance for amino acid replacements, and alternative sources of NAG and NCG in the body. We also evaluated whether the small genomic footprint of the NAGS catalytic domain might play a role. The small number of patients diagnosed with NAGSD could result from the absence of specific disease biomarkers and/or short NAGS catalytic domain. We screened for sequence variants in NAGS regulatory regions in patients suspected of having NAGSD and found a novel NAGS regulatory element in the first intron of the NAGS gene. We applied the same datamining approach to identify regulatory elements in the remaining urea cycle genes. In addition to the known promoters and enhancers of each gene, we identified several novel regulatory elements in their upstream regions and first introns. The identification of cis-regulatory elements of urea cycle genes and their associated transcription factors holds promise for uncovering shared mechanisms governing urea cycle gene expression and potentially lead to new treatments for urea cycle disorders. This article is protected by copyright. All rights reserved.

Datamining approaches for examining the low prevalence of N-acetylglutamate synthase deficiency and understanding transcriptional regulation of urea cycle genes.

Article	Year
Cell type specific isolation of primary astrocytes and microglia from adult mouse spinal cord. Journal of neuroscience methods\| PubMed ID: 35460698	2022
B cell depletion modulates glial responses and enhances blood vessel integrity in a model of multiple sclerosis. Neurobiology of disease\| PubMed ID: 37709209	2023
Datamining approaches for examining the low prevalence of N-acetylglutamate synthase deficiency and understanding transcriptional regulation of urea cycle genes. Journal of inherited metabolic disease\| PubMed ID: 37847851	2023

Julie J. Ahn

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