Isolation of Pure Astrocytes and Microglia from the Adult Mouse Spinal Cord For In Vitro Assays and Transcriptomic Studies.

Astrocytes and microglia play pivotal roles in central nervous system development, injury responses, and neurodegenerative diseases. These highly dynamic cells exhibit rapid responses to environmental changes and display significant heterogeneity in terms of morphology, transcriptional profiles, and functions. While our understanding of the functions of glial cells in health and disease has advanced substantially, there remains a need for in vitro, cell-specific analyses conducted in the context of insults or injuries to comprehensively characterize distinct cell populations. Isolating cells from the adult mouse offers several advantages over cell lines or neonatal animals, as it allows for the analysis of cells under pathological conditions and at specific time points. Furthermore, focusing on spinal cord-specific isolation, excluding brain involvement, enables research into spinal cord pathologies, including experimental autoimmune encephalomyelitis, spinal cord injury, and amyotrophic lateral sclerosis. This protocol presents an efficient method for isolating astrocytes and microglia from the adult mouse spinal cord, facilitating immediate or future analysis with potential applications in functional, molecular, or proteomic downstream studies.

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

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 leading to new treatments for urea cycle disorders.

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

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.

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

BACKGROUND: 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. NEW METHOD: We have developed an efficient and reliable protocol for isolation of astrocytes and microglia from the adult mouse spinal cord, which can be easily manipulated for immediate or future analyses. This method involves (1) rapid tissue dissociation; (2) cell release after myelin debris removal; (3) magnetic-activated cell sorting; and (4) optional downstream molecular and functional analyses. RESULTS: High levels of viability and purity of the cells were confirmed after isolation. More importantly, characterization of cells verified their ability to proliferate and respond to external stimuli for potential use in downstream molecular and functional assays. COMPARISON WITH EXISTING METHOD(S): Long-term culture of cells isolated from neonatal animals and cell type specific isolation from the brain have been successful; however, isolation of spinal cord cells from adult mice has been challenging due to the large amount of myelin and limited size of the tissue compared to the brain. Our method allows for efficient isolation of astrocytes and microglia from spinal cord alone and includes simple modifications to allow for various downstream applications. CONCLUSIONS: This technique will be a valuable tool to better understand the functions of astrocytes and microglia in spinal cord function and pathology.

B Cells in Neuroinflammation: New Perspectives and Mechanistic Insights.

In recent years, the role of B cells in neurological disorders has substantially expanded our perspectives on mechanisms of neuroinflammation. The success of B cell-depleting therapies in patients with CNS diseases such as neuromyelitis optica and multiple sclerosis has highlighted the importance of neuroimmune crosstalk in inflammatory processes. While B cells are essential for the adaptive immune system and antibody production, they are also major contributors of pro- and anti-inflammatory cytokine responses in a number of inflammatory diseases. B cells can contribute to neurological diseases through peripheral immune mechanisms, including production of cytokines and antibodies, or through CNS mechanisms following compartmentalization. Emerging evidence suggests that aberrant pro- or anti-inflammatory B cell populations contribute to neurological processes, including glial activation, which has been implicated in the pathogenesis of several neurodegenerative diseases. In this review, we summarize recent findings on B cell involvement in neuroinflammatory diseases and discuss evidence to support pathogenic immunomodulatory functions of B cells in neurological disorders, highlighting the importance of B cell-directed therapies.