Biology subjects

Bitarafan, S.

Publications and source records attributed to Bitarafan, S..

4 recordsLinked to original sources

bioRxiv2023

Profiling the neuroimmune cascade in 3xTg mice exposed to successive mild traumatic brain injuries

Repetitive mild traumatic brain injuries (rmTBI) sustained within a window of vulnerability can result in long term cognitive deficits, depression, and eventual neurodegeneration associated with tau pathology, amyloid beta (A{beta}) plaques, gliosis, and neuronal and functional loss. However, we have limited understanding of how successive injuries acutely affect the brain to result in these devastating long-term consequences. In the current study, we addressed the question of how repeated injuries affect the brain in the acute phase of injury (<24hr) by exposing the 3xTg-AD mouse model of tau and A{beta} pathology to successive (1x, 3x, 5x) once-daily weight drop closed-head injuries and quantifying immune markers, pathological markers, and transcriptional profiles at 30min, 4hr, and 24hr after each injury. We used young adult mice (2-4 months old) to model the effects of rmTBI relevant to young adult athletes, and in the absence of significant tau and A{beta} pathology. Importantly, we identified pronounced sexual dimorphism, with females eliciting more differentially expressed proteins after injury compared to males. Specifically, females showed: 1) a single injury caused a decrease in neuron-enriched genes inversely correlated with inflammatory protein expression as well as an increase in AD-related genes within 24hr, 2) each injury significantly increased expression of a group of cortical cytokines (IL-1, IL-1{beta}, IL-2, IL-9, IL-13, IL-17, KC) and MAPK phospho-proteins (phospho-Atf2, phospho-Mek1), several of which were co-labeled with neurons and correlated with phospho-tau, and 3) repetitive injury caused increased expression of genes associated with astrocyte reactivity and immune function. Collectively our data suggest that neurons respond to a single injury within 24h, while other cell types including astrocytes transition to inflammatory phenotypes within days of repetitive injury.

neuroscience↗

bioRxiv2022

Cellular proteomic profiling using proximity labelling by TurboID-NES in microglial and neuronal cell lines.

1.0Different brain cell types play distinct roles in brain development and disease. Molecular characterization of cell-specific mechanisms using cell type-specific approaches at the protein (proteomic) level, can provide biological and therapeutic insights. To overcome the barriers of conventional isolation-based methods for cell type-specific proteomics, in vivo proteomic labeling with proximity dependent biotinylation of cytosolic proteins using biotin ligase TurboID, coupled with mass spectrometry (MS) of labeled proteins, has emerged as a powerful strategy for cell type-specific proteomics in the native state of cells without need for cellular isolation. To complement in vivo proximity labeling approaches, in vitro studies are needed to ensure that cellular proteomes using the TurboID approach are representative of the whole cell proteome, and capture cellular responses to stimuli without disruption of cellular processes. To address this, we generated murine neuroblastoma (N2A) and microglial (BV2) lines stably expressing cytosolic TurboID to biotinylate the cellular proteome for downstream purification and analysis using MS. TurboID-mediated biotinylation captured 59% of BV2 and 65% of N2A proteomes under homeostatic conditions. TurboID expression and biotinylation minimally impacted homeostatic cellular proteomes of BV2 and N2A cells, and did not affect cytokine production or mitochondrial respiration in BV2 cells under resting or lipopolysaccharide (LPS)-stimulated conditions. These included endo-lysosome, translation, vesicle and signaling proteins in BV2 microglia, and synaptic, neuron projection and microtubule proteins in N2A neurons. The effect of LPS treatment on the microglial proteome was captured by MS analysis of biotinylated proteins (>500 differentially-abundant proteins) including increased canonical pro-inflammatory proteins (Il1a, Irg1, Oasl1) and decrease anti-inflammatory proteins (Arg1, Mgl2).

neuroscience↗

bioRxiv2022

Systematic delineation of signaling and epigenomic mechanisms underlying microglia inflammatory activity in acute and chronic brain pathologies

Microglia promptly mount an inflammatory response following detection of infectious agents or injuries in the central nervous system. Such function fundamentally depends upon dynamic modulation of gene expression. However, the signaling and epigenomic mechanisms that regulate the transcriptional process underlying microglial inflammatory activity are not well understood. To address this, we used RNA-seq, ChIP-seq and ATAC-seq to delineate gene signatures and activity across the repertoire of genomic regulatory elements of microglia engaged in acute and chronic neuroinflammatory activity. Systematic interrogations of the microglial population over time during a systemic inflammatory response revealed a coordinated, sequential activation of multiple gene programs associated with defense response, translation and cell cycling. Activation of these programs occurred in parallel with gain and loss of activity at 4,080 and 3,119 genomic cis-regulatory elements, respectively. Furthermore, computational analyses identified key transcriptional regulators, including Ets, AP-1, C/epb, Nf-{kappa}B, Irf, Runx, c-Myc and E2f family members, that display differential propensity for activity at gene promoters and promoter-distal cis-regulatory elements. Gene expression analyses also suggested that the transcriptional process likely contribute to the effective activity of numerous transcriptional regulators through the modulation of their mRNA levels. Finally, characterization of CD11c-positive microglia that emerge with chronic demyelinating brain lesions suggested that Egr2, Mef2 members and E-box-binding factors such as Tfeb and Mitf contribute to the enhanced phagosomal activity of this inflammatory subset. Loss-of-function experiments validated that Mef2a in microglia is necessary for the acquisition of the CD11c-positive phenotype. Collectively, these results demonstrate that the inflammatory activity of microglia arises through an intricate, ultimately context-dependent, interplay between signaling pathways, genomic regulatory elements and the transcriptional machinery.

neuroscience↗

bioRxiv2021

Cell type-specific biotin labeling in vivo resolves regional neuronal proteomic differences in mouse brain

Isolation and proteomic profiling of brain cell types, particularly neurons, pose several technical challenges which limit our ability to resolve distinct cellular phenotypes in neurological diseases. Therefore, we generated a novel mouse line that enables cell type-specific expression of a biotin ligase, TurboID, via Cre-lox strategy for in vivo proximity-dependent biotinylation of proteins. Using adenoviral-based and transgenic approaches, we show striking protein biotinylation in neuronal cell bodies and axons throughout the mouse brain. We quantified more than 2,000 neuron-derived proteins following enrichment that mapped to numerous subcellular compartments. Synaptic, transmembrane transporters, ion channel subunits, and disease-relevant druggable targets were among the most significantly enriched proteins. Remarkably, we resolved brain region-specific proteomic profiles of Camk2a neurons with distinct functional molecular signatures and disease associations that may underlie regional neuronal vulnerability. Leveraging the neuronal specificity of this in vivo biotinylation strategy, we used an antibody-based approach to uncover regionally unique patterns of neuron-derived signaling phospho-proteins and cytokines, particularly in the cortex and cerebellum. Our work provides a proteomic framework to investigate cell type-specific mechanisms driving physiological and pathological states of the brain as well as complex tissues beyond the brain.

neuroscience↗