Biology subjects

Bitarafan, S.

Publications and source records attributed to Bitarafan, S..

10 recordsLinked to original sources

bioRxiv2025

A Feature Learning Model Identifies Predictive Attributes of Mesenchymal Stromal Cell Efficacy

The therapeutic efficacy of human mesenchymal stromal cells (hMSCs) is highly variable, limiting their clinical translation for musculoskeletal diseases and other regenerative medicine applications. There is a poor understanding of the critical quality attributes correlating to therapeutic efficacy of hMSCs. To address this challenge, we analyzed pre-clinical in vitro secretome profiles and in vivo therapeutic efficacy of hMSCs from multiple human donors. hMSCs from different donors showed significant differences between donors in therapeutic efficacy when assessed in a rat post-traumatic osteoarthritis (OA) model. A partial least squares feature learning model was trained to evaluate differences between more and less therapeutic donor hMSCs by examining cytokine secretion profiles, to predict donor-specific therapeutic outcomes. More therapeutic hMSCs exhibited increased secretion of GM-CSF, GRO, IL-4, and PDGF-AA, whereas less therapeutic donors had higher TNF-, IL-6, and MCP-1 secretion. The cytokine profile was accompanied by evaluation of MAPK pathway, which revealed distinct differences in phospho-protein signaling between more and less therapeutic hMSC secretome profiles. Pharmacological inhibition of JNK signaling in more therapeutic donor cells decreased hMSC secretion of the key therapeutic associated cytokines and shifted hMSC secretome towards a less therapeutic profile. Prospective validation of cells from additional donors demonstrated significant correlations between predicted and observed pre-clinical in vivo efficacy to attenuate OA. This approach identifies critical quality attributes enabling consistent prediction of therapeutic potency, thereby addressing a major barrier to scalable and effective cell therapies. These findings advance precision cell-based therapies and offer a framework for standardized donor screening in clinical applications. SummaryA feature learning model was developed, trained, and validated to identify critical quality attributes of MSCs that predict therapeutic potency.

bioengineering↗

bioRxiv2025

Multi-tier signaling and epigenomic reprogramming orchestrate microglial inflammatory states and functions associated with demyelination

The extensive heterogeneity of microglia inflammatory states accompanying neurodegenerative diseases underscores the complex molecular mechanisms that regulate these cells. Here, we report on transcriptional mechanisms that control microglial inflammatory polarization associated with brain demyelination in mice. Using flow cytometry, microscopy and RNA-seq, we identified two dominant, functionally distinct states inflammatory microglia: Clec7a+CD229+CD11c-microglia that are prone to proliferation and that transcribe high levels of extracellular matrix-associated genes, and Clec7a+CD229+CD11c+ microglia characterized by inflammatory tissue-remodeling and antigen presentation gene signatures. Epigenomic analyses implicated genome-wide nucleosome remodeling to the polarization process, driven by state-associated hierarchal stimulation of pro-inflammatory transcription factors, as well as re-calibration of Mef2 homeostatic input. Mechanistically, we confirmed relevance for Trem2, Mef2a and Egr2 to the microglial inflammatory polarization and demyelinating processes. Therefore, distinct configurations of signaling input cooperate with epigenetic mechanisms to reprogram the transcriptional output of microglia to enable their inflammatory functions.

neuroscience↗

bioRxiv2025

Sensory neurostimulation promotes stress resilience with frequency-specificity

Chronic stress is a major risk factor for neuropsychiatric disorders, acting via increased neuroinflammation and disrupted synaptic plasticity. While non-invasive visual or audiovisual neurostimulation (AV flicker) at 40Hz has been shown to modulate brain immune signaling and improve cognitive performance in mouse models of Alzheimers disease, its effects in the context of stress remain unknown. Here we show that AV flicker protects against stress-induced behavioral, microglial, astrocytic, and synaptic changes in a sex- and frequency-specific manner. Male and female mice underwent 28 days of chronic unpredictable stress with concomitant daily AV flicker exposure at 10Hz, 20Hz, or 40Hz. Stress-induced behaviors were most effectively mitigated by 10Hz AV flicker in males and 40Hz AV flicker in females. In the medial prefrontal cortex, AV flicker normalized the balance of mature and immature dendritic spines and counteracted stress-induced molecular changes in neurons, microglia, and astrocytes, including in key neuropsychiatric risk genes. These findings show that frequency optimized AV flicker induces resilience to chronic stress.

neuroscience↗

bioRxiv2025

Identification of novel Kv1.3 channel-interacting proteins using proximity labelling in T-cells

Potassium channels regulate membrane potential, calcium flux, cellular activation and effector functions of adaptive and innate immune cells. The voltage-activated Kv1.3 channel is an important regulator of T cell-mediated autoimmunity and microglia-mediated neuroinflammation. Kv1.3 channels, via protein-protein interactions, are localized with key immune proteins and pathways, enabling functional coupling between K+ efflux and immune mechanisms. To gain insights into proteins and pathways that interact with Kv1.3 channels, we applied a proximity-labeling proteomics approach to characterize protein interactors of the Kv1.3 channel in activated T-cells. Biotin ligase TurboID was fused to either N or C termini of Kv1.3, stably expressed in Jurkat T cells and biotinylated proteins in proximity to Kv1.3 were enriched and quantified by mass spectrometry. We identified over 1,800 Kv1.3 interactors including known interactors (beta-integrins, Stat1) although majority were novel. We found that the N-terminus of Kv1.3 preferentially interacts with protein synthesis and protein trafficking machinery, while the C-terminus interacts with immune signaling and cell junction proteins. T- cell Kv1.3 interactors included 335 cell surface, T-cell receptor complex, mitochondrial, calcium and cytokine-mediated signaling pathway and lymphocyte migration proteins. 178 Kv1.3 interactors in T-cells also represent genetic risk factors of T cell-mediated autoimmunity, including STIM1, which was further validated using co-immunoprecipitation. Our studies reveal novel proteins and molecular pathways that interact with Kv1.3 channels in adaptive (T-cell) and innate immune (microglia), providing a foundation for how Kv1.3 channels may regulate immune mechanisms in autoimmune and neurological diseases.

cell biology↗

bioRxiv2024

Microglia Morphological Response to Mesenchymal Stromal Cell Extracellular Vesicles Demonstrates EV Therapeutic Potential for Modulating Neuroinflammation

BackgroundMesenchymal stromal cell derived extracellular vesicles (MSC-EVs) are a promising therapeutic for neuroinflammation. MSC-EVs can interact with microglia, the resident immune cells of the brain, to exert their immunomodulatory effects. In response to inflammatory cues, such as cytokines, microglia undergo phenotypic changes indicative of their function e.g. morphology and secretion. However, these changes in response to MSC-EVs are not well understood. Additionally, no disease-relevant screening tools to assess MSC-EV bioactivity exist, which has further impeded clinical translation. Here, we developed a quantitative, high throughput morphological profiling approach to assess the response of microglia to neuroinflammation-relevant signals and whether this morphological response can be used to indicate the bioactivity of MSC-EVs. ResultsUsing an immortalized human microglia cell-line, we observed increased size (perimeter, major axis length) and complexity (form factor) upon stimulation with interferon-gamma (IFN-{gamma}) and tumor necrosis factor-alpha (TNF-). Upon treatment with MSC-EVs, the overall morphological score (determined using principal component analysis) shifted towards the unstimulated morphology, indicating that MSC-EVs are bioactive and modulate microglia. The morphological effects of MSC-EVs in TNF-{gamma}/IFN- stimulated cells were concomitant with reduced secretion of 14 chemokines/cytokines (e.g. CXCL6, CXCL9) and increased secretion of 12 chemokines/cytokines (e.g. CXCL8, CXCL10). Proteomic analysis of cell lysates revealed significant increases in 192 proteins (e.g. HIBADH, MEAK7, LAMC1) and decreases in 257 proteins (e.g. PTEN, TOM1, MFF) with MSC-EV treatment. Of note, many of these proteins are involved in regulation of cell morphology and migration. Gene Set Variation Analysis revealed upregulation of pathways associated with immune response, such as regulation of cytokine production, immune cell infiltration (e.g. T cells, NK cells) and morphological changes (e.g. Semaphorin, RHO/Rac signaling). Additionally, changes in microglia mitochondrial morphology were measured suggesting that MSC-EV modulate mitochondrial metabolism. ConclusionThis study comprehensively demonstrates the effects of MSC-EVs on human microglial morphology, cytokine secretion, cellular proteome, and mitochondrial content. Our high-throughput, rapid, low-cost morphological approach enables screening of MSC-EV batches and manufacturing conditions to enhance EV function and mitigate EV functional heterogeneity in a disease relevant manner. This approach is highly generalizable and can be further adapted and refined based on selection of the disease-relevant signal, target cell, and therapeutic product.

bioengineering↗

bioRxiv2024

Frequency and duration of sensory flicker controls astrocyte and neuron specific transcriptional profiles in 5xFAD mice

BackgroundCurrent clinical trials are investigating gamma frequency sensory stimulation as a potential therapeutic strategy for Alzheimers disease, yet we lack a comprehensive picture of the effects of this stimulation on multiple aspects of brain function. While most prior research has focused on gamma frequency sensory stimulation, we previously showed that exposing mice to visual flickering stimulation increased MAPK and NF{kappa}B signaling in the visual cortex in a manner dependent on duration and frequency of sensory stimulation exposure. Because these pathways control multiple neuronal and glial functions and are differentially activated based on the duration and frequency of flicker stimulation, we aimed to define the transcriptional effects of different frequencies and durations of flicker stimulation on multiple brain functions. MethodsWe exposed 5xFAD mice to different frequencies of audio/visual flicker stimulation (constant light, 10Hz, 20Hz, 40Hz) for durations of 0.5hr, 1hr, or 4hr, then used bulk RNAseq to profile transcriptional changes within the visual cortex and hippocampus tissues. Using weighted gene co-expression network analysis, we identified modules of co-expressed genes controlled by frequency and/or duration of stimulation. ResultsWithin the visual cortex, we found that all stimulation frequencies caused fast activation of a module of immune genes within 1hr and slower suppression of synaptic genes after 4hrs of stimulation. Interestingly, all frequencies of stimulation led to slow suppression of astrocyte specific gene sets, while activation of neuronal gene sets was frequency and duration specific. In contrast, in the hippocampus, immune and synaptic modules were suppressed based on the frequency of stimulation. Specifically,10Hz activated a module of genes associated with mitochondrial function, metabolism, and synaptic translation while 10Hz rapidly suppressed a module of genes linked to neurotransmitter activity. ConclusionCollectively, our data indicate that the frequency and duration of flicker stimulation controls immune, neuronal, and metabolic genes in multiple regions of the brain affected by Alzheimers disease. Flicker stimulation may thus represent a potential therapeutic strategy that can be tuned based on the brain region and the specific cellular process to be modulated.

systems biology↗

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↗