Biology subjects

Bagchi, P.

Publications and source records attributed to Bagchi, P..

17 recordsLinked to original sources

bioRxiv2025

Neuroinflammatory Stress Preferentially Impacts Synaptic MAPK Signaling and Mitochondria in Excitatory Neurons

BackgroundUnderstanding synapse-specific effects of neuroinflammation can provide mechanistic and therapeutically relevant insights across the spectrum of neurological diseases. MethodsWe applied neuron-specific proteomic biotinylation in vivo, differential centrifugation of brain for crude synaptosome enrichment (P2 fraction) and mass spectrometry (MS) analysis of biotinylated proteins to derive native-state proteomes of Camk2a-positive neurons and their corresponding P2 synaptic compartments. Next, in an in vivo model of systemic lipopolysaccharide (LPS) dosing, we examined the effects of neuroinflammation on whole neuron and synaptic compartments using a combination of MS, network analysis, confirmatory biochemical and ultrastructural assays and integrative approaches across our mouse-derived and existing human datasets. ResultsUltrastructural and biochemical analyses of P2 fractions verified enrichment in synaptic elements, including synaptic vesicles and mitochondria. MS of biotinylated proteins from Camk2a-specific bulk brain homogenates (whole neuron) and P2 fractions (synaptosome) showed enrichment of >1000 proteins, consistent with neuron-specific biotinylation, also confirmed by immunofluorescence microscopy. Camk2a-specific synaptic proteome revealed molecular signatures related to mitochondrial function, synaptic transmission, protein translation. LPS-treated mice displayed body weight loss and neuroinflammation, characterized by glial activation, increased pro-inflammatory cytokine levels and upregulated expression of Alzheimers disease (AD)-related microglial genes. LPS-induced neuroinflammation exerted distinct effects on the synaptic proteome, including increased mitochondrial and reduced cytoskeletal-synaptic proteins, while suppressed synaptic MAPK signaling. Importantly, these changes were not observed at the whole neuron level, indicating unique vulnerability of the synapse to neuroinflammation. In line with synapse proteomic and signaling changes, LPS altered the ultrastructure of asymmetric synapses, suggesting dysregulation of excitatory neurotransmission. Co-expression network analysis of Camk2a neuronal proteins further resolved mitochondria- and synapse-specific protein modules, some of which were neuroinflammation-dependent. Neuroinflammation increased levels of a mitochondria-enriched module, and decreased levels of a pre-synaptic vesicle module, without impacting a post-synaptic membrane module. LPS-dependent mitochondrial and LPS-independent post-synaptic modules in mouse neurons mapped to post-mortem human AD brain proteomic modules which were decreased in cases with AD dementia and positively correlated to cognitive function, including pro-resilience markers for AD. ConclusionOur findings using native-state proteomics of Camk2a neurons combined with synaptosome enrichment identify proteome-level mechanisms of early synaptic vulnerability to neuroinflammation relevant to AD.

neuroscience↗

bioRxiv2025

Progressive Supranuclear Palsy PERK haplotype B selectively translates DLX1 promoting tau toxicity

The unfolded protein response (UPR) sensor PERK exists in two haplotypes termed A and B. PERK-B uniquely confers increased risk for tauopathies like progressive supranuclear palsy (PSP), but the mechanisms distinguishing its function from PERK-A and contributing to its association with tau pathology are not known. Here, we developed a controlled cellular model for a pair-wise comparison of the two PERK haplotypes, finding their UPR functions nearly indistinguishable. However, a careful examination employing puromycin-based proteomics revealed that a subset of mRNA translation events were permissible under PERK-B, but not PERK-A, dependent UPR. Critically, one of the targets that escaped PERK-B suppression was the transcription factor DLX1, which has been genetically linked to PSP risk. Here, we found a shift in the solubility of DLX1 in human PSP brain tissue, and report that silencing of DLX-1 reduced the aggregation of tau in mammalian cells. Furthermore, silencing of the fly homolog of DLX1 was sufficient to decrease tau-induced toxicity, in vivo. Our results detail the haplotype-specific PERK-B/DLX-1 pathway as a novel driver of tau pathology in cells, flies, and likely human brain, revealing new insights into PSP pathogenesis and potential therapeutic targets.

neuroscience↗

bioRxiv2025

Multiplex Proteomics of Lewy Body Dementia Reveals Cerebrospinal Fluid Biomarkers of Disease Pathology and Progression

Lewy body dementia (LBD), which encompasses Parkinsons disease dementia (PDD) and Dementia with Lewy bodies (DLB), lacks established biofluid markers of its complex clinical and neuropathological heterogeneity. Multiplex proteomic tools, such as the recently developed NUcleic acid Linked ImmunoSandwich Assay (NULISATM), can assess a diverse array of neurodegenerative targets and address these biomarker gaps. We used this platform to analyze 852 baseline and longitudinal cerebrospinal fluid (CSF) samples from control, PD, and DLB subjects from the Parkinsons Disease Biomarker Program (PDBP). We then examined LBD protein signatures across different clinical diagnoses, patterns of cognitive progression, and underlying neuropathologies. Of the 124 markers analyzed, 54 were significantly altered in PD with cognitive impairment and/or DLB compared to controls, many serving as informative clinical classifiers in machine learning models. We also identified 15 proteins with significant baseline and/or longitudinal changes linked to cognitive progression. To identify neuropathological signatures, we stratified 209 cases by the presence of underlying synuclein and amyloid-beta (A{beta}) pathology using CSF alpha-synuclein seed amplification assay (Syn-SAA) results and NULISA pTau-217 levels, respectively. Select synaptic markers were decreased relative to synucleinopathy but increased relative to A{beta} accumulation, complicating their interpretation in cases with co-pathology. These neuropathological analyses were replicated in an independent cohort of 917 cases from the Alzheimers Disease Neuroimaging Initiative (ADNI). Overall, these results highlight the utility of multiplex proteomic analysis in the identification of candidate CSF biomarkers of disease heterogeneity in LBD. One Sentence SummaryThis multiplex proteomic analysis using the recently released NULISA platform revealed unique targets of Lewy body dementia progression and neuropathology.

systems biology↗

bioRxiv2025

Simultaneous profiling of native-state proteomes and transcriptomes of neural cell types using proximity labeling

Deep molecular phenotyping of cells at transcriptomic and proteomic levels is an essential first step to understanding cellular contributions to development, aging, injury, and disease. Since proteome and transcriptome level abundances only modestly correlate with each other, complementary profiling of both is needed. We report a novel method called simultaneous protein and RNA -omics (SPARO) to capture the cell type-specific transcriptome and proteome simultaneously from both in vitro and in vivo experimental model systems. This method leverages the ability of biotin ligase, TurboID, to biotinylate cytosolic proteins including ribosomal and RNA-binding proteins, which allows enrichment of biotinylated proteins for proteomics as well as protein-associated RNA for transcriptomics. We validated this approach first using well-controlled in vitro systems to verify that the proteomes and transcriptomes obtained reflect the ground truth, bulk proteomes and transcriptomes. We also show that the effect of a biological stimulus (e.g., neuroinflammatory activation by lipopolysaccharide) can be faithfully captured. We also applied this approach to obtain native-state proteomes and transcriptomes from two key neural cell types, astrocytes and neurons, thereby validating the in vivo application of SPARO. Next, we used these data to interrogate protein-mRNA concordance and discordance across these cell types, providing insights into groups of molecular processes that exhibit uniform or cell type-specific patterns of mRNA-protein discordance.

neuroscience↗

bioRxiv2025

Proximity labeling of the Tau repeat domain enriches RNA-binding proteins that are altered in Alzheimer's disease and related tauopathies

In Alzheimers disease (AD) and other tauopathies, tau dissociates from microtubules and forms toxic aggregates that contribute to neurodegeneration. Although some of the pathological interactions of tau have been identified from postmortem brain tissue, these studies are limited by their inability to capture transient interactions. To investigate the interactome of aggregate-prone fragments of tau, we applied an in vitro proximity labeling technique using split TurboID biotin ligase (sTurbo) fused with the tau microtubule repeat domain (TauRD), a core region implicated in tau aggregation. We characterized sTurbo TauRD co-expression, robust enzyme activity and nuclear and cytoplasmic localization in a human cell line. Following enrichment of biotinylated proteins and mass spectrometry, we identified over 700 TauRD interactors. Gene ontology analysis of enriched TauRD interactors highlighted processes often dysregulated in tauopathies, including spliceosome complexes, RNA-binding proteins (RBPs), and nuclear speckles. The disease relevance of these interactors was supported by integrating recombinant TauRD interactome data with human AD tau interactome datasets and protein co-expression networks from individuals with AD and related tauopathies. This revealed an overlap with the TauRD interactome and several modules enriched with RBPs and increased in AD and Progressive Supranuclear Palsy (PSP). These findings emphasize the importance of nuclear pathways in tau pathology, such as RNA splicing and nuclear-cytoplasmic transport and establish the sTurbo TauRD system as a valuable tool for exploring the tau interactome.

neuroscience↗

bioRxiv2024

Biochemical and neurophysiological effects of deficiency of the mitochondrial import protein TIMM50

TIMM50, an essential TIM23 complex subunit, is suggested to facilitate the import of [~]60% of the mitochondrial proteome. In this study, we characterized a TIMM50 disease causing mutation in human fibroblasts and noted significant decreases in TIM23 core protein levels (TIMM50, TIMM17A/B, and TIMM23). Strikingly, TIMM50 deficiency had no impact on the steady state levels of most of its putative substrates, suggesting that even low levels of a functional TIM23 complex are sufficient to maintain the majority of TIM23 complex-dependent mitochondrial proteome. As TIMM50 mutations have been linked to severe neurological phenotypes, we aimed to characterize TIMM50 defects in manipulated mammalian neurons. TIMM50 knockdown in mouse neurons had a minor effect on the steady state level of most of the mitochondrial proteome, supporting the results observed in patient fibroblasts. Amongst the few affected TIM23 substrates, a decrease in the steady state level of components of the intricate oxidative phosphorylation and mitochondrial ribosome complexes was evident. This led to declined respiration rates in fibroblasts and neurons, reduced cellular ATP levels and defective mitochondrial trafficking in neuronal processes, possibly contributing to the developmental defects observed in patients with TIMM50 disease. Finally, increased electrical activity was observed in TIMM50 deficient mice neuronal cells, which correlated with reduced levels of KCNJ10 and KCNA2 plasma membrane potassium channels, likely underlying the patients epileptic phenotype.

biochemistry↗

bioRxiv2024

Proximity labeling proteomics reveals Kv1.3 potassium channel immune interactors in microglia.

Microglia are the resident immune cells of the brain and regulate the brains inflammatory state. In neurodegenerative diseases, microglia transition from a homeostatic state to a state referred to as disease associated microglia (DAM). DAM express higher levels of proinflammatory signaling, like STAT1 and TLR2, and show transitions in mitochondrial activity toward a more glycolytic response. Inhibition of Kv1.3 decreases the proinflammatory signature of DAM, though how Kv1.3 influences the response is unknown. Our goal was to establish the potential proteins interacting with Kv1.3 during the TLR4-mendiated transition to DAM. We utilized TurboID, a biotin ligase, fused to Kv1.3 to evaluate the potential interacting proteins with Kv1.3 via mass spectrometry in BV-2 microglia during an immune response. Electrophysiology, western blots, and flow cytometry were used to evaluate Kv1.3 channel presence and TurboID biotinylation activity. We hypothesized that Kv1.3 contains domain-specific interactors that vary during an TLR4-induced inflammatory response, some of which are dependent on the PDZ-binding domain on the C-terminus. We determined that the N-terminus of Kv1.3 is responsible for trafficking Kv1.3 to the cell surface and mitochondria (e.g. NUNDC, TIMM50). The C-terminus interacts with immune signaling proteins in an LPS-induced inflammatory response (e.g. STAT1, TLR2, and C3). There are 70 proteins that rely on the c-terminal PDZ-binding domain to interact with Kv1.3 (i.e. ND3, Snx3, and Sun1). Overall, we highlight that the Kv1.3 potassium channel functions beyond outward flux of potassium in an inflammatory context and contributes to activity of key immune signaling proteins, such as STAT1 and C3. MAIN POINTSKv1.3 channels are highly abundant in pro-inflammatory microglia in neurological diseases. Kv1.3 channels may regulate microglial functions by interacting with other proteins via its N and C terminal domains. Using proximity-based proteomics, we identified several novel proteins that interact with the N and C terminus of Kv1.3 channels, some of which are domain-specific. Kv1.3 channels in microglia interact with many immune signaling proteins, including Tlr2, Stat1 and integrins. Under homeostatic conditions, the N-terminus of Kv1.3 interacts with proteins involved in protein trafficking, to the cell surface and mitochondria. The PDZ-binding region was an important determinant of the C terminal interactome. During an LPS-induced inflammatory response, the C-terminus of Kv1.3 uniquely interacts with immune and signaling proteins of disease relevance, including STAT1

neuroscience↗

bioRxiv2023

Proteomic signaling of dual specificity phosphatase 4 (DUSP4) in Alzheimer's disease

DUSP4 is a member of the DUSP (Dual-Specificity Phosphatase) subfamily that is selective to the mitogen-activated protein kinases (MAPK) and has been implicated in a range of biological processes and functions in Alzheimers disease (AD). In this study, we utilized stereotactic delivery of adeno-associated virus (AAV)-DUSP4 to overexpress DUSP4 in the dorsal hippocampus of 5xFAD and wildtype (WT) mice, then used mass spectrometry (MS)-based proteomics along with label-free quantification to profile the proteome and phosphoproteome in the hippocampus. We identified patterns of protein expression and phosphorylation that are modulated in 5xFAD mice and examined the sex-specific impact of DUSP4 overexpression on the 5xFAD proteome/phosphoproteome. In 5xFAD mice, a substantial number of proteins were up- or down-regulated in both male and female mice in comparison to age and sex-matched WT mice, many of which are involved in AD-related biological processes, such as the activated immune response or suppression of synaptic activities. Upon DUSP4 overexpression, significantly regulated proteins were found in pathways that were suppressed, such as the immune response, in male 5xFAD mice. In contrast, such a shift was absent in female mice. For the phosphoproteome, we detected an array of phosphorylation sites that are regulated in 5xFAD compared to WT, and are modulated by DUSP4 overexpression in each sex. Interestingly, the changes in 5xFAD- and DUSP4-associated phosphorylation occurred in opposite directions. Strikingly, both the 5xFAD- and DUSP4-associated phosphorylation changes were found for the most part in neurons, and play key roles in neuronal processes and synaptic function. Site-centric pathway analysis revealed that both the 5xFAD- and DUSP4-associated phosphorylation sites were enriched for a number of kinase sets in female, but only a limited number of sets of kinases in male mice. Taken together, our results suggest that male and female 5xFAD mice respond to DUSP4 overexpression via shared and sex-specific molecular mechanisms, which might underly similar reductions in amyloid pathology in both sexes, while learning deficits were reduced in only females with DUSP4 overexpression. Finally, we validated our findings with the sex-specific AD-associated proteomes in human cohorts and further developed DUSP4-centric proteomic network models and signaling maps for each sex.

bioinformatics↗

bioRxiv2023

A Conserved Switch Controls Virulence, Sporulation, and Motility in C. difficile

Spore formation is required for environmental survival and transmission of the human enteropathogenic Clostridioides difficile. In all bacterial spore formers, sporulation is regulated through activation of the master response regulator, Spo0A. However, the factors and mechanisms that directly regulate C. difficile Spo0A activity are not defined. In the well-studied Bacillus species, Spo0A is directly inactivated by Spo0E, a small phosphatase. To understand Spo0E function in C. difficile, we created a null mutation of the spo0E ortholog and assessed sporulation and physiology. The spo0E mutant produced significantly more spores, demonstrating Spo0E represses C. difficile sporulation. Unexpectedly, the spo0E mutant also exhibited increased motility and toxin production, and enhanced virulence in animal infections. We uncovered that Spo0E interacts with both Spo0A and the toxin and motility regulator, RstA. Direct interactions between Spo0A, Spo0E, and RstA constitute a previously unknown molecular switch that coordinates sporulation with motility and toxin production. Reinvestigation of Spo0E function in B. subtilis revealed that Spo0E induced motility, demonstrating Spo0E regulation of motility and sporulation among divergent species. Further, we found that Spo0E orthologs are widespread among prokaryotes, suggesting that Spo0E performs conserved regulatory functions in diverse bacteria.

microbiology↗

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

Dynamic in vivo mapping of the methylproteome using a chemoenzymatic approach

Dynamic protein post-translation methylation is essential for cellular function, highlighted by the essential role of methylation in transcriptional regulation and its aberrant dysregulation in diseases including cancer. This underscores the importance of cataloging the cellular methylproteome. However, comprehensive analysis of the methylproteome remains elusive due to limitations in current enrichment and analysis pipelines. Here, we employ an L-Methionine analogue, ProSeMet, that is chemoenzymatically converted to the SAM analogue ProSeAM in cells and in vivo to tag proteins with a biorthogonal alkyne that can be directly detected via LC-MS/MS, or functionalized for subsequent selective enrichment and LC-MS/MS identification. Without enrichment, we identify lysine mono-, di-, and trimethylation, histidine methylation, and arginine methylation with site specific resolution on proteins including heat shock protein HSPA8, for which methylation is implicated in human disease. With enrichment, we identify 486 proteins known to be methylated and 221 proteins with novel methylation sites encompassing diverse cellular functions. Systemic ProSeMet delivery in mice pseudomethylates proteins across organ systems with blood-brain barrier penetrance and identifies site-specific pseudomethylation in vivo with LC-MS/MS. Leveraging these pipelines to define the cellular methylproteome may have broad applications for understanding the methylproteome in the context of disease.

biochemistry↗

bioRxiv2022

Defining the Ligand-dependent Interactome of the Sigma 1 Receptor

Sigma 1 Receptor (S1R) is a therapeutic target for a wide spectrum of pathological conditions ranging from neurodegenerative diseases to cancer and COVID-19. S1R is ubiquitously expressed throughout the visceral organs, nervous, immune and cardiovascular systems. It is proposed to function as a ligand-dependent molecular chaperone that modulates multiple intracellular signaling pathways. The purpose of this study was to define the S1R interactome under native conditions and upon binding to well-characterized ligands. This was accomplished by fusing the biotin ligase, Apex2, to the C terminus of S1R. Cells stably expressing S1R-Apex or a GFP-Apex control were used to map specific protein interactions. Biotinylated proteins were labeled under native conditions and in a ligand dependent manner, then purified and identified using quantitative mass spectrometry. Under native conditions, S1R biotinylates over 200 novel proteins, many of which localize within the endomembrane system (ER, Golgi, secretory vesicles) and function within the secretory pathway. Under conditions of cellular exposure to either S1R agonist or antagonist, results show enrichment of proteins integral to secretion, extracellular matrix formation, and cholesterol biosynthesis. Notably, Proprotein Convertase Subtilisin/Kexin Type 9 (PCSK9) displays increased binding to S1R under conditions of treatment with Haloperidol, a well-known S1R antagonist; whereas Low density lipoprotein receptor (LDLR) binds more efficiently to S1R upon treatment with (+)-Pentazocine ((+)-PTZ), a classical S1R agonist. Our results are consistent with the postulated role of S1R as an intracellular chaperone and further suggest important and novel functionalities related to cholesterol metabolism and biosynthesis.

cell biology↗

bioRxiv2022

Candesartan treatment preserves learning and working memory in female TgF344-AD rats

BackgroundTargeting the renin angiotensin system, especially with angiotensin receptor II blockers (ARB), and related vascular dysfunction is a promising therapeutic intervention for cognitive impairment including Alzheimers Disease (AD). The underlying mechanisms of the effects of ARB is unclear. This study sought to examine if treatment with candesartan, an ARB, affects neurobehavioral manifestation and the underlying neuro- and vascular mechanisms in male and female TgF344-AD rats. MethodsCandesartan or vehicle was administered to TgF344-AD rats (n=127) daily from 12-months to 18-months of age. Behavioral assays (spontaneous alternation test, novel object recognition, water radial arm maze) and neuropathologic assessment were completed along with brain proteome and measures of contractility in 12- and 18-month rat brains. ResultsUntreated 18-month TgF344-AD showed impairments in learning and increased perseverative working memory errors on the water radial arm maze (WRAM). These behavioral changes were corrected with candesartan treatment in female rats only. Treatment with candesartan was also associated with improved vascular reactivity and reduced blood pressure in both wild type and TgF344-AD male and female rats. Although there was no effect on amyloid-{beta}, treatment with candesartan reduced whole brain clusterin, an AD-risk associated protein, and GFAP in female TgF344-AD. ConclusionsOur results demonstrate that candesartan administered in the early stages of AD has a sexual dimorphic response in Tgf344-AD rat, where it reduced cognitive disturbances only in female TgF344-AD rat. These effects appear to be independent of changes in blood pressure and amyloid-{beta} reduction and are likely mediated through mechanisms related to clusterin and GFAP pathways.

neuroscience↗

bioRxiv2021

Stable Flow-induced Expression of KLK10 Inhibits Endothelial Inflammation and Atherosclerosis

IntroductionAtherosclerosis preferentially occurs in arterial regions exposed to disturbed blood flow (d-flow), while regions exposed to stable flow (s-flow) are protected. The proatherogenic and atheroprotective effects of d-flow and s-flow are mediated in part by the global changes in endothelial cell gene expression, which regulates endothelial dysfunction, inflammation, and atherosclerosis. Previously, we identified Kallikrein-Related Peptidase 10 (KLK10, a secreted serine protease) as a flow-sensitive gene in arterial endothelial cells, but its role in endothelial biology and atherosclerosis was unknown. Methods and ResultsHere, we show that KLK10 is upregulated under s-flow conditions and downregulated under d-flow conditions using in vivo mouse models and in vitro studies with cultured endothelial cells (ECs). Single-cell RNA sequencing (scRNAseq) and scATAC sequencing (scATACseq) study using the partial carotid ligation mouse model showed flow-regulated KLK10 expression at the epigenomic and transcription levels. Functionally, KLK10 protected against d-flow-induced inflammation and permeability dysfunction in human artery ECs (HAECs). Further, treatment of mice in vivo with rKLK10 decreased arterial endothelial inflammation in d-flow regions. Additionally, rKLK10 injection or ultrasound-mediated transfection of KLK10-expressing plasmids inhibited atherosclerosis in ApoE-/- mice. Studies using pharmacological inhibitors and siRNAs revealed that the anti-inflammatory effects of KLK10 were mediated by a Protease Activated Receptors (PAR1/2)-dependent manner. However, unexpectedly, KLK10 did not cleave the PARs. Through a proteomics study, we identified HTRA1 (High-temperature requirement A serine peptidase 1), which bound and cleaved KLK10. Further, siRNA knockdown of HTRA1 prevented KLK10s anti-inflammatory and barrier protective function in HAECs, suggesting that HTRA1 regulates KLK10 function. Moreover, KLK10 expression was significantly reduced in human coronary arteries with advanced atherosclerotic plaques compared to those with less severe plaques. ConclusionKLK10 is a flow-sensitive endothelial protein and, in collaboration with HTRA1, serves as an anti-inflammatory, barrier-protective, and anti-atherogenic factor.

cell biology↗

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↗

bioRxiv2021

Proximity-based labeling reveals DNA damage-induced N-terminal phosphorylation of fused in sarcoma (FUS) leads to distinct changes in the FUS protein interactome.

Cytoplasmic accumulation of the RNA/DNA binding protein, fused in sarcoma (FUS), into inclusions is a common hallmark of frontotemporal lobar degeneration (FTLD) and amyotrophic lateral sclerosis (ALS) pathology. We have previously shown that DNA damage can trigger the cytoplasmic accumulation of an N-terminally phosphorylated FUS. However, the functional consequences of N-terminal FUS phosphorylation are unknown. To gain insight into this question, we utilized proximity-dependent biotin labeling via ascorbate peroxidase 2 (APEX2) paired with mass-spectrometry (MS) to investigate whether N-terminal phosphorylation shifts the FUS protein-protein interaction network (interactome), and subsequently, its function. We report the first comparative analysis of the interactomes for three FUS variants: homeostatic wild-type FUS (FUS WT), a phosphomimetic variant of FUS (a proxy for N-terminally phosphorylated FUS, FUS PM), and a toxic FUS P525L mutant (a mutation that causes juvenile ALS, FUS P525L). Data are available via ProteomeXchange with identifier PXD026578. We demonstrate that compared to FUS WT and FUS P525L, the FUS PM interactome uniquely enriches for a set of cytoplasmic proteins that mediate mRNA metabolism and translation and nuclear proteins involved in spliceosome and DNA repair functions, respectively. We further identify and validate three proteins, VPS35, MOV10, and CLTA, as novel interacting partners of all three FUS variants. Lastly, we provide functional evidence that N-terminally phosphorylated FUS may disrupt homeostatic translation and steady state levels of specific mRNA transcripts. Taken together, these results highlight phosphorylation as a unique modulator of the FUS interactome and function.

neuroscience↗

bioRxiv2021

Differential expression of striatal proteins in a mouse model of DOPA-responsive dystonia reveals shared mechanisms among dystonic disorders

Dystonia is characterized by involuntary muscle contractions that cause debilitating twisting movements and postures. Although basal ganglia dysfunction is implicated in many forms of dystonia, the underlying mechanisms are unclear. Therefore, to reveal abnormal striatal cellular processes and pathways implicated in dystonia, we used an unbiased proteomic approach in a knockin mouse model of DOPA-responsive dystonia, a model in which the striatum is known to play a central role in the expression of dystonia. Fifty-seven of the 1805 proteins identified were differentially regulated in DOPA-responsive dystonia mice compared to control mice. Most differentially regulated proteins were associated with gene ontology terms that implicated either mitochondrial or synaptic dysfunction whereby proteins associated with mitochondrial function were generally over-represented whereas proteins associated with synaptic function were largely under-represented. Remarkably, nearly 20% of the differentially regulated proteins identified in our screen are associated with pathogenic variants that cause inherited dystonic disorders in humans suggesting shared mechanisms across many different forms of dystonia.

neuroscience↗