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Srivastava, U.

Publications and source records attributed to Srivastava, U..

6 recordsLinked to original sources

Pathogenic tau in the mouse locus coeruleus induces noradrenergic hyperactivity and neuropsychiatric phenotypes reminiscent of early Alzheimers disease

Alzheimers disease (AD), though defined as a cognitive disorder, often presents neuropsychiatric symptoms such as anxiety, depression, agitation and sleep disruptions years before the onset of frank memory impairment. An early pathological feature is the accumulation of hyperphosphorylated "pretangle" tau (pTau) in the locus coeruleus (LC), the brains primary source of norepinephrine (NE). While clinical studies link LC pTau burden to behavioral abnormalities, causal mechanisms remain unclear. We developed a translationally-relevant mouse model that recapitulates the LC-first phenomenon using cell type-specific viral expression of pathogenic P364S mutant human tau in LC neurons. Three months post-infusion, pTau accumulation induced anxiety-and compulsive-like behaviors and reduced sleep spindles without altering overall sleep architecture. Consistent with the behavioral phenotypes, electrophysiological recordings revealed significant increases in spontaneous and evoked firing of LC neurons, accompanied by robust astrocytic reactivity with no apparent cell death. Transcriptomic analysis identified upregulation of Hcn2 and downregulation of Clic6, suggesting changes in neuronal excitability. To further define molecular mechanisms, we developed a cell type-specific proteomics approach, which showed synaptic and metabolic alterations associated with LC-specific tau pathology. Early anxiety-like behaviors observed at 3 months diminished at later timepoints (6-9 months) and were replaced by anxiolytic characteristics. These findings demonstrate that pTau triggers phenotypes reflective of LC-NE hyperactivity in the early stages of AD pathogenesis, laying the foundation for the development of LC-based disease-modifying therapies to address neuropsychiatric manifestations.

neuroscience↗

Deeper neuronal and glial proteomic insights using an optimized pipeline for proximity labeling proteomics

Proximity-based proteomics using TurboID has enabled cell-type-specific profiling without the need for cell purification, although major bottlenecks in sample lysis, biotinylated protein enrichment, digestion, and mass spectrometry (MS) parameters have limited depth of proteome coverage. Here, we systematically optimized these variables using TurboID-based labeling of BV2 microglia in vitro and brain astrocytes in vivo to define conditions that maximize proteome coverage. In microglia, the optimized protocol using 8 M urea lysis with on-bead S-Trap digestion and data-independent acquisition MS (DIA-MS) identified 4,016 proteins, double the depth of prior studies, and revealed metabolic, ribosomal, lipid-processing, autophagy, and trafficking signatures. Brain astrocyte proteomes were best recovered using SDS lysis with S-Trap digestion and DIA-MS, yielding a proteome of over 3,600 highly enriched proteins, twice the depth of prior astrocyte-TurboID studies. The expanded astrocyte proteomes captured canonical astrocyte markers as well as membrane-associated, vesicular trafficking, and presynaptic protein signatures, consistent with labeling of astrocyte-neuron interface regions, including proteins involved in receptor signaling, lipid metabolism, and plasticity at tripartite synapses, and several AD risk proteins. The increased peptide recovery following S-Trap digestion allowed the reduction of starting material to 20 {micro}g protein for DIA-MS, and enabled multiplexed tandem mass tag (TMT-MS) proteomics using even smaller samples. When applied to synaptosomes enriched from mouse brains with neuronal TurboID labeling, our pipeline identified a synapse-specific proteome of 2,529 proteins, revealing synaptic, mitochondrial and disease-relevant signatures not detectable in prior studies. By tackling critical bottlenecks from tissue processing to MS, our optimized pipelines enable cell-type and compartment-specific proximity-labeling proteomics to obtain comprehensive biological and disease-relevant insights across various biological fields.

neuroscience↗

Kv1.3 inhibition alleviates neuropathology via neuroinflammatory and resilience pathways in a mouse model of Aβ pathology

Inhibition of voltage-gated potassium channel Kv1.3 is a therapeutic strategy to curb microglia-mediated neuroinflammation in neurodegeneration, although the cellular and signaling mechanisms of disease-modification by Kv1.3 blockers are unclear. In this study, we delineate protective mechanisms of Kv1.3 blockade in a mouse model of Alzheimers disease (AD) pathology using comprehensive transcriptomics and proteomics profiling of brain, corresponding with neuropathological effects of two translationally relevant Kv1.3 blockers, namely small molecule PAP-1 and peptide ShK-223. Following 3 months of treatment, both molecules reduced Ab plaque burden. Single nuclear RNA seq (snRNA seq) of brain nuclei showed that PAP-1 disproportionately impacted oligodendrocytes and microglia and increased crosstalk between neurons and astrocytes with endothelial cells. In contrast, ShK-223 had pronounced effects on glutamatergic neurons and astrocytes. Both blockers increased expression of myelination genes in oligodendrocytes and synaptic genes in neurons. Neuroprotective effects of PAP-1 were further confirmed by bulk brain transcriptomics and proteomics whereby PAP-1 increased levels of synaptic, cognitive resilience and mitochondrial proteins, while decreasing glial and immune pathways including STAT1/3 phosphorylation. Using proximity labeling and co-immunoprecipitation, we found that Kv1.3 interacts with STAT1/3 in microglia. Using microglial cell lines and primary microglia, we discovered a preferential functional coupling between Kv1.3 and type 2 but not type 1 IFN signaling. Brain-level disease modification by Kv1.3 blockade was reflected in the cerebrospinal fluid (CSF) via reduced levels of neurofilament-light (NEFL) and resilience protein RPH3A, both of which are increased in human AD CSF. Together, this study demonstrates functional links between Kv1.3 channels and type 2 IFN signaling and reveals distinct cellular effects of Kv1.3 blockers in AD pathology that correspond with reduced neuropathology and neuroinflammation, augmentation of resilience and neuro-vascular pathways, along with biomarkers of therapeutic effect.

neuroscience↗

The protein interactome of the Neuron Specific Gene family (NSG1-3)

The Neuron-Specific Gene (NSG) family members (NSG1-3) play critical and diverse roles in neuronal protein trafficking, but their precise molecular functions remain poorly understood. Here, we employed proximity-labeling proteomics to map the interactomes of each NSG protein. Unlabeled mass spectrometry identified over 1,000 significantly enriched interactors compared with a cytoplasmic control, revealing substantial overlap between NSG1 and NSG2, and a more divergent profile for NSG3. Gene ontology and KEGG pathway analysis confirmed established associations with glutamatergic synapses and endosomal trafficking, while also uncovering unexpected links to presynaptic machinery, inhibitory synapses, and endoplasmic reticulum-associated protein translation, particularly for NSG3. Reciprocal biotinylation patterns and co-immunoprecipitation revealed novel heteromeric complex formation between NSG1 and NSG2, with limited interactions involving NSG3. All three NSGs biotinylated core AMPA receptor subunits and auxiliary proteins, while NSG1 and NSG2 also associated with NMDA receptors, GABA receptor subunits, as well as multiple presynaptic proteins. Moreover, NSG1 and NSG2 specifically biotinylated components of multiunit tethering complexes including neuron-specific retromer, and biotinylated a preponderance of ADAM10 substrates, reinforcing their role in proteolytic processing. Finally, despite the relatively divergent interactomes of NSG1 and NSG2 compared to NSG3, all family members robustly biotinylated amyloid precursor protein (APP), suggesting possible synergistic or competitive interactions that could shape APP proteolytic processing and/or trafficking. Together, these data provide a comprehensive systems-level view of NSG protein interactions, establishing a molecular framework for future investigations into NSG-mediated neural plasticity and disease mechanisms.

neuroscience↗

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↗

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↗