bioRxiv Science⌕ Search

Biology subjects

Kour, D.

Publications and source records attributed to Kour, D..

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↗

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↗

Bisdemethoxycurcumin mitigates Alzheimer disease pathology through autophagy-mediated reduction of senescence and amyloid beta

AD pathology is accompanied by increased senescence and reduced levels of autophagy in the brain. We investigated whether pharmacologically inducing autophagy could alter the senescent phenotype and help ameliorate AD pathology. We discovered that Bisdemethoxycurcumin (BDMC), a natural compound found in Curcuma longa, stimulates autophagy in primary astrocytes. We found that autophagy and senescence exhibit an inverse relationship in aging astrocytes, with increased expression of senescent proteins and downregulation of autophagic proteins. However, treatment of aged astrocytes with BDMC reversed the senescent phenotype by ameliorating the impaired autophagy. Interestingly, the senescent phenotype persisted when autophagy was downregulated by knockdown of AMPK. Additionally, BDMC-induced autophagy aided in the removal of amyloid beta that was administered externally to the astrocytes. Further, to validate these results in a mouse model of AD, we confirmed that BDMC can significantly penetrate the blood-brain barrier (BBB) in mice. Therefore, we administered 50 and 100 mg/kg b.w. of BDMC to transgenic 3xTg-AD mice for two months. In their hippocampus, the Control 3xTg-AD animals showed more senescent cells and lower autophagy levels. In contrast, autophagic proteins were significantly upregulated while senescence indicators, such as senescence-associated secretory phenotype (SASP) proteins, were sharply downregulated in the brain of treated animals. Additionally, we discovered that the treated mices hippocampus had a significantly lower amyloid beta load. These molecular changes in the brain were ultimately reflected in the improved working memory and neuromuscular coordination behavior of mice treated with BDMC. This study warrants further evaluation of BDMC for the management of AD. Graphical Abstract O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=139 SRC="FIGDIR/small/654834v1_ufig1.gif" ALT="Figure 1"> View larger version (53K): org.highwire.dtl.DTLVardef@1176bbforg.highwire.dtl.DTLVardef@a2e2cdorg.highwire.dtl.DTLVardef@1d824f5org.highwire.dtl.DTLVardef@1628a30_HPS_FORMAT_FIGEXP M_FIG This illustration was created by using biorender.com C_FIG

pharmacology and toxicology↗

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↗

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↗