Biology subjects

Espinosa-Garcia, C.

Publications and source records attributed to Espinosa-Garcia, C..

9 recordsLinked to original sources

bioRxiv2026

Network analysis of mouse brain proteomes resolves molecular and cellular endophenotypes of aging and Aβ pathology

Aging and Alzheimer's disease (AD) pathology exert both shared and distinct effects on the brain, yet their relative contributions to molecular alterations remain poorly defined. Here we performed tandem mass tag proteomics across the lifespan in wild-type and 5xFAD mice and integrated these data with human brain proteomic networks to resolve aging- and amyloid-beta (A{beta}-associated molecular programs. We quantified 8,535 proteins and identified 51 co-expression modules representing neuronal, mitochondrial, glial, vascular and proteostatic pathways. Aging emerged as the predominant driver of proteomic remodeling, with widespread alterations in RNA metabolism, protein translation, and neuronal pathways occurring independently of A{beta} pathology. In contrast, A{beta} selectively modified a subset of age-dependent processes, including suppression of mitochondrial ATP biosynthetic machinery and amplification of astrocytic and microglial activation states. These key aging and disease-related molecular signatures were highly conserved in human AD brain proteomes. Together, our findings establish aging as the primary determinant of brain proteome organization while revealing specific mechanisms through which A{beta} pathology reshapes age-associated biological processes. Our results also highlight molecular mechanisms of human aging and AD pathology that can be modeled in mouse models.

neuroscience↗

bioRxiv2026

Chronic Sleep Disruption Activates the Transcriptional Unfolded Protein Response and Induces Compartment-Specific Proteomic Changes in Mice

Sleep disruption (SD) is associated with adverse brain health and increased risk of dementia, including Alzheimer's disease (AD), yet the pathological mechanisms underlying this association are not fully understood. Preclinical studies have shown that acute SD impairs cognition by altering vulnerable synapses (molecular composition, ultrastructure, and function). However, the cumulative effects of chronic SD on the adult brain and its vulnerable synapses, that might contribute to AD risk, remain poorly defined. Here, we investigated the impact of chronic SD on brain transcriptomic signatures and subcellular compartment-specific proteomic profiles to identify genes, proteins, and signaling pathways that might confer AD risk. Chronic SD induced a modest transcriptional stress response globally, whereas alterations were more pronounced in the synaptosomal (synapse-enriched P2 fractions) and whole-brain proteomes. Remarkably, chronic SD elicited unique proteomic alterations at the synaptic compartment were distinct from whole-brain effects, including increased proteins involved in synaptic vesicle endocytosis, regulation of dendritic spine morphogenesis, and regulation of long-term synaptic depression, compared to increased proteins involved in immune/inflammatory, glial responses and neuronal function at the whole-brain level. Consistent with the chronic SD-induced molecular signatures, we observed subtle ultrastructural changes in vulnerable CA1 hippocampal synapses, including reduced pre-synapse size and decreased synaptic vesicle density, which suggest that chronic SD modifies synaptic transmission and plasticity. Together, our findings indicate that chronic SD differentially impacts the brain and the synaptic proteome, nominating candidate therapeutic targets and signaling pathways for future mechanistic studies of chronic SD and how SD contributes to increased AD risk.

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bioRxiv2026

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.

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bioRxiv2026

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↗

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

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.

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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

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bioRxiv2023

Tofacitinib effects profound seizure suppression through mimicking EZH2 antagonism of a pro-inflammatory gene network in epilepsy.

All current drug treatments for epilepsy, a neurological disorder affecting over 50 million people(1, 2) merely treat symptoms, and a third of patients do not respond to medication. There are no disease modifying treatments that may be administered briefly to patients to enduringly eliminate spontaneous seizures and reverse cognitive deficits(3, 4). Applying network approaches to rodent models and human temporal lobectomy samples at both whole tissue and single-nuclei resolutions, we observe the well-characterized pattern of rapid induction and subsequent quenching exhibited of the JAK/STAT pathway within days of epileptogenic insult. This is followed by a resurgent activation weeks to months later with the onset of spontaneous seizures. Targeting the first wave of activation after epileptic insult does not prevent disease. However, brief inhibition of the second wave with CP690550 (Tofacitinib) (5, 6) enduringly suppresses seizures, rescues deficits in spatial memory, and alleviates epilepsy-associated histopathological alterations. Seizure suppression lasts for at least 2 months after the final dose. Using discovery-based transcriptomic analysis across models of epilepsy and validation of putative mechanisms with human data, we demonstrate a powerful approach to identifying disease modifying targets; this may be useful for other neurological disorders. With this approach, we find that reignition of inflammatory JAK/STAT3 signaling in chronic epilepsy opens a window for disease modification with the FDA-approved, orally available drug CP690550.

neuroscience↗

bioRxiv2023

Native-state proteomics of Parvalbumin interneurons identifies novel molecular signatures and metabolic vulnerabilities to early Alzheimer's disease pathology

One of the earliest pathophysiological perturbations in Alzheimers Disease (AD) may arise from dysfunction of fast-spiking parvalbumin (PV) interneurons (PV-INs). Defining early protein-level (proteomic) alterations in PV-INs can provide key biological and translationally relevant insights. Here, we use cell-type-specific in vivo biotinylation of proteins (CIBOP) coupled with mass spectrometry to obtain native-state proteomes of PV interneurons. PV-INs exhibited proteomic signatures of high metabolic, mitochondrial, and translational activity, with over-representation of causally linked AD genetic risk factors. Analyses of bulk brain proteomes indicated strong correlations between PV-IN proteins with cognitive decline in humans, and with progressive neuropathology in humans and mouse models of A{beta} pathology. Furthermore, PV-IN-specific proteomes revealed unique signatures of increased mitochondrial and metabolic proteins, but decreased synaptic and mTOR signaling proteins in response to early A{beta} pathology. PV-specific changes were not apparent in whole-brain proteomes. These findings showcase the first native state PV-IN proteomes in mammalian brain, revealing a molecular basis for their unique vulnerabilities in AD.

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