Biology subjects

McClatchy, D.

Publications and source records attributed to McClatchy, D..

8 recordsLinked to original sources

bioRxiv2026

Quantitation of Spatial Proteoforms in Alzheimer's Disease

Alzheimers disease (AD) pathogenesis involves complex, multifactorial changes to the brain proteome that conventional unfractionated analyses may obscure. Proteins frequently occupy multiple subcellular compartments as spatial proteoforms, yet the contribution of aberrant protein localization to AD pathogenesis remains poorly understood. To address this, we fractionated post-mortem human hippocampi from 13 AD and 14 non-AD individuals into four subcellular fractions and quantified 6,123 proteins by TMT-LC-MS. Although 75% of proteins were detected in more than one fraction, 78% of significant AD-associated alterations were restricted to a single fraction, demonstrating that subcellular localization is a primary determinant of disease vulnerability. Discordant abundance patterns between fractions revealed retromer complex mislocalization, nuclear transport dysfunction, and insoluble protein accumulation, with the endosomal-lysosomal and protein folding pathways most consistently perturbed. To examine how these perturbations evolve with disease progression, we applied the QUAD strategy to measure protein degradation in two fractions of APPswePS1delta9 mouse cortex at 2, 5, and 12 months. Degradation rates diverged between fractions and genotypes in an age-dependent manner, and cross-dataset comparison identified six proteins altered at the earliest pre-pathological timepoint, implicating vesicle transport and proteostasis disruption as initiating features of AD. These findings establish spatial proteoforms as essential units of pathogenic analysis and reveal disease-relevant signals invisible to bulk tissue approaches.

neuroscience↗

bioRxiv2024

Acid cleavable biotin-alkyne improves sensitivity for direct detection of biotin labeled peptides in BONCAT analysis.

BONCAT (Biorthogonal noncanonical amino acid tagging) is a labeling strategy that covalently adds a biotin-alkyne (BA) to methionine analogs via a click reaction. When methionine analogs are incorporated into a proteome, enrichment of the BA-labeled proteins allows the detection of newly synthesized proteins (NSP) by mass spectrometry. We previously reported that using our Direct Detection of Biotin-containing Tags (DidBIT) strategy, protein identifications and confidence are increased by enriching for BA-peptides instead of BA-proteins. We compared cleavable BA (DADPS) and uncleavable BA in the identification and TMT quantification of NSP. More than fifty percent more proteins were identified and quantified using DADPS than with uncleavable BA. Interrogation of the data revealed that multiple factors are responsible for the superior performance of DADPS.

biochemistry↗

bioRxiv2024

Activity-dependent synthesis of Emerin gates neuronal plasticity by regulating proteostasis

Neurons dynamically regulate their proteome in response to sensory input, a key process underlying experience-dependent plasticity. We characterized the visual experience-dependent nascent proteome within a brief, defined time window after stimulation using an optimized metabolic labeling approach. Visual experience induced cell type-specific and age-dependent alterations in the nascent proteome, including proteostasis-related processes. We identified Emerin as the top activity-induced candidate plasticity protein and demonstrated that its rapid activity-induced synthesis is transcription-independent. In contrast to its nuclear localization and function in myocytes, activity-induced neuronal Emerin is abundant in the endoplasmic reticulum and broadly inhibits protein synthesis, including translation regulators and synaptic proteins. Downregulating Emerin shifted the dendritic spine population from predominantly mushroom morphology to filopodia and decreased network connectivity. In mice, decreased Emerin reduced visual response magnitude and impaired visual information processing. Our findings support an experience-dependent feed-forward role for Emerin in temporally gating neuronal plasticity by negatively regulating translation.

neuroscience↗

bioRxiv2024

DiDBiT-TMT: A novel method to quantify changes in the proteomic landscape induced by neural plasticity

Withdrawal StatementThe authors have withdrawn this manuscript because during the peer review process in the journal, it was discovered that one of the processed proteomic data groups (the chemical LTP group) was mistakenly processed from the raw data for the norepinephrine group, resulting in an apparent complete overlap between the two conditions. For a correct analysis, please refer to the currently published peer-reviewed publication through the following DOI: https://doi.org/10.1021/acs.jproteome.4c00180 Therefore, the authors do not wish this preprint to be cited as reference for the project. If you have any questions, please contact the corresponding author.

neuroscience↗

bioRxiv2023

In vivo mapping of protein-protein interactions of schizophrenia risk factors generates an interconnected disease network.

Genetic analyses of Schizophrenia (SCZ) patients have identified thousands of risk factors. In silico protein-protein interaction (PPI) network analysis has provided strong evidence that disrupted PPI networks underlie SCZ pathogenesis. In this study, we performed in vivo PPI analysis of several SCZ risk factors in the rodent brain. Using endogenous antibody immunoprecipitations coupled to mass spectrometry (MS) analysis, we constructed a SCZ network comprising 1612 unique PPI with a 5% FDR. Over 90% of the PPI were novel, reflecting the lack of previous PPI MS studies in brain tissue. Our SCZ PPI network was enriched with known SCZ risk factors, which supports the hypothesis that an accumulation of disturbances in selected PPI networks underlies SCZ. We used Stable Isotope Labeling in Mammals (SILAM) to quantitate phencyclidine (PCP) perturbations in the SCZ network and found that PCP weakened most PPI but also led to some enhanced or new PPI. These findings demonstrate that quantitating PPI in perturbed biological states can reveal alterations to network biology.

neuroscience↗

bioRxiv2020

A Temporal Quantitative Profiling of Newly Synthesized Proteins during Aβ Accumulation

Accumulation of aggregated amyloid beta (A{beta}) in the brain is believed to impair multiple cellular pathways and play a central role in Alzheimers disease pathology. But how this process is regulated remains unclear. In theory, measuring protein synthesis is the most direct way to evaluate a cells response to stimuli, but to date there have been few reliable methods to do this. To identify the protein regulatory network during the development of A{beta} deposition in AD, we applied a new proteomic technique to quantitate newly synthesized protein (NSP) changes in the cerebral cortex and hippocampus of 2, 5 and 9-month-old APP/PS1 AD transgenic mice. This bioorthogonal non-canonical amino acid tagging (BONCAT) analysis combined PALM (Pulse Azidohomoalanine Labeling in Mammals) and HILAQ (Heavy Isotope Labeled AHA Quantitation) to reveal a comprehensive dataset of NSPs prior to and post A{beta} deposition, including the identification of proteins not previously associated with AD, and demonstrated that the pattern of differentially expressed NSPs is age-dependent. We also found dysregulated vesicle transportation networks including endosomal subunits, coat protein complex I (COPI) and mitochondrial respiratory chain throughout all timepoints and two brain regions. These results point to a pathological dysregulation of vesicle transportation that occurs prior to A{beta} accumulation and the onset of AD symptoms, which may progressively impact the entire protein network and thereby drive neurodegeneration. This study illustrates key pathway regulation responses to the development of AD pathogenesis by directly measuring the changes of protein synthesis and provides unique insights into the mechanisms that underlie AD.

neuroscience↗

bioRxiv2017

Role of the Visual Experience-Dependent Nascent Proteome in Neuronal Plasticity

Experience-dependent synaptic plasticity refines brain circuits during development. To identify novel protein synthesis-dependent mechanisms contributing to experience-dependent plasticity, we conducted a quantitative proteomic screen of the nascent proteome in response to visual experience in Xenopus optic tectum using bio-orthogonal metabolic labeling (BONCAT). We identified 83 differentially synthesized candidate plasticity proteins (CPPs). The CPPs form strongly interconnected networks and are annotated to a variety of biological functions, including RNA splicing, protein translation, and chromatin remodeling. Functional analysis of select CPPs revealed the requirement for eukaryotic initiation factor 3 subunit A (eIF3A), fused in sarcoma (FUS), and ribosomal protein s17 (RPS17) in experience-dependent structural plasticity in tectal neurons and behavioral plasticity in tadpoles. These results demonstrate that the nascent proteome is dynamic in response to visual experience and that de novo synthesis of machinery that regulates RNA splicing and protein translation is required for experience-dependent plasticity.\n\nHighlightsO_LIThe nascent proteome changes dynamically in response to visual experience\nC_LIO_LINewly synthesized candidate plasticity proteins were identified using MS/MS\nC_LIO_LIVisual experience induces de novo synthesis of synaptic and cytoskeletal proteins\nC_LIO_LISynthesis of RNA splicing and translation machinery is required for plasticity\nC_LI\n\neTOC BlurbLiu et al. show that the nascent proteome changes dynamically in response to plasticity-inducing visual experience. Functional analysis reveals that visual experience-dependent synthesis of RNA splicing and protein translation machinery is required for plasticity.

neuroscience↗

bioRxiv2017

Exosomes regulate Neurogenesis and Circuit Assembly in a Model of Rett Syndrome

Exosomes are thought to be secreted by all cells in the body and to be involved in intercellular communication. Here, we tested whether neural exosomes regulate the development of neural circuits and whether exosome-mediated signaling may be aberrant in the neurodevelopmental disorder Rett Syndrome (RTT). Quantitative proteomic analysis comparing exosomes from human induced pluripotent stem cells (hiPSC) - derived RTT patient or control neural cultures indicates that control exosomes contain signaling components capable of influencing neuronal development and function, which are lacking in RTT exosomes. Moreover, treatment with control exosomes rescues neuron number, apoptosis, synaptic puncta and synchronized firing phenotypes of MeCP2 knockdown in human primary neurons, indicating that exosomes have the capacity to influence neural development and may be a promising avenue to treat neurodevelopmental disorders like Rett Syndrome.

neuroscience↗