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In vitro studies have established the prevalent theory that the mitochondrial kinase PINK1 protects neurodegeneration by removing damaged mitochondria in Parkinson's disease (PD). However, difficulty in detecting endogenous PINK1 protein in rodent brains and cell lines has prevented the rigorous investigation of the in vivo role of PINK1. Here we report that PINK1 kinase form is selectively expressed in the human and monkey brains. CRISPR/Cas9-mediated deficiency of PINK1 causes similar neurodegeneration in the brains of fetal and adult monkeys as well as cultured monkey neurons without affecting mitochondrial protein expression and morphology. Importantly, PINK1 mutations in the primate brain and human cells reduce protein phosphorylation that is important for neuronal function and survival. Our findings suggest that PINK1 kinase activity rather than its mitochondrial function is essential for the neuronal survival in the primate brains and that its kinase dysfunction could be involved in the pathogenesis of PD., (© 2021. The Author(s).)

Angelman syndrome (AS) is a severe neurodevelopmental disorder caused by maternal mutation and paternal imprinting of the gene encoding UBE3A, an E3 ubiquitin ligase. Although several potential target proteins of UBE3A have been reported, how these proteins regulate neuronal development remains unclear. We performed a large-scale quantitative proteomic analysis using stable-isotope labeling of amino acids in mammals (SILAM) in mice with maternal Ube3a mutation. We identified huntingtin (Htt)-associated protein (HAP1), a protein that is involved in Huntington's disease (HD), as a new target of UBE3A. We demonstrate that HAP1 regulates autophagy at the initiation stage by promoting PtdIns3K complex formation and enhancing its activity. HAP1 also co-localized with MAP1LC3 (LC3) and other proteins involved in autophagosome expansion. As a result, HAP1 increased autophagy flux. Strikingly, knocking down of HAP1 alleviated aberrant autophagy in primary neurons from AS mice. Concordantly, treatment of AS neurons with an autophagy inhibitor alleviated the reduction in density of dendritic spines. Furthermore, autophagy inhibition in AS mice partially alleviated a social interaction deficit as shown in open field test. Thus, our results identify HAP1 as an in vivo UBE3A target that contributes to deregulated autophagy and synaptic dysfunction in the central nervous system of AS mouse., (Copyright © 2019 Elsevier Inc. All rights reserved.)

Mutations of X-linked gene Methyl CpG binding protein 2 (MECP2) are the major causes of Rett syndrome (RTT), a severe neurodevelopmental disorder. Duplications of MECP2-containing genomic segments lead to severe autistic symptoms in human. MECP2-coding protein methyl-CpG-binding protein 2 (MeCP2) is involved in transcription regulation, microRNA processing and mRNA splicing. However, molecular mechanisms underlying the involvement of MeCP2 in mRNA splicing in neurons remain largely elusive. In this work we found that the majority of MeCP2-associated proteins are involved in mRNA splicing using mass spectrometry analysis with multiple samples from Mecp2-null rat brain, mouse primary neuron and human cell lines. We further showed that Mecp2 knockdown in cultured cortical neurons led to widespread alternations of mRNA alternative splicing. Analysis of ChIP-seq datasets indicated that MeCP2-regulated exons display specific epigenetic signatures, with DNA modification 5-hydroxymethylcytosine (5hmC) and histone modification H3K4me3 are enriched in down-regulated exons, while the H3K36me3 signature is enriched in exons up-regulated in Mecp2-knockdown neurons comparing to un-affected neurons. Functional analysis reveals that genes containing MeCP2-regulated exons are mainly involved in synaptic functions and mRNA splicing. These results suggested that MeCP2 regulated mRNA splicing through interacting with 5hmC and epigenetic changes in histone markers, and provide functional insights of MeCP2-mediated mRNA splicing in the nervous system.

Neuronal activity influences genes involved in circuit development and information processing. However, the molecular basis of this process remains poorly understood. We found that HDAC4, a histone deacetylase that shuttles between the nucleus and cytoplasm, controls a transcriptional program essential for synaptic plasticity and memory. The nuclear import of HDAC4 and its association with chromatin is negatively regulated by NMDA receptors. In the nucleus, HDAC4 represses genes encoding constituents of central synapses, thereby affecting synaptic architecture and strength. Furthermore, we show that a truncated form of HDAC4 encoded by an allele associated with mental retardation is a gain-of-function nuclear repressor that abolishes transcription and synaptic transmission despite the loss of the deacetylase domain. Accordingly, mice carrying a mutant that mimics this allele exhibit deficits in neurotransmission, spatial learning, and memory. These studies elucidate a mechanism of experience-dependent plasticity and define the biological role of HDAC4 in the brain., (Copyright © 2012 Elsevier Inc. All rights reserved.)

Many neurological disorders are caused by perturbations during brain development, but these perturbations cannot be readily identified until there is comprehensive description of the development process. In this study, we performed mass spectrometry analysis of the synaptosomal and mitochondrial fractions from three rat brain regions at four postnatal time points. To quantitate our analysis, we employed (15)N labeled rat brains using a technique called SILAM (stable isotope labeling in mammals). We quantified 167429 peptides and identified over 5000 statistically significant changes during development including known disease-associated proteins. Global analysis revealed distinct trends between the synaptic and nonsynaptic mitochondrial proteomes and common protein networks between regions each consisting of a unique array of expression patterns. Finally, we identified novel regulators of neurodevelopment that possess the identical temporal pattern of known regulators of neurodevelopment. Overall, this study is the most comprehensive quantitative analysis of the developing brain proteome to date, providing an important resource for neurobiologists.

Terminally differentiated primary cells represent a valuable in vitro model to study signaling events associated within a specific tissue. Quantitative proteomic methods using metabolic labeling in primary cells encounter labeling efficiency issues hindering the use of these cells. Here we developed a method to quantify the proteome and phosphoproteome of cultured neurons using (15)N-labeled brain tissue as an internal standard and applied this method to determine how an inhibitor of an excitatory neural transmitter receptor, phencyclidine (PCP), affects the global phosphoproteome of cortical neurons. We identified over 10,000 phosphopeptides and made accurate quantitative measurements of the neuronal phosphoproteome after neuronal inhibition. We show that short PCP treatments lead to changes in phosphorylation for 7% of neuronal phosphopeptides and that prolonged PCP treatment alters the total levels of several proteins essential for synaptic transmission and plasticity and leads to a massive reduction in the synaptic strength of inhibitory synapses. The results provide valuable insights into the dynamics of molecular networks implicated in PCP-mediated NMDA receptor inhibition and sensorimotor deficits.

Mass spectrometry-based proteomics is increasingly used to address basic and clinical questions in biomedical research through studies of differential protein expression, protein-protein interactions, and posttranslational modifications. The complex structural and functional organization of the human brain warrants the application of high-throughput, systematic approaches to understand the functional alterations under normal physiological conditions and the perturbations of neurological diseases. This primer focuses on shotgun-proteomics-based tandem mass spectrometry for the identification of proteins in a complex mixture. It describes the basic concepts of protein differential expression analysis and posttranslational modification analysis and discusses several strategies to improve the coverage of the proteome.

Protein phosphorylation is a globally adopted and tightly controlled post-translational modification, and represents one of the most important molecular switching mechanisms that govern the entire spectrum of biological processes. In the central nervous system, it has been demonstrated that phosphorylation of key proteins mediating chromatin remodeling and gene transcription plays an important role controlling brain development, synaptogenesis, learning and memory. Many studies have focused on large scale identification of phosphopeptides in brain tissue. These studies have identified phosphorylation site specific motifs useful for predicting protein kinase substrates. In this study, we applied a previously developed quantitative approach, stable isotope labeling of amino acids in mammals (SILAM), to quantify changes in the phosphorylation of nuclear proteins between a postnatal day one (p1) and a p45 rat brain cortex. Using a 15N labeled rat brain as an internal standard, we quantified 705 phosphopeptides in the p1 cortex and 1477 phosphopeptides in the p45 cortex, which translates to 380 and 585 phosphoproteins in p1 and p45 cortex, respectively. Bioinformatic analysis of the differentially modified phosphoproteins revealed that phosphorylation is upregulated on multiple components of chromatin remodeling complexes in the p1 cortex. Taken together, we demonstrated for the first time the usefulness of employing stable isotope labeled rat tissue for global quantitative phosphorylation analysis.

Large-scale proteomic analysis of the mammalian brain has been successfully performed with mass spectrometry techniques, such as Multidimensional Protein Identification Technology (MudPIT), to identify hundreds to thousands of proteins. Strategies to efficiently quantify protein expression levels in the brain in a large-scale fashion, however, are lacking. Here, we demonstrate a novel quantification strategy for brain proteomics called SILAM (Stable Isotope Labeling in Mammals). We utilized a 15N metabolically labeled rat brain as an internal standard to perform quantitative MudPIT analysis on the synaptosomal fraction of the cerebellum during post-natal development. We quantified the protein expression level of 1138 proteins in four developmental time points, and 196 protein alterations were determined to be statistically significant. Over 50% of the developmental changes observed have been previously reported using other protein quantification techniques, and we also identified proteins as potential novel regulators of neurodevelopment. We report the first large-scale proteomic analysis of synaptic development in the cerebellum, and we demonstrate a useful quantitative strategy for studying animal models of neurological disease. [ABSTRACT FROM AUTHOR]