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It is of critical importance to our understanding of Alzheimer's disease (AD) pathology to determine how key pathological factors are interconnected and implicated in nerve cell death, clinical symptoms, and disease progression. The formation of extracellular beta-amyloid (Aß) plaques is the major pathological hallmark of AD and Aß has been suggested to be a critical inducer of AD, driving disease pathogenesis. Exactly how Aß plaque formation begins and how ongoing plaque deposition proceeds and initiates subsequent neurotoxic mechanisms is not well understood. The primary aim of our research is to elucidate the biochemical processes underlying early Aß plaque formation in brain tissue. We recently introduced a chemical imaging paradigm based on mass spectrometry imaging (MSI) and metabolic isotope labelling to follow stable isotope labelling kinetics (iSILK) in vivo to track the in vivo build-up and deposition of Aß. Herein, knock-in Aß mouse models (App NL-F ) that develop Aß pathology gradually are metabolically labeled with stable isotopes. This chemical imaging approach timestamps amyloid plaques during the period of initial deposition allowing the fate of aggregating Aß species from before and during the earliest events of plaque pathology through plaque maturation to be tracked. To identify the molecular and cellular response to plaque maturation, we integrated iSILK with single plaque transcriptomics performed on adjacent tissue sections. This enabled changes in gene expression to be tracked as a function of plaque age (as encoded in the Aß peptide isotopologue pattern) distinct from changes due to the chronological age or pathological severity. This approach identified that plaque age correlates negatively with gene expression patterns associated with synaptic function as early as in 10-month-old animals but persists into 18 months. Finally, we integrated hyperspectral confocal microscopy into our multiomic approach to image amyloid structural isomers, revealing a positive correlation between plaque age and amyloid structural maturity. This analysis identified three categories of plaques, each with a distinct impact on the surrounding microenvironment. Here, we identified that older, more compact plaques were associated with the most significant synapse loss and toxicity. These data show how isotope-encoded MS imaging can be used to delineate Aß toxicity dynamics in vivo. Moreover, we show for the first time a functional integration of dynamic MSI, structural plaque imaging and whole genome-wide spatial transcriptomics at the single plaque level. This multiomic approach offers an unprecedented combination of temporal and spatial resolution enabling a description of the earliest events of precipitating amyloid pathology and how Aß modulates synaptotoxic mechanisms.
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Perineuronal nets (PNNs), a specialized form of extra cellular matrix (ECM), surround numerous neurons in the CNS and allow synaptic connectivity through holes in its structure. We hypothesize that PNNs serve as gatekeepers that guard and protect synaptic territory and thus may stabilize an engram circuit. We present high-resolution and 3D EM images of PNN-engulfed neurons in mice brains, showing that synapses occupy the PNN holes and that invasion of other cellular components is rare. PNN constituents in mice brains are long-lived and can be eroded faster in an enriched environment, while synaptic proteins have a high turnover rate. Preventing PNN erosion by using pharmacological inhibition of PNN-modifying proteases or matrix metalloproteases 9 (MMP9) knockout mice allowed normal fear memory acquisition but diminished long-term memory stabilization, supporting the above hypothesis.
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Matriz Extracelular , Neurônios , Sinapses , Animais , Sinapses/metabolismo , Matriz Extracelular/metabolismo , Camundongos , Neurônios/metabolismo , Camundongos Knockout , Metaloproteinase 9 da Matriz/metabolismo , Encéfalo/metabolismo , Camundongos Endogâmicos C57BL , Medo/fisiologia , Rede Nervosa/fisiologia , Rede Nervosa/metabolismo , Rede Nervosa/efeitos dos fármacosRESUMO
Protein misfolding and aggregation are cardinal features of neurodegenerative disease (NDD) and they contribute to pathophysiology by both loss-of-function (LOF) and gain-of-function (GOF) mechanisms. This is well exemplified by TDP-43 which aggregates and mislocalizes in several NDDs. The depletion of nuclear TDP-43 leads to reduction in its normal function in RNA metabolism and the cytoplasmic accumulation of TDP-43 leads to aberrant protein homeostasis. A modifier screen found that loss of rad23 suppressed TDP-43 pathology in invertebrate and tissue culture models. Here we show in a mouse model of TDP-43 pathology that genetic or antisense oligonucleotide (ASO)-mediated reduction in rad23a confers benefits on survival and behavior, histological hallmarks of disease and reduction of mislocalized and aggregated TDP-43. This results in improved function of the ubiquitin-proteasome system (UPS) and correction of transcriptomic alterations evoked by pathologic TDP-43. RAD23A-dependent remodeling of the insoluble proteome appears to be a key event driving pathology in this model. As TDP-43 pathology is prevalent in both familial and sporadic NDD, targeting RAD23A may have therapeutic potential.
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Painful diabetic neuropathy (PDN) is a challenging complication of diabetes with patients experiencing a painful and burning sensation in their extremities. Existing treatments provide limited relief without addressing the underlying mechanisms of the disease. PDN involves the gradual degeneration of nerve fibers in the skin. Keratinocytes, the most abundant epidermal cell type, are closely positioned to cutaneous nerve terminals, suggesting the possibility of bi-directional communication. Exosomes are small extracellular vesicles released from many cell types that mediate cell to cell communication. The role of keratinocyte-derived exosomes (KDEs) in influencing signaling between the skin and cutaneous nerve terminals and their contribution to the genesis of PDN has not been explored. In this study, we characterized KDEs in a well-established high-fat diet (HFD) mouse model of PDN using primary adult mouse keratinocyte cultures. We obtained highly enriched KDEs through size exclusion chromatography and then analyzed their molecular cargo using proteomic analysis and small RNA sequencing. We found significant differences in the protein and microRNA content of HFD KDEs compared to KDEs obtained from control mice on a regular diet (RD), including pathways involved in axon guidance and synaptic transmission. Additionally, using an in vivo conditional extracellular vesicle (EV) reporter mouse model, we demonstrated that epidermal-originating GFP-tagged KDEs are retrogradely trafficked into the DRG neuron cell body. Overall, our study presents a potential novel mode of communication between keratinocytes and DRG neurons in the skin, revealing a possible role for KDEs in contributing to the axonal degeneration that underlies neuropathic pain in PDN. Moreover, this study presents potential therapeutic targets in the skin for developing more effective, disease-modifying, and better-tolerated topical interventions for patients suffering from PDN, one of the most common and untreatable peripheral neuropathies.
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Neurotropic alphaherpesviruses, including herpes simplex virus type 1 and pseudorabies virus, establish a lifelong presence within the peripheral nervous system of their mammalian hosts. Upon entering cells, two conserved tegument proteins, pUL36 and pUL37, traffic DNA-containing capsids to nuclei. These proteins support long-distance retrograde axonal transport and invasion of the nervous system in vivo. To better understand how pUL36 and pUL37 function, recombinant viral particles carrying BioID2 fused to these proteins were produced to biotinylate cellular proteins in their proximity (<10 nm) during infection. Eighty-six high-confidence host proteins were identified by mass spectrometry and subsequently targeted by CRISPR-Cas9 gene editing to assess their contributions to early infection. Proteins were identified that both supported and antagonized infection in immortalized human epithelial cells. The latter included zyxin, a protein that localizes to focal adhesions and regulates actin cytoskeletal dynamics. Zyxin knockout cells were hyper-permissive to infection and could be rescued with even modest expression of GFP-zyxin. These results provide a resource for studies of the virus-cell interface and identify zyxin as a novel deterrent to alphaherpesvirus infection.IMPORTANCENeuroinvasive alphaherpesviruses are highly prevalent with many members found across mammals [e.g., herpes simplex virus type 1 (HSV-1) in humans and pseudorabies virus in pigs]. HSV-1 causes a range of clinical manifestations from cold sores to blindness and encephalitis. There are no vaccines or curative therapies available for HSV-1. A fundamental feature of these viruses is their establishment of lifelong infection of the nervous system in their respective hosts. This outcome is possible due to a potent neuroinvasive property that is coordinated by two proteins: pUL36 and pUL37. In this study, we explore the cellular protein network in proximity to pUL36 and pUL37 during infection and examine the impact of knocking down the expression of these proteins upon infection.
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Biotina , Humanos , Biotina/metabolismo , Zixina/metabolismo , Zixina/genética , Animais , Linhagem Celular , Herpesvirus Humano 1/genética , Herpesvirus Humano 1/fisiologia , Herpesvirus Suídeo 1/genética , Herpesvirus Suídeo 1/fisiologia , Interações Hospedeiro-Patógeno , Alphaherpesvirinae/genética , Alphaherpesvirinae/metabolismo , Sistemas CRISPR-Cas , Células Epiteliais/virologia , Células Epiteliais/metabolismoRESUMO
Combinatorial expression of postsynaptic proteins underlies synapse diversity within and between neuron types. Thus, characterization of neuron-type-specific postsynaptic proteomes is key to obtaining a deeper understanding of discrete synaptic properties and how selective dysfunction manifests in synaptopathies. To overcome the limitations associated with bulk measures of synaptic protein abundance, we developed a biotin proximity protein tagging probe to characterize neuron-type-specific postsynaptic proteomes in vivo. We found Shank3 protein isoforms are differentially expressed by direct and indirect pathway spiny projection neurons (dSPNs and iSPNs). Investigation of Shank3B-/- mice lacking exons 13-16 within the Shank3 gene, reveal distinct Shank3 protein isoform expression in iSPNs and dSPNs. In Shank3B-/- striatum, Shank3E and Shank3NT are expressed by dSPNs but are undetectable in iSPNs. Proteomic analysis indicates significant and selective alterations in the postsynaptic proteome of Shank3B-/- iSPNs. Correspondingly, the deletion of exons 13-16 diminishes dendritic spine density, reduces spine head diameter, and hampers corticostriatal synaptic transmission in iSPNs. Remarkably, reintroducing Shank3E in adult Shank3B-/- iSPNs significantly rectifies the observed dendritic spine morphological and corticostriatal synaptic transmission deficits. We report unexpected cell-type specific synaptic protein isoform expression which could play a key causal role in specifying synapse diversity and selective synapse dysfunction in synaptopathies.
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Corpo Estriado , Camundongos Knockout , Proteínas dos Microfilamentos , Proteínas do Tecido Nervoso , Neurônios , Proteômica , Sinapses , Animais , Masculino , Camundongos , Corpo Estriado/metabolismo , Corpo Estriado/patologia , Espinhas Dendríticas/metabolismo , Camundongos Endogâmicos C57BL , Proteínas dos Microfilamentos/metabolismo , Proteínas do Tecido Nervoso/metabolismo , Proteínas do Tecido Nervoso/genética , Neurônios/metabolismo , Isoformas de Proteínas/metabolismo , Proteoma/metabolismo , Proteômica/métodos , Sinapses/metabolismo , FemininoRESUMO
The recently discovered HAPSTR1 protein broadly oversees cellular stress responses. This function requires HUWE1, a ubiquitin ligase that paradoxically marks HAPSTR1 for degradation, but much about this pathway remains unclear. Here, leveraging multiplexed proteomics, we find that HAPSTR1 enables nuclear localization of HUWE1 with implications for nuclear protein quality control. We show that HAPSTR1 is tightly regulated and identify ubiquitin ligase TRIP12 and deubiquitinase USP7 as upstream regulators titrating HAPSTR1 stability. Finally, we generate conditional Hapstr1 knockout mice, finding that Hapstr1-null mice are perinatal lethal, adult mice depleted of Hapstr1 have reduced fitness, and primary cells explanted from Hapstr1-null animals falter in culture coincident with HUWE1 mislocalization and broadly remodeled signaling. Notably, although HAPSTR1 potently suppresses p53, we find that Hapstr1 is essential for life even in mice lacking p53. Altogether, we identify novel components and functional insights into the conserved HAPSTR1-HUWE1 pathway and demonstrate its requirement for mammalian life.
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Proteína Supressora de Tumor p53 , Ubiquitina-Proteína Ligases , Animais , Camundongos , Mamíferos/metabolismo , Proteínas Nucleares/metabolismo , Transdução de Sinais/genética , Proteína Supressora de Tumor p53/genética , Proteína Supressora de Tumor p53/metabolismo , Ubiquitina/metabolismo , Ubiquitina-Proteína Ligases/genética , Ubiquitina-Proteína Ligases/metabolismo , Ubiquitinação/genética , Proteínas Supressoras de Tumor/genética , Proteínas Supressoras de Tumor/metabolismo , Peptídeos e Proteínas de Sinalização Intracelular/genética , Peptídeos e Proteínas de Sinalização Intracelular/metabolismoRESUMO
In neurons, it is commonly assumed that mitochondrial replication only occurs in the cell body, after which the mitochondria must travel to the neuron's periphery. However, while mitochondrial DNA replication has been observed to occur away from the cell body, the specific mechanisms involved remain elusive. Using EdU-labelling in mouse primary neurons, we developed a tool to determine the mitochondrial replication rate. Taking of advantage of microfluidic devices, we confirmed that mitochondrial replication also occurs locally in the periphery of neurons. To achieve this, mitochondria require de novo nuclear-encoded, but not mitochondrial-encoded protein translation. Following a proteomic screen comparing synaptic with non-synaptic mitochondria, we identified two elongation factors - eEF1A1 and TUFM - that were upregulated in synaptic mitochondria. We found that mitochondrial replication is impaired upon the downregulation of eEF1A1, and this is particularly relevant in the periphery of neurons.
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Given the complexity of nervous tissues, understanding neurochemical pathophysiology puts high demands on bioanalytical techniques with respect to specificity and sensitivity. Mass spectrometry imaging (MSI) has evolved to become an important, biochemical imaging technology for spatial biology in biological and translational research. The technique facilitates comprehensive, sensitive elucidation of the spatial distribution patterns of drugs, lipids, peptides, and small proteins in situ. Matrix-assisted laser desorption ionization (MALDI)-based MSI is the dominating modality due to its broad applicability and fair compromise of selectivity, sensitivity price, throughput, and ease of use. This is particularly relevant for the analysis of spatial lipid patterns, where no other comparable spatial profiling tools are available. Understanding spatial lipid biology in nervous tissue is therefore a key and emerging application area of MSI research. The aim of this review is to give a concise guide through the MSI workflow for lipid imaging in central nervous system (CNS) tissues and essential parameters to consider while developing and optimizing MSI assays. Further, this review provides a broad overview of key developments and applications of MALDI MSI-based spatial neurolipidomics to map lipid dynamics in neuronal structures, ultimately contributing to a better understanding of neurodegenerative disease pathology.
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Doenças Neurodegenerativas , Humanos , Espectrometria de Massas por Ionização e Dessorção a Laser Assistida por Matriz , Doenças Neurodegenerativas/diagnóstico por imagem , Fluxo de Trabalho , Encéfalo/diagnóstico por imagem , LipídeosRESUMO
Extracellular vesicles (EVs) facilitate intercellular communication by transferring cargo between cells in a variety of tissues. However, how EVs achieve cell-type-specific intercellular communication is still largely unknown. We found that Notch1 and Notch2 proteins are expressed on the surface of neuronal EVs that have been generated in response to neuronal excitatory synaptic activity. Notch ligands bind these EVs on the neuronal plasma membrane, trigger their internalization, activate the Notch signaling pathway, and drive the expression of Notch target genes. The generation of these neuronal EVs requires the endosomal sorting complex required for transport-associated protein Alix. Adult Alix conditional knockout mice have reduced hippocampal Notch signaling activation and glutamatergic synaptic protein expression. Thus, EVs facilitate neuron-to-neuron communication via the Notch receptor-ligand system in the brain.
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Vesículas Extracelulares , Neurônios , Animais , Camundongos , Ligantes , Transporte Proteico , Transdução de Sinais , Camundongos KnockoutRESUMO
Efficient protein turnover is essential for cellular homeostasis and organ function. Loss of proteostasis is a hallmark of aging culminating in severe dysfunction of protein turnover. To investigate protein turnover dynamics as a function of age, we performed continuous in vivo metabolic stable isotope labeling in mice along the aging continuum. First, we discovered that the brain proteome uniquely undergoes dynamic turnover fluctuations during aging compared to heart and liver tissue. Second, trends in protein turnover in the brain proteome during aging showed sex-specific differences that were tightly tied to cellular compartments. Next, parallel analyses of the insoluble proteome revealed that several cellular compartments experience hampered turnover, in part due to misfolding. Finally, we found that age-associated fluctuations in proteasome activity were associated with the turnover of core proteolytic subunits, which was recapitulated by pharmacological suppression of proteasome activity. Taken together, our study provides a proteome-wide atlas of protein turnover across the aging continuum and reveals a link between the turnover of individual proteasome subunits and the age-associated decline in proteasome activity.
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Complexo de Endopeptidases do Proteassoma , Proteoma , Masculino , Feminino , Animais , Camundongos , Complexo de Endopeptidases do Proteassoma/metabolismo , Proteoma/metabolismo , Envelhecimento/metabolismo , Proteólise , Encéfalo/metabolismo , Mamíferos , Marcação por IsótopoRESUMO
Numerous rare variants that cause neurodevelopmental disorders (NDDs) occur within genes encoding synaptic proteins, including ionotropic glutamate receptors. However, in many cases, it remains unclear how damaging missense variants affect brain function. We determined the physiological consequences of an NDD causing missense mutation in the GRIK2 kainate receptor (KAR) gene, that results in a single amino acid change p.Ala657Thr in the GluK2 receptor subunit. We engineered this mutation in the mouse Grik2 gene, yielding a GluK2(A657T) mouse, and studied mice of both sexes to determine how hippocampal neuronal function is disrupted. Synaptic KAR currents in hippocampal CA3 pyramidal neurons from heterozygous A657T mice exhibited slow decay kinetics, consistent with incorporation of the mutant subunit into functional receptors. Unexpectedly, CA3 neurons demonstrated elevated action potential spiking because of downregulation of the small-conductance Ca2+ activated K+ channel (SK), which mediates the post-spike afterhyperpolarization. The reduction in SK activity resulted in increased CA3 dendritic excitability, increased EPSP-spike coupling, and lowered the threshold for the induction of LTP of the associational-commissural synapses in CA3 neurons. Pharmacological inhibition of SK channels in WT mice increased dendritic excitability and EPSP-spike coupling, mimicking the phenotype in A657T mice and suggesting a causative role for attenuated SK activity in aberrant excitability observed in the mutant mice. These findings demonstrate that a disease-associated missense mutation in GRIK2 leads to altered signaling through neuronal KARs, pleiotropic effects on neuronal and dendritic excitability, and implicate these processes in neuropathology in patients with genetic NDDs.SIGNIFICANCE STATEMENT Damaging mutations in genes encoding synaptic proteins have been identified in various neurodevelopmental disorders, but the functional consequences at the cellular and circuit level remain elusive. By generating a novel knock-in mutant mouse, this study examined the role of a pathogenic mutation in the GluK2 kainate receptor (KAR) subunit, a subclass of ionotropic glutamate receptors. Analyses of hippocampal CA3 pyramidal neurons determined elevated action potential firing because of an increase in dendritic excitability. Increased dendritic excitability was attributable to reduced activity of a Ca2+ activated K+ channel. These results indicate that a pathogenic KAR mutation results in dysregulation of dendritic K+ channels, which leads to an increase in synaptic integration and backpropagation of action potentials into distal dendrites.
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Mutação de Sentido Incorreto , Receptores de Ácido Caínico , Masculino , Feminino , Humanos , Camundongos , Animais , Receptores de Ácido Caínico/genética , Receptores de Ácido Caínico/metabolismo , Neurônios/fisiologia , Hipocampo/fisiologia , Células Piramidais/fisiologiaRESUMO
The mechanisms contributing to age-related deterioration of the female reproductive system are complex, however aberrant protein homeostasis is a major contributor. We elucidated exceptionally stable proteins, structures, and macromolecules that persist in mammalian ovaries and gametes across the reproductive lifespan. Ovaries exhibit localized structural and cell-type specific enrichment of stable macromolecules in both the follicular and extrafollicular environments. Moreover, ovaries and oocytes both harbor a panel of exceptionally long-lived proteins, including cytoskeletal, mitochondrial, and oocyte-derived proteins. The exceptional persistence of these long-lived molecules suggest a critical role in lifelong maintenance and age-dependent deterioration of reproductive tissues.
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Mutations in SOD1 cause amyotrophic lateral sclerosis (ALS) through gain-of-function effects, yet the mechanisms by which misfolded mutant SOD1 (mutSOD1) protein impairs human motor neurons (MNs) remain unclear. Here, we use induced-pluripotent-stem-cell-derived MNs coupled to metabolic stable isotope labeling and mass spectrometry to investigate proteome-wide degradation dynamics. We find several proteins, including the ALS-causal valosin-containing protein (VCP), which predominantly acts in proteasome degradation and autophagy, that degrade slower in mutSOD1 relative to isogenic control MNs. The interactome of VCP is altered in mutSOD1 MNs in vitro, while VCP selectively accumulates in the affected motor cortex of ALS-SOD1 patients. Overexpression of VCP rescues mutSOD1 toxicity in MNs in vitro and in a C. elegans model in vivo, in part due to its ability to modulate the degradation of insoluble mutSOD1. Our results demonstrate that VCP contributes to mutSOD1-dependent degeneration, link two distinct ALS-causal genes, and highlight selective protein degradation impairment in ALS pathophysiology.
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Esclerose Lateral Amiotrófica , Células-Tronco Pluripotentes Induzidas , Animais , Humanos , Esclerose Lateral Amiotrófica/genética , Esclerose Lateral Amiotrófica/metabolismo , Superóxido Dismutase-1/genética , Superóxido Dismutase-1/metabolismo , Proteoma/metabolismo , Proteína com Valosina/metabolismo , Células-Tronco Pluripotentes Induzidas/metabolismo , Caenorhabditis elegans/metabolismo , Neurônios Motores/metabolismo , Homeostase , MutaçãoRESUMO
BACKGROUND: The accumulation of amyloid beta (Aß) peptides in fibrils is prerequisite for Alzheimer's disease (AD). Our understanding of the proteins that promote Aß fibril formation and mediate neurotoxicity has been limited due to technical challenges in isolating pure amyloid fibrils from brain extracts. METHODS: To investigate how amyloid fibrils form and cause neurotoxicity in AD brain, we developed a robust biochemical strategy. We benchmarked the success of our purifications using electron microscopy, amyloid dyes, and a large panel of Aß immunoassays. Tandem mass-spectrometry based proteomic analysis workflows provided quantitative measures of the amyloid fibril proteome. These methods allowed us to compare amyloid fibril composition from human AD brains, three amyloid mouse models, transgenic Aß42 flies, and Aß42 seeded cultured neurons. RESULTS: Amyloid fibrils are primarily composed by Aß42 and unexpectedly harbor Aß38 but generally lack Aß40 peptides. Multidimensional quantitative proteomics allowed us to redefine the fibril proteome by identifying 20 new amyloid-associated proteins. Notably, we confirmed 57 previously reported plaque-associated proteins. We validated a panel of these proteins as bona fide amyloid-interacting proteins using antibodies and orthogonal proteomic analysis. One metal-binding chaperone metallothionein-3 is tightly associated with amyloid fibrils and modulates fibril formation in vitro. Lastly, we used a transgenic Aß42 fly model to test if knock down or over-expression of fibril-interacting gene homologues modifies neurotoxicity. Here, we could functionally validate 20 genes as modifiers of Aß42 toxicity in vivo. CONCLUSIONS: These discoveries and subsequent confirmation indicate that fibril-associated proteins play a key role in amyloid formation and AD pathology.
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Doença de Alzheimer , Amiloide , Humanos , Animais , Camundongos , Peptídeos beta-Amiloides , Proteoma , Proteômica , Proteínas Amiloidogênicas , EncéfaloRESUMO
Parkin-mediated mitophagy has been studied extensively, but whether mutations in parkin contribute to Parkinson's disease pathogenesis through alternative mechanisms remains unexplored. Using patient-derived dopaminergic neurons, we found that phosphorylation of parkin by Ca2+/calmodulin-dependent protein kinase 2 (CaMK2) at Ser9 leads to activation of parkin in a neuronal-activity-dependent manner. Activated parkin ubiquitinates synaptojanin-1, facilitating its interaction with endophilin A1 and synaptic vesicle recycling. Neurons from PD patients with mutant parkin displayed defective recycling of synaptic vesicles, leading to accumulation of toxic oxidized dopamine that was attenuated by boosting endophilin A1 expression. Notably, combined heterozygous parkin and homozygous PTEN-induced kinase 1 (PINK1) mutations led to earlier disease onset compared with homozygous mutant PINK1 alone, further underscoring a PINK1-independent role for parkin in contributing to disease. Thus, this study identifies a pathway for selective activation of parkin at human dopaminergic synapses and highlights the importance of this mechanism in the pathogenesis of Parkinson's disease.
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Neurônios Dopaminérgicos , Doença de Parkinson , Humanos , Neurônios Dopaminérgicos/metabolismo , Mutação , Doença de Parkinson/metabolismo , Proteínas Quinases/genética , Proteínas Quinases/metabolismo , Vesículas Sinápticas/metabolismo , Ubiquitina-Proteína Ligases/genética , Ubiquitina-Proteína Ligases/metabolismoRESUMO
Rare genetic variants in ANK2, which encodes ankyrin-B, are associated with neurodevelopmental disorders (NDDs); however, their pathogenesis is poorly understood. We find that mice with prenatal deletion in cortical excitatory neurons and oligodendrocytes (Ank2-/-:Emx1-Cre), but not with adolescent deletion in forebrain excitatory neurons (Ank2-/-:CaMKIIα-Cre), display severe spontaneous seizures, increased mortality, hyperactivity, and social deficits. Calcium imaging of cortical slices from Ank2-/-:Emx1-Cre mice shows increased neuronal calcium event amplitude and frequency, along with network hyperexcitability and hypersynchrony. Quantitative proteomic analysis of cortical synaptic membranes reveals upregulation of dendritic spine plasticity-regulatory proteins and downregulation of intermediate filaments. Characterization of the ankyrin-B interactome identifies interactors associated with autism and epilepsy risk factors and synaptic proteins. The AMPA receptor antagonist, perampanel, restores cortical neuronal activity and partially rescues survival in Ank2-/-:Emx1-Cre mice. Our findings suggest that synaptic proteome alterations resulting from Ank2 deletion impair neuronal activity and synchrony, leading to NDDs-related behavioral impairments.
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Anquirinas , Prosencéfalo , Proteoma , Convulsões , Animais , Camundongos , Anquirinas/genética , Cálcio , Fenótipo , Prosencéfalo/fisiopatologia , Proteoma/genética , Proteômica , Convulsões/genética , Camundongos KnockoutRESUMO
Orchestration of protein production and degradation and the regulation of protein lifetimes play a central role in many basic biological processes. Nearly all mammalian proteins are replenished by protein turnover in waves of synthesis and degradation. Protein lifetimes in vivo are typically measured in days, but a small number of extremely long-lived proteins (ELLPs) persist for months or even years. ELLPs are rare in all tissues but are enriched in tissues containing terminally differentiated post-mitotic cells and extracellular matrix. Consistently, emerging evidence suggests that the cochlea may be particularly enriched in ELLPs. Damage to ELLPs in specialized cell types, such as crystallin in the lens cells of the eye, causes organ failure such as cataracts. Similarly, damage to cochlear ELLPs is likely to occur with many insults, including acoustic overstimulation, drugs, anoxia, and antibiotics, and may play an underappreciated role in hearing loss. Furthermore, hampered protein degradation may contribute to acquired hearing loss. In this review, I highlight our knowledge of the lifetimes of cochlear proteins with an emphasis on ELLPs and the potential contribution that impaired cochlear protein degradation has on acquired hearing loss and the emerging relevance of ELLPs.
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Surdez , Perda Auditiva Provocada por Ruído , Animais , Perda Auditiva Provocada por Ruído/etiologia , Cóclea/metabolismo , Surdez/metabolismo , MamíferosRESUMO
Perineuronal nets (PNN), a specialized form of ECM (?), surround numerous neurons in the CNS and allow synaptic connectivity through holes in its structure. We hypothesis that PNNs serve as gatekeepers that guard and protect synaptic territory, and thus may stabilize an engram circuit. We present high-resolution, and 3D EM images of PNN- engulfed neurons showing that synapses occupy the PNN holes, and that invasion of other cellular components are rare. PNN constituents are long-lived and can be eroded faster in an enriched environment, while synaptic proteins have high turnover rate. Preventing PNN erosion by using pharmacological inhibition of PNN-modifying proteases or MMP9 knockout mice allowed normal fear memory acquisition but diminished remote-memory stabilization, supporting the above hypothesis. Significance: In this multidisciplinary work, we challenge the hypothesis that the pattern of holes in the perineuronal nets (PNN) hold the code for very-long-term memories. The scope of this work might lead us closer to the understanding of how we can vividly remember events from childhood to death bed. We postulate that the PNN holes hold the code for the engram. To test this hypothesis, we used three independent experimental strategies; high-resolution 3D electron microscopy, Stable Isotop Labeling in Mammals (SILAM) for proteins longevity, and pharmacologically and genetically interruption of memory consolidation in fear conditioning experiments. All of these experimental results did not dispute the PNN hypothesis.