Several neurodegenerative diseases including Alzheimer's Disease (AD) are characterized by ubiquitin‐positive pathological protein aggregates. Here, an immunoaffinity approach is utilized to enrich ...ubiquitylated isopeptides after trypsin digestion from five AD and five age‐matched control postmortem brain tissues. Label‐free MS‐based proteomic analysis identifies 4291 unique ubiquitylation sites mapping to 1682 unique proteins. Differential enrichment analysis shows that over 800 ubiquitylation sites are significantly altered between AD and control cases. Of these, ≈80% are increased in AD, including seven poly ubiquitin linkages, which is consistent with proteolytic stress and high burden of ubiquitylated pathological aggregates in AD. The microtubule associated protein Tau, the core component of neurofibrillary tangles, has the highest number of increased sites of ubiquitylation per any protein in AD. Tau poly ubiquitylation from AD brain homogenates is confirmed by reciprocal co‐immunoprecipitation and by affinity capture using tandem ubiquitin binding entities. Co‐modified peptides, with both ubiquitylation and phosphorylation sites, are also enriched in AD. Notably, many of the co‐modified peptides mapped to Tau within KXGS motifs in the microtubule binding region suggesting that crosstalk between phosphorylation and ubiquitylation occurs on Tau in AD. Overall, these findings highlight the utility of MS to map ubiquitylated substrates in human brain and provides insight into mechanisms underlying pathological protein posttranslational modification in AD.
In the central nervous system (CNS), microglia are innate immune mononuclear phagocytes (CNS MPs) that can phagocytose infectious particles, apoptotic cells, neurons, and pathological protein ...aggregates, such as Aβ in Alzheimer's disease (AD). While CD11b+CD45low microglia account for the majority of CNS MPs, a small population of CD11b+CD45high CNS MPs is also recognized in AD that surround Aβ plaques. These transcriptionally and pathologically unique CD45high cells have unclear origin and undefined phagocytic characteristics. We have comprehensively validated rapid flow cytometric assays of bulk-phase and amyloid β fibril (fAβ) phagocytosis and applied these to study acutely isolated CNS MPs. Using these methods, we provide novel insights into differential abilities of CD11b+ CD45low and CD45high CNS MPs to phagocytose macroparticles and fAβ under normal, acute, and chronic neuroinflammatory states. CD45high CNS MPs also highly upregulate TREM2, CD11c, and several disease-associated microglia signature genes and have a higher phagocytic capacity for Aβ as compared to CD45low microglia in the 5xFAD mouse model of AD that becomes more apparent with aging. Our data suggest an overall pro-phagocytic and protective role for CD11b+CD45high CNS MPs in neurodegeneration, which if promoted, could be beneficial.
Robust and accessible biomarkers that can capture the heterogeneity of Alzheimer's disease and its diverse pathological processes are urgently needed. Here, we undertook an investigation of ...Alzheimer's disease cerebrospinal fluid (CSF) and plasma from the same subjects (n=18 control, n=18 AD) using three different proteomic platforms-SomaLogic SomaScan, Olink proximity extension assay, and tandem mass tag-based mass spectrometry-to assess which protein markers in these two biofluids may serve as reliable biomarkers of AD pathophysiology observed from unbiased brain proteomics studies. Median correlation of overlapping protein measurements across platforms in CSF (r~0.7) and plasma (r~0.6) was good, with more variability in plasma. The SomaScan technology provided the most measurements in plasma. Surprisingly, many proteins altered in AD CSF were found to be altered in the opposite direction in plasma, including important members of AD brain co-expression modules. An exception was SMOC1, a key member of the brain matrisome module associated with amyloid-β deposition in AD, which was found to be elevated in both CSF and plasma. Protein co-expression analysis on greater than 7000 protein measurements in CSF and 9500 protein measurements in plasma across all proteomic platforms revealed strong changes in modules related to autophagy, ubiquitination, and sugar metabolism in CSF, and endocytosis and the matrisome in plasma. Cross-platform and cross-biofluid proteomics represents a promising approach for AD biomarker development.
We generated an online brain pQTL resource for 7,376 proteins through the analysis of genetic and proteomic data derived from post-mortem samples of the dorsolateral prefrontal cortex of 330 older ...adults. The identified pQTLs tend to be non-synonymous variation, are over-represented among variants associated with brain diseases, and replicate well (77%) in an independent brain dataset. Comparison to a large study of brain eQTLs revealed that about 75% of pQTLs are also eQTLs. In contrast, about 40% of eQTLs were identified as pQTLs. These results are consistent with lower pQTL mapping power and greater evolutionary constraint on protein abundance. The latter is additionally supported by observations of pQTLs with large effects’ tending to be rare, deleterious, and associated with proteins that have evidence for fewer protein-protein interactions. Mediation analyses using matched transcriptomic and proteomic data provided additional evidence that pQTL effects are often, but not always, mediated by mRNA. Specifically, we identified roughly 1.6 times more mRNA-mediated pQTLs than mRNA-independent pQTLs (550 versus 341). Our pQTL resource provides insight into the functional consequences of genetic variation in the human brain and a basis for novel investigations of genetics and disease.
Alzheimer's disease (AD) affects half the US population over the age of 85 and is universally fatal following an average course of 10 years of progressive cognitive disability. Genetic and ...genome-wide association studies (GWAS) have identified about 33 risk factor genes for common, late-onset AD (LOAD), but these risk loci fail to account for the majority of affected cases and can neither provide clinically meaningful prediction of development of AD nor offer actionable mechanisms. This cohort study generated large-scale matched multi-Omics data in AD and control brains for exploring novel molecular underpinnings of AD. Specifically, we generated whole genome sequencing, whole exome sequencing, transcriptome sequencing and proteome profiling data from multiple regions of 364 postmortem control, mild cognitive impaired (MCI) and AD brains with rich clinical and pathophysiological data. All the data went through rigorous quality control. Both the raw and processed data are publicly available through the Synapse software platform.
Cancer genome and transcriptome analyses advanced our understanding of cancer biology. We performed transcriptome analysis of all known genes of peptidases also called proteases and their endogenous ...inhibitors in glioblastoma multiforme (GBM), which is one of the most aggressive and deadly types of brain cancers, where unbalanced proteolysis is associated with tumor progression.
Comparisons were performed between the transcriptomics of primary GBM tumors and unmatched non-malignant brain tissue, and between GBM cell lines (U87-MG and U373) and a control human astrocyte cell line (NHA). Publicly-available data sets and our own datasets were integrated and normalized using bioinformatics tools to reveal protease and protease inhibitor genes with deregulated expression in both malignant versus non-malignant tissues and cells.
Of the 311 protease genes identified to be differentially expressed in both GBM tissues and cells, 5 genes were highly overexpressed, 2 genes coding for non-peptidase homologues transferrin receptor (TFRC) and G protein-coupled receptor 56 (GPR56), as well as 3 genes coding for the proteases endoplasmic reticulum aminopeptidase 2 (ERAP2), glutamine-fructose-6-phosphate transaminase 2 (GFPT2) and cathepsin K (CTSK), whereas one gene, that of the serine protease carboxypeptidase E (CPE) was strongly reduced in expression. Seventy five protease inhibitor genes were differentially expressed, of which 3 genes were highly overexpressed, the genes coding for stefin B (CSTB), peptidase inhibitor 3 (PI3 also named elafin) and CD74. Seven out of 8 genes (except CSTB) were validated using RT-qPCR in GBM cell lines. CTSK overexpression was validated using RT-qPCR in GBM tissues as well. Cathepsin K immunohistochemical staining and western blotting showed that only proteolytically inactive proforms of cathepsin K were overexpressed in GBM tissues and cells.
The presence of high levels of inactive proforms of cathepsin K in GBM tissues and cells indicate that in GBM the proteolytic/collagenolytic role is not its primary function but it plays rather a different yet unknown role.
Celotno besedilo
Dostopno za:
DOBA, IZUM, KILJ, NUK, PILJ, PNG, SAZU, SIK, UILJ, UKNU, UL, UM, UPUK
Our understanding of Alzheimer's disease (AD) pathophysiology remains incomplete. Here we used quantitative mass spectrometry and coexpression network analysis to conduct the largest proteomic study ...thus far on AD. A protein network module linked to sugar metabolism emerged as one of the modules most significantly associated with AD pathology and cognitive impairment. This module was enriched in AD genetic risk factors and in microglia and astrocyte protein markers associated with an anti-inflammatory state, suggesting that the biological functions it represents serve a protective role in AD. Proteins from this module were elevated in cerebrospinal fluid in early stages of the disease. In this study of >2,000 brains and nearly 400 cerebrospinal fluid samples by quantitative proteomics, we identify proteins and biological processes in AD brains that may serve as therapeutic targets and fluid biomarkers for the disease.
Genome-wide association studies (GWAS) have identified many risk loci for Alzheimer's disease (AD)
, but how these loci confer AD risk is unclear. Here, we aimed to identify loci that confer AD risk ...through their effects on brain protein abundance to provide new insights into AD pathogenesis. To that end, we integrated AD GWAS results with human brain proteomes to perform a proteome-wide association study (PWAS) of AD, followed by Mendelian randomization and colocalization analysis. We identified 11 genes that are consistent with being causal in AD, acting via their cis-regulated brain protein abundance. Nine replicated in a confirmation PWAS and eight represent new AD risk genes not identified before by AD GWAS. Furthermore, we demonstrated that our results were independent of APOE e4. Together, our findings provide new insights into AD pathogenesis and promising targets for further mechanistic and therapeutic studies.
TAR DNA-binding protein 43 (TDP-43) is a major component within ubiquitin-positive inclusions of a number of neurodegenerative diseases that increasingly are considered as TDP-43 proteinopathies. ...Identities of other inclusion proteins associated with TDP-43 aggregation remain poorly defined. In this study, we identify and quantitate 35 co-aggregating proteins in the detergent-resistant fraction of HEK-293 cells in which TDP-43 or a particularly aggregate prone variant, TDP-S6, were enriched following overexpression, using stable isotope-labeled (SILAC) internal standards and liquid chromatography coupled to tandem mass spectrometry (LC-MS/MS). We also searched for differential post-translational modification (PTM) sites of ubiquitination. Four sites of ubiquitin conjugation to TDP-43 or TDP-S6 were confirmed by dialkylated GST-TDP-43 external reference peptides, occurring on or near RNA binding motif (RRM) 1. RRM-containing proteins co-enriched in cytoplasmic granular structures in HEK-293 cells and primary motor neurons with insoluble TDP-S6, including cytoplasmic stress granule associated proteins G3BP, PABPC1, and eIF4A1. Proteomic evidence for TDP-43 co-aggregation with paraspeckle markers RBM14, PSF and NonO was also validated by western blot and by immunocytochemistry in HEK-293 cells. An increase in peptides from methylated arginine-glycine-glycine (RGG) RNA-binding motifs of FUS/TLS and hnRNPs was found in the detergent-insoluble fraction of TDP-overexpressing cells. Finally, TDP-43 and TDP-S6 detergent-insoluble species were reduced by mutagenesis of the identified ubiquitination sites, even following oxidative or proteolytic stress. Together, these findings define some of the aggregation partners of TDP-43, and suggest that TDP-43 ubiquitination influences TDP-43 oligomerization.
Celotno besedilo
Dostopno za:
DOBA, IZUM, KILJ, NUK, PILJ, PNG, SAZU, SIK, UILJ, UKNU, UL, UM, UPUK
Deposition of insoluble protein aggregates is a hallmark of neurodegenerative diseases. The universal presence of β-amyloid and tau in Alzheimer’s disease (AD) has facilitated advancement of the ...amyloid cascade and tau hypotheses that have dominated AD pathogenesis research and therapeutic development. However, the underlying etiology of the disease remains to be fully elucidated. Here we report a comprehensive study of the human brain-insoluble proteome in AD by mass spectrometry. We identify 4,216 proteins, among which 36 proteins accumulate in the disease, including U1-70K and other U1 small nuclear ribonucleoprotein (U1 snRNP) spliceosome components. Similar accumulations in mild cognitive impairment cases indicate that spliceosome changes occur in early stages of AD. Multiple U1 snRNP subunits form cytoplasmic tangle-like structures in AD but not in other examined neurodegenerative disorders, including Parkinson disease and frontotemporal lobar degeneration. Comparison of RNA from AD and control brains reveals dysregulated RNA processing with accumulation of unspliced RNA species in AD, including myc box-dependent-interacting protein 1, clusterin, and presenilin-1 . U1-70K knockdown or antisense oligonucleotide inhibition of U1 snRNP increases the protein level of amyloid precursor protein. Thus, our results demonstrate unique U1 snRNP pathology and implicate abnormal RNA splicing in AD pathogenesis.
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