Genetic background influences the 5XFAD Alzheimer\u27s disease mouse model brain proteome.

There is an urgent need to improve the translational validity of Alzheimer’s disease (AD) mouse models. Introducing genetic background diversity in AD mouse models has been proposed as a way to increase validity and enable the discovery of previously uncharacterized genetic contributions to AD susceptibility or resilience. However, the extent to which genetic background influences the mouse brain proteome and its perturbation in AD mouse models is unknown. In this study, we crossed the 5XFAD AD mouse model on a C57BL/6J (B6) inbred background with the DBA/2J (D2) inbred background and analyzed the effects of genetic background variation on the brain proteome in F1 progeny. Both genetic background and 5XFAD transgene insertion strongly affected protein variance in the hippocampus and cortex (n = 3,368 proteins). Protein co-expression network analysis identified 16 modules of highly co-expressed proteins common across the hippocampus and cortex in 5XFAD and non- transgenic mice. Among the modules strongly influenced by genetic background were those related to small molecule metabolism and ion transport. Modules strongly influenced by the 5XFAD transgene were related to lysosome/stress responses and neuronal synapse/signaling. The modules with the strongest relationship to human disease—neuronal synapse/signaling and lysosome/stress response—were not significantly influenced by genetic background. However, other modules in 5XFAD that were related to human disease, such as GABA synaptic signaling and mitochondrial membrane modules, were influenced by genetic background. Most disease-related modules were more strongly correlated with AD genotype in the hippocampus compared with the cortex. Our findings suggest that the genetic diversity introduced by crossing B6 and D2 inbred backgrounds influences proteomic changes related to disease in the 5XFAD model, and that proteomic analysis of other genetic backgrounds in transgenic and knock-in AD mouse models is warranted to capture the full range of molecular heterogeneity in genetically diverse models of AD

Proteomic Analysis of Hippocampal Dentate Granule Cells in Frontotemporal Lobar Degeneration: Application of Laser Capture Technology

Frontotemporal lobar degeneration (FTLD) is the most common cause of dementia with pre-senile onset, accounting for as many as 20% of cases. A common subset of FTLD cases is characterized by the presence of ubiquitinated inclusions in vulnerable neurons (FTLD-U). While the pathophysiological mechanisms underlying neurodegeneration in FTLD-U have not yet been elucidated, the presence of inclusions in this disease indicates enhanced aggregation of one or several proteins. Moreover, these inclusions suggest altered expression, processing, or degradation of proteins during FTLD-U pathogenesis. Thus, one approach to understanding disease mechanisms is to delineate the molecular changes in protein composition in FTLD-U brain. Using a combined approach consisting of laser capture microdissection (LCM) and high-resolution liquid chromatography-tandem mass spectrometry (LC–MS/MS), we identified 1252 proteins in hippocampal dentate granule cells excised from three post-mortem FTLD-U and three unaffected control cases processed in parallel. Additionally, we employed a labeling-free quantification technique to compare the abundance of the identified proteins between FTLD-U and control cases. Quantification revealed 54 proteins with selective enrichment in FTLD-U, including TAR–DNA binding protein 43 (TDP-43), a recently identified component of ubiquitinated inclusions. Moreover, 19 proteins were selectively decreased in FTLD-U. Subsequent immunohistochemical analysis of TDP-43 and three additional protein candidates suggests that our proteomic profiling of FTLD-U dentate granule cells reveals both inclusion-associated proteins and non-aggregated disease-specific proteins. Application of LCM is a valuable tool in the molecular analysis of complex tissues, and its application in the proteomic characterization of neurodegenerative disorders such as FTLD-U may be used to identify proteins altered in disease

Large-scale proteomic analysis of human brain identifies proteins associated with cognitive trajectory in advanced age

In advanced age, some individuals maintain a stable cognitive trajectory while others experience a rapid decline. Such variation in cognitive trajectory is only partially explained by traditional neurodegenerative pathologies. Hence, to identify new processes underlying variation in cognitive trajectory, we perform an unbiased proteome-wide association study of cognitive trajectory in a discovery (n = 104) and replication cohort (n = 39) of initially cognitively unimpaired, longitudinally assessed older-adult brain donors. We find 579 proteins associated with cognitive trajectory after meta-analysis. Notably, we present evidence for increased neuronal mitochondrial activities in cognitive stability regardless of the burden of traditional neuropathologies. Furthermore, we provide additional evidence for increased synaptic abundance and decreased inflammation and apoptosis in cognitive stability. Importantly, we nominate proteins associated with cognitive trajectory, particularly the 38 proteins that act independently of neuropathologies and are also hub proteins of protein co-expression networks, as promising targets for future mechanistic studies of cognitive trajectory.Accelerating Medicine Partnership for AD [U01AG046161, U01 AG061357]; Emory Alzheimer's Disease Research Center [P50 AG025688]; NINDS Emory Neuroscience Core [P30 NS055077]; intramural program of the National Institute on Aging (NIA); Alzheimer's Association; Alzheimer's Research UK; Michael J. Fox Foundation for Parkinson's Research; Weston Brain Institute Biomarkers Across Neurodegenerative Diseases Grant [11060]; National Institute of Neurological Disorders and Stroke [U24 NS072026]; National Institute on Aging [P30 AG19610]; Arizona Department of Health Services [211002]; Arizona Biomedical Research Commission [4001, 0011, 05-901, 1001]; [R01 AG056533]; [R01 AG053960]; [U01 MH115484]; [I01 BX003853]; [IK2 BX001820]; [R01 AG061800]; [R01 AG057911]Open access journalThis item from the UA Faculty Publications collection is made available by the University of Arizona with support from the University of Arizona Libraries. If you have questions, please contact us at [email protected]

Effects of APOE Genotype on Brain Proteomic Network and Cell Type Changes in Alzheimer's Disease

Polymorphic alleles in the apolipoprotein E (APOE) gene are the main genetic determinants of late-onset Alzheimer's disease (AD) risk. Individuals carrying the APOE E4 allele are at increased risk to develop AD compared to those carrying the more common E3 allele, whereas those carrying the E2 allele are at decreased risk for developing AD. How ApoE isoforms influence risk for AD remains unclear. To help fill this gap in knowledge, we performed a comparative unbiased mass spectrometry-based proteomic analysis of post-mortem brain cortical tissues from pathologically-defined AD or control cases of different APOE genotypes. Control cases (n = 10) were homozygous for the common E3 allele, whereas AD cases (n = 24) were equally distributed among E2/3, E3/3, and E4/4 genotypes. We used differential protein expression and co-expression analytical approaches to assess how changes in the brain proteome are related to APOE genotype. We observed similar levels of amyloid-β, but reduced levels of neurofibrillary tau, in E2/3 brains compared to E3/3 and E4/4 AD brains. Weighted co-expression network analysis revealed 33 modules of co-expressed proteins, 12 of which were significantly different by APOE genotype in AD. The modules that were significantly different by APOE genotype were associated with synaptic transmission and inflammation, among other biological processes. Deconvolution and analysis of brain cell type changes revealed that the E2 allele suppressed homeostatic and disease-associated cell type changes in astrocytes, microglia, oligodendroglia, and endothelia. The E2 allele-specific effect on brain cell type changes was validated in a separate cohort of 130 brains. Our systems-level proteomic analyses of AD brain reveal alterations in the brain proteome and brain cell types associated with allelic variants in APOE, and suggest further areas for investigation into the upstream mechanisms that drive ApoE-associated risk for AD

Meta-Analysis of the Alzheimer\u27s Disease Human Brain Transcriptome and Functional Dissection in Mouse Models.

We present a consensus atlas of the human brain transcriptome in Alzheimer\u27s disease (AD), based on meta-analysis of differential gene expression in 2,114 postmortem samples. We discover 30 brain coexpression modules from seven regions as the major source of AD transcriptional perturbations. We next examine overlap with 251 brain differentially expressed gene sets from mouse models of AD and other neurodegenerative disorders. Human-mouse overlaps highlight responses to amyloid versus tau pathology and reveal age- and sex-dependent expression signatures for disease progression. Human coexpression modules enriched for neuronal and/or microglial genes broadly overlap with mouse models of AD, Huntington\u27s disease, amyotrophic lateral sclerosis, and aging. Other human coexpression modules, including those implicated in proteostasis, are not activated in AD models but rather following other, unexpected genetic manipulations. Our results comprise a cross-species resource, highlighting transcriptional networks altered by human brain pathophysiology and identifying correspondences with mouse models for AD preclinical studies

Chromatin, SF-1, and CtBP structural and post-translational modifications induced by ACTH/cAMP accelerate CYP17 transcription rate

CYP17 is an ACTH/cAMP inducible gene in the human adrenal cortex encoding a cytochrome P450 enzyme with sterol 17α-hydroxylase activity and 17,20 lyase activity essential for biosynthesis of cortisol and androgens. Studies carried out during the past decade have shown that acclerated transcription of inducible eukaryotic genes involves sequential chromatin modifications by cooperative promoter-specific transcription factors and the class of proteins called transcriptional coregulators. In the present work, we aimed to first identify important chromatin modifications and chromatin modifying complexes at the CYP17 transcription start site and nearby steroidogenic factor-1 (SF-1) binding site. Then, we asked what modifications to SF-1 occur during the interaction of this nuclear receptor with the CYP17 promoter, and what their function may be. Finally, we asked how ACTH/cAMP signaling affects SF-1-containing chromatin-modifying complexes during the early phase of transcriptional induction of CYP17. Results from chromatin immunoprecipitation (ChIP) and mammalian two hybrid experiments identified complexes including one comprised of SF-1, steroid receptor coactivator-1 (SRC-1), and the histone acetyltransferase general control nonderepressed 5 (GCN5) as cAMP-inducible, but sensitive to the SF-1 antagonist sphingosine, and able to act in stimulating CYP17 transcription. Moreover, ATPases on the promoter coincided with manipulation of nucleosome histone H2 dimer content. Next, we found that SF-1 phosphorylation by glycogen synthase kinase 3beta (GSK3beta), reciprocal dephosphorylation by phosphatase(s), and acetylation by GCN5 at nearby sites at the ligand binding pocket opening were required for efficient CYP17 transcription. This leads us to propose that ligand binding to SF-1 is controlled by these post-translational modifications. Finally, we determined that the corepressors E1A C-terminal binding proteins (CtBP) 1 and 2 are protein kinase A (PKA) targets and are sensitive to PKA-dependent NADH accumulation. These effects of PKA activation by ACTH/cAMP in adrenal cortex cells enforce CYP17 transcription concomitant with dimerization of CtBP1 and CtBP2.Ph.D.Committee Chair: Marion B. Sewer; Committee Member: Alfred H. Merrill, Jr.; Committee Member: Donald F. Doyle; Committee Member: Dr. Edward T. Morgan; Committee Member: Kirill S. Lobache

Comparative proteomic analysis highlights metabolic dysfunction in α-synucleinopathy

© 2020, The Author(s). The synaptic protein α-synuclein is linked through genetics and neuropathology to the pathogenesis of Parkinson’s disease and related disorders. However, the mechanisms by which α-synuclein influences disease onset and progression are incompletely understood. To identify pathogenic pathways and therapeutic targets we performed proteomic analysis in a highly penetrant new Drosophila model of α-synucleinopathy. We identified 476 significantly upregulated and 563 significantly downregulated proteins in heads from α-synucleinopathy model flies compared to controls. We then used multiple complementary analyses to identify and prioritize genes and pathways within the large set of differentially expressed proteins for functional studies. We performed Gene Ontology enrichment analysis, integrated our proteomic changes with human Parkinson’s disease genetic studies, and compared the α-synucleinopathy proteome with that of tauopathy model flies, which are relevant to Alzheimer’s disease and related disorders. These approaches identified GTP cyclohydrolase (GCH1) and folate metabolism as candidate mediators of α-synuclein neurotoxicity. In functional validation studies, we found that the knockdown of Drosophila Gch1 enhanced locomotor deficits in α-synuclein transgenic flies, while folate supplementation protected from α-synuclein toxicity. Our integrative analysis suggested that mitochondrial dysfunction was a common downstream mediator of neurodegeneration. Accordingly, Gch1 knockdown enhanced metabolic dysfunction in α-synuclein transgenic fly brains while folate supplementation partially normalized brain bioenergetics. Here we outline and implement an integrative approach to identify and validate potential therapeutic pathways using comparative proteomics and genetics and capitalizing on the facile genetic and pharmacological tools available in Drosophila

Transcriptome Analysis Identifies Tumor Immune Microenvironment Signaling Networks Supporting Metastatic Castration-Resistant Prostate Cancer

Prostate cancer (PCa) is the second most common cause of cancer death in American men. Metastatic castration-resistant prostate cancer (mCRPC) is the most lethal form of PCa and preferentially metastasizes to the bones through incompletely understood molecular mechanisms. Herein, we processed RNA sequencing data from patients with mCRPC (n = 60) and identified 14 gene clusters (modules) highly correlated with mCRPC bone metastasis. We used a novel combination of weighted gene co-expression network analysis (WGCNA) and upstream regulator and gene ontology analyses of clinically annotated transcriptomes to identify the genes. The cyan module (M14) had the strongest positive correlation (0.81, p = 4 × 10−15) with mCRPC bone metastasis. It was associated with two significant biological pathways through KEGG enrichment analysis (parathyroid hormone synthesis, secretion, and action and protein digestion and absorption). In particular, we identified 10 hub genes (ALPL, PHEX, RUNX2, ENPP1, PHOSPHO1, PTH1R, COL11A1, COL24A1, COL22A1, and COL13A1) using cytoHubba of Cytoscape. We also found high gene expression for collagen formation, degradation, absorption, cell-signaling peptides, and bone regulation processes through Gene Ontology (GO) enrichment analysis