Alzheimer’s disease (ad) is a devastating neurological disorder characterized by changes in cell-type proportions and consequently marked alterations of the transcriptome. Here we use a data-driven systems biology meta-analytical approach across three human ad cohorts, encompassing six cortical brain regions, and integrate with multi-scale datasets comprising of DNA methylation, histone acetylation, transcriptome- and genome-wide association studies, and quantitative trait loci to further characterize the genetic architecture of ad. We perform co-expression network analysis across more than twelve hundred human brain samples, identifying robust ad-associated dysregulation of the transcriptome, unaltered in normal human aging. We assess the cell-type specificity of ad gene co-expression changes and estimate cell-type proportion changes in human ad by integrating co-expression modules with single-cell transcriptome data generated from 27 321 nuclei from human postmortem prefrontal cortical tissue. We also show that genetic variants of ad are enriched in a microglial ad-associated module and identify key transcription factors regulating co-expressed modules. Additionally, we validate our results in multiple published human ad gene expression datasets, which can be easily accessed using our online resource (https://swaruplab.bio.uci.edu/consensusAD).
Biological systems are immensely complex, organized into a multi-scale hierarchy of functional units based on tightly-regulated interactions between distinct molecules, cells, organs, and organisms. While experimental methods enable transcriptome-wide measurements across millions of cells, the most ubiquitous bioinformatic tools do not support systems-level analysis. Here we present hdWGCNA, a comprehensive framework for analyzing co-expression networks in high dimensional transcriptomics data such as single-cell and spatial RNA-seq. hdWGCNA provides built-in functions for network inference, gene module identification, functional gene enrichment analysis, statistical tests for network reproducibility, and data visualization. In addition to conventional single-cell RNA-seq, hdWGCNA is capable of performing isoform-level network analysis using long-read single-cell data. We showcase hdWGCNA using publicly available single-cell datasets from Autism spectrum disorder and Alzheimer's disease brain samples, identifying disease-relevant co-expression network modules in specific cell populations. hdWGCNA is directly compatible with Seurat, a widely-used R package for single-cell and spatial transcriptomics analysis, and we demonstrate the scalability of hdWGCNA by analyzing a dataset containing nearly one million cells.
Targeting of molecular pathways involved in the cell-to-cell propagation of pathological tau species is a novel approach for development of disease-modifying therapies that could block tau pathology and attenuate cognitive decline in patients with Alzheimer's disease and other tauopathies. We discovered cambinol through a screening effort and show that it is an inhibitor of cell-to-cell tau propagation. Our in vitro data demonstrate that cambinol inhibits neutral sphingomyelinase 2 (nSMase2) enzyme activity in dose response fashion, and suppresses extracellular vesicle (EV) production while reducing tau seed propagation. Our in vivo testing with cambinol shows that it can reduce the nSMase2 activity in the brain after oral administration. Our molecular docking and simulation analysis reveals that cambinol can target the DK-switch in the nSMase2 active site.
Synaptic transfer of tau has long been hypothesized from the human pathology pattern and has been demonstrated in vitro and in vivo, but the precise mechanisms remain unclear. Extracellular vesicles such as exosomes have been suggested as a mechanism, but not all tau is exosomal. The present experiments use a novel flow cytometry assay to quantify depolarization of synaptosomes by KCl after loading with FM2–10, which induces a fluorescence reduction associated with synaptic vesicle release; the degree of reduction in cryopreserved human samples equaled that seen in fresh mouse synaptosomes. Depolarization induced the release of vesicles in the size range of exosomes, along with tetraspanin markers of extracellular vesicles. A number of tau peptides were released, including tau oligomers; released tau was primarily unphosphorylated and C-terminal truncated, with Aβ release just above background. When exosomes were immunopurified from release supernatants, a prominent tau band showed a dark smeared appearance of SDS-stable oligomers along with the exosomal marker syntenin-1, and these exosomes induced aggregation in the HEK tau biosensor assay. However, the flow-through did not seed aggregation. Size exclusion chromatography of purified released exosomes shows faint signals from tau in the same fractions that show a CD63 band, an exosomal size signal, and seeding activity. Crude synaptosomes from control, tauopathy, and AD cases demonstrated lower seeding in tauopathy compared to AD that is correlated with the measured Aβ42 level. These results show that AD synapses release exosomal tau that is C-terminal-truncated, oligomeric, and with seeding activity that is enhanced by Aβ. Taken together with previous findings, these results are consistent with a direct prion-like heterotypic seeding of tau by Aβ within synaptic terminals, with subsequent loading of aggregated tau onto exosomes that are released and competent for tau seeding activity.
The present work examines the hypothesis that apoE receptors mediate uptake of apoE/Ab complex into synaptic terminals. Western blot analysis shows multiple SDS-stable assemblies in synaptosomes from human AD cortex; apoE/Ab complex was markedly increased in AD compared with aged control samples. Complex formation between apoE and Ab was confirmed by coimmunoprecipitation experiments. The apoE receptors lowdensity lipoprotein receptor (LDLR) and LDLR-related protein 1 (LRP1) were quantified in synaptosomes using flow cytometry, revealing up-regulation of LRP1 in early-and late-stage AD. Dual-labeling flow cytometry analysis of LRP1-and LDLR positives indicate most (approximately 65%) of LDLR and LRP1 is associated with postsynaptic density-95 (PSD-95)epositive synaptosomes, indicating that remaining LRP1 and LDLR receptors are exclusively presynaptic. Flow cytometry analysis of Nile red labeling revealed a reduction in cholesterol esters in AD synaptosomes. Dual-labeling experiments showed apoE and Ab concentration into LDLR and LRP1-positive synaptosomes, along with free and esterified cholesterol. Synaptic Ab was increased by apoE4 in control and AD samples. These results are consistent with uptake of apoE/Ab complex and associated lipids into synaptic terminals, with subsequent Ab clearance in control synapses and accumulation in AD synapses.
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