Whole genome gene expression analysis of a genomic mouse model of tauopathy
ABSTRACT: The goal of this study was to assess the number and types of differentially expressed genes in a transgenic (genomic) mouse model of tau pathology. Notably, we crossed previously described genomic mouse line called ‘8c’ that expresses all isoforms of human tau protein (Duff et al., Neurobiology of Disease, 2000;7(2):87) with a mouse tau knockout line (Dawson et al., J Cell Sci 2001;114:1179) and generated humanized mice line called ‘hTau Dk/Dk’. These mice are complete knockout of endogenous mouse tau and express all six isoforms of human tau driven by the endogenous human tau promoter. We performed microarray analysis in the hippocampal tissue from six-month old hTau Dk/Dk mice (n=3) and compared it with age-matched non-transgenic control group. Both the lines are in C57BL/6J genetic background.
Project description:The microtubule associated protein Tau (MAPT) expressed in neurons is involved in microtubules stabilization, cell morphogenesis and axonal transport. In pathological conditions, Tau assembles into high molecular weight assemblies leading to neuropathological Tau deposits, the hallmark of several neurodegenerative diseases collectively named “tauopathies”, including Alzheimer’s disease. These pathologic Tau assemblies are released by affected neuronal cells and taken up by naïve neighbor cells, propagate from one cell to another and amplify by seeding the aggregation of endogenous Tau. In order to identify plasma membrane proteins exposed extracellularly that interact with extracellularly applied fibrillar Tau assemblies, we exposed pure-neuronal cultures to fibrillar Tau for 10 min, pulled down the associated proteins, and identified them using a proteomic-based approach. Of the six Tau isoforms produced by alternative splicing of the MAPT gene and differing from each other by the presence or absence of one or two inserts in the N-terminal part (0N, 1N or 2N) of the protein and by the presence of either three or four repeated microtubule binding motifs in the protein C-terminal part (3R or 4R), we have expressed and purified 1N3R and 1N4R Tau isoforms and further assembled them into 1N3R and 1N4R Tau fibrils. Using pull-down of whole cell lysates and mass spectrometry, we have identified proteins interacting with extracellularly applied Tau fibrils. We have performed two different experiments with either 1N3R Tau fibrils or 1N4R Tau fibrils (condition 1 and condition 2 respectively). Each condition consists in six experimental replicates of cells exposed 10 min to Tau fibrils and of the non-treated cells used as controls.
Project description:A study of differential gene expression in the hippocampus of CV mice (C57Bl x VM/Dk) inoculated with ME7 scrapie to determine the molecular events that accompany the scrapie-associated hippocampal neuropathology. Analysis was performed at 170 days post infection, a timepoint that coincides with severe hippocampal neuronal loss.
Project description:Using a high-end mass spectrometry, we screened phosphoproteins and phosphopeptides in four types of Alzheimer's disease (AD) mouse models and human AD postmortem brains. We identified commonly changed phosphoproteins in multiple models and also determined phosphoproteins related to initiation of Abeta deposition in the mouse brain. We put the proteins on experimentally verified protein-protein interaction databases. Surprisingly most of the core phosphoproteins were directly connected, and they formed a functional network linked to synaptic spine formation. The change of the core network started at a preclinical stage even before histological Abeta deposition. Systems biology analyses suggested phosphorylation of MARCKS by over-activated kinases including PKCs and CaMKs initiates synapse pathology.
Project description: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.
Project description:BRAF(V600E) is a frequent mutation in colon cancer. We have analysed transcriptional effects of BRAF(V600E) in the intestinal epithelium of transgenic mice that harbour an inducible BRAF(V600E) transgene. Mice used in this experiment were compound transgenic for a stem-cell specific Lgr5-EGFP Reporter and either an inducible TdTomato-2A-BRAF(V600E) transgene or an inducibe TdTomato-luciferase transgene. Transgenes were induced by doxycycline (4mg/ml) treatment provided in a 1% sucrose solution in the drinking water of transgenic mice for 24h. Intestinal crypts were isolated by filtering (70um) following a PBS/EDTA incubation step. Single cell suspensions were made by digestion of crypts in 500ug/ml trypsine and 0.8u/ml DNAseI for approx. 20min at RT. Cell populations for profiling were isolated by FACS, using an Aria SOPR (BD), equipped with a 70um nozzle.
Project description:Alzheimer's disease (AD) and most of other tauopathies are incurable neurodegenerative diseases with unpleasant symptoms and consequences. The common hallmark of all these diseases is tau pathology but its connection with disease progress has not been completely understood so far. Therefore, uncovering novel tau interacting partners and pathology affected molecular pathways can reveal the causes of diseases as well as potential targets for development of AD treatment. Despite of large amount of known tau interacting partners, limited number of studies focused on in vivo tau interactions in disease or healthy conditions are available. Here, we applied an in vivo crosslinking approach, capable of capturing weak and transient protein-protein interactions, to a unique transgenic rat model of progressive tau pathology similar to human AD. We have identified 175 potential novel and known tau interacting proteins by MALDI-TOF mass spectrometry. Several of the most promising candidates for potential drug development were selected for validation by coimmunoprecipitation and colocalization experiments in animal and cellular models.
Project description:Tau is a microtubule-associated protein that ensures neuronal shape and function. Besides, Tau is a central player in Alzheimer’s disease (AD) and related Tauopathies where it is found aggregated in degenerating neurons. Mechanisms leading to Tau pathology and its progression are far from being elucidated. Among Tau species found in AD brains, several yet unidentified truncated Tau fragments are showed. A major step forward in the understanding of the role of Tau truncation would be to identify the precise cleavage sites of Tau species. This key step is mandatory to generate appropriate experimental tools in order to investigate the impact of each identified truncated-species on Tau function/dysfunction. Here, we achieved an optimized proteomics approach and succeed in identifying a number of new N-terminally truncated-Tau species from human brain. As N-terminal residues of these fragments are located broadly across Tau sequence, one could expect to have different effects on Tau. We initiated cell-based functional studies by analyzing biochemical characteristics of two N-terminally truncated Tau species starting at residues Met11 and Gln124 (accordingly to the longest Tau isoform) regarding Tau microtubule function. Our results surprisingly showed that the ability of Tau to bind and stabilize microtubules was greater when the first 123 residues are truncated, suggesting that Tau N-terminus would have a role in regulation of microtubule stabilization. Overall, future studies based on our new N-terminally truncated-Tau species will provide new knowledge on the role of truncation in Tau biology as well as in AD pathological process.
Project description:To understand at molecular level how aggregation of Tau modulates its interactions with proteins we reveal key determinant for derailing Tau protein network. By performing quantitative AP-MS we define a new set of interactors which bind Tau upon fibril formation. These interactors contain disordered regions with a unique amino acidic footprint. Such regions are enriched in positively charged Arginines which can engage aberrant binding to Tau fibrils through their guanidinium group via pi-stacking. We also find that the Hsp90 chaperone stalls formation of Tau fibrils and reshapes their abnormal interactome.
Project description:Protein interactions of the tau protein are of interest in efforts to decipher the mechanisms of cell death in Alzheimer Disease (AD), a subset of frontotemporal dementias (FTD) and other tauopathies. We recently reported on extensive interactions of tau with the ribonucleoproteome and chaperones. A confounder of this prior work was that it probed steady-state interactions of plasmid-encoded four-repeat (4R) tau in dividing cells. Since then, we genome-edited a genomic safe harbor locus in two human neuronal cell lines, namely IMR-32 and ReNcell VM, creating inducible EGFP tagged wild-type and P301L tau models. We expressed balanced levels of 3R- and 4R-tau and interrogated tau protein interactions at specific times following its induction. In addition to its association with the ribonucleoproteome, tau was observed to interact in these models with proteins that escaped prior investigations.