Leducq Center Against Small Vessel Disease

Network of genes/proteins driving vessel pathology in CADASIL/CARASIL

The GOM proteome will be identified, and a combination of immunohistochemistry, immuno-electron microscopy, and immunoblotting of human and murine CADASIL samples together with co-immunoprecipitation, GST-pull-down and computational analysis will be employed to rank the proteins and map protein-protein interactions to build the GOM interactome (Joutel). RNA-Seq in conjunction with RT-PCR analyses will identify genes that are differentially expressed between TgPAC-Notch3^R169C and WT mice and will uncover molecular changes shared with Notch3 gain and loss-of-function models. The role of top candidates identified by proteomic (starting with Timp3 and vitronectin) and genomic analyses in pathogenesis (Joutel) and arteriolar function (Nelson) will be explored by decreasing or increasing their expression level using loss-of-function and gain-of-function alleles, respectively, singly and in interbreedings with the TgPAC-Notch3R169C line.

In vitro studies have shown that HtrA1 promotes the intracellular degradation of the TGF-ß latency-associated peptide (LAP)²⁶. First, the ability of the related HtrA proteases to cleave LAP will be examined. Second, taking advantage of an established cellular assay and employing an unbiased proteomic approach based on terminal amine isotopic labeling of substrates, we will identify additional substrates of HtrA1 (collaboration w/M. Mann, Max Planck Institute, Munich). Candidate substrates will be validated by assessing their expression level in HtrA1 WT versus KO samples. Their contribution to CARASIL pathogenesis will be further explored, starting with the analysis of TGF-ß signaling in cell and tissues samples from young and aged HtrA1 KO mice using a combination of immunostaining, immunoblotting, and real-time RT-PCR analyses (Dichgans).

We will also test the hypothesis that CADASIL and CARASIL share common pathways. The transcriptome and proteome from CADASIL and CARASIL brain vessels will be defined, and common up- or down-regulated transcripts and proteins will be identified. Possible genetic interactions between HtrA1 and mutant Notch3 will be explored by generating and analyzing TgNotch3^R169C mice lacking one of the two alleles of HtrA1 (Joutel, Dichgans). Genes of pathways common to CADASIL and CARASIL are strong candidate contributors to multifactorial SVD.