Identifying Candidate Genes And Molecular Targets For Alopecia Areata Treatment In C3H/HeJ Mouse Model

Alopecia Areata (AA) is a disfiguring human autoimmune skin disease with a complex genetic basis that targets hair follicles in the actively growing anagen phase of the hair cycle and is associated with other systemic autoimmune diseases. AA can affect nearly 2% of the general population at some time during their lives. We have extensively characterized the C₃H/HeJ mice that spontaneously develop an AA-like disease that very closely resembles the adult onset form of human AA. Using existing DNA from a quantitative trait locus (QTL) study done previously using simple sequence length repeat markers (SSLPs), we will identify the candidate genes for susceptibility using single nucleotide polymorphisms (SNPs) technology that was not available for the earlier study. Using new quantitative QPCR technology and Affymetrix Gene Array analyses, we identified molecular pathways for the pathogenesis of AA. Concurrently, haplotype association mapping approaches are used, building on other strains we know develop an AA-like skin disease, to validate susceptibility loci, identify new loci, and predict which crosses are necessary to further resolve the intervals. In so doing, we will be able to resolve the mouse genetic intervals and identify and test candidate genes, identify new loci for saturation mapping approaches in the human genome-wide scans, and develop molecular tools that will have value in the drug and diagnostic screening approaches. Discoveries using this model continue to contribute to a better understanding of human AA.一,Experimental induction of AA in C3H/HeJ mice using full-thickness skin graftIn order to study the mechanism of AA, we induced AA successfully in C3H/HeJ mice using full-thickness skin graft . Mice received skin grafts and were necropsied at five-week intervals after surgery. Histopathology, transmission electron microscopy, and indirect immunofluorescence studies were done on the graft site and at distant sites to follow progression of AA based on types of infiltrating cells. Most mice received AA skin graft developed hair loss after surgery and almost lost all the hair till the 20 weeks after surgery. Anagen but not telogen hair follicles are affected. We found CD8+ /CD4+ T cells infiltrated in/around the follicle. CD8+ T cells dominated during the disease progression. We conclude that AA is an autoimmune skin disease with a complex genetic basis that targets hair follicles in the actively growing anagen phase of the hair cycle. C3H/HeJ mouse model is a useful tool for studing AA mechanisms. Another benefit of the graft model is the production of readily available, reproducible, and predictable induction of AA in mice for therapeutic screening studies.二,Identifies molecular pathway and drug target for AA treatment in C3H/HeJ mouse modelIn order to identifiy molecular pathway and drug target for AA treatment, we used the Affymetrix? gene expression tools (GeneChip Mouse Genome 430 2.0 Array) combined with Ingenuity Pathways Analysis? software and QPCR technology, we evaluated mice at 5, 10, 15, and 20 weeks after skin grafting and mice with ntaneous AA compared to age and gender matched controls. This approach identified numerous molecular networks involved in the pathogenesis of AA, including activation of Cd8+ T cells, Cd4+ T cells, and NK cells. We conclude thatâ‘ CD8a,b and CD4 upregulated at different time point after skin surgery which confirmed AA is a cells mediate autoimmune disease。②CXCL9,10,11,CCL5 upregulated, these data suggest that chemokine may play important role in AA.â‘¢More importantly, this approach identified dysregulated genes at specific time points in the evolution of AA whose protein products are known targets for FDA-approved drugs. Very few dysregulated genes, especially those that were drug targets, were significantly upregulated at all stages, suggesting that treatments need to be designed for specific stages of AA not just the general diagnosis of AA as is currently done. This information provides a priority list for screening drugs for efficacy in treating AA and will help design pharmacologists design new single and combination therapies for AA patients.三,Haplotype association mapping identifies Tap2 as a candidate gene in C3H/HeJ mouse AA.AA is a disfiguring, complex genetic, human autoimmune skin disease targeting actively growing hair follicles. In order to continue to narrow down genetic intervole and identify canditate gene in mouse AA, we used Mouse Phenome Database ( http://phenome.jax.org ) combined with Ingenuity Pathways Analysis? software and QPCR and two inbred mouse strains, C3H/HeJ (and many of its substrains) and A/J develop a spontaneous alopecia areata-like disease. Haplotype association mapping, done by screening SNPs within the four Alaa1-4 QTL intervals found for C3H/HeJ AA, done by searching for groups of 3 or more contiguous SNPs identical between C3H/HeJ and A/J but different for C57BL/6J and DBA/2J strains, identified the transporter 2, ATP-binding cassette sub-family B (Tap2) gene as one of the genes within the highest QTL (Alaa1) on chr. 17. Gene expression studies (Affymetrix) performed on skin from mice receiving skin grafts from affected vs. normal mice at 5, 10, 15, and 20 weeks after grafting as well as for mice with spontaneous AA vs. normal skin revealed a steady increase of over 3 fold in Tap2 and Tap binding protein (Tapbp) expression. The homologous genes in humans are located on chr. 6 where a QTL for AA susceptibility was also mapped. The TAPs select from the generated peptides, some that theu then translocate from the cytosol into the lumen of the RER. The Tapbp helps to draw the nascent class I molecules near the TAPs for loading of the translocated peptides into the peptide-binding groove of the class I molecules. This is an important new observation.