Dissecting the mechanisms of disease of COL4A1 and COL4A2 mutations

The best characterised collagen IV disease is Alport syndrome, caused by α3.α4.α5(IV) mutations. Mutations affecting α1(IV)2α2(IV) can cause diseases, such as haemorrhagic stroke, aneurysm, haematuria, proteinuria, cataracts, vascular tortuosity and iris hypoplasia. Recent data has shown that α and COL4A2 mutations may be more common in the population than previously anticipated. However, the mechanisms of disease of all collagen IV mutations are poorly understood. The aim of this thesis was to investigate the disease mechanism in a selection of COL4A1, COL4A2 and COL4A5 mutations. Collagen IV is folded in the endoplasmic reticulum (ER) then secreted to provide the major structural component of the basement membrane (BM). As such, a combination of ex situ biopsies, in vitro cell cultures and an in vivo mouse model provided an excellent platform for investigating the intracellular and extracellular effects of mutant trimers. Primary dermal fibroblasts containing COL4A2 and COL4A5 mutations, and a Col4a1 mouse model were treated with the FDA approved chemical chaperone phenyl-4-butyric acid (PBA) to determine its effects on intracellular ER retention of trimers (which can activate ER stress), extracellular trimer incorporation into the BM, and disease phenotypes. In vitro and ex situ investigation of a COL4A2 mutation suggested that intracellular α1(IV)2α2(IV) accumulation and ER stress associated with severe BM defects and the disease. Intracellular accumulation and ER stress were reduced by PBA treatment, and treatment of a Col4a1 mouse model displayed elevated α1(IV)2α2(IV) incorporation onto the BM. In vivo PBA treatment did not exacerbate any defects, rescued some disease phenotypes, and had no effect on others. In addition, one of five COL4A5 mutations analysed in vitro displayed intracellular α3.α4.α5(IV) accumulation and ER stress that were ameliorated by PBA. Collectively these data suggest that ER stress and/or reduced α1(IV)2α2(IV) BM incorporation underlie some collagen IV pathologies, and provide novel evidence that they may be amenable to chemical chaperone therapy. More investigation is now needed to further dissect the relative contributions of ER stress and BM defects to collagen IV diseases