HTRA1 expression and functionality in HTRA1 mutation carriers CARRIERS

High temperature requirement A1 (HTRA1) belongs to heat shock-induced serine proteases and is ubiquitously expressed in normal human adult tissues. HTRA1 plays a modulatory role in various cell processes, particularly regulates the transforming growth factor-β (TGF-ß) signalling. Biallelic mutations in HTRA1 lead to cerebral autosomal recessive arteriopathy with subcortical infarcts and leukoencephalopathy (CARASIL), a rare cerebral small vessel disease (CSVD). Nowadays, fifteen HTRA1 mutations have been identified. Recent data reported that heterozygous HTRA1 mutations seem to be linked to familial CSVD of unknown aetiology, which is characterized by a later age at onset. These data suggest that HTRA1 mutation could behave as autosomal recessive or dominant mutation. Our aim is to obtain further data about the pathogenic effect of various heterozygous HTRA1 mutations. We compared expression profiles of HTRA1 and intermediaries of TGF-β signalling proteins both in heterozygous carriers, with missense and stop codon HTRA1 mutations, and in heterozygous and homozygous mouse embryonic fibroblasts harbouring HTRA1-R274Q mutation. Moreover, we used heterozygous and homozygous murine models harbouring HTRA1-R274Q in order to evaluate in vivo the effects of mutant HTRA1. Further, we performed supplementary studies to evaluate the possibility of a dominant negative effect on HTRA1-WT by HTRA1-mutants, and the possibility torescue HTRA1-protease activity in homozygous HTRA1-R274Q carriers. The cell lysates and culture medium of cultured cells were used to analyse the expression pattern of both HTRA1 and intermediaries of TGF-β signalling proteins by western blot and immunofluorescence analysis. RNAs extracted from cultured cells and from mice tissues were used to analyse HTRA1-RNA and CTGF-RNA expression level by RT-qPCR. We found a ∼50% reduction in HTRA1 expression in human fibroblasts carrying heterozygous HTRA1-mutations compared to control. RT-qPCR analysis confirmed these data for two of the analysed subjects, whilst showed no significant reduction in the remaining carriers compared to control. Analysis of the murine models showed that there is no alteration of HTRA1-RNA expression nor in heterozygous nor in homozygous HTRA1-R274Q mice. No significant alteration of Smad2/3 phosphorylation and CTGF expression, down- and up-stream intermediaries of TGF-β signalling pathway, respectively, were found, suggesting that dysfunction of TGF-β signalling in fibroblasts might not contribute to the pathogenesis of CARASIL and CSVD linked to heterozygous HTRA1 mutations reported in this study. Heterozygous and homozygous HTRA1-R274Q murine cells displayed an increased fibronectin accumulation of 10-fold and 40-fold, respectively, than HTRA1-WT cells, suggesting that even the heterozygous HTRA1-mutations could be enough to cause deleterious phenotypic alterations in brain small vessels. HTRA1-WT protease activity did not display any remarkable alteration in presence of HTRA1-mutants.These findings seem to rule out a dominant negative effect in HTRA1 mutations we investigated. Finally, MEFs transfected with the rescue protein give an outcome similar to that obtained with MEFs transfected with HTRA1-WT, opening actual possibility to rescue the functionality of HTRA1-mutants. In conclusion, our results seem to suggest that CSVD, linked to heterozygous HTRA1 mutations, may occur in the presence of ~50% expression of the protein. Progressive tissue damage accumulation in small vessels leading to delayed and milder clinical expression with later onset with respect to classical CARASIL phenotype may be hypothesized. Moreover, data collection on heterozygous HTRA1 mutants is still limited, so investigation on the HTRA1 expression and activity, in cells from a wider number of subjects harboring different heterozygous HTRA1 missense mutations, could be helpful to verify a possible correlation between specific aminoacidic variations and particular protein alterations.