Molecular mechanisms of recessive and dominant mutations in the small vessel disease-related high temperature requirement protease HTRA1

脑小血管疾病（SVD）是中风和痴呆的主要原因，但治疗选择仍然非常有限。孟德尔条件在定义SVD的分子、细胞和病理生理学基础方面起了重要作用。伴有皮质下梗死和白质脑病的常染色体隐性遗传性脑动脉病（CARASIL）是一种隐性遗传性早发性家族性SVD，由高温需要蛋白HTRA 1基因的功能缺失突变引起。HTRA 1是一种分泌型蛋白酶，以三聚体和更高级的寡聚体形式组装，形成成熟的具有蛋白水解活性的复合物。我们以前的研究结果表明，SVD相关的HTRA 1突变导致分子多样性的影响，包括但不限于组装缺陷。与其他人合作，我们进一步发现HTRA 1杂合突变与常染色体显性迟发性SVD相关，但隐性和显性HTRA 1突变的区别特征仍有待确定。本申请解决了三个主要目的：i）确定致病性突变对HTRA 1功能的关键分子和细胞方面的影响（mRNA稳定性、蛋白质稳定性、三聚体组装、ECM整合、底物识别）; ii）研究杂合HTRA 1突变是否对酶功能具有显性负效应并确定潜在机制; iii）探索恢复HTRA 1功能的策略，作为未来靶向治疗的潜在基础。这一目标是由我们的初步结果，表明它是可能的，以恢复个别突变的酶活性的动机。为了实现这些目标，我们将采用各种遗传工具，生物化学技术和细胞生物学方法，这些方法将应用于患者和对照受试者的转染细胞和原代细胞。无论具体结果如何，该项目将为CARASIL的关键机制以及HTRA 1相关SVD的常染色体显性形式提供新的见解。此外，该项目可能为携带具有确定分子特性的突变的患者开辟治疗前景。

Cerebral small vessel diseases (SVDs) are a major cause of both stroke and dementia but therapeutic options are still very limited. Mendelian conditions have been instrumental in defining the molecular, cellular, and pathophysiological basis of SVDs. Cerebral autosomal recessive arteriopathy with subcortical infarcts and leukoencephalopathy (CARASIL) is a recessive early onset familial SVD caused by loss-of-function mutations in the high temperature requirement protein HTRA1 gene. HTRA1, a secreted protease, assembles as trimers and higher order oligomers to form a mature and proteolytically active complex. Our previous results indicate that SVD-related mutations in HTRA1 result in molecularly diverse effects including but not limited to assembly defects. In collaboration with others, we further found that heterozygous mutations in HTRA1 associate with autosomal dominant late onset SVD but the distinguishing features of recessive and dominant HTRA1 mutations remain to be defined. The current application addresses three major aims: i) to determine the effects of pathogenic mutations on key molecular and cellular aspects of HTRA1 function (mRNA stability, protein stability, trimer assembly, ECM integration, substrate recognition); ii) to investigate, whether heterozygous HTRA1 mutations have a dominant negative effect on enzyme function and determine the underlying mechanisms; iii) to explore strategies to restore HTRA1 function as a potential basis for future targeted therapies. This aim is motivated by our preliminary results indicating it is be possible to restore enzymatic activity for individual mutations. To achieve these aims we will employ a variety of genetic tools, biochemical techniques and cell biology approaches, which will be applied to both transfected cells and primary cells from patients and control subjects. Regardless of the specific outcomes this project will provide novel insights into key mechanisms of CARASIL as well as autosomal dominant forms of HTRA1-related SVD. In addition, the project might open a therapeutic perspective for patients carrying mutations with defined molecular properties.