Advanced conformational changes in NOTCH3 and cerebrovascular disease severity

摘要 神经退行性疾病是退伍军人死亡和永久残疾的主要原因; 脑血管疾病在神经退行性疾病的进展中起着巨大的作用， 包括阿尔茨海默病和相关痴呆症（ADRD）在内的疾病。就其本身而言， 脑疾病可导致一种形式的ADRD、血管性认知障碍和痴呆 （VCID）。与阿尔茨海默病、脑血管疾病等疾病合作， 显著增加了痴呆症的速度和严重程度。不幸的是，有效的 目前还没有针对脑血管疾病的治疗方法。因此，在 是揭示脑血管疾病分子机制的一个未满足的需求。最 脑血管疾病的常见遗传原因是常染色体显性遗传性脑动脉病 皮质下梗死和痴呆（CADASIL）。CADASIL是一个杰出的模型， 了解脑血管的病理学，并由NOTCH 3突变引起， 预测改变半胱氨酸数。我们发现了一系列的变化， 与CADASIL突变相关的NOTCH 3构型。首先，CADASIL中的NOTCH 3 更可能含有多个还原半胱氨酸（一种称为mrc-N3的形式）。第二，NOTCH 3 在N-末端发生裂解，释放出41个氨基酸 NOTCH 3 N-末端片段（NTF）。基于驱动NTF的分子过程 在新的实验中，我们描述了第二次切割事件的鉴定， 预测产生第二个41个氨基酸的肽NTF 2。针对NTF 2产生的抗体 证明CADASIL血管富含这种裂解产物。此外，一部小说 已经鉴定了一种单克隆抗体，其识别NTF 2的构象， 在血管子集（NTF 2 *）中表达。在本提案中，我们将检验以下假设： NTF 2 * 与CADASIL血管的晚期病理学相关。我们还将测试 NTF 2 * 从表面到深层白色物质呈梯度沉积，这将指向 NTF 2 * 可能是血管疾病进展的可预测标志物。

ABSTRACT Neurodegenerative disease is a major cause of death and permanent disability in Veterans; vascular disease of the brain plays an outsized role in the progression of neurodegenerative diseases including Alzheimer’s Disease and Related Dementias (ADRD). On its own, vascular disease of the brain can cause one form of ADRD, vascular cognitive impairment and dementia (VCID). In collaboration with disorders such as Alzheimer’s disease, brain vascular disease significantly augments the pace and severity of dementing disorders. Unfortunately, effective treatments that target vascular disease of the brain are currently not available. As such, there is an unmet need to uncover the molecular mechanisms of brain vascular disease. The most common inherited cause of brain vascular disease is cerebral autosomal dominant arteriopathy with subcortical infarcts and dementia (CADASIL). CADASIL is an outstanding model for understanding pathology of brain blood vessels and is caused by mutations in NOTCH3 that are predicted to alter cysteine number. We have discovered a sequence of changes in the configuration of NOTCH3 in association with CADASIL mutations. First, NOTCH3 in CADASIL is more likely to harbor multiple reduced cysteines (a form called mrc-N3). Second, NOTCH3 that is multiply reduced undergoes cleavage at the N-terminus that releases a 41-amino acid peptide, the NOTCH3 N-terminal fragment (NTF). Based on the molecular process driving NTF generation, in new experiments, we describe the identification of a second cleavage event that is predicted to result in a second 41-amino acid peptide NTF2. Antibodies generated to NTF2 demonstrate that CADASIL vessels are enriched in this cleavage product. Moreover, a novel monoclonal antibody has been identified that recognizes a conformation of NTF2 that is only expressed in a subset of vessels (NTF2*). In this proposal, we will test the hypothesis that NTF2* is associated with advanced pathology of CADASIL blood vessels. We will also test if NTF2* is deposited in a gradient from the surface to the deep white matter, which would point to the possibility that NTF2* is a predictable marker for vascular disease progression.