A CADASIL-mutated Notch 3 receptor exhibits impaired intracellular trafficking and maturation but normal ligand-induced signaling

Notch receptors are single transmembrane receptors that contain a large number of epidermal growth factor-like repeats (EGF repeats) in their extracellular domains. Mutations in the EGF repeats of the human Notch 3 receptor lead to the vascular dementia disease Cerebral Autosomal Dominant Arteriopathy with Subcortical Infarcts and Leukoencephalopathy (CADASIL). The vast majority of CADASIL mutations are missense mutations removing or inserting cysteine residues in the EGF repeats, but it is not yet clear whether these mutations primarily affect receptor trafficking, maturation, andor signaling. To address this issue, we have generated and analyzed stable cell lines expressing either wild-type murine Notch 3 (mNotch 3) or the mutant mNotch 3(R142C), which corresponds to the prevalent CADASIL form of Notch 3, Notch 3(R141C) in humans. We find that a lower proportion of mNotch 3(R142C) is expressed in the site 1-cleaved configuration, and that reduced amounts of mNotch 3(R142C) appear at the cell surface, as compared with wild-type mNotch 3. This observation is accompanied by a higher propensity for mNotch 3(R142C) to form intracellular aggregates, which may be a result of increased accumulation or slowed transport in the secretory pathway. In contrast to the impaired cell surface expression, mNotch 3(R142C) signals equally well in response to Delta 1 and Jagged 1 as wild-type mNotch 3. Taken together, these data suggest that trafficking and localization rather than signaling of mNotch 3 are affected in mNotch 3(R142C).     

Two Japanese CADASIL families with a R141C mutation in the Notch3 gene.

Cerebral autosomal dominant arteriopathy with subcortical infarcts and leukoencephalopathy (CADASIL) is a rare hereditary disease characterized by recurrent transient ischemic attacks (TIA) and strokes, and vascular dementia with Notch3 gene mutations as the cause of the disease. To date, there are only a few Japanese families ever reported with a mutation in the gene. Here, we report two more Japanese CADASIL families carrying a missense mutation in the Notch3 gene (R141C) with a unique lesion in the corpus callosum. This is the first report of two unrelated Japanese CADASIL families with a R141C mutation in the Notch3 gene. Although the disease is very rare among the Japanese population, our result suggests a possible relationship of this particular mutation (R141C) with the lesions of the corpus callosum.     

Arg133Cys mutation of Notch3 in two unrelated Japanese families with CADASIL

OBJECTIVE: More than 80 unrelated, but all Caucasian, patients with cerebral autosomal dominant arteriopathy with subcortical infarcts and leukoencephalopathy (CADASIL), originating from various communities around the world, have been molecularly identified. To clarify the occurrence of CADASIL in Orientals, we investigated Japanese families presenting as CADASIL. METHODS: We performed the PCR-SSCP and sequence analyses using genomic DNA, isolated from venous blood of participants under informed consent. PATIENTS: We identified two unrelated Japanese families with CADASIL, including 5 affected members through 2 generations. RESULTS: Each of the affected individuals developed recurrent strokes without risk factors resulting in progressive dementia, pseudobulbar palsy, and gait disturbances which started after the fifth decade of life. Although affected individuals had no vascular risk factors, they showed various degrees of narrowing of retinal arteries. Their MRI/CTs showed characteristics of the disease; bilateral small infarcts in the thalamus, basal ganglia, brain stem, and deep white matter in addition to the findings of leukoaraiosis. On SPECT imaging, there was severe hypoperfusion in the cortex as well as in the white matter. Ultrastructural studies revealed an abnormal deposition of granular osmiophilic materials (GOM) within the basal lamina of pericytes in muscular capillaries. On PCR-SSCP and sequence analyses, a heterozygous Arg133Cys mutation was present, in the affected individuals, in the exon 4 of Notch3 gene which is the hot spot region for CADASIL mutations in Caucasian families. None of the non-affected members nor the 50 Japanese normal controls revealed this mutation. CONCLUSION: Thus, our results confirm that CADASIL is a geographically widespread disorder caused by a Notch3 mutation.     

Cerebral autosomal dominant arteriopathy with subcortical infarcts and leukoencephalopathy

Cerebral autosomal dominant arteriopathy with subcortical infarcts and leukoencephalopathy (CADASIL) is an inherited autosomal dominant condition characterized by migrane, recurrent stroke, subcortical dementia, and pseudobulbar palsy. It begins with migraine with aura in -33% of patients. CADASIL is commonly overlooked or misdiagnosed owing to its recent identification. The pathological hallmark of angiopathy is the presence of multiple, small, deep cerebral infarcts, leucoencephalopathy, and nonatherorosclerotic, nonamyloid angiopathy involving mainly small, deep perforating cerebral arteries. Changes also are present in vascular smooth muscle cells and consist in the presence of granular osmiophilic material (GOM). The defective gene in CADASIL is Notch 3, which encodes a large transmembrane receptor. Magnetic resonance imaging shows high intensity signal lesions, often confluent, and areas of cystic degeneration of subcortical white matter and basal ganglia. Diagnostic strategies in CADASIL are matter of discussions because the electron microscopic demonstration of GOM was reported in 100% of symptomatic patients of French authors, but only in 45% of a British study. GOMs are not present in presymptomatic patients.     

Cerebral Autosomal Dominant Arteriopathy with Subcortical Infarcts and Leukoencephalopathy

Cerebral autosomal dominant arteriopathy with subcortical infarcts and leukoencephalopathy (CADASIL) is an inherited autosomal dominant condition characterized by migrane, recurrent stroke, subcortical dementia, and pseudobulbar palsy. It begins with migraine with aura in ～33% of patients. CADASIL is commonly overlooked or misdiagnosed owing to its recent identification. The pathological hallmark of angiopathy is the presence of multiple, small, deep cerebral infarcts, leucoencephalopathy, and nonatherorosclerotic, nonamyloid angiopathy involving mainly small, deep perforating cerebral arteries. Changes also are present in vascular smooth muscle cells and consist in the presence of granular osmiophilic material (GOM). The defective gene in CADASIL is Notch 3, which encodes a large transmembrane receptor. Magnetic resonance imaging shows high intensity signal lesions, often confluent, and areas of cystic degeneration of subcortical white matter and basal ganglia. Diagnostic strategies in CADASIL are matter of discussions because the electron microscopic demonstration of GOM was reported in 100% of symptomatic patients of French authors, but only in 45% of a British study. GOMs are not present in presymptomatic patients.     

CADASIL Notch3 mutant proteins localize to the cell surface and bind ligand

Cerebral autosomal-dominant arteriopathy with subcortical infarcts and leukoencephalopathy (CADASIL) is a vascular dementia arising from abnormal arteriolar vascular smooth muscle cells. CADASIL results from mutations in Notch3 that alter the number of cysteine residues in the extracellular epidermal growth factor-like repeats, important for ligand binding. It is not known whether CADASIL mutations lead to loss or gain of Notch3 receptor function. To examine the functional consequences of CADASIL mutations, we engineered 4 CADASIL-like mutations into rat Notch3 and have shown that the presence of an unpaired cysteine does not impair cell-surface expression or ligand binding.     

Linking Notch signaling to ischemic stroke

Vascular smooth muscle cells (SMCs) have been implicated in the pathophysiology of stroke, the third most common cause of death and the leading cause of long-term neurological disability in the world. However, there is little insight into the underlying cellular pathways that link SMC function to brain ischemia susceptibility. Using a hitherto uncharacterized knockout mouse model of Notch 3, a Notch signaling receptor paralogue highly expressed in vascular SMCs, we uncover a striking susceptibility to ischemic stroke upon challenge. Cellular and molecular analyses of vascular SMCs derived from these animals associate Notch 3 activity to the expression of specific gene targets, whereas genetic rescue experiments unambiguously link Notch 3 function in vessels to the ischemic phenotype.     

伴皮层下梗死和白质脑病的常染色体显性遗传性脑动脉病家系研究

目的探讨伴皮层下梗死和白质脑病的常染色体显性遗传性脑动脉病(CADASIL)的临床特征、病理与基因改变及诊断方法。方法调查先证者及其家族的发病情况及遗传方式,对其临床表现、影像学特征、病理学及分子遗传学等方面进行研究。结果先证者中年起病,临床上主要表现为反复发作的缺血性卒中,波动渐进性记忆、认知功能减退,痴呆。磁共振显示皮层下多发腔隙性或小灶性梗死,脑深部白质广泛稀疏,呈现典型的O’Sullivin征。皮肤血管活检：电镜下见小动脉基底膜增厚,其中存在嗜锇颗粒物质沉积;基因测序显示Notch-3基因第4号外显子突变。全家系5代,已有4代15人呈临床或亚临床发病,符合常染色体显性遗传。结论该家系的临床、影像、病理及分子遗传学特征符合CADASIL诊断标准。该病凭皮肤活检和基因测序而不经脑组织活检,可以在生前确诊。     

中国河南4个CADASIL家系NOTCH3基因R607C突变和临床特征

目的 探讨伴皮质下梗死和白质脑病的常染色体显性遗传性脑动脉病(cerebral autosomal dominant arteriopathy with subcortical infarcts and leukoencephalopathy,CADASIL)家系的NOTCH3基因突变和临床特征.方法 回顾性分析中国河南4个CADASIL先证者的临床和发病特征,并调查其家系中其他成员的发病情况,对患者和部分家系成员进行NOTCH3基因第3、4、11、18号外显子突变检测和结果分析.结果 基因测序显示,4个家系共6例患者和1例突变携带者存在NOTCH3基因第11号外显子R607C突变,均符合CADASIL临床特征.3例患者伴有血管危险因素.临床出现卒中者均伴有单侧肢体无力,有完整头颅MRI资料的5例患者均存在丘脑梗死.结论 在4个存在R607C突变的CADASIL家系中,6例CADASIL患者的临床特征相似,但不同家系成员之间存在个体化差异;影像学检查在CADASIL的诊断中具有重要作用;高血压、糖尿病等血管危险因素可存在于CADASIL患者.     

CADASIL with a novel mutation in exon 7 of NOTCH3 (C388Y)

We report a 38-year-old Japanese woman who had cerebral autosomal dominant arteriopathy with subcortical infarcts and leukoencephalopathy (CADASIL) with a novel mutation (TGT to TAT) at nucleotide position 1241 (C388Y) in exon 7 of the Notch3 gene (NOTCH3). Immunostaining of a skin biopsy with a Notch3 monoclonal antibody is a beneficial method for the screening of CADASIL, particularly in the case of rare mutations outside the mutation hotspots in NOTCH3 as shown in this patient.     

伴皮质下梗死和白质脑病的常染色体显性遗传性脑动脉病

近年来的研究表明,以家族遗传方式起病,中年发病且逐渐进展的缺血性卒中样病程,弥漫性多发性白质病变,明确的MRI白质异常信号以及病理学明确的小动脉病变是伴皮质下梗死和白质脑病的常染色体显性遗传性脑动脉病(cerebral autosomal dominant arteripathy with subcortical infarcts and leukoencephalopathy,CADASIL)的基本特征.分子遗传学研究表明,Notch3基因的多种点突变与cADASIL有关.基因诊断与外周组织活检相结合有可能是CADASIL最有价值的生前诊断手段.从发病机制、临床表现等多个方面研究CADASIL,有助于提高临床诊断率.     

Apolipoprotein A5 gene IVS3+G476A allelic variant confers susceptibility for development of ischemic stroke

BACKGROUND: T-1131C, T1259C and IVS3+G476A are naturally occurring variants of the apolipoprotein A5 (APOA5) gene and their possible impact on the development of ischemic stroke was investigated in the present study. METHODS AND RESULTS: PCR-RFLP assays were used to determine the distributions of the APOA5 alleles in small-vessel, large-vessel and mixed subgroups of 378 patients and in 131 stroke-free control subjects. Increased triglyceride levels were found in subjects carrying -1131C, 1259C, IVS3+476A alleles in all stroke groups and in the controls. The -1131C and IVS3+476A alleles, but not the T1259C variant, showed significant accumulation in all stroke subgroups. Logistic regression analysis adjusted for age, gender, body mass index, total cholesterol level, ischemic heart disease, hypertension, diabetes mellitus, smoking-and drinking habits revealed that the IVS3+476A allele represents independent susceptibility factor for stroke (odds ratio for small-vessel: 4.748; large-vessel: 3.905; mixed: 2.926; overall: 3.644 at 95% confidence interval; p<0.05), we could also confirm the previously verified pathogenic role of the -1131C variant. CONCLUSIONS: All of the 3 APOA5 variants are associated with elevated triglycerides, but only the -1131C and the IVS3+476A alleles confer risk for all types of ischemic stroke; such an association could not be detected for the 1259C allele.     

CADASIL

作为卒中、痴呆和偏头痛的病因,伴有皮质下梗死和白质脑病的常染色体显性遗传性脑动脉病(CADASIL)越来越受到人们的重视。Notch3基因突变是CADASIL的分子遗传学基础。目前,基因突变检测和皮肤肌肉组织活检是诊断CADASIL的主要手段。     

Notch signaling in vascular morphogenesis

PURPOSE OF REVIEW: This review highlights recent developments in the role of the Notch signaling pathway during vascular morphogenesis, angiogenesis, and vessel homeostasis. RECENT FINDINGS: Studies conducted over the past 4 years have significantly advanced the understanding of the effect of Notch signaling on vascular development. Major breakthroughs have elucidated the role of Notch in arterial versus venular specification and have placed this pathway downstream of vascular endothelial growth factor. SUMMARY: An emerging hallmark of the Notch signaling pathway is its nearly ubiquitous participation in cell fate decisions that affect several tissues, including epithelial, neuronal, hematopoietic, and muscle. The vascular compartment has been the latest addition to the list of tissues known to be regulated by Notch. Unraveling the contribution of Notch signaling to blood vessel formation has resulted principally from gain-of-function and loss-of-function experiments in mouse and zebrafish. During the past 4 years, these mechanistic studies have revealed that Notch is required for the successful completion of several steps during vascular morphogenesis and differentiation. In addition, the findings that Notch mutations are linked to some late-onset hereditary vascular pathologic conditions suggest the added contribution of this signaling pathway to vascular homeostasis.     

CADASIL的研究进展

伴有皮质下梗死和白质脑病的常染色体显性遗传性脑动脉病 (CADASIL)与Notch3基因突变有关 ,该基因的突变引起一系列的生化、病理生理学和血流动力学改变。CADASIL的临床表现复杂多样 ,只有与神经影像学检查、皮肤活检和基因检测结合起来才能做出正确诊断。     

Inducible inactivation of Notch1 causes nodular regenerative hyperplasia in mice

The discovery that the human Jagged1 gene (JAG1) is the Alagille syndrome disease gene indicated that Notch signaling has an important role in bile duct homeostasis. The functional study of this signaling pathway has been difficult because mice with targeted mutations in Jagged1, Notch1, or Notch2 have an embryonic lethal phenotype. We have previously generated mice with inducible Notch1 disruption using an interferon-inducible Cre-recombinase transgene in combination with the loxP flanked Notch1 gene. We used this conditional Notch1 knockout mouse model to investigate the role of Notch1 signaling in liver cell proliferation and differentiation. Deletion of Notch1 did not result in bile duct paucity, but, surprisingly, resulted in a continuous proliferation of hepatocytes. In conclusion, within weeks after Notch1 inactivation, the mice developed nodular regenerative hyperplasia without vascular changes in the liver.     

Notch ligand Delta-like 1 is essential for postnatal arteriogenesis

Growth of functional arteries is essential for the restoration of blood flow to ischemic organs. Notch signaling regulates arterial differentiation upstream of ephrin-B2 during embryonic development, but its role during postnatal arteriogenesis is unknown. Here, we identify the Notch ligand Delta-like 1 (Dll1) as an essential regulator of postnatal arteriogenesis. Dll1 expression was specifically detected in arterial endothelial cells, but not in venous endothelial cells or capillaries. During ischemia-induced arteriogenesis endothelial Dll1 expression was strongly induced, Notch signaling activated and ephrin-B2 upregulated, whereas perivascular cells expressed proangiogenic vascular endothelial growth factor, and the ephrin-B2 activator EphB4. In heterozygous Dll1 mutant mice endothelial Notch activation and ephrin-B2 induction after hindlimb ischemia were absent, arterial collateral growth was abrogated and recovery of blood flow was severely impaired, but perivascular vascular endothelial growth factor and EphB4 expression was unaltered. In vitro, angiogenic growth factors synergistically activated Notch signaling by induction of Dll1, which was necessary and sufficient to regulate ephrin-B2 expression and to induce ephrin-B2 and EphB4-dependent branching morphogenesis in human arterial EC. Thus, Dll1-mediated Notch activation regulates ephrin-B2 expression and postnatal arteriogenesis.     

Research actuality in the genetics of stroke

Stroke can be viewed as a paradigm for late-onset, complex polygenic diseases. There are two main clinical phenotypes for stroke: ischemic stroke, responsible for 80-90% of stroke events, and hemorrhagic stroke, responsible for the remaining 10-20%. Stroke may either be the outcome of a number of monogenic disorders or, more commonly, a polygenic multifactorial disease. Cerebral autosomal dominant arteriopathy with subcortical infarcts and leucoencephalopathy (CADASIL), due to mutations in the Notch 3 gene, is the best example of monogenic pathology leading to stroke. The identification of individual causative mutations for polygenic stroke is problematic due to the complexity of such condition. The two main methods of genetic investigation are linkage analyses and association studies, each with advantages and limitations. Associations with polymorphisms in a variety of candidate genes have been investigated, including hemostatic genes, genes controlling homocystein metabolism, the angiotensin-converting enzyme gene, and the endothelial nitric oxide synthase gene. The combination of linkage and association approaches has led to the identification of the first putative gene associated with common polygenic stroke, PDE4D, mapped to chromosome 5q21. The biological revolution of the past years, spurred by the Human Genome Project, promises the advent of novel technologies supported by bioinformatics, which will transform the study of polygenic disorders such as stroke. Understanding the causes of stroke and its effect will allow definition of high-risk populations and make possible specific programs of primary and secondary prevention as well as new therapeutic approaches where prevention has failed.     

Research Actuality in the Genetics of Stroke

Stroke can be viewed as a paradigm for late-onset, complex polygenic diseases. There are two main clinical phenotypes for stroke: ischemic stroke, responsible for 80–90% of stroke events, and hemorrhagic stroke, responsible for the remaining 10–20%. Stroke may either be the outcome of a number of monogenic disorders or, more commonly, a polygenic multifactorial disease. Cerebral autosomal dominant arteriopathy with subcortical infarcts and leucoencephalopathy (CADASIL), due to mutations in the Notch 3 gene, is the best example of monogenic pathology leading to stroke. The identification of individual causative mutations for polygenic stroke is problematic due to the complexity of such condition. The two main methods of genetic investigation are linkage analyses and association studies, each with advantages and limitations. Associations with polymorphisms in a variety of candidate genes have been investigated, including hemostatic genes, genes controlling homocystein metabolism, the angiotensin-converting enzyme gene, and the endothelial nitric oxide synthase gene. The combination of linkage and association approaches has led to the identification of the first putative gene associated with common polygenic stroke, PDE4D, mapped to chromosome 5q21. The biological revolution of the past years, spurred by the Human Genome Project, promises the advent of novel technologies supported by bioinformatics, which will transform the study of polygenic disorders such as stroke. Understanding the causes of stroke and its effect will allow definition of high-risk populations and make possible specific programs of primary and secondary prevention as well as new therapeutic approaches where prevention has failed.