Myopathic changes caused by protein aggregates in adult-onset spinal muscular atrophy.

Spinal muscular atrophy (SMA), an autosomal-recessive lower motor neuron disease, causes progressive proximal muscle waste and weakness. It remains unclear whether myopathic changes are involved in pathogenesis. We encountered a patient with adult-onset SMA caused by a homozygous deletion in exon 7 of the survival motor neuron 1 (SMN1) gene who had had four copies of SMN2 exon 7. Muscle biopsy showed neurogenic features of groups of atrophic fibers, fiber-type grouping, and pyknotic nuclear clumps associated with fibers with rimmed vacuoles. Immunohistochemistry revealed sarcoplasmic aggregates of phosphorylated TDP-43 and p62 but not SMN. This study demonstrated myopathic changes with the accumulation of phosphorylated p62 and TDP-43 in the muscles of a patient with SMA, suggesting that abnormal protein aggregation may be involved in myopathic pathology.

Novel dot-blot assay for detection of vascular Notch3 aggregates in patients with CADASIL.

To detect vascular Notch3 extracellular domain aggregates in CADASIL, we developed a novel dot-blot assay with both autopsy and biopsy skin samples. We obtained samples from 11 patients with CADASIL and 12 control patients, and we performed dot-blot analyses by using sequential biochemical tissue extractions with three different antibodies against specific regions of the Notch3 extracellular domain. We also analyzed clinical features and vascular accumulations of Notch3 by immunohistochemistry. Via the dot-blot assay with the antibody against the C-terminal region of the Notch3 extracellular domain, we successfully detected Notch3 extracellular domain aggregates in skin tissue homogenates obtained from patients with CADASIL. Our novel method may therefore aid the diagnosis of CADASIL.

Serum amyloid P component: A novel potential player in vessel degeneration in CADASIL.

In cerebral autosomal dominant arteriopathy with subcortical infarcts and leukoencephalopathy (CADASIL), granular osmiophilic material (GOM) may play some roles in inducing cerebrovascular events. To elucidate the pathogenesis of CADASIL, we used laser microdissection and liquid chromatography-tandem mass spectrometry to analyze cerebrovascular lesions of patients with CADASIL for GOM. The analyses detected serum amyloid P component (SAP), annexin A2, and periostin as the proteins with the largest increase in the samples, which also demonstrated NOTCH3. For the three proteins, anti-human SAP antibody had the strongest reaction in the lesions where the anti-human NOTCH3 antibody showed positive staining. Moreover, immunofluorescence staining with the two antibodies clearly showed co-localization of SAP and NOTCH3. mRNA analyses indicated no positive SAP expression in the brain materials, which suggested that the source of SAP found in the GOM was only the liver. A solid phase enzyme-linked immunosorbent assay confirmed the binding of SAP with NOTCH3. Serum SAP concentrations were neither up-regulated nor down-regulated in CADASIL patients, when compared with those in control subjects. SAP may play an important role in GOM formation although precise mechanisms remain to be elucidated.

Sushi repeat-containing protein 1: a novel disease-associated molecule in cerebral amyloid angiopathy.

Sporadic cerebral amyloid angiopathy (CAA) is characterized by cerebrovascular amyloid beta (Aß) deposits and causes cerebral hemorrhage and dementia. The exact molecules that co-accumulate with cerebrovascular Aß deposits are still not fully known. In our study here, we performed proteomic analyses with microdissected leptomeningeal arteries and cerebral neocortical arterioles from 8 cases with severe CAA, 12 cases with mild CAA, and 10 control cases without CAA, and we determined the levels of highly expressed proteins in cerebral blood vessels in CAA. We focused on sushi repeat-containing protein 1 (SRPX1), which is specifically expressed in CAA-affected cerebral blood vessels. Because SRPX1, which is known as a tumor suppressor gene, reportedly induced apoptosis in tumor cells, we hypothesized that SRPX1 may play an important role in Aß-induced apoptosis in CAA. Immunohistochemical studies revealed that SRPX1 co-accumulated with Aß deposits in cerebral blood vessels of all autopsied cases with severe CAA. In contrast, no SRPX1 co-accumulated with Aß deposits in senile plaques. Furthermore, we demonstrated that both Aß40 and Aß42 bound to SRPX1 in vitro and enhanced SRPX1 expression in primary cultures of cerebrovascular smooth muscle cells. SRPX1 enhanced caspase activity induced by Aß40. Knockdown of SRPX1, in contrast, reduced the formation of Aß40 accumulations and the activity of caspase in cultured cerebrovascular smooth muscle cells. SRPX1 may thus be a novel molecule that is up-regulated in cerebrovascular Aß deposits and that may increase Aß-induced cerebrovascular degeneration in CAA.

[Skin Biopsy is a Useful Tool for the Diagnosis of Atypical CADASIL: A Case Report].

A 57-year-old man developed migraine at the age of 25 years. Thereafter, he developed depression at the age of 50 years, and was admitted to a psychiatric hospital at the age of 54 years because of deteriorating depression. He returned to his work after receiving treatment for depression; however, he made mistakes several times in his work. He was referred to our hospital for further neurological evaluation. The results of the neurological examination performed on admission were unremarkable. His Mini Mental State Examination (MMSE) score was 24/30, and neuropsycological evaluations revealed executive dysfunction. There was no family history of dementia or cerebral infarction. Magnetic resonance fluid attenuated inversion recovery (MR FLAIR) image of the brain showed hyperintense lesions around the lateral ventricle without involvement in the temporal pole and external capsule. Despite a lack of family history of dementia and cerebral infarction and non-specific brain MRI findings, his history of headache and depression were suggestive of cerebral autosomal dominant arteriopathy with subcortical infarcts and leukoencephalopathy (CADASIL). Therefore, skin biopsy was performed; electron microscopy of the biopsied sample revealed granular osmiophilic material deposits. Genetic analysis of the NOTCH3 gene showed a missense mutation with substitution of R427C in exon 8, i.e., out of the hot-spot, exon 3, and 4. Thus, skin biopsy is a useful tool for diagnosing atypical CADASIL.

Genotypic and phenotypic spectrum of CADASIL in Japan: the experience at a referral center in Kumamoto University from 1997 to 2014.

This study elucidates the genotypic and phenotypic spectrum and histopathological findings related to cerebral autosomal dominant arteriopathy with subcortical infarcts and leukoencephalopathy (CADASIL) in Japan. For this single-center retrospective observational study, we enrolled 215 patients who were clinically suspected of having CADASIL and were examined at Kumamoto University from 1997 to 2014, and we diagnosed CADASIL in 70 patients. We found 19 different NOTCH3 mutations in the patients, with the NOTCH3 Arg133Cys mutation being found most frequently. We also found the Arg75Pro mutation, a cysteine-sparing NOTCH3 mutation. CADASIL patients with this Arg75Pro mutation were frequently found throughout Japan, and fewer patients with the Arg75Pro mutation showed MRI hyperintensity in the anterior temporal pole compared with patients with other NOTCH3 mutations. Significantly more CADASIL patients with the NOTCH3 Arg133Cys mutation had hyperintensity in the external capsule compared with CADASIL patients with the other mutations not including the NOTCH3 Arg75Pro mutation. We also showed postmortem pathological findings of the first Japanese CADASIL case with the NOTCH3 Arg133Cys mutation, and histopathological findings of fresh frozen skin biopsy specimens of CADASIL patients. In conclusions, the spectrum of NOTCH3 mutations in Japanese CADASIL patients may be partially explained by founder effects. Genotype-phenotype correlations may exist in CADASIL, which should be considered so as to make an accurate diagnosis of CADASIL in each population. Fresh frozen skin biopsy specimens may aid detection of Notch3 deposits on vascular walls for an improved diagnosis of CADASIL.