Takashi Kurashige

Immunopositivity for ESCRT-III subunit CHMP2B in granulovacuolar degeneration of neurons in the Alzheimer's disease hippocampus

Endosomal sorting complex required for transport (ESCRT)-III subunit charged multivesicular body protein 2B (CHMP2B) is involved in the degradation of proteins in the endocytic and autophagic pathways. Mutations in the CHMP2B gene are reportedly associated with frontotemporal dementia (FTD) and amyotrophic lateral sclerosis (ALS) characterised by accumulation of ubiquitinated protein aggregates in affected neurons, suggesting a relationship between protein accumulation and efficient autophagic degradation. This study investigated CHMP2B immunoreactivity in the hippocampus of patients with Alzheimers disease (AD), revealing intense labeling of intraneuronal dot-like structures by antibody to CHMP2B. Since the morphological characteristics of these granular structures were compatible with those of granulovacuolar degeneration (GVD), a hallmark of AD pathology, immunohistochemical study using anti-CHMP2B antibody was performed using AD and control brain sections to investigate whether this antibody can be used as a GVD label. The number and percentage of hippocampal neurons with CHMP2B-positive granules were higher in AD cases and CHMP2B-positive granules corresponded to GVD. Anti-CHMP2B antibody detected a single 28-kDa band on Western blotting using control and AD specimens. This antibody clearly and intensely detected GVD over the hippocampus and entorhinal and transentorhinal cortices. These findings suggest that researchers will be able to use CHMP2B as a molecular label for studying GVD.

Statins and myotoxic effects associated with anti-3-hydroxy-3-methylglutaryl-coenzyme A reductase autoantibodies: an observational study in Japan

AbstractStatins have a variety of myotoxic effects and can trigger the development of inflammatory myopathies or myasthenia gravis (MG) mediated by immunomodulatory properties. Autoantibodies to 3-hydroxy-3-methylglutaryl-coenzyme A reductase (HMGCR) have been identified in patients with statin-associated myopathy. The purpose of the present study is to develop an enzyme-linked immunosorbent assay (ELISA) of anti-HMGCR antibodies and to elucidate the clinical significance of anti-HMGCR antibodies in Japanese patients with inflammatory myopathies or MG.We enrolled 75 patients with inflammatory myopathies, who were all negative for anti-signal recognition particle and anti-aminoacyl transfer RNA synthetase antibodies. They were referred to Keio University and National Center of Neurology and Psychiatry between October 2010 and September 2012. We also studied 251 patients with MG who were followed at the MG Clinic at Keio University Hospital. Anti-HMGCR antibodies were detected by ELISA. We investigated demographic, clinical, radiological, and histological findings associated with anti-HMGCR antibodies.We established the anti-HMGCR ELISA with the recombinant protein. Protein immunoprecipitation detected autoantigens corresponding to HMGCR. Immunohistochemistry using muscle biopsy specimens revealed regenerating muscle fibers clearly stained by polyclonal anti-HMGCR antibodies and patients’ serum. Anti-HMGCR autoantibodies were specifically detected in 8 patients with necrotizing myopathy. The seropositivity rate in the necrotizing myopathy patients was significantly higher than those in the patients with other histological diagnoses of inflammatory myopathies (31% vs 2%, P = 0.001). Statins were administered in only 3 of the 8 anti-HMGCR-positive patients. Myopathy associated with anti-HMGCR antibodies showed mild limb weakness and favorable response to immunotherapy. All 8 patients exhibited increased signal intensities on short T1 inversion recovery of muscle MRI. Of the 251 patients with MG, 23 were administered statins at the onset of MG. One late-onset MG patient experienced MG worsening after 4-wk treatment with atorvastatin. However, anti-HMGCR antibodies were not detected in the 251 MG patients except for one early-onset MG patient with no history of statin therapy.Anti-HMGCR antibodies are a relevant clinical marker of necrotizing myopathy with or without statin exposure, but they are not associated with the onset or deterioration of MG.

Granulovacuolar Degenerations Appear in Relation to Hippocampal Phosphorylated Tau Accumulation in Various Neurodegenerative Disorders

Background Granulovacuolar degeneration (GVD) is one of the pathological hallmarks of Alzheimers disease (AD), and it is defined as electron-dense granules within double membrane-bound cytoplasmic vacuoles. Several lines of evidence have suggested that GVDs appear within hippocampal pyramidal neurons in AD when phosphorylated tau begins to aggregate into early-stage neurofibrillary tangles. The aim of this study is to investigate the association of GVDs with phosphorylated tau pathology to determine whether GVDs and phosphorylated tau coexist among different non-AD neurodegenerative disorders. Methods An autopsied series of 28 patients with a variety of neurodegenerative disorders and 9 control patients were evaluated. Standard histological stains along with immunohistochemistry using protein markers for GVD and confocal microscopy were utilized. Results The number of neurons with GVDs significantly increased with the level of phosphorylated tau accumulation in the hippocampal regions in non-AD neurodegenerative disorders. At the cellular level, diffuse staining for phosphorylated tau was detected in neurons with GVDs. Conclusions Our data suggest that GVDs appear in relation to hippocampal phosphorylated tau accumulation in various neurodegenerative disorders, while the presence of phosphorylated tau in GVD-harbouring neurons in non-AD neurodegenerative disorders was indistinguishable from age-related accumulation of phosphorylated tau. Although GVDs in non-AD neurodegenerative disorders have not been studied thoroughly, our results suggest that they are not incidental findings, but rather they appear in relation to phosphorylated tau accumulation, further highlighting the role of GVD in the process of phosphorylated tau accumulation.

Localization of CHMP2B‐immunoreactivity in the brainstem of Lewy body disease

Alpha‐synuclein (αS) is one of the major constituents of Lewy bodies (LBs). Several lines of evidence suggest that the autophagy‐lysosome pathway (ALP) is involved in the removal of αS. We have previously reported that granulovacuolar degeneration (GVD) in neurons involved a subunit of the endosomal sorting complexes required for transport (ESCRT). In this study, we examined the association between alpha‐synucleinopathy and autophagy through immunohistochemical analysis of charged multivesicular body protein 2B (CHMP2B), a component of the ESCRT‐pathway. We examined the brainstems of 17 patients with Parkinsons disease (PD), incidental Lewy body disease (ILBD), multiple system atrophy (MSA), and Alzheimers disease (AD) immunohistochemically using antibodies against phosphorylated αS (pαS), phosphorylated tau and CHMP2B. LBs and a proportion of glial cytoplasmic inclusions (GCIs) were immunopositive for pαS and CHMP2B. Neurons containing CHMP2B‐immunoreactive granules were detected in PD and ILBD, but not in MSA and AD brains. CHMP2B immunoreactivity was increased in the dorsal motor nucleus of the vagus nerve (DMNX) in PD and ILBD brains, relative to that in MSA and AD. These findings indicate that the ESCRT‐pathway is implicated in the formation of αS inclusions, especially in PD and ILBD.

Cyclin-dependent kinase 5 immunoreactivity for granulovacuolar degeneration.

In addition to senile plaque and neurofibrillary tangles, granulovacuolar degeneration is a hallmark of Alzheimer’s disease. A number of tau kinases, such as c-jun N-terminal kinase (JNK), glycogen-synthase kinase-3&bgr; (GSK3&bgr;), and casein kinase 1 (CK1), have been reported to be markers of granulovacuolar degeneration. In addition, cyclin-dependent kinase 5 (CDK5), which phosphorylates tau, has been shown to be abundantly expressed in neurofibrillary tangles in the hippocampus. CDK5 has a unique staining pattern, and therefore, has the potential to be a novel marker for granulovacuolar degeneration. In this study, we investigated the ability of CDK5 to be a marker for granulovacuolar degeneration using immunohistochemical analysis. Four Alzheimer’s disease cases, three myotonic dystrophy (MyD) cases, and three control cases were subjected to immunohistochemistry and immunofluorescent techniques using anti-CDK5, anti-charged multivesicular body protein 2B (CHMP2B), anti-pSmad2/3, anti-ubiquitin (Ub), anti-phospho-TDP-43 and AT8 antibodies. Some CDK5-positive granules were morphologically similar to granulovacuolar degeneration intraluminal granules, and these granules overlapped with those immunopositive for pSmad2/3, Ub and phospho-TDP-43 established granulovacuolar degeneration markers. Moreover, CDK5-positive granulovacuolar degeneration and phosphorylated tau colocalized in pyramidal neurons in Alzheimer’s disease and MyD cases. The numbers of CDK5-positive granules showed an inverse relationship with the degree of mature neurofibrillary tangles in each cell, as was the case with CHMP2B-positive granulovacuolar degeneration granules and neurofibrillary tangles. The presence of tau kinases including CDK5 in granulovacuolar degeneration might implicate that granulovacuolar degeneration is structurally involved in tau modification.

Phosphatidylinositol-4,5-bisphosphate is enriched in granulovacuolar degeneration bodies and neurofibrillary tangles

Among the pathological findings in Alzheimers disease (AD), the temporal and spatial profiles of granulovacuolar degeneration (GVD) bodies are characteristic in that they seem to be related to those of neurofibrillary tangles (NFTs), suggesting a common mechanism underlying the pathogenesis of these structures. Flotillin‐1, a marker of lipid rafts, accumulates in lysosomes of tangle‐bearing neurones in AD patients. In addition, recent reports have shown that GVD bodies accumulate at the nexus of the autophagic and endocytic pathways. The aim of this study was to elucidate the distribution of the lipid component of lipid rafts, phosphatidylinositol‐4,5‐bisphosphate [PtdIns(4,5)P2], in AD and other neurodegenerative disorders.

Characterization and distribution of adaptor protein containing a PH domain, PTB domain and leucine zipper motif (APPL1) in Alzheimer’s disease hippocampus: an immunohistochemical study

Adaptor protein containing a PH domain, PTB domain and leucine zipper motif (APPL1) is emerging as a critical regulator of various cellular processes in non-neuronal cells as well as in neurons where it localizes to dendritic spines and synapses. It regulates the development of these structures in hippocampal neurons. Although memory impairment in Alzheimers disease (AD) has been attributed to disruption of synaptic plasticity, there is scant information on this protein in the human brain. In the present study, we immunohistochemically characterized the localization of APPL1 in AD and control brains. APPL1 accumulated perisomatically as granules around neurons within vulnerable sectors of the hippocampus (CA1 and subiculum) in AD brain, whilst APPL1-positive granules were rarely identified in control brains derived from elderly individuals with no known cognitive impairment. Interestingly, in the AD hippocampus, APPL1 also co-localized with perisomatic granules (non-plaque dystrophic dendrites) expressing glutamate receptor 2 and ubiquitin, suggesting the possible involvement of APPL1 in the synaptic modifications in AD. Thus, the immunohistochemical distribution of APPL1 in AD brain was distinct from that in non-AD control brains, suggesting that signaling via APPL1 might play a critical role in the memory impairment in AD.

Elevated urinary β2 microglobulin in the first identified Japanese family afflicted by X-linked myopathy with excessive autophagy.

Here we report what is to our knowledge the first identified Japanese family afflicted by X-linked myopathy with excessive autophagy. The index case is a 52-year-old man with almost 40years of progressive proximal muscle weakness. High urinary β2 microglobulin, normal serum β2 microglobulin, autophagic vacuoles with sarcolemmal features, and a hemizygous c.164-7T>G mutation in the VMA21 gene were found. His two maternal uncles had similar clinicopathological findings. High urinary β2 microglobulin without obvious renal dysfunction might result from decreased urine acidification in the distal convoluted tubules caused by the VMA21 gene mutation. These findings might prove to be useful as a preliminary marker suggestive of X-linked myopathy with excessive autophagy.

Detecting gene mutations in Japanese Alzheimer's patients by semiconductor sequencing

Alzheimers disease (AD) is the most common form of dementia. To date, several genes have been identified as the cause of AD, including PSEN1, PSEN2, and APP. The association between APOE and late-onset AD has also been reported. We here used a bench top next-generation sequencer, which uses an integrated semiconductor device, detects hydrogen ions, and operates at a high-speed using nonoptical technology. We examined 45 Japanese AD patients with positive family histories, and 29 sporadic patients with early onset (<60-year-old). Causative mutations were detected in 5 patients in the familial group (11%). Three patients had a known heterozygous missense mutation in the PSEN1 gene (p.H163R). Two patients from 1 family had a novel heterozygous missense mutation in the PSEN1 gene (p.F386L). In the early onset group, 1 patient carrying homozygous APOEε4 had a novel heterozygous missense mutation in the PSEN2 gene (p.T421M). Approximately 43% patients were APOEε4 positive in our study. This new sequencing technology is useful for detecting genetic variations in familial AD.

Molecular markers for granulovacuolar degeneration are present in rimmed vacuoles.

Background Rimmed vacuoles (RVs) are round-oval cytoplasmic inclusions, detected in muscle cells of patients with myopathies, such as inclusion body myositis (IBM) and distal myopathy with RVs (DMRV). Granulovacuolar degeneration (GVD) bodies are spherical vacuoles containing argentophilic and hematoxyphilic granules, and are one of the pathological hallmarks commonly found in hippocampal pyramidal neurons of patients with aging-related neurodegenerative diseases, such as Alzheimers disease and Parkinsons disease. These diseases are common in the elderly and share some pathological features. Therefore, we hypothesized that mechanisms of vacuolar formation in RVs and GVD bodies are common despite their role in two differing pathologies. We explored the components of RVs by immunohistochemistry, using antibodies for GVD markers. Methods Subjects included one AD case, eight cases of sporadic IBM, and three cases of DMRV. We compared immunoreactivity and staining patterns for GVD markers. These markers included: (1) tau-modifying proteins (caspase 3, cyclin-dependent kinase 5 [CDK5], casein kinase 1δ [CK1δ], and c-jun N-terminal kinase [JNK]), (2) lipid raft-associated materials (annexin 2, leucine-rich repeat kinase 2 [LRRK2], and flotillin-1), and (3) other markers (charged multi-vesicular body protein 2B [CHMP2B] and phosphorylated transactive response DNA binding protein-43 [pTDP43]) in both GVD bodies and RVs. Furthermore, we performed double staining of each GVD marker with pTDP43 to verify the co-localization. Results GVD markers, including lipid raft-associated proteins and tau kinases, were detected in RVs. CHMP2B, pTDP43, caspase 3, LRRK2, annexin 2 and flotillin-1 were detected on the rim and were diffusely distributed in the cytoplasm of RV-positive fibers. CDK5, CK1δ and JNK were detected only on the rim. In double staining experiments, all GVD markers colocalized with pTDP43 in RVs. Conclusions These results suggest that RVs of muscle cells and GVD bodies of neurons share a number of molecules, such as raft-related proteins and tau-modifying proteins.