Showing papers by "Kunikazu Tanji published in 2014"

Accumulation of the sigma-1 receptor is common to neuronal nuclear inclusions in various neurodegenerative diseases.

Abstract: The sigma-1 receptor (SIGMAR1) is now known to be one of the endoplasmic reticulum (ER) chaperones, which participate in the degradation of misfolded proteins in cells via the ER-related degradation machinery linked to the ubiquitin-proteasome pathway. Mutations of the SIGMAR1 gene are implicated in the pathogenesis of familial frontotemporal lobar degeneration and motor neuron disease. Involvement of ER dysfunction in the formation of inclusion bodies in various neurodegenerative diseases has also become evident. We performed immunohistochemical staining to clarify the localization of SIGMAR1 in the brains of patients with neurodegenerative disorders, including trans-activation response DNA protein 43 (TDP-43) proteinopathy, tauopathy, α-synucleinopathy, polyglutamine disease and intranuclear inclusion body disease (INIBD). Double-immunocytofluorescence and Western blot analyses of cultured cells were also performed to investigate the role of SIGMAR1 using a specific exportin 1 inhibitor, leptomycin B and an ER stress inducer, thapsigargin. SIGMAR1 was consistently shown to be co-localized with neuronal nuclear inclusions in TDP-43 proteinopathy, five polyglutamine diseases and INIBD, as well as in intranuclear Marinesco bodies in aged normal controls. Cytoplasmic inclusions in neurons and glial cells were unreactive for SIGMAR1. In cultured cells, immunocytofluorescent study showed that leptomycin B and thapsigargin were shown to sequester SIGMAR1 within the nucleus, acting together with p62. This finding was also supported by immunoblot analysis. These results indicate that SIGMAR1 might shuttle between the nucleus and the cytoplasm. Neurodegenerative diseases characterized by neuronal nuclear inclusions might utilize the ER-related degradation machinery as a common pathway for the degradation of aberrant proteins.

Phosphorylation of serine 349 of p62 in Alzheimer’s disease brain

Abstract: Extensive research on p62 has established its role in oxidative stress, protein degradation and in several diseases such as Paget’s disease of the bone, frontotemporal lobar degeneration and amyotrophic lateral sclerosis. Importantly, previous studies showed that p62 binds directly to Keap1, which is a ubiquitin E3 ligase responsible for degrading Nrf2. Indeed, colocalisation of p62 and Keap1 occurs in tumorigenesis and neurodegeneration. A serine (S) residue in the Keap1-interacting region of p62 is phosphorylated in hepatocellular carcinoma, and this phosphorylation contributes to tumour growth through the higher affinity of p62 to Keap1. However, it remains largely unknown whether p62 is phosphorylated in the Keap1-interacting region under neurodegenerative conditions. To answer this question, we generated an antibody against phosphorylated S349 (P-S349) of p62 and showed that S349 is phosphorylated following disruption of protein degradation. In particular, the ratio of P-S349 to total p62 levels was significantly increased in the brains with Alzheimer’s disease (AD) compared with controls. We also compared the reactivity of the P-S349 antibody with P-S403 of p62 and showed that these two phosphorylated sites on p62 cause different responses with proteasome inhibition and show distinct localisation patterns in AD brains. In addition to disruption of protein degradation systems, activation of oxidative stress can induce P-S349. These results support the hypothesis that disruption of protein degradation systems and sustained activation of the Keap1-Nrf2 system occur in the brains with AD.

ALS-associated protein FIG4 is localized in Pick and Lewy bodies, and also neuronal nuclear inclusions, in polyglutamine and intranuclear inclusion body diseases

Abstract: FIG4 is a phosphatase that regulates intracellular vesicle trafficking along the endosomal-lysosomal pathway. Mutations of FIG4 lead to the development of Charcot-Marie-Tooth disease type 4J and amyotrophic lateral sclerosis (ALS). Moreover, ALS-associated proteins (transactivation response DNA protein 43 (TDP-43), fused in sarcoma (FUS), optineurin, ubiquilin-2, charged mutivesicular body protein 2b (CHMP2B) and valosin-containing protein) are involved in inclusion body formation in several neurodegenerative diseases. Using immunohistochemistry, we examined the brains and spinal cords of patients with various neurodegenerative diseases, including sporadic TDP-43 proteinopathy (ALS and frontotemporal lobar degeneration). TDP-43 proteinopathy demonstrated no FIG4 immunoreactivity in neuronal inclusions. However, FIG4 immunoreactivity was present in Pick bodies in Pick's disease, Lewy bodies in Parkinson's disease and dementia with Lewy bodies, neuronal nuclear inclusions in polyglutamine and intranuclear inclusion body diseases, and Marinesco and Hirano bodies in aged control subjects. These findings suggest that FIG4 is not incorporated in TDP-43 inclusions and that it may have a common role in the formation or degradation of neuronal cytoplasmic and nuclear inclusions in several neurodegenerative diseases.

An autopsy case of incipient Pick's disease: Immunohistochemical profile of early-stage Pick body formation

Abstract: There is little immunohistochemical information about the early stage of Pick body formation, due to the extremely limited opportunities of studying Pick's disease at the incipient or subclinical stage. We report a 62-year-old man without any clinical manifestations of Pick's disease, who died of B-cell lymphoma of the brainstem. Post mortem examination revealed many Pick bodies without obvious neuronal loss mainly in the left frontal and temporal lobes. Three brains of patients with typical Pick's disease (disease duration: 7, 11 and 16 years) were also examined. Pick bodies were immunopositive for phosphorylated tau and 3-repeat tau, and less consistently for p62 in both incipient and typical cases. In the incipient case, borderline positivity for ubiquitin was evident in only a few Pick bodies, whereas in the typical cases many Pick bodies showed obvious positivity for ubiquitin. These findings suggest that Pick bodies are rarely ubiquitinated in the early stage of Pick body formation.

FUS colocalizes with polyglutamine, but not with TDP-43 in neuronal intranuclear inclusions in spinocerebellar ataxia type 2.

Abstract: The classification of neurodegenerative diseases is predicated on elucidation of the pathogenic protein species that define each disease or family of diseases. According to the protein-based classification, frontotemporal lobar degeneration (FTLD) is divided into four categories: FTLD-tau, FTLD-TDP, FTLD-FUS and FTLD-UPS. There appear to be mutually exclusive reciprocal staining pattern of neuronal cytoplasmic inclusions (NCIs) for either tau, TDP-43 or FUS [1]. Moreover, FUS is a component of neuronal intranuclear inclusions (NIIs) in polyglutamine (polyQ) diseases, including Huntington’s disease [2], spinocerebellar ataxia type 1 (SCA1) [1] and SCA3 [1]. Woulfe et al. assumed the reciprocal relationship with respect to FUS and TDP-43 staining in neurodegenerative disorders in which NIIs are a hallmark [1]. Recently, Toyoshima et al. demonstrated extensive TDP-43 pathology in a case of SCA2, one of the polyQ diseases, characterized by TDP-43-positive NCIs, NIIs and glial cytoplasmic inclusions [3], in which no apparent overlap of TDP-43 and polyQ was noted. An immunohistochemical investigation was conducted to determine whether FUS exists in TDP-43-positive or polyQ-positive NIIs in this case. Here we report that FUS colocalized with polyQ, but not with TDP-43 in NIIs in SCA2. Paraffin-embedded sections from a case of SCA2 [3] were immunostained using monoclonal antibodies against phosphorylated TDP-43 (pTDP-43) (Cosmo Bio Co., Ltd, Tokyo, Japan; 1:1000), polyQ (1C2; Chemicon, Temecula, CA, USA; 1:1000) and ubiquitin (1B3; MBL, Nagoya, Japan; 1:2000), and polyclonal antibodies against TDP-43 (nTDP-43) (10782-1-AP; ProteinTec Group, Inc., Chicago, IL, USA; 1:4000) and FUS (Sigma-Aldrich HPA008784, St. Louis, MO, USA; 1:1000). Selected sections were doubleimmunolabelled with anti-FUS (1:200) and either antipTDP-43 (1:400) or anti-polyQ (1:200) antibodies. This study was approved by the Institutional Ethics Committee of Hirosaki University Graduate School of Medicine. The distribution and frequency of NCIs and NIIs immunoreactive for polyQ, pTDP-43 and FUS are shown in Table 1. Two types of eosinophilic NIIs were found in sections stained with haematoxylin and eosin (H&E): cat’s eye-shaped (Figure 1a) or round to oval NIIs (Figure 1b). The former was mainly detected in medium-sized neurones in the striatum and the latter in the claustrum and cerebral cortex. Ubiquitin immunohistochemistry revealed coarse granular (Figure 1c) or round NCIs (Figure 1d). Moreover, cat’s eye-shaped NIIs (Figure 1e) or round to oval NIIs (Figure 1f) were observed. Immunostaining with antibody against pTDP-43 revealed linear wisp-like (Figure 1g), round (Figure 1h) and skeinlike NCIs (Figure 1i). Neuropil threads were also noted (Figure 1h,j; arrowheads). pTDP-43-positive, cat’s eyeshaped NIIs were observed in neurones in the striatum (Figure 1j), but not in the claustrum. Although pTDP-43positive NCIs and NIIs were also positive for nTDP-43, the nucleoplasm and cytoplasm of inclusion-bearing neurones showed no nTDP-43 immunoreactivity (data not shown). FUS immunohistochemistry revealed coarse granular or dot-like NCIs (Figure 1k) in the cerebral cortex and various subcortical areas. The distribution of FUS-positive round to oval NIIs (Figure 1l) was similar to that of polyQ-positive NIIs (Table 1). The nucleoplasm and cytoplasm of inclusion-bearing neurones showed no FUS immunoreactivity (Figure 1k,l). Sequential staining of the same sections with H&E and anti-polyQ antibody revealed that anti-polyQ antibody did not immunolabel cat’s eye-shaped NIIs (Figure 1 m,n), but immunostained round to oval NIIs and coarse granular NCIs (Figure 1o,p). Double-labelling immunofluorescence demonstrated that cat’s eye-shaped pTDP-43-positive NIIs were negative for FUS (Figure 2a–c). FUS-positive, oval NIIs were negative for pTDP-43 (Figure 2d–f) and polyQ-positive, oval NIIs and granular NCIs were positive for FUS (Figure 2g– i). More than 50 inclusions were examined for each immunostaining. Toyoshima et al. demonstrated that TDP-43-positive linear NCIs and polyQ-positive granular NCIs coexisted in the same neurones in the red nucleus in a case of SCA2 [3]. However, colocalization of TDP-43 and polyQ was not a feature in these neurones. In the present study, we further demonstrated no overlap of TDP-43 and polyQ in NIIs in SCA2. The morphological profile of TDP-43-

Multiple system atrophy and autophagy

Abstract: Macroautophagy is a dynamic process whereby cytoplasmic molecules are sequestered within autophagosomes. There exist two groups of mammalian autophagy-related gene (Atg) 8 homologues (LC3 and GABARAPs), which play essential role in autophagosomal formation. We determined whether Atg8 homologues are affected in Lewy body disease (LBD) and multiple system atrophy (MSA). The level of LC3 was increased in an insoluble fraction from the brain of patients with LBD, whereas the level of GABARAPs was decreased in LBD. The level of matured GABARAPs was significantly decreased in the cerebellum of MSA, and that the higher levels of matured and lipidated LC3 were detected in detergent-insoluble fraction of MSA. Furthermore, immunohistochemical staining revealed that both LC3 and GABARAPs were localized in Lewy bodies and glial cytoplasmic inclusions in MSA were positive for LC3. These findings suggest that autophagic function is impaired through alteration of Atg8 homologues in LBD and MSA. Autophagy-enhancing strategies can therefore have therapeutic efficacy for various neurodegenerative diseases including LBD and MSA.

Ubiquitin-negative, eosinophilic neuronal cytoplasmic inclusions associated with stress granules and autophagy: an immunohistochemical investigation of two cases.

Abstract: Identification of the proteinaceous components of the pathological inclusions is an important step in understanding the associated disease mechanisms. We immunohistochemically examined two previously reported cases with eosinophilic neuronal cytoplasmic inclusions (NCIs)(case 1, Mori et al. Neuropathology 2010; 30: 648–53; case 2, Kojima et al. Acta Pathol Jpn 1990; 40: 785–91) using 67 antibodies against proteins related to cytoskeletal constituents, ubiquitin-proteasome system, autophagy-lysosome pathway and stress granule formation. Regional distribution pattern of eosinophilic NCIs in case 1 was substantially different from that in case 2. However, NCIs in both cases were immunonegative for ubiquitin and p62 and were immunopositive for stress granule markers as well as autophagy-related proteins, including valosin-containing protein. Considering that eukaryotic stress granules are cleared by autophagy and valosin-containing protein function, our findings suggest that eosinophilic NCIs in the present two cases may represent the process of autophagic clearance of stress granules.