Showing papers by "Kunikazu Tanji published in 2012"

Autophagic adapter protein NBR1 is localized in Lewy bodies and glial cytoplasmic inclusions and is involved in aggregate formation in α-synucleinopathy.

Abstract: Macroautophagy is a dynamic process whereby cytoplasmic components are initially sequestered within autophagosomes. Recent studies have shown that the autophagosome membrane can selectively recognize ubiquitinated proteins and organelles through interaction with adapter proteins such as p62 and NBR1. Both proteins are structurally similar at the amino acid level, and bind with ubiquitin and ubiquitinated proteins. Although p62 is incorporated into a wide spectrum of pathological inclusions in various neurodegenerative diseases, abnormalities of NBR1 have not been reported in these diseases. Our immunohistochemical examination revealed that the vast majority of Lewy bodies (LBs) in Parkinson’s disease and dementia with LBs (DLB) as well as of glial cytoplasmic inclusions in multiple system atrophy (MSA) were positive for NBR1. Neuronal and glial inclusions in tauopathies and TAR DNA-binding protein of 43 kDa proteinopathies were rarely immunolabeled, or were unstained. Using cultured cells bearing LB-like inclusions, formation of α-synuclein aggregates was repressed in cells with NBR1 knockdown. Immunoblot analysis showed that the level of NBR1 was significantly increased by 2.5-fold in MSA, but not in DLB. These findings suggest that NBR1 is involved in the formation of cytoplasmic inclusions in α-synucleinopathy.

p62/sequestosome 1 binds to TDP-43 in brains with frontotemporal lobar degeneration with TDP-43 inclusions.

Abstract: Ubiquitin-positive cytoplasmic inclusions are consistently found in various neurodegenerative diseases. As with ubiquitin, anti-p62/SQSTM1 (referred to as p62) antibody clearly immunostains these inclusions. p62 has a ubiquitin-associated domain at the carboxyl terminus and thereby interacts with ubiquitinated and misfolded proteins. Here we immunoprecipitated endogenous p62 in the cerebral cortex from patients with frontotemporal lobar degeneration with TDP-43 inclusions (FTLD-TDP) and found that p62 coimmunoprecipitated several proteins, including TDP-43, which is a major disease protein in FTLD-TDP. Unexpectedly, p62 immunoprecipitated a smaller amount of TDP-43 in FTLD-TDP compared with controls. Further analyses showed that p62 physiologically binds to TDP-43 and likely is involved in degradation of TDP-43 with 35-kDa, but not full-length TDP-43. Our results suggest that the interaction of TDP-43 and p62 is disrupted and may participate in the pathogenesis of TDP-43 proteinopathy.

Ubiquilin immunoreactivity in cytoplasmic and nuclear inclusions in synucleinopathies, polyglutamine diseases and intranuclear inclusion body disease.

Abstract: Ubiquilin-1 (UBQLN1), a member of the ubiquitin-like protein family (UBQLN1-4), is associated with neurofibrillary tangles in Alzheimer’s disease (AD) and with Lewy bodies (LBs) in Parkinson’s disease (PD) [7]. Mutations in UBQLN2 cause dominant X-linked amyotrophic lateral sclerosis (ALS) [4]. UBQLN2-immunoreactive neuronal cytoplasmic inclusions (NCIs) are found in the hippocampus and spinal cord in ALS with or without UBQLN2 mutation. Moreover, a distinct pattern of UBQLN2 pathology is seen in cases of ALS and frontotemporal lobar degeneration with TDP-43-positive inclusions (FTLDTDP) showing C9ORF72-hexanucleotide repeat expansion [2], which is the most common genetic abnormality in ALS/ FTLD [3, 12]. Here we report that UBQLN2 immunoreactivity is present in cytoplasmic and nuclear inclusions in various neurodegenerative diseases. Post-mortem cases of sporadic ALS (n = 5), FTLDTDP type B (n = 4), PD (n = 5), neocortical-type DLB (n = 5), multiple system atrophy (MSA; n = 5), AD (n = 5), Pick’s disease (n = 4), progressive supranuclear palsy (n = 4), corticobasal degeneration (n = 4), argyrophilic grain disease (n = 4), Huntington’s disease (HD; n = 3), dentatorubral-pallidoluysian atrophy (DRPLA; n = 5), spinal and bulbar muscular atrophy (SBMA; n = 3), spinocerebellar ataxia type 1 (SCA1; n = 3), SCA2 (n = 1), SCA3 (n = 5), intranuclear inclusion body disease (INIBD; n = 5) and controls (n = 5) were utilized. Immunohistochemistry was performed as described previously [10] with the following antibodies: UBQLN2, UBQLN1, phosphorylated a-synuclein, phosphorylated tau, ubiquitin, polyglutamine and TDP-43 (Online Resource). The total number of inclusions immunostained with each antibody was counted in contiguous sections. In controls, neuronal nuclei were weakly immunolabeled with anti-UBQLN2 (Fig. 1a). UBQLN2-immunoreactive NCIs were found in the temporal cortex in FTLD-TDP (25 % relative to TDP-43-positive inclusions) as well as in the spinal cord in ALS (14 %) (Fig. 1b). In PD/DLB, both brainstem type and cortical LBs were intensely stained (Fig. 1c, d). Contiguous sections stained with anti-UBQLN2 and anti-a-synuclein revealed that 21 % of brainstem-type LBs and 48 % of cortical LBs were positive for UBQLN2. In MSA, 82 % of glial cytoplasmic inclusions (GCIs) were positive for UBQLN2 (Fig. 1e). In HD, DRPLA, SBMA, SCA1-3 and INIBD, more than 95 % of neuronal nuclear inclusions (NNIs) were strongly immunolabeled (Fig. 1f–l). In addition, Marinesco bodies (MBs) F. Mori (&) K. Tanji S. Odagiri K. Wakabayashi Department of Neuropathology, Institute of Brain Science, Hirosaki University Graduate School of Medicine, 5 Zaifu-cho, Hirosaki 036-8562, Japan e-mail: neuropal@cc.hirosaki-u.ac.jp

Optineurin immunoreactivity in neuronal nuclear inclusions of polyglutamine diseases (Huntington's, DRPLA, SCA2, SCA3) and intranuclear inclusion body disease.

Abstract: Optineurin (OPTN) is a ubiquitous cytoplasmic protein with various functions including membrane trafficking and signal transduction [9]. OPTN mutations are associated with adult-onset glaucoma and familial and sporadic amyotrophic lateral sclerosis (ALS) [4]. OPTN immunoreactivity has been reported in a variety of ubiquitinated inclusions including ALS-associated neuronal cytoplasmic inclusions, neurofibrillary tangles in Alzheimer’s disease, Pick bodies in Pick disease, Lewy bodies in Parkinson’s disease and glial cytoplasmic inclusions in multiple system atrophy [3, 6]. With respect to polyglutamine diseases, neuronal nuclear inclusions (NNIs) in Huntington’s disease (HD) and spinocerebellar ataxia type 3 (SCA3) are reported to be immunonegative for OPTN [2]. However, Schwab et al. [7] have contended that NNIs in HD are immunopositive for OPTN. We report that OPTN immunoreactivity is present in NNIs in various polyglutamine diseases and intranuclear inclusion body disease (INIBD). A total of 30 post-mortem cases were utilized in the present study; these included cases of HD (n = 3), dentatorubral-pallidoluysian atrophy (DRPLA; n = 5), SCA2 (n = 1), SCA3 (n = 5), SCA6 (n = 2), SCA17 (n = 1), spinal and bulbar muscular atrophy (SBMA; n = 3), INIBD (n = 5) and normal control subjects (n = 5). The diagnoses of polyglutamine diseases were confirmed genetically and histopathologically. Immunohistochemical analysis was carried out using formalin-fixed, paraffinembedded sections from the basal ganglia in HD, SCA2 and SCA17, the pons in DRPLA and SCA3, the cerebellum in SCA6, the spinal cord in SBMA, the frontal lobe in INIBD, and the cortical and subcortical regions in control subjects. Anti-ubiquitin (1B3; MBL, Nagoya, Japan), antipolyglutamine (1C2; Chemicon, Temecula, CA, USA) and anti-human OPTN C terminus (#100000; Cayman CHEMICAL, Ann Arbor, MI, USA) [2, 7, 8] were used as primary antibodies. In controls, anti-OPTN antibody weakly immunolabeled the neuronal cytoplasm in a diffuse granular pattern, and neuronal nuclei were negative for OPTN (Fig. 1a). OPTN-immunoreactive NNIs were found in all the cases of HD, DRPLA, SCA2 and SCA3 (Fig. 1b–e). Intranuclear inclusions in neurons, but not in glial cells, in INIBD were also immunopositive for OPTN (Fig. 1f). Double immunofluorescence analyses revealed that OPTN immunoreactivity was found in 69% of NNIs in HD, 46% in DRPLA, 17% in SCA2, 55% in SCA3 and 50% in INIBD (Fig. 2). Although ubiquitinated NNIs were seen in SCA17 and SBMA, these inclusions were F. Mori (&) K. Tanji K. Wakabayashi Department of Neuropathology, Institute of Brain Science, Hirosaki University Graduate School of Medicine, 5 Zaifu-cho, Hirosaki 036-8562, Japan e-mail: neuropal@cc.hirosaki-u.ac.jp

Immunohistochemical analysis of Marinesco bodies, using antibodies against proteins implicated in the ubiquitin-proteasome system, autophagy and aggresome formation

Abstract: Marinesco bodies (MBs) are spherical eosinophilic intranuclear inclusions in pigmented neurons in the substantia nigra and locus ceruleus. Previous immunohistochemical studies have shown that MBs are positive for ubiquitin, p62 and SUMO-1, suggesting the involvement of ubiquitination and related proteins in the formation or disaggregation of MBs. However, the involvement is not thoroughly understood. Therefore, we immunohistochemically examined the midbrain from five control subjects ranged from 53 to 84 years old. MBs were positive for various proteins implicated in the ubiquitin-proteasome system (ubiquitin, p62, EDD1, NEDD8, NUB1, SUMO-1 and SUMO-2), aggresome formation (HDAC6) and autophagy (ubiquitin, p62, LC3, GABARAP and GATE-16). These findings suggest that proteins related to ubiquitination, proteasomal degradation and autophagy are involved in the formation or disaggregation of MBs.

FUS immunoreactivity of neuronal and glial intranuclear inclusions in intranuclear inclusion body disease.

Abstract: F. Mori, K. Tanji, T. Kon, S. Odagiri, M. Hattori, Y. Hoshikawa, C. Kono, K. Yasui, S. Yokoi, Y. Hasegawa, M. Yoshida and K. Wakabayashi (2012) Neuropathology and Applied Neurobiology38, 322–328 FUS immunoreactivity of neuronal and glial intranuclear inclusions in intranuclear inclusion body disease Aims: Recent studies have shown that fused-in-sarcoma (FUS) protein is a component of ‘neuronal’ intranuclear inclusion bodies (INIBs) in the brains of patients with intranuclear inclusion body disease (INIBD). However, the extent and frequency of FUS-immunoreactive structures in INIBD are uncertain. Methods: We immunohistochemically examined the brain, spinal cord and peripheral ganglia from five patients with INIBD and five control subjects, using anti-FUS antibodies. Results: In controls, the nuclei of both neurones and glial cells were intensely immunolabelled with anti-FUS and neuronal cytoplasm was weakly positive for FUS. In INIBD, neuronal and glial INIBs in the brain and spinal cord were positive for FUS. FUS-positive INIBs were also found in the peripheral ganglia. The proportion of FUS-positive neuronal INIBs relative to the total number of inclusion-bearing neurones ranged from 55.6% to 83.3% (average 73.2%) and that of FUS-positive glial INIBs ranged from 45.9% to 85.7% (average 62.7%). The nucleus and cytoplasm of inclusion-bearing neurones and glial cells showed no FUS immunoreactivity. Conclusions: These findings suggest that FUS is incorporated into INIBs in both neurones and glial cells and that loss of normal FUS immunoreactivity may result from reduced protein expression and/or sequestration within inclusions.

Ubiquitin-related proteins in neuronal and glial intranuclear inclusions in intranuclear inclusion body disease

Abstract: Recent studies have shown that eosinophilic intranuclear inclusions (INI) in the brain of patients with intranuclear inclusion body disease (INIBD) are immunopositive for ubiquitin and ubiquitin-related proteins (URP). However, the extent and frequency of URP-immunoreactive inclusions in INIBD are uncertain. We immunohistochemically examined the brain, spinal cord and dorsal root ganglia from five patients with INIBD, using a virtual slide system with sequential staining of the same sections with hematoxylin and eosin and by immunolabeling with antibodies against ubiquitin and URP (NEDD8, NUB1, SUMO-1 and SUMO-2). Intranuclear inclusions were widely distributed in neurons and glial cells in all the cases. Sequential staining revealed that 100% of INI in neurons and glial cells were positive for ubiquitin. Moreover, the majority or a significant proportion of INI were positive for NEDD8, NUB1, SUMO-1 and SUMO-2. However, the proportions of NEDD8-, NUB1- and SUMO-1-positive inclusions were significantly higher in neurons than in glial cells (P < 0.05). These findings suggest that proteins related to ubiquitination and proteasomal degradation are involved in the formation of INI in INIBD.

Status epilepticus associated with extensive axonal swelling in the unilateral cerebral cortex and hippocampus

Abstract: Axonal swelling can appear in the cerebral white matter, thalamus and brainstem in dying patients after status epilepticus (SE), in most of whom there is evidence of increased intracranial pressure [1,2]. However, axonal swelling has previously been reported in the unilateral thalamus in a case of hemiconvulsion-hemiplegia, suggesting that thalamic dysfunction disrupts the thalamocortical circuit [1]. Recently, we encountered a woman who presented with prolonged SE for 7 days and died on the 24th day after seizure onset. Post-mortem examination revealed distinct cerebral lesions, compatible with the histological changes seen in patients with SE. Moreover, a large number of axonal swellings were also evident in these lesions. To our knowledge, this is the first demonstration of extensive axonal swelling associated with SE. A 72-year-old woman, without a history of convulsion or epilepsy, complained of appetite loss and general fatigue. On the following day (Day 1), she suffered a generalized tonic-clonic convulsion. At the time of hospitalization, she showed SE. Electroencephalogram (EEG) showed epileptiform discharges predominantly in the left hemisphere. A cranial computed tomography (CT) scan demonstrated no abnormality. Despite intravenous administration of diazepam, the seizures could not be controlled. On Day 7, she was transferred to the Department of Neurology in a comatose state (Glasgow Coma Scale score, 3). EEG showed periodic lateralized epileptiform discharges in the left occipito-temporal region. She was intubated and placed on a respirator. Intravenous administration of midazolam and oral administration of sodium valproate and clobazam were started, and the seizures were relieved on Day 7. On Day 9, magnetic resonance imaging showed hyperintense lesions in the left parietal, medial temporal and insular cortices. Since limbic encephalitis was suspected, steroid pulse therapy was administered from Day 12 to Day 14. On Day 14, the patient regained consciousness (Glasgow Coma Scale score, 11). She then received steroid pulse therapy again from Day 18 to Day 20. On Day 20, she was extubated and was able to respond to verbal commands (Glasgow Coma Scale score, 14). On Day 21, EEG showed generalized slow waves (2 Hz) and spikes in the left hemisphere, and on Day 24 she died suddenly. General autopsy was performed 2 h after death, but the cause of death could not be confirmed. The slightly atrophic brain weighed 1028 g. Coronal hemisphere sections were taken at the levels of the frontal lobe, basal ganglia, thalamus and occipital lobe, bilaterally. The midbrain, pons, medulla oblongata and cerebellum were also sampled. Histological examination revealed severe neuronal loss with reactive astrocytosis and macrophage infiltration in the CA1, CA3 and CA4 regions of the left hippocampus (Figure 1a,b), which was consistent with the distribution pattern of neuronal damage observed in patients with SE [3]. Red neurones were also scattered in this area. In addition, cortical laminar necrosis with many red neurones, one of the features of prolonged focal SE [4], was found in the left parietal lobe (Figure 1c,d). Similar lesions were also found in the medial temporal and occipital cortices on the left side. Interestingly, the unilateral distribution of cerebral cortical lesions was quite similar to that of cortical laminar necrosis in two patients who presented with prolonged focal SE [4]. Furthermore, a large number of axonal swellings (axonal bulbs) were found in these lesions. Although swollen axons were widely distributed in the cortical and subcortical regions including the thalamus, nucleus basalis of Meynert and cerebellar dentate nucleus (Figure 1e–h), they were most numerous in the stratum oriens of the left hippocampus, corresponding to the proximal axons of hippocampal pyramidal neurones. No axonal swellings were present in the cerebral white matter. Axonal bulbs were positive for phosphorylated and non-phosphorylated neurofilament, but the majority were negative for b-amyloid precursor protein (bAPP) (Figure 1i–k). Figure 2 shows the distribution and severity of neuronal loss and axonal swellings, predominantly in the left hemisphere, in the present case, indicating that the changes were secondary to epileptogenic neuronal damage and different from global hypoxia or hypoglycaemia. There was no evidence of neurodegenerative disease.

Abnormal tau deposition in neurons, but not in glial cells in the cerebral tissue surrounding arteriovenous malformation.

Abstract: We report an autopsy case of arteriovenous malformation (AVM) of the right frontal lobe in a 50-year-old man, in whom post mortem examination revealed massive tau deposition in the affected cerebral cortex. The patient was diagnosed as having AVM at the age of 21 years, and died of unknown cause at the age of 50 years. Immunostaining with anti-phosphorylated tau antibody (AT8) revealed many NFTs and neuropil threads, but not glial tau accumulation, in the right frontal cortex surrounding the AVM. The NFTs and neuropil threads contained both 3-repeat and 4-repeat tau. Ultrastructurally, the NFTs consisted of paired helical filaments. In the other brain areas, a few NFTs were found in the parahippocampal gyrus. There was no amyloid deposition in the brain. A variety of disease conditions, including brain tumor, viral encephalitis, angioma and cervical spondylotic myelopathy, have been reported to show Alzheimer-type NFTs. The present findings indicate that abnormal tau deposition can occur in neurons, but not in glial cells, of the affected cerebral cortex surrounding AVM.