Saori Odagiri

The Lewy Body in Parkinson’s Disease and Related Neurodegenerative Disorders

The histopathological hallmark of Parkinson’s disease (PD) is the presence of fibrillar aggregates referred to as Lewy bodies (LBs), in which α-synuclein is a major constituent. Pale bodies, the precursors of LBs, may serve the material for that LBs continue to expand. LBs consist of a heterogeneous mixture of more than 90 molecules, including PD-linked gene products (α-synuclein, DJ-1, LRRK2, parkin, and PINK-1), mitochondria-related proteins, and molecules implicated in the ubiquitin–proteasome system, autophagy, and aggresome formation. LB formation has been considered to be a marker for neuronal degeneration because neuronal loss is found in the predilection sites for LBs. However, recent studies have indicated that nonfibrillar α-synuclein is cytotoxic and that fibrillar aggregates of α-synuclein (LBs and pale bodies) may represent a cytoprotective mechanism in PD.

Keap1 Is Localized in Neuronal and Glial Cytoplasmic Inclusions in Various Neurodegenerative Diseases

Abstract Oxidative stress has been proposed as a potential mechanism for neurodegenerative diseases, such as Alzheimer disease (AD), Parkinson disease (PD), and amyotrophic lateral sclerosis (ALS). In response to oxidative stress, the levels of numerous cytoprotectiveproducts are increased via alteration of the Kelch-like ECH-associatedprotein 1 (Keap1) and NF-E2–related factor 2 (Nrf2) system. One of the Nrf2 targets, p62, has been known to be incorporated into a wide spectrum of cytoplasmic inclusions in neurodegenerative diseases and interact with Keap1. However, it remains unclear whether Keap1 is associated with the pathogenesis of neurodegenerative diseases. In this study, we investigated the relationship between p62 and Keap1 in the brains of patients with AD, PD, dementia with Lewy bodies (DLB), and ALS. Biochemical analysesshowed that p62 and Keap1 interacted with each other in AD andDLB brains and were extracted into similar detergent-soluble and -insoluble fractions. Pathologic examination demonstrated that anti-Keap1 antibodies immunostained Lewy bodies in PD and DLB, neurofibrillary tangles in AD, and skeinlike inclusions in ALS. Further analysis showed that the levels of common Nrf2 target genes were increased in AD compared with those in controls. However, there were no statistical significances in the levels of Nrf2 target genes in DLB relative to controls. Our pathologic and biochemical results suggest a molecular basis for stress response to be involved in the formation of cytoplasmic inclusions observed in several neurodegenerative diseases.

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

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

Alteration of autophagosomal proteins in the brain of multiple system atrophy

Autophagosomal formation is an initial step for macroautophagy. Similar to the yeast autophagy-related gene 8 (ATG8), mammalian ATG8 is responsible for autophagosomal formation, and categorized into LC3 and GABARAPs/GATE-16. Recent studies have shown that impairment of the autophagy-lysosome system is associated with formation of cytoplasmic inclusions observed in various neurodegenerative disorders including Parkinsons disease (PD) and dementia with Lewy bodies (DLB). Although abnormal α-synuclein accumulation is a cardinal neuropathological feature in PD, DLB and multiple system atrophy (MSA), it is unclear whether autophagy is altered in MSA. We here demonstrated that the level of matured GABARAPs was significantly decreased in the cerebellum of MSA relative to controls, and that the higher levels of matured and lipidated LC3 were detected in detergent-insoluble fraction of MSA. Immunohistochemical analysis showed that the vast majority of glial cytoplasmic inclusions, a hallmark of MSA, were positive for LC3, whereas they were unstained or barely stained with anti-GABARAPs or anti-GATE-16 antibodies. Our data suggest that autophagy maturation is impaired through the repressed levels of autophagosomal proteins in MSA.

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

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.

Brain expression level and activity of HDAC6 protein in neurodegenerative dementia.

Histone deacetylase 6 (HDAC6) is a multifunctional cytoplasmic protein that plays an especially critical role in the formation of aggresomes, where aggregates of excess protein are deposited. Previous immunohistochemical studies have shown that HDAC6 accumulates in Lewy bodies in Parkinsons disease and dementia with Lewy bodies (DLB) as well as in glial cytoplasmic inclusions in multiple system atrophy (MSA). However, it is uncertain whether the level and activity of HDAC6 are altered in the brains of patients with neurodegenerative dementia. In the present study, we demonstrated that the level of HDAC6 was not altered in the temporal cortex of patients with Alzheimers disease and DLB in comparison with controls. In contrast, the level of HDAC6 was significantly increased in the temporal cortex of patients with frontotemporal lobar degeneration with TDP-43 inclusions (FTLD-TDP) and in the cerebellar white matter of patients with MSA. However, the level of acetylated α-tubulin, one of the substrates of HDAC6, was not altered in FTLD-TDP and MSA relative to controls. These findings suggest that the induced level of HDAC6 in the brain is insufficient for manifestation of its activity in FTLD-TDP and MSA.

Autophagy-related proteins (p62, NBR1 and LC3) in intranuclear inclusions in neurodegenerative diseases.

Incorporation of ubiquitin and ubiquitin-related proteins including p62 into neuronal intranuclear inclusions (NIIs) has been reported in a variety of neurodegenerative diseases. However, involvement of autophagy-specific proteins (NBR1 and LC3) in NIIs has not been mentioned. We immunohistochemically examined the brain of patients with Machado-Joseph disease (MJD; n=5), dentatorubral-pallidoluysian atrophy (DRPLA; n=5) and intranuclear inclusion body disease (INIBD; n=5), using antibodies against ubiquitin, p62, NBR1 and LC3. The proportion of p62-, NBR1- and LC3-positive inclusions relative to the number of ubiquitin-positive inclusions was calculated in each case. NIIs were positive for p62 in MJD (19.3%), DRPLA (49.7%) and INIBD (99.8%). As for autophagy-specific proteins, NIIs were positive for NBR1 in MJD (4.2%), DRPLA (5.5%) and INIBD (13.2%) and negative for LC3 in MJD, DRPLA and INIBD, except for one case of INIBD. These findings suggest that autophagy-lysosome pathway is not involved in the formation/degradation of NIIs.

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

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

p62 Deficiency Enhances α-Synuclein Pathology in Mice

In Lewy body disease (LBD) such as dementia with LBs and Parkinsons disease, several lines of evidence show that disrupted proteolysis occurs. p62/SQSTM1 (p62) is highly involved with intracellular proteolysis and is a component of ubiquitin‐positive inclusions in various neurodegenerative disorders. However, it is not clear whether p62 deficiency affects inclusion formation and abnormal protein accumulation. To answer this question, we used a mouse model of LBD that lacks p62, and found that LB‐like inclusions were observed in transgenic mice that overexpressed α‐synuclein (Tg mice) with or without the p62 protein. p62 deficiency enhanced α‐synuclein pathology with regard to the number of inclusions and staining intensity compared with Tg mice that expressed p62. To further investigate the molecular mechanisms associated with the loss of p62 in Tg mice, we assessed the mRNA and protein levels of several molecules, and found that the neighbor of the brca1 gene (NBr1), which is functionally and structurally similar to p62, is increased in Tg mice without p62 compared with control Tg mice. These findings suggest that p62 and NBR1 affect the pathogenesis of neurodegenerative diseases through the cooperative modulation of α‐synuclein aggregation.

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

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.