Shinji Higashi

Concurrence of TDP-43, tau and α-synuclein pathology in brains of Alzheimer's disease and dementia with Lewy bodies

TAR-DNA-binding protein 43 (TDP-43) has been identified as a major component protein of ubiquitin-positive inclusions in brains from patients with frontotemporal lobar degeneration with ubiquitin-positive inclusions (FTLD-U) and amyotrophic lateral sclerosis. To obtain the precise prevalence of TDP-43 pathology in neurodegenerative disorders, we examined brains from patients with tauopathies and synucleinopathies as well as FTLD-U using immunohistochemical analysis. Consequently, TDP-43-positive inclusions within neurons and oligodendroglia were found in brains from patients with Alzheimers disease (AD) and dementia with Lewy bodies (DLB) in addition to FTLD-U, but not with Parkinsons disease, Picks disease, progressive supranuclear palsy, corticobasal degeneration or FTDP-17. The amygdala and hippocampus that were vulnerable to tau or alpha-synuclein pathology demonstrated more severe TDP-43 pathology in AD and DLB cases than in FTLD-U cases. In contrast, in the frontal cortex and basal ganglia that were vulnerable to TDP-43 pathology in FTLD-U, TDP-43 pathology was not observed in AD and DLB cases. Thus, the neuroanatomical distribution of TDP-43 pathology in AD and DLB cases was obviously different from that in FTLD-U cases. Furthermore, a subset of TDP-43-positive inclusions co-existed with neurofibrillary tangles (NFTs) or Lewy bodies (LBs) in the same neurons. Upon double-immunofluorescent labeling analysis, TDP-43 was hardly superimposed with tau, while TDP-43 was partially superimposed with alpha-synuclein, suggesting that neither NFTs nor LBs themselves show TDP-43 immunoreactivity and that TDP-43 pathology found in this study may be related in some way to AD and LB pathology. This study will provide a more in-depth understanding of the various pathogenic pathways leading to neurodegenerative disorders.

Localization of Parkinson's disease-associated LRRK2 in normal and pathological human brain

Mutations in the LRRK2 gene cause autosomal dominant, late-onset parkinsonism, which presents with pleomorphic pathology including alpha-synucleopathy. To promote our understanding of the biological role of LRRK2 in the brain we examined the distribution of LRRK2 mRNA and protein in postmortem human brain tissue from normal and neuropathological subjects. In situ hybridization and immunohistochemical analysis demonstrate the expression and localization of LRRK2 to various neuronal populations in brain regions implicated in Parkinsons disease (PD) including the cerebral cortex, caudate-putamen and substantia nigra pars compacta. Immunofluorescent double labeling studies additionally reveal the prominent localization of LRRK2 to cholinergic-, calretinin- and GABA(B) receptor 1-positive, dopamine-innervated, neuronal subtypes in the caudate-putamen. The distribution of LRRK2 in brain tissue from sporadic PD and dementia with Lewy bodies (DLB) subjects was also examined. In PD brains, LRRK2 immunoreactivity localized to nigral neuronal processes is dramatically reduced which reflects the disease-associated loss of dopaminergic neurons in this region. However, surviving nigral neurons occasionally exhibit LRRK2 immunostaining of the halo structure of Lewy bodies. Moreover, LRRK2 immunoreactivity is not associated with Lewy neurites or with cortical Lewy bodies in sporadic PD and DLB brains. These observations indicate that LRRK2 is not a primary component of Lewy bodies and does not co-localize with mature fibrillar alpha-synuclein to a significant extent. The localization of LRRK2 to key neuronal populations throughout the nigrostriatal dopaminergic pathway is consistent with the involvement of LRRK2 in the molecular pathogenesis of familial and sporadic parkinsonism.

Expression and localization of Parkinson's disease-associated leucine-rich repeat kinase 2 in the mouse brain

Mutations in the gene encoding leucine‐rich repeat kinase 2 (LRRK2) have been identified as the cause of familial Parkinsons disease (PD) at the PARK8 locus. To begin to understand the physiological role of LRRK2 and its involvement in PD, we have investigated the distribution of LRRK2 mRNA and protein in the adult mouse brain. In situ hybridization studies indicate sites of mRNA expression throughout the mouse brain, with highest levels of expression detected in forebrain regions, including the cerebral cortex and striatum, intermediate levels observed in the hippocampus and cerebellum, and low levels in the thalamus, hypothalamus and substantia nigra. Immunohistochemical studies demonstrate localization of LRRK2 protein to neurones in the cerebral cortex and striatum, and to a variety of interneuronal subtypes in these regions. Furthermore, expression of LRRK2 mRNA in the striatum of VMAT2‐deficient mice is unaltered relative to wild‐type littermate controls despite extensive dopamine depletion in this mouse model of parkinsonism. Collectively, our results demonstrate that LRRK2 is present in anatomical brain regions of direct relevance to the pathogenesis of PD, including the nigrostriatal dopaminergic pathway, in addition to other regions unrelated to PD pathology, and is likely to play an important role in the normal function of telencephalic forebrain neurones and other neuronal populations.

Abnormal Localization of Leucine-Rich Repeat Kinase 2 to the Endosomal-Lysosomal Compartment in Lewy Body Disease

Missense mutations in the leucine-rich repeat kinase 2 (LRRK2) gene are the most common causes of both familial and sporadic forms of Parkinson disease and are also associated with diverse pathological alterations. The mechanisms whereby LRRK2 mutations cause these pathological phenotypes are unknown. We used immunohistochemistry with 3 distinct anti-LRRK2 antibodies to characterize the expression of LRRK2 in the brains of 21 subjects with various neurodegenerative disorders and 7 controls. The immunoreactivity of LRRK2 was localized in a subset of brainstem-type Lewy bodies (LBs) but not in cortical-type LBs, tau-positive inclusions, or TAR-DNA-binding protein-43-positive inclusions. The immunoreactivity of LRRK2 frequently appeared as enlarged granules or vacuoles within neurons of affected brain regions, including the substantia nigra, amygdala, and entorhinal cortex in patients with Parkinson disease or dementia with LBs. The volumes of LRRK2-positive granular structures in neurons of the entorhinal cortex were significantly increased in dementia with LBs brains compared with age-matched control brains (p < 0.05). Double immunolabeling demonstrated that these LRRK2-positive granular structures frequently colocalized with the late-endosomal marker Rab7B and occasionally with the lysosomal marker, the lysosomal-associated membrane protein 2. These results suggest that LRRK2 normally localizes to the endosomal-lysosomal compartment within morphologically altered neurons in neurodegenerative diseases, particularly in the brains of patients with LB diseases.

A novel 4EHP-GIGYF2 translational repressor complex is essential for mammalian development

ABSTRACT The binding of the eukaryotic initiation factor 4E (eIF4E) to the mRNA 5′ cap structure is a rate-limiting step in mRNA translation initiation. eIF4E promotes ribosome recruitment to the mRNA. In Drosophila, the eIF4E homologous protein (d4EHP) forms a complex with binding partners to suppress the translation of distinct mRNAs by competing with eIF4E for binding the 5′ cap structure. This repression mechanism is essential for the asymmetric distribution of proteins and normal embryonic development in Drosophila. In contrast, the physiological role of the mammalian 4EHP (m4EHP) was not known. In this study, we have identified the Grb10-interacting GYF protein 2 (GIGYF2) and the zinc finger protein 598 (ZNF598) as components of the m4EHP complex. GIGYF2 directly interacts with m4EHP, and this interaction is required for stabilization of both proteins. Disruption of the m4EHP-GIGYF2 complex leads to increased translation and perinatal lethality in mice. We propose a model by which the m4EHP-GIGYF2 complex represses translation of a subset of mRNAs during embryonic development, as was previously reported for d4EHP.

Genetic Association between SORL1 Polymorphisms and Alzheimer’s Disease in a Japanese Population

Background/Aims: It has recently been shown that the neuronal sortilin-related receptor (SORL1) plays an important role in the pathogenesis of Alzheimer’s disease (AD). Methods: To investigate whether variations around the SORL1 gene are associated with AD, 7 single-nucleotide polymorphisms (SNPs) were genotyped using TaqMan® technology with 180 AD patients and 130 age-matched controls. Results: Our results confirmed the strong linkage disequilibrium among the 7 SNPs studied. However, our study failed to detect any association between the SNPs and AD. We could not confirm any synergetic interaction between the SNPs and apolipoprotein E in our AD patients either. Conclusion: Further genetic studies are needed to clarify the relationship between the SORL1 gene and AD.

Distribution of cerebral amyloid deposition and its relevance to clinical phenotype in Lewy body dementia.

Parkinsons disease dementia (PDD) and dementia with Lewy bodies (DLB) are clinically distinguished based only on the duration of parkinsonism prior to dementia. It is known that there is considerable pathological overlap between these two conditions, but the pathological difference between them remains unknown. We evaluated Alzheimer-type pathology in 30 brains of patients with Lewy body dementia using standardized methods based on those of the Brain-Net Europe (BNE) Consortium. Only 2 of 13 PDD cases (15%) showed Aβ-immunoreactive pathology in the midbrain (amyloid phase IV). In contrast, 12 of 17 DLB cases (71%) exhibited midbrain involvement. Four of the DLB cases (24%) but none of the PDD cases exhibited Aβ-immunoreactive pathology in the cerebellum (amyloid phase V). The ratio of cases with subtentorial involvement of amyloid deposition was significantly higher in DLB than in PDD. The median of amyloid phases was significantly greater in DLB than in PDD, but there was no difference in neurofibrillary tangle (NFT) Braak stages or in Lewy body scores. When patients were classified according to whether dementia or parkinsonism had occurred first, the rate of dementia having occurred first was significantly greater in amyloid phase IV and V than in phase 0-I, with phase III in the middle, though there was no significant difference in median NFT Braak stage or mean Lewy body score associated with amyloid phase. These results suggest that amyloid deposition may contribute to the timing of the onset of dementia relative to that of parkinsonism in Lewy body dementia.

Localization of MAP1-LC3 in Vulnerable Neurons and Lewy Bodies in Brains of Patients With Dementia With Lewy Bodies

There is emerging evidence implicating a role for the autophagy-lysosome pathway in the pathogenesis of Lewy body disease. We investigated potential neuropathologic and biochemical alterations of autophagy-lysosome pathway-related proteins in the brains of patients with dementia with Lewy bodies (DLB), Alzheimer disease (AD), and control subjects using antibodies against Ras-related protein Rab-7B (Rab7B), lysosomal-associated membrane protein 2 (LAMP2), and microtubule-associated protein 1A/1B light chain 3 (LC3). In DLB, but not in control brains, there were large Rab7B-immunoreactive endosomal granules. LC3 immunoreactivity was increased in vulnerable areas of DLB brains relative to that in control brains; computerized cell counting analysis revealed that LC3 levels were greater in the entorhinal cortex and amygdala of DLB brains than in controls. Rab7B levels were increased, and LAMP2 levels were decreased in the entorhinal cortex of DLB brains. In contrast, only a decrease in LAMP2 levels versus controls was found in AD brains. LC3 widely colocalized with several types of Lewy pathology; LAMP2 localized to the periphery or outside of brainstem-type Lewy bodies; Rab7B did not colocalize with Lewy pathology. Immunoblot analysis demonstrated specific accumulation of the autophagosomal LC3-II isoform in detergent-insoluble fractions from DLB brains. These results support apotential role for the autophagy-lysosome pathway in the pathogenesis of DLB.

Diffuse occipital hypometabolism on [18F]‐FDG PET scans in patients with idiopathic REM sleep behavior disorder: Prodromal dementia with Lewy bodies?

Background:  Previous longitudinal studies have revealed that specific patterns on [18F]‐fluoro‐d‐glucose (FDG) positron emission tomography (PET) scans in patients with amnesic mild cognitive impairment can predict Alzheimers disease (AD). However, the significance of particular patterns on [18F]‐FDG PET scans in prodromal patients with dementia with Lewy bodies (DLB) remains unclear.

TDP-43 associates with stalled ribosomes and contributes to cell survival during cellular stress.

TAR DNA‐binding protein 43 (TDP‐43) has emerged as an important contributor to amyotrophic lateral sclerosis and frontotemporal lobar degeneration. To understand the physiological roles of TDP‐43 in the complex translational regulation mechanisms, we exposed cultured cells to oxidative stress induced by sodium arsenite (ARS) for different periods of time, leading to non‐lethal or sublethal injury. Polysome profile analysis revealed that ARS‐induced stress caused the association of TDP‐43 with stalled ribosomes via binding to mRNA, which was not found under the steady‐state condition. When the cells were exposed to short‐term/non‐lethal stress, TDP‐43 associating with ribosomes localized to stress granules (SGs); this association was transient because it was immediately dissolved by the removal of the stress. In contrast, when the cells were exposed to long‐term/sublethal stress, TDP‐43 was excluded from SGs and shifted to the heavy fractions independent of any binding to mRNA. In these severely stressed cells, biochemical alterations of TDP‐43, such as increased insolubility and disulfide bond formation, were irreversible. TDP‐43 was finally phosphorylated via the ARS‐induced c‐jun N‐terminal kinase pathway. In TDP‐43‐silenced cells, stalled mRNA and poly (A)+ RNA stability was disturbed and cytotoxicity increased under sublethal stress. Thus, TDP‐43 associates with stalled ribosomes and contributes to cell survival during cellular stress.