Tsuyoshi Ishida

Aberrant microRNA expression in the brains of neurodegenerative diseases: miR-29a decreased in Alzheimer disease brains targets neurone navigator 3.

M. Shioya, S. Obayashi, H. Tabunoki, K. Arima, Y. Saito, T. Ishida and J. Satoh (2010) Neuropathology and Applied Neurobiology36, 320–330u2028Aberrant microRNA expression in the brains of neurodegenerative diseases: miR‐29a decreased in Alzheimer disease brains targets neurone navigator 3

TMEM119 marks a subset of microglia in the human brain

Microglia are resident myeloid cells of the central nervous system (CNS), activated in the brains of various neurological diseases. Microglia are ontogenetically and functionally distinct from monocyte‐derived macrophages that infiltrate the CNS under pathological conditions. However, a lack of specific markers that distinguish resident microglia from circulating blood‐derived macrophages in human brain tissues hampers accurate evaluation of microglial contributions to the human brain pathology. By comparative analysis of five comprehensive microglial transcriptome datasets, we identified an evolutionarily conserved protein TMEM119 as the most promising candidate for human microglial markers. TMEM119 was expressed on immortalized human microglia, in which the expression levels were not elevated by exposure to lipopolysaccharide, IFNγ, IL‐4, IL‐13 or TGFβ1. Notably, TMEM119 immunoreactivity was expressed exclusively on a subset of Iba1+ CD68+ microglia with ramified and amoeboid morphologies in the brains of neurodegenerative diseases, such as Alzheimers disease (AD), whereas Iba1+ CD68+ infiltrating macrophages do not express TMEM119 in demyelinating lesions of multiple sclerosis and necrotic lesions of cerebral infarction. TMEM119 mRNA levels were elevated in AD brains, although the protein levels were not significantly different between AD and non‐AD cases by western blot and morphometric analyses. TMEM119‐positive microglia did not consistently express polarized markers for M1 (CD80) or M2 (CD163, CD209) in AD brains. These results suggest that TMEM119 serves as a reliable microglial marker that discriminates resident microglia from blood‐derived macrophages in the human brain.

Immunohistochemical characterization of microglia in Nasu-Hakola disease brains

Nasu‐Hakola disease (NHD) is a rare autosomal recessive disorder, characterized by progressive presenile dementia and formation of multifocal bone cysts, caused by genetic mutations of DNAX‐activation protein 12 (DAP12) or triggering receptor expressed on myeloid cells 2 (TREM2). TREM2 and DAP12 constitute a receptor/adapter signaling complex expressed on osteoclasts, dendritic cells (DC), macrophages and microglia. Previous studies using knockout mice and mouse brain cell cultures suggest that a loss‐of‐function of DAP12/TREM2 in microglia plays a central role in the neuropathological manifestation of NHD. However, there exist no immunohistochemical studies that focus attention on microglia in NHD brains. To elucidate a role of microglia in the pathogenesis of NHD, we searched NHD‐specific biomarkers and characterized their expression on microglia in NHD brains. Here, we identified allograft inflammatory factor 1 (AIF1, Iba1) and sialic acid binding Ig‐like lectin 1 (SIGLEC1) as putative NHD‐specific biomarkers by bioinformatics analysis of microarray data of NHD DC. We studied three NHD and eight control brains by immunohistochemistry with a panel of 16 antibodies, including those against Iba1 and SIGLEC1. We verified the absence of DAP12 expression in NHD brains and the expression of DAP12 immunoreactivity on ramified microglia in control brains. Unexpectedly, TREM2 was not expressed on microglia but expressed on a small subset of intravascular monocytes/macrophages in control and NHD brains. In the cortex of NHD brains, we identified accumulation of numerous Iba1‐positive microglia to an extent similar to control brains, while SIGLEC1 was undetectable on microglia in all the brains examined. These observations indicate that human microglia in brain tissues do not express TREM2 and DAP12‐deficient microglia are preserved in NHD brains, suggesting that the loss of DAP2/TREM2 function in microglia might not be primarily responsible for the neuropathological phenotype of NHD.

PLD3 is accumulated on neuritic plaques in Alzheimer's disease brains

IntroductionRecently, a whole-exome sequencing (WES) study showed that a rare variant rs145999145 composed of p.Val232Met located in exon 7 of the phospholipase D3 (PLD3) gene confers a doubled risk for late-onset Alzheimer’s disease (AD). Knockdown of PLD3 elevates the levels of extracellular amyloid-beta (Aβ), suggesting that PLD3 acts as a negative regulator of Aβ precursor protein (APP) processing. However, the precise cellular location and distribution of PLD3 in AD brains remain largely unknown.MethodsBy quantitative RT-PCR (qPCR), western blot, immunohistochemistry, and bioinformatics analysis, we studied PLD3 expression patterns and levels in a series of AD and control brains, including amyotrophic lateral sclerosis, Parkinson’s disease, multiple system atrophy, and non-neurological cases.ResultsThe levels of PLD3 mRNA and protein expression were reduced modestly in AD brains, compared with those in non-AD brains. In all brains, PLD3 was expressed constitutively in cortical neurons, hippocampal pyramidal and granular neurons but not in glial cells. Notably, PLD3 immunoreactivity was accumulated on neuritic plaques in AD brains. We identified the human granulin (GRN) gene encoding progranulin (PRGN) as one of most significant genes coexpressed with PLD3 by bioinformatics database search. PLD3 was actually coexpressed and interacted with PGRN both in cultured cells in vitro and in AD brains in vivo.ConclusionsWe identified an intense accumulation of PLD3 on neuritic plaques coexpressed with PGRN in AD brains, suggesting that PLD3 plays a key role in the pathological processes of AD.

Dystrophic neurites express C9orf72 in Alzheimer's disease brains

IntroductionChromosome 9 open reading frame 72 (C9orf72) is an evolutionarily conserved protein with unknown function, expressed at high levels in the brain. An expanded hexanucleotide GGGGCC repeat located in the first intron of the C9orf72 gene represents the most common genetic cause of familial frontotemporal dementia (FTD) and amyotrophic lateral sclerosis (ALS). Previous studies by immunohistochemistry with two different anti-C9orf72 antibodies named sc-138763 and HPA023873 showed that C9orf72 is expressed chiefly in the cytoplasm of neurons, and is concentrated in the synaptic terminals in the brains of FTD/ALS with or without C9orf72 repeat expansion as well as those of controls. At present, a pathological role of C9orf72 in the process of neurodegeneration remains unknown.MethodsUsing immunohistochemistry we studied C9orf72 expression in the frontal cortex and the hippocampus of six Alzheimers disease (AD) and 13 control cases, including ALS, Parkinsons disease, multiple system atrophy, and non-neurological cases.ResultsThe HPA023873 antibody showed a cross-reactivity to glial fibrillary acidic protein, and therefore stained intensely reactive astrocytes in AD and non-AD brains. Both sc-138763 and HPA023873 antibodies labeled the neuronal cytoplasm and the neuropil with variable intensities, and intensely stained a cluster of p62-negative, UBQLN1-positive swollen neurites, which were distributed in the CA1 region and the molecular layer in the hippocampus of both AD and non-AD brains. Most notably, both of these antibodies reacted strongly with dystrophic neurites accumulated on senile plaques in AD brains.ConclusionThese results suggest a general role of C9orf72 in the process of neurodegeneration in a range of human neurodegenerative diseases.

Accumulation of a repulsive axonal guidance molecule RGMa in amyloid plaques: a possible hallmark of regenerative failure in Alzheimer's disease brains.

J. Satoh, H. Tabunoki, T. Ishida, Y. Saito and K. Arima (2013) Neuropathology and Applied Neurobiology39, 109–120

Immunohistochemical characterization of γ-secretase activating protein expression in Alzheimer's disease brains

J. Satoh, H. Tabunoki, T. Ishida, Y. Saito and K. Arima (2012) Neuropathology and Applied Neurobiology38, 132–141

Phosphorylated Syk expression is enhanced in Nasu‐Hakola disease brains

Nasu‐Hakola disease (NHD) is a rare autosomal recessive disorder, characterized by progressive presenile dementia and formation of multifocal bone cysts, caused by a loss‐of‐function mutation of DNAX‐activation protein 12 (DAP12) or triggering receptor expressed on myeloid cells 2 (TREM2). TREM2 and DAP12 constitute a receptor/adaptor complex on myeloid cells. The post‐receptor signals are transmitted via rapid phosphorylation of the immunoreceptor tyrosine‐based activating motif (ITAM) of DAP12, mediated by Src protein tyrosine kinases, followed by binding of phosphorylated ITAM to Src homology 2 (SH2) domains of spleen tyrosine kinase (Syk), resulting in autophosphorylation of the activation loop of Syk. To elucidate the molecular mechanism underlying the pathogenesis of NHD, we investigated Syk expression and activation in the frontal cortex and the hippocampus of three NHD and eight control brains by immunohistochemistry. In NHD brains, the majority of neurons expressed intense immunoreactivities for Syk and Y525/Y526‐phosphorylated Syk (pSyk) chiefly located in the cytoplasm, while more limited populations of neurons expressed Src. The levels of pSyk expression were elevated significantly in NHD brains compared with control brains. In both NHD and control brains, substantial populations of microglia and macrophages expressed pSyk, while the great majority of reactive astrocytes and myelinating oligodendrocytes did not express pSyk, Syk or Src. These observations indicate that neuronal expression of pSyk was greatly enhanced in the cerebral cortex and the hippocampus of NHD brains, possibly via non‐TREM2/DAP12 signaling pathways involved in Syk activation.

LC3, an autophagosome marker, is expressed on oligodendrocytes in Nasu-Hakola disease brains.

BackgroundNasu-Hakola disease (NHD) is a rare autosomal recessive disorder characterized by sclerosing leukoencephalopathy and multifocal bone cysts, caused by a loss-of-function mutation of either DAP12 or TREM2. TREM2 and DAP12 constitute a receptor/adaptor signaling complex expressed exclusively on osteoclasts, dendritic cells, macrophages, and microglia. Neuropathologically, NHD exhibits profound loss of myelin and accumulation of axonal spheroids, accompanied by intense gliosis accentuated in the white matter of the frontal and temporal lobes. At present, the molecular mechanism responsible for development of leukoencephalopathy in NHD brains remains totally unknown.MethodsBy immunohistochemistry, we studied the expression of microtubule-associated protein 1 light chain 3 (LC3), an autophagosome marker, in 5 NHD and 12 control brains.ResultsIn all NHD brains, Nogo-A-positive, CNPase-positive oligodendrocytes surviving in the non-demyelinated white matter intensely expressed LC3. They also expressed ubiquitin, ubiquilin-1, and histone deacetylase 6 (HDAC6) but did not express Beclin 1 or sequestosome 1 (p62). Substantial numbers of axonal spheroids were also labeled with LC3 in NHD brains. In contrast, none of oligodendrocytes expressed LC3 in control brains. Furthermore, surviving oligodendrocytes located at the demyelinated lesion edge of multiple sclerosis (MS) did not express LC3, whereas infiltrating Iba1-positive macrophages and microglia intensely expressed LC3 in MS lesions.ConclusionsThese results propose a novel hypothesis that aberrant regulation of autophagy might induce oligodendrogliopathy causative of leukoencephalopathy in NHD brains.

Ubiquilin-1 immunoreactivity is concentrated on Hirano bodies and dystrophic neurites in Alzheimer's disease brains.

Ubiquilin‐1 acts as an adaptor protein that mediates the translocation of polyubiquitinated proteins to the proteasome for degradation. Although previous studies suggested a key role of ubiquilin‐1 in the pathogenesis of Alzheimers disease (AD), a direct relationship between ubiquilin‐1 and Hirano bodies in AD brains remains unknown.