Norikazu Hara

Serum microRNA miR-501-3p as a potential biomarker related to the progression of Alzheimer’s disease

MicroRNAs (miRNAs) are attractive molecules to utilize as one of the blood-based biomarkers for neurodegenerative disorders such as Alzheimer’s disease (AD) because miRNAs are relatively stable in biofluid, including serum or plasma. To determine blood miRNA biomarkers for AD with next-generation sequencing genome-wide, we first surveyed 45 serum samples. These came from 27 AD patients and 18 controls (discovery set) that underwent autopsy within two weeks after their serum sampling and were neuropathologically diagnosed. We found that three miRNAs, hsa-miR-501-3p, hsa-let-7f-5p, and hsa-miR-26b-5p, were significantly deregulated between the AD samples and the controls. The deregulation for hsa-miR-501-3p was further confirmed by quantitative reverse transcription polymerase chain reaction (PCR) in a validation set composed of 36 clinically diagnosed AD patients and 22 age-matched cognitively normal controls with a sensitivity and specificity of 53% and 100%, respectively (area under the curve = 0.82). Serum hsa-miR-501-3p levels were downregulated in AD patients, and its lower levels significantly correlated with lower Mini-Mental State Examination scores. Contrary to its serum levels, we found that hsa-miR-501-3p was remarkably upregulated in the same donors’ AD brains obtained at autopsy from the discovery set. The hsa-miR-501-3p overexpression in cultured cells, which mimicked the hsa-miR-501-3p upregulation in the AD brains, induced significant downregulation of 128 genes that overrepresented the Gene Ontology terms, DNA replication, and the mitotic cell cycle. Our results suggest that hsa-miR-501-3p is a novel serum biomarker that presumably corresponds to pathological events occurring in AD brains.

Genes associated with the progression of neurofibrillary tangles in Alzheimer’s disease

The spreading of neurofibrillary tangles (NFTs), intraneuronal aggregates of highly phosphorylated microtubule-associated protein tau, across the human brain is correlated with the cognitive severity of Alzheimer’s disease (AD). To identify genes relevant to NFT expansion defined by the Braak stage, we conducted whole-genome exon array analysis with an exploratory sample set consisting of 213 human post-mortem brain tissue specimens from the entorinal, temporal and frontal cortices of 71 brain-donor subjects: Braak NFT stages 0 (N=13), I–II (N=20), III–IV (N=19) and V–VI (N=19). We identified eight genes, RELN, PTGS2, MYO5C, TRIL, DCHS2, GRB14, NPAS4 and PHYHD1, associated with the Braak stage. The expression levels of three genes, PHYHD1, MYO5C and GRB14, exhibited reproducible association on real-time quantitative PCR analysis. In another sample set, including control subjects (N=30), and in patients with late-onset AD (N=37), dementia with Lewy bodies (N=17) and Parkinson disease (N=36), the expression levels of two genes, PHYHD1 and MYO5C, were obviously associated with late-onset AD. Protein–protein interaction network analysis with a public database revealed that PHYHD1 interacts with MYO5C via POT1, and PHYHD1 directly interacts with amyloid beta-peptide 42. It is thus likely that functional failure of PHYHD1 and MYO5C could lead to AD development.

Genome-Wide Target Analyses of Otx2 Homeoprotein in Postnatal Cortex

Juvenile brain has a unique time window, or critical period, in which neuronal circuits are remodeled by experience. Mounting evidence indicates the importance of neuronal circuit rewiring in various neurodevelopmental disorders of human cognition. We previously showed that Otx2 homeoprotein, essential for brain formation, is recaptured during postnatal maturation of parvalbumin-positive interneurons (PV cells) to activate the critical period in mouse visual cortex. Cortical Otx2 is the only interneuron-enriched transcription factor known to regulate the critical period, but its downstream targets remain unknown. Here, we used ChIP-seq (chromatin immunoprecipitation sequencing) to identify genome-wide binding sites of Otx2 in juvenile mouse cortex, and interneuron-specific RNA-seq to explore the Otx2-dependent transcriptome. Otx2-bound genes were associated with human diseases such as schizophrenia as well as critical periods. Of these genes, expression of neuronal factors involved in transcription, signal transduction and mitochondrial function was moderately and broadly affected in Otx2-deficient interneurons. In contrast to reported binding sites in the embryo, genes encoding potassium ion transporters such as KV3.1 had juvenile cortex-specific binding sites, suggesting that Otx2 is involved in regulating fast-spiking properties during PV cell maturation. Moreover, transcripts of oxidative resistance-1 (Oxr1), whose promoter has Otx2 binding sites, were markedly downregulated in Otx2-deficient interneurons. Therefore, an important role of Otx2 may be to protect the cells from the increased oxidative stress in fast-spiking PV cells. Our results suggest that coordinated expression of Otx2 targets promotes PV cell maturation and maintains its function in neuronal plasticity and disease.

Loss of kallikrein‐related peptidase 7 exacerbates amyloid pathology in Alzheimer's disease model mice

Deposition of amyloid‐β (Aβ) as senile plaques is one of the pathological hallmarks in the brains of Alzheimers disease (AD) patients. In addition, glial activation has been found in AD brains, although the precise pathological role of astrocytes remains unclear. Here, we identified kallikrein‐related peptidase 7 (KLK7) as an astrocyte‐derived Aβ degrading enzyme. Expression of KLK7 mRNA was significantly decreased in the brains of AD patients. Ablation of Klk7 exacerbated the thioflavin S‐positive Aβ pathology in AD model mice. The expression of Klk7 was upregulated by Aβ treatment in the primary astrocyte, suggesting that Klk7 is homeostatically modulated by Aβ‐induced responses. Finally, we found that the Food and Drug Administration‐approved anti‐dementia drug memantine can increase the expression of Klk7 and Aβ degradation activity specifically in the astrocytes. These data suggest that KLK7 is an important enzyme in the degradation and clearance of deposited Aβ species by astrocytes involved in the pathogenesis of AD.

Systematic review and meta-analysis of Japanese familial Alzheimer's disease and FTDP-17

Mutations in APP, PSEN1 and PSEN2 as the genetic causes of familial Alzheimer’s disease (FAD) have been found in various ethnic populations. A substantial number of FAD pedigrees with mutations have been reported in the Japanese population; however, it remains unclear whether the genetic and clinical features of FAD in the Japanese population differ from those in other populations. To address this issue, we conducted a systematic review and meta-analysis of Japanese FAD and frontotemporal dementia with parkinsonism linked to chromosome 17 (FTDP-17) by literature search. Using this analysis, we identified 39 different PSEN1 mutations in 140 patients, 5 APP mutations in 35 patients and 16 MAPT mutations in 84 patients. There was no PSEN2 mutation among Japanese patients. The age at onset in Japanese FAD patients with PSEN1 mutations was significantly younger than that in patients with APP mutations. Kaplan–Meier analysis revealed that patients with MAPT mutations showed a shorter survival than patients with PSEN1 or APP mutations. Patients with mutations in different genes exhibit characteristic clinical presentations, suggesting that mutations in causative genes may modify the clinical presentations. By collecting and cataloging genetic and clinical information on Japanese FAD and FTDP-17, we developed an original database designated as Japanese Familial Alzheimers Disease Database, which is accessible at http://alzdb.bri.niigata-u.ac.jp/.

Enhancer variants associated with Alzheimer\'s disease affect gene expression via chromatin looping

Background: Genome-wide association studies (GWASs) have identified single-nucleotide polymorphisms (SNPs) that may be genetic factors underlying Alzheimer’s disease (AD). However, how these AD-associated SNPs (AD SNPs) contribute to the pathogenesis of this disease is poorly understood because most of them are located in non-coding regions, such as introns and intergenic regions. Previous studies reported that some disease-associated SNPs affect regulatory elements including enhancers. We hypothesized that non-coding AD SNPs are located in enhancers and affect gene expression levels via chromatin loops. Results: We examined enhancer locations that were predicted in 127 human tissues or cell types, including ten brain tissues, and identified chromatin-chromatin interactions by Hi-C experiments. We report the following findings: (1) nearly 30% of non-coding AD SNPs are located in enhancers; (2) expression quantitative trait locus (eQTL) genes affected by non-coding AD SNPs within enhancers are associated with amyloid beta clearance, synaptic transmission, and immune responses; (3) 95% of the AD SNPs located in enhancers co-localize with their eQTL genes in topologically associating domains suggesting that regulation may occur through chromatin higher-order structures; (4) rs1476679 spatially contacts the promoters of eQTL genes via CTCF-CTCF interactions; (5) the effect of other AD SNPs such as rs7364180 is likely to be, at least in part, indirect through regulation of transcription factors that in turn regulate AD associated genes. Conclusion: Our results suggest that non-coding AD SNPs may affect the function of enhancers thereby influencing the expression levels of surrounding or distant genes via chromatin loops. This result may explain how some non-coding AD SNPs contribute to AD pathogenesis.

Identification and functional characterization of novel mutations including frameshift mutation in exon 4 of CSF1R in patients with adult-onset leukoencephalopathy with axonal spheroids and pigmented glia

ObjectiveAdult-onset leukoencephalopathy with axonal spheroids and pigmented glia (ALSP) is caused by mutations in CSF1R. Pathogenic mutations in exons 12–22 including coding sequence of the tyrosine kinase domain (TKD) of CSF1R were previously identified. We aimed to identify CSF1R mutations in patients who were clinically suspected of having ALSP and to determine the pathogenicity of novel CSF1R variants.MethodsSixty-one patients who fulfilled the diagnostic criteria of ALSP were included in this study. Genetic analysis of CSF1R was performed for all the coding exons. The haploinsufficiency of CSF1R was examined for frameshift mutations by RT-PCR. Ligand-dependent autophosphorylation of CSF1R was examined in cells expressing CSF1R mutants.ResultsWe identified ten variants in CSF1R including two novel frameshift, five novel missense, and two known missense mutations as well as one known missense variant. Eight mutations were located in TKD. One frameshift mutation (p.Pro104LeufsTer8) and one missense variant (p.His362Arg) were located in the extracellular domain. RT-PCR analysis revealed that the frameshift mutation of p.Pro104LeufsTer8 caused nonsense-mediated mRNA decay. Functional assay revealed that none of the mutations within TKD showed autophosphorylation of CSF1R. The p.His362Arg variant located in the extracellular domain showed comparable autophosphorylation of CSF1R to the wild type, suggesting that this variant is not likely pathogenic.ConclusionsThe detection of the CSF1R mutation outside of the region-encoding TKD may extend the genetic spectrum of ALSP with CSF1R mutations. Mutational analysis of all the coding exons of CSF1R should be considered for patients clinically suspected of having ALSP.

MYELIN ALTERATION MAY PRECEDE NEUROPATHOLOGICAL CHANGES ASSOCIATED WITH ALZHEIMER’S DISEASE: RESULTS FROM RNA-SEQ ANALYSIS USING AUTOPSIED BRAINS

Background: The majority of Late Onset Alzheimer’s Disease (LOAD) GWAS associated SNPs are in noncoding regions of the genome, suggesting regulatory function. In addition, changes in gene expression in LOAD vs. healthy control brains have been described, and several groups reported expression quantitative trait loci (eQTLs) within LOAD associated regions. However, these disease and expression associations represent indirect links that may be attributed to other variants in high linkage disequilibrium with the associated tagging variants. Our goal was to define regulatory elements in the vicinity of the LOAD associated regions that are likely to influence the expression of genes important in LOAD etiology. Towards this goal we applied a bioinformatics approach using public databases for functional annotations. These analyses represent the first step in a global strategy to identify causal variants involved in LOAD. Methods:Genomic regions 60.5Mb surrounding LOAD GWAS-associated SNPs from the were integrated with data from The Roadmap Epigenomics Mapping Consortium for chromatin state segmentation (25-state model) to identify potential active enhancers for specific brain tissue vulnerable in LOAD. This data was aligned with CTCF transcription factor (TF) binding sites determined by ChIP-seq data from ENCODE to identify 3D chromatin structure, looping, between distal enhancer elements and promoter regions. Results:A total of 494 genes map within the defined LOAD GWAS regions. As an example, across the 1Mb region tagged by rs3865444 (CD33) we found 4 enhancer segments (brain hippocampus middle) that include CTCF ChIP-seq peaks. The CTCF signals mapped to the promoters of 4 genes (KLK6, KLK10, IGLON5 and SPACA6), suggesting that these enhancers are likely to regulate these target genes. Conclusions:We describe a valuable resource for testing the hypothesis that causal variants are positioned in regulatory elements of critical LOAD genes, and contribute directly to LOAD susceptibility by affecting gene regulation. These results will inform experimental work for direct validation of regulatory function using model systems such as isogenic iPSC-derived models generated by CRISPR/Cas9 genome editing. Our study is a foundational step in a larger strategy for progressing from GWAS association signals to target genes and specific causal variants for LOAD.

Co-existence of spastic paraplegia-30 with novel KIF1A mutation and spinocerebellar ataxia 31 with intronic expansion of BEAN and TK2 in a family

Hereditary spastic paraplegia (HSP) is a clinically and genetically heterogeneous neurodegenerative disorder, which is characterized by the spasticity of the lower limbs due to pyramidal tract dysfunction. More than 70 different genetic HSP forms have been reported [1]. Spastic paraplegia-30 (SPG30; OMIM #610357) is an autosomal recessive form of HSP and is characterized by childhood-onset, progressive spastic paraplegia [2–4]. SPG30 has been shown to be caused by a homozygous missense mutation in KIF1A [3,4]. KIF1A encodes the neuronspecific motor protein involved in anterograde axonal transport [5]. Spinocerebellar ataxia 31 (SCA31; OMIM #117210) is an autosomal dominant, adult-onset neurodegenerative disorder that exhibits predominantly pure cerebellar ataxia and is caused by an abnormal pentanucleotide repeat expansion in an intron shared by BEAN and TK2 [6]. Here we describe a patient with SPG30, who is carrying a novel homozygous KIF1A mutation and is in a Japanese family in which multiple members are affected with SCA31.

IDENTIFICATION OF SERUM MICRORNA AS A POTENTIAL BIOMARKER RELATED TO THE PROGRESSION OF ALZHEIMER’S DISEASE

P3-201 IDENTIFICATION OF SERUM MICRORNA AS A POTENTIAL BIOMARKER RELATED TO THE PROGRESSION OFALZHEIMER’S DISEASE Ryozo Kuwano, Norikazu Hara, Masataka Kikuchi, Akinori Miyashita, Hiroyuki Hatsuta, Yuko Saito, Kensaku Kasuga, Shigeo Murayama, Takeshi Ikeuchi, Asahigawaso Research Institute, Asahigawaso Medical-Welfare Center, Okayama, Japan; Brain Research Institute, NiigataUniversity, Niigata, Japan; Graduate School ofMedicine, Osaka University, Osaka, Japan; Tokyo Metropolitan Geriatric Hospital and Institute of Gerontology, Tokyo, Japan; National Center of Neurology and Psychiatry, Kodaira, Japan. Contact e-mail: ryosun@jidouin.jp