RESUMO
ABCC9 genetic polymorphisms are associated with increased risk for various human diseases including hippocampal sclerosis of aging. The main goals of this study were 1 > to detect the ABCC9 variants and define the specific 3' untranslated region (3'UTR) for each variant in human brain, and 2 > to determine whether a polymorphism (rs704180) associated with risk for hippocampal sclerosis of aging pathology is also associated with variation in ABCC9 transcript expression and/or splicing. Rapid amplification of ABCC9 cDNA ends (3'RACE) provided evidence of novel 3' UTR portions of ABCC9 in human brain. In silico and experimental studies were performed focusing on the single nucleotide polymorphism, rs704180. Analyses from multiple databases, focusing on rs704180 only, indicated that this risk allele is a local expression quantitative trait locus (eQTL). Analyses of RNA from human brains showed increased ABCC9 transcript levels in individuals with the risk genotype, corresponding with enrichment for a shorter 3' UTR which may be more stable than variants with the longer 3' UTR. MicroRNA transfection experiments yielded results compatible with the hypothesis that miR-30c causes down-regulation of SUR2 transcripts with the longer 3' UTR. Thus we report evidence of complex ABCC9 genetic regulation in brain, which may be of direct relevance to human disease. ABCC9 gene variants are associated with increased risk for hippocampal sclerosis of aging (HS-Aging--a prevalent brain disease with symptoms that mimic Alzheimer's disease). We describe novel ABCC9 variants in human brain, corresponding to altered 3'UTR length, which could lead to targeting by miR-30c. We also determined that the HS-Aging risk mutation is associated with variation in ABCC9 transcript expression.
Assuntos
Envelhecimento/patologia , Encefalopatias/genética , Hipocampo/patologia , Doenças Neurodegenerativas/genética , Receptores de Sulfonilureias/genética , Idoso de 80 Anos ou mais , Encefalopatias/patologia , Feminino , Regulação da Expressão Gênica/genética , Predisposição Genética para Doença , Humanos , Masculino , MicroRNAs/genética , Doenças Neurodegenerativas/patologia , Reação em Cadeia da Polimerase , Polimorfismo de Nucleotídeo Único , Locos de Características Quantitativas , Esclerose/genética , Esclerose/patologiaRESUMO
Granulin (GRN, or progranulin) is a protein involved in wound repair, inflammation, and neoplasia. GRN has also been directly implicated in frontotemporal dementia and may contribute to Alzheimer's disease pathogenesis. However, GRN regulation expression is poorly understood. A high-throughput experimental microRNA assay showed that GRN is the strongest target for miR-107 in human H4 neuroglioma cells. miR-107 has been implicated in Alzheimer's disease pathogenesis, and sequence elements in the open reading frame-rather than the 3' untranslated region-of GRN mRNA are recognized by miR-107 and are highly conserved among vertebrate species. To better understand the mechanism of this interaction, FLAG-tagged Argonaute constructs were used following miR-107 transfection. GRN mRNA interacts preferentially with Argonaute 2. In vitro and in vivo studies indicate that regulation of GRN by miR-107 may be functionally important. Glucose supplementation in cultured cells that leads to increased miR-107 levels also results in decreased GRN expression, including changes in cell compartmentation and decreased secretion of GRN protein. This effect was eliminated following miR-107 transfection. We also tested a mouse model where miR-107 has been shown to be down-regulated. In brain tissue subjacent to 1.0 mm depth controlled cortical impact, surviving hippocampal neurons show decreased miR-107 with augmentation of neuronal GRN expression. These findings indicate that miR-107 contributes to GRN expression regulation with implications for brain disorders.
Assuntos
Lesões Encefálicas/metabolismo , Peptídeos e Proteínas de Sinalização Intercelular/metabolismo , MicroRNAs/metabolismo , Doenças Neurodegenerativas/metabolismo , Animais , Proteínas Argonautas , Sequência de Bases , Encéfalo/anatomia & histologia , Encéfalo/metabolismo , Encéfalo/patologia , Lesões Encefálicas/genética , Lesões Encefálicas/patologia , Células Cultivadas , Fatores de Iniciação em Eucariotos/genética , Fatores de Iniciação em Eucariotos/metabolismo , Glucose/metabolismo , Humanos , Peptídeos e Proteínas de Sinalização Intercelular/genética , Masculino , Camundongos , Camundongos Endogâmicos C57BL , MicroRNAs/genética , Análise em Microsséries , Doenças Neurodegenerativas/genética , Doenças Neurodegenerativas/patologia , Progranulinas , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Alinhamento de SequênciaRESUMO
MicroRNAs (miRNAs) play key roles in gene expression regulation in both healthy and disease brains. To better understand those roles, it is necessary to characterize the miRNAs that are expressed in particular cell types under a range of conditions. In situ hybridization (ISH) can demonstrate cell- and lamina-specific patterns of miRNA expression that would be lost in tissue-level expression profiling. In the present study, ISH was performed with special focus on the human entorhinal cortex (EC) and transentorhinal cortex (TEC). The TEC is the area of the cerebral cortex that first develops neurofibrillary tangles in Alzheimer's disease (AD). However, the reason for TEC's special vulnerability to AD-type pathology is unknown. MiRNA ISH was performed on three human brains with well-characterized clinical and pathological parameters. Locked nucleic acid ISH probes were used referent to miR-107, miR-124, miR-125b, and miR-320. In order to correlate the ISH data with AD pathology, the ISH staining was compared with near-adjacent slides processed using Thioflavine stains. Not all neurons or cortical lamina stain with equal intensity for individual miRNAs. As with other areas of brain, the TEC and EC have characteristic miRNA expression patterns. MiRNA ISH is among the first methods to show special staining characteristics of cells and laminae of the human TEC.