RESUMEN
Hippocampal CA1 pyramidal neurons, a major component of the medial temporal lobe memory circuit, are selectively vulnerable during the progression of Alzheimer's disease (AD). The cellular mechanism(s) underlying degeneration of these neurons and the relationship to cognitive performance remains largely undefined. Here, we profiled neurotrophin and neurotrophin receptor gene expression within microdissected CA1 neurons along with regional hippocampal dissections from subjects who died with a clinical diagnosis of no cognitive impairment (NCI), mild cognitive impairment (MCI), or AD using laser capture microdissection (LCM), custom-designed microarray analysis, and qPCR of CA1 subregional dissections. Gene expression levels were correlated with cognitive test scores and AD neuropathology criteria. We found a significant downregulation of several neurotrophin genes (e.g., Gdnf, Ngfb, and Ntf4) in CA1 pyramidal neurons in MCI compared to NCI and AD subjects. In addition, the neurotrophin receptor transcripts TrkB and TrkC were decreased in MCI and AD compared to NCI. Regional hippocampal dissections also revealed select neurotrophic gene dysfunction providing evidence for vulnerability within the hippocampus proper during the progression of dementia. Downregulation of several neurotrophins of the NGF family and cognate neurotrophin receptor (TrkA, TrkB, and TrkC) genes correlated with antemortem cognitive measures including the Mini-Mental State Exam (MMSE), a composite global cognitive score (GCS), and Episodic, Semantic, and Working Memory, Perceptual Speed, and Visuospatial domains. Significant correlations were found between select neurotrophic expression downregulation and neuritic plaques (NPs) and neurofibrillary tangles (NFTs), but not diffuse plaques (DPs). These data suggest that dysfunction of neurotrophin signaling complexes have profound negative sequelae within vulnerable hippocampal cell types, which play a role in mnemonic and executive dysfunction during the progression of AD.
Asunto(s)
Enfermedad de Alzheimer/patología , Disfunción Cognitiva/patología , Hipocampo/patología , Factores de Crecimiento Nervioso/metabolismo , Células Piramidales/patología , Receptores de Factor de Crecimiento Nervioso/metabolismo , Anciano , Anciano de 80 o más Años , Enfermedad de Alzheimer/metabolismo , Región CA1 Hipocampal/metabolismo , Región CA1 Hipocampal/patología , Disfunción Cognitiva/metabolismo , Progresión de la Enfermedad , Femenino , Hipocampo/metabolismo , Humanos , Masculino , Células Piramidales/metabolismoRESUMEN
Epidemiological findings suggest that diabetic individuals are at a greater risk for developing Alzheimer's disease (AD). To examine the mechanisms by which diabetes mellitus (DM) may contribute to AD pathology in humans, we examined brain tissue from streptozotocin-treated type 1 diabetic adult male vervet monkeys receiving twice-daily exogenous insulin injections for 8-20 weeks. We found greater inhibitory phosphorylation of insulin receptor substrate 1 in each brain region examined of the diabetic monkeys when compared with controls, consistent with a pattern of brain insulin resistance that is similar to that reported in the human AD brain. Additionally, a widespread increase in phosphorylated tau was seen, including brain areas vulnerable in AD, as well as relatively spared structures, such as the cerebellum. An increase in active ERK1/2 was also detected, consistent with DM leading to changes in tau-kinase activity broadly within the brain. In contrast to these widespread changes, we found an increase in soluble amyloid-ß (Aß) levels that was restricted to the temporal lobe, with the greatest increase seen in the hippocampus. Consistent with this localized Aß increase, a hippocampus-restricted decrease in the protein and mRNA for the Aß-degrading enzyme neprilysin (NEP) was found, whereas various Aß-clearing and -degrading proteins were unchanged. Thus, we document multiple biochemical changes in the insulin-controlled DM monkey brain that can link DM with the risk of developing AD, including dysregulation of the insulin-signaling pathway, changes in tau phosphorylation, and a decrease in NEP expression in the hippocampus that is coupled with a localized increase in Aß. SIGNIFICANCE STATEMENT: Given that diabetes mellitus (DM) appears to increase the risk of developing Alzheimer's disease (AD), understanding the mechanisms by which DM promotes AD is important. We report that DM in a nonhuman primate brain leads to changes in the levels or posttranslational processing of proteins central to AD pathobiology, including tau, amyloid-ß (Aß), and the Aß-degrading protease neprilysin. Additional evidence from this model suggests that alterations in brain insulin signaling occurred that are reminiscent of insulin signaling pathway changes seen in human AD. Thus, in an in vivo model highly relevant to humans, we show multiple alterations in the brain resulting from DM that are mechanistically linked to AD risk.
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Péptidos beta-Amiloides/metabolismo , Química Encefálica , Diabetes Mellitus Tipo 1/metabolismo , Hipocampo/metabolismo , Resistencia a la Insulina , Neprilisina/metabolismo , Proteínas tau/metabolismo , Animales , Chlorocebus aethiops , Diabetes Mellitus Experimental/metabolismo , Hígado/metabolismo , Masculino , Fosforilación , Transducción de SeñalRESUMEN
An additional copy of the beta-amyloid precursor protein (APP) gene causes early-onset Alzheimer's disease (AD) in trisomy 21 (DS). Endosome dysfunction develops very early in DS and AD and has been implicated in the mechanism of neurodegeneration. Here, we show that morphological and functional endocytic abnormalities in fibroblasts from individuals with DS are reversed by lowering the expression of APP or beta-APP-cleaving enzyme 1 (BACE-1) using short hairpin RNA constructs. By contrast, endosomal pathology can be induced in normal disomic (2N) fibroblasts by overexpressing APP or the C-terminal APP fragment generated by BACE-1 (betaCTF), all of which elevate the levels of betaCTFs. Expression of a mutant form of APP that cannot undergo beta-cleavage had no effect on endosomes. Pharmacological inhibition of APP gamma-secretase, which markedly reduced Abeta production but raised betaCTF levels, also induced AD-like endosome dysfunction in 2N fibroblasts and worsened this pathology in DS fibroblasts. These findings strongly implicate APP and the betaCTF of APP, and exclude Abeta and the alphaCTF, as the cause of endocytic pathway dysfunction in DS and AD, underscoring the potential multifaceted value of BACE-1 inhibition in AD therapeutics.
Asunto(s)
Enfermedad de Alzheimer/metabolismo , Secretasas de la Proteína Precursora del Amiloide/metabolismo , Precursor de Proteína beta-Amiloide/metabolismo , Ácido Aspártico Endopeptidasas/metabolismo , Síndrome de Down/metabolismo , Endosomas/metabolismo , Interferencia de ARN , Adolescente , Adulto , Enfermedad de Alzheimer/complicaciones , Enfermedad de Alzheimer/tratamiento farmacológico , Enfermedad de Alzheimer/genética , Secretasas de la Proteína Precursora del Amiloide/genética , Péptidos beta-Amiloides/metabolismo , Ácido Aspártico Endopeptidasas/genética , Células Cultivadas , Niño , Preescolar , Síndrome de Down/complicaciones , Síndrome de Down/genética , Fibroblastos/metabolismo , Humanos , Lactante , Transporte de Proteínas , Superóxido Dismutasa/genética , Superóxido Dismutasa/metabolismo , Superóxido Dismutasa-1 , Adulto JovenRESUMEN
To evaluate molecular signatures of an individual cell type in comparison to the associated region relevant towards understanding the pathogenesis of Alzheimer's disease (AD), CA1 pyramidal neurons and the surrounding hippocampal formation were microaspirated via laser capture microdissection (LCM) from neuropathologically confirmed AD and age-matched control (CTR) subjects as well as from wild type mouse brain using single population RNA amplification methodology coupled with custom-designed microarray analysis with real-time quantitative polymerase-chain reaction (qPCR) validation. CA1 pyramidal neurons predominantly displayed downregulation of classes of transcripts related to synaptic transmission in AD versus CTR. Regional hippocampal dissections displayed downregulation of several overlapping genes found in the CA1 neuronal population related to neuronal expression, as well as upregulation of select transcripts indicative of admixed cell types including glial-associated markers and immediate-early and cell death genes. Gene level distributions observed in CA1 neurons and regional hippocampal dissections in wild type mice paralleled expression mosaics seen in postmortem human tissue. Microarray analysis was validated in qPCR studies using human postmortem brain tissue and CA1 sector and regional hippocampal dissections obtained from a mouse model of AD/Down syndrome (Ts65Dn mice) and normal disomic (2N) littermates. Classes of transcripts that have a greater percentage of the overall hybridization signal intensity within single neurons tended to be genes related to neuronal communication. The converse was also found, as classes of transcripts such as glial-associated markers were under represented in CA1 pyramidal neuron expression profiles relative to regional hippocampal dissections. These observations highlight a dilution effect that is likely to occur in conventional regional microarray and qPCR studies. Thus, single population studies of specific neurons and intrinsic circuits will likely yield informative gene expression profile data that may be subthreshold and/or underrepresented in regional studies with an admixture of cell types.
Asunto(s)
Enfermedad de Alzheimer/genética , Región CA1 Hipocampal/metabolismo , Células Piramidales/metabolismo , Anciano , Anciano de 80 o más Años , Enfermedad de Alzheimer/metabolismo , Enfermedad de Alzheimer/patología , Animales , Región CA1 Hipocampal/patología , Regulación hacia Abajo , Femenino , Expresión Génica , Perfilación de la Expresión Génica , Humanos , Captura por Microdisección con Láser , Masculino , Ratones , Persona de Mediana Edad , Células Piramidales/patologíaRESUMEN
In vitro studies have shown that cystatin C (CysC) is neuroprotective. Here we demonstrate that CysC is neuroprotective in vivo, in a mouse model of the inherited neurodegenerative disorder, progressive myoclonic epilepsy type 1 (EPM1). Loss-of-function mutations in the cystatin B (CysB) gene, an intracellular cysteine protease inhibitor, lead to this human disease. A CysB-knockout (CysBKO) mouse model develops symptoms that mimic EPM1. CysB deficiency in these mice results in enhanced cathepsin B and D activities, indicating lysosomal dysfunction. We show that expression of CysC is enhanced in the brains of CysBKO mice. Crossbreeding of CysBKO mice with either CysC-overexpressing transgenic mice or CysC-knockout mice demonstrates that clinical symptoms and neuropathologies, including motor coordination disorder, cerebellar atrophy, neuronal loss in the cerebellum and cerebral cortex, and gliosis caused by CysB deficiency, are rescued by CysC overexpression and exacerbated by CysC deficiency. Thus, CysC effectively rescues the CysB loss-of-function mutations, facilitating the reversal of pathophysiological changes and suggesting a novel therapeutic intervention for patients with EPM1 and other neurodegenerative disorders.
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Cistatina B/metabolismo , Cistatina C/metabolismo , Epilepsias Mioclónicas Progresivas/metabolismo , Epilepsias Mioclónicas Progresivas/patología , Neuronas/metabolismo , Neuronas/patología , Animales , Encéfalo/metabolismo , Encéfalo/patología , Catepsina B/genética , Catepsina B/metabolismo , Catepsina D/genética , Catepsina D/metabolismo , Cistatina B/genética , Cistatina C/genética , Modelos Animales de Enfermedad , Femenino , Gliosis/patología , Humanos , Lisosomas/enzimología , Masculino , Ratones , Ratones Noqueados , Ratones Transgénicos , Degeneración Nerviosa/metabolismo , Degeneración Nerviosa/patología , Prueba de Desempeño de Rotación con Aceleración ConstanteRESUMEN
Terminal continuation (TC) RNA amplification was developed originally to reproducibly and inexpensively amplify RNA. The TC RNA amplification method has been improved further by obviating second strand DNA synthesis, a cost-effective protocol that takes less time to perform with fewer manipulations required for RNA amplification. Results demonstrate that TC RNA amplification without second strand synthesis does not differ from the original protocol using RNA harvested from mouse brain and from hippocampal neurons obtained via laser capture microdissection from postmortem human brains. The modified TC RNA amplification method can discriminate single cell gene expression profiles between normal control and Alzheimer's disease hippocampal neurons indistinguishable from the original protocol. Thus, TC RNA amplification without second strand synthesis is a reproducible, time- and cost-effective method for RNA amplification from minute amounts of input RNA, and is compatible with microaspiration strategies and subsequent microarray analysis as well as quantitative real-time PCR.
Asunto(s)
Perfilación de la Expresión Génica/métodos , Técnicas de Amplificación de Ácido Nucleico/métodos , Análisis de Secuencia por Matrices de Oligonucleótidos/métodos , ARN/análisis , Anciano , Anciano de 80 o más Años , Enfermedad de Alzheimer/genética , Animales , Encéfalo/metabolismo , Encéfalo/patología , Química Encefálica/genética , Femenino , Expresión Génica/genética , Humanos , Masculino , Ratones , Ratones Endogámicos C57BL , Microdisección/métodos , ARN/síntesis química , Reproducibilidad de los ResultadosRESUMEN
BACKGROUND: Fibers containing galanin (GAL) enlarge and hyperinnervate cholinergic basal forebrain (CBF) nucleus basalis (NB) neurons in late-stage Alzheimer's disease (AD), yet the physiological consequences of this phenomenon are unclear. OBJECTIVE: To determine whether GAL hyperinnervation of cholinergic NB neurons modulates the expression of genes critical to cholinergic transmission [e.g. acetylcholine (ACh) metabolism and ACh receptors] in AD. METHODS: Single-cell gene expression profiling was used to compare cholinergic mRNA levels in non-GAL-hyperinnervated NB neurons in tissue autopsied from cases classified as having no cognitive impairment (NCI) or late-stage AD (AD/GAL-) and in GAL-hyperinnervated (AD/GAL+) NB neurons from the same AD subjects. RESULTS: AD/GAL+ cells displayed a significant upregulation in choline acetyltransferase (ChAT) mRNA expression compared to NCI and AD/GAL- cells. CONCLUSION: GAL fiber hyperinnervation of cholinergic NB neurons upregulates the expression of ChAT, the synthetic enzyme for ACh, suggesting that GAL regulates the cholinergic tone of CBF neurons in AD.
Asunto(s)
Enfermedad de Alzheimer/enzimología , Colina O-Acetiltransferasa/biosíntesis , Fibras Colinérgicas/enzimología , Galanina/fisiología , Regulación Enzimológica de la Expresión Génica/fisiología , Neuronas/enzimología , Prosencéfalo/enzimología , Regulación hacia Arriba/fisiología , Enfermedad de Alzheimer/genética , Colina O-Acetiltransferasa/genética , HumanosRESUMEN
A novel methodology named terminal continuation (TC) RNA amplification has been developed to amplify RNA from minute amounts of starting material. Utility of the TC RNA amplification method is demonstrated with two new modifications including obviating the need for second strand synthesis, and purifying the amplification template using column filtration prior to in vitro transcription (IVT). Using four low concentrations of RNA extracted from mouse brain (1, 10, 25 and 50 ng), one round TC RNA amplification was compared to one round amplified antisense RNA (aRNA) in conjunction with column filtration and drop dialysis purification. The TC RNA amplification without second strand synthesis performed extremely well on custom-designed cDNA array platforms, and column filtration was found to provide higher positive detection of individual clones when hybridization signal intensity was subtracted from corresponding negative control hybridization signal levels. Results indicate that TC RNA amplification without second strand synthesis, in conjunction with column filtration, is an excellent method for RNA amplification from extremely small amounts of input RNA from mouse brain and postmortem human brain, and is compatible with microaspiration strategies and subsequent microarray analysis.
RESUMEN
BACKGROUND: Dysfunction of basocortical cholinergic projection neurons of the nucleus basalis (NB) correlates with cognitive deficits in Alzheimer disease (AD). Nucleus basalis neurons receive cholinergic inputs and express nicotinic acetylcholine receptors (nAChRs) and muscarinic AChRs (mAChRs), which may regulate NB neuron activity in AD. Although alterations in these AChRs occur in the AD cortex, there is little information detailing whether defects in nAChR and mAChR gene expression occur in cholinergic NB neurons during disease progression. OBJECTIVE: To determine whether nAChR and mAChR gene expression is altered in cholinergic NB neurons during the progression of AD. DESIGN: Individual NB neurons from subjects diagnosed ante mortem as having no cognitive impairment (NCI), mild cognitive impairment (MCI), or mild to moderate AD were analyzed by single-cell AChR expression profiling via custom-designed microarrays. SETTING: Academic research. PARTICIPANTS: Participants were members of the Rush Religious Orders Study cohort. MAIN OUTCOME MEASURES: Real-time quantitative polymerase chain reaction was performed to validate microarray findings. RESULTS: Cholinergic NB neurons displayed a statistically significant up-regulation of alpha7 nAChR messenger RNA expression in subjects with mild to moderate AD compared with those with NCI and MCI (P<.001). No differences were found for other nAChR and mAChR subtypes across the cohort. Expression levels of alpha7 nAChRs were inversely associated with Global Cognitive Score and with Mini-Mental State Examination performance. CONCLUSIONS: Up-regulation of alpha7 nAChRs may signal a compensatory response to maintain basocortical cholinergic activity during AD progression. Alternatively, putative competitive interactions of this receptor with beta-amyloid may provide a pathogenic mechanism for NB dysfunction. Increasing NB alpha7 nAChR expression may serve as a marker for the progression of AD.
Asunto(s)
Enfermedad de Alzheimer/metabolismo , Neuronas/metabolismo , Sistema Nervioso Parasimpático/metabolismo , Receptores Nicotínicos/biosíntesis , Anciano , Anciano de 80 o más Años , Enfermedad de Alzheimer/patología , Ganglios Basales/metabolismo , Ganglios Basales/patología , Encéfalo/patología , Trastornos del Conocimiento/metabolismo , Trastornos del Conocimiento/patología , Estudios de Cohortes , ADN Complementario/biosíntesis , ADN Complementario/genética , Femenino , Regulación de la Expresión Génica/fisiología , Marcadores Genéticos , Humanos , Inmunohistoquímica , Masculino , Neuronas/patología , Pruebas Neuropsicológicas , Análisis de Secuencia por Matrices de Oligonucleótidos , Sistema Nervioso Parasimpático/patología , Escalas de Valoración Psiquiátrica , ARN Mensajero/biosíntesis , ARN Mensajero/genética , Receptores Nicotínicos/genética , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Regulación hacia Arriba , Receptor Nicotínico de Acetilcolina alfa 7RESUMEN
The development of gene array techniques to quantify expression levels of dozens to thousands of genes simultaneously within selected tissue samples from control and diseased brain has enabled researchers to generate expression profiles of vulnerable neuronal populations in several neurodegenerative diseases, including Alzheimer's disease, Parkinson's disease, schizophrenia, multiple sclerosis, and Creutzfeld-Jakob disease. Intriguingly, gene expression analysis reveals that vulnerable brain regions in many of these diseases share putative pathogenetic alterations in common classes of genes, including decrements in synaptic transcript levels and increments in immune response transcripts. Thus, gene expression profiles of diseased neuronal populations may reveal mechanistic clues to the molecular pathogenesis underlying various neurological diseases and aid in identifying potential therapeutic targets. This chapter will review how regional and single cell gene array technologies have advanced our understanding of the genetics of human neurological disease.
Asunto(s)
Encéfalo/patología , Dermatoglifia del ADN , Perfilación de la Expresión Génica , Enfermedades Neurodegenerativas/genética , Neuronas/patología , Animales , Encéfalo/fisiología , Dermatoglifia del ADN/métodos , Expresión Génica , Perfilación de la Expresión Génica/métodos , Humanos , Neuronas/fisiología , Análisis de Secuencia por Matrices de Oligonucleótidos/métodosRESUMEN
Defective autophagy contributes to Alzheimer disease (AD) pathogenesis although evidence is conflicting on whether multiple stages are impaired. Here, for the first time, we have comprehensively evaluated the entire autophagic process specifically in CA1 pyramidal neurons of hippocampus from early and late-stage AD subjects and nondemented controls. CA1 neurons aspirated by laser capture microdissection were analyzed using a custom-designed microarray comprising 578 neuropathology- and neuroscience-associated genes. Striking upregulation of autophagy-related genes, exceeding that of other gene ontology groups, reflected increases in autophagosome formation and lysosomal biogenesis beginning at early AD stages. Upregulated autophagosome formation was further indicated by elevated gene and protein expression levels for autophagosome components and increased LC3-positive puncta. Increased lysosomal biogenesis was evidenced by activation of MiTF/TFE family transcriptional regulators, particularly TFE3 (transcription factor binding to IGHM enhancer 3) and by elevated expression of their target genes and encoded proteins. Notably, TFEB (transcription factor EB) activation was associated more strongly with glia than neurons. These findings establish that autophagic sequestration is both competent and upregulated in AD. Autophagosome-lysosome fusion is not evidently altered. Despite this early disease response, however, autophagy flux is progressively impeded due to deficient substrate clearance, as reflected by autolysosomal accumulation of LC3-II and SQSTM1/p62 and expansion of autolysosomal size and total area. We propose that sustained induction of autophagy in the face of progressively declining lysosomal clearance of substrates explains the uncommonly robust autophagic pathology and neuritic dystrophy implicated in AD pathogenesis.
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Autofagia , Región CA1 Hipocampal/patología , Lisosomas/metabolismo , Neuritas/patología , Anciano , Anciano de 80 o más Años , Enfermedad de Alzheimer/genética , Enfermedad de Alzheimer/patología , Autofagosomas/metabolismo , Factores de Transcripción Básicos con Cremalleras de Leucinas y Motivos Hélice-Asa-Hélice/metabolismo , Región CA1 Hipocampal/metabolismo , Catepsina D/metabolismo , Demografía , Femenino , Regulación de la Expresión Génica , Humanos , Masculino , Proteínas Asociadas a Microtúbulos , Persona de Mediana Edad , Neuritas/metabolismoRESUMEN
The hippocampus contains several distinct cell types that are interconnected by a well-characterized series of synaptic circuits. To evaluate molecular and cellular signatures of individual cell types within the normal adult human hippocampal formation, expression profile analysis was performed on individual CA1 and CA3 pyramidal neurons using a novel single cell RNA amplification methodology coupled with custom-designed cDNA array analysis. Populations of CA1 and CA3 neurons were also compared with regional dissections of the hippocampus from the same tissue sections. Molecular fingerprint comparison of cresyl violet-stained CA1 and CA3 pyramidal neurons microaspirated from the hippocampus of normal control subjects indicated significant differences in relative expression levels for approximately 16% (20 of 125) genes evaluated on the custom-designed cDNA array platform. Significant differences were observed for several transcripts relevant to the structure and function of hippocampal neurons, including specific glutamate receptors, gamma-aminobutyric acid (GABA) A receptors, cytoskeletal elements, dopamine receptors, and immediate-early genes. Compared with the regional assessment of gene expression, both CA1 and CA3 neurons displayed a relative enrichment of classes of transcripts that included glutamate receptors, transporters, and interacting proteins, GABA receptors and transporters, synaptic-related markers, and catecholamine receptors and transporters. In contrast, the regional hippocampal dissection had an increased level of gene expression for cytoskeletal elements as well as glial-associated markers. Expression profile analysis illustrates the importance of evaluating individual cellular populations within a functional circuit and may help define elements that confer unique properties to individual populations of hippocampal neurons under normal and diseased conditions.
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Diferenciación Celular/genética , Perfilación de la Expresión Génica , Hipocampo/metabolismo , Células Piramidales/metabolismo , Anciano , Proteínas del Citoesqueleto/metabolismo , Dermatoglifia del ADN , Hipocampo/citología , Humanos , Técnicas In Vitro , Masculino , Enfermedades Neurodegenerativas/genética , Enfermedades Neurodegenerativas/metabolismo , Análisis de Secuencia por Matrices de Oligonucleótidos , Células Piramidales/citología , ARN/análisis , Valores de ReferenciaRESUMEN
The signature sequence amplification method (SSAM) described herein is an approach for amplifying noncoding RNA (ncRNA), microRNA (miRNA), and small polynucleotide sequences. A key point of the SSAM technology is the generation of signature sequences. The signature sequences include target sequences (miRNA, ncRNA, and/or any small polynucleotide sequence) flanked by two DNA fragments. Target sequences can be amplified through DNA synthesis, RNA synthesis, or the combination of DNA and RNA synthesis. The amplification of signature sequences provides an efficient and reproducible mechanism to determine the presence or absence of the target miRNAs/ncRNAs, to analyze the quantities of the miRNAs in biological samples, and for miRNA/ncRNA profiling.
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MicroARNs/análisis , Técnicas de Amplificación de Ácido Nucleico/métodos , ARN no Traducido/análisis , Animales , Perfilación de la Expresión Génica/métodos , Humanos , Ratones , MicroARNs/química , ARN no Traducido/químicaRESUMEN
Clinical neuropathologic studies suggest that the selective vulnerability of hippocampal CA1 pyramidal projection neurons plays a key role in the onset of cognitive impairment during the early phases of Alzheimer's disease (AD). Disruption of this neuronal population likely affects hippocampal pre- and postsynaptic efficacy underlying episodic memory circuits. Therefore, identifying perturbations in the expression of synaptic gene products within CA1 neurons prior to frank AD is crucial for the development of disease modifying therapies. Here we used custom-designed microarrays to examine progressive alterations in synaptic gene expression within CA1 neurons in cases harvested from the Rush Religious Orders Study who died with a clinical diagnosis of no cognitive impairment (NCI), mild cognitive impairment (MCI, a putative prodromal AD stage), or mild/moderate AD. Quantitative analysis revealed that 21 out of 28 different transcripts encoding regulators of synaptic function were significantly downregulated (1.4-1.8 fold) in CA1 neurons in MCI and AD compared to NCI, whereas synaptic transcript levels were not significantly different between MCI and AD. The downregulated transcripts encoded regulators of presynaptic vesicle trafficking, including synaptophysin and synaptogyrin, regulators of vesicle docking and fusion/release, such as synaptotagmin and syntaxin 1, and regulators of glutamatergic postsynaptic function, including PSD-95 and synaptopodin. Clinical pathologic correlation analysis revealed that downregulation of these synaptic markers was strongly associated with poorer antemortem cognitive status and postmortem AD pathological criteria such as Braak stage, NIA-Reagan, and CERAD diagnosis. In contrast to the widespread loss of synaptic gene expression observed in CA1 neurons in MCI, transcripts encoding ß-amyloid precursor protein (APP), APP family members, and regulators of APP metabolism were not differentially regulated in CA1 neurons across the clinical diagnostic groups. Taken together, these data suggest that CA1 synaptic gene dysregulation occurs early in the cascade of pathogenic molecular events prior to the onset of AD, which may form the basis for novel pharmacological treatment approaches for this dementing disorder. This article is part of a Special Issue entitled 'Neurodegenerative Disorders'.
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Enfermedad de Alzheimer/metabolismo , Región CA1 Hipocampal/metabolismo , Disfunción Cognitiva/metabolismo , Células Piramidales/metabolismo , Anciano de 80 o más Años , Femenino , Expresión Génica , Humanos , Masculino , Reacción en Cadena de la Polimerasa , Índice de Severidad de la Enfermedad , Análisis de Matrices TisularesRESUMEN
The higher incidence rate of Alzheimer's disease (AD) in elderly women indicates that gender plays a role in AD pathogenesis. Evidence from clinical and pharmacologic studies, neuropathological examinations, and models of hormone replacement therapy suggest that cholinergic basal forebrain (CBF) cortical projection neurons within the nucleus basalis (NB), which mediate memory and attention and degenerate in AD, may be preferentially vulnerable in elderly women compared to men. CBF neurons depend on nerve growth factor (NGF) and their cognate receptors (trkA and p75(NTR)) for their survival and maintenance. We recently demonstrated a shift in the balance of NGF and its receptors toward cell death mechanisms during the progression of AD. To address whether gender affects NGF signaling system expression within the CBF, we used single cell RNA amplification and custom microarray technologies to compare gene expression profiles of single cholinergic NB neurons in tissue specimens from male and female members of the Religious Orders Study who died with a clinical diagnosis of no cognitive impairment (NCI), mild cognitive impairment (MCI), or mild/moderate AD. p75(NTR) expression within male cholinergic NB neurons was unchanged across clinical diagnosis, whereas p75(NTR) mRNA levels in female NB neurons exhibited a â¼40% reduction in AD compared to NCI. Male AD subjects displayed a â¼45% reduction in trkA mRNA levels within NB neurons compared to NCI and MCI. In contrast, NB neuronal trkA expression in females was reduced â¼50% in both MCI and AD compared to NCI. Reduced trkA mRNA levels were associated with poorer global cognitive performance and higher Braak scores in the female subjects. In addition, we found a female-selective reduction in GluR2 AMPA glutamate receptor subunit expression in NB neurons in AD. These data suggest that cholinergic NB neurons in females may be at greater risk for degeneration during the progression of AD and support the concept of gender-specific therapeutic interventions during the preclinical stages of the disease.
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Enfermedad de Alzheimer/fisiopatología , Núcleo Basal de Meynert/metabolismo , Neuronas Colinérgicas/metabolismo , Factor de Crecimiento Nervioso/genética , Receptores de Glutamato/genética , Caracteres Sexuales , Anciano , Anciano de 80 o más Años , Enfermedad de Alzheimer/genética , Enfermedad de Alzheimer/patología , Núcleo Basal de Meynert/patología , Núcleo Basal de Meynert/fisiopatología , Muerte Celular/genética , Neuronas Colinérgicas/patología , Neuronas Colinérgicas/fisiología , Progresión de la Enfermedad , Regulación hacia Abajo/genética , Femenino , Humanos , Masculino , Factor de Crecimiento Nervioso/biosíntesis , Factor de Crecimiento Nervioso/fisiología , Receptor de Factor de Crecimiento Nervioso/genética , Receptor trkA/genética , Receptores AMPA/genética , Receptores de Glutamato/biosíntesis , Receptores de Glutamato/fisiología , Factores de Riesgo , Distribución por SexoRESUMEN
This study was designed to understand molecular and cellular mechanisms underlying aggressive behaviors in mice exposed to repeated interactions in their homecage with conspecifics. A resident-intruder procedure was employed whereby two males were allowed to interact for 10 min trials, and aggressive and/or submissive behaviors (e.g., degree of attacking, biting, chasing, grooming, rearing, or upright posture) were assessed. Following 10 days of behavioral trials, brains were removed and dissected into specific regions including the cerebellum, frontal cortex, hippocampus, midbrain, pons, and striatum. Gene expression analysis was performed using real-time quantitative polymerase-chain reaction (qPCR) for catechol-O-methyltransferase (COMT) and tyrosine hydroxylase (TH). Compared to naive control mice, significant up regulation of COMT expression of residents was observed in the cerebellum, frontal cortex, hippocampus, midbrain, and striatum; in all of these brain regions the COMT expression of residents was also significantly higher than that of intruders. The intruders also had a significant down regulation (compared to naive control mice) within the hippocampus, indicating a selective decrease in COMT expression in the hippocampus of submissive subjects. Immunoblot analysis confirmed COMT up regulation in the midbrain and hippocampus of residents and down regulation in intruders. qPCR analysis of TH expression indicated significant up regulation in the midbrain of residents and concomitant down regulation in intruders. These findings implicate regionally- and behaviorally-specific regulation of COMT and TH expression in aggressive and submissive behaviors. Additional molecular and cellular characterization of COMT, TH, and other potential targets is warranted within this animal model of aggression.
Asunto(s)
Agresión/fisiología , Encéfalo/metabolismo , Catecol O-Metiltransferasa/metabolismo , Regulación de la Expresión Génica/fisiología , Predominio Social , Tirosina 3-Monooxigenasa/metabolismo , Análisis de Varianza , Animales , Encéfalo/anatomía & histología , Immunoblotting , Masculino , Ratones , Ratones Endogámicos C57BL , Reacción en Cadena en Tiempo Real de la PolimerasaRESUMEN
Endocytic system dysfunction is one of the earliest disturbances that occur in Alzheimer's disease (AD), and may underlie the selective vulnerability of cholinergic basal forebrain (CBF) neurons during the progression of dementia. Herein we report that genes regulating early and late endosomes are selectively upregulated within CBF neurons in mild cognitive impairment (MCI) and AD. Specifically, upregulation of rab4, rab5, rab7, and rab27 was observed in CBF neurons microdissected from postmortem brains of individuals with MCI and AD compared to age-matched control subjects with no cognitive impairment (NCI). Upregulated expression of rab4, rab5, rab7, and rab27 correlated with antemortem measures of cognitive decline in individuals with MCI and AD. qPCR validated upregulation of these select rab GTPases within microdissected samples of the basal forebrain. Moreover, quantitative immunoblot analysis demonstrated upregulation of rab5 protein expression in the basal forebrain of subjects with MCI and AD. The elevation of rab4, rab5, and rab7 expression is consistent with our recent observations in CA1 pyramidal neurons in MCI and AD. These findings provide further support that endosomal pathology accelerates endocytosis and endosome recycling, which may promote aberrant endosomal signaling and neurodegeneration throughout the progression of AD.
Asunto(s)
Enfermedad de Alzheimer/enzimología , Núcleo Basal de Meynert/enzimología , Neuronas Colinérgicas/enzimología , Disfunción Cognitiva/enzimología , Regulación hacia Arriba/fisiología , Proteínas de Unión al GTP rab/biosíntesis , Proteínas de Unión al GTP rab4/biosíntesis , Proteínas de Unión al GTP rab5/biosíntesis , Anciano , Anciano de 80 o más Años , Enfermedad de Alzheimer/fisiopatología , Núcleo Basal de Meynert/fisiopatología , Disfunción Cognitiva/fisiopatología , Femenino , Humanos , Masculino , Proteínas de Unión al GTP rab/genética , Proteínas rab27 de Unión a GTP , Proteínas de Unión al GTP rab4/genética , Proteínas de Unión al GTP rab5/genética , Proteínas de Unión a GTP rab7RESUMEN
Endocytic alterations are one of the earliest changes to occur in Alzheimer's disease (AD), and are hypothesized to be involved in the selective vulnerability of specific neuronal populations during the progression of AD. Previous microarray and real-time quantitative PCR experiments revealed an upregulation of the early endosomal effector rab5 and the late endosome constituent rab7 in the hippocampus of people with mild cognitive impairment (MCI) and AD. To assess whether these select rab GTPase gene expression changes are reflected in protein levels within selectively vulnerable brain regions (basal forebrain, frontal cortex, and hippocampus) and relatively spared areas (cerebellum and striatum), we performed immunoblot analysis using antibodies directed against rab5 and rab7 on postmortem human brain tissue harvested from cases with a premortem clinical diagnosis of no cognitive impairment (NCI), MCI, and AD. Results indicate selective upregulation of both rab5 and rab7 levels within basal forebrain, frontal cortex, and hippocampus in MCI and AD, which also correlated with Braak staging. In contrast, no differences in protein levels were found in the less vulnerable cerebellum and striatum. These regional immunoblot assays are consistent with single cell gene expression data, and provide protein-based evidence for endosomal markers contributing to the vulnerability of cell types within selective brain regions during the progression of AD.
Asunto(s)
Enfermedad de Alzheimer , Encéfalo/metabolismo , Trastornos del Conocimiento , Regulación hacia Arriba/fisiología , Proteínas de Unión al GTP rab/metabolismo , Proteínas de Unión al GTP rab5/metabolismo , Anciano , Anciano de 80 o más Años , Enfermedad de Alzheimer/metabolismo , Enfermedad de Alzheimer/patología , Enfermedad de Alzheimer/fisiopatología , Encéfalo/patología , Trastornos del Conocimiento/metabolismo , Trastornos del Conocimiento/patología , Trastornos del Conocimiento/fisiopatología , Femenino , Humanos , Masculino , Proteínas de Unión a GTP rab7RESUMEN
BACKGROUND: Endocytic dysfunction and neurotrophin signaling deficits may underlie the selective vulnerability of hippocampal neurons during the progression of Alzheimer's disease (AD), although there is little direct in vivo and biochemical evidence to support this hypothesis. METHODS: Microarray analysis of hippocampal CA1 pyramidal neurons acquired via laser capture microdissection was performed using postmortem brain tissue. Validation was achieved using real-time quantitative polymerase chain reaction and immunoblot analysis. Mechanistic studies were performed using human fibroblasts subjected to overexpression with viral vectors or knockdown via small interference RNA. RESULTS: Expression levels of genes regulating early endosomes (rab5) and late endosomes (rab7) are selectively upregulated in homogeneous populations of CA1 neurons from individuals with mild cognitive impairment and AD. The levels of these genes are selectively increased as antemortem measures of cognition decline during AD progression. Hippocampal quantitative polymerase chain reaction and immunoblot analyses confirmed increased levels of these transcripts and their respective protein products. Elevation of select rab GTPases regulating endocytosis paralleled the downregulation of genes encoding the neurotrophin receptors TrkB and TrkC. Overexpression of rab5 in cells suppressed TrkB expression, whereas knockdown of TrkB expression did not alter rab5 levels, suggesting that TrkB downregulation is a consequence of endosomal dysfunction associated with elevated rab5 levels in early AD. CONCLUSIONS: These data support the hypothesis that neuronal endosomal dysfunction is associated with preclinical AD. Increased endocytic pathway activity, driven by elevated rab GTPase expression, may result in long-term deficits in hippocampal neurotrophic signaling and represent a key pathogenic mechanism underlying AD progression.
Asunto(s)
Enfermedad de Alzheimer/genética , Enfermedad de Alzheimer/metabolismo , Región CA1 Hipocampal/metabolismo , Endosomas/metabolismo , Neuronas/metabolismo , Análisis de Secuencia por Matrices de Oligonucleótidos/métodos , Anciano de 80 o más Años , Línea Celular Transformada , Progresión de la Enfermedad , Regulación hacia Abajo , Femenino , Perfilación de la Expresión Génica/métodos , Técnicas de Silenciamiento del Gen/métodos , Humanos , Masculino , Receptor trkB/metabolismo , Receptor trkC/metabolismo , Regulación hacia Arriba , Proteínas de Unión al GTP rab/metabolismo , Proteínas de Unión al GTP rab5/metabolismo , Proteínas de Unión a GTP rab7RESUMEN
Fibers containing galanin (GAL) hyperinnervate cholinergic basal forebrain (CBF) nucleus basalis neurons in late stage Alzheimer's disease (AD), yet the molecular consequences of this phenomenon are unknown. To determine whether GAL alters the expression of genes critical to CBF cell survival in AD, single cell microarray analysis was used to determine mRNA levels within nucleus basalis neurons lacking GAL innervation from subjects who died with a clinical diagnosis of no cognitive impairment (NCI) compared to nucleus basalis neurons from AD cases either lacking GAL hyperinnervation (AD/GAL-) or those displaying prominent GAL hyperinnervation (AD/GAL+). Levels of mRNAs encoding putatively neuroprotective proteins such as the GluR2 Ca(2)-impermeable glutamate receptor subunit, superoxide dismutase 2, and the GLUT2 glucose transporter were significantly decreased in AD/GAL- nucleus basalis neurons compared to NCI and AD/GAL+ neurons. By contrast, mRNAs encoding calpain catalytic and regulatory subunits, which may contribute to cell death in AD, were increased in AD/GAL- compared to NCI and AD/GAL+ neurons. Hence, GAL fiber hyperinnervation appears to preserve the expression of genes subserving multiple neuroprotective pathways suggesting that GAL overexpression regulates CBF neuron survival in AD.