RESUMO
Genetic, epidemiologic, and biochemical evidence suggests that predisposition to Alzheimer's disease (AD) may arise from altered cholesterol metabolism, although the molecular pathways that may link cholesterol to AD phenotypes are only partially understood. Here, we perform a phenotypic screen for pTau accumulation in AD-patient iPSC-derived neurons and identify cholesteryl esters (CE), the storage product of excess cholesterol, as upstream regulators of Tau early during AD development. Using isogenic induced pluripotent stem cell (iPSC) lines carrying mutations in the cholesterol-binding domain of APP or APP null alleles, we found that while CE also regulate Aß secretion, the effects of CE on Tau and Aß are mediated by independent pathways. Efficacy and toxicity screening in iPSC-derived astrocytes and neurons showed that allosteric activation of CYP46A1 lowers CE specifically in neurons and is well tolerated by astrocytes. These data reveal that CE independently regulate Tau and Aß and identify a druggable CYP46A1-CE-Tau axis in AD.
Assuntos
Doença de Alzheimer/metabolismo , Peptídeos beta-Amiloides/metabolismo , Colesterol/metabolismo , Células-Tronco Pluripotentes Induzidas/metabolismo , Neurônios/metabolismo , Proteínas tau/metabolismo , Doença de Alzheimer/patologia , Animais , Células Cultivadas , Camundongos , Camundongos Endogâmicos C57BLRESUMO
Reprogramming somatic cells to induced pluripotent stem cells (iPSCs) offers the possibility of studying the molecular mechanisms underlying human diseases in cell types difficult to extract from living patients, such as neurons and cardiomyocytes. To date, studies have been published that use small panels of iPSC-derived cell lines to study monogenic diseases. However, to study complex diseases, where the genetic variation underlying the disorder is unknown, a sizable number of patient-specific iPSC lines and controls need to be generated. Currently the methods for deriving and characterizing iPSCs are time consuming, expensive, and, in some cases, descriptive but not quantitative. Here we set out to develop a set of simple methods that reduce cost and increase throughput in the characterization of iPSC lines. Specifically, we outline methods for high-throughput quantification of surface markers, gene expression analysis of in vitro differentiation potential, and evaluation of karyotype with markedly reduced cost.
Assuntos
Variação Genética , Ensaios de Triagem em Larga Escala/métodos , Células-Tronco Pluripotentes Induzidas/metabolismo , Cariotipagem/métodos , Miócitos Cardíacos/metabolismo , Neurônios/metabolismo , Biomarcadores/metabolismo , Diferenciação Celular , Linhagem Celular , Reprogramação Celular/genética , Análise Custo-Benefício , Genótipo , Ensaios de Triagem em Larga Escala/economia , Ensaios de Triagem em Larga Escala/instrumentação , Humanos , Células-Tronco Pluripotentes Induzidas/citologia , Cariotipagem/economia , Miócitos Cardíacos/citologia , Neurônios/citologia , FenótipoRESUMO
Predisposition to sporadic Alzheimer's disease (SAD) involves interactions between a person's unique combination of genetic variants and the environment. The molecular effect of these variants may be subtle and difficult to analyze with standard in vitro or in vivo models. Here we used hIPSCs to examine genetic variation in the SORL1 gene and possible contributions to SAD-related phenotypes in human neurons. We found that human neurons carrying SORL1 variants associated with an increased SAD risk show a reduced response to treatment with BDNF, at the level of both SORL1 expression and APP processing. shRNA knockdown of SORL1 demonstrates that the differences in BDNF-induced APP processing between genotypes are dependent on SORL1 expression. We propose that the variation in SORL1 expression induction by BDNF is modulated by common genetic variants and can explain how genetic variation in this one locus can contribute to an individual's risk of developing SAD.
Assuntos
Doença de Alzheimer/genética , Células-Tronco Pluripotentes Induzidas/fisiologia , Proteínas Relacionadas a Receptor de LDL/genética , Proteínas de Membrana Transportadoras/genética , Neurônios/fisiologia , Proteína Amiloide A Sérica/metabolismo , Fator Neurotrófico Derivado do Encéfalo/metabolismo , Linhagem Celular , Análise Mutacional de DNA/métodos , Regulação da Expressão Gênica/genética , Interação Gene-Ambiente , Predisposição Genética para Doença , Genótipo , Humanos , Fenótipo , Polimorfismo Genético , Transporte Proteico/genética , RNA Interferente Pequeno/genética , Fatores de RiscoRESUMO
IMPORTANCE: Although considerable effort has been expended developing drug candidates for Alzheimer disease, none have yet succeeded owing to the lack of efficacy or to safety concerns. One potential shortcoming of current approaches to Alzheimer disease drug discovery and development is that they rely primarily on transformed cell lines and animal models that substantially overexpress wild-type or mutant proteins. It is possible that drug development failures thus far are caused in part by the limits of these approaches, which do not accurately reveal how drug candidates will behave in naive human neuronal cells. OBJECTIVE: To analyze purified neurons derived from human induced pluripotent stem cells from patients carrying 3 different presenilin 1 (PS1) mutations and nondemented control individuals in the absence of any overexpression. We tested the efficacy of γ-secretase inhibitor and γ-secretase modulator (GSM) in neurons derived from both normal control and 3 PS1 mutations (A246E, H163R, and M146L). DESIGN, SETTING, AND PARTICIPANTS: Adult human skin biopsies were obtained from volunteers at the Alzheimer Disease Research Center, University of California, San Diego. Cell cultures were treated with γ-secretase inhibitor or GSM. Comparisons of total ß-amyloid (Aß) and Aß peptides 38, 40, and 42 in the media were made between vehicle- vs drug-treated cultures. MAIN OUTCOMES AND MEASURES: Soluble Aß levels in the media were measured by enzyme-linked immunosorbent assay. RESULTS: As predicted, mutant PS1 neurons exhibited an elevated Aß42:Aß40 ratio (P < .05) at the basal state as compared with the nondemented control neurons. Treatment with a potent non-nonsteroidal anti-inflammatory druglike GSM revealed a new biomarker signature that differs from all previous cell types and animals tested. This new signature was the same in both the mutant and control neurons and consisted of a reduction in Aß42, Aß40, and Aß38 and in the Aß42:Aß40 ratio, with no change in the total Aß levels. CONCLUSIONS AND RELEVANCE: This biomarker discrepancy is likely due to overexpression of amyloid precursor protein in the transformed cellular models. Our results suggest that biomarker signatures obtained with such models are misleading and that human neurons derived from human induced pluripotent stem cells provide a unique signature that will more accurately reflect drug response in human patients and in cerebrospinal fluid biomarker changes observed during GSM treatment.
Assuntos
Secretases da Proteína Precursora do Amiloide/antagonistas & inibidores , Secretases da Proteína Precursora do Amiloide/metabolismo , Peptídeos beta-Amiloides/metabolismo , Células-Tronco Pluripotentes Induzidas/citologia , Neurônios/metabolismo , Fragmentos de Peptídeos/metabolismo , Presenilina-1/genética , Alanina/análogos & derivados , Alanina/farmacologia , Peptídeos beta-Amiloides/efeitos dos fármacos , Anti-Inflamatórios/farmacologia , Azepinas/farmacologia , Biomarcadores/metabolismo , Heterozigoto , Humanos , Mutação/genética , Neurônios/enzimologia , Fragmentos de Peptídeos/efeitos dos fármacosRESUMO
Presenilins, the catalytic components of the γ-secretase complex, are upstream regulators of multiple cellular pathways via regulation of gene transcription. However, the underlying mechanisms and the genes regulated by these pathways are poorly characterized. In this study, we identify Tequila and its mammalian ortholog Prss12 as genes negatively regulated by presenilins in Drosophila larval brains and mouse embryonic fibroblasts, respectively. Prss12 encodes the serine protease neurotrypsin, which cleaves the heparan sulfate proteoglycan agrin. Altered neurotrypsin activity causes serious synaptic and cognitive defects; despite this, the molecular processes regulating neurotrypsin expression and activity are poorly understood. Using γ-secretase drug inhibitors and presenilin mutants in mouse embryonic fibroblasts, we found that a mature γ-secretase complex was required to repress neurotrypsin expression and agrin cleavage. We also determined that PSEN1 endoproteolysis or processing of well known γ-secretase substrates was not essential for this process. At the transcriptional level, PSEN1/2 removal induced cyclic AMP response element-binding protein (CREB)/CREB-binding protein binding, accumulation of activating histone marks at the neurotrypsin promoter, and neurotrypsin transcriptional and functional up-regulation that was dependent on GSK3 activity. Upon PSEN1/2 reintroduction, this active epigenetic state was replaced by a methyl CpG-binding protein 2 (MeCP2)-containing repressive state and reduced neurotrypsin expression. Genome-wide analysis revealed hundreds of other mouse promoters in which CREB binding is similarly modulated by the presence/absence of presenilins. Our study thus identifies Tequila and neurotrypsin as new genes repressed by presenilins and reveals a novel mechanism used by presenilins to modulate CREB signaling based on controlling CREB recruitment.
Assuntos
Agrina/metabolismo , Proteína de Ligação ao Elemento de Resposta ao AMP Cíclico/metabolismo , Presenilina-1/metabolismo , Presenilina-2/metabolismo , Serina Endopeptidases/genética , Serina Endopeptidases/metabolismo , Secretases da Proteína Precursora do Amiloide/metabolismo , Animais , Células Cultivadas , Drosophila/genética , Drosophila/metabolismo , Proteínas de Drosophila/genética , Proteínas de Drosophila/metabolismo , Regulação da Expressão Gênica , Humanos , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Transgênicos , Modelos Biológicos , Presenilina-1/deficiência , Presenilina-1/genética , Presenilina-2/deficiência , Presenilina-2/genética , Regiões Promotoras Genéticas , Transdução de SinaisRESUMO
Our understanding of Alzheimer's disease pathogenesis is currently limited by difficulties in obtaining live neurons from patients and the inability to model the sporadic form of the disease. It may be possible to overcome these challenges by reprogramming primary cells from patients into induced pluripotent stem cells (iPSCs). Here we reprogrammed primary fibroblasts from two patients with familial Alzheimer's disease, both caused by a duplication of the amyloid-ß precursor protein gene (APP; termed APP(Dp)), two with sporadic Alzheimer's disease (termed sAD1, sAD2) and two non-demented control individuals into iPSC lines. Neurons from differentiated cultures were purified with fluorescence-activated cell sorting and characterized. Purified cultures contained more than 90% neurons, clustered with fetal brain messenger RNA samples by microarray criteria, and could form functional synaptic contacts. Virtually all cells exhibited normal electrophysiological activity. Relative to controls, iPSC-derived, purified neurons from the two APP(Dp) patients and patient sAD2 exhibited significantly higher levels of the pathological markers amyloid-ß(1-40), phospho-tau(Thr 231) and active glycogen synthase kinase-3ß (aGSK-3ß). Neurons from APP(Dp) and sAD2 patients also accumulated large RAB5-positive early endosomes compared to controls. Treatment of purified neurons with ß-secretase inhibitors, but not γ-secretase inhibitors, caused significant reductions in phospho-Tau(Thr 231) and aGSK-3ß levels. These results suggest a direct relationship between APP proteolytic processing, but not amyloid-ß, in GSK-3ß activation and tau phosphorylation in human neurons. Additionally, we observed that neurons with the genome of one sAD patient exhibited the phenotypes seen in familial Alzheimer's disease samples. More generally, we demonstrate that iPSC technology can be used to observe phenotypes relevant to Alzheimer's disease, even though it can take decades for overt disease to manifest in patients.
Assuntos
Doença de Alzheimer/metabolismo , Doença de Alzheimer/patologia , Células-Tronco Pluripotentes Induzidas/metabolismo , Células-Tronco Pluripotentes Induzidas/patologia , Neurônios/metabolismo , Idoso de 80 Anos ou mais , Secretases da Proteína Precursora do Amiloide/antagonistas & inibidores , Secretases da Proteína Precursora do Amiloide/metabolismo , Peptídeos beta-Amiloides/metabolismo , Precursor de Proteína beta-Amiloide/genética , Precursor de Proteína beta-Amiloide/metabolismo , Astrócitos/citologia , Biomarcadores/metabolismo , Células Cultivadas , Reprogramação Celular , Técnicas de Cocultura , Endossomos/metabolismo , Ativação Enzimática , Feminino , Fibroblastos/citologia , Fibroblastos/metabolismo , Quinase 3 da Glicogênio Sintase/metabolismo , Humanos , Masculino , Pessoa de Meia-Idade , Modelos Biológicos , Neurônios/efeitos dos fármacos , Neurônios/patologia , Fragmentos de Peptídeos/metabolismo , Fosfoproteínas/metabolismo , Fosforilação/efeitos dos fármacos , Inibidores de Proteases/farmacologia , Proteólise , Sinapsinas/metabolismo , Proteínas tau/metabolismoRESUMO
The etiology of sporadic Alzheimer disease (AD) is largely unknown, although evidence implicates the pathological hallmark molecules amyloid beta (Aß) and phosphorylated Tau. Work in animal models suggests that altered axonal transport caused by Kinesin-1 dysfunction perturbs levels of both Aß and phosphorylated Tau in neural tissues, but the relevance of Kinesin-1 dependent functions to the human disease is unknown. To begin to address this issue, we generated human embryonic stem cells (hESC) expressing reduced levels of the kinesin light chain 1 (KLC1) Kinesin-1 subunit to use as a source of human neural cultures. Despite reduction of KLC1, undifferentiated hESC exhibited apparently normal colony morphology and pluripotency marker expression. Differentiated neural cultures derived from KLC1-suppressed hESC contained neural rosettes but further differentiation revealed obvious morphological changes along with reduced levels of microtubule-associated neural proteins, including Tau and less secreted Aß, supporting the previously established connection between KLC1, Tau and Aß. Intriguingly, KLC1-suppressed neural precursors (NPs), isolated using a cell surface marker signature known to identify cells that give rise to neurons and glia, unlike control cells, failed to proliferate. We suggest that KLC1 is required for normal human neural differentiation, ensuring proper metabolism of AD-associated molecules APP and Tau and for proliferation of NPs. Because impaired APP metabolism is linked to AD, this human cell culture model system will not only be a useful tool for understanding the role of KLC1 in regulating the production, transport and turnover of APP and Tau in neurons, but also in defining the essential function(s) of KLC1 in NPs and their progeny. This knowledge should have important implications for human neurodevelopmental and neurodegenerative diseases.
Assuntos
Precursor de Proteína beta-Amiloide/metabolismo , Diferenciação Celular , Células-Tronco Embrionárias/metabolismo , Proteínas Associadas aos Microtúbulos/metabolismo , Neurônios/metabolismo , Antígenos de Superfície/metabolismo , Western Blotting , Linhagem Celular , Proliferação de Células , Células-Tronco Embrionárias/citologia , Citometria de Fluxo , Humanos , Cariotipagem , Cinesinas , Proteínas Associadas aos Microtúbulos/genética , Células-Tronco Neurais/citologia , Células-Tronco Neurais/metabolismo , Neuritos/metabolismo , Neurônios/citologia , Fator 3 de Transcrição de Octâmero/metabolismo , Células-Tronco Pluripotentes/citologia , Células-Tronco Pluripotentes/metabolismo , Interferência de RNA , Proteínas tau/metabolismoRESUMO
The unique architecture of neurons requires the establishment and maintenance of polarity, which relies in part on microtubule-based transport to deliver essential cargo into dendrites. To test different models of differential motor protein regulation and to understand how different compartments in neurons are supplied with necessary functional proteins, we studied mechanisms of dendritic transport, using Drosophila as a model system. Our data suggest that dendritic targeting systems in Drosophila and mammals are evolutionarily conserved, since mammalian cargoes are moved into appropriate domains in Drosophila. In a genetic screen for mutants that mislocalize the dendritic marker human transferrin receptor (hTfR), we found that kinesin heavy chain (KHC) may function as a dendritic motor. Our analysis of dendritic and axonal phenotypes of KHC loss-of-function clones revealed a role for KHC in maintaining polarity of neurons, as well as ensuring proper axonal outgrowth. In addition we identified adenomatous polyposis coli 1 (APC1) as an interaction partner of KHC in controlling directed transport and modulating kinesin function in neurons.
Assuntos
Dendritos/metabolismo , Proteínas de Drosophila/metabolismo , Drosophila melanogaster/citologia , Cinesinas/metabolismo , Larva/citologia , Transporte Proteico , Vesículas Transportadoras/metabolismo , Animais , Animais Geneticamente Modificados , Subunidade Apc1 do Ciclossomo-Complexo Promotor de Anáfase , Axônios/metabolismo , Polaridade Celular , Forma Celular , Proteínas de Drosophila/genética , Drosophila melanogaster/genética , Drosophila melanogaster/metabolismo , Proteínas de Fluorescência Verde/metabolismo , Humanos , Cinesinas/genética , Larva/genética , Larva/metabolismo , Mutação , Neurônios/metabolismo , Receptores da Transferrina/metabolismo , Proteínas Recombinantes de Fusão/metabolismoRESUMO
Transport of cellular and neuronal vesicles, organelles, and other particles along microtubules requires the molecular motor protein dynein (Mallik and Gross, 2004). Critical to dynein function is dynactin, a multiprotein complex commonly thought to be required for dynein attachment to membrane compartments (Karki and Holzbaur, 1999). Recent work also has found that mutations in dynactin can cause the human motor neuron disease amyotrophic lateral sclerosis (Puls et al., 2003). Thus, it is essential to understand the in vivo function of dynactin. To test directly and rigorously the hypothesis that dynactin is required to attach dynein to membranes, we used both a Drosophila mutant and RNA interference to generate organisms and cells lacking the critical dynactin subunit, actin-related protein 1. Contrary to expectation, we found that apparently normal amounts of dynein associate with membrane compartments in the absence of a fully assembled dynactin complex. In addition, anterograde and retrograde organelle movement in dynactin deficient axons was completely disrupted, resulting in substantial changes in vesicle kinematic properties. Although effects on retrograde transport are predicted by the proposed function of dynactin as a regulator of dynein processivity, the additional effects we observed on anterograde transport also suggest potential roles for dynactin in mediating kinesin-driven transport and in coordinating the activity of opposing motors (King and Schroer, 2000).
Assuntos
Membrana Celular/metabolismo , Proteínas de Drosophila/metabolismo , Dineínas/metabolismo , Proteínas Associadas aos Microtúbulos/metabolismo , Subunidades Proteicas/metabolismo , Animais , Transporte Biológico , Células Cultivadas , Proteínas de Drosophila/genética , Drosophila melanogaster , Complexo Dinactina , Dineínas/genética , Humanos , Proteínas Associadas aos Microtúbulos/genética , Fenótipo , Subunidades Proteicas/genética , Interferência de RNA , Proteínas Recombinantes de Fusão/genética , Proteínas Recombinantes de Fusão/metabolismoRESUMO
Drosophila melanogaster is a widely used model organism for genetic dissection of developmental processes. To exploit its full potential for studying the genetic basis of male fertility, we performed a large-scale screen for male-sterile (ms) mutations. From a collection of 12,326 strains carrying ethyl-methanesulfonate-treated, homozygous viable second or third chromosomes, 2216 ms lines were identified, constituting the largest collection of ms mutations described to date for any organism. Over 2000 lines were cytologically characterized and, of these, 81% failed during spermatogenesis while 19% manifested postspermatogenic processes. Of the phenotypic categories used to classify the mutants, the largest groups were those that showed visible defects in meiotic chromosome segregation or cytokinesis and those that failed in sperm individualization. We also identified 62 fertile or subfertile lines that showed high levels of chromosome loss due to abnormal mitotic or meiotic chromosome transmission in the male germ line or due to paternal chromosome loss in the early embryo. We argue that the majority of autosomal genes that function in male fertility in Drosophila are represented by one or more alleles in the ms collection. Given the conservation of molecular mechanisms underlying important cellular processes, analysis of these mutations should provide insight into the genetic networks that control male fertility in Drosophila and other organisms, including humans.