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1.
Cell Rep ; 19(10): 1967-1976, 2017 06 06.
Artigo em Inglês | MEDLINE | ID: mdl-28591569

RESUMO

Alzheimer's disease is characterized by intracerebral deposition of ß-amyloid (Aß). While Aß40 is the most abundant form, neurotoxicity is mainly mediated by Aß42. Sequential cleavage of amyloid precursor protein (APP) by ß- and γ-secretases gives rise to full-length Aß (Aß1-x) and N-terminally truncated Aß' (Aß11-x) whereas cleavage by α- and γ-secretases leads to the shorter p3 peptides (Aß17-x). We uncovered significantly higher ratios of 42- versus 40-ending variants for Aß and Aß' than for p3 secreted by mouse neurons and human induced pluripotent stem cell (iPSC)-derived neurons or produced in a cell-free γ-secretase assay with recombinant APP-CTFs. The 42:40 ratio was highest for Aß', followed by Aß and then p3. Mass spectrometry analysis of APP intracellular domains revealed differential processing of APP-C83, APP-C89, and APP-C99 by γ-secretase already at the ε-cleavage stage. This mechanistic insight could aid in developing substrate-targeted modulators of APP-C99 processing to specifically lower the Aß42:Aß40 ratio without compromising γ-secretase function.


Assuntos
Doença de Alzheimer/metabolismo , Secretases da Proteína Precursora do Amiloide/metabolismo , Peptídeos beta-Amiloides/metabolismo , Neurônios/metabolismo , Fragmentos de Peptídeos/metabolismo , Doença de Alzheimer/patologia , Animais , Humanos , Células-Tronco Pluripotentes Induzidas/metabolismo , Células-Tronco Pluripotentes Induzidas/patologia , Camundongos , Camundongos Endogâmicos ICR , Neurônios/patologia
3.
EMBO J ; 34(17): 2237-54, 2015 Sep 02.
Artigo em Inglês | MEDLINE | ID: mdl-26105073

RESUMO

MicroRNAs (miRNAs) are important regulators of neuronal development, network connectivity, and synaptic plasticity. While many neuronal miRNAs were previously shown to modulate neuronal morphogenesis, little is known regarding the regulation of miRNA function. In a large-scale functional screen, we identified two novel regulators of neuronal miRNA function, Nova1 and Ncoa3. Both proteins are expressed in the nucleus and the cytoplasm of developing hippocampal neurons. We found that Nova1 and Ncoa3 stimulate miRNA function by different mechanisms that converge on Argonaute (Ago) proteins, core components of the miRNA-induced silencing complex (miRISC). While Nova1 physically interacts with Ago proteins, Ncoa3 selectively promotes the expression of Ago2 at the transcriptional level. We further show that Ncoa3 regulates dendritic complexity and dendritic spine maturation of hippocampal neurons in a miRNA-dependent fashion. Importantly, both the loss of miRNA activity and increased dendrite complexity upon Ncoa3 knockdown were rescued by Ago2 overexpression. Together, we uncovered two novel factors that control neuronal miRISC function at the level of Ago proteins, with possible implications for the regulation of synapse development and plasticity.


Assuntos
Proteínas Argonautas/biossíntese , Regulação da Expressão Gênica/fisiologia , MicroRNAs/biossíntese , Neurônios/metabolismo , Coativador 3 de Receptor Nuclear/metabolismo , Proteínas de Ligação a RNA/metabolismo , Animais , Proteínas Argonautas/genética , Células HEK293 , Humanos , MicroRNAs/genética , Antígeno Neuro-Oncológico Ventral , Neurônios/citologia , Coativador 3 de Receptor Nuclear/genética , Proteínas de Ligação a RNA/genética , Ratos , Ratos Sprague-Dawley
4.
Cell Rep ; 5(6): 1536-51, 2013 Dec 26.
Artigo em Inglês | MEDLINE | ID: mdl-24373285

RESUMO

Alzheimer's disease (AD) is characterized by cerebral deposition of ß-amyloid (Aß) peptides, which are generated from amyloid precursor protein (APP) by ß- and γ-secretases. APP and the secretases are membrane associated, but whether membrane trafficking controls Aß levels is unclear. Here, we performed an RNAi screen of all human Rab-GTPases, which regulate membrane trafficking, complemented with a Rab-GTPase-activating protein screen, and present a road map of the membrane-trafficking events regulating Aß production. We identify Rab11 and Rab3 as key players. Although retromers and retromer-associated proteins control APP recycling, we show that Rab11 controlled ß-secretase endosomal recycling to the plasma membrane and thus affected Aß production. Exome sequencing revealed a significant genetic association of Rab11A with late-onset AD, and network analysis identified Rab11A and Rab11B as components of the late-onset AD risk network, suggesting a causal link between Rab11 and AD. Our results reveal trafficking pathways that regulate Aß levels and show how systems biology approaches can unravel the molecular complexity underlying AD.


Assuntos
Doença de Alzheimer/metabolismo , Peptídeos beta-Amiloides/metabolismo , Proteínas rab de Ligação ao GTP/metabolismo , Secretases da Proteína Precursora do Amiloide/genética , Secretases da Proteína Precursora do Amiloide/metabolismo , Ácido Aspártico Endopeptidases/genética , Ácido Aspártico Endopeptidases/metabolismo , Membrana Celular/metabolismo , Endossomos/metabolismo , Exoma , Proteínas Ativadoras de GTPase/genética , Proteínas Ativadoras de GTPase/metabolismo , Células HeLa , Humanos , Transporte Proteico , Proteólise , RNA Interferente Pequeno/genética , Proteínas rab de Ligação ao GTP/genética , Proteínas rab3 de Ligação ao GTP/genética , Proteínas rab3 de Ligação ao GTP/metabolismo
5.
Curr Opin Genet Dev ; 21(4): 491-7, 2011 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-21561760

RESUMO

The regulation of synapse formation and plasticity in the developing and adult brain underlies a complex interplay of intrinsic genetic programs and extrinsic factors. Recent research identified microRNAs (miRNAs), a class of small non-coding RNAs, as a new functional layer in this regulatory network. Within only a few years, a network of synaptic miRNAs and their target genes has been extensively characterized, highlighting the importance of this mechanism for synapse development and physiology. Very recent data further provide insight into activity-dependent regulation of miRNAs, thereby connecting miRNAs with adaptive processes of neural circuits. First direct links between miRNA dysfunction and synaptic pathologies are emerging, raising the interest in these molecules as potential biomarkers and therapeutic targets in neurological disorders.


Assuntos
Encéfalo/metabolismo , MicroRNAs/metabolismo , Sinapses/metabolismo , Regiões 3' não Traduzidas , Animais , Encéfalo/embriologia , Encéfalo/crescimento & desenvolvimento , Regulação da Expressão Gênica no Desenvolvimento , Humanos , MicroRNAs/genética , Pequeno RNA não Traduzido/metabolismo , Sinapses/genética
6.
Nat Cell Biol ; 11(6): 705-16, 2009 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-19465924

RESUMO

The microRNA pathway has been implicated in the regulation of synaptic protein synthesis and ultimately in dendritic spine morphogenesis, a phenomenon associated with long-lasting forms of memory. However, the particular microRNAs (miRNAs) involved are largely unknown. Here we identify specific miRNAs that function at synapses to control dendritic spine structure by performing a functional screen. One of the identified miRNAs, miR-138, is highly enriched in the brain, localized within dendrites and negatively regulates the size of dendritic spines in rat hippocampal neurons. miR-138 controls the expression of acyl protein thioesterase 1 (APT1), an enzyme regulating the palmitoylation status of proteins that are known to function at the synapse, including the alpha(13) subunits of G proteins (Galpha(13)). RNA-interference-mediated knockdown of APT1 and the expression of membrane-localized Galpha(13) both suppress spine enlargement caused by inhibition of miR-138, suggesting that APT1-regulated depalmitoylation of Galpha(13) might be an important downstream event of miR-138 function. Our results uncover a previously unknown miRNA-dependent mechanism in neurons and demonstrate a previously unrecognized complexity of miRNA-dependent control of dendritic spine morphogenesis.


Assuntos
Espinhas Dendríticas , MicroRNAs/metabolismo , Sinapses , Tioléster Hidrolases/metabolismo , Animais , Sequência de Bases , Linhagem Celular , Espinhas Dendríticas/enzimologia , Espinhas Dendríticas/ultraestrutura , Subunidades alfa G12-G13 de Proteínas de Ligação ao GTP/metabolismo , Perfilação da Expressão Gênica , Hipocampo/citologia , Humanos , Lipoilação , Camundongos , Camundongos Endogâmicos C57BL , MicroRNAs/genética , Dados de Sequência Molecular , Morfogênese , Neurônios/citologia , Neurônios/metabolismo , Análise de Sequência com Séries de Oligonucleotídeos , Ratos , Receptores de Glutamato/metabolismo , Sinapses/metabolismo , Sinapses/ultraestrutura , Tioléster Hidrolases/antagonistas & inibidores , Tioléster Hidrolases/genética
7.
EMBO J ; 28(6): 697-710, 2009 Mar 18.
Artigo em Inglês | MEDLINE | ID: mdl-19197241

RESUMO

Neuronal activity orchestrates the proper development of the neuronal circuitry by regulating both transcriptional and post-transcriptional gene expression programmes. How these programmes are coordinated, however, is largely unknown. We found that the transcription of miR379-410, a large cluster of brain-specific microRNAs (miRNAs), is induced by increasing neuronal activity in primary rat neurons. Results from chromatin immunoprecipitation and luciferase reporter assays suggest that binding of the transcription factor myocyte enhancing factor 2 (Mef2) upstream of miR379-410 is necessary and sufficient for activity-dependent transcription of the cluster. Mef2-induced expression of at least three individual miRNAs of the miR379-410 cluster is required for activity-dependent dendritic outgrowth of hippocampal neurons. One of these miRNAs, the dendritic miR-134, promotes outgrowth by inhibiting translation of the mRNA encoding for the translational repressor Pumilio2. In summary, we have described a novel regulatory pathway that couples activity-dependent transcription to miRNA-dependent translational control of gene expression during neuronal development.


Assuntos
Dendritos/metabolismo , Proteínas de Domínio MADS/metabolismo , MicroRNAs/genética , Família Multigênica , Fatores de Regulação Miogênica/metabolismo , Organogênese , Proteínas de Ligação a RNA/metabolismo , Transcrição Gênica , Animais , Sequência de Bases , Sítios de Ligação , Regulação para Baixo , Regulação da Expressão Gênica no Desenvolvimento , Humanos , Fatores de Transcrição MEF2 , Dados de Sequência Molecular , Proteínas de Ligação a RNA/genética , Ratos , Ratos Sprague-Dawley
8.
Biochim Biophys Acta ; 1779(8): 471-8, 2008 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-18194678

RESUMO

The development and function of the nervous system is orchestrated by a plethora of gene regulatory mechanisms. MicroRNAs (miRNAs), an abundant class of small non-coding RNAs, are emerging as important post-transcriptional regulators of gene expression in the brain. MiRNAs function at all stages of neuronal development, ranging from the initial specification of neuronal cell types to the formation and plasticity of synaptic connections between individual neurons. Moreover, links between miRNA dysfunction and neurological diseases become more and more apparent. The study of this novel layer of gene regulation therefore promises to enrich our knowledge of brain function and pathology.


Assuntos
Encéfalo/metabolismo , MicroRNAs/fisiologia , Neurônios/fisiologia , Animais , Axônios/metabolismo , Encéfalo/embriologia , Encéfalo/crescimento & desenvolvimento , Encefalopatias/genética , Encefalopatias/metabolismo , Humanos , Plasticidade Neuronal
9.
J Comp Neurol ; 478(2): 176-88, 2004 Oct 11.
Artigo em Inglês | MEDLINE | ID: mdl-15349978

RESUMO

Animal models of focal ischemic infarcts reveal an impaired GABAergic (gamma-aminobutyric acid) neurotransmission. GABA, the main inhibitory neurotransmitter, is primarily taken up by specific sodium-dependent transporters. As these transporters play a crucial role in maintaining levels of GABA concentration, they may be functionally involved in ischemic processes. We investigated whether the mRNA and protein expression of GAT-1, the dominant neuronal GABA transporter, is altered after cortical infarct induced by photothrombosis in Wistar rats. In situ hybridization was performed to analyze GAT-1 mRNA-positive cells in cortical brain regions and the hippocampus. The lesion dramatically raised the number of GABA transporter mRNA-expressing cells in all investigated cortical regions. Double-labeling studies with a general neuronal marker and a marker for astrocytes revealed that cells expressing GAT-1 mRNA after photothrombosis are neurons. The mRNA expression pattern of all hippocampal subfields remained unchanged. In contrast, cortical GAT-1 protein density was only slightly affected and surprisingly in the opposite way. In the primary and secondary somatosensory cortex, density values were significantly reduced. Immunoreactivity was not altered in all investigated hippocampal areas. We found a marked discordance between the increased number of cells expressing GAT-1 mRNA in the cortex and the reduced tissue GAT-1 protein content. Focal brain ischemia obviously triggers mechanisms that interfere with GAT-1 transcriptional regulation and protein synthesis or turnover.


Assuntos
Encéfalo/metabolismo , Infarto Cerebral/metabolismo , Proteínas de Membrana Transportadoras/biossíntese , Neurônios/metabolismo , Animais , Infarto Cerebral/fisiopatologia , Modelos Animais de Doenças , Proteínas da Membrana Plasmática de Transporte de GABA , Processamento de Imagem Assistida por Computador , Imuno-Histoquímica , Hibridização In Situ , Masculino , RNA Mensageiro/análise , Ratos , Ratos Wistar
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