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1.
Nature ; 518(7538): 236-9, 2015 Feb 12.
Artigo em Inglês | MEDLINE | ID: mdl-25607368

RESUMO

In the healthy adult brain synapses are continuously remodelled through a process of elimination and formation known as structural plasticity. Reduction in synapse number is a consistent early feature of neurodegenerative diseases, suggesting deficient compensatory mechanisms. Although much is known about toxic processes leading to synaptic dysfunction and loss in these disorders, how synaptic regeneration is affected is unknown. In hibernating mammals, cooling induces loss of synaptic contacts, which are reformed on rewarming, a form of structural plasticity. We have found that similar changes occur in artificially cooled laboratory rodents. Cooling and hibernation also induce a number of cold-shock proteins in the brain, including the RNA binding protein, RBM3 (ref. 6). The relationship of such proteins to structural plasticity is unknown. Here we show that synapse regeneration is impaired in mouse models of neurodegenerative disease, in association with the failure to induce RBM3. In both prion-infected and 5XFAD (Alzheimer-type) mice, the capacity to regenerate synapses after cooling declined in parallel with the loss of induction of RBM3. Enhanced expression of RBM3 in the hippocampus prevented this deficit and restored the capacity for synapse reassembly after cooling. RBM3 overexpression, achieved either by boosting endogenous levels through hypothermia before the loss of the RBM3 response or by lentiviral delivery, resulted in sustained synaptic protection in 5XFAD mice and throughout the course of prion disease, preventing behavioural deficits and neuronal loss and significantly prolonging survival. In contrast, knockdown of RBM3 exacerbated synapse loss in both models and accelerated disease and prevented the neuroprotective effects of cooling. Thus, deficient synapse regeneration, mediated at least in part by failure of the RBM3 stress response, contributes to synapse loss throughout the course of neurodegenerative disease. The data support enhancing cold-shock pathways as potential protective therapies in neurodegenerative disorders.


Assuntos
Temperatura Baixa , Resposta ao Choque Frio/fisiologia , Doenças Neurodegenerativas/metabolismo , Doenças Neurodegenerativas/patologia , Plasticidade Neuronal , Fármacos Neuroprotetores , Proteínas de Ligação a RNA/metabolismo , Sinapses/metabolismo , Doença de Alzheimer/metabolismo , Animais , Proteínas e Peptídeos de Choque Frio/metabolismo , Modelos Animais de Doenças , Hibernação/fisiologia , Hipocampo/metabolismo , Masculino , Camundongos , Príons/fisiologia , Proteínas de Ligação a RNA/genética , Regeneração
2.
Nature ; 485(7399): 507-11, 2012 May 06.
Artigo em Inglês | MEDLINE | ID: mdl-22622579

RESUMO

The mechanisms leading to neuronal death in neurodegenerative disease are poorly understood. Many of these disorders, including Alzheimer's, Parkinson's and prion diseases, are associated with the accumulation of misfolded disease-specific proteins. The unfolded protein response is a protective cellular mechanism triggered by rising levels of misfolded proteins. One arm of this pathway results in the transient shutdown of protein translation, through phosphorylation of the α-subunit of eukaryotic translation initiation factor, eIF2. Activation of the unfolded protein response and/or increased eIF2α-P levels are seen in patients with Alzheimer's, Parkinson's and prion diseases, but how this links to neurodegeneration is unknown. Here we show that accumulation of prion protein during prion replication causes persistent translational repression of global protein synthesis by eIF2α-P, associated with synaptic failure and neuronal loss in prion-diseased mice. Further, we show that promoting translational recovery in hippocampi of prion-infected mice is neuroprotective. Overexpression of GADD34, a specific eIF2α-P phosphatase, as well as reduction of levels of prion protein by lentivirally mediated RNA interference, reduced eIF2α-P levels. As a result, both approaches restored vital translation rates during prion disease, rescuing synaptic deficits and neuronal loss, thereby significantly increasing survival. In contrast, salubrinal, an inhibitor of eIF2α-P dephosphorylation, increased eIF2α-P levels, exacerbating neurotoxicity and significantly reducing survival in prion-diseased mice. Given the prevalence of protein misfolding and activation of the unfolded protein response in several neurodegenerative diseases, our results suggest that manipulation of common pathways such as translational control, rather than disease-specific approaches, may lead to new therapies preventing synaptic failure and neuronal loss across the spectrum of these disorders.


Assuntos
Fator de Iniciação 2 em Eucariotos/química , Fator de Iniciação 2 em Eucariotos/metabolismo , Doenças Neurodegenerativas/metabolismo , Fosfoproteínas/metabolismo , Príons/metabolismo , Biossíntese de Proteínas , Proteínas Repressoras/metabolismo , Animais , Morte Celular/efeitos dos fármacos , Cinamatos/farmacologia , Fator de Iniciação 2 em Eucariotos/análise , Hipocampo/citologia , Hipocampo/metabolismo , Hipocampo/patologia , Estimativa de Kaplan-Meier , Camundongos , Camundongos Endogâmicos C57BL , Doenças Neurodegenerativas/etiologia , Doenças Neurodegenerativas/patologia , Neurônios/efeitos dos fármacos , Neurônios/patologia , Fármacos Neuroprotetores , Fosfoproteínas/análise , Fosforilação , Proteínas PrPSc/análise , Proteínas PrPSc/metabolismo , Proteínas PrPSc/toxicidade , Doenças Priônicas/patologia , Príons/biossíntese , Príons/genética , Biossíntese de Proteínas/efeitos dos fármacos , Dobramento de Proteína/efeitos dos fármacos , Proteína Fosfatase 1/genética , Proteína Fosfatase 1/metabolismo , Proteínas Repressoras/análise , Proteínas Repressoras/química , Sinapses/efeitos dos fármacos , Sinapses/metabolismo , Sinapses/patologia , Transmissão Sináptica/efeitos dos fármacos , Tioureia/análogos & derivados , Tioureia/farmacologia , Resposta a Proteínas não Dobradas/fisiologia
3.
Methods Mol Biol ; 2725: 121-129, 2024.
Artigo em Inglês | MEDLINE | ID: mdl-37856021

RESUMO

Volume electron microscopy technologies such as serial block face scanning electron microscopy (SBF-SEM) allow the characterization of tissue organization and cellular content in three dimensions at nanoscale resolution. Here, we describe the procedure to process and image an air-liquid interface culture of human or mouse airway epithelial cells for visualization of the multiciliated epithelium by SBF-SEM in vertical or horizontal cross section.


Assuntos
Imageamento Tridimensional , Microscopia Eletrônica de Volume , Animais , Humanos , Camundongos , Imageamento Tridimensional/métodos , Microscopia Eletrônica de Varredura , Epitélio , Células Epiteliais
4.
Nat Commun ; 12(1): 4920, 2021 08 13.
Artigo em Inglês | MEDLINE | ID: mdl-34389715

RESUMO

Malignant mesothelioma (MpM) is an aggressive, invariably fatal tumour that is causally linked with asbestos exposure. The disease primarily results from loss of tumour suppressor gene function and there are no 'druggable' driver oncogenes associated with MpM. To identify opportunities for management of this disease we have carried out polysome profiling to define the MpM translatome. We show that in MpM there is a selective increase in the translation of mRNAs encoding proteins required for ribosome assembly and mitochondrial biogenesis. This results in an enhanced rate of mRNA translation, abnormal mitochondrial morphology and oxygen consumption, and a reprogramming of metabolic outputs. These alterations delimit the cellular capacity for protein biosynthesis, accelerate growth and drive disease progression. Importantly, we show that inhibition of mRNA translation, particularly through combined pharmacological targeting of mTORC1 and 2, reverses these changes and inhibits malignant cell growth in vitro and in ex-vivo tumour tissue from patients with end-stage disease. Critically, we show that these pharmacological interventions prolong survival in animal models of asbestos-induced mesothelioma, providing the basis for a targeted, viable therapeutic option for patients with this incurable disease.


Assuntos
Mesotelioma Maligno/genética , Oncogenes/genética , Biossíntese de Proteínas/genética , RNA Mensageiro/genética , Animais , Amianto , Humanos , Alvo Mecanístico do Complexo 1 de Rapamicina/antagonistas & inibidores , Alvo Mecanístico do Complexo 1 de Rapamicina/metabolismo , Alvo Mecanístico do Complexo 2 de Rapamicina/antagonistas & inibidores , Alvo Mecanístico do Complexo 2 de Rapamicina/metabolismo , Mesotelioma Maligno/induzido quimicamente , Mesotelioma Maligno/metabolismo , Camundongos Endogâmicos C57BL , Camundongos Knockout , Mitocôndrias/genética , Mitocôndrias/metabolismo , Naftiridinas/farmacologia , Polirribossomos/efeitos dos fármacos , Polirribossomos/metabolismo , Biossíntese de Proteínas/efeitos dos fármacos , RNA Mensageiro/metabolismo , Células Tumorais Cultivadas
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