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1.
Mol Psychiatry ; 26(10): 5592-5607, 2021 10.
Artigo em Inglês | MEDLINE | ID: mdl-33144711

RESUMO

Although APP metabolism is being intensively investigated, a large fraction of its modulators is yet to be characterized. In this context, we combined two genome-wide high-content screenings to assess the functional impact of miRNAs and genes on APP metabolism and the signaling pathways involved. This approach highlighted the involvement of FERMT2 (or Kindlin-2), a genetic risk factor of Alzheimer's disease (AD), as a potential key modulator of axon guidance, a neuronal process that depends on the regulation of APP metabolism. We found that FERMT2 directly interacts with APP to modulate its metabolism, and that FERMT2 underexpression impacts axonal growth, synaptic connectivity, and long-term potentiation in an APP-dependent manner. Last, the rs7143400-T allele, which is associated with an increased AD risk and localized within the 3'UTR of FERMT2, induced a downregulation of FERMT2 expression through binding of miR-4504 among others. This miRNA is mainly expressed in neurons and significantly overexpressed in AD brains compared to controls. Altogether, our data provide strong evidence for a detrimental effect of FERMT2 underexpression in neurons and insight into how this may influence AD pathogenesis.


Assuntos
Doença de Alzheimer , Doença de Alzheimer/genética , Precursor de Proteína beta-Amiloide/genética , Humanos , Proteínas de Membrana , Proteínas de Neoplasias , Plasticidade Neuronal/genética , Neurônios , Fatores de Risco
2.
Acta Neuropathol ; 141(1): 39-65, 2021 01.
Artigo em Inglês | MEDLINE | ID: mdl-33079262

RESUMO

Several lines of recent evidence indicate that the amyloid precursor protein-derived C-terminal fragments (APP-CTFs) could correspond to an etiological trigger of Alzheimer's disease (AD) pathology. Altered mitochondrial homeostasis is considered an early event in AD development. However, the specific contribution of APP-CTFs to mitochondrial structure, function, and mitophagy defects remains to be established. Here, we demonstrate in neuroblastoma SH-SY5Y cells expressing either APP Swedish mutations, or the ß-secretase-derived APP-CTF fragment (C99) combined with ß- and γ-secretase inhibition, that APP-CTFs accumulation independently of Aß triggers excessive mitochondrial morphology alteration (i.e., size alteration and cristae disorganization) associated with enhanced mitochondrial reactive oxygen species production. APP-CTFs accumulation also elicit basal mitophagy failure illustrated by enhanced conversion of LC3, accumulation of LC3-I and/or LC3-II, non-degradation of SQSTM1/p62, inconsistent Parkin and PINK1 recruitment to mitochondria, enhanced levels of membrane and matrix mitochondrial proteins, and deficient fusion of mitochondria with lysosomes. We confirm the contribution of APP-CTFs accumulation to morphological mitochondria alteration and impaired basal mitophagy in vivo in young 3xTgAD transgenic mice treated with γ-secretase inhibitor as well as in adeno-associated-virus-C99 injected mice. Comparison of aged 2xTgAD and 3xTgAD mice indicates that, besides APP-CTFs, an additional contribution of Aß to late-stage mitophagy activation occurs. Importantly, we report on mitochondrial accumulation of APP-CTFs in human post-mortem sporadic AD brains correlating with mitophagy failure molecular signature. Since defective mitochondria homeostasis plays a pivotal role in AD pathogenesis, targeting mitochondrial dysfunctions and/or mitophagy by counteracting early APP-CTFs accumulation may represent relevant therapeutic interventions in AD.


Assuntos
Doença de Alzheimer/patologia , Precursor de Proteína beta-Amiloide/genética , Precursor de Proteína beta-Amiloide/metabolismo , Encéfalo/patologia , Mitocôndrias/patologia , Mitocôndrias/ultraestrutura , Mitofagia/genética , Idoso , Idoso de 80 Anos ou mais , Doença de Alzheimer/metabolismo , Secretases da Proteína Precursora do Amiloide/antagonistas & inibidores , Secretases da Proteína Precursora do Amiloide/metabolismo , Animais , Ácido Aspártico Endopeptidases/antagonistas & inibidores , Ácido Aspártico Endopeptidases/metabolismo , Autopsia , Linhagem Celular , Feminino , Humanos , Potencial da Membrana Mitocondrial , Camundongos , Mitocôndrias/metabolismo , Fragmentos de Peptídeos/genética , Fragmentos de Peptídeos/metabolismo , Espécies Reativas de Oxigênio/metabolismo
4.
Acta Neuropathol ; 134(5): 749-767, 2017 11.
Artigo em Inglês | MEDLINE | ID: mdl-28631094

RESUMO

The mechanisms underlying ryanodine receptor (RyR) dysfunction associated with Alzheimer disease (AD) are still not well understood. Here, we show that neuronal RyR2 channels undergo post-translational remodeling (PKA phosphorylation, oxidation, and nitrosylation) in brains of AD patients, and in two murine models of AD (3 × Tg-AD, APP +/- /PS1 +/-). RyR2 is depleted of calstabin2 (KFBP12.6) in the channel complex, resulting in endoplasmic reticular (ER) calcium (Ca2+) leak. RyR-mediated ER Ca2+ leak activates Ca2+-dependent signaling pathways, contributing to AD pathogenesis. Pharmacological (using a novel RyR stabilizing drug Rycal) or genetic rescue of the RyR2-mediated intracellular Ca2+ leak improved synaptic plasticity, normalized behavioral and cognitive functions and reduced Aß load. Genetically altered mice with congenitally leaky RyR2 exhibited premature and severe defects in synaptic plasticity, behavior and cognitive function. These data provide a mechanism underlying leaky RyR2 channels, which could be considered as potential AD therapeutic targets.


Assuntos
Doença de Alzheimer/metabolismo , Cálcio/metabolismo , Transtornos Cognitivos/metabolismo , Processamento de Proteína Pós-Traducional , Canal de Liberação de Cálcio do Receptor de Rianodina/metabolismo , Doença de Alzheimer/patologia , Animais , Sinalização do Cálcio , Transtornos Cognitivos/patologia , Proteínas Quinases Dependentes de AMP Cíclico/metabolismo , Feminino , Humanos , Masculino , Aprendizagem em Labirinto/fisiologia , Camundongos , Camundongos Transgênicos , Estresse Oxidativo/fisiologia , Fosforilação , Reconhecimento Psicológico/fisiologia , Retículo Sarcoplasmático/metabolismo
5.
Acta Neuropathol ; 133(6): 955-966, 2017 06.
Artigo em Inglês | MEDLINE | ID: mdl-27933404

RESUMO

Genome-wide association studies (GWASs) have identified 19 susceptibility loci for Alzheimer's disease (AD). However, understanding how these genes are involved in the pathophysiology of AD is one of the main challenges of the "post-GWAS" era. At least 123 genes are located within the 19 susceptibility loci; hence, a conventional approach (studying the genes one by one) would not be time- and cost-effective. We therefore developed a genome-wide, high-content siRNA screening approach and used it to assess the functional impact of gene under-expression on APP metabolism. We found that 832 genes modulated APP metabolism. Eight of these genes were located within AD susceptibility loci. Only FERMT2 (a ß3-integrin co-activator) was also significantly associated with a variation in cerebrospinal fluid Aß peptide levels in 2886 AD cases. Lastly, we showed that the under-expression of FERMT2 increases Aß peptide production by raising levels of mature APP at the cell surface and facilitating its recycling. Taken as a whole, our data suggest that FERMT2 modulates the AD risk by regulating APP metabolism and Aß peptide production.


Assuntos
Precursor de Proteína beta-Amiloide/metabolismo , Proteínas de Membrana/genética , Proteínas de Membrana/metabolismo , Proteínas de Neoplasias/genética , Proteínas de Neoplasias/metabolismo , RNA Interferente Pequeno/genética , Doença de Alzheimer/genética , Doença de Alzheimer/metabolismo , Doença de Alzheimer/patologia , Animais , Biomarcadores/líquido cefalorraquidiano , Membrana Celular/metabolismo , Córtex Cerebral/metabolismo , Córtex Cerebral/patologia , Loci Gênicos , Predisposição Genética para Doença , Estudo de Associação Genômica Ampla , Células HEK293 , Hipocampo/metabolismo , Hipocampo/patologia , Humanos , Neurônios/metabolismo , Neurônios/patologia , Interferência de RNA , Ratos
7.
Cell Mol Life Sci ; 73(1): 217-36, 2016 Jan.
Artigo em Inglês | MEDLINE | ID: mdl-26202697

RESUMO

Membrane-type 5-matrix metalloproteinase (MT5-MMP) is a proteinase mainly expressed in the nervous system with emerging roles in brain pathophysiology. The implication of MT5-MMP in Alzheimer's disease (AD), notably its interplay with the amyloidogenic process, remains elusive. Accordingly, we crossed the genetically engineered 5xFAD mouse model of AD with MT5-MMP-deficient mice and examined the impact of MT5-MMP deficiency in bigenic 5xFAD/MT5-MMP(-/-) mice. At early stages (4 months) of the pathology, the levels of amyloid beta peptide (Aß) and its amyloid precursor protein (APP) C-terminal fragment C99 were largely reduced in the cortex and hippocampus of 5xFAD/MT5-MMP(-/-), compared to 5xFAD mice. Reduced amyloidosis in bigenic mice was concomitant with decreased glial reactivity and interleukin-1ß (IL-1ß) levels, and the preservation of long-term potentiation (LTP) and spatial learning, without changes in the activity of α-, ß- and γ-secretases. The positive impact of MT5-MMP deficiency was still noticeable at 16 months of age, as illustrated by reduced amyloid burden and gliosis, and a better preservation of the cortical neuronal network and synaptophysin levels in bigenic mice. MT5-MMP expressed in HEKswe cells colocalized and co-immunoprecipitated with APP and significantly increased the levels of Aß and C99. MT5-MMP also promoted the release of a soluble APP fragment of 95 kDa (sAPP95) in HEKswe cells. sAPP95 levels were significantly reduced in brain homogenates of 5xFAD/MT5-MMP(-/-) mice, supporting altogether the idea that MT5-MMP influences APP processing. MT5-MMP emerges as a new pro-amyloidogenic regulator of APP metabolism, whose deficiency alleviates amyloid pathology, neuroinflammation and cognitive decline.


Assuntos
Doença de Alzheimer/enzimologia , Doença de Alzheimer/fisiopatologia , Hipocampo/enzimologia , Hipocampo/fisiopatologia , Metaloproteinases da Matriz Associadas à Membrana/metabolismo , Doença de Alzheimer/genética , Doença de Alzheimer/patologia , Secretases da Proteína Precursora do Amiloide/análise , Secretases da Proteína Precursora do Amiloide/metabolismo , Peptídeos beta-Amiloides/análise , Peptídeos beta-Amiloides/metabolismo , Precursor de Proteína beta-Amiloide/análise , Precursor de Proteína beta-Amiloide/metabolismo , Animais , Cognição , Feminino , Deleção de Genes , Células HEK293 , Hipocampo/metabolismo , Hipocampo/patologia , Humanos , Potenciação de Longa Duração , Masculino , Metaloproteinases da Matriz Associadas à Membrana/análise , Metaloproteinases da Matriz Associadas à Membrana/genética , Camundongos Endogâmicos C57BL , Camundongos Transgênicos , Aprendizagem Espacial
8.
Acta Neuropathol ; 132(2): 257-276, 2016 08.
Artigo em Inglês | MEDLINE | ID: mdl-27138984

RESUMO

Endosomal-autophagic-lysosomal (EAL) dysfunction is an early and prominent neuropathological feature of Alzheimers's disease, yet the exact molecular mechanisms contributing to this pathology remain undefined. By combined biochemical, immunohistochemical and ultrastructural approaches, we demonstrate a link between EAL pathology and the intraneuronal accumulation of the ß-secretase-derived ßAPP fragment (C99) in two in vivo models, 3xTgAD mice and adeno-associated viral-mediated C99-infected mice. We present a pathological loop in which the accumulation of C99 is both the effect and causality of impaired lysosomal-autophagic function. The deleterious effect of C99 was found to be linked to its aggregation within EAL-vesicle membranes leading to disrupted lysosomal proteolysis and autophagic impairment. This effect was Aß independent and was even exacerbated when γ-secretase was pharmacologically inhibited. No effect was observed in inhibitor-treated wild-type animals suggesting that lysosomal dysfunction was indeed directly linked to C99 accumulation. In some brain areas, strong C99 expression also led to inflammatory responses and synaptic dysfunction. Taken together, this work demonstrates a toxic effect of C99 which could underlie some of the early-stage anatomical hallmarks of Alzheimer's disease pathology. Our work also proposes molecular mechanisms likely explaining some of the unfavorable side-effects associated with γ-secretase inhibitor-directed therapies.


Assuntos
Doença de Alzheimer/patologia , Secretases da Proteína Precursora do Amiloide/metabolismo , Precursor de Proteína beta-Amiloide/metabolismo , Encéfalo/patologia , Neurônios/metabolismo , Doença de Alzheimer/metabolismo , Peptídeos beta-Amiloides/metabolismo , Animais , Autofagia/fisiologia , Encéfalo/metabolismo , Modelos Animais de Doenças , Endossomos/metabolismo , Lisossomos/metabolismo , Camundongos Endogâmicos C57BL , Camundongos Transgênicos , Neurônios/patologia
9.
Cell Death Dis ; 15(5): 367, 2024 May 28.
Artigo em Inglês | MEDLINE | ID: mdl-38806484

RESUMO

Mitochondria dysfunctions and mitophagy failure have been associated with several Alzheimer's disease (AD) related molecular actors including amyloid beta (Aß) and recently the amyloid precursor protein-C terminal fragments (APP-CTFs). The efficacy of the mitophagy process in neurons relies on regulated mitochondrial transport along axons involving a complex molecular machinery. The contribution of the amyloid precursor protein (APP) and its derived fragments to the mitochondrial transport machinery alterations in AD have not been investigated before. We report herein a change of the expression of mitochondrial transport proteins (SNPH and Miro1), motor adapters (TRANK1 and TRAK2), and components of the dynein and kinesin motors (i.e., IC1,2 and Kif5 (A, B, C) isoforms) by endogenous APP and by overexpression of APP carrying the familial Swedish mutation (APPswe). We show that APP-CTFs and Aß concomitantly regulate the expression of a set of transport proteins as demonstrated in APPswe cells treated with ß- and γ-secretase inhibitors and in cells Knock-down for presenilin 1 and 2. We further report the impact of APP-CTFs on the expression of transport proteins in AAV-injected C99 mice brains. Our data also indicate that both Aß oligomers (Aßo) and APP-CTFs impair the colocalization of mitochondria and transport proteins. This has been demonstrated in differentiated SH-SY5Y naive cells treated with Aßo and in differentiated SH-SY5Y and murine primary neurons expressing APPswe and treated with the γ-secretase inhibitor. Importantly, we uncover that the expression of a set of transport proteins is modulated in a disease-dependent manner in 3xTgAD mice and in human sporadic AD brains. This study highlights molecular mechanisms underlying mitochondrial transport defects in AD that likely contribute to mitophagy failure and disease progression.


Assuntos
Doença de Alzheimer , Precursor de Proteína beta-Amiloide , Mitocôndrias , Doença de Alzheimer/metabolismo , Doença de Alzheimer/patologia , Doença de Alzheimer/genética , Precursor de Proteína beta-Amiloide/metabolismo , Precursor de Proteína beta-Amiloide/genética , Animais , Mitocôndrias/metabolismo , Humanos , Camundongos , Camundongos Transgênicos , Neurônios/metabolismo , Peptídeos beta-Amiloides/metabolismo , Proteínas Mitocondriais/metabolismo , Proteínas Mitocondriais/genética , Secretases da Proteína Precursora do Amiloide/metabolismo , Cinesinas/metabolismo , Transporte Biológico , Mitofagia , Proteínas do Tecido Nervoso , Proteínas rho de Ligação ao GTP , Peptídeos e Proteínas de Sinalização Intracelular
10.
J Neurosci ; 32(46): 16243-1655a, 2012 Nov 14.
Artigo em Inglês | MEDLINE | ID: mdl-23152608

RESUMO

Triple-transgenic mice (3xTgAD) overexpressing Swedish-mutated ß-amyloid precursor protein (ßAPP(swe)), P310L-Tau (Tau(P301L)), and physiological levels of M146V-presenilin-1 (PS1(M146V)) display extracellular amyloid-ß peptides (Aß) deposits and Tau tangles. More disputed is the observation that these mice accumulate intraneuronal Aß that has been linked to synaptic dysfunction and cognitive deficits. Here, we provide immunohistological, genetic, and pharmacological evidences for early, age-dependent, and hippocampus-specific accumulation of the ß-secretase-derived ßAPP fragment C99 that is observed from 3 months of age and enhanced by pharmacological blockade of γ-secretase. Notably, intracellular Aß is only detectable several months later and appears, as is the case of C99, in enlarged cathepsin B-positive structures, while extracellular Aß deposits are detected ~12 months of age and beyond. Early C99 production occurs mainly in the CA1/subicular interchange area of the hippocampus corresponding to the first region exhibiting plaques and tangles in old mice. Furthermore, the comparison of 3xTgAD mice with double-transgenic mice bearing the ßAPP(swe) and Tau(P301L) mutations but expressing endogenous PS1 (2xTgAD) demonstrate that C99 accumulation is not accounted for by a loss of function triggered by PS1 mutation that would have prevented C99 secondary cleavage by γ-secretase. Together, our work identifies C99 as the earliest ßAPP catabolite and main contributor to the intracellular ßAPP-related immunoreactivity in 3xTgAD mice, suggesting its implication as an initiator of the neurodegenerative process and cognitive alterations taking place in this mouse model.


Assuntos
Secretases da Proteína Precursora do Amiloide/metabolismo , Peptídeos beta-Amiloides/fisiologia , Precursor de Proteína beta-Amiloide/fisiologia , Hipocampo/patologia , Interneurônios/patologia , Fragmentos de Peptídeos/fisiologia , Envelhecimento/fisiologia , Secretases da Proteína Precursora do Amiloide/antagonistas & inibidores , Precursor de Proteína beta-Amiloide/química , Animais , Western Blotting , Eletroforese em Gel de Poliacrilamida , Ensaio de Imunoadsorção Enzimática , Hipocampo/enzimologia , Hipocampo/crescimento & desenvolvimento , Hipocampo/metabolismo , Imuno-Histoquímica , Imunoprecipitação , Camundongos , Camundongos Transgênicos , Fragmentos de Peptídeos/química , Presenilina-1/genética , Presenilina-1/metabolismo , Espectrometria de Massas por Ionização e Dessorção a Laser Assistida por Matriz , Proteínas tau/genética , Proteínas tau/metabolismo
11.
J Biol Chem ; 287(7): 5021-32, 2012 Feb 10.
Artigo em Inglês | MEDLINE | ID: mdl-22184125

RESUMO

In physiological conditions, both ß-amyloid precursor protein (ßAPP) and cellular prion (PrP(c)) undergo similar disintegrin-mediated α-secretase cleavage yielding N-terminal secreted products referred to as soluble amyloid precursor protein-α (sAPPα) and N1, respectively. We recently demonstrated that N1 displays neuroprotective properties by reducing p53-dependent cell death both in vitro and in vivo. In this study, we examined the potential of N1 as a neuroprotector against amyloid ß (Aß)-mediated toxicity. We first show that both recombinant sAPPα and N1, but not its inactive parent fragment N2, reduce staurosporine-stimulated caspase-3 activation and TUNEL-positive cell death by lowering p53 promoter transactivation and activity in human cells. We demonstrate that N1 also lowers toxicity, cell death, and p53 pathway exacerbation triggered by Swedish mutated ßAPP overexpression in human cells. We designed a CHO cell line overexpressing the London mutated ßAPP (APP(LDN)) that yields Aß oligomers. N1 protected primary cultured neurons against toxicity and cell death triggered by oligomer-enriched APP(LDN)-derived conditioned medium. Finally, we establish that N1 also protects neurons against oligomers extracted from Alzheimer disease-affected brain tissues. Overall, our data indicate that a cellular prion catabolite could interfere with Aß-associated toxicity and that its production could be seen as a cellular protective mechanism aimed at compensating for an sAPPα deficit taking place at the early asymptomatic phase of Alzheimer disease.


Assuntos
Doença de Alzheimer/metabolismo , Secretases da Proteína Precursora do Amiloide/metabolismo , Precursor de Proteína beta-Amiloide/metabolismo , Peptídeos/metabolismo , Proteínas PrPC/metabolismo , Multimerização Proteica , Doença de Alzheimer/patologia , Secretases da Proteína Precursora do Amiloide/genética , Precursor de Proteína beta-Amiloide/genética , Animais , Células CHO , Caspase 3/genética , Caspase 3/metabolismo , Morte Celular , Cricetinae , Cricetulus , Ativação Enzimática/efeitos dos fármacos , Ativação Enzimática/genética , Inibidores Enzimáticos/farmacologia , Células HEK293 , Humanos , Camundongos , Neurônios/metabolismo , Neurônios/patologia , Peptídeos/genética , Proteínas PrPC/genética , Estaurosporina/farmacologia
12.
Cells ; 12(24)2023 12 08.
Artigo em Inglês | MEDLINE | ID: mdl-38132122

RESUMO

The SORL1 gene encodes LR11/SorLA, a protein that binds ß-amyloid precursor protein (APP) and drives its intracellular trafficking. SORL1 mutations, occurring frequently in a subset of familial cases of Alzheimer's disease (AD), have been documented, but their pathogenic potential is not yet clear and questions remain concerning their putative influence on the physiopathological processing of APP. We have assessed the influence of two SORL1 mutations that were described as likely disease-causing and that were associated with either benign (SorLA924) or severe (SorLA511) AD phenotypes. We examined the influence of wild-type and mutants SorLA in transiently transfected HEK293 cells expressing either wild-type or Swedish mutated APP on APP expression, secreted Aß and sAPPα levels, intracellular Aß 40 and Aß42 peptides, APP-CTFs (C99 and C83) expressions, α-, ß- and γ-secretases expressions and activities as well as Aß and CTFs-degrading enzymes. These paradigms were studied in control conditions or after pharmacological proteasomal modulation. We also established stably transfected CHO cells expressing wild-type SorLA and established the colocalization of APP and either wild-type or mutant SorLA. SorLA mutations partially disrupt co-localization of wild-type sorLA with APP. Overall, although we mostly confirmed previous data concerning the influence of wild-type SorLA on APP processing, we were unable to evidence significant alterations triggered by our set of SorLA mutants, whatever the cells or pharmacological conditions examined. Our study , however, does not rule out the possibility that other AD-linked SORL1 mutations could indeed affect APP processing, and that pathogenic mutations examined in the present study could interfere with other cellular pathways/triggers in AD.


Assuntos
Doença de Alzheimer , Precursor de Proteína beta-Amiloide , Animais , Cricetinae , Humanos , Doença de Alzheimer/metabolismo , Peptídeos beta-Amiloides/metabolismo , Precursor de Proteína beta-Amiloide/genética , Precursor de Proteína beta-Amiloide/metabolismo , Cricetulus , Células HEK293 , Proteínas Relacionadas a Receptor de LDL/metabolismo , Proteínas de Membrana Transportadoras/genética , Mutação/genética
13.
J Biol Chem ; 286(33): 29192-29206, 2011 Aug 19.
Artigo em Inglês | MEDLINE | ID: mdl-21586567

RESUMO

The α-secretases A disintegrin and metalloprotease 10 (ADAM10) and ADAM17 trigger constitutive and regulated processing of the cellular prion protein (PrP(c)) yielding N1 fragment. The latter depends on protein kinase C (PKC)-coupled M1/M3 muscarinic receptor activation and subsequent phosphorylation of ADAM17 on its intracytoplasmic threonine 735. Here we show that regulated PrP(c) processing and ADAM17 phosphorylation and activation are controlled by the extracellular-regulated kinase-1/MAP-ERK kinase (ERK1/MEK) cascade. Thus, reductions of ERK1 or MEK activities by dominant-negative analogs, pharmacological inhibition, or genetic ablation all impair N1 secretion, whereas constitutively active proteins increase N1 recovery in the conditioned medium. Interestingly, we also observed an ERK1-mediated enhanced expression of PrP(c). We demonstrate that the ERK1-associated increase in PrP(c) promoter transactivation and mRNA levels involve transcription factor AP-1 as a downstream effector. Altogether, our data identify ERK1 as an important regulator of PrP(c) cellular homeostasis and indicate that this kinase exerts a dual control of PrP(c) levels through transcriptional and post-transcriptional mechanisms.


Assuntos
Secretases da Proteína Precursora do Amiloide/metabolismo , Proteína Quinase 3 Ativada por Mitógeno/metabolismo , Proteínas PrPC/metabolismo , Regiões Promotoras Genéticas/fisiologia , Ativação Transcricional/fisiologia , Proteínas ADAM/genética , Proteínas ADAM/metabolismo , Proteína ADAM10 , Proteína ADAM17 , Secretases da Proteína Precursora do Amiloide/genética , Animais , Células HEK293 , Humanos , Proteínas de Membrana/genética , Proteínas de Membrana/metabolismo , Camundongos , Camundongos Knockout , Proteína Quinase 3 Ativada por Mitógeno/genética , Fosforilação/fisiologia , Proteínas PrPC/genética , RNA Mensageiro/biossíntese , RNA Mensageiro/genética , Receptor Muscarínico M1/genética , Receptor Muscarínico M1/metabolismo , Receptor Muscarínico M3/genética , Receptor Muscarínico M3/metabolismo , Fator de Transcrição AP-1/genética , Fator de Transcrição AP-1/metabolismo
14.
Neurobiol Aging ; 71: 21-31, 2018 11.
Artigo em Inglês | MEDLINE | ID: mdl-30071370

RESUMO

The triple transgenic mouse model (3×TgAD: APPswe, TauP301L, PS1M146V) recapitulates both amyloid ß (Aß)- and tau-related lesions as well as synaptic and memory deficits. In these mice, we reported an early apathy-like behavior and alterations in synaptic plasticity appearing concomitantly with intraneuronal accumulation of C99 in the subiculum. To delineate the genuine contribution of C99 on the above phenotypes, we generated double transgenic mice (2×TgAD: APPswe, TauP301L) that accumulate C99 without Aß deposition or hyperphosphorylation of tau and compared them to 3×TgAD mice. Here, we show that both TgAD mice display similar decreases in long-term potentiation and in spontaneous locomotor activity measured by actimetry suggesting that the synaptic alterations and the apathy-like behavior were likely linked to C99 rather than Aß. However, spatial learning alterations, assessed by the Morris water maze task, are more pronounced in 3×TgAD than in 2×TgAD, suggesting that both Aß and C99 contribute to defects in the acquisition of spatial information. Finally, even if similar results are observed in males, cognitive and non-cognitive deficits are more severe in females.


Assuntos
Doença de Alzheimer/metabolismo , Precursor de Proteína beta-Amiloide/metabolismo , Apatia/fisiologia , Potenciação de Longa Duração , Neurônios/metabolismo , Aprendizagem Espacial/fisiologia , Sinapses/fisiologia , Precursor de Proteína beta-Amiloide/genética , Animais , Modelos Animais de Doenças , Feminino , Hipocampo/metabolismo , Locomoção , Masculino , Camundongos Transgênicos , Fragmentos de Peptídeos/genética , Fragmentos de Peptídeos/metabolismo , Fenótipo , Fosforilação , Proteínas tau/genética , Proteínas tau/metabolismo
15.
J Alzheimers Dis ; 55(4): 1549-1570, 2017.
Artigo em Inglês | MEDLINE | ID: mdl-27911326

RESUMO

Alteration of mitochondria-associated membranes (MAMs) has been proposed to contribute to the pathogenesis of Alzheimer's disease (AD). We studied herein the subcellular distribution, the processing, and the protein interactome of the amyloid-ß protein precursor (AßPP) and its proteolytic products in MAMs. We reveal that AßPP and its catabolites are present in MAMs in cellular models overexpressing wild type AßPP or AßPP harboring the double Swedish or London familial AD mutations, and in brains of transgenic mice model of AD. Furthermore, we evidenced that both ß- and γ-secretases are present and harbor AßPP processing activities in MAMs. Interestingly, cells overexpressing APPswe show increased ER-mitochondria contact sites. We also document increased neutral lipid accumulation linked to Aß production and reversed by inhibiting ß- or γ-secretases. Using a proteomic approach, we show that AßPP and its catabolites interact with key proteins of MAMs controlling mitochondria and ER functions. These data highlight the role of AßPP processing and proteomic interactome in MAMs deregulation taking place in AD.


Assuntos
Precursor de Proteína beta-Amiloide/metabolismo , Membrana Celular/metabolismo , Mitocôndrias/metabolismo , Secretases da Proteína Precursora do Amiloide/metabolismo , Peptídeos beta-Amiloides/genética , Peptídeos beta-Amiloides/metabolismo , Precursor de Proteína beta-Amiloide/genética , Animais , Células CHO , Linhagem Celular Tumoral , Membrana Celular/efeitos dos fármacos , Membrana Celular/ultraestrutura , Cricetulus , Complexo IV da Cadeia de Transporte de Elétrons/metabolismo , Inibidores Enzimáticos/farmacologia , Regulação da Expressão Gênica/efeitos dos fármacos , Regulação da Expressão Gênica/fisiologia , Humanos , Imunoprecipitação , Camundongos , Camundongos Transgênicos , Microscopia Eletrônica , Mitocôndrias/efeitos dos fármacos , Mitocôndrias/ultraestrutura , Mutação/genética , Neuroblastoma/patologia , Presenilina-1/genética , Presenilina-1/metabolismo , Pirazóis/farmacologia , Quinolinas/farmacologia , Canal de Liberação de Cálcio do Receptor de Rianodina/metabolismo , Espectrometria de Massas por Ionização e Dessorção a Laser Assistida por Matriz , Transfecção , Canal de Ânion 1 Dependente de Voltagem/metabolismo
16.
Sci Rep ; 6: 37436, 2016 11 17.
Artigo em Inglês | MEDLINE | ID: mdl-27853315

RESUMO

The aspartyl protease ß-site APP cleaving enzyme, BACE1, is the rate-limiting enzyme involved in the production of amyloid-ß peptide, which accumulates in both sporadic and familial cases of Alzheimer's disease and is at the center of gravity of the amyloid cascade hypothesis. In this context, unravelling the molecular mechanisms controlling BACE1 expression and activity in both physiological and pathological conditions remains of major importance. We previously demonstrated that Aß controlled BACE1 transcription in an NFκB-dependent manner. Here, we delineate an additional cellular pathway by which natural and synthetic Aß42 oligomers enhance active X-box binding protein XBP-1s. XBP-1s lowers BACE1 expression and activity indirectly, via the up-regulation of the ubiquitin-ligase HRD1 that acts as an endogenous down-regulator of BACE1. Thus, we delineate a novel pathway by which cells could compensate for Aß42 oligomers production and thus, associated toxicity, by triggering a compensatory mechanism aimed at lowering BACE-1-mediated Aß production by a molecular cascade involving XBP-1s and HRD1. It thus identifies HRD1 as a potential target for a novel Aß-centered therapeutic strategy.


Assuntos
Secretases da Proteína Precursora do Amiloide/genética , Peptídeos beta-Amiloides/farmacologia , Ácido Aspártico Endopeptidases/genética , Neurônios/efeitos dos fármacos , Fragmentos de Peptídeos/farmacologia , Ubiquitina-Proteína Ligases/genética , Proteína 1 de Ligação a X-Box/genética , Secretases da Proteína Precursora do Amiloide/metabolismo , Animais , Ácido Aspártico Endopeptidases/metabolismo , Linhagem Celular , Linhagem Celular Tumoral , Cicloeximida/farmacologia , Fibroblastos/citologia , Fibroblastos/efeitos dos fármacos , Fibroblastos/metabolismo , Regulação da Expressão Gênica/efeitos dos fármacos , Células HEK293 , Humanos , Luciferases/genética , Luciferases/metabolismo , Camundongos , Neurônios/citologia , Neurônios/metabolismo , Plasmídeos/química , Plasmídeos/metabolismo , Inibidores da Síntese de Proteínas/farmacologia , Proteínas Proto-Oncogênicas c-myc/genética , Proteínas Proto-Oncogênicas c-myc/metabolismo , Proteínas Recombinantes de Fusão/genética , Proteínas Recombinantes de Fusão/metabolismo , Transdução de Sinais , Transfecção , Ubiquitina-Proteína Ligases/metabolismo , Proteína 1 de Ligação a X-Box/metabolismo
17.
Front Mol Neurosci ; 9: 163, 2016.
Artigo em Inglês | MEDLINE | ID: mdl-28119565

RESUMO

We previously reported that deficiency of membrane-type five matrix metalloproteinase (MT5-MMP) prevents amyloid pathology in the cortex and hippocampus of 5xFAD mice, and ameliorates the functional outcome. We have now investigated whether the integrity of another important area affected in Alzheimer's disease (AD), the frontal cortex, was also preserved upon MT5-MMP deficiency in 4-month old mice at prodromal stages of the pathology. We used the olfactory H-maze (OHM) to show that learning impairment associated with dysfunctions of the frontal cortex in 5xFAD was prevented in bigenic 5xFAD/MT5-MMP-/- mice. The latter exhibited concomitant drastic reductions of amyloid beta peptide (Aß) assemblies (soluble, oligomeric and fibrillary) and its immediate precursor, C99. Simultaneously, astrocyte reactivity and tumor necrosis factor alpha (TNF-α) levels were also lowered. Moreover, MT5-MMP deficiency induced a decrease in N-terminal soluble fragments of amyloid precursor protein (APP), including soluble APPα (sAPPα), sAPPß and the MT5-MMP-linked fragment of 95 kDa, sAPP95. However, the lack of MT5-MMP did not affect the activity of ß- and γ-secretases. In cultured HEKswe cells, transiently expressed MT5-MMP localized to early endosomes and increased the content of APP and Aß40 in these organelles, as well as Aß levels in cell supernatants. This is the first evidence that the pro-amyloidogenic features of MT5-MMP lie, at least in part, on the ability of the proteinase to promote trafficking into one of the amyloidogenic subcellular loci. Together, our data further support the pathogenic role of MT5-MMP in AD and that its inhibition improves the functional and pathological outcomes, in this case in the frontal cortex. These data also support the idea that MT5-MMP could become a novel therapeutic target in AD.

18.
EBioMedicine ; 9: 278-292, 2016 Jul.
Artigo em Inglês | MEDLINE | ID: mdl-27333034

RESUMO

Although several ADAMs (A disintegrin-like and metalloproteases) have been shown to contribute to the amyloid precursor protein (APP) metabolism, the full spectrum of metalloproteases involved in this metabolism remains to be established. Transcriptomic analyses centred on metalloprotease genes unraveled a 50% decrease in ADAM30 expression that inversely correlates with amyloid load in Alzheimer's disease brains. Accordingly, in vitro down- or up-regulation of ADAM30 expression triggered an increase/decrease in Aß peptides levels whereas expression of a biologically inactive ADAM30 (ADAM30(mut)) did not affect Aß secretion. Proteomics/cell-based experiments showed that ADAM30-dependent regulation of APP metabolism required both cathepsin D (CTSD) activation and APP sorting to lysosomes. Accordingly, in Alzheimer-like transgenic mice, neuronal ADAM30 over-expression lowered Aß42 secretion in neuron primary cultures, soluble Aß42 and amyloid plaque load levels in the brain and concomitantly enhanced CTSD activity and finally rescued long term potentiation alterations. Our data thus indicate that lowering ADAM30 expression may favor Aß production, thereby contributing to Alzheimer's disease development.


Assuntos
Proteínas ADAM/metabolismo , Peptídeos beta-Amiloides/metabolismo , Catepsina D/metabolismo , Proteínas ADAM/antagonistas & inibidores , Proteínas ADAM/genética , Doença de Alzheimer/metabolismo , Doença de Alzheimer/patologia , Sequência de Aminoácidos , Animais , Encéfalo/metabolismo , Encéfalo/patologia , Catepsina D/química , Linhagem Celular Tumoral , Regulação para Baixo/efeitos dos fármacos , Células HEK293 , Humanos , Lisossomos/metabolismo , Macrolídeos/farmacologia , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Transgênicos , Microscopia de Fluorescência , Técnicas de Patch-Clamp , Pepstatinas/farmacologia , Interferência de RNA , RNA Interferente Pequeno/metabolismo
19.
Neurobiol Aging ; 35(7): 1570-81, 2014 Jul.
Artigo em Inglês | MEDLINE | ID: mdl-24495834

RESUMO

The ß-amyloid precursor protein undergoes cleavages by ß- and γ-secretasses yielding amyloid-ß peptides (Aß) that accumulate in Alzheimer's disease. Subsequently, Aß peptides are targets of additional truncations or endoproteolytic cleavages explaining the diversity of Aß-related fragments recovered in cell media or pathologic human fluids. Here, we focused on Aß1-34 (Aß34) that has been detected both in vitro and in vivo and that derives from the hydrolysis of Aß by ß-secretase. We have obtained and fully characterized by immunologic and biochemical approaches, a polyclonal antibody that specifically recognizes the C-terminus of Aßx-34. We present immunohistochemical evidence for the presence of Aßx-34 in the brain of 3xTg mice and Alzheimer's disease-affected human brains. Finally, we demonstrate a neprilysin-mediated degradation process of Aß34 and the ability of synthetic Aß34 to protect HEK cells overexpressing either wild type or Swedish-mutated ß-amyloid precursor protein from apoptosis.


Assuntos
Doença de Alzheimer/genética , Peptídeos beta-Amiloides/metabolismo , Encéfalo/metabolismo , Fragmentos de Peptídeos/metabolismo , Doença de Alzheimer/metabolismo , Secretases da Proteína Precursora do Amiloide/metabolismo , Secretases da Proteína Precursora do Amiloide/fisiologia , Precursor de Proteína beta-Amiloide/metabolismo , Animais , Apoptose/genética , Ácido Aspártico Endopeptidases/metabolismo , Caspase 3/metabolismo , Modelos Animais de Doenças , Ensaio de Imunoadsorção Enzimática , Células HEK293 , Humanos , Camundongos , Camundongos Transgênicos , Neprilisina/fisiologia
20.
J Mol Cell Biol ; 5(2): 132-42, 2013 Apr.
Artigo em Inglês | MEDLINE | ID: mdl-23359614

RESUMO

We previously established that besides its canonical function as E3-ubiquitin ligase, parkin also behaves as a transcriptional repressor of p53. Here we show that parkin differently modulates presenilin-1 and presenilin-2 expression and functions at transcriptional level. Thus, parkin enhances/reduces the protein expression, promoter activity and mRNA levels of presenilin-1 and presenilin-2, respectively, in cells and in vivo. This parkin-associated function is independent of its ubiquitin-ligase activity and remains unrelated to its capacity to repress p53. Accordingly, physical interaction of endogenous or overexpressed parkin with presenilins promoters is demonstrated by chromatin immunoprecipitation assays (ChIP). Furthermore, we identify a consensus sequence, the deletion of which abolishes parkin-dependent modulation of presenilins-1/2 and p53 promoter activities. Interestingly, electrophoretic mobility shift assays (EMSA) revealed a physical interaction between this consensus sequence and wild-type but not mutated parkin. Finally, we demonstrate that the RING1-IBR-RING2 domain of parkin harbors parkin's potential to modulate presenilins promoters. This transcriptional control impacts on presenilins-associated phenotypes, since parkin increases presenilin-1-associated γ-secretase activity and reduces presenilin-2-linked caspase-3 activation. Overall, our data delineate a promoter responsive element targeted by parkin that drives differential regulation of presenilin-1 and presenilin-2 transcription with functional consequences for γ-secretase activity and cell death.


Assuntos
Presenilina-1/metabolismo , Presenilina-2/metabolismo , Regiões Promotoras Genéticas/fisiologia , Transcrição Gênica/fisiologia , Ubiquitina-Proteína Ligases/metabolismo , Secretases da Proteína Precursora do Amiloide/genética , Secretases da Proteína Precursora do Amiloide/metabolismo , Animais , Caspase 3/genética , Caspase 3/metabolismo , Morte Celular/fisiologia , Ativação Enzimática/fisiologia , Camundongos , Camundongos Knockout , Complexo Repressor Polycomb 1/genética , Complexo Repressor Polycomb 1/metabolismo , Presenilina-1/genética , Presenilina-2/genética , Proteína Supressora de Tumor p53/biossíntese , Proteína Supressora de Tumor p53/genética , Ubiquitina-Proteína Ligases/genética
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