RESUMEN
Mitochondrial dysfunctions are central players in Alzheimer's disease (AD). In addition, impairments in mitophagy, the process of selective mitochondrial degradation by autophagy leading to a gradual accumulation of defective mitochondria, have also been reported to occur in AD. We provide an updated overview of the recent discoveries and advancements on mitophagic molecular dysfunctions in AD-derived fluids and cells as well as in AD brains. We discuss studies using AD cellular and animal models that have unraveled the contribution of relevant AD-related proteins (Tau, Aß, APP-derived fragments and APOE) in mitophagy failure. In accordance with the important role of impaired mitophagy in AD, we report on various therapeutic strategies aiming at stimulating mitophagy in AD and we summarize the benefits of these potential therapeutic strategies in human clinical trials.
Asunto(s)
Enfermedad de Alzheimer , Animales , Humanos , Enfermedad de Alzheimer/metabolismo , Mitofagia/fisiología , Autofagia/genética , Mitocondrias/metabolismo , Modelos Animales de Enfermedad , Péptidos beta-Amiloides/metabolismoRESUMEN
The processing of the amyloid precursor protein (APP) is one of the key events contributing to Alzheimer's disease (AD) etiology. Canonical cleavages by ß- and γ-secretases lead to Aß production which accumulate in amyloid plaques. Recently, the matrix metalloprotease MT5-MMP, referred to as η-secretase, has been identified as a novel APP cleaving enzyme producing a transmembrane fragment, ηCTF that undergoes subsequent cleavages by α- and ß-secretases yielding the Aηα and AÎ·ß peptides, respectively. The functions and contributions of ηCTF and its related fragments to AD pathology are poorly understood. In this study, we designed a novel immunological probe referred to as ηCTF-NTer antibody that specifically interacts with the N-terminal part of ηCTF targeting ηCTF, Aηα, AÎ·ß but not C99, C83 and Aß. We examined the fate and localization of ηCTF fragment in various cell models and in mice. We found that overexpressed ηCTF undergoes degradation in the proteasomal and autophagic pathways and accumulates mainly in the Golgi and in endosomes. Moreover, we observed the presence of ηCTF in small extracellular vesicles purified from neuroblastoma cells or from mouse brains expressing ηCTF. Importantly, the expression of ηCTF in fibroblasts devoid on APP leads to Aß production demonstrating its contribution to the amyloidogenic pathway. Finally, we observed an ηCTF-like immunoreactivity around amyloid plaques and an age-dependent accumulation of ηCTF in the triple-transgenic mouse AD model. Thus, our study suggests that the ηCTF fragment likely contributes to AD pathology by its exosomal spreading and involvement in Aß production.
Asunto(s)
Enfermedad de Alzheimer , Vesículas Extracelulares , Ratones , Animales , Precursor de Proteína beta-Amiloide/genética , Precursor de Proteína beta-Amiloide/metabolismo , Secretasas de la Proteína Precursora del Amiloide/genética , Secretasas de la Proteína Precursora del Amiloide/metabolismo , Placa Amiloide , Enfermedad de Alzheimer/metabolismo , Ratones Transgénicos , Endosomas/metabolismo , Vesículas Extracelulares/metabolismo , Péptidos beta-Amiloides/metabolismoRESUMEN
The amyloid cascade hypothesis, which proposes a prominent role for full-length amyloid ß peptides in Alzheimer's disease, is currently being questioned. In addition to full-length amyloid ß peptide, several N-terminally truncated fragments of amyloid ß peptide could well contribute to Alzheimer's disease setting and/or progression. Among them, pyroGlu3-amyloid ß peptide appears to be one of the main components of early anatomical lesions in Alzheimer's disease-affected brains. Little is known about the proteolytic activities that could account for the N-terminal truncations of full-length amyloid ß, but they appear as the rate-limiting enzymes yielding the Glu3-amyloid ß peptide sequence that undergoes subsequent cyclization by glutaminyl cyclase, thereby yielding pyroGlu3-amyloid ß. Here, we investigated the contribution of dipeptidyl peptidase 4 in Glu3-amyloid ß peptide formation and the functional influence of its genetic depletion or pharmacological blockade on spine maturation as well as on pyroGlu3-amyloid ß peptide and amyloid ß 42-positive plaques and amyloid ß 42 load in the triple transgenic Alzheimer's disease mouse model. Furthermore, we examined whether reduction of dipeptidyl peptidase 4 could rescue learning and memory deficits displayed by these mice. Our data establish that dipeptidyl peptidase 4 reduction alleviates anatomical, biochemical, and behavioral Alzheimer's disease-related defects. Furthermore, we demonstrate that dipeptidyl peptidase 4 activity is increased early in sporadic Alzheimer's disease brains. Thus, our data demonstrate that dipeptidyl peptidase 4 participates in pyroGlu3-amyloid ß peptide formation and that targeting this peptidase could be considered as an alternative strategy to interfere with Alzheimer's disease progression.
Asunto(s)
Enfermedad de Alzheimer , Animales , Encéfalo/metabolismo , Dipeptidil Peptidasa 4 , Modelos Animales de Enfermedad , Humanos , Ratones , Placa AmiloideRESUMEN
One of the main components of senile plaques in Alzheimer's disease (AD)-affected brain is the Aß peptide species harboring a pyroglutamate at position three pE3-Aß. Several studies indicated that pE3-Aß is toxic, prone to aggregation and serves as a seed of Aß aggregation. The cyclisation of the glutamate residue is produced by glutaminyl cyclase, the pharmacological and genetic reductions of which significantly alleviate AD-related anatomical lesions and cognitive defects in mice models. The cyclisation of the glutamate in position 3 requires prior removal of the Aß N-terminal aspartyl residue to allow subsequent biotransformation. The enzyme responsible for this rate-limiting catalytic step and its relevance as a putative trigger of AD pathology remained yet to be established. Here, we identify aminopeptidase A as the main exopeptidase involved in the N-terminal truncation of Aß and document its key contribution to AD-related anatomical and behavioral defects. First, we show by mass spectrometry that human recombinant aminopeptidase A (APA) truncates synthetic Aß1-40 to yield Aß2-40. We demonstrate that the pharmacological blockade of APA with its selective inhibitor RB150 restores the density of mature spines and significantly reduced filopodia-like processes in hippocampal organotypic slices cultures virally transduced with the Swedish mutated Aß-precursor protein (ßAPP). Pharmacological reduction of APA activity and lowering of its expression by shRNA affect pE3-42Aß- and Aß1-42-positive plaques and expressions in 3xTg-AD mice brains. Further, we show that both APA inhibitors and shRNA partly alleviate learning and memory deficits observed in 3xTg-AD mice. Importantly, we demonstrate that, concomitantly to the occurrence of pE3-42Aß-positive plaques, APA activity is augmented at early Braak stages in sporadic AD brains. Overall, our data indicate that APA is a key enzyme involved in Aß N-terminal truncation and suggest the potential benefit of targeting this proteolytic activity to interfere with AD pathology.
Asunto(s)
Enfermedad de Alzheimer/metabolismo , Enfermedad de Alzheimer/fisiopatología , Péptidos beta-Amiloides/metabolismo , Encéfalo/metabolismo , Disfunción Cognitiva/metabolismo , Glutamil Aminopeptidasa/metabolismo , Animales , Encéfalo/patología , Línea Celular , Modelos Animales de Enfermedad , Glutamil Aminopeptidasa/antagonistas & inhibidores , Glutamil Aminopeptidasa/fisiología , Hipocampo/metabolismo , Humanos , Ratones , Ratones Endogámicos C57BL , Ratones Transgénicos , Placa Amiloide/patologíaRESUMEN
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.
Asunto(s)
Enfermedad de Alzheimer/patología , Precursor de Proteína beta-Amiloide/genética , Precursor de Proteína beta-Amiloide/metabolismo , Encéfalo/patología , Mitocondrias/patología , Mitocondrias/ultraestructura , Mitofagia/genética , Anciano , Anciano de 80 o más Años , Enfermedad de Alzheimer/metabolismo , Secretasas de la Proteína Precursora del Amiloide/antagonistas & inhibidores , Secretasas de la Proteína Precursora del Amiloide/metabolismo , Animales , Ácido Aspártico Endopeptidasas/antagonistas & inhibidores , Ácido Aspártico Endopeptidasas/metabolismo , Autopsia , Línea Celular , Femenino , Humanos , Potencial de la Membrana Mitocondrial , Ratones , Mitocondrias/metabolismo , Fragmentos de Péptidos/genética , Fragmentos de Péptidos/metabolismo , Especies Reactivas de Oxígeno/metabolismoRESUMEN
Alzheimer's disease (AD) is a multifactorial neurodegenerative pathology characterized by a progressive decline of cognitive functions. Alteration of various signaling cascades affecting distinct subcellular compartment functions and their communication likely contribute to AD progression. Among others, the alteration of the physical association between the endoplasmic reticulum (ER) and mitochondria, also reffered as mitochondria-associated membranes (MAMs), impacts various cellular housekeeping functions such as phospholipids-, glucose-, cholesterol-, and fatty-acid-metabolism, as well as calcium signaling, which are all altered in AD. Our review describes the physical and functional proteome crosstalk between the ER and mitochondria and highlights the contribution of distinct molecular components of MAMs to mitochondrial and ER dysfunctions in AD progression. We also discuss potential strategies targeting MAMs to improve mitochondria and ER functions in AD.
Asunto(s)
Enfermedad de Alzheimer/metabolismo , Retículo Endoplásmico/metabolismo , Membranas Mitocondriales/metabolismo , Animales , Estrés del Retículo Endoplásmico/fisiología , Humanos , Mitocondrias/metabolismoRESUMEN
Alteration of ryanodine receptor (RyR)-mediated calcium (Ca2+) signaling has been reported in Alzheimer disease (AD) models. However, the molecular mechanisms underlying altered RyR-mediated intracellular Ca2+ release in AD remain to be fully elucidated. We report here that RyR2 undergoes post-translational modifications (phosphorylation, oxidation, and nitrosylation) in SH-SY5Y neuroblastoma cells expressing the ß-amyloid precursor protein (ßAPP) harboring the familial double Swedish mutations (APPswe). RyR2 macromolecular complex remodeling, characterized by depletion of the regulatory protein calstabin2, resulted in increased cytosolic Ca2+ levels and mitochondrial oxidative stress. We also report a functional interplay between amyloid ß (Aß), ß-adrenergic signaling, and altered Ca2+ signaling via leaky RyR2 channels. Thus, post-translational modifications of RyR occur downstream of Aß through a ß2-adrenergic signaling cascade that activates PKA. RyR2 remodeling in turn enhances ßAPP processing. Importantly, pharmacological stabilization of the binding of calstabin2 to RyR2 channels, which prevents Ca2+ leakage, or blocking the ß2-adrenergic signaling cascade reduced ßAPP processing and the production of Aß in APPswe-expressing SH-SY5Y cells. We conclude that targeting RyR-mediated Ca2+ leakage may be a therapeutic approach to treat AD.
Asunto(s)
Péptidos beta-Amiloides/metabolismo , Señalización del Calcio , Neuronas/enzimología , Procesamiento Proteico-Postraduccional , Receptores Adrenérgicos beta 2/metabolismo , Canal Liberador de Calcio Receptor de Rianodina/metabolismo , Antagonistas de Receptores Adrenérgicos beta 2/farmacología , Enfermedad de Alzheimer/enzimología , Enfermedad de Alzheimer/genética , Enfermedad de Alzheimer/metabolismo , Enfermedad de Alzheimer/patología , Péptidos beta-Amiloides/genética , Precursor de Proteína beta-Amiloide/genética , Precursor de Proteína beta-Amiloide/metabolismo , Señalización del Calcio/efectos de los fármacos , Línea Celular Tumoral , Proteínas Quinasas Dependientes de AMP Cíclico/química , Proteínas Quinasas Dependientes de AMP Cíclico/metabolismo , Activación Enzimática/efectos de los fármacos , Humanos , Mutación , Proteínas del Tejido Nervioso/agonistas , Proteínas del Tejido Nervioso/química , Proteínas del Tejido Nervioso/genética , Proteínas del Tejido Nervioso/metabolismo , Neuronas/efectos de los fármacos , Neuronas/metabolismo , Neuronas/patología , Oxidación-Reducción , Estrés Oxidativo/efectos de los fármacos , Fosforilación/efectos de los fármacos , Multimerización de Proteína/efectos de los fármacos , Procesamiento Proteico-Postraduccional/efectos de los fármacos , Proteolisis/efectos de los fármacos , Receptores Adrenérgicos beta 2/química , Receptores Adrenérgicos beta 2/genética , Proteínas Recombinantes/química , Proteínas Recombinantes/metabolismo , Canal Liberador de Calcio Receptor de Rianodina/química , Proteínas de Unión a Tacrolimus/antagonistas & inhibidores , Proteínas de Unión a Tacrolimus/metabolismoRESUMEN
Deviation of the ambient temperature is one of the most ubiquitous stimuli that continuously affect mammals' skin. Although the role of the warmth receptors in epidermal homeostasis (EH) was elucidated in recent years, the mystery of the keratinocyte mild-cold sensor remains unsolved. Here we report the cloning and characterization of a new functional epidermal isoform of the transient receptor potential M8 (TRPM8) mild-cold receptor, dubbed epidermal TRPM8 (eTRPM8), which is localized in the keratinocyte endoplasmic reticulum membrane and controls mitochondrial Ca(2+) concentration ([Ca(2+)]m). In turn, [Ca(2+)]m modulates ATP and superoxide (O2(·-)) synthesis in a cold-dependent manner. We report that this fine tuning of ATP and O2(·-) levels by cooling controls the balance between keratinocyte proliferation and differentiation. Finally, to ascertain eTRPM8's role in EH in vivo we developed a new functional knockout mouse strain by deleting the pore domain of TRPM8 and demonstrated that eTRPM8 knockout impairs adaptation of the epidermis to low temperatures.
Asunto(s)
Diferenciación Celular/fisiología , Proliferación Celular/fisiología , Frío , Epidermis/metabolismo , Queratinocitos/citología , Isoformas de Proteínas/fisiología , Canales Catiónicos TRPM/fisiología , Adenosina Trifosfato/metabolismo , Animales , Calcio/metabolismo , Canales de Calcio/metabolismo , Células Cultivadas , Humanos , Ratones , Ratones Noqueados , Datos de Secuencia Molecular , Superóxidos/metabolismoRESUMEN
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.
Asunto(s)
Enfermedad de Alzheimer/metabolismo , Calcio/metabolismo , Trastornos del Conocimiento/metabolismo , Procesamiento Proteico-Postraduccional , Canal Liberador de Calcio Receptor de Rianodina/metabolismo , Enfermedad de Alzheimer/patología , Animales , Señalización del Calcio , Trastornos del Conocimiento/patología , Proteínas Quinasas Dependientes de AMP Cíclico/metabolismo , Femenino , Humanos , Masculino , Aprendizaje por Laberinto/fisiología , Ratones , Ratones Transgénicos , Estrés Oxidativo/fisiología , Fosforilación , Reconocimiento en Psicología/fisiología , Retículo Sarcoplasmático/metabolismoRESUMEN
Among the new players at the endoplasmic reticulum (ER)-mitochondria interface regulating interorganelle calcium signaling, those specifically involved during ER stress are not known at present. We report here that the truncated variant of the sarcoendoplasmic reticulum Ca(2+)-ATPase 1 (S1T) amplifies ER stress through the PERK-eIF2alpha-ATF4-CHOP pathway. S1T, which is localized in the ER-mitochondria microdomains, determines ER Ca(2+) depletion due to increased Ca(2+) leak, an increased number of ER-mitochondria contact sites, and inhibition of mitochondria movements. This leads to increased Ca(2+) transfer to mitochondria in both resting and stimulated conditions and activation of the mitochondrial apoptotic pathway. Interestingly, S1T knockdown was shown to prevent ER stress, mitochondrial Ca(2+) overload, and subsequent apoptosis. Thus, by bridging ER stress to apoptosis through increased ER-mitochondria Ca(2+) transfer, S1T acts as an essential determinant of cellular fate.
Asunto(s)
Apoptosis , Calcio/metabolismo , Retículo Endoplásmico/enzimología , Retículo Endoplásmico/patología , Mitocondrias/enzimología , Mutación/genética , ATPasas Transportadoras de Calcio del Retículo Sarcoplásmico/metabolismo , Factor de Transcripción Activador 4/metabolismo , Apoptosis/efectos de los fármacos , Secuencia de Bases , Brefeldino A/farmacología , Retículo Endoplásmico/efectos de los fármacos , Retículo Endoplásmico/ultraestructura , Inducción Enzimática/efectos de los fármacos , Factor 2 Eucariótico de Iniciación/metabolismo , Células HeLa , Homeostasis/efectos de los fármacos , Humanos , Isoenzimas/metabolismo , Mitocondrias/efectos de los fármacos , Mitocondrias/ultraestructura , Membranas Mitocondriales/efectos de los fármacos , Membranas Mitocondriales/enzimología , Datos de Secuencia Molecular , Elementos de Respuesta/genética , ATPasas Transportadoras de Calcio del Retículo Sarcoplásmico/biosíntesis , eIF-2 Quinasa/metabolismoRESUMEN
Mitochondrial dysfunctions are key features of Alzheimer's disease (AD). The occurrence of these disturbances in the peripheral cells of AD patients and their potential correlation with disease progression are underinvestigated. We studied mitochondrial structure, function and mitophagy in fibroblasts from healthy volunteers and AD patients at the prodromal (AD-MCI) or demented (AD-D) stages. We carried out correlation studies with clinical cognitive scores, namely, (i) Mini-Mental State Examination (MMSE) and (ii) Dementia Rating-Scale Sum of Boxes (CDR-SOB), and with (iii) amyloid beta (Aß) plaque burden (PiB-PET imaging) and (iv) the accumulation of peripheral amyloid precursor protein C-terminal fragments (APP-CTFs). We revealed alterations in mitochondrial structure as well as specific mitochondrial dysfunction signatures in AD-MCI and AD-D fibroblasts and revealed that defective mitophagy and autophagy are linked to impaired lysosomal activity in AD-D fibroblasts. We reported significant correlations of a subset of these dysfunctions with cognitive decline, AD-related clinical hallmarks and peripheral APP-CTFs accumulation. This study emphasizes the potential use of peripheral cells for investigating AD pathophysiology.
Asunto(s)
Enfermedad de Alzheimer , Fibroblastos , Mitocondrias , Mitofagia , Humanos , Enfermedad de Alzheimer/patología , Enfermedad de Alzheimer/metabolismo , Enfermedad de Alzheimer/diagnóstico por imagen , Fibroblastos/patología , Fibroblastos/metabolismo , Anciano , Femenino , Mitocondrias/patología , Mitocondrias/metabolismo , Masculino , Mitofagia/fisiología , Persona de Mediana Edad , Anciano de 80 o más Años , Precursor de Proteína beta-Amiloide/metabolismo , Precursor de Proteína beta-Amiloide/genética , Disfunción Cognitiva/patología , Disfunción Cognitiva/metabolismo , Autofagia/fisiologíaRESUMEN
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.
Asunto(s)
Enfermedad de Alzheimer , Precursor de Proteína beta-Amiloide , Mitocondrias , Enfermedad de Alzheimer/metabolismo , Enfermedad de Alzheimer/patología , Enfermedad de Alzheimer/genética , Precursor de Proteína beta-Amiloide/metabolismo , Precursor de Proteína beta-Amiloide/genética , Animales , Mitocondrias/metabolismo , Humanos , Ratones , Ratones Transgénicos , Neuronas/metabolismo , Péptidos beta-Amiloides/metabolismo , Proteínas Mitocondriales/metabolismo , Proteínas Mitocondriales/genética , Secretasas de la Proteína Precursora del Amiloide/metabolismo , Cinesinas/metabolismo , Transporte Biológico , Mitofagia , Proteínas del Tejido Nervioso , Proteínas de Unión al GTP rho , Péptidos y Proteínas de Señalización IntracelularRESUMEN
In Alzheimer disease (AD), the perturbation of the endoplasmic reticulum (ER) calcium (Ca²âº) homeostasis has been linked to presenilins, the catalytic core in γ-secretase complexes cleaving the amyloid precursor protein (APP), thereby generating amyloid-ß (Aß) peptides. Here we investigate whether APP contributes to ER Ca²âº homeostasis and whether ER Ca²âº could in turn influence Aß production. We show that overexpression of wild-type human APP (APP(695)), or APP harboring the Swedish double mutation (APP(swe)) triggers increased ryanodine receptor (RyR) expression and enhances RyR-mediated ER Ca²âº release in SH-SY5Y neuroblastoma cells and in APP(swe)-expressing (Tg2576) mice. Interestingly, dantrolene-induced lowering of RyR-mediated Ca²âº release leads to the reduction of both intracellular and extracellular Aß load in neuroblastoma cells as well as in primary cultured neurons derived from Tg2576 mice. This Aß reduction can be accounted for by decreased Thr-668-dependent APP phosphorylation and ß- and γ-secretases activities. Importantly, dantrolene diminishes Aß load, reduces Aß-related histological lesions, and slows down learning and memory deficits in Tg2576 mice. Overall, our data document a key role of RyR in Aß production and learning and memory performances, and delineate RyR-mediated control of Ca²âº homeostasis as a physiological paradigm that could be targeted for innovative therapeutic approaches.
Asunto(s)
Enfermedad de Alzheimer/complicaciones , Enfermedad de Alzheimer/metabolismo , Péptidos beta-Amiloides/metabolismo , Trastornos de la Memoria/etiología , Trastornos de la Memoria/metabolismo , Canal Liberador de Calcio Receptor de Rianodina/metabolismo , Enfermedad de Alzheimer/genética , Aminofenoles/uso terapéutico , Secretasas de la Proteína Precursora del Amiloide/genética , Secretasas de la Proteína Precursora del Amiloide/metabolismo , Precursor de Proteína beta-Amiloide/genética , Precursor de Proteína beta-Amiloide/metabolismo , Análisis de Varianza , Animales , Encéfalo/citología , Cafeína/farmacología , Calcio/metabolismo , Bloqueadores de los Canales de Calcio/uso terapéutico , Células Cultivadas , Citosol/efectos de los fármacos , Citosol/metabolismo , Dantroleno/farmacología , Modelos Animales de Enfermedad , Embrión de Mamíferos , Retículo Endoplásmico/efectos de los fármacos , Retículo Endoplásmico/metabolismo , Retículo Endoplásmico/patología , Inhibidores Enzimáticos/uso terapéutico , Conducta Exploratoria/efectos de los fármacos , Regulación de la Expresión Génica/efectos de los fármacos , Regulación de la Expresión Génica/genética , Humanos , Receptores de Inositol 1,4,5-Trifosfato/genética , Receptores de Inositol 1,4,5-Trifosfato/metabolismo , Maleimidas/uso terapéutico , Aprendizaje por Laberinto/efectos de los fármacos , Potenciales de la Membrana/efectos de los fármacos , Potenciales de la Membrana/genética , Proteínas de la Membrana/metabolismo , Trastornos de la Memoria/tratamiento farmacológico , Ratones , Ratones Transgénicos , Relajantes Musculares Centrales/farmacología , Mutación/genética , Proteínas del Tejido Nervioso/metabolismo , Neuroblastoma/patología , Neuronas/efectos de los fármacos , Neuronas/fisiología , Técnicas de Placa-Clamp , Fragmentos de Péptidos/metabolismo , Fosforilación/efectos de los fármacos , Fosforilación/genética , Placa Amiloide/metabolismo , Placa Amiloide/patología , Purinas/uso terapéutico , ARN Mensajero/metabolismoRESUMEN
Anatomical lesions in Alzheimer disease-affected brains mainly consist of senile plaques, inflammation stigmata, and oxidative stress. The nuclear factor-κB (NF-κB) is a stress-activated transcription factor that is activated around senile plaques. We have assessed whether NF-κB could be differentially regulated at physiological or supraphysiological levels of amyloid ß (Aß) peptides. Under these experimental conditions, we delineated the putative NF-κB-dependent modulation of all cellular participants in Aß production, namely its precursor ßAPP (ß-amyloid precursor protein) and the ß- and γ-secretases, the two enzymatic machines involved in Aß genesis. Under physiological conditions, NF-κB lowers the transcriptional activity of the promoters of ßAPP, ß-secretase (ß-site APP-cleaving enzyme 1, BACE1), and of the four protein components (Aph-1, Pen-2, nicastrin, presenilin-1, or presenilin-2) of the γ-secretase in HEK293 cells. This was accompanied by a reduction of both protein levels and enzymatic activities, thereby ultimately yielding lower amounts of Aß and AICD (APP intracellular domain). In stably transfected Swedish ßAPP-expressing HEK293 cells triggering supraphysiological concentrations of Aß peptides, NF-κB activates the transcription of ßAPP, BACE1, and some of the γ-secretase members and increases protein expression and enzymatic activities, resulting in enhanced Aß production. Our pharmacological approach using distinct NF-κB kinase modulators indicates that both NF-κB canonical and alternative pathways are involved in the control of Aß production. Overall, our data demonstrate that under physiological conditions, NF-κB triggers a repressive effect on Aß production that contributes to maintaining its homeostasis, while NF-κB participates in a degenerative cycle where Aß would feed its own production under pathological conditions.
Asunto(s)
Secretasas de la Proteína Precursora del Amiloide/metabolismo , Péptidos beta-Amiloides/metabolismo , Precursor de Proteína beta-Amiloide/metabolismo , FN-kappa B/farmacología , Secretasas de la Proteína Precursora del Amiloide/genética , Precursor de Proteína beta-Amiloide/genética , Ácido Aspártico Endopeptidasas/genética , Ácido Aspártico Endopeptidasas/metabolismo , Western Blotting , Línea Celular , Expresión Génica/efectos de los fármacos , Humanos , Glicoproteínas de Membrana/genética , Glicoproteínas de Membrana/metabolismo , Regiones Promotoras Genéticas/genéticaRESUMEN
Morphological alterations of the endosomal compartment have been widely described in post-mortem brains from Alzheimer's disease (AD) patients and subjects with Down syndrome (DS) who are at high risk for AD. Immunostaining with antibodies against endosomal markers such as Early Endosome Antigen 1 (EEA1) revealed increased size of EEA1-positive puncta. In DS, peripheral cells such as peripheral blood mononuclear cells (PBMCs) and fibroblasts, share similar phenotype even in the absence of AD. We previously found that PBMCs from AD patients have larger EEA1-positive puncta, correlating with brain amyloid load. Here we analysed the endosomal compartment of fibroblasts from a very well characterised cohort of AD patients (IMABio3) who underwent thorough clinical, imaging and biomarkers assessments. Twenty-one subjects were included (7 AD with mild cognitive impairment (AD-MCI), 7 AD with dementia (AD-D) and 7 controls) who had amyloid-PET at baseline (PiB) and neuropsychological tests at baseline and close to skin biopsy. Fibroblasts isolated from skin biopsies were immunostained with anti-EEA1 antibody and imaged using a spinning disk microscope. Endosomal compartment ultrastructure was also analysed by electron microscopy. All fibroblast lines were genotyped and their AD risk factors identified. Our results show a trend to an increased EEA1-positive puncta volume in fibroblasts from AD-D as compared to controls (p.adj = 0.12) and reveal enhanced endosome area in fibroblasts from AD-MCI and AD-AD versus controls. Larger puncta size correlated with PiB retention in different brain areas and with worse cognitive scores at the time of biopsy as well as faster decline from baseline to the time of biopsy. Finally, we identified three genetic risk factors for AD (ABCA1, COX7C and MYO15A) that were associated with larger EEA1 puncta volume. In conclusion, the endosomal compartment in fibroblasts could be used as cellular peripheral biomarker for both amyloid deposition and cognitive decline in AD patients.
Asunto(s)
Enfermedad de Alzheimer , Disfunción Cognitiva , Humanos , Amiloide , Péptidos beta-Amiloides , Endosomas/patología , Fibroblastos , Leucocitos Mononucleares , Tomografía de Emisión de PositronesRESUMEN
Desbuquois dysplasia is a severe condition characterized by short stature, joint laxity, scoliosis, and advanced carpal ossification with a delta phalanx. Studying nine Desbuquois families, we identified seven distinct mutations in the Calcium-Activated Nucleotidase 1 gene (CANT1), which encodes a soluble UDP-preferring nucleotidase belonging to the apyrase family. Among the seven mutations, four were nonsense mutations (Del 5' UTR and exon 1, p.P245RfsX3, p.S303AfsX20, and p.W125X), and three were missense mutations (p.R300C, p.R300H, and p.P299L) responsible for the change of conserved amino acids located in the seventh nucleotidase conserved region (NRC). The arginine substitution at position 300 was identified in five out of nine families. The specific function of CANT1 is as yet unknown, but its substrates are involved in several major signaling functions, including Ca2+ release, through activation of pyrimidinergic signaling. Importantly, using RT-PCR analysis, we observed a specific expression in chondrocytes. We also found electron-dense material within distended rough endoplasmic reticulum in the fibroblasts of Desbuquois patients. Our findings demonstrate the specific involvement of a nucleotidase in the endochondral ossification process.
Asunto(s)
Enfermedades del Desarrollo Óseo/genética , Calcio/metabolismo , Mutación , Nucleotidasas/genética , Regiones no Traducidas 5' , Adolescente , Adulto , Secuencia de Aminoácidos , Sustitución de Aminoácidos , Arginina/metabolismo , Enfermedades del Desarrollo Óseo/diagnóstico por imagen , Células Cultivadas , Preescolar , Condrocitos/metabolismo , Cromosomas Humanos Par 17 , Codón sin Sentido , Consanguinidad , Retículo Endoplásmico Rugoso/ultraestructura , Exones , Resultado Fatal , Femenino , Fibroblastos/ultraestructura , Homocigoto , Humanos , Lactante , Recién Nacido , Masculino , Datos de Secuencia Molecular , Mutación Missense , Núcleo Familiar , ARN Mensajero/metabolismo , RadiografíaRESUMEN
Alzheimer's disease (AD) is a neurodegenerative pathology representing a socioeconomic challenge, however, the complex mechanism behind the disease is not yet fully understood [...].
Asunto(s)
Enfermedad de Alzheimer/fisiopatología , Enfermedad de Alzheimer/terapia , Señalización del Calcio , Animales , Calcio/metabolismo , Humanos , Plasticidad Neuronal , Neuronas/metabolismoRESUMEN
Parkinson disease (PD)-affected brains show consistent endoplasmic reticulum (ER) stress and mitophagic dysfunctions. The mechanisms underlying these perturbations and how they are directly linked remain a matter of questions. XBP1 is a transcription factor activated upon ER stress after unconventional splicing by the nuclease ERN1/IREα thereby yielding XBP1s, whereas PINK1 is a kinase considered as the sensor of mitochondrial physiology and a master gatekeeper of mitophagy process. We showed that XBP1s transactivates PINK1 in human cells, primary cultured neurons and mice brain, and triggered a pro-mitophagic phenotype that was fully dependent of endogenous PINK1. We also unraveled a PINK1-dependent phosphorylation of XBP1s that conditioned its nuclear localization and thereby, governed its transcriptional activity. PINK1-induced XBP1s phosphorylation occurred at residues reminiscent of, and correlated to, those phosphorylated in substantia nigra of sporadic PD-affected brains. Overall, our study delineated a functional loop between XBP1s and PINK1 governing mitophagy that was disrupted in PD condition.Abbreviations: 6OHDA: 6-hydroxydopamine; baf: bafilomycin A1; BECN1: beclin 1; CALCOCO2/NDP52: calcium binding and coiled-coil domain 2; CASP3: caspase 3; CCCP: carbonyl cyanide chlorophenylhydrazone; COX8A: cytochrome c oxidase subunit 8A; DDIT3/CHOP: DNA damage inducible transcript 3; EGFP: enhanced green fluorescent protein; ER: endoplasmic reticulum; ERN1/IRE1α: endoplasmic reticulum to nucleus signaling 1; FACS: fluorescence-activated cell sorting; HSPD1/HSP60: heat shock protein family D (Hsp60) member 1; MAP1LC3/LC3: microtubule associated protein 1 light chain 3; MFN2: mitofusin 2; OPTN: optineurin; PD: Parkinson disease; PINK1: PTEN-induced kinase 1; PCR: polymerase chain reaction:; PRKN: parkin RBR E3 ubiquitin protein ligase; XBP1s [p-S61A]: XBP1s phosphorylated at serine 61; XBP1s [p-T48A]: XBP1s phosphorylated at threonine 48; shRNA: short hairpin RNA, SQSTM1/p62: sequestosome 1; TIMM23: translocase of inner mitochondrial membrane 23; TM: tunicamycin; TMRM: tetramethyl rhodamine methylester; TOMM20: translocase of outer mitochondrial membrane 20; Toy: toyocamycin; TP: thapsigargin; UB: ubiquitin; UB (S65): ubiquitin phosphorylated at serine 65; UPR: unfolded protein response, XBP1: X-box binding protein 1; XBP1s: spliced X-box binding protein 1.
Asunto(s)
Mitofagia , Enfermedad de Parkinson , Proteínas Quinasas/metabolismo , Proteína 1 de Unión a la X-Box/metabolismo , Animales , Autofagia , Endorribonucleasas , Ratones , Mitofagia/genética , Enfermedad de Parkinson/genética , Fosforilación , Proteínas Serina-Treonina QuinasasRESUMEN
Pathologic calcium (Ca2+) signaling linked to Alzheimer's Disease (AD) involves the intracellular Ca2+ release channels/ryanodine receptors (RyRs). RyRs are macromolecular complexes where the protein-protein interactions between RyRs and several regulatory proteins impact the channel function. Pharmacological and genetic approaches link the destabilization of RyRs macromolecular complexes to several human pathologies including brain disorders. In this review, we discuss our recent data, which demonstrated that enhanced neuronal RyR2-mediated Ca2+ leak in AD is associated with posttranslational modifications (hyperphosphorylation, oxidation, and nitrosylation) leading to RyR2 macromolecular complex remodeling, and dissociation of the stabilizing protein Calstabin2 from the channel. We describe RyR macromolecular complex structure and discuss the molecular mechanisms and signaling cascade underlying neuronal RyR2 remodeling in AD. We provide evidence linking RyR2 dysfunction with ß-adrenergic signaling cascade that is altered in AD. RyR2 remodeling in AD leads to histopathological lesions, alteration of synaptic plasticity, learning and memory deficits. Targeting RyR macromolecular complex remodeling should be considered as a new therapeutic window to treat/or prevent AD setting and/or progression.