RESUMO
PSENEN/PEN2 is the smallest subunit of the γ-secretase complex, an intramembrane protease that cleaves proteins within their transmembrane domains. Mutations in components of the γ-secretase underlie familial Alzheimer disease. In addition to its proteolytic activity, supplementary, γ-secretase independent, functions in the macroautophagy/autophagy-lysosome system have been proposed. Here, we screened for PSENEN-interacting proteins and identified CLN3. Mutations in CLN3 are causative for juvenile neuronal ceroid lipofuscinosis, a rare lysosomal storage disorder considered the most common neurodegenerative disease in children. As mutations in the PSENEN and CLN3 genes cause different neurodegenerative diseases, understanding shared cellular functions of both proteins might be pertinent for understanding general cellular mechanisms underlying neurodegeneration. We hypothesized that CLN3 modulates γ-secretase activity and that PSENEN and CLN3 play associated roles in the autophagy-lysosome system. We applied CRISPR gene-editing and obtained independent isogenic HeLa knockout cell lines for PSENEN and CLN3. Following previous studies, we demonstrate that PSENEN is essential for forming a functional γ-secretase complex and is indispensable for γ-secretase activity. In contrast, CLN3 does not modulate γ-secretase activity to a significant degree. We observed in PSENEN- and CLN3-knockout cells corresponding alterations in the autophagy-lysosome system. These include reduced activity of lysosomal enzymes and lysosome number, an increased number of autophagosomes, increased lysosome-autophagosome fusion, and elevated levels of TFEB (transcription factor EB). Our study strongly suggests converging roles of PSENEN and CLN3 in the autophagy-lysosome system in a γ-secretase activity-independent manner, supporting the idea of common cytopathological processes underlying different neurodegenerative diseases.Abbreviations: Aß, amyloid-beta; AD, Alzheimer disease; APP, amyloid precursor protein; ATP5MC, ATP synthase membrane subunit c; DQ-BSA, dye-quenched bovine serum albumin; ER, endoplasmic reticulum; GFP, green fluorescent protein; ICC, immunocytochemistry; ICD, intracellular domain; JNCL, juvenile neuronal ceroid lipofuscinosis; KO, knockout; LC3, microtubule associated protein 1 light chain 3; NCL, neuronal ceroid lipofuscinoses; PSEN, presenilin; PSENEN/PEN2: presenilin enhancer, gamma-secretase subunit; TAP, tandem affinity purification; TEV, tobacco etch virus; TF, transferrin; WB, Western blot; WT, wild type.
Assuntos
Doença de Alzheimer , Lipofuscinoses Ceroides Neuronais , Doença de Alzheimer/genética , Doença de Alzheimer/metabolismo , Secretases da Proteína Precursora do Amiloide/genética , Secretases da Proteína Precursora do Amiloide/metabolismo , Autofagia/genética , Criança , Humanos , Lisossomos/metabolismo , Glicoproteínas de Membrana/genética , Glicoproteínas de Membrana/metabolismo , Proteínas de Membrana/metabolismo , Chaperonas Moleculares/metabolismo , Lipofuscinoses Ceroides Neuronais/genética , Lipofuscinoses Ceroides Neuronais/metabolismo , Presenilinas/genética , Presenilinas/metabolismo , Fatores de Transcrição/metabolismoRESUMO
Mitochondrial dysfunction plays a pivotal role in the Alzheimer's Disease (AD) pathology. Disrupted mitochondrial dynamics (i.e., fusion/fission balance), which are essential for normal mitochondria structure and function, are documented in AD. Caveolin-1 (Cav-1), a membrane/lipid raft (MLR) scaffolding protein regulates metabolic pathways in several different cell types such as hepatocytes and cancer cells. Previously, we have shown decreased expression of Cav-1 in the hippocampus of 9-month (m) old PSAPP mice, while hippocampal overexpression of neuron-targeted Cav-1 using the synapsin promoter (i.e., SynCav1) preserved cognitive function, neuronal morphology, and synaptic ultrastructure in 9 and 12 m PSAPP mice. Considering the central role of energy production in maintaining normal neuronal and synaptic function and survival, the present study reveals that PSAPP mice exhibit disrupted mitochondrial distribution, morphometry, and respiration. In contrast, SynCav1 mitigates mitochondrial damage and loss and enhances mitochondrial respiration. Furthermore, by examining mitochondrial dynamics, we found that PSAPP mice showed a significant increase in the phosphorylation of mitochondrial dynamin-related GTPase protein (DRP1), resulting in excessive mitochondria fragmentation and dysfunction. In contrast, hippocampal delivery of SynCav1 significantly decreased p-DRP1 and augmented the level of the mitochondrial fusion protein, mitofusin1 (Mfn1) in PSAPP mice, a molecular event, which may mechanistically explain for the preserved balance of mitochondria fission/fusion and metabolic resilience in 12 m PSAPP-SynCav1 mice. Our data demonstrate the critical role for Cav-1 in maintaining normal mitochondrial morphology and function through affecting mitochondrial dynamics and explain a molecular and cellular mechanism underlying the previously reported neuroprotective and cognitive preservation induced by SynCav1 in PSAPP mouse model of AD.
Assuntos
Doença de Alzheimer/terapia , Caveolina 1/metabolismo , Modelos Animais de Doenças , Hipocampo/fisiologia , Mitocôndrias/fisiologia , Neurônios/fisiologia , Fármacos Neuroprotetores/administração & dosagem , Sinapsinas/metabolismo , Doença de Alzheimer/etiologia , Doença de Alzheimer/patologia , Precursor de Proteína beta-Amiloide/genética , Animais , Caveolina 1/genética , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Transgênicos , Fosforilação , Presenilinas/genética , Sinapsinas/genéticaRESUMO
Presenilin proteins make the catalytic component of γ-secretase, a multiprotein transmembrane protease, and are type II transmembrane proteins. Amyloid protein, Notch, and beta catenin are among more than 90 substrates of Presenilins. Mutations in Presenilins lead to defects in proteolytic cleavage of its substrate resulting in some of the most devastating pathological conditions including Alzheimer disease (AD), developmental disorders, and cancer. In addition to catalytic roles, Presenilin protein is also shown to be involved in many non-catalytic roles, i.e., calcium homeostasis, regulation of autophagy, and protein trafficking, etc. These proteolytic proteins are highly conserved and are present in almost all the major eukaryotic groups. Studies, performed on a wide variety of organisms ranging from human to unicellular dictyostelium, have shown the important catalytic and non-catalytic roles of Presenilins. In this study, we infer the evolutionary patterns and history of Presenilins as well as of other γ-secretase proteins. We show that Presenilins are the most ancient of the γ-secretase proteins and that Presenilins may have their origin in the last common ancestor (LCA) of Eukaryotes. We also demonstrate that Presenilin proteins generally lack diversifying selection during the course of their evolution. Through evolutionary trace analysis, we show that Presenilin protein sites that undergo mutations in Familial Alzheimer disease, are highly conserved in metazoans. Finally, we discuss the evolutionary, physiological, and pathological implications of our findings and propose that the evolutionary profile of Presenilins supports the loss of function hypothesis of AD pathogenesis.
Assuntos
Doença de Alzheimer , Evolução Molecular , Presenilinas/genética , Secretases da Proteína Precursora do Amiloide/genética , Dictyostelium , HumanosRESUMO
Alzheimer's disease (AD) is the most frequent type of dementia affecting memory, thinking and behaviour. The major hallmark of the disease is pathological neurodegeneration due to abnormal aggregation of Amyloid beta (Aß) peptides generated by ß- and γ-secretases via amyloidogenic pathway. Purpose of the current study was to evaluate the effects of theasaponin E1 on the inhibition of Aß producing ß-, γ-secretases (BACE1, PS1 and NCT) and acetylcholinesterase and activation of the non-amyloidogenic APP processing α-secretase (ADAM10). Additionally, theasaponin E1 effects on Aß degrading and clearing proteins neprilysin and insulin degrading enzyme (IDE). The effect of theasaponin E1 on these crucial enzymes was investigated by RT-PCR, ELISA, western blotting and fluorometric assays using mouse neuroblastoma cells (SweAPP N2a). theasaponin E1 was extracted and purified from green tea seed extract via HPLC, and N2a cells were treated with different concentrations for 24 h. Gene and protein expression in the cells were measured to determine the effects of activation and/or inhibition of theasaponin E1 on ß- and γ-secretases, neprilysin and IDE. Results demonstrated that theasaponin E1 significantly reduced Aß concentration by activation of the α-secretase and neprilysin. The activities of ß- and γ-secretase were reduced in a dose-dependent manner due to downregulation of BACE1, presenilin, and nicastrin. Similarly, theasaponin E1 significantly reduced the activity of acetylcholinesterase. Overall, from the results it is concluded that green tea seed extracted saponin E1 possess therapeutic significance as a neuroprotective natural product recommended for the treatment of Alzheimer's disease.
Assuntos
Camellia sinensis/química , Regulação da Expressão Gênica/efeitos dos fármacos , Neurônios/efeitos dos fármacos , Fármacos Neuroprotetores/farmacologia , Ácido Oleanólico/análogos & derivados , Saponinas/farmacologia , Proteína ADAM10/genética , Proteína ADAM10/metabolismo , Acetilcolinesterase/genética , Acetilcolinesterase/metabolismo , Secretases da Proteína Precursora do Amiloide/antagonistas & inibidores , Secretases da Proteína Precursora do Amiloide/genética , Secretases da Proteína Precursora do Amiloide/metabolismo , Peptídeos beta-Amiloides/antagonistas & inibidores , Peptídeos beta-Amiloides/genética , Peptídeos beta-Amiloides/metabolismo , Animais , Ácido Aspártico Endopeptidases/antagonistas & inibidores , Ácido Aspártico Endopeptidases/genética , Ácido Aspártico Endopeptidases/metabolismo , Linhagem Celular Tumoral , Relação Dose-Resposta a Droga , Insulisina/antagonistas & inibidores , Insulisina/genética , Insulisina/metabolismo , Glicoproteínas de Membrana/antagonistas & inibidores , Glicoproteínas de Membrana/genética , Glicoproteínas de Membrana/metabolismo , Proteínas de Membrana/agonistas , Proteínas de Membrana/genética , Proteínas de Membrana/metabolismo , Camundongos , Neprilisina/antagonistas & inibidores , Neprilisina/genética , Neprilisina/metabolismo , Neurônios/metabolismo , Neurônios/patologia , Fármacos Neuroprotetores/isolamento & purificação , Ácido Oleanólico/isolamento & purificação , Ácido Oleanólico/farmacologia , Extratos Vegetais/química , Presenilinas/antagonistas & inibidores , Presenilinas/genética , Presenilinas/metabolismo , Saponinas/isolamento & purificação , Sementes/química , Chá/químicaRESUMO
Presenilins (PS) are the catalytic components of γ-secretase complexes that mediate intramembrane proteolysis. Mutations in the PS genes are a major cause of familial early-onset Alzheimer disease and affect the cleavage of the amyloid precursor protein, thereby altering the production of the amyloid ß-peptide. However, multiple additional protein substrates have been identified, suggesting pleiotropic functions of γ-secretase. Here, we demonstrate that inhibition of γ-secretase causes dysregulation of cellular lipid homeostasis, including up-regulation of liver X receptors, and complex changes in the cellular lipid composition. Genetic and pharmacological inhibition of γsecretase leads to strong accumulation of cytoplasmic lipid droplets, associated with increased levels of acylglycerols, but lowered cholesteryl esters. Furthermore, accumulation of lipid droplets was augmented by increasing levels of amyloid precursor protein C-terminal fragments, indicating a critical involvement of this γ-secretase substrate. Together, these data provide a mechanism that functionally connects γ-secretase activity to cellular lipid metabolism. These effects were also observed in human astrocytic cells, indicating an important function of γ-secretase in cells critical for lipid homeostasis in the brain.
Assuntos
Secretases da Proteína Precursora do Amiloide/metabolismo , Ésteres do Colesterol/metabolismo , Glicerídeos/metabolismo , Gotículas Lipídicas/metabolismo , Receptores X do Fígado/metabolismo , Transdução de Sinais/efeitos dos fármacos , Transdução de Sinais/genética , Doença de Alzheimer/genética , Doença de Alzheimer/metabolismo , Secretases da Proteína Precursora do Amiloide/antagonistas & inibidores , Secretases da Proteína Precursora do Amiloide/genética , Animais , Astrócitos/metabolismo , Encéfalo/metabolismo , Linhagem Celular Tumoral , Diaminas/farmacologia , Fibroblastos/metabolismo , Deleção de Genes , Homeostase/efeitos dos fármacos , Homeostase/genética , Humanos , Camundongos , Camundongos Knockout , Presenilinas/deficiência , Presenilinas/genética , Tiazóis/farmacologia , Transfecção , Regulação para Cima/efeitos dos fármacos , Regulação para Cima/genéticaRESUMO
Aluminum (Al) is an important environmental metal factor that can be potentially associated with pathological changes leading to neurotoxicity. The silkworm, Bombyx mori, is an important economic insect and has also been used as a model organism in various research areas. However, the toxicity of Al on silkworm physiology has not been reported. Here, we comprehensively investigate the toxic effects of Al on the silkworm, focusing on its effects on viability and development, superoxide dismutase (SOD) activity, and the expression of presenilin and cAMP response element-binding protein (CREB) in BmE cells and silkworm larvae. BmE cell viability decreased after treatment with aluminum chloride (AlCl3 ) in both dose- and time-dependent manners. When AlCl3 solution was injected into newly hatched fifth instar larvae, both larval weight gain and survival rate were significantly decreased in a manner correlating with AlCl3 dose and developmental stage. Furthermore, when BmE cells and silkworm larvae were exposed to AlCl3 , SOD activity decreased significantly relative to the control group, whereas presenilin expression increased more than twofold. Additionally, CREB and phosphorylated CREB (p-CREB) expression in the heads of fifth instar larvae decreased by 28.0% and 50.0%, respectively. These results indicate that Al inhibits the growth and development of silkworms in vitro and in vivo, altering SOD activity and the expressions of presenilin, CREB, and p-CREB. Our data suggest that B. mori can serve as a model animal for studying Al-induced neurotoxicity or neurodegeneration.
Assuntos
Compostos de Alumínio/toxicidade , Alumínio/toxicidade , Bombyx/efeitos dos fármacos , Cloretos/toxicidade , Poluentes Ambientais/toxicidade , Proteínas de Insetos/genética , Neurotoxinas/toxicidade , Cloreto de Alumínio , Animais , Peso Corporal/efeitos dos fármacos , Bombyx/enzimologia , Bombyx/genética , Bombyx/crescimento & desenvolvimento , Linhagem Celular , Sobrevivência Celular/efeitos dos fármacos , Proteína de Ligação ao Elemento de Resposta ao AMP Cíclico/genética , Proteína de Ligação ao Elemento de Resposta ao AMP Cíclico/metabolismo , Proteínas de Insetos/metabolismo , Larva/efeitos dos fármacos , Larva/enzimologia , Larva/genética , Longevidade/efeitos dos fármacos , Presenilinas/genética , Presenilinas/metabolismo , Superóxido Dismutase/genética , Superóxido Dismutase/metabolismoRESUMO
Alzheimer's disease (AD) is a major neurodegenerative disease, associated with the hallmark proteinacious constituent called amyloid beta (Aß) of senile plaques. Moreover, it is already established that metals (particularly copper, zinc and iron) have a key role in the pathogenesis of AD. In order to reduce the Aß plaque burden and overcome the side effects from the synthetic inhibitors, the current study was designed to focus on direct inhibition of with or without metal-induced Aß fibril formation and aggregation by using olive biophenols. Exposure of neuroblastoma (SH-SY5Y) cells with Aß42 resulted in decrease of cell viability and morphological changes might be due to severe increase in the reactive oxygen species (ROS). The pre-treated SH-SY5Y cells with olive biophenols were able to attenuate cell death caused by Aß42, copper- Aß42, and [laevodihydroxyphenylalanine (l-DOPA)] l-DOPA-Aß42-induced toxicity after 24 h of treatment. Oleuropein, verbascoside and rutin were the major anti-amyloidogenic compounds. Transgenic mice (APPswe/PS1dE9) received 50 mg/kg of oleuropein containing olive leaf extracts (OLE) or control diet from 7 to 23 weeks of age. Treatment mice (OLE) were showed significantly reduced amyloid plaque deposition (p < 0.001) in cortex and hippocampus as compared to control mice. Our findings provide a basis for considering natural and low cost biophenols from olive as a promising candidate drug against AD. Further studies warrant to validate and determine the anti-amyloid mechanism, bioavailability as well as permeability of olive biophenols against blood brain barrier in AD.
Assuntos
Doença de Alzheimer/tratamento farmacológico , Glucosídeos/farmacologia , Iridoides/farmacologia , Fármacos Neuroprotetores/farmacologia , Fenóis/farmacologia , Rutina/farmacologia , Doença de Alzheimer/genética , Precursor de Proteína beta-Amiloide/genética , Animais , Linhagem Celular Tumoral , Glucosídeos/uso terapêutico , Humanos , Glucosídeos Iridoides , Iridoides/uso terapêutico , Masculino , Camundongos , Neurônios/efeitos dos fármacos , Neurônios/metabolismo , Fármacos Neuroprotetores/uso terapêutico , Olea/química , Fenóis/uso terapêutico , Presenilinas/genética , Rutina/uso terapêuticoAssuntos
Fucosiltransferases/genética , Glucosiltransferases/genética , Doenças do Cabelo/genética , Hiperpigmentação/genética , Queratina-5/genética , Mutação , Presenilinas/genética , Dermatopatias Genéticas/genética , Dermatopatias Papuloescamosas/genética , Marcadores Genéticos/genética , Doenças do Cabelo/patologia , Folículo Piloso/patologia , Humanos , Hiperpigmentação/patologia , Receptores Notch/genética , Transdução de Sinais/genética , Dermatopatias Genéticas/patologia , Dermatopatias Papuloescamosas/patologiaRESUMO
Alzheimer's disease is the most common form of dementia accounting for 50-60% of all dementia cases. This chapter briefly reviews the history of Alzheimer's disease and provides an overview of the clinical syndromes associated with Alzheimer pathology and their associated neuroimaging findings. This chapter also reviews the neuropathology and genetics of Alzheimer's disease and concludes by discussing current work undertaken to identify suitable in vivo biomarkers for the disease.
Assuntos
Doença de Alzheimer/fisiopatologia , Afasia/fisiopatologia , Encéfalo/fisiopatologia , Cognição , Memória , Transportadores de Cassetes de Ligação de ATP/genética , Proteínas Adaptadoras de Transdução de Sinal/genética , Doença de Alzheimer/diagnóstico por imagem , Doença de Alzheimer/genética , Doença de Alzheimer/patologia , Precursor de Proteína beta-Amiloide/genética , Compostos de Anilina , Apolipoproteína E4/genética , Atrofia , Encéfalo/diagnóstico por imagem , Encéfalo/patologia , Antígenos HLA/genética , Humanos , Imageamento por Ressonância Magnética , Neuroimagem , Proteínas Nucleares/genética , Tomografia por Emissão de Pósitrons , Presenilinas/genética , Receptores de Complemento 3b/genética , Tiazóis , Tomografia Computadorizada de Emissão de Fóton Único , Tomografia Computadorizada por Raios X , Proteínas Supressoras de Tumor/genéticaRESUMO
BACKGROUND: Mesenchymal stem cells (MSCs) show therapeutic efficacy in many different age-related degenerative diseases, including Alzheimer's disease. Very little is currently known about whether or not aging impacts the transplantation efficiency of MSCs. METHODS: In this study, we investigated the distribution of intravenously transplanted syngeneic MSCs derived from young and aged mice into young, aged, and transgenic APP/PS1 Alzheimer's disease mice. MSCs from male donors were transplanted into female mice and their distribution pattern was monitored by PCR using Y-chromosome specific probes. Biodistribution of transplanted MSCs in the brains of APP/PS1 mice was additionally confirmed by immunofluorescence and confocal microscopy. RESULTS: Four weeks after transplantation into young mice, young MSCs were found in the lung, axillary lymph nodes, blood, kidney, bone marrow, spleen, liver, heart, and brain cortex. In contrast, young MSCs that were transplanted into aged mice were only found in the brain cortex. In both young and aged mouse recipients, transplantation of aged MSCs showed biodistribution only in the blood and spleen. Although young transplanted MSCs only showed neuronal distribution in the brain cortex in young mice, they exhibited a wide neuronal distribution pattern in the brains of APP/PS1 mice and were found in the cortex, cerebellum, hippocampus, olfactory bulb, and brainstem. The immunofluorescent signal of both transplanted MSCs and resident microglia was robust in the brains of APP/PS1 mice. Monocyte chemoattractant-1 levels were lowest in the brain cortex of young mice and were significantly increased in APP/PS1 mice. Within the hippocampus, monocyte chemoattractant-1 levels were significantly higher in aged mice compared with younger and APP/PS1 mice. CONCLUSIONS: We demonstrate in vivo that MSC biodistribution post transplantation is detrimentally affected by aging and neuronal health. Aging of both the recipient and the donor MSCs used attenuates transplantation efficiency. Clinically, our data would suggest that aged MSCs should not be used for transplantation and that transplantation of MSCs into aged patients will be less efficacious.
Assuntos
Doença de Alzheimer/terapia , Encéfalo/crescimento & desenvolvimento , Movimento Celular , Transplante de Células-Tronco Mesenquimais , Células-Tronco Mesenquimais/fisiologia , Neurônios/metabolismo , Doença de Alzheimer/genética , Peptídeos beta-Amiloides/genética , Animais , Encéfalo/citologia , Encéfalo/metabolismo , Células Cultivadas , Quimiocina CCL2/genética , Quimiocina CCL2/metabolismo , Feminino , Masculino , Células-Tronco Mesenquimais/citologia , Células-Tronco Mesenquimais/metabolismo , Camundongos , Camundongos Endogâmicos C57BL , Neurônios/citologia , Presenilinas/genética , Baço/citologia , Baço/crescimento & desenvolvimento , Baço/metabolismoRESUMO
Presenilin is a catalytic subunit of γ-secretase, which hydrolyzes several transmembrane proteins within the lipid bilayer, together with binding cofactors such as nicastrin, Aph-1, and Pen-2. However, the structural basis as well as molecular mechanism of this unusual proteolytic process remains unknown. We have analyzed the structure and function relationships of presenilin using the substituted-cysteine accessibility method (SCAM), which enables identification of the hydrophilic environment by the accessibility of sulfhydryl reagents to cysteine residues introduced at a desired position. In combination with small molecule inhibitors/modulators and cross-linking experiments, we were able to identify certain residues and regions of presenilin that contribute to its intramembrane-cleaving activity. In addition, we revealed the structural dynamics of the transmembrane domains of presenilin during the formation of the complex and its proteolytic process. The SCAM provides new insights into the relationship between the structure and activity of presenilin, and is useful for probing the protein dynamics of the membrane-embedded enzymes.
Assuntos
Secretases da Proteína Precursora do Amiloide/química , Biologia Molecular/métodos , Presenilinas/química , Relação Estrutura-Atividade , Substituição de Aminoácidos/genética , Secretases da Proteína Precursora do Amiloide/genética , Domínio Catalítico/genética , Cisteína/química , Cisteína/genética , Inibidores Enzimáticos/química , Humanos , Interações Hidrofóbicas e Hidrofílicas/efeitos dos fármacos , Bicamadas Lipídicas/química , Bicamadas Lipídicas/metabolismo , Presenilinas/genética , Presenilinas/metabolismo , Domínios Proteicos/efeitos dos fármacos , Estrutura Terciária de Proteína , Bibliotecas de Moléculas Pequenas/químicaRESUMO
Despite existing knowledge about the role of the A Disintegrin and Metalloproteinase 10 (ADAM10) as the α-secretase involved in the non-amyloidogenic processing of the amyloid precursor protein (APP) and Notch signalling we have only limited information about its regulation. In this study, we have identified ADAM10 interactors using a split ubiquitin yeast two hybrid approach. Tetraspanin 3 (Tspan3), which is highly expressed in the murine brain and elevated in brains of Alzheimer´s disease (AD) patients, was identified and confirmed to bind ADAM10 by co-immunoprecipitation experiments in mammalian cells in complex with APP and the γ-secretase protease presenilin. Tspan3 expression increased the cell surface levels of its interacting partners and was mainly localized in early and late endosomes. In contrast to the previously described ADAM10-binding tetraspanins, Tspan3 did not affect the endoplasmic reticulum to plasma membrane transport of ADAM10. Heterologous Tspan3 expression significantly increased the appearance of carboxy-terminal cleavage products of ADAM10 and APP, whereas N-cadherin ectodomain shedding appeared unaffected. Inhibiting the endocytosis of Tspan3 by mutating a critical cytoplasmic tyrosine-based internalization motif led to increased surface expression of APP and ADAM10. After its downregulation in neuroblastoma cells and in brains of Tspan3-deficient mice, ADAM10 and APP levels appeared unaltered possibly due to a compensatory increase in the expression of Tspans 5 and 7, respectively. In conclusion, our data suggest that Tspan3 acts in concert with other tetraspanins as a stabilizing factor of active ADAM10, APP and the γ-secretase complex at the plasma membrane and within the endocytic pathway.
Assuntos
Proteína ADAM10/genética , Secretases da Proteína Precursora do Amiloide/genética , Precursor de Proteína beta-Amiloide/genética , Endossomos/metabolismo , Proteínas de Membrana/genética , Presenilinas/genética , Tetraspaninas/genética , Proteína ADAM10/metabolismo , Secretases da Proteína Precursora do Amiloide/metabolismo , Precursor de Proteína beta-Amiloide/metabolismo , Animais , Encéfalo/metabolismo , Química Encefálica , Caderinas/genética , Caderinas/metabolismo , Linhagem Celular Tumoral , Membrana Celular/metabolismo , Endocitose , Endossomos/química , Regulação da Expressão Gênica , Células HEK293 , Humanos , Proteínas de Membrana/metabolismo , Camundongos , Proteínas do Tecido Nervoso/genética , Proteínas do Tecido Nervoso/metabolismo , Neurônios/citologia , Neurônios/metabolismo , Presenilinas/metabolismo , Ligação Proteica , Transporte Proteico , Receptores Notch/genética , Receptores Notch/metabolismo , Transdução de Sinais , Tetraspaninas/metabolismo , Técnicas do Sistema de Duplo-HíbridoRESUMO
The γ-secretase protease and associated regulated intramembrane proteolysis play an important role in controlling receptor-mediated intracellular signaling events, which have a central role in Alzheimer disease, cancer progression, and immune surveillance. An increasing number of γ-secretase substrates have a role in cytokine signaling, including the IL-6 receptor, IL-1 receptor type I, and IL-1 receptor type II. In this study, we show that following TNF-converting enzyme-mediated ectodomain shedding of TNF type I receptor (TNFR1), the membrane-bound TNFR1 C-terminal fragment is subsequently cleaved by γ-secretase to generate a cytosolic TNFR1 intracellular domain. We also show that clathrin-mediated internalization of TNFR1 C-terminal fragment is a prerequisite for efficient γ-secretase cleavage of TNFR1. Furthermore, using in vitro and in vivo model systems, we show that in the absence of presenilin expression and γ-secretase activity, TNF-mediated JNK activation was prevented, assembly of the TNFR1 pro-apoptotic complex II was reduced, and TNF-induced apoptosis was inhibited. These observations demonstrate that TNFR1 is a γ-secretase substrate and suggest that γ-secretase cleavage of TNFR1 represents a new layer of regulation that links the presenilins and the γ-secretase protease to pro-inflammatory cytokine signaling.
Assuntos
Secretases da Proteína Precursora do Amiloide/metabolismo , Apoptose , Receptores Tipo I de Fatores de Necrose Tumoral/metabolismo , Fator de Necrose Tumoral alfa/metabolismo , Animais , Linhagem Celular , Quimiocina CXCL1/metabolismo , Ativação Enzimática , Deleção de Genes , Humanos , MAP Quinase Quinase 4/metabolismo , Camundongos Endogâmicos C57BL , Camundongos Knockout , Presenilinas/genética , Presenilinas/metabolismo , Proteólise , Transdução de SinaisRESUMO
Melatonin is involved in the control of various physiological functions, such as sleep, cell growth and free radical scavenging. The ability of melatonin to behave as an antioxidant, together with the fact that the Alzheimer-related amyloid ß-peptide (Aß) triggers oxidative stress through hydroxyl radical-induced cell death, suggests that melatonin could reduce Alzheimer's pathology. Although the exact etiology of Alzheimer's disease (AD) remains to be established, excess Aß is believed to be the primary contributor to the dysfunction and degeneration of neurons that occurs in AD. Aß peptides are produced via the sequential cleavage of ß-secretase ß-site APP-cleaving enzyme 1 (BACE1) and γ-secretase (PS1/PS2), while α-secretase (ADAM10) prevents the production of Aß peptides. We hypothesized that melatonin could inhibit BACE1 and PS1/PS2 and enhance ADAM10 expression. Using the human neuronal SH-SY5Y cell line, we found that melatonin inhibited BACE1 and PS1 and activated ADAM10 mRNA level and protein expression in a concentration-dependent manner and mediated via melatonin G protein-coupled receptors. Melatonin inhibits BACE1 and PS1 protein expressions through the attenuation of nuclear factor-κB phosphorylation (pNF-κB). Moreover, melatonin reduced BACE1 promoter transactivation and consequently downregulated ß-secretase catalytic activity. The present data show that melatonin is not only a potential regulator of ß/γ-secretase but also an activator of α-secretase expression through the activation of protein kinase C, thereby favoring the nonamyloidogenic pathway over the amyloidogenic pathway. Altogether, our findings suggest that melatonin may be a potential therapeutic agent for reducing the risk of AD in humans.
Assuntos
Proteínas ADAM/metabolismo , Secretases da Proteína Precursora do Amiloide/metabolismo , Peptídeos beta-Amiloides/metabolismo , Ácido Aspártico Endopeptidases/metabolismo , Melatonina/farmacologia , Proteínas de Membrana/metabolismo , Neuroblastoma/metabolismo , Receptores de Melatonina/metabolismo , Proteínas ADAM/genética , Proteína ADAM10 , Secretases da Proteína Precursora do Amiloide/genética , Ácido Aspártico Endopeptidases/genética , Linhagem Celular Tumoral , Expressão Gênica/efeitos dos fármacos , Expressão Gênica/genética , Humanos , Proteínas de Membrana/genética , Neuroblastoma/genética , Presenilinas/genética , Presenilinas/metabolismo , Receptores de Melatonina/genéticaRESUMO
Alzheimer's disease (AD) is a major cause of dementia in the elderly. Although early-onset (familial) AD is attributed to gene mutations, the cause for late-onset (sporadic) AD, which accounts for 95% of AD cases, is unknown. In this study, we show that exposure of 6-week-old amyloid beta precursor protein (APP)/presenilin (PS1) overexpressing mice, a well-established animal model of AD, and nontransgenic littermates to a cyclic O3 exposure protocol, which mimics environmental exposure episodes, accelerated learning/memory function loss in male APP/PS1 mice but not in female APP/PS1 mice or nontransgenic littermates. Female APP/PS1 mice had higher brain levels of amyloid beta peptide (Aß42) and Aß40, compared with male APP/PS1 mice; O3 exposure, however, had no significant effect on brain Aß load in either male or female mice. Our results further show that male APP/PS1 mice had lower levels of antioxidants (glutathione and ascorbate) and experienced augmented induction of NADPH oxidases, lipid peroxidation, and neuronal apoptosis upon O3 exposure, compared with female APP/PS1 mice. No significant effect of O3 on any of these parameters was detected in nontransgenic littermates. In vitro studies further show that 4-hydroxynonenal, a lipid peroxidation product which was increased in the plasma and cortex/hippocampus of O3-exposed male APP/PS1 mice, induced neuroblastoma cell apoptosis. Together, the results suggest that O3 exposure per se may not cause AD but can synergize with genetic risk factors to accelerate the pathophysiology of AD in genetically predisposed populations. The results also suggest that males may be more sensitive to O3-induced neuropathophysiology than females due to lower levels of antioxidants.
Assuntos
Doença de Alzheimer/induzido quimicamente , Doença de Alzheimer/psicologia , Transtornos da Memória/induzido quimicamente , Transtornos da Memória/psicologia , Doenças do Sistema Nervoso/induzido quimicamente , Doenças do Sistema Nervoso/psicologia , Ozônio/toxicidade , Peptídeos beta-Amiloides/biossíntese , Peptídeos beta-Amiloides/genética , Animais , Antioxidantes/metabolismo , Apoptose/efeitos dos fármacos , Química Encefálica/efeitos dos fármacos , Feminino , Peroxidação de Lipídeos/efeitos dos fármacos , Masculino , Camundongos , NADPH Oxidases/metabolismo , Neurônios/efeitos dos fármacos , Neurônios/patologia , Fragmentos de Peptídeos/biossíntese , Fragmentos de Peptídeos/genética , Presenilinas/biossíntese , Presenilinas/genética , Caracteres SexuaisRESUMO
BACKGROUND: Familial Alzheimer's disease (FAD) mutations in presenilin (PS) modulate PS/γ-secretase activity and therefore contribute to AD pathogenesis. Previously, we found that PS/γ-secretase cleaves voltage-gated sodium channel ß2-subunits (Navß2), releases the intracellular domain of Navß2 (ß2-ICD), and thereby, increases intracellular sodium channel α-subunit Nav1.1 levels. Here, we tested whether FAD-linked PS1 mutations modulate Navß2 cleavages and Nav1.1 levels. OBJECTIVE: It was the aim of this study to analyze the effects of PS1-linked FAD mutations on Navß2 processing and Nav1.1 levels in neuronal cells. METHODS: We first generated B104 rat neuroblastoma cells stably expressing Navß2 and wild-type PS1 (wtPS1), PS1 with one of three FAD mutations (E280A, M146L or ΔE9), or PS1 with a non-FAD mutation (D333G). Navß2 processing and Nav1.1 protein and mRNA levels were then analyzed by Western blot and real-time RT-PCR, respectively. RESULTS: The FAD-linked E280A mutation significantly decreased PS/γ-secretase-mediated processing of Navß2 as compared to wtPS1 controls, both in cells and in a cell-free system. Nav1.1 mRNA and protein levels, as well as the surface levels of Nav channel α-subunits, were also significantly reduced in PS1(E280A) cells. CONCLUSION: Our data indicate that the FAD-linked PS1(E280A) mutation decreases Nav channel levels by partially inhibiting the PS/γ-secretase-mediated cleavage of Navß2 in neuronal cells.
Assuntos
Mutação , Neurônios/metabolismo , Presenilinas/genética , Canais de Sódio Disparados por Voltagem/genética , Canais de Sódio Disparados por Voltagem/metabolismo , Secretases da Proteína Precursora do Amiloide/metabolismo , Animais , Western Blotting , Células Cultivadas , Ratos , Reação em Cadeia da Polimerase em Tempo Real , Reação em Cadeia da Polimerase Via Transcriptase Reversa , TransfecçãoRESUMO
Within axons, molecular motors transport essential components required for neuronal growth and viability. Although many levels of control and regulation must exist for proper anterograde and retrograde transport of vital proteins, little is known about these mechanisms. We previously showed that presenilin (PS), a gene involved in Alzheimer's disease (AD), influences kinesin-1 and dynein function in vivo. Here, we show that these PS-mediated effects on motor protein function are via a pathway that involves glycogen synthase kinase-3ß (GSK-3ß). PS genetically interacts with GSK-3ß in an activity-dependent manner. Excess of active GSK-3ß perturbed axonal transport by causing axonal blockages, which were enhanced by reduction of kinesin-1 or dynein. These GSK-3ß-mediated axonal defects do not appear to be caused by disruptions or alterations in microtubules (MTs). Excess of non-functional GSK-3ß did not affect axonal transport. Strikingly, GSK-3ß-activity-dependent axonal transport defects were enhanced by reduction of PS. Collectively, our findings suggest that PS and GSK-3ß are required for normal motor protein function. Our observations propose a model, in which PS likely plays a role in regulating GSK-3ß activity during transport. These findings have important implications for our understanding of the complex regulatory machinery that must exist in vivo and how this system is coordinated during the motility of vesicles within axons.
Assuntos
Transporte Axonal/fisiologia , Dineínas/metabolismo , Quinase 3 da Glicogênio Sintase/metabolismo , Cinesinas/metabolismo , Presenilinas/metabolismo , Animais , Animais Geneticamente Modificados , Linhagem Celular , Drosophila , Epistasia Genética , Feminino , Genótipo , Quinase 3 da Glicogênio Sintase/genética , Glicogênio Sintase Quinase 3 beta , Humanos , Masculino , Atividade Motora/genética , Presenilinas/genética , Transdução de SinaisRESUMO
The Coxsackievirus and Adenovirus Receptor (CAR) is a cell adhesion molecule originally characterized as a virus receptor but subsequently shown to be involved in physiological processes such as neuronal and heart development, epithelial tight junction integrity, and tumour suppression. Proteolysis of cell adhesion molecules and a wide variety of other cell surface proteins serves as a mechanism for protein turnover and, in some cases, cell signaling. Metalloproteases such as A Disintegrin and Metalloprotease (ADAM) family members cleave cell surface receptors to release their substrates' ectodomains, while the presenilin/ɣ-secretase complex mediates regulated intramembrane proteolysis (RIP), releasing intracellular domain fragments from the plasma membrane. In the case of some substrates such as Notch and amyloid precursor protein (APP), the released intracellular domains enter the nucleus to modulate gene expression. We report that CAR ectodomain is constitutively shed from glioma cells and developing neurons, and is also shed when cells are treated with the phorbol ester phorbol 12-myristate 13-acetate (PMA) and the calcium ionophore ionomycin. We identified ADAM10 as a sheddase of CAR using assays involving shRNA knockdown and rescue, overexpression of wild-type ADAM10 and inhibition of ADAM10 activity by addition of its prodomain. In vitro peptide cleavage, mass spectrometry and mutagenesis revealed the amino acids M224 to L227 of CAR as the site of ADAM10-mediated ectodomain cleavage. CAR also undergoes RIP by the presenilin/γ-secretase complex, and the intracellular domain of CAR enters the nucleus. Ectodomain shedding is a prerequisite for RIP of CAR. Thus, CAR belongs to the increasing list of cell surface molecules that undergo ectodomain shedding and that are substrates for ɣ-secretase-mediated RIP.
Assuntos
Secretases da Proteína Precursora do Amiloide/metabolismo , Membrana Celular/metabolismo , Proteína de Membrana Semelhante a Receptor de Coxsackie e Adenovirus/metabolismo , Presenilinas/metabolismo , Proteólise , Proteínas ADAM/genética , Proteínas ADAM/metabolismo , Proteína ADAM10 , Secretases da Proteína Precursora do Amiloide/genética , Animais , Carcinógenos/farmacologia , Linhagem Celular Tumoral , Membrana Celular/genética , Proteína de Membrana Semelhante a Receptor de Coxsackie e Adenovirus/genética , Células HEK293 , Humanos , Proteínas de Membrana/genética , Proteínas de Membrana/metabolismo , Camundongos , Mutagênese Sítio-Dirigida , Neurônios/citologia , Neurônios/metabolismo , Presenilinas/genética , Estrutura Terciária de Proteína , Acetato de Tetradecanoilforbol/farmacologiaRESUMO
Neurons and other cells require intracellular transport of essential components for viability and function. Previous work has shown that while net amyloid precursor protein (APP) transport is generally anterograde, individual vesicles containing APP move bi-directionally. This discrepancy highlights our poor understanding of the in vivo regulation of APP-vesicle transport. Here, we show that reduction of presenilin (PS) or suppression of gamma-secretase activity substantially increases anterograde and retrograde velocities for APP vesicles. Strikingly, PS deficiency has no effect on an unrelated cargo vesicle class containing synaptotagmin, which is powered by a different kinesin motor. Increased velocities caused by PS or gamma-secretase reduction require functional kinesin-1 and dynein motors. Together, our findings suggest that a normal function of PS is to repress kinesin-1 and dynein motor activity during axonal transport of APP vesicles. Furthermore, our data suggest that axonal transport defects induced by loss of PS-mediated regulatory effects on APP-vesicle motility could be a major cause of neuronal and synaptic defects observed in Alzheimer's Disease (AD) pathogenesis. Thus, perturbations of APP/PS transport could contribute to early neuropathology observed in AD, and highlight a potential novel therapeutic pathway for early intervention, prior to neuronal loss and clinical manifestation of disease.
Assuntos
Precursor de Proteína beta-Amiloide/metabolismo , Transporte Axonal , Dineínas/metabolismo , Cinesinas/metabolismo , Neurônios/fisiologia , Presenilinas/metabolismo , Vesículas Transportadoras/metabolismo , Doença de Alzheimer/genética , Doença de Alzheimer/metabolismo , Doença de Alzheimer/patologia , Secretases da Proteína Precursora do Amiloide/metabolismo , Precursor de Proteína beta-Amiloide/genética , Animais , Drosophila/genética , Drosophila/metabolismo , Dineínas/genética , Cinesinas/genética , Larva/genética , Larva/metabolismo , Camundongos , Camundongos Transgênicos , Neurônios/metabolismo , Presenilinas/genética , Transporte Proteico/fisiologia , Vesículas Transportadoras/química , Vesículas Transportadoras/genéticaRESUMO
Mutations in presenilins (PS1 and PS2) have been linked to the pathogenesis of early onset familial Alzheimer's disease. Presenilins function as the catalytic component of the γ-secretase protease complexes responsible for the cleavage of the amyloid precursor protein (APP), subsequent generation of amyloid-ß and associated amyloid plaques in Alzheimer's disease. Biochemical and genetic studies have revealed that through interactions with several proteins, the presenilins are functionally involved in a range of cellular processes, including the regulation of intracellular calcium homeostasis. Our group has previously reported an association between presenilins and members of the tumour necrosis factor receptor-associated factor (TRAF) family of proteins. In this study we further investigated the association between TRAF6, an E3 ubiquitin ligase, and the presenilins. Here we show that the presenilin full-length holoproteins are novel substrates of TRAF6-mediated Lysine-63-linked ubiquitination. Interestingly, co-expression of catalytically active TRAF6 with the presenilins leads to decreased turnover of PS1 full-length holoprotein accompanying elevated presenilin protein levels. Similarly, while overexpression of TRAF6 increases presenilin holoprotein levels and ubiquitination in HEK293 cells, expression of catalytically deficient TRAF6 or TRAF6-deficiency leads to a reduction in presenilin protein levels and reduced PS1 ubiquitination. We also demonstrate that TRAF6 induces PS1 gene transcription in a JNK-dependent manner. Notably, we reveal that TRAF6-mediated ubiquitination of presenilin does not affect γ-secretase enzyme activity, but may regulate presenilin function in calcium signalling. Taken together, we propose that presenilins are novel substrates for TRAF6-mediated K63-linked ubiquitination and that ubiquitination of presenilins by TRAF6 increases presenilin holoprotein levels and in conditions in which TRAF6 ubiquitination of presenilins is reduced results in reduction of calcium release from the endoplasmic reticulum.