RESUMO
Alzheimer's disease (AD) is a devastating neurodegenerative disorder characterized by pathological brain lesions and a decline in cognitive function. ß-Amyloid peptides (Aß), derived from proteolytic processing of amyloid precursor protein (APP), play a central role in AD pathogenesis. ß-Site APP cleaving enzyme 1 (BACE1), the transmembrane aspartyl protease which initiates Aß production, is axonally transported in neurons and accumulates in dystrophic neurites near cerebral amyloid deposits in AD. BACE1 is modified by S-palmitoylation at four juxtamembrane cysteine residues. S-palmitoylation is a dynamic posttranslational modification that is important for trafficking and function of several synaptic proteins. Here, we investigated the in vivo significance of BACE1 S-palmitoylation through the analysis of knock-in mice with cysteine-to-alanine substitution at the palmitoylated residues (4CA mice). BACE1 expression, as well as processing of APP and other neuronal substrates, was unaltered in 4CA mice despite the lack of BACE1 S-palmitoylation and reduced lipid raft association. Whereas steady-state Aß levels were similar, synaptic activity-induced endogenous Aß production was not observed in 4CA mice. Furthermore, we report a significant reduction of cerebral amyloid burden and BACE1 accumulation in dystrophic neurites in the absence of BACE1 S-palmitoylation in mouse models of AD amyloidosis. Studies in cultured neurons suggest that S-palmitoylation is required for dendritic spine localization and axonal targeting of BACE1. Finally, the lack of BACE1 S-palmitoylation mitigates cognitive deficits in 5XFAD mice. Using transgenic mouse models, these results demonstrate that intrinsic posttranslational S-palmitoylation of BACE1 has a significant impact on amyloid pathogenesis and the consequent cognitive decline.
Assuntos
Doença de Alzheimer/metabolismo , Secretases da Proteína Precursora do Amiloide/metabolismo , Amiloide/metabolismo , Ácido Aspártico Endopeptidases/metabolismo , Transtornos da Memória/metabolismo , Peptídeos beta-Amiloides/metabolismo , Precursor de Proteína beta-Amiloide/metabolismo , Proteínas Amiloidogênicas/metabolismo , Amiloidose/metabolismo , Animais , Axônios/metabolismo , Encéfalo/metabolismo , Modelos Animais de Doenças , Feminino , Lipoilação/fisiologia , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Transgênicos , Neurônios/metabolismo , Processamento de Proteína Pós-Traducional/fisiologiaRESUMO
Numerous physiological functions, including a role as a cell surface receptor, have been ascribed to Alzheimer's disease-associated amyloid precursor protein (APP). However, detailed analysis of intracellular signaling mediated by APP in neurons has been lacking. Here, we characterized intrinsic signaling associated with membrane-bound APP C-terminal fragments, which are generated following APP ectodomain release by α- or ß-secretase cleavage. We found that accumulation of APP C-terminal fragments or expression of membrane-tethered APP intracellular domain results in adenylate cyclase-dependent activation of PKA (protein kinase A) and inhibition of GSK3ß signaling cascades, and enhancement of axodendritic arborization in rat immortalized hippocampal neurons, mouse primary cortical neurons, and mouse neuroblastoma. We discovered an interaction between BBXXB motif of APP intracellular domain and the heterotrimeric G-protein subunit Gα(S), and demonstrate that Gα(S) coupling to adenylate cyclase mediates membrane-tethered APP intracellular domain-induced neurite outgrowth. Our study provides clear evidence that APP intracellular domain can have a nontranscriptional role in regulating neurite outgrowth through its membrane association. The novel functional coupling of membrane-bound APP C-terminal fragments with Gα(S) signaling identified in this study could impact several brain functions such as synaptic plasticity and memory formation.
Assuntos
Precursor de Proteína beta-Amiloide/fisiologia , Subunidades alfa Gs de Proteínas de Ligação ao GTP/fisiologia , Membranas Intracelulares/fisiologia , Transdução de Sinais/fisiologia , Adenilil Ciclases/metabolismo , Adenilil Ciclases/fisiologia , Sequência de Aminoácidos , Precursor de Proteína beta-Amiloide/química , Animais , Células COS , Linhagem Celular Transformada , Linhagem Celular Tumoral , Membrana Celular/química , Membrana Celular/fisiologia , Proliferação de Células , Chlorocebus aethiops , Feminino , Subunidades alfa Gs de Proteínas de Ligação ao GTP/química , Membranas Intracelulares/química , Masculino , Camundongos , Dados de Sequência Molecular , Neuritos/fisiologia , Estrutura Terciária de Proteína , RatosRESUMO
Sequential cleavage of amyloid precursor protein by ß- and γ-secretases generates ß-amyloid peptides (Aß), which accumulate in the brains of patients with Alzheimer's disease. We recently identified S-palmitoylation of two γ-secretase subunits, APH1 and nicastrin. S-Palmitoylation is an essential posttranslational modification for the proper trafficking and function of many neuronal proteins. In cultured cell lines, lack of S-palmitoylation causes instability of nascent APH1 and nicastrin but does not affect γ-secretase processing of amyloid precursor protein. To determine the importance of γ-secretase S-palmitoylation for Aß deposition in the brain, we generated transgenic mice coexpressing human wild-type or S-palmitoylation-deficient APH1aL and nicastrin in neurons in the forebrain. We found that lack of S-palmitoylation did not impair the ability of APH1aL and nicastrin to form enzymatically active protein complexes with endogenous presenilin 1 and PEN2 or affect the localization of γ-secretase subunits in dendrites and axons of cortical neurons. When we crossed these mice with 85Dbo transgenic mice, which coexpress familial Alzheimer's disease-causing amyloid precursor protein and presenilin 1 variants, we found that coexpression of wild-type or mutant APH1aL and nicastrin led to marked stabilization of transgenic presenilin 1 in the brains of double-transgenic mice. Interestingly, we observed a moderate, but significant, reduction in amyloid deposits in the forebrain of mice expressing S-palmitoylation-deficient γ-secretase subunits compared with mice overexpressing wild-type subunits, as well as a reduction in the levels of insoluble Aß(40-42). These results indicate that γ-secretase S-palmitoylation modulates Aß deposition in the brain.
Assuntos
Doença de Alzheimer/metabolismo , Secretases da Proteína Precursora do Amiloide/deficiência , Peptídeos beta-Amiloides/metabolismo , Endopeptidases/deficiência , Lipoilação/fisiologia , Glicoproteínas de Membrana/deficiência , Doença de Alzheimer/genética , Doença de Alzheimer/patologia , Sequência de Aminoácidos , Secretases da Proteína Precursora do Amiloide/biossíntese , Secretases da Proteína Precursora do Amiloide/fisiologia , Peptídeos beta-Amiloides/antagonistas & inibidores , Peptídeos beta-Amiloides/fisiologia , Animais , Endopeptidases/biossíntese , Endopeptidases/fisiologia , Lipoilação/genética , Glicoproteínas de Membrana/biossíntese , Glicoproteínas de Membrana/fisiologia , Proteínas de Membrana , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Transgênicos , Dados de Sequência Molecular , Fragmentos de Peptídeos/metabolismo , Fragmentos de Peptídeos/fisiologia , Placa Amiloide/metabolismo , Placa Amiloide/patologiaRESUMO
The γ-secretase protein complex executes the intramembrane proteolysis of amyloid precursor protein (APP), which releases Alzheimer disease ß-amyloid peptide. In addition to APP, γ-secretase also cleaves several other type I membrane protein substrates including Notch1 and N-cadherin. γ-Secretase is made of four integral transmembrane protein subunits: presenilin (PS), nicastrin, APH1, and PEN2. Multiple lines of evidence indicate that a heteromer of PS-derived N- and C-terminal fragments functions as the catalytic subunit of γ-secretase. Only limited information is available on the domains within each subunit involved in the recognition and recruitment of diverse substrates and the transfer of substrates to the catalytic site. Here, we performed mutagenesis of two domains of PS1, namely the first luminal loop domain (LL1) and the second transmembrane domain (TM2), and analyzed PS1 endoproteolysis as well as the catalytic activities of PS1 toward APP, Notch, and N-cadherin. Our results show that distinct residues within LL1 and TM2 domains as well as the length of the LL1 domain are critical for PS1 endoproteolysis, but not for PS1 complex formation with nicastrin, APH1, and PEN2. Furthermore, our experimental PS1 mutants formed γ-secretase complexes with distinct catalytic properties toward the three substrates examined in this study; however, the mutations did not affect PS1 interaction with the substrates. We conclude that the N-terminal LL1 and TM2 domains are critical for PS1 endoproteolysis and the coordination between the putative substrate-docking site and the catalytic core of the γ-secretase.
Assuntos
Secretases da Proteína Precursora do Amiloide/metabolismo , Presenilina-1/metabolismo , Secretases da Proteína Precursora do Amiloide/genética , Precursor de Proteína beta-Amiloide , Animais , Caderinas/genética , Caderinas/metabolismo , Domínio Catalítico/fisiologia , Glicoproteínas de Membrana/genética , Glicoproteínas de Membrana/metabolismo , Camundongos , Camundongos Knockout , Mutagênese , Mutação , Presenilina-1/genética , Estrutura Terciária de Proteína , Receptor Notch1/genética , Receptor Notch1/metabolismo , Especificidade por Substrato/fisiologiaRESUMO
Amyloid precursor protein (APP) and its metabolites play key roles in Alzheimer's disease (AD) pathophysiology. Whereas short amyloid-ß (Aß) peptides derived from APP are pathogenic, the APP holoprotein serves multiple purposes in the nervous system through its cell adhesion and receptor-like properties. Our studies focused on the signaling mediated by the APP cytoplasmic tail. We investigated whether sustained APP signaling during brain development might favor neuronal plasticity and memory process through a direct interaction with the heterotrimeric G-protein subunit GαS (stimulatory G-protein alpha subunit). Our results reveal that APP possesses autonomous regulatory capacity within its intracellular domain that promotes APP cell surface residence, precludes Aß production, facilitates axodendritic development, and preserves cellular substrates of memory. Altogether, these events contribute to strengthening cognitive functions and are sufficient to modify the course of AD pathology.
Assuntos
Doença de Alzheimer/metabolismo , Precursor de Proteína beta-Amiloide/metabolismo , Memória , Neurogênese , Transdução de Sinais , Precursor de Proteína beta-Amiloide/química , Animais , Encéfalo/crescimento & desenvolvimento , Encéfalo/metabolismo , Células Cultivadas , Feminino , Subunidades alfa Gs de Proteínas de Ligação ao GTP/metabolismo , Células HEK293 , Humanos , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Plasticidade Neuronal , Domínios ProteicosRESUMO
Mutations in the genes encoding presenilins (PS1 and PS2) account for the majority of cases of early-onset Alzheimer's disease. PS1 and PS2 form the catalytic center of gamma-secretase, an enzyme responsible for intramembraneous proteolysis of several type I transmembrane proteins. Many gamma-secretase substrates are coupled to intracellular signaling events such as cAMP-response element binding protein and Rac1/p21-activated kinase pathways, which are associated with synaptic function. Here, we have examined the activation of these pathways in neurons lacking PS1 expression or gamma-secretase activity. We found evidence for heightened steady-state activation of cAMP-response element binding protein, Rac1, and p21-activated kinase signaling in PS-deficient neurons. Our study highlights the importance of PS-dependent proteolytic cleavage of gamma-secretase substrates in regulating neuronal signal transduction.
Assuntos
Proteína de Ligação ao Elemento de Resposta ao AMP Cíclico/metabolismo , Neurônios/metabolismo , Neuropeptídeos/metabolismo , Presenilina-1/genética , Quinases Ativadas por p21/metabolismo , Proteínas rac de Ligação ao GTP/metabolismo , Secretases da Proteína Precursora do Amiloide/metabolismo , Animais , Células Cultivadas , Feminino , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Neurônios/citologia , Presenilina-1/metabolismo , Transdução de Sinais/fisiologia , Proteínas rac1 de Ligação ao GTPRESUMO
Sequential processing of amyloid precursor protein by beta- and gamma-secretases generates Alzheimer's disease (AD)-associated beta-amyloid peptides. Recently it was reported that the transmembrane protein p23/TMP21 associates with gamma-secretase, and negatively regulates beta-amyloid production. Despite the link between p23 function and AD pathogenesis, the expression of p23 has not been examined in the brain. Here, we describe the detailed immunohistochemical characterization of p23 expression in rodent and human brain. We report that p23 is co-expressed with gamma-secretase subunits in select neuronal cell populations in rodent brain. Interestingly, the steady-state level of p23 in the brain is high during embryonic development and then declines after birth. Furthermore, the steady-state p23 levels are reduced in the brains of individuals with AD. We conclude that p23 is expressed in neurons throughout the brain and the decline in p23 expression during postnatal development may significantly contribute to enhanced beta-amyloid production in the adult brain.
Assuntos
Proteínas de Membrana/metabolismo , Adulto , Idoso , Idoso de 80 Anos ou mais , Doença de Alzheimer/metabolismo , Doença de Alzheimer/patologia , Animais , Animais Recém-Nascidos , Química Encefálica/genética , Linhagem Celular Tumoral , Células Cultivadas , Cerebelo/química , Cerebelo/metabolismo , Lobo Frontal/química , Lobo Frontal/metabolismo , Células HeLa , Hipocampo/química , Hipocampo/metabolismo , Humanos , Masculino , Proteínas de Membrana/biossíntese , Proteínas de Membrana/genética , Camundongos , Pessoa de Meia-Idade , Proteínas de Transporte Nucleocitoplasmático , Ratos , Ratos Sprague-DawleyRESUMO
Presenilin (PS) plays an essential role in intramembranous gamma-secretase processing of amyloid precursor protein (APP) and several membrane-bound proteins. Here we report that selective accumulation of a membrane-tethered deleted in colorectal cancer (DCC) derivative (DCC-alpha) correlates with extensive neurite outgrowth in transfected neuroblastoma cells and axodendritic connectivity associated with increased spine density in cortical neurons derived from PS1(-/-) embryos, as well as wild-type neurons treated with gamma-secretase inhibitors. cAMP-dependent signaling was also increased in both the neuroblastoma and cortical neuron systems. As a physiological consequence of increases in axodendritic connectivity and in the magnitude of cAMP-dependent signaling, short- and long-term glutamatergic synaptic transmission was enhanced in PS-deficient neurons. Together, these results demonstrate for the first time that PS-mediated gamma-secretase activity attenuates receptor-mediated intracellular signaling pathways that are critical in regulating glutamatergic synaptic transmission and memory processes.
Assuntos
Proteínas de Membrana/metabolismo , Memória/fisiologia , Neurônios/metabolismo , Sistemas do Segundo Mensageiro/fisiologia , Transmissão Sináptica/fisiologia , Secretases da Proteína Precursora do Amiloide , Animais , Ácido Aspártico Endopeptidases/antagonistas & inibidores , Ácido Aspártico Endopeptidases/metabolismo , Carbamatos/farmacologia , Carbazóis/farmacologia , Moléculas de Adesão Celular/metabolismo , Linhagem Celular Tumoral/efeitos dos fármacos , Linhagem Celular Tumoral/metabolismo , Linhagem Celular Tumoral/ultraestrutura , Membrana Celular/metabolismo , Colforsina/farmacologia , AMP Cíclico/fisiologia , Proteínas Quinases Dependentes de AMP Cíclico/metabolismo , Receptor DCC , Dipeptídeos/farmacologia , Endopeptidases , Potenciais Pós-Sinápticos Excitadores/efeitos dos fármacos , Potenciais Pós-Sinápticos Excitadores/fisiologia , Genes DCC , Ácido Glutâmico/fisiologia , Indóis/farmacologia , Lidocaína/análogos & derivados , Lidocaína/farmacologia , Proteínas de Membrana/deficiência , Proteínas de Membrana/genética , Proteínas de Membrana/fisiologia , Memória/efeitos dos fármacos , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Neuritos/metabolismo , Neuritos/ultraestrutura , Neuroblastoma/patologia , Neurônios/efeitos dos fármacos , Piperazinas/farmacologia , Presenilina-1 , Processamento de Proteína Pós-Traducional , Pirróis/farmacologia , Quinoxalinas/farmacologia , Ratos , Receptores de Superfície Celular , Proteínas Recombinantes de Fusão/metabolismo , Sistemas do Segundo Mensageiro/efeitos dos fármacos , Transmissão Sináptica/efeitos dos fármacos , Transfecção , Proteínas Supressoras de Tumor/metabolismoRESUMO
Amyloid precursor protein (APP) and its metabolites play a key role in Alzheimer's disease pathogenesis. The idea that APP may function as a receptor has gained momentum based on its structural similarities to type I transmembrane receptors and the identification of putative APP ligands. We review the recent experimental evidence in support of this notion and discuss how this concept is viewed in the field. Specifically, we focus on the structural and functional characteristics of APP as a cell surface receptor, and on its interaction with adaptors and signaling proteins. We also address the importance of APP function as a receptor in Alzheimer's disease etiology and discuss how this function might be potentially important for the development of novel therapeutic approaches.
Assuntos
Precursor de Proteína beta-Amiloide/metabolismo , Doença de Alzheimer/metabolismo , Animais , HumanosRESUMO
Presenilin 1 (PS1) is an essential γ-secretase component, the enzyme responsible for amyloid precursor protein (APP) intramembraneous cleavage. Mutations in PS1 lead to dominant-inheritance of early-onset familial Alzheimer's disease (FAD). Although expression of FAD-linked PS1 mutations enhances toxic Aß production, the importance of other APP metabolites and γ-secretase substrates in the etiology of the disease has not been confirmed. We report that neurons expressing FAD-linked PS1 variants or functionally deficient PS1 exhibit enhanced axodendritic outgrowth due to increased levels of APP intracellular C-terminal fragment (APP-CTF). APP expression is required for exuberant neurite outgrowth and hippocampal axonal sprouting observed in knock-in mice expressing FAD-linked PS1 mutation. APP-CTF accumulation initiates CREB signaling cascade through an association of APP-CTF with Gαs protein. We demonstrate that pathological PS1 loss-of-function impinges on neurite formation through a selective APP gain-of-function that could impact on axodendritic connectivity and contribute to aberrant axonal sprouting observed in AD patients.
Assuntos
Precursor de Proteína beta-Amiloide/metabolismo , Proteínas Mutantes/genética , Proteínas Mutantes/metabolismo , Neurônios/metabolismo , Presenilina-1/genética , Presenilina-1/metabolismo , Animais , Expressão Gênica , Técnicas de Introdução de Genes , Humanos , CamundongosRESUMO
Abnormal accumulation of ß-secretase (BACE1) in dystrophic neurites and presynaptic ß-amyloid (Aß) production contribute to Alzheimer's disease pathogenesis. Little, however, is known about BACE1 sorting and dynamic transport in neurons. We investigated BACE1 trafficking in hippocampal neurons using live-cell imaging and selective labeling. We report that transport vesicles containing internalized BACE1 in dendrites undergo exclusive retrograde transport toward the soma, whereas they undergo bidirectional transport in axons. Unidirectional dendritic transport requires Eps15-homology-domain-containing (EHD) 1 and 3 protein function. Furthermore, loss of EHD function compromises dynamic axonal transport and overall BACE1 levels in axons. EHD1/3 colocalize with BACE1 and APP ß-C-terminal fragments in hippocampal mossy fiber terminals, and their depletion in neurons significantly attenuates Aß levels. These results demonstrate unidirectional endocytic transport of a dendritic cargo and reveal a role for EHD proteins in neuronal BACE1 transcytosis and Aß production, processes that are highly relevant for Alzheimer's disease.
Assuntos
Doença de Alzheimer/metabolismo , Secretases da Proteína Precursora do Amiloide/metabolismo , Peptídeos beta-Amiloides/metabolismo , Ácido Aspártico Endopeptidases/metabolismo , Transporte Axonal , Proteínas de Transporte/metabolismo , Dendritos/metabolismo , Proteínas de Transporte Vesicular/metabolismo , Animais , Proteínas de Transporte/genética , Células Cultivadas , Células HEK293 , Células HeLa , Hipocampo/citologia , Hipocampo/metabolismo , Humanos , Camundongos , Transporte Proteico , Proteínas de Transporte Vesicular/genéticaRESUMO
BACKGROUND: p23 belongs to the highly conserved p24 family of type I transmembrane proteins, which participate in the bidirectional protein transport between the endoplasmic reticulum and Golgi apparatus. Mammalian p23 has been shown to interact with γ-secretase complex, and modulate secretory trafficking as well as intramembranous processing of amyloid precursor protein in cultured cells. Negative modulation of ß-amyloid production by p23 in cultured cell lines suggested that elevation of p23 expression in neurons might mitigate cerebral amyloid burden. RESULTS: We generated several lines of transgenic mice expressing human p23 in neurons under the control of Thy-1.2 promoter. We found that even a 50% increase in p23 levels in the central nervous system of mice causes post-natal growth retardation, severe neurological problems characterized by tremors, seizure, ataxia, and uncoordinated movements, and premature death. The severity of the phenotype closely correlated with the level of p23 overexpression in multiple transgenic lines. While the number and general morphology of neurons in Hup23 mice appeared to be normal throughout the brain, abnormal non-Golgi p23 localization was observed in a subset of neurons with high transgene expression in brainstem. Moreover, detailed immunofluorescence analysis revealed marked proliferation of astrocytes, activation of microglia, and thinning of myelinated bundles in brainstem of Hup23 mice. CONCLUSIONS: These results demonstrate that proper level of p23 expression is critical for neuronal function, and perturbing p23 function by overexpression initiates a cascade of cellular reactions in brainstem that leads to severe motor deficits and other neurological problems, which culminate in premature death. The neurological phenotype observed in Hup23 mice highlights significant adverse effects associated with manipulating neuronal expression of p23, a previously described negative modulator of γ-secretase activity and ß-amyloid production. Moreover, our report has broader relevance to molecular mechanisms in several neurodegenerative diseases as it highlights the inherent vulnerability of the early secretory pathway mechanisms that ensure proteostasis in neurons.
Assuntos
Proteínas de Membrana/metabolismo , Atividade Motora/fisiologia , Transtornos dos Movimentos/fisiopatologia , Neurônios/metabolismo , Precursor de Proteína beta-Amiloide/metabolismo , Animais , Encéfalo/anatomia & histologia , Encéfalo/metabolismo , Células Cultivadas , Humanos , Proteínas de Membrana/genética , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Transgênicos , Transtornos dos Movimentos/patologia , Proteína Básica da Mielina/metabolismo , Neurônios/citologia , Neurônios/patologia , Proteínas de Transporte NucleocitoplasmáticoRESUMO
Mutations in PSEN genes, which encode presenilin proteins, cause familial early-onset Alzheimer's disease (AD). Transgenic mouse models based on coexpression of familial AD-associated presenilin and amyloid precursor protein variants successfully mimic characteristic pathological features of AD, including plaque formation, synaptic dysfunction, and loss of memory. Presenilins function as the catalytic subunit of gamma-secretase, the enzyme that catalyzes intramembraneous proteolysis of amyloid precursor protein to release beta-amyloid peptides. Familial AD-associated mutations in presenilins alter the site of gamma-secretase cleavage in a manner that increases the generation of longer and highly fibrillogenic beta-amyloid peptides. In addition to amyloid precursor protein, gamma-secretase catalyzes intramembrane proteolysis of many other substrates known to be important for synaptic function. This paper focuses on how various animal models have enabled us to elucidate the physiological importance of diverse gamma-secretase substrates, including amyloid precursor protein and discusses their roles in the context of cellular signaling and synaptic function.
RESUMO
Cerebral deposition of beta-amyloid (Abeta) peptides is a pathological hallmark of Alzheimer disease. Intramembranous proteolysis of amyloid precursor protein by a multiprotein gamma-secretase complex generates Abeta. Previously, it was reported that CD147, a glycoprotein that stimulates production of matrix metalloproteinases (MMPs), is a subunit of gamma-secretase and that the levels of secreted Abeta inversely correlate with CD147 expression. Here, we show that the levels and localization of CD147 in fibroblasts, as well as postnatal expression and distribution in brain, are distinct from those of integral gamma-secretase subunits. Notably, we show that although depletion of CD147 increased extracellular Abeta levels in intact cells, membranes isolated from CD147-depleted cells failed to elevate Abeta production in an in vitro gamma-secretase assay. Consistent with an extracellular source that modulates Abeta metabolism, synthetic Abeta was degraded more rapidly in the conditioned medium of cells overexpressing CD147. Moreover, modulation of CD147 expression had no effect on epsilon-site cleavage of amyloid precursor protein and Notch1 receptor. Collectively, our results demonstrate that CD147 modulates Abeta levels not by regulating gamma-secretase activity, but by stimulating extracellular degradation of Abeta. In view of the known function of CD147 in MMP production, we postulate that CD147 expression influences Abeta levels by an indirect mechanism involving MMPs that can degrade extracellular Abeta.
Assuntos
Precursor de Proteína beta-Amiloide/metabolismo , Basigina/metabolismo , Metaloproteinases da Matriz/metabolismo , Doença de Alzheimer/genética , Doença de Alzheimer/metabolismo , Doença de Alzheimer/patologia , Secretases da Proteína Precursora do Amiloide/metabolismo , Animais , Basigina/genética , Linhagem Celular , Cerebelo/metabolismo , Cerebelo/patologia , Regulação da Expressão Gênica/genética , Humanos , Camundongos , Receptor Notch1/genética , Receptor Notch1/metabolismoRESUMO
Gamma-secretase facilitates the regulated intramembrane proteolysis of select type I membrane proteins that play diverse physiological roles in multiple cell types and tissue. In this study, we used biochemical approaches to examine the distribution of amyloid precursor protein (APP) and several additional gamma-secretase substrates in membrane microdomains. We report that APP C-terminal fragments (CTFs) and gamma-secretase reside in Lubrol WX detergent-insoluble membranes (DIM) of cultured cells and adult mouse brain. APP CTFs that accumulate in cells lacking gamma-secretase activity preferentially associate with DIM. Cholesterol depletion and magnetic immunoisolation studies indicate recruitment of APP CTFs into cholesterol- and sphingolipid-rich lipid rafts, and co-residence of APP CTFs, PS1, and syntaxin 6 in DIM patches derived from the trans-Golgi network. Photoaffinity cross-linking studies provided evidence for the preponderance of active gamma-secretase in lipid rafts of cultured cells and adult brain. Remarkably, unlike the case of APP, CTFs derived from Notch1, Jagged2, deleted in colorectal cancer (DCC), and N-cadherin remain largely detergent-soluble, indicative of their spatial segregation in non-raft domains. In embryonic brain, the majority of PS1 and nicastrin is present in Lubrol WX-soluble membranes, wherein the CTFs derived from APP, Notch1, DCC, and N-cadherin also reside. We suggest that gamma-secretase residence in non-raft membranes facilitates proteolysis of diverse substrates during embryonic development but that the translocation of gamma-secretase to lipid rafts in adults ensures processing of certain substrates, including APP CTFs, while limiting processing of other potential substrates.
Assuntos
Ácido Aspártico Endopeptidases/metabolismo , Membrana Celular/enzimologia , Lipídeos de Membrana/metabolismo , Fatores Etários , Secretases da Proteína Precursora do Amiloide , Precursor de Proteína beta-Amiloide/metabolismo , Animais , Encéfalo/embriologia , Encéfalo/enzimologia , Linhagem Celular Tumoral , Colesterol/metabolismo , Endopeptidases , Deleção de Genes , Proteína Jagged-2 , Microdomínios da Membrana/enzimologia , Proteínas de Membrana/genética , Proteínas de Membrana/metabolismo , Camundongos , Neuroblastoma , Polietilenoglicóis , Presenilina-1 , Presenilina-2 , Proteínas Qa-SNARE , Receptor Notch1 , Receptores de Superfície Celular/metabolismo , Esfingolipídeos/metabolismo , Especificidade por Substrato , Fatores de Transcrição/metabolismoRESUMO
Mutations in the genes encoding presenilin 1 (PS1) and presenilin 2 (PS2) account for the majority of the cases of familial early-onset Alzheimer's disease (FAD). Presenilins (PSs) facilitate the intramembraneous cleavage of amyloid precursor protein (APP), coined gamma-secretase cleavage, which generates beta-amyloid peptides (A beta). Considerable evidence suggests that FAD-linked PS variants exert their pathogenic influence by selectively elevating the levels of highly fibrillogenic A beta 42 peptides. In addition, numerous other functions have been ascribed to PSs based on subcellular localization, protein interactions, loss of function studies, and intramembraneous gamma-secretase cleavage of growing number of substrates. This review summarizes the diverse physiological functions that are regulated by PSs beyond APP metabolism.