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1.
J Biol Chem ; 294(12): 4477-4487, 2019 03 22.
Artigo em Inglês | MEDLINE | ID: mdl-30692199

RESUMO

Alzheimer's disease (AD) is pathologically characterized by the deposition of the ß-amyloid (Aß) peptide in senile plaques in the brain, leading to neuronal dysfunction and eventual decline in cognitive function. Genome-wide association studies have identified the bridging integrator 1 (BIN1) gene within the second most significant susceptibility locus for late-onset AD. BIN1 is a member of the amphiphysin family of proteins and has reported roles in the generation of membrane curvature and endocytosis. Endocytic dysfunction is a pathological feature of AD, and endocytosis of the amyloid precursor protein is an important step in its subsequent cleavage by ß-secretase (BACE1). In vitro evidence implicates BIN1 in endosomal sorting of BACE1 and Aß generation in neurons, but a role for BIN1 in this process in vivo is yet to be described. Here, using biochemical and immunohistochemistry analyses we report that a 50% global reduction of BIN1 protein levels resulting from a single Bin1 allele deletion in mice does not change BACE1 levels or localization in vivo, nor does this reduction alter the production of endogenous murine Aß in nontransgenic mice. Furthermore, we found that reduction of BIN1 levels in the 5XFAD mouse model of amyloidosis does not alter Aß deposition nor behavioral deficits associated with cerebral amyloid burden. Finally, a conditional BIN1 knockout in excitatory neurons did not alter BACE1, APP, C-terminal fragments derived from BACE1 cleavage of APP, or endogenous Aß levels. These results indicate that BIN1 function does not regulate Aß generation in vivo.


Assuntos
Proteínas Adaptadoras de Transdução de Sinal/genética , Doença de Alzheimer/metabolismo , Peptídeos beta-Amiloides/metabolismo , Precursor de Proteína beta-Amiloide/metabolismo , Predisposição Genética para Doença , Proteínas do Tecido Nervoso/genética , Proteínas Supressoras de Tumor/genética , Doença de Alzheimer/genética , Secretases da Proteína Precursora do Amiloide/metabolismo , Animais , Ácido Aspártico Endopeptidases/metabolismo , Encéfalo/metabolismo , Modelos Animais de Doenças , Endocitose , Endossomos/metabolismo , Feminino , Humanos , Masculino , Camundongos , Camundongos Knockout
2.
Proc Natl Acad Sci U S A ; 114(45): E9665-E9674, 2017 11 07.
Artigo em Inglês | MEDLINE | ID: mdl-29078331

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/fisiologia
3.
J Neurochem ; 149(3): 399-412, 2019 05.
Artigo em Inglês | MEDLINE | ID: mdl-30664241

RESUMO

The generation of the amyloid-ß (Aß) peptides from the amyloid precursor protein (APP) through sequential proteolysis by ß- and γ-secretases is a key pathological event in the initiation and propagation of Alzheimer's disease. Aß and the transcriptionally active APP intracellular domain are generated preferentially from the APP695 isoform compared to the longer APP751 isoform. As the Aß and amyloid precursor protein intracellular domain produced from cleavage of APP695 and APP751 are identical we hypothesised that the two isoforms have differences within their interactomes which mediate the differential processing of the two isoforms. To investigate this, we applied a proteomics-based approach to identify differences in the interactomes of the APP695 and APP751 isoforms. Using stable isotope labelling of amino acids in cell culture and quantitative proteomics, we compared the interactomes of APP695 and APP751 expressed in human SH-SY5Y cells. Through this approach, we identified enrichment of proteins involved in mitochondrial function, the nuclear pore and nuclear transport specifically in the APP695 interactome. Further interrogation of the APP interactome and subsequent experimental validation (co-immunoprecipitation and siRNA knockdown) revealed GAP43 as a specific modulator of APP751 proteolysis, altering Aß generation. Our data indicate that interrogation of the APP interactome can be exploited to identify proteins which influence APP proteolysis and Aß production in an isoform dependent-manner. Cover Image for this issue: doi: 10.1111/jnc.14504.


Assuntos
Precursor de Proteína beta-Amiloide/metabolismo , Linhagem Celular Tumoral , Humanos , Mitocôndrias/metabolismo , Neurônios/metabolismo , Isoformas de Proteínas , Proteômica
4.
J Biol Chem ; 291(37): 19235-44, 2016 09 09.
Artigo em Inglês | MEDLINE | ID: mdl-27474742

RESUMO

Proteolysis of the amyloid precursor protein (APP) liberates various fragments including the proposed initiator of Alzheimer disease-associated dysfunctions, amyloid-ß. However, recent evidence suggests that the accepted view of APP proteolysis by the canonical α-, ß-, and γ-secretases is simplistic, with the discovery of a number of novel APP secretases (including δ- and η-secretases, alternative ß-secretases) and additional metabolites, some of which may also cause synaptic dysfunction. Furthermore, various proteins have been identified that interact with APP and modulate its cleavage by the secretases. Here, we give an overview of the increasingly complex picture of APP proteolysis.


Assuntos
Doença de Alzheimer/metabolismo , Secretases da Proteína Precursora do Amiloide/metabolismo , Precursor de Proteína beta-Amiloide/metabolismo , Proteólise , Animais , Humanos
6.
Cell Rep ; 30(10): 3520-3535.e7, 2020 03 10.
Artigo em Inglês | MEDLINE | ID: mdl-32160554

RESUMO

BIN1, a member of the BAR adaptor protein family, is a significant late-onset Alzheimer disease risk factor. Here, we investigate BIN1 function in the brain using conditional knockout (cKO) models. Loss of neuronal Bin1 expression results in the select impairment of spatial learning and memory. Examination of hippocampal CA1 excitatory synapses reveals a deficit in presynaptic release probability and slower depletion of neurotransmitters during repetitive stimulation, suggesting altered vesicle dynamics in Bin1 cKO mice. Super-resolution and immunoelectron microscopy localizes BIN1 to presynaptic sites in excitatory synapses. Bin1 cKO significantly reduces synapse density and alters presynaptic active zone protein cluster formation. Finally, 3D electron microscopy reconstruction analysis uncovers a significant increase in docked and reserve pools of synaptic vesicles at hippocampal synapses in Bin1 cKO mice. Our results demonstrate a non-redundant role for BIN1 in presynaptic regulation, thus providing significant insights into the fundamental function of BIN1 in synaptic physiology relevant to Alzheimer disease.


Assuntos
Proteínas Adaptadoras de Transdução de Sinal/metabolismo , Consolidação da Memória , Proteínas do Tecido Nervoso/metabolismo , Neurônios/metabolismo , Neurotransmissores/metabolismo , Terminações Pré-Sinápticas/metabolismo , Proteínas Supressoras de Tumor/metabolismo , Animais , Encéfalo/metabolismo , Potenciais Pós-Sinápticos Excitadores , Camundongos Endogâmicos C57BL , Camundongos Knockout , Neurônios/ultraestrutura , Terminações Pré-Sinápticas/ultraestrutura , Reconhecimento Psicológico , Proteínas SNARE/metabolismo , Aprendizagem Espacial
7.
Sci Rep ; 9(1): 11416, 2019 08 06.
Artigo em Inglês | MEDLINE | ID: mdl-31388055

RESUMO

The development of cardiovascular disease is intimately linked to elevated levels of low-density lipoprotein (LDL) cholesterol in the blood. Hepatic LDL receptor (LDLR) levels regulate the amount of plasma LDL. We identified the secreted zinc metalloproteinase, bone morphogenetic protein 1 (BMP1), as responsible for the cleavage of human LDLR within its extracellular ligand-binding repeats at Gly171↓Asp172. The resulting 120 kDa membrane-bound C-terminal fragment (CTF) of LDLR had reduced capacity to bind LDL and when expressed in LDLR null cells had compromised LDL uptake as compared to the full length receptor. Pharmacological inhibition of BMP1 or siRNA-mediated knockdown prevented the generation of the 120 kDa CTF and resulted in an increase in LDL uptake into cells. The 120 kDa CTF was detected in the livers from humans and mice expressing human LDLR. Collectively, these results identify that BMP1 regulates cellular LDL uptake and may provide a target to modulate plasma LDL cholesterol.


Assuntos
Proteína Morfogenética Óssea 1/metabolismo , Lipoproteínas LDL/metabolismo , Receptores de LDL/metabolismo , Animais , Aterosclerose/sangue , Aterosclerose/tratamento farmacológico , Aterosclerose/metabolismo , Aterosclerose/patologia , Biópsia , Proteína Morfogenética Óssea 1/antagonistas & inibidores , Proteína Morfogenética Óssea 1/genética , Células CHO , Cricetulus , Técnicas de Silenciamento de Genes , Células Hep G2 , Humanos , Lipoproteínas LDL/sangue , Fígado/química , Fígado/metabolismo , Fígado/patologia , Camundongos , Camundongos Transgênicos , Oxidiazóis/farmacologia , Proteólise/efeitos dos fármacos , RNA Interferente Pequeno/metabolismo , Receptores de LDL/análise , Receptores de LDL/genética , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo
8.
Brain Pathol ; 29(4): 485-501, 2019 07.
Artigo em Inglês | MEDLINE | ID: mdl-30506549

RESUMO

Bridging integrator 1 (BIN1) is the most significant late-onset Alzheimer's disease (AD) susceptibility locus identified via genome-wide association studies. BIN1 is an adaptor protein that regulates membrane dynamics in the context of endocytosis and membrane remodeling. An increase in BIN1 expression and changes in the relative levels of alternatively spliced BIN1 isoforms have been reported in the brains of patients with AD. BIN1 can bind to Tau, and an increase in BIN1 expression correlates with Tau pathology. In contrast, the loss of BIN1 expression in cultured cells elevates Aß production and Tau propagation by insfluencing endocytosis and recycling. Here, we show that BIN1 accumulates adjacent to amyloid deposits in vivo. We found an increase in insoluble BIN1 and a striking accrual of BIN1 within and near amyloid deposits in the brains of multiple transgenic models of AD. The peri-deposit aberrant BIN1 localization was conspicuously different from the accumulation of APP and BACE1 within dystrophic neurites. Although BIN1 is highly expressed in mature oligodendrocytes, BIN1 association with amyloid deposits occurred in the absence of the accretion of other oligodendrocyte or myelin proteins. Finally, super-resolution microscopy and immunogold electron microscopy analyses highlight the presence of BIN1 in proximity to amyloid fibrils at the edges of amyloid deposits. These results reveal the aberrant accumulation of BIN1 is a feature associated with AD amyloid pathology. Our findings suggest a potential role for BIN1 in extracellular Aß deposition in vivo that is distinct from its well-characterized function as an adaptor protein in endocytosis and membrane remodeling.


Assuntos
Proteínas Adaptadoras de Transdução de Sinal/metabolismo , Doença de Alzheimer/patologia , Proteínas Nucleares/metabolismo , Placa Amiloide/patologia , Proteínas Supressoras de Tumor/metabolismo , Proteínas Adaptadoras de Transdução de Sinal/fisiologia , Doença de Alzheimer/metabolismo , Amiloide/metabolismo , Peptídeos beta-Amiloides/metabolismo , Precursor de Proteína beta-Amiloide/metabolismo , Amiloidose/patologia , Animais , Encéfalo/patologia , Modelos Animais de Doenças , Feminino , Estudo de Associação Genômica Ampla , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Transgênicos , Neurogênese/fisiologia , Proteínas Nucleares/fisiologia , Placa Amiloide/metabolismo , Transdução de Sinais , Proteínas Supressoras de Tumor/fisiologia , Proteínas tau/metabolismo
10.
Matters (Zur) ; 20172017 Jan.
Artigo em Inglês | MEDLINE | ID: mdl-29479533

RESUMO

BIN1 is the second most significant Alzheimer's disease (AD) risk factor gene identified through genome-wide association studies. BIN1 is an adaptor protein that can bind to several proteins including c-Myc, clathrin, adaptor protein-2 and dynamin. BIN1 is widely expressed in the brain and peripheral tissue as ubiquitous and tissue-specific alternatively spliced isoforms that regulate membrane dynamics and endocytosis in multiple cell types. The function of BIN1 in the brain and the mechanism(s) by which AD-associated BIN1 alleles increase the risk for the disease are not known. BIN1 has been shown to interact with Tau and two studies reported a positive correlation between BIN1 expression and neurofibrillary tangle pathology in AD. However, an inverse correlation between BIN1 expression and Tau propagation has also been reported. Moreover, there have been conflicting reports on whether BIN1 is present in tangles. A recent study characterized predominant BIN1 expression in mature oligodendrocytes in the gray matter and the white matter in rodent, and the human brain. Here, we have examined BIN1 localization in the brains of patients with AD using immunohistochemistry and immunofluorescence techniques to analyze BIN1 cellular expression in relation to cellular markers and pathological lesions in AD. We report that BIN1 immunoreactivity in human AD is not associated with neurofibrillary tangles or senile plaques. Moreover, our results show that BIN1 is not expressed by resting and activated microglia, astrocytes, or macrophages in human AD. In accordance with a recent report, low-level de novo BIN1 expression can be observed in a subset of neurons in the AD brain. Further investigations are warranted to understand the complex cellular mechanisms underlying the observed correlation between BIN1 expression and the severity of tangle pathology in AD.

11.
Mol Neurodegener ; 11(1): 59, 2016 08 03.
Artigo em Inglês | MEDLINE | ID: mdl-27488240

RESUMO

BACKGROUND: Genome-wide association studies have identified BIN1 within the second most significant susceptibility locus in late-onset Alzheimer's disease (AD). BIN1 undergoes complex alternative splicing to generate multiple isoforms with diverse functions in multiple cellular processes including endocytosis and membrane remodeling. An increase in BIN1 expression in AD and an interaction between BIN1 and Tau have been reported. However, disparate descriptions of BIN1 expression and localization in the brain previously reported in the literature and the lack of clarity on brain BIN1 isoforms present formidable challenges to our understanding of how genetic variants in BIN1 increase the risk for AD. METHODS: In this study, we analyzed BIN1 mRNA and protein levels in human brain samples from individuals with or without AD. In addition, we characterized the BIN1 expression and isoform diversity in human and rodent tissue by immunohistochemistry and immunoblotting using a panel of BIN1 antibodies. RESULTS: Here, we report on BIN1 isoform diversity in the human brain and document alterations in the levels of select BIN1 isoforms in individuals with AD. In addition, we report striking BIN1 localization to white matter tracts in rodent and the human brain, and document that the large majority of BIN1 is expressed in mature oligodendrocytes whereas neuronal BIN1 represents a minor fraction. This predominant non-neuronal BIN1 localization contrasts with the strict neuronal expression and presynaptic localization of the BIN1 paralog, Amphiphysin 1. We also observe upregulation of BIN1 at the onset of postnatal myelination in the brain and during differentiation of cultured oligodendrocytes. Finally, we document that the loss of BIN1 significantly correlates with the extent of demyelination in multiple sclerosis lesions. CONCLUSION: Our study provides new insights into the brain distribution and cellular expression of an important risk factor associated with late-onset AD. We propose that efforts to define how genetic variants in BIN1 elevate the risk for AD would behoove to consider BIN1 function in the context of its main expression in mature oligodendrocytes and the potential for a role of BIN1 in the membrane remodeling that accompanies the process of myelination.


Assuntos
Proteínas Adaptadoras de Transdução de Sinal/metabolismo , Doença de Alzheimer/metabolismo , Proteínas Nucleares/metabolismo , Oligodendroglia/metabolismo , Proteínas Supressoras de Tumor/metabolismo , Substância Branca/metabolismo , Proteínas Adaptadoras de Transdução de Sinal/genética , Adulto , Idoso , Idoso de 80 Anos ou mais , Feminino , Estudo de Associação Genômica Ampla , Humanos , Masculino , Pessoa de Meia-Idade , Neurogênese/genética , Proteínas Nucleares/genética , Proteínas Supressoras de Tumor/genética , Substância Branca/patologia , Proteínas tau/metabolismo
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