RESUMO
γ-Secretases are a family of intramembrane-cleaving proteases involved in various signaling pathways and diseases, including Alzheimer's disease (AD). Cells co-express differing γ-secretase complexes, including two homologous presenilins (PSENs). We examined the significance of this heterogeneity and identified a unique motif in PSEN2 that directs this γ-secretase to late endosomes/lysosomes via a phosphorylation-dependent interaction with the AP-1 adaptor complex. Accordingly, PSEN2 selectively cleaves late endosomal/lysosomal localized substrates and generates the prominent pool of intracellular Aß that contains longer Aß; familial AD (FAD)-associated mutations in PSEN2 increased the levels of longer Aß further. Moreover, a subset of FAD mutants in PSEN1, normally more broadly distributed in the cell, phenocopies PSEN2 and shifts its localization to late endosomes/lysosomes. Thus, localization of γ-secretases determines substrate specificity, while FAD-causing mutations strongly enhance accumulation of aggregation-prone Aß42 in intracellular acidic compartments. The findings reveal potentially important roles for specific intracellular, localized reactions contributing to AD pathogenesis.
Assuntos
Doença de Alzheimer/patologia , Secretases da Proteína Precursora do Amiloide/análise , Peptídeos beta-Amiloides/metabolismo , Fragmentos de Peptídeos/metabolismo , Presenilina-2/análise , Complexo 1 de Proteínas Adaptadoras/metabolismo , Doença de Alzheimer/genética , Doença de Alzheimer/metabolismo , Motivos de Aminoácidos , Secretases da Proteína Precursora do Amiloide/metabolismo , Animais , Linhagem Celular Tumoral , Endossomos/química , Humanos , Lisossomos/química , Camundongos , Presenilina-1/análise , Presenilina-1/química , Presenilina-1/genética , Presenilina-1/metabolismo , Presenilina-2/química , Presenilina-2/genética , Presenilina-2/metabolismo , Ratos , Especificidade por SubstratoRESUMO
Directed conversion of mature human cells, as from fibroblasts to neurons, is of potential clinical utility for neurological disease modeling as well as cell therapeutics. Here, we describe the efficient generation of human-induced neuronal (hiN) cells from adult skin fibroblasts of unaffected individuals and Alzheimer's patients, using virally transduced transcription regulators and extrinsic support factors. hiN cells from unaffected individuals display morphological, electrophysiological, and gene expression profiles that typify glutamatergic forebrain neurons and are competent to integrate functionally into the rodent CNS. hiN cells from familial Alzheimer disease (FAD) patients with presenilin-1 or -2 mutations exhibit altered processing and localization of amyloid precursor protein (APP) and increased production of Aß, relative to the source patient fibroblasts or hiN cells from unaffected individuals. Together, our findings demonstrate directed conversion of human fibroblasts to a neuronal phenotype and reveal cell type-selective pathology in hiN cells derived from FAD patients.
Assuntos
Doença de Alzheimer/patologia , Transdiferenciação Celular , Medicina Regenerativa/métodos , Pele/citologia , Doença de Alzheimer/genética , Precursor de Proteína beta-Amiloide/metabolismo , Células Cultivadas , Fibroblastos/citologia , Humanos , Neurônios/metabolismo , Presenilina-1/metabolismo , Presenilina-2/metabolismoRESUMO
γ-secretase processing of amyloid precursor protein (APP) has long been of interest in the pathological progression of Alzheimer's disease (AD) due to its role in the generation of amyloid-ß. The catalytic component of the enzyme is the presenilins of which there are two homologues, Presenilin-1 (PS1) and Presenilin-2 (PS2). The field has focussed on the PS1 form of this enzyme, as it is typically considered the more active at APP processing. However, much of this work has been completed without appropriate consideration of the specific levels of protein expression of PS1 and PS2. We propose that expression is an important factor in PS1- and PS2-γ-secretase activity, and that when this is considered, PS1 does not have greater activity than PS2. We developed and validated tools for quantitative assessment of PS1 and PS2 protein expression levels to enable the direct comparison of PS in exogenous and endogenous expression systems, in HEK-293 PS1 and/or PS2 knockout cells. We show that exogenous expression of Myc-PS1-NTF is 5.5-times higher than Myc-PS2-NTF. Quantitating endogenous PS protein levels, using a novel PS1/2 fusion standard we developed, showed similar results. When the marked difference in PS1 and PS2 protein levels is considered, we show that compared to PS1-γ-secretase, PS2-γ-secretase has equal or more activity on APP and Notch1. This study has implications for understanding the PS1- and PS2-specific contributions to substrate processing, and their potential influence in AD pathogenesis.
Assuntos
Doença de Alzheimer , Secretases da Proteína Precursora do Amiloide , Presenilina-2 , Humanos , Doença de Alzheimer/genética , Peptídeos beta-Amiloides/metabolismo , Precursor de Proteína beta-Amiloide/metabolismo , Secretases da Proteína Precursora do Amiloide/genética , Secretases da Proteína Precursora do Amiloide/metabolismo , Endopeptidases/metabolismo , Células HEK293 , Presenilina-1/genética , Presenilina-1/metabolismo , Presenilina-2/genética , Presenilina-2/metabolismoRESUMO
The γ-secretase complex catalyzes the intramembrane cleavage of C99, a carboxy-terminal fragment of the amyloid precursor protein. Two paralogs of its catalytic subunit presenilin (PS1 and PS2) are expressed which are autocatalytically cleaved into an N-terminal and a C-terminal fragment during maturation of γ-secretase. In this study, we compared the efficiency and specificity of C99 cleavage by PS1- and PS2-containing γ-secretases. Mass spectrometric analysis of cleavage products obtained in cell-free and cell-based assays revealed that the previously described lower amyloid-ß (Aß)38 generation by PS2 is accompanied by a reciprocal increase in Aß37 production. We further found PS1 and PS2 to show different preferences in the choice of the initial cleavage site of C99. However, the differences in Aß38 and Aß37 generation appear to mainly result from altered subsequent stepwise cleavage of Aß peptides. Apart from these differences in cleavage specificity, we confirmed a lower efficiency of initial C99 cleavage by PS2 using a detergent-solubilized γ-secretase system. By investigating chimeric PS1/2 molecules, we show that the membrane-embedded, nonconserved residues of the N-terminal fragment mainly account for the differential cleavage efficiency and specificity of both presenilins. At the level of individual transmembrane domains (TMDs), TMD3 was identified as a major modulator of initial cleavage site specificity. The efficiency of endoproteolysis strongly depends on nonconserved TMD6 residues at the interface to TMD2, i.e., at a putative gate of substrate entry. Taken together, our results highlight the role of individual presenilin TMDs in the cleavage of C99 and the generation of Aß peptides.
Assuntos
Secretases da Proteína Precursora do Amiloide , Presenilina-1 , Presenilina-2 , Humanos , Doença de Alzheimer/metabolismo , Peptídeos beta-Amiloides/genética , Peptídeos beta-Amiloides/metabolismo , Precursor de Proteína beta-Amiloide/metabolismo , Secretases da Proteína Precursora do Amiloide/genética , Secretases da Proteína Precursora do Amiloide/metabolismo , Presenilina-1/química , Presenilina-1/genética , Presenilina-1/metabolismo , Presenilina-2/química , Presenilina-2/genética , Presenilina-2/metabolismo , Domínios ProteicosRESUMO
The link between cholesterol homeostasis and cleavage of the amyloid precursor protein (APP), and how this relationship relates to Alzheimer's disease (AD) pathogenesis, is still unknown. Cellular cholesterol levels are regulated through crosstalk between the plasma membrane (PM), where most cellular cholesterol resides, and the endoplasmic reticulum (ER), where the protein machinery that regulates cholesterol levels resides. The intracellular transport of cholesterol from the PM to the ER is believed to be activated by a lipid-sensing peptide(s) in the ER that can cluster PM-derived cholesterol into transient detergent-resistant membrane domains (DRMs) within the ER, also called the ER regulatory pool of cholesterol. When formed, these cholesterol-rich domains in the ER maintain cellular homeostasis by inducing cholesterol esterification as a mechanism of detoxification while attenuating its de novo synthesis. In this manuscript, we propose that the 99-aa C-terminal fragment of APP (C99), when delivered to the ER for cleavage by γ-secretase, acts as a lipid-sensing peptide that forms regulatory DRMs in the ER, called mitochondria-associated ER membranes (MAM). Our data in cellular AD models indicates that increased levels of uncleaved C99 in the ER, an early phenotype of the disease, upregulates the formation of these transient DRMs by inducing the internalization of extracellular cholesterol and its trafficking from the PM to the ER. These results suggest a novel role for C99 as a mediator of cholesterol disturbances in AD, potentially explaining early hallmarks of the disease.
Assuntos
Doença de Alzheimer/metabolismo , Secretases da Proteína Precursora do Amiloide/metabolismo , Precursor de Proteína beta-Amiloide/genética , Precursor de Proteína beta-Amiloide/metabolismo , Membrana Celular/metabolismo , Colesterol/metabolismo , Retículo Endoplasmático/metabolismo , Doença de Alzheimer/enzimologia , Doença de Alzheimer/genética , Animais , Linhagem Celular , Colesterol/biossíntese , Retículo Endoplasmático/genética , Fibroblastos/metabolismo , Técnicas de Silenciamento de Genes , Inativação Gênica , Humanos , Células-Tronco Pluripotentes Induzidas , Metabolismo dos Lipídeos , Lipidômica , Camundongos , Mitocôndrias/metabolismo , Presenilina-1/genética , Presenilina-1/metabolismo , Presenilina-2/genética , Presenilina-2/metabolismo , Domínios Proteicos , RNA Interferente Pequeno , Esfingomielina Fosfodiesterase/metabolismoRESUMO
BACKGROUND: Widescale evidence points to the involvement of glia and immune pathways in the progression of Alzheimer's disease (AD). AD-associated iPSC-derived glial cells show a diverse range of AD-related phenotypic states encompassing cytokine/chemokine release, phagocytosis and morphological profiles, but to date studies are limited to cells derived from PSEN1, APOE and APP mutations or sporadic patients. The aim of the current study was to successfully differentiate iPSC-derived microglia and astrocytes from patients harbouring an AD-causative PSEN2 (N141I) mutation and characterise the inflammatory and morphological profile of these cells. METHODS: iPSCs from three healthy control individuals and three familial AD patients harbouring a heterozygous PSEN2 (N141I) mutation were used to derive astrocytes and microglia-like cells and cell identity and morphology were characterised through immunofluorescent microscopy. Cellular characterisation involved the stimulation of these cells by LPS and Aß42 and analysis of cytokine/chemokine release was conducted through ELISAs and multi-cytokine arrays. The phagocytic capacity of these cells was then indexed by the uptake of fluorescently-labelled fibrillar Aß42. RESULTS: AD-derived astrocytes and microglia-like cells exhibited an atrophied and less complex morphological appearance than healthy controls. AD-derived astrocytes showed increased basal expression of GFAP, S100ß and increased secretion and phagocytosis of Aß42 while AD-derived microglia-like cells showed decreased IL-8 secretion compared to healthy controls. Upon immunological challenge AD-derived astrocytes and microglia-like cells showed exaggerated secretion of the pro-inflammatory IL-6, CXCL1, ICAM-1 and IL-8 from astrocytes and IL-18 and MIF from microglia. CONCLUSION: Our study showed, for the first time, the differentiation and characterisation of iPSC-derived astrocytes and microglia-like cells harbouring a PSEN2 (N141I) mutation. PSEN2 (N141I)-mutant astrocytes and microglia-like cells presented with a 'primed' phenotype characterised by reduced morphological complexity, exaggerated pro-inflammatory cytokine secretion and altered Aß42 production and phagocytosis.
Assuntos
Doença de Alzheimer , Células-Tronco Pluripotentes Induzidas , Humanos , Astrócitos/metabolismo , Microglia/metabolismo , Células-Tronco Pluripotentes Induzidas/metabolismo , Interleucina-8/metabolismo , Doença de Alzheimer/genética , Doença de Alzheimer/metabolismo , Citocinas/metabolismo , Fenótipo , Peptídeos beta-Amiloides/metabolismo , Presenilina-2/genética , Presenilina-2/metabolismoRESUMO
Presenilin, a transmembrane protein primarily known for its role in Alzheimer's disease (AD) as part of the γ-secretase complex, has garnered increased attention due to its multifaceted functions in various cellular processes. Recent investigations have unveiled a plethora of functions beyond its amyloidogenic role. This review aims to provide a comprehensive overview of presenilin's diverse roles in AD and other neurodegenerative disorders. It includes a summary of well-known substrates of presenilin, such as its involvement in amyloid precursor protein (APP) processing and Notch signaling, along with other functions. Additionally, it highlights newly discovered functions, such as trafficking function, regulation of ferritin expression, apolipoprotein E (ApoE) secretion, the interaction of ApoE and presenilin, and the Aß42-to-Aß40-converting activity of ACE. This updated perspective underscores the evolving landscape of presenilin research, emphasizing its broader impact beyond established pathways. The incorporation of these novel findings accentuates the dynamic nature of presenilin's involvement in cellular processes, further advancing our comprehension of its multifaceted roles in neurodegenerative disorders. By synthesizing evidence from a range of studies, this review sheds light on the intricate web of presenilin functions and their implications in health and disease.
Assuntos
Doença de Alzheimer , Doenças Neurodegenerativas , Humanos , Doença de Alzheimer/metabolismo , Peptídeos beta-Amiloides/metabolismo , Doenças Neurodegenerativas/etiologia , Secretases da Proteína Precursora do Amiloide/metabolismo , Presenilina-1/genética , Presenilina-1/metabolismo , Precursor de Proteína beta-Amiloide/metabolismo , Apolipoproteínas E , Presenilina-2/genética , Presenilina-2/metabolismoRESUMO
Presenilin proteins (PS1 and PS2) represent the catalytic subunit of γ-secretase and play a critical role in the generation of the amyloid ß (Aß) peptide and the pathogenesis of Alzheimer disease (AD). However, PS proteins also exert multiple functions beyond Aß generation. In this study, we examine the individual roles of PS1 and PS2 in cellular cholesterol metabolism. Deletion of PS1 or PS2 in mouse models led to cholesterol accumulation in cerebral neurons. Cholesterol accumulation was also observed in the lysosomes of embryonic fibroblasts from Psen1-knockout (PS1-KO) and Psen2-KO (PS2-KO) mice and was associated with decreased expression of the Niemann-Pick type C1 (NPC1) protein involved in intracellular cholesterol transport in late endosomal/lysosomal compartments. Mass spectrometry and complementary biochemical analyses also revealed abnormal N-glycosylation of NPC1 and several other membrane proteins in PS1-KO and PS2-KO cells. Interestingly, pharmacological inhibition of N-glycosylation resulted in intracellular cholesterol accumulation prominently in lysosomes and decreased NPC1, thereby resembling the changes in PS1-KO and PS2-KO cells. In turn, treatment of PS1-KO and PS2-KO mouse embryonic fibroblasts (MEFs) with the chaperone inducer arimoclomol partially normalized NPC1 expression and rescued lysosomal cholesterol accumulation. Additionally, the intracellular cholesterol accumulation in PS1-KO and PS2-KO MEFs was prevented by overexpression of NPC1. Collectively, these data indicate that a loss of PS function results in impaired protein N-glycosylation, which eventually causes decreased expression of NPC1 and intracellular cholesterol accumulation. This mechanism could contribute to the neurodegeneration observed in PS KO mice and potentially to the pathogenesis of AD.
Assuntos
Colesterol , Fibroblastos , Lisossomos , Proteína C1 de Niemann-Pick , Presenilina-1 , Presenilina-2 , Animais , Camundongos , Doença de Alzheimer/metabolismo , Doença de Alzheimer/genética , Doença de Alzheimer/patologia , Colesterol/metabolismo , Fibroblastos/metabolismo , Glicosilação , Peptídeos e Proteínas de Sinalização Intracelular/metabolismo , Peptídeos e Proteínas de Sinalização Intracelular/genética , Lisossomos/metabolismo , Camundongos Knockout , Neurônios/metabolismo , Presenilina-1/genética , Presenilina-1/metabolismo , Presenilina-2/metabolismo , Presenilina-2/genéticaRESUMO
Alzheimer's disease (AD) is a debilitating dementia characterized by progressive memory loss and aggregation of amyloid-ß (Aß) protein into amyloid plaques in patient brains. Mutations in presenilin (PS) lead to abnormal generation of Aß, which is the major cause of familial AD (FAD), and apolipoprotein E4 (ApoE4) is the major genetic risk factor for sporadic AD (SAD) onset. However, whether dysfunction of PS is involved in the pathogenesis of SAD is largely unknown. We found that ApoE secretion was completely abolished in PS-deficient cells and markedly decreased by inhibition of γ-secretase activity. Blockade of γ-secretase activity by a γ-secretase inhibitor, DAPT, decreased ApoE secretion, suggesting an important role of γ-secretase activity in ApoE secretion. Reduced ApoE secretion is also observed in nicastrin-deficient cells with reduced γ-secretase activity. PS deficiency enhanced nuclear translocation of ApoE and binding of ApoE to importin α4, a nuclear transport receptor. Moreover, the expression of PS mutants in PS-deficient cells suppressed the restoration effects on ApoE secretion compared with the expression of wild-type PS. Plasma ApoE levels were lower in FAD patients carrying PS1 mutations compared with normal control subjects. Our findings suggest a novel role of PS contributing to the pathogenesis of SAD by regulating ApoE secretion.SIGNIFICANCE STATEMENT Familial AD (FAD) typically results from mutations in the genes encoding amyloid precursor protein, presenilin 1 (PS1), or PS2. Many PS mutants have been found to exert impaired γ-secretase activity and increased amyloid-ß 42 (Aß42)/Aß40 ratio, which induce early amyloid deposition and FAD. On the other hand, apolipoprotein E4 (ApoE4) is the major genetic risk factor for sporadic AD (SAD) and contributes to AD pathogenesis because it has reduced Aß clearance capability compared with ApoE3 and ApoE2. FAD and SAD have long been considered to be caused by these two independent mechanisms; however, for the first time, we demonstrated that PS is essential for ApoE secretion and PS mutants affected ApoE secretion in vitro and in human samples, suggesting a novel mechanism by which PS is also involved in SAD pathogenesis.
Assuntos
Doença de Alzheimer , Apolipoproteína E4 , Presenilina-1 , Presenilina-2 , Humanos , Doença de Alzheimer/genética , Doença de Alzheimer/metabolismo , Peptídeos beta-Amiloides/genética , Peptídeos beta-Amiloides/metabolismo , Precursor de Proteína beta-Amiloide/genética , Precursor de Proteína beta-Amiloide/metabolismo , Secretases da Proteína Precursora do Amiloide/genética , Secretases da Proteína Precursora do Amiloide/metabolismo , Apolipoproteína E4/genética , Apolipoproteína E4/metabolismo , Mutação , Presenilina-1/genética , Presenilina-1/metabolismo , Presenilina-2/genética , Presenilina-2/metabolismoRESUMO
Previously, we investigated gene expression in a high aldehyde dehydrogenase 1 expression (ALDH1high) population of urothelial carcinoma (UC) cells as UC cancer stem-like cells (CSCs)/cancer-initiating cells (CICs) and found that NRG1 expression was upregulated in ALDH1high cells. NRG1 is a trophic factor that contains an epidermal growth factor (EGF)-like domain that signals by stimulating ERBB receptor tyrosine kinases and the cytoplasmic domain. NRG1 has been determined to be involved in frequent gene fusions with other partners in several malignancies and has a role in carcinogenesis through the NRG1 EGF-like domain and its cognitive receptor ERBBs. We thus aimed to elucidate the function of NRG1 in UC CSCs/CICs in this study. Both NRG1α and NRG1-ß1 were preferentially expressed in ALDH1high cells compared with ALDH1low cells; however, siRNA experiments revealed that NRG1-ß1 but not NRG1-α has a role in sphere formation. The EGF-like domain of NRG1 had a role in sphere formation of UC cells to some extent but was not essential. The intracellular domain of NRG1 did not have a role in sphere-formation. Inhibition of γ-secretase suppressed sphere formation. These findings indicate that cleavage of NRG1-ß1 by γ-secretase plays an important role in UC CSC/CIC proliferation; however, the downstream targets of NRG1-ß1 remain elusive.
Assuntos
Secretases da Proteína Precursora do Amiloide/genética , Células-Tronco Neoplásicas/metabolismo , Neuregulina-1/genética , Esferoides Celulares/metabolismo , Neoplasias Urológicas/genética , Urotélio/metabolismo , Secretases da Proteína Precursora do Amiloide/metabolismo , Apoptose/genética , Linhagem Celular Tumoral , Sobrevivência Celular/genética , Regulação Neoplásica da Expressão Gênica , Humanos , Neuregulina-1/metabolismo , Presenilina-1/genética , Presenilina-1/metabolismo , Presenilina-2/genética , Presenilina-2/metabolismo , Interferência de RNA , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Neoplasias Urológicas/metabolismo , Urotélio/patologiaRESUMO
Early-onset Alzheimer's disease (AD) is associated with variants in amyloid precursor protein (APP) and presenilin (PSEN) 1 and 2. It is increasingly recognized that patients with AD experience undiagnosed focal seizures. These AD patients with reported seizures may have worsened disease trajectory. Seizures in epilepsy can also lead to cognitive deficits, neuroinflammation, and neurodegeneration. Epilepsy is roughly three times more common in individuals aged 65 and older. Due to the numerous available antiseizure drugs (ASDs), treatment of seizures has been proposed to reduce the burden of AD. More work is needed to establish the functional impact of seizures in AD to determine whether ASDs could be a rational therapeutic strategy. The efficacy of ASDs in aged animals is not routinely studied, despite the fact that the elderly represents the fastest growing demographic with epilepsy. This leaves a particular gap in understanding the discrete pathophysiological overlap between hyperexcitability and aging, and AD more specifically. Most of our preclinical knowledge of hyperexcitability in AD has come from mouse models that overexpress APP. While these studies have been invaluable, other drivers underlie AD, e.g. PSEN2. A diversity of animal models should be more frequently integrated into the study of hyperexcitability in AD, which could be particularly beneficial to identify novel therapies. Specifically, AD-associated risk genes, in particular PSENs, altogether represent underexplored contributors to hyperexcitability. This review assesses the available studies of ASDs administration in clinical AD populations and preclinical studies with AD-associated models and offers a perspective on the opportunities for further therapeutic innovation.
Assuntos
Doença de Alzheimer/tratamento farmacológico , Anticonvulsivantes/uso terapêutico , Epilepsia/tratamento farmacológico , Convulsões/tratamento farmacológico , Envelhecimento/fisiologia , Doença de Alzheimer/epidemiologia , Doença de Alzheimer/fisiopatologia , Animais , Comorbidade , Epilepsia/epidemiologia , Epilepsia/fisiopatologia , Humanos , Mutação , Presenilina-1/genética , Presenilina-1/metabolismo , Presenilina-2/genética , Presenilina-2/metabolismo , Convulsões/epidemiologia , Convulsões/fisiopatologiaRESUMO
Alzheimer's disease is a common neurodegenerative disease in the elderly population and a leading cause of dementia. Genetics and environmental risk factors were considered to play a major role in the onset of the disease. This study aimed to examine the correlation between different metals levels and the gene expression in Alzheimer's patients with age-matched control subjects. Non- essential metals were measured in the whole blood due to its higher concentration in red blood corpuscles (RBCs) and essential biometals in the serum samples of Alzheimer's disease (AD) by using Inductively coupled plasma optical emission spectroscopy (ICP-OES) that allows the analysis and detection of the different elements at low levels. Gene expression level was performed by quantitative real-time PCR (qRT-PCR). In this study, the levels of Lead and Arsenic metals were not detected in the AD patient samples. Cadmium, Mercury, and Aluminum were found higher in cases as compared to controls with 0.009240 ± 0.0007707 (P = < 0.0001), 0.02332 ± 0.001041 (P = < 0.0001), and 0.09222 ± 0.02804 (P = 0.0087) respectively. Essential biometal like copper was higher 0.1274 ± 0.02453 (P = 0.0254) in cases, while iron 0.1117 ± 0.009599 (P = 0.0304) and zinc 0.03800 ± 0.003462 mg/L were found significantly lower as compared to controls. All targeted genes such as APP, PSEN1, PSEN2, and APOE4 were found up-regulated in AD patients. We concluded that there was no significant correlation between metals dyshomeostasis and gene expressions in this study.
Assuntos
Doença de Alzheimer/metabolismo , Expressão Gênica , Metais/sangue , Idoso , Alumínio/sangue , Apolipoproteínas/genética , Apolipoproteínas/metabolismo , Apolipoproteínas E/genética , Apolipoproteínas E/metabolismo , Cádmio/sangue , Cobre/sangue , Feminino , Humanos , Ferro/sangue , Masculino , Doenças Neurodegenerativas/metabolismo , Presenilina-1/genética , Presenilina-1/metabolismo , Presenilina-2/genética , Presenilina-2/metabolismo , Zinco/sangueRESUMO
Alzheimer's disease (AD) is the most frequent cause of dementia in the elderly. Few cases are familial (FAD), due to autosomal dominant mutations in presenilin-1 (PS1), presenilin-2 (PS2) or amyloid precursor protein (APP). The three proteins are involved in the generation of amyloid-beta (Aß) peptides, providing genetic support to the hypothesis of Aß pathogenicity. However, clinical trials focused on the Aß pathway failed in their attempt to modify disease progression, suggesting the existence of additional pathogenic mechanisms. Ca2+ dysregulation is a feature of cerebral aging, with an increased frequency and anticipated age of onset in several forms of neurodegeneration, including AD. Interestingly, FAD-linked PS1 and PS2 mutants alter multiple key cellular pathways, including Ca2+ signaling. By generating novel tools for measuring Ca2+ in living cells, and combining different approaches, we showed that FAD-linked PS2 mutants significantly alter cell Ca2+ signaling and brain network activity, as summarized below.
Assuntos
Doença de Alzheimer , Idoso , Doença de Alzheimer/genética , Peptídeos beta-Amiloides/genética , Peptídeos beta-Amiloides/metabolismo , Encéfalo/metabolismo , Homeostase , Humanos , Presenilina-1/genética , Presenilina-1/metabolismo , Presenilina-2/genética , Presenilina-2/metabolismoRESUMO
Perinatal asphyxia is mainly a brain disease leading to the development of neurodegeneration, in which a number of peripheral lesions have been identified; however, little is known about the expression of key genes involved in amyloid production by peripheral cells, such as lymphocytes, during the development of hypoxic-ischemic encephalopathy. We analyzed the gene expression of the amyloid protein precursor, ß-secretase, presenilin 1 and 2 and hypoxia-inducible factor 1-α by RT-PCR in the lymphocytes of post-asphyxia and control neonates. In all examined periods after asphyxia, decreased expression of the genes of the amyloid protein precursor, ß-secretase and hypoxia-inducible factor 1-α was noted in lymphocytes. Conversely, expression of presenilin 1 and 2 genes decreased on days 1-7 and 8-14 but increased after survival for more than 15 days. We believe that the expression of presenilin genes in lymphocytes could be a potential biomarker to determine the severity of the post-asphyxia neurodegeneration or to identify the underlying factors for brain neurodegeneration and get information about the time they occurred. This appears to be the first worldwide data on the role of the presenilin 1 and 2 genes associated with Alzheimer's disease in the dysregulation of neonatal lymphocytes after perinatal asphyxia.
Assuntos
Asfixia/patologia , Linfócitos/patologia , Presenilina-1/metabolismo , Presenilina-2/metabolismo , Asfixia/genética , Asfixia/metabolismo , Estudos de Casos e Controles , Feminino , Regulação da Expressão Gênica , Humanos , Recém-Nascido , Linfócitos/metabolismo , Masculino , Presenilina-1/genética , Presenilina-2/genéticaRESUMO
Proteolytic processing of amyloid precursor protein (APP) plays a critical role in the pathogenesis of Alzheimer's disease (AD). Sequential cleavage of APP by ß and γ secretases leads to the generation of Aß40 (non-amyloidogenic) and Aß42 (amyloidogenic) peptides. Presenilin-1 (PS1) or presenilin-2 (PS2) play the role of a catalytic subunit of γ-secretase. Multiple familial AD (FAD) mutations in APP, PS1, or PS2 result in an increased Aß42:Aß40 ratio and the accumulation of toxic Aß42 oligomers and plaques in patient brains. In this study, we perform molecular modeling of the APP complex with γ-secretase and analyze potential effects of FAD mutations in APP and PS1. We noticed that all FAD mutations in the APP transmembrane domain are predicted to cause an increase in the local disorder of its secondary structure. Based on structural analysis of known γ-secretase structures, we propose that APP can form a complex with γ-secretase in 2 potential conformations-M1 and M2. In conformation, the M1 transmembrane domain of APP forms a contact with the perimembrane domain that follows transmembrane domain 6 (TM6) in the PS1 structure. In conformation, the M2 transmembrane domain of APP forms a contact with transmembrane domain 7 (TM7) in the PS1 structure. By analyzing the effects of PS1-FAD mutations on the local protein disorder index, we discovered that these mutations increase the conformational flexibility of M2 and reduce the conformational flexibility of M1. Based on these results, we propose that M2 conformation, but not M1 conformation, of the γ secretase complex with APP leads to the amyloidogenic (Aß42-generating) processing of APP. Our model predicts that APP processing in M1 conformation is favored by curved membranes, such as the membranes of early endosomes. In contrast, APP processing in M2 conformation is likely to be favored by relatively flat membranes, such as membranes of late endosomes and plasma membranes. These predictions are consistent with published biochemical analyses of APP processing at different subcellular locations. Our results also suggest that specific inhibitors of Aß42 production could be potentially developed by selectively targeting the M2 conformation of the γ secretase complex with APP.
Assuntos
Secretases da Proteína Precursora do Amiloide/metabolismo , Precursor de Proteína beta-Amiloide/metabolismo , Presenilina-1/metabolismo , Presenilina-2/metabolismo , Doença de Alzheimer/genética , Doença de Alzheimer/metabolismo , Secretases da Proteína Precursora do Amiloide/química , Secretases da Proteína Precursora do Amiloide/genética , Precursor de Proteína beta-Amiloide/química , Precursor de Proteína beta-Amiloide/genética , Humanos , Modelos Moleculares , Mutação , Presenilina-1/química , Presenilina-1/genética , Presenilina-2/química , Presenilina-2/genética , Conformação Proteica , Domínios Proteicos , Estabilidade ProteicaRESUMO
γ-secretase is an intramembrane protease complex consisting of nicastrin, presenilin-1/2, APH-1a/b, and Pen-2. Hydrolysis of the 99-residue transmembrane fragment of amyloid precursor protein (APP-C99) by γ-secretase produces ß-amyloid (Aß) peptides. Pathogenic mutations in PSEN1 and PSEN2, which encode the catalytic subunit presenilin-1/2 of γ-secretase, lead to familial Alzheimer's disease in an autosomal dominant manner. However, the underlying mechanism of how the mutant PSEN gene may affect the function of the WT allele remains to be elucidated. Here we report that each of the loss-of-function γ-secretase variants that carries a PSEN1 mutation suppresses the protease activity of the WT γ-secretase on Aß production. Each of these γ-secretase variants forms a stable oligomer with the WT γ-secretase in vitro in the presence of the detergent CHAPSO {3-[(3-cholamidopropyl)dimethylammonio]-2-hydroxy-1-propanesulfonate}, but not digitonin. Importantly, robust protease activity of γ-secretase is detectable in the presence of CHAPSO, but not digitonin. These experimental observations suggest a dominant negative effect of the γ-secretase, in which the protease activity of WT γ-secretase is suppressed by the loss-of-function γ-secretase variants through hetero-oligomerization. The relevance of this finding to the genesis of Alzheimer's disease is critically evaluated.
Assuntos
Secretases da Proteína Precursora do Amiloide/genética , Peptídeos beta-Amiloides/genética , Proteínas de Membrana/genética , Fragmentos de Peptídeos/genética , Presenilina-1/genética , Presenilina-2/genética , Doença de Alzheimer/genética , Doença de Alzheimer/metabolismo , Doença de Alzheimer/patologia , Secretases da Proteína Precursora do Amiloide/química , Secretases da Proteína Precursora do Amiloide/metabolismo , Peptídeos beta-Amiloides/metabolismo , Ácidos Cólicos/química , Clonagem Molecular , Detergentes/química , Digitonina/química , Endopeptidases , Expressão Gênica , Células HeLa , Humanos , Proteínas de Membrana/química , Proteínas de Membrana/metabolismo , Modelos Biológicos , Mutação , Fragmentos de Peptídeos/metabolismo , Peptídeo Hidrolases/química , Peptídeo Hidrolases/genética , Peptídeo Hidrolases/metabolismo , Presenilina-1/química , Presenilina-1/metabolismo , Presenilina-2/química , Presenilina-2/metabolismo , Multimerização Proteica , Proteínas Recombinantes/química , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismoRESUMO
Alzheimer's disease (AD) is the most common form of dementia. Even though most AD cases are sporadic, a small percentage is familial due to autosomal dominant mutations in amyloid precursor protein (APP), presenilin-1 (PSEN1), and presenilin-2 (PSEN2) genes. AD mutations contribute to the generation of toxic amyloid ß (Aß) peptides and the formation of cerebral plaques, leading to the formulation of the amyloid cascade hypothesis for AD pathogenesis. Many drugs have been developed to inhibit this pathway but all these approaches currently failed, raising the need to find additional pathogenic mechanisms. Alterations in cellular calcium (Ca2+) signaling have also been reported as causative of neurodegeneration. Interestingly, Aß peptides, mutated presenilin-1 (PS1), and presenilin-2 (PS2) variously lead to modifications in Ca2+ homeostasis. In this contribution, we focus on PS2, summarizing how AD-linked PS2 mutants alter multiple Ca2+ pathways and the functional consequences of this Ca2+ dysregulation in AD pathogenesis.
Assuntos
Doença de Alzheimer/metabolismo , Sinalização do Cálcio , Presenilina-2/metabolismo , Doença de Alzheimer/genética , Doença de Alzheimer/patologia , Precursor de Proteína beta-Amiloide/genética , Precursor de Proteína beta-Amiloide/metabolismo , Humanos , Presenilina-1/genética , Presenilina-1/metabolismo , Presenilina-2/genéticaRESUMO
A chronic neuroinflammatory response has been considered as a critical pathogenesis promoting neurodegenerative progression in Alzheimer's disease (AD). During neuroinflammatory process, microglia are excessively activated and simultaneously release numerous pro-inflammatory mediators that cause synaptic dysfunction in the forebrain prior to neuronal degeneration and memory deficits in AD. Thus, prevention of neuroinflammation-mediated synaptic dysfunction may be a potential therapeutic approach against neurodegenerative disorders. Trans-cinnamaldehyde (TCA) is a primary bioactive component derived from the stem bark of Cinnamomum cassia, and it possesses potent anti-inflammatory and neuroprotective activities in in vivo and in vitro experiments. However, the in-depth molecular mechanisms of TCA underlying anti-neuroinflammatory and neuroprotective effects on memory deficits in AD are still unclear. The presenilin 1 and 2 conditional double knockout (PS cDKO) mice exhibit AD-like phenotypes including obvious neuroinflammatory responses and synaptic dysfunction and memory deficits. Here, PS cDKO were used to evaluate the potential neuroprotective effects of TCA against neuroinflammation-mediated dementia by performing behavioral tests, electrophysiological recordings and molecular biology analyses. We observed that TCA treatment reversed abnormal expression of synaptic proteins and tau hyperphosphorylation in the hippocampus and prefrontal cortex of PS cDKO mice. TCA treatment also ameliorated NMDA receptor (NMDAR) dysfunction including impaired NMDAR-mediated responses and long-term potentiation (LTP) induction in the hippocampus of PS cDKO mice. Moreover, TCA possesses an ability to suppress neuroinflammatory responses by diminishing microglial activation and levels of pro-inflammatory mediators in the hippocampus and prefrontal cortex of PS cDKO mice. Importantly, improving NMDAR dysfunction and memory deficits of PS cDKO mice was due to the inhibition of neuroinflammatory responses through TCA's interruptive effect on the nuclear factor kappa B (NF-κB) signaling pathway. Therefore, TCA may be a potential anti-neuroinflammatory agent for deterring neurodegenerative progression of AD.
Assuntos
Acroleína/análogos & derivados , Doença de Alzheimer/metabolismo , NF-kappa B/metabolismo , Acroleína/metabolismo , Acroleína/farmacologia , Animais , Feminino , Hipocampo/metabolismo , Proteínas I-kappa B/metabolismo , Potenciação de Longa Duração/fisiologia , Masculino , Transtornos da Memória/fisiopatologia , Camundongos , Camundongos Knockout , Microglia/metabolismo , N-Metilaspartato , Fármacos Neuroprotetores/metabolismo , Fármacos Neuroprotetores/farmacologia , Presenilina-1/genética , Presenilina-1/metabolismo , Presenilina-2/genética , Presenilina-2/metabolismo , Receptores de N-Metil-D-Aspartato/metabolismo , Transdução de Sinais/efeitos dos fármacosRESUMO
The two presenilin-1 (PS1) and presenilin-2 (PS2) homologs are the catalytic core of the γ-secretase complex, which has a major role in cell fate decision and Alzheimer's disease (AD) progression. Understanding the precise contribution of PS1- and PS2-dependent γ-secretases to the production of ß-amyloid peptide (Aß) from amyloid precursor protein (APP) remains an important challenge to design molecules efficiently modulating Aß release without affecting the processing of other γ-secretase substrates. To that end, we studied PS1- and PS2-dependent substrate processing in murine cells lacking presenilins (PSs) (PS1KO, PS2KO or PS1-PS2 double-KO noted PSdKO) or stably re-expressing human PS1 or PS2 in an endogenous PS-null (PSdKO) background. We characterized the processing of APP and Notch on both endogenous and exogenous substrates, and we investigated the effect of pharmacological inhibitors targeting the PSs activity (DAPT and L-685,458). We found that murine PS1 γ-secretase plays a predominant role in APP and Notch processing when compared to murine PS2 γ-secretase. The inhibitors blocked more efficiently murine PS2- than murine PS1-dependent processing. Human PSs, especially human PS1, expression in a PS-null background efficiently restored APP and Notch processing. Strikingly, and contrary to the results obtained on murine PSs, pharmacological inhibitors appear to preferentially target human PS1- than human PS2-dependent γ-secretase activity.
Assuntos
Secretases da Proteína Precursora do Amiloide/metabolismo , Presenilina-1/metabolismo , Presenilina-2/metabolismo , Peptídeos beta-Amiloides/metabolismo , Animais , Biocatálise , Fibroblastos/metabolismo , Humanos , Camundongos Knockout , Receptores Notch/metabolismo , Especificidade por SubstratoRESUMO
Oestrogen receptor É (ERÉ) is overexpressed in two-thirds of all breast cancers and involves in development and breast cancer progression. Although ERÉ-positive breast cancer could be effective treated by endocrine therapy, the endocrine resistance is still an urgent clinical problem. Thus, further understanding of the underlying mechanisms ERÉ signalling is critical in dealing with endocrine resistance in breast cancer patients. MCF-7 and T47D breast cancer cell lines are used to carry out the molecular biological experiments. Western blot is used to assess the relative protein level of ERÉ, RNF168 and actin. Real-time PCR is used the measure the relative ERÉ-related gene mRNA level. Luciferase assay is used to measure the relative ERÉ signalling activity. Chromatin immunoprecipitation is used to measure the RNF168 binding affinity to ERÉ promoter regions. WST assay and flow cytometry are used to measure the cell proliferation capacity. We use Student's t test and one-way ANOVA test for statistical data analysis. Here, we report an important role in ERÉ-positive breast cancer cells for RNF168 protein in supporting cell proliferation by driving the transcription of ERÉ. RNF168 is highly expressed in breast cancer samples, compared with normal breast tissue. In patients with breast cancer, RNF168 expression level is correlated with poor endocrine treatment outcome. Depletion of RNF168 causes decreased cell proliferation in MCF-7 and T47D cells. Besides, depletion RNF168 reduced mRNA level of ERÉ and its target genes, such as PS2 and GREB1. Chromatin immunoprecipitation revealed that ERÉ transcription is associated with RNF168 recruitment to ERÉ promoter region, suggesting that transcriptional regulation is one mechanism by which RNF168 regulates ERÉ mRNA level and ERÉ signalling in breast cancer cells. RNF168 is required for ERÉ-positive breast cancer cell proliferation and facilitate ERÉ signalling activity possibly through promoting transcription of ERÉ.