RESUMEN
Mutations in the Presenilin (PSEN) genes are the most common cause of early-onset familial Alzheimer's disease (FAD). Studies in cell culture, in vitro biochemical systems, and knockin mice showed that PSEN mutations are loss-of-function mutations, impairing γ-secretase activity. Mouse genetic analysis highlighted the importance of Presenilin (PS) in learning and memory, synaptic plasticity and neurotransmitter release, and neuronal survival, and Drosophila studies further demonstrated an evolutionarily conserved role of PS in neuronal survival during aging. However, molecular pathways that interact with PS in neuronal survival remain unclear. To identify genetic modifiers that modulate PS-dependent neuronal survival, we developed a new DrosophilaPsn model that exhibits age-dependent neurodegeneration and increases of apoptosis. Following a bioinformatic analysis, we tested top ranked candidate genes by selective knockdown (KD) of each gene in neurons using two independent RNAi lines in Psn KD models. Interestingly, 4 of the 9 genes enhancing neurodegeneration in Psn KD flies are involved in lipid transport and metabolism. Specifically, neuron-specific KD of lipophorin receptors, lpr1 and lpr2, dramatically worsens neurodegeneration in Psn KD flies, and overexpression of lpr1 or lpr2 does not alleviate Psn KD-induced neurodegeneration. Furthermore, lpr1 or lpr2 KD alone also leads to neurodegeneration, increased apoptosis, climbing defects, and shortened lifespan. Lastly, heterozygotic deletions of lpr1 and lpr2 or homozygotic deletions of lpr1 or lpr2 similarly lead to age-dependent neurodegeneration and further exacerbate neurodegeneration in Psn KD flies. These findings show that LpRs modulate Psn-dependent neuronal survival and are critically important for neuronal integrity in the aging brain.
Asunto(s)
Enfermedad de Alzheimer , Drosophila , Animales , Ratones , Drosophila/genética , Drosophila/metabolismo , Presenilinas/genética , Presenilinas/metabolismo , Encéfalo/metabolismo , Enfermedad de Alzheimer/genética , Envejecimiento/genéticaRESUMEN
Cleavage of the transmembrane domain (TMD) of amyloid-ß precursor protein (APP) by γ-secretase, an intramembrane aspartyl protease, generates Aß peptides of various lengths that form plaques in the brains of Alzheimer's disease patients. Although the debate has not been finally resolved whether these plaques trigger the onset of Alzheimer's or are side products, disease-related mutations suggest their implication in the etiology of the dementia. These occur both in presenilin, the catalytic subunit of γ-secretase, and in the TMD of APP. Despite two seminal cryo-electron microscopy structures that show the complex of γ-secretase with its substrates APP and Notch, the mechanism of γ-secretase is not yet fully understood. Especially on which basis it selects its substrates is still an enigma. The presenilin homolog PSH from the archaeon Methanoculleus marisnigri JR1 (MCMJR1) is catalytically active without accessory proteins in contrast to γ-secretase making it an excellent model for studies of the basic cleavage process. We here focused on the cell-free expression of PSH screening a range of conditions. Cleavage assays to verify the activity show that not only the yield, but mainly the activity of the protease depends on the careful selection of expression conditions. Optimal results were found for a cell-free expression at relatively low temperature, 20 °C, employing cell lysates prepared from E. coli Rosetta cells. To speed up protein preparation for immediate functional assays, a crude purification protocol was developed. This allows to produce ready-made PSH in a fast and efficient manner in less than two days.
Asunto(s)
Enfermedad de Alzheimer , Secretasas de la Proteína Precursora del Amiloide , Humanos , Secretasas de la Proteína Precursora del Amiloide/genética , Secretasas de la Proteína Precursora del Amiloide/química , Secretasas de la Proteína Precursora del Amiloide/metabolismo , Microscopía por Crioelectrón , Escherichia coli/genética , Escherichia coli/metabolismo , Precursor de Proteína beta-Amiloide/genética , Precursor de Proteína beta-Amiloide/química , Precursor de Proteína beta-Amiloide/metabolismo , Presenilinas/química , Presenilinas/metabolismo , Enfermedad de Alzheimer/genética , Péptidos beta-AmiloidesRESUMEN
Formation of the PEN2-ATP6AP1 complex induced by the binding of metformin to PEN2 results in the inhibition of v-ATPase activity and in the recruitment of AXIN/LKB1 to lysosomes, which in turn results in the phosphorylation and activation of AMPK.
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Metformina , ATPasas de Translocación de Protón Vacuolares , Humanos , Metformina/farmacología , Metformina/uso terapéutico , Proteínas Serina-Treonina Quinasas/metabolismo , Proteínas Quinasas Activadas por AMP/metabolismo , Lisosomas/metabolismo , Presenilinas/metabolismo , ATPasas de Translocación de Protón Vacuolares/metabolismoRESUMEN
Kainate receptors (KARs) form a family of ionotropic glutamate receptors that regulate the activity of neuronal networks by both presynaptic and postsynaptic mechanisms. Their implication in pathologies is well documented for epilepsy. The higher prevalence of epileptic symptoms in Alzheimer's disease (AD) patients questions the role of KARs in AD. Here we investigated whether the synaptic expression and function of KARs was impaired in mouse models of AD. We addressed this question by immunostaining and electrophysiology at synapses between mossy fibers and CA3 pyramidal cells, in which KARs are abundant and play a prominent physiological role. We observed a decrease of the immunostaining for GluK2 in the stratum lucidum in CA3, and of the amplitude and decay time of synaptic currents mediated by GluK2-containing KARs in an amyloid mouse model (APP/PS1) of AD. Interestingly, a similar phenotype was observed in CA3 pyramidal cells in male and female mice with a genetic deletion of either presenilin or APP/APLP2 as well as in organotypic cultures treated with γ-secretase inhibitors. Finally, the GluK2 protein interacts with full-length and C-terminal fragments of APP. Overall, our data suggest that APP stabilizes KARs at synapses, possibly through a transsynaptic mechanism, and this interaction is under the control the γ-secretase proteolytic activity of presenilin.SIGNIFICANCE STATEMENT Synaptic impairment correlates strongly with cognitive deficits in Alzheimer's disease (AD). In this context, many studies have addressed the dysregulation of AMPA and NMDA ionotropic glutamate receptors. Kainate receptors (KARs), which form the third family of iGluRs, represent an underestimated actor in the regulation of neuronal circuits and have not yet been examined in the context of AD. Here we provide evidence that synaptic KARs are markedly impaired in a mouse model of AD. Additional experiments indicate that the γ-secretase activity of presenilin acting on the amyloid precursor protein controls synaptic expression of KAR. This study clearly indicates that KARs should be taken into consideration whenever addressing synaptic dysfunction and related cognitive deficits in the context of AD.
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Secretasas de la Proteína Precursora del Amiloide , Ácido Kaínico , Presenilina-1 , Receptores de Ácido Kaínico , Animales , Femenino , Masculino , Ratones , Enfermedad de Alzheimer/genética , Enfermedad de Alzheimer/metabolismo , Precursor de Proteína beta-Amiloide/genética , Precursor de Proteína beta-Amiloide/metabolismo , Secretasas de la Proteína Precursora del Amiloide/metabolismo , Ácido Kaínico/farmacología , Fibras Musgosas del Hipocampo/fisiología , Presenilina-1/metabolismo , Presenilinas/metabolismo , Receptores de Ácido Kaínico/metabolismo , Sinapsis/fisiología , Receptor de Ácido Kaínico GluK2RESUMEN
Recent evidence has shown that presenilin enhancer 2 (Pen2; Psenen) plays an essential role in corticogenesis by regulating the switch of apical progenitors (APs) to basal progenitors (BPs). The hippocampus is a brain structure required for advanced functions, including spatial navigation, learning and memory. However, it remains unknown whether Pen2 is important for hippocampal morphogenesis. To address this question, we generated Pen2 conditional knockout (cKO) mice, in which Pen2 is inactivated in neural progenitor cells (NPCs) in the hippocampal primordium. We showed that Pen2 cKO mice exhibited hippocampal malformation and decreased population of NPCs in the neuroepithelium of the hippocampus. We found that deletion of Pen2 neither affected the proliferative capability of APs nor the switch of APs to BPs in the hippocampus, and that it caused enhanced transition of APs to neurons. We demonstrated that expression of the Notch1 intracellular domain (N1ICD) significantly increased the population of NPCs in the Pen2 cKO hippocampus. Collectively, this study uncovers a crucial role for Pen2 in the maintenance of NPCs during hippocampal development.
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Hipocampo , Neuronas , Animales , Hipocampo/metabolismo , Ratones , Ratones Endogámicos C57BL , Ratones Noqueados , Neurogénesis/genética , Neuronas/metabolismo , Presenilinas/metabolismoRESUMEN
Cleavage of membrane proteins in the lipid bilayer by intramembrane proteases is crucial for health and disease. Although different lipid environments can potently modulate their activity, how this is linked to their structural dynamics is unclear. Here, we show that the carboxy-peptidase-like activity of the archaeal intramembrane protease PSH, a homolog of the Alzheimer's disease-associated presenilin/γ-secretase is impaired in micelles and promoted in a lipid bilayer. Comparative molecular dynamics simulations revealed that important elements for substrate binding such as transmembrane domain 6a of PSH are more labile in micelles and stabilized in the lipid bilayer. Moreover, consistent with an enhanced interaction of PSH with a transition-state analog inhibitor, the bilayer promoted the formation of the enzyme's catalytic active site geometry. Our data indicate that the lipid environment of an intramembrane protease plays a critical role in structural stabilization and active site arrangement of the enzyme-substrate complex thereby promoting intramembrane proteolysis.
Cutting proteins into pieces is a crucial process in the cell, allowing several important processes to take place, including cell differentiation (which allows cells to develop into specific types), cell death, protein quality control, or even where in the cell a protein will end up. However, the specialized proteins that carry out this task, known as proteases, can also be involved in the development of disease. For example, in the brain, a protease called γ-secretase cuts up the amyloid-ß protein precursor, producing toxic forms of amyloid-ß peptides that are widely believed to cause Alzheimer's disease. Proteases like γ-secretase carry out their role in the membrane, the layer of fats (also known as lipids) that forms the outer boundary of the cell. The environment in this area of the cell can influence the activity of proteases, but it is poorly understood how this happens. One way to address this question would be to compare the activity of γ-secretase in the lipid environment of the membrane to its activity when it is entirely surrounded by different molecules, such as detergent molecules. Unfortunately, γ-secretase is not active when it is removed from its lipid environment by a detergent, making it difficult to perform this comparison. To overcome this issue, Feilen et al. chose to study PSH, a protease similar to γ-secretase that produces the same amyloid-ß peptides but remains active in detergent. When Feilen et al. mixed PSH with lipid molecules like those found in the membrane and amyloid-ß precursor protein, PSH produced amyloid-ß peptides including those that are thought to cause Alzheimer's. However, when a detergent was substituted for the lipid molecules this led to longer amyloid-ß peptides than usual, indicating that PSH was not able to cut proteins as effectively. The change in environment appeared to reduce PSH's ability to progressively trim small segments from the peptides. Computer modelling of the protease's structure in lipids versus detergent supported the experimental findings: the model predicted that the areas of PSH important for recognizing and cutting other proteins would be more stable in the membrane compared to the detergent. These results indicate that the cell membrane plays a vital role in the stability of the active regions of proteases that are cleaving in this environment. In the future, this could help to better understand how changes to the lipid molecules in the membrane may contribute to the activity of γ-secretase and its role in Alzheimer's disease.
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Enfermedad de Alzheimer , Presenilinas , Secretasas de la Proteína Precursora del Amiloide/metabolismo , Precursor de Proteína beta-Amiloide/metabolismo , Archaea , Proteínas Arqueales , Dominio Catalítico , Humanos , Membrana Dobles de Lípidos , Micelas , Presenilina-1/metabolismo , Presenilinas/química , Presenilinas/metabolismo , ProteolisisRESUMEN
Mutations in presenilin 1 and 2 (PS1 and PS2) cause autosomal dominant familial Alzheimer's disease (FAD). Ferroptosis has been implicated as a mechanism of neurodegeneration in AD since neocortical iron burden predicts Alzheimer's disease (AD) progression. We found that loss of the presenilins dramatically sensitizes multiple cell types to ferroptosis, but not apoptosis. FAD causal mutations of presenilins similarly sensitizes cells to ferroptosis. The presenilins promote the expression of GPX4, the selenoprotein checkpoint enzyme that blocks ferroptosis by quenching the membrane propagation of lethal hydroperoxyl radicals. Presenilin γ-secretase activity cleaves Notch-1 to signal LRP8 expression, which then controls GPX4 expression by regulating the supply of selenium into the cell since LRP8 is the uptake receptor for selenoprotein P. Selenium uptake is thus disrupted by presenilin FAD mutations, suppressing GPX4 expression. Therefore, presenilin mutations may promote neurodegeneration by derepressing ferroptosis, which has implications for disease-modifying therapeutics.
Asunto(s)
Enfermedad de Alzheimer , Ferroptosis , Selenio , Humanos , Enfermedad de Alzheimer/genética , Enfermedad de Alzheimer/metabolismo , Secretasas de la Proteína Precursora del Amiloide/genética , Secretasas de la Proteína Precursora del Amiloide/metabolismo , Ferroptosis/genética , Mutación/genética , Presenilina-1/genética , Presenilina-1/metabolismo , Presenilinas/metabolismoRESUMEN
Presenilin (PSEN)/γ-secretase is a protease complex responsible for the proteolytic processing of numerous substrates. These substrates include the amyloid precursor protein (APP), the cleavage of which by γ-secretase results in the production of ß-amyloid (Aß) peptides. However, exactly where within the neuron γ-secretase processes APP C99 to generate Aß and APP intracellular domain (AICD) is still not fully understood. Here, we employ novel Förster resonance energy transfer (FRET)-based multiplexed imaging assays to directly "visualize" the subcellular compartment(s) in which γ-secretase primarily cleaves C99 in mouse cortex primary neurons (from both male and female embryos). Our results demonstrate that γ-secretase processes C99 mainly in LysoTracker-positive low-pH compartments. Using a new immunostaining protocol which distinguishes Aß from C99, we also show that intracellular Aß is significantly accumulated in the same subcellular loci. Furthermore, we found functional correlation between the endo-lysosomal pH and cellular γ-secretase activity. Taken together, our findings are consistent with Aß being produced from C99 by γ-secretase within acidic compartments such as lysosomes and late endosomes in living neurons.SIGNIFICANCE STATEMENT Alzheimer's disease (AD) genetics and histopathology highlight the importance of amyloid precursor protein (APP) processing by γ-secretase in pathogenesis. For the first time, this study has enabled us to directly "visualize" that γ-secretase processes C99 mainly in acidic compartments such as late endosomes and lysosomes in live neurons. Furthermore, we uncovered that intracellular ß-amyloid (Aß) is significantly accumulated in the same subcellular loci. Emerging evidence proposes the great importance of the endo-lysosomal pathway in mechanisms of misfolded proteins propagation (e.g., Tau, α-Syn). Therefore, the predominant processing of C99 and enrichment of Aß in late endosomes and lysosomes may be critical events in the molecular cascade leading to AD.
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Secretasas de la Proteína Precursora del Amiloide/metabolismo , Precursor de Proteína beta-Amiloide/metabolismo , Endosomas/metabolismo , Lisosomas/metabolismo , Neuronas/metabolismo , Presenilinas/metabolismo , Animales , Femenino , Masculino , RatonesRESUMEN
Disruption of the presenilin (ps) genes are the major genetic cause of familial Alzheimer's disease. The silkworm, Bombyx mori (B. mori), is an important model insect. The ps homologue gene in B. mori was identified and characterized. However, the role of ps in B. mori was poorly understood. Here, we found that Bmps was ubiquitously expressed in all the tested tissues during metamorphosis. In the current study, loss-of-function analysis of Bmps was performed by the binary transgenic CRISPR/cas9 system. Compared with the wild type, the developmental time of ∆Bmps animals were significantly delayed. In addition, ∆Bmps showed abnormal appendage including antenna, leg, wing and eye during pupal and adult stages. RNA-seq analysis indicated that apoptosis and proliferation related pathways were affected in ∆Bmps. Moreover, the Hippo pathway was affected by Bmps depletion in brain and wing disc. Our results suggest that PS is essential for maintaining the dynamic balance of apoptosis and proliferation during metamorphosis.
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Bombyx , Animales , Bombyx/metabolismo , Proteínas de Insectos/genética , Proteínas de Insectos/metabolismo , Larva , Metamorfosis Biológica , Presenilinas/metabolismo , Pupa/genética , Pupa/metabolismoRESUMEN
PSENEN/PEN2 is the smallest subunit of the γ-secretase complex, an intramembrane protease that cleaves proteins within their transmembrane domains. Mutations in components of the γ-secretase underlie familial Alzheimer disease. In addition to its proteolytic activity, supplementary, γ-secretase independent, functions in the macroautophagy/autophagy-lysosome system have been proposed. Here, we screened for PSENEN-interacting proteins and identified CLN3. Mutations in CLN3 are causative for juvenile neuronal ceroid lipofuscinosis, a rare lysosomal storage disorder considered the most common neurodegenerative disease in children. As mutations in the PSENEN and CLN3 genes cause different neurodegenerative diseases, understanding shared cellular functions of both proteins might be pertinent for understanding general cellular mechanisms underlying neurodegeneration. We hypothesized that CLN3 modulates γ-secretase activity and that PSENEN and CLN3 play associated roles in the autophagy-lysosome system. We applied CRISPR gene-editing and obtained independent isogenic HeLa knockout cell lines for PSENEN and CLN3. Following previous studies, we demonstrate that PSENEN is essential for forming a functional γ-secretase complex and is indispensable for γ-secretase activity. In contrast, CLN3 does not modulate γ-secretase activity to a significant degree. We observed in PSENEN- and CLN3-knockout cells corresponding alterations in the autophagy-lysosome system. These include reduced activity of lysosomal enzymes and lysosome number, an increased number of autophagosomes, increased lysosome-autophagosome fusion, and elevated levels of TFEB (transcription factor EB). Our study strongly suggests converging roles of PSENEN and CLN3 in the autophagy-lysosome system in a γ-secretase activity-independent manner, supporting the idea of common cytopathological processes underlying different neurodegenerative diseases.Abbreviations: Aß, amyloid-beta; AD, Alzheimer disease; APP, amyloid precursor protein; ATP5MC, ATP synthase membrane subunit c; DQ-BSA, dye-quenched bovine serum albumin; ER, endoplasmic reticulum; GFP, green fluorescent protein; ICC, immunocytochemistry; ICD, intracellular domain; JNCL, juvenile neuronal ceroid lipofuscinosis; KO, knockout; LC3, microtubule associated protein 1 light chain 3; NCL, neuronal ceroid lipofuscinoses; PSEN, presenilin; PSENEN/PEN2: presenilin enhancer, gamma-secretase subunit; TAP, tandem affinity purification; TEV, tobacco etch virus; TF, transferrin; WB, Western blot; WT, wild type.
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Enfermedad de Alzheimer , Lipofuscinosis Ceroideas Neuronales , Enfermedad de Alzheimer/genética , Enfermedad de Alzheimer/metabolismo , Secretasas de la Proteína Precursora del Amiloide/genética , Secretasas de la Proteína Precursora del Amiloide/metabolismo , Autofagia/genética , Niño , Humanos , Lisosomas/metabolismo , Glicoproteínas de Membrana/genética , Glicoproteínas de Membrana/metabolismo , Proteínas de la Membrana/metabolismo , Chaperonas Moleculares/metabolismo , Lipofuscinosis Ceroideas Neuronales/genética , Lipofuscinosis Ceroideas Neuronales/metabolismo , Presenilinas/genética , Presenilinas/metabolismo , Factores de Transcripción/metabolismoRESUMEN
Mutations in the presenilin (PS/PSEN) genes encoding the catalytic components of γ-secretase accelerate amyloid-ß (Aß) and tau pathologies in familial Alzheimer's disease (AD). Although the mechanisms by which these mutations affect Aß are well defined, the precise role PS/γ-secretase on tau pathology in neurodegeneration independently of Aß is largely unclear. Here we report that neuronal PS deficiency in conditional knockout (cKO) mice results in age-dependent brain atrophy, inflammatory responses and accumulation of pathological tau in neurons and glial cells. Interestingly, genetic inactivation of presenilin 1 (PS1) or both PS genes in mutant human Tau transgenic mice exacerbates memory deficits by accelerating phosphorylation and aggregation of tau in excitatory neurons of vulnerable AD brain regions (e.g., hippocampus, cortex and amygdala). Remarkably, neurofilament (NF) light chain (NF-L) and phosphorylated NF are abnormally accumulated in the brain of Tau mice lacking PS. Synchrotron infrared microspectroscopy revealed aggregated and oligomeric ß-sheet structures in amyloid plaque-free PS-deficient Tau mice. Hippocampal-dependent memory deficits are associated with synaptic tau accumulation and reduction of pre- and post-synaptic proteins in Tau mice. Thus, partial loss of PS/γ-secretase in neurons results in temporal- and spatial-dependent tau aggregation associated with memory deficits and neurodegeneration. Our findings show that tau phosphorylation and aggregation are key pathological processes that may underlie neurodegeneration caused by familial AD-linked PSEN mutations.
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Encéfalo/patología , Neuronas/patología , Presenilinas/genética , Agregación Patológica de Proteínas/genética , Proteínas tau/metabolismo , Enfermedad de Alzheimer/genética , Enfermedad de Alzheimer/metabolismo , Animales , Femenino , Humanos , Masculino , Ratones , Ratones Endogámicos C57BL , Ratones Noqueados , Ratones Transgénicos , Fosforilación , Presenilinas/metabolismoRESUMEN
The ß-amyloid peptide (Aß) is found as amyloid fibrils in senile plaques, a typical hallmark of Alzheimer's disease (AD). However, intermediate soluble oligomers of Aß are now recognized as initiators of the pathogenic cascade leading to AD. Studies using recombinant Aß have shown that hexameric Aß in particular acts as a critical nucleus for Aß self-assembly. We recently isolated hexameric Aß assemblies from a cellular model, and demonstrated their ability to enhance Aß aggregation in vitro. Here, we report the presence of similar hexameric-like Aß assemblies across several cellular models, including neuronal-like cell lines. In order to better understand how they are produced in a cellular context, we investigated the role of presenilin-1 (PS1) and presenilin-2 (PS2) in their formation. PS1 and PS2 are the catalytic subunits of the γ-secretase complex that generates Aß. Using CRISPR-Cas9 to knockdown each of the two presenilins in neuronal-like cell lines, we observed a direct link between the PS2-dependent processing pathway and the release of hexameric-like Aß assemblies in extracellular vesicles. Further, we assessed the contribution of hexameric Aß to the development of amyloid pathology. We report the early presence of hexameric-like Aß assemblies in both transgenic mice brains exhibiting human Aß pathology and in the cerebrospinal fluid of AD patients, suggesting hexameric Aß as a potential early AD biomarker. Finally, cell-derived hexameric Aß was found to seed other human Aß forms, resulting in the aggravation of amyloid deposition in vivo and neuronal toxicity in vitro.
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Enfermedad de Alzheimer/metabolismo , Péptidos beta-Amiloides/metabolismo , Encéfalo/metabolismo , Placa Amiloide/metabolismo , Presenilinas/metabolismo , Enfermedad de Alzheimer/patología , Animales , Encéfalo/patología , Células CHO , Línea Celular Tumoral , Cricetulus , Fibroblastos/metabolismo , Humanos , Ratones , Ratones Transgénicos , Neuronas/metabolismo , Neuronas/patología , Placa Amiloide/patologíaRESUMEN
Metabolic dysfunction and protein aggregation are common characteristics that occur in age-related neurodegenerative disease. However, the mechanisms underlying these abnormalities remain poorly understood. We have found that mutations in the gene encoding presenilin in Caenorhabditis elegans, sel-12, results in elevated mitochondrial activity that drives oxidative stress and neuronal dysfunction. Mutations in the human presenilin genes are the primary cause of familial Alzheimer's disease. Here, we demonstrate that loss of SEL-12/presenilin results in the hyperactivation of the mTORC1 pathway. This hyperactivation is caused by elevated mitochondrial calcium influx and, likely, the associated increase in mitochondrial activity. Reducing mTORC1 activity improves proteostasis defects and neurodegenerative phenotypes associated with loss of SEL-12 function. Consistent with high mTORC1 activity, we find that SEL-12 loss reduces autophagosome formation, and this reduction is prevented by limiting mitochondrial calcium uptake. Moreover, the improvements of proteostasis and neuronal defects in sel-12 mutants due to mTORC1 inhibition require the induction of autophagy. These results indicate that mTORC1 hyperactivation exacerbates the defects in proteostasis and neuronal function in sel-12 mutants and demonstrate a critical role of presenilin in promoting neuronal health.
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Enfermedad de Alzheimer/genética , Proteínas de Caenorhabditis elegans/metabolismo , Caenorhabditis elegans/genética , Calcio/metabolismo , Diana Mecanicista del Complejo 1 de la Rapamicina/metabolismo , Mitocondrias/metabolismo , Enfermedades Neurodegenerativas/genética , Presenilinas/metabolismo , Animales , Enfermedades Neurodegenerativas/patología , Transducción de SeñalRESUMEN
[Formula: see text]-Secretase is an enzyme known to cleave multiple substrates within their transmembrane domains, with the amyloid precursor protein of Alzheimer's Disease among the most prominent examples. The activity of [Formula: see text]-secretase strictly depends on the membrane cholesterol content, yet the mechanistic role of cholesterol in the substrate binding and cleavage remains unclear. In this work, we used all-atom molecular dynamics simulations to examine the role of cholesterol in the initial binding of a direct precursor of [Formula: see text]-amyloid polypeptides by [Formula: see text]-secretase. We showed that in cholesterol-rich membranes, both the substrate and the enzyme region proximal to the active site induce a local membrane thinning. With the free energy methods we found that in the presence of cholesterol the substrate binds favorably to the identified exosite, while cholesterol depletion completely abolishes the binding. To explain these findings, we directly examined the role of hydrophobic mismatch in the substrate binding to [Formula: see text]-secretase, showing that increased membrane thickness results in higher propensity of the enzyme to bind substrates. Therefore, we propose that cholesterol promotes substrate binding to [Formula: see text]-secretase by increasing the membrane thickness, which leads to the negative hydrophobic mismatch between the membrane and binding partners.
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Secretasas de la Proteína Precursora del Amiloide/metabolismo , Precursor de Proteína beta-Amiloide/metabolismo , Colesterol/metabolismo , Interacciones Hidrofóbicas e Hidrofílicas , Simulación de Dinámica Molecular , Presenilinas/metabolismoRESUMEN
The simultaneous accumulation of multiple pathological proteins, such as hyperphosphorylated tau (hp-tau) and phosphorylated α-synuclein (p-αSyn), has been reported in the brains of patients with various neurodegenerative diseases. We previously demonstrated that hp-tau-dependent p-αSyn accumulation was associated with the activation of GSK-3ß in the brains of P301L tau transgenic mice. To confirm the effects of another mutant tau on p-αSyn accumulation in vivo, we herein examined the brains of PS19 mice that overexpress human P301S mutant tau. Immunohistochemically, hp-tau and p-αSyn aggregates were detected in the same neuronal cells in the cerebrum and brain stem of aged PS19 mice. A semiquantitative analysis showed a positive correlation between hp-tau and p-αSyn accumulation. Furthermore, an activated form of GSK-3ß was detected within cells containing both hp-tau and p-αSyn aggregates in PS19 mice. Western blotting showed a decrease in inactivated PP2A levels in PS19 mice. The present results suggest that the overexpression of human P301S mutant tau induces p-αSyn accumulation that is accompanied by not only GSK-3ß, but also PP2A activation in PS19 mice, and highlight the synergic effects between tau and αSyn in the pathophysiology of neurodegenerative diseases that show the codeposition of tau and αSyn.
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Glucógeno Sintasa Quinasa 3 beta/metabolismo , Proteína Fosfatasa 2/metabolismo , Tauopatías/metabolismo , alfa-Sinucleína/metabolismo , Animales , Encéfalo/metabolismo , Encéfalo/patología , Ratones , Mutación , Presenilinas/genética , Presenilinas/metabolismo , Tauopatías/genética , Tauopatías/patología , alfa-Sinucleína/genéticaRESUMEN
This chapter outlines a protocol developed to prepare a purified deuterated membrane protein for a small-angle neutron scattering (SANS) experiment. SANS is a noninvasive technique well suited to studying membrane protein solution structures, and deuteration enhances the signal from the protein over the background (Breyton et al., Eur Phys J E Soft Matter 36 (7):71, 2013; Garg et al., Biophys J 101 (2):370-377, 2011). We present our workflow: transformation of our plasmid into E. coli, cell growth and expression of our deuterated protein, membrane isolation, detergent solubilization, protein purification, purity assessment, and final preparation for SANS.
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Escherichia coli/crecimiento & desarrollo , Presenilinas/química , Presenilinas/genética , Detergentes/química , Escherichia coli/genética , Humanos , Modelos Moleculares , Difracción de Neutrones , Plásmidos/genética , Presenilinas/aislamiento & purificación , Presenilinas/metabolismo , Estructura Secundaria de Proteína , Proteínas Recombinantes/química , Proteínas Recombinantes/aislamiento & purificación , Proteínas Recombinantes/metabolismo , Dispersión del Ángulo Pequeño , Transformación Bacteriana , Flujo de TrabajoRESUMEN
Alzheimer's disease (AD) is an age-related neurodegenerative disease and characterized by dementia, memory decline, loss of learning and cognitive disorder. The main pathological features of AD are the deposition of amyloid plaques and the formation of neurofibrillary tangles (NFTs) in the brain. The current anti-AD drugs have shown unsatisfactory therapeutic results. Due to the complications and unclear pathogenesis, AD is still irreversible and incurable. Among several hypotheses proposed by the academic community, the amyloid cascade is widely recognized by scholars and supported by a large amount of evidences. However, controversy over pathogenic factors has also been ongoing. Increasing evidence has shown that amyloid-ß (Aß) and especially amyloid-ß oligomers (AßOs) are highly neurotoxic and pathogenic agents that damage neurons, mediate various receptors in the downstream pathways, and ultimately lead to learning and cognitive dysfunction. However, efforts in developing inhibitors of Aß or amyloid-ß precursor protein (APP) have all failed to yield good clinical results. More recently, it has been demonstrated that sigma receptors, including sigma-1 and sigma-2 subtypes, may play critical roles in the regulation of binding and metabolism of AßOs in neuron cells and the pathophysiology of AD. Thus, sigma receptor ligands are being recognized as promising therapeutic agents for treating or ameliorating AD. This article will review the pathophysiology of AD and highlight the sigma ligands that display the capability of preventing or even reversing Aß- and AßOs-induced neurotoxicity and blocking the signal transduction caused by AßOs.
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Enfermedad de Alzheimer/metabolismo , Péptidos beta-Amiloides/metabolismo , Precursor de Proteína beta-Amiloide/metabolismo , Neuronas/metabolismo , Receptores sigma/agonistas , Receptores sigma/metabolismo , Enfermedad de Alzheimer/fisiopatología , Animales , Humanos , Ligandos , Enfermedades Neuroinflamatorias/metabolismo , Presenilinas/metabolismo , Proteínas tau/metabolismo , Receptor Sigma-1RESUMEN
Signal peptides help newly synthesized proteins reach the cell membrane or be secreted. As part of a biological process key to immune response and surveillance in humans, and associated with diseases, for example, Alzheimer, remnant signal peptides and other transmembrane segments are proteolyzed by the intramembrane aspartyl protease (IAP) enzyme family. Here, we identified IAP orthologs throughout the tree of life. In addition to eukaryotes, IAPs are encoded in metabolically diverse archaea from a wide range of environments. We found three distinct clades of archaeal IAPs: (a) Euryarchaeota (eg, halophilic Halobacteriales, methanogenic Methanosarcinales and Methanomicrobiales, marine Poseidoniales, acidophilic Thermoplasmatales, hyperthermophilic Archaeoglobus spp.), (b) DPANN, and (c) Bathyarchaeota, Crenarchaeota, and Asgard. IAPs were also present in bacterial genomes from uncultivated members of Candidate Phylum Radiation, perhaps due to horizontal gene transfer from DPANN archaeal lineages. Sequence analysis of the catalytic motif YD GXGD (where X is any amino acid) in IAPs from archaea and bacteria reveals WD in Lokiarchaeota and many residue types in the X position. Gene neighborhood analysis in halophilic archaea shows IAP genes near corrinoid transporters (btuCDF genes). In marine Euryarchaeota, a putative BtuF-like domain is found in N-terminus of the IAP gene, suggesting a role for these IAPs in metal ion cofactor or other nutrient scavenging. Interestingly, eukaryotic IAP family members appear to have evolved either from Euryarchaeota or from Asgard archaea. Taken together, our phylogenetic and bioinformatics analysis should prompt experiments to probe the biological roles of IAPs in prokaryotic secretomes.
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Proteasas de Ácido Aspártico/genética , Bacterias/genética , Crenarchaeota/genética , Euryarchaeota/genética , Nanoarchaeota/genética , Presenilinas/genética , Secuencia de Aminoácidos , Proteasas de Ácido Aspártico/química , Proteasas de Ácido Aspártico/metabolismo , Bacterias/clasificación , Bacterias/enzimología , Evolución Biológica , Dominio Catalítico , Biología Computacional/métodos , Secuencia Conservada , Crenarchaeota/clasificación , Crenarchaeota/enzimología , Euryarchaeota/clasificación , Euryarchaeota/enzimología , Expresión Génica , Humanos , Isoenzimas/química , Isoenzimas/genética , Isoenzimas/metabolismo , Nanoarchaeota/clasificación , Nanoarchaeota/enzimología , Filogenia , Presenilinas/química , Presenilinas/metabolismo , Conformación Proteica en Hélice alfa , Conformación Proteica en Lámina beta , Señales de Clasificación de Proteína/genética , Alineación de Secuencia , Análisis de Secuencia de Proteína , Homología de Secuencia de AminoácidoRESUMEN
BACKGROUND: Mutations in the PSEN1 and PSEN2 genes are the major cause of familial Alzheimer's disease. Previous studies demonstrated that Presenilin (PS), the catalytic subunit of γ-secretase, is required for survival of excitatory neurons in the cerebral cortex during aging. However, the role of PS in inhibitory interneurons had not been explored. METHODS: To determine PS function in GABAergic neurons, we generated inhibitory neuron-specific PS conditional double knockout (IN-PS cDKO) mice, in which PS is selectively inactivated by Cre recombinase expressed under the control of the endogenous GAD2 promoter. We then performed behavioral, biochemical, and histological analyses to evaluate the consequences of selective PS inactivation in inhibitory neurons. RESULTS: IN-PS cDKO mice exhibit earlier mortality and lower body weight despite normal food intake and basal activity. Western analysis of protein lysates from various brain sub-regions of IN-PS cDKO mice showed significant reduction of PS1 levels and dramatic accumulation of γ-secretase substrates. Interestingly, IN-PS cDKO mice develop age-dependent loss of GABAergic neurons, as shown by normal number of GAD67-immunoreactive interneurons in the cerebral cortex at 2-3 months of age but reduced number of cortical interneurons at 9 months. Moreover, age-dependent reduction of Parvalbumin- and Somatostatin-immunoreactive interneurons is more pronounced in the neocortex and hippocampus of IN-PS cDKO mice. Consistent with these findings, the number of apoptotic cells is elevated in the cerebral cortex of IN-PS cDKO mice, and the enhanced apoptosis is due to dramatic increases of apoptotic interneurons, whereas the number of apoptotic excitatory neurons is unaffected. Furthermore, progressive loss of interneurons in the cerebral cortex of IN-PS cDKO mice is accompanied with astrogliosis and microgliosis. CONCLUSION: Our results together support a cell-autonomous role of PS in the survival of cortical interneurons during aging. Together with earlier studies, these findings demonstrate a universal, essential requirement of PS in the survival of both excitatory and inhibitory neurons during aging.
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Factores de Edad , Encéfalo/metabolismo , Interneuronas/metabolismo , Presenilinas/metabolismo , Envejecimiento/metabolismo , Animales , Corteza Cerebral/citología , Hipocampo/metabolismo , Ratones , Presenilina-1/metabolismoRESUMEN
The increasing detection of infections of Trypanosoma cruzi, the etiological agent of Chagas disease, in non-endemic regions beyond Latin America has risen to be a major public health issue. With an impact in the millions of people, current treatments rely on antiquated drugs that produce severe side effects and are considered nearly ineffective for the chronic phase. The minimal progress in the development of new drugs highlights the need for advances in basic research on crucial biochemical pathways in T. cruzi to identify new targets. Here, we report on the T. cruzi presenilin-like transmembrane aspartyl enzyme, a protease of the aspartic class in a unique phylogenetic subgroup with T. vivax separate from protozoans. Computational analyses suggest it contains nine transmembrane domains and an active site with the characteristic PALP motif of the A22 family. Multiple linear B-cell epitopes were identified by SPOT-synthesis analysis with Chagasic patient sera. Two were chosen to generate rabbit antisera, whose signal was primarily localized to the flagellar pocket, intracellular vesicles, and endoplasmic reticulum in parasites by whole-cell immunofluorescence. The results suggest that the parasitic presenilin-like enzyme could have a role in the secretory pathway and serve as a target for the generation of new therapeutics specific to the T. cruzi.