RESUMO
Expansion of CAG trinucleotide repeats in ATXN1 causes spinocerebellar ataxia type 1 (SCA1), a neurodegenerative disease that impairs coordination and cognition. While ATXN1 is associated with increased Alzheimer's disease (AD) risk, CAG repeat number in AD patients is not changed. Here, we investigated the consequences of ataxin-1 loss of function and discovered that knockout of Atxn1 reduced CIC-ETV4/5-mediated inhibition of Bace1 transcription, leading to increased BACE1 levels and enhanced amyloidogenic cleavage of APP, selectively in AD-vulnerable brain regions. Elevated BACE1 expression exacerbated Aß deposition and gliosis in AD mouse models and impaired hippocampal neurogenesis and olfactory axonal targeting. In SCA1 mice, polyglutamine-expanded mutant ataxin-1 led to the increase of BACE1 post-transcriptionally, both in cerebrum and cerebellum, and caused axonal-targeting deficit and neurodegeneration in the hippocampal CA2 region. These findings suggest that loss of ataxin-1 elevates BACE1 expression and Aß pathology, rendering it a potential contributor to AD risk and pathogenesis.
Assuntos
Doença de Alzheimer/patologia , Secretases da Proteína Precursora do Amiloide/metabolismo , Ataxina-1/metabolismo , Encéfalo/metabolismo , Doença de Alzheimer/metabolismo , Secretases da Proteína Precursora do Amiloide/genética , Precursor de Proteína beta-Amiloide/metabolismo , Animais , Ataxina-1/deficiência , Ataxina-1/genética , Encéfalo/patologia , Região CA2 Hipocampal/metabolismo , Região CA2 Hipocampal/patologia , Proteínas de Ligação a DNA/genética , Proteínas de Ligação a DNA/metabolismo , Modelos Animais de Doenças , Feminino , Frequência do Gene , Humanos , Masculino , Camundongos , Camundongos Transgênicos , Neurogênese , Proteínas Proto-Oncogênicas c-ets/genética , Proteínas Proto-Oncogênicas c-ets/metabolismo , Fatores de Transcrição/genética , Fatores de Transcrição/metabolismo , Transcrição Gênica , Repetições de Trinucleotídeos/genética , Regulação para CimaRESUMO
Cleavage of membrane-anchored proteins by ADAM (a disintegrin and metalloproteinase) endopeptidases plays a key role in a wide variety of biological signal transduction and protein turnover processes. Among ADAM family members, ADAM10 stands out as particularly important because it is both responsible for regulated proteolysis of Notch receptors and catalyzes the non-amyloidogenic α-secretase cleavage of the Alzheimer's precursor protein (APP). We present here the X-ray crystal structure of the ADAM10 ectodomain, which, together with biochemical and cellular studies, reveals how access to the enzyme active site is regulated. The enzyme adopts an unanticipated architecture in which the C-terminal cysteine-rich domain partially occludes the enzyme active site, preventing unfettered substrate access. Binding of a modulatory antibody to the cysteine-rich domain liberates the catalytic domain from autoinhibition, enhancing enzymatic activity toward a peptide substrate. Together, these studies reveal a mechanism for regulation of ADAM activity and offer a roadmap for its modulation.
Assuntos
Proteína ADAM10/química , Secretases da Proteína Precursora do Amiloide/química , Proteínas de Membrana/química , Proteólise , Proteína ADAM10/metabolismo , Secretases da Proteína Precursora do Amiloide/metabolismo , Cristalografia por Raios X , Humanos , Proteínas de Membrana/metabolismo , Modelos Moleculares , Receptores Notch/metabolismo , Transdução de SinaisRESUMO
Alzheimer's disease (AD)-linked mutations in Presenilins (PSEN) and the amyloid precursor protein (APP) lead to production of longer amyloidogenic Aß peptides. The shift in Aß length is fundamental to the disease; however, the underlying mechanism remains elusive. Here, we show that substrate shortening progressively destabilizes the consecutive enzyme-substrate (E-S) complexes that characterize the sequential γ-secretase processing of APP. Remarkably, pathogenic PSEN or APP mutations further destabilize labile E-S complexes and thereby promote generation of longer Aß peptides. Similarly, destabilization of wild-type E-S complexes by temperature, compounds, or detergent promotes release of amyloidogenic Aß. In contrast, E-Aßn stabilizers increase γ-secretase processivity. Our work presents a unifying model for how PSEN or APP mutations enhance amyloidogenic Aß production, suggests that environmental factors may increase AD risk, and provides the theoretical basis for the development of γ-secretase/substrate stabilizing compounds for the prevention of AD.
Assuntos
Doença de Alzheimer/enzimologia , Doença de Alzheimer/genética , Precursor de Proteína beta-Amiloide/metabolismo , Proteínas de Membrana/metabolismo , Peptídeo Hidrolases/metabolismo , Presenilina-1/metabolismo , Precursor de Proteína beta-Amiloide/química , Animais , Encéfalo/metabolismo , Encéfalo/patologia , Linhagem Celular , Endopeptidases , Estabilidade Enzimática , Feminino , Células HEK293 , Humanos , Proteínas de Membrana/química , Proteínas de Membrana/genética , Camundongos , Modelos Moleculares , Mutação , Peptídeo Hidrolases/química , Peptídeo Hidrolases/genética , Presenilina-1/química , Presenilina-1/genéticaRESUMO
Human apolipoprotein E (ApoE) apolipoprotein is primarily expressed in three isoforms (ApoE2, ApoE3, and ApoE4) that differ only by two residues. ApoE4 constitutes the most important genetic risk factor for Alzheimer's disease (AD), ApoE3 is neutral, and ApoE2 is protective. How ApoE isoforms influence AD pathogenesis, however, remains unclear. Using ES-cell-derived human neurons, we show that ApoE secreted by glia stimulates neuronal Aß production with an ApoE4 > ApoE3 > ApoE2 potency rank order. We demonstrate that ApoE binding to ApoE receptors activates dual leucine-zipper kinase (DLK), a MAP-kinase kinase kinase that then activates MKK7 and ERK1/2 MAP kinases. Activated ERK1/2 induces cFos phosphorylation, stimulating the transcription factor AP-1, which in turn enhances transcription of amyloid-ß precursor protein (APP) and thereby increases amyloid-ß levels. This molecular mechanism also regulates APP transcription in mice in vivo. Our data describe a novel signal transduction pathway in neurons whereby ApoE activates a non-canonical MAP kinase cascade that enhances APP transcription and amyloid-ß synthesis.
Assuntos
Precursor de Proteína beta-Amiloide/genética , Apolipoproteínas E/metabolismo , Sistema de Sinalização das MAP Quinases , Doença de Alzheimer/metabolismo , Animais , Células-Tronco Embrionárias/citologia , Células-Tronco Embrionárias/metabolismo , Fibroblastos/metabolismo , Humanos , Camundongos , Neurônios/metabolismo , Isoformas de Proteínas/metabolismoRESUMO
Sequential proteolysis of the amyloid precursor protein (APP) by γ-secretases generates amyloid-ß (Aß) peptides and defines the proportion of short-to-long Aß peptides, which is tightly connected to Alzheimer's disease (AD) pathogenesis. Here, we study the mechanism that controls substrate processing by γ-secretases and Aß peptide length. We found that polar interactions established by the APPC99 ectodomain (ECD), involving but not limited to its juxtamembrane region, restrain both the extent and degree of γ-secretases processive cleavage by destabilizing enzyme-substrate interactions. We show that increasing hydrophobicity, via mutation or ligand binding, at APPC99 -ECD attenuates substrate-driven product release and rescues the effects of Alzheimer's disease-associated pathogenic γ-secretase and APP variants on Aß length. In addition, our study reveals that APPC99 -ECD facilitates the paradoxical production of longer Aßs caused by some γ-secretase inhibitors, which act as high-affinity competitors of the substrate. These findings assign a pivotal role to the substrate ECD in the sequential proteolysis by γ-secretases and suggest it as a sweet spot for the potential design of APP-targeting compounds selectively promoting its processing by these enzymes.
Assuntos
Doença de Alzheimer , Precursor de Proteína beta-Amiloide , Humanos , Precursor de Proteína beta-Amiloide/genética , Precursor de Proteína beta-Amiloide/metabolismo , Peptídeos beta-Amiloides/metabolismo , Secretases da Proteína Precursora do Amiloide/genética , Secretases da Proteína Precursora do Amiloide/metabolismo , Doença de Alzheimer/metabolismo , ProteóliseRESUMO
Disease-associated variants identified from genome-wide association studies (GWASs) frequently map to non-coding areas of the genome such as introns and intergenic regions. An exclusive reliance on gene-agnostic methods of genomic investigation could limit the identification of relevant genes associated with polygenic diseases such as Alzheimer disease (AD). To overcome such potential restriction, we developed a gene-constrained analytical method that considers only moderate- and high-risk variants that affect gene coding sequences. We report here the application of this approach to publicly available datasets containing 181,388 individuals without and with AD and the resulting identification of 660 genes potentially linked to the higher AD prevalence among Africans/African Americans. By integration with transcriptome analysis of 23 brain regions from 2,728 AD case-control samples, we concentrated on nine genes that potentially enhance the risk of AD: AACS, GNB5, GNS, HIPK3, MED13, SHC2, SLC22A5, VPS35, and ZNF398. GNB5, the fifth member of the heterotrimeric G protein beta family encoding Gß5, is primarily expressed in neurons and is essential for normal neuronal development in mouse brain. Homozygous or compound heterozygous loss of function of GNB5 in humans has previously been associated with a syndrome of developmental delay, cognitive impairment, and cardiac arrhythmia. In validation experiments, we confirmed that Gnb5 heterozygosity enhanced the formation of both amyloid plaques and neurofibrillary tangles in the brains of AD model mice. These results suggest that gene-constrained analysis can complement the power of GWASs in the identification of AD-associated genes and may be more broadly applicable to other polygenic diseases.
Assuntos
Doença de Alzheimer , Subunidades beta da Proteína de Ligação ao GTP , Camundongos , Humanos , Animais , Doença de Alzheimer/genética , Doença de Alzheimer/metabolismo , Estudo de Associação Genômica Ampla , Emaranhados Neurofibrilares/metabolismo , Fenótipo , Genômica , Peptídeos beta-Amiloides/genética , Encéfalo/metabolismo , Membro 5 da Família 22 de Carreadores de Soluto/genética , Membro 5 da Família 22 de Carreadores de Soluto/metabolismo , Subunidades beta da Proteína de Ligação ao GTP/genética , Subunidades beta da Proteína de Ligação ao GTP/metabolismoRESUMO
Alzheimer's disease is associated with increased levels of amyloid beta (Aß) generated by sequential intracellular cleavage of amyloid precursor protein (APP) by membrane-bound secretases. However, the spatial and temporal APP cleavage events along the trafficking pathways are poorly defined. Here, we use the Retention Using Selective Hooks (RUSH) to compare in real time the anterograde trafficking and temporal cleavage events of wild-type APP (APPwt) with the pathogenic Swedish APP (APPswe) and the disease-protective Icelandic APP (APPice). The analyses revealed differences in the trafficking profiles and processing between APPwt and the APP familial mutations. While APPwt was predominantly processed by the ß-secretase, BACE1, following Golgi transport to the early endosomes, the transit of APPswe through the Golgi was prolonged and associated with enhanced amyloidogenic APP processing and Aß secretion. A 20°C block in cargo exit from the Golgi confirmed ß- and γ-secretase processing of APPswe in the Golgi. Inhibition of the ß-secretase, BACE1, restored APPswe anterograde trafficking profile to that of APPwt. APPice was transported rapidly through the Golgi to the early endosomes with low levels of Aß production. This study has revealed different intracellular locations for the preferential cleavage of APPwt and APPswe and Aß production, and the Golgi as the major processing site for APPswe, findings relevant to understand the molecular basis of Alzheimer's disease.
Assuntos
Doença de Alzheimer , Precursor de Proteína beta-Amiloide , Humanos , 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 , Peptídeos beta-Amiloides/metabolismo , Doença de Alzheimer/genética , Doença de Alzheimer/metabolismo , Suécia , Ácido Aspártico Endopeptidases/genética , Ácido Aspártico Endopeptidases/metabolismo , MutaçãoRESUMO
Alzheimer's disease (AD) pathogenesis has been linked to the accumulation of longer, aggregation-prone amyloid ß (Aß) peptides in the brain. Γ-secretases generate Aß peptides from the amyloid precursor protein (APP). Γ-secretase modulators (GSMs) promote the generation of shorter, less-amyloidogenic Aßs and have therapeutic potential. However, poorly defined drug-target interactions and mechanisms of action have hampered their therapeutic development. Here, we investigate the interactions between the imidazole-based GSM and its target γ-secretase-APP using experimental and in silico approaches. We map the GSM binding site to the enzyme-substrate interface, define a drug-binding mode that is consistent with functional and structural data, and provide molecular insights into the underlying mechanisms of action. In this respect, our analyses show that occupancy of a γ-secretase (sub)pocket, mediating binding of the modulator's imidazole moiety, is sufficient to trigger allosteric rearrangements in γ-secretase as well as stabilize enzyme-substrate interactions. Together, these findings may facilitate the rational design of new modulators of γ-secretase with improved pharmacological properties.
Assuntos
Doença de Alzheimer , Secretases da Proteína Precursora do Amiloide , Humanos , Secretases da Proteína Precursora do Amiloide/metabolismo , Precursor de Proteína beta-Amiloide/metabolismo , Peptídeos beta-Amiloides/metabolismo , Inibidores e Moduladores de Secretases gama , Doença de Alzheimer/metabolismo , Imidazóis/uso terapêuticoRESUMO
Soluble amyloid precursor protein-alpha (sAPPα) is a multi-functional brain-derived protein that has neuroprotective, neurogenic and neurotropic properties. Moreover, it is known to facilitate synaptic function and promote neural repair. These properties suggest sAPPα may be useful as a therapeutic agent for the treatment of neurological diseases characterized by synaptic failure and neuronal loss, such as occurs in Alzheimer's disease, and for neural repair following traumatic brain injury and stroke. However, sAPPα's relatively large size and the difficulty of ongoing delivery of therapeutics to the brain mean this is not currently practicable. Importantly, however, sAPPα is composed of several neuroactive domains that each possess properties that collectively are remarkably similar to those of sAPPα itself. Here, we review the molecular structure of sAPPα and identify the domains that contribute to its overall functionality. Four peptide motifs present as possible targets for therapeutic development. We review their physiochemical and neuroactive properties, both within sAPPα and as isolated peptides, and discuss their potential for future development as multipurpose therapeutic agents for the treatment of Alzheimer's disease and other disorders of neuronal function. Further, we discuss the role of heparin binding sites, found within sAPPα's structure and overlapping with the neuroactive domains, as sites for interactions with effector proteins and synaptic receptors. The potential role of the neuroactive peptides known as Cationic Arginine-Rich Peptides (CARPs) as neuroprotective motifs is also reviewed. Mechanisms of peptide delivery to the brain are briefly discussed. Finally, we summarise the potential benefits and pitfalls of using the isolated peptides, either individually or in combination, for the treatment of neurological diseases.
Assuntos
Doença de Alzheimer , Precursor de Proteína beta-Amiloide , Humanos , Precursor de Proteína beta-Amiloide/metabolismo , Doença de Alzheimer/tratamento farmacológico , Doença de Alzheimer/metabolismo , Peptídeos beta-Amiloides/metabolismo , Encéfalo/metabolismo , NeuroproteçãoRESUMO
Alzheimer's disease (AD) is a progressive neurodegenerative disorder characterized by dysregulation of the expression and processing of the amyloid precursor protein (APP). Protein quality control systems are dedicated to remove faulty and deleterious proteins to maintain cellular protein homeostasis (proteostasis). Identidying mechanisms underlying APP protein regulation is crucial for understanding AD pathogenesis. However, the factors and associated molecular mechanisms regulating APP protein quality control remain poorly defined. In this study, we show that mutant APP with its mitochondrial-targeting sequence ablated exhibited predominant endoplasmic reticulum (ER) distribution and led to aberrant ER morphology, deficits in locomotor activity, and shortened lifespan. We searched for regulators that could counteract the toxicity caused by the ectopic expression of this mutant APP. Genetic removal of the ribosome-associated quality control (RQC) factor RACK1 resulted in reduced levels of ectopically expressed mutant APP. By contrast, gain of RACK1 function increased mutant APP level. Additionally, overexpression of the ER stress regulator (IRE1) resulted in reduced levels of ectopically expressed mutant APP. Mechanistically, the RQC related ATPase VCP/p97 and the E3 ubiquitin ligase Hrd1 were required for the reduction of mutant APP level by IRE1. These factors also regulated the expression and toxicity of ectopically expressed wild type APP, supporting their relevance to APP biology. Our results reveal functions of RACK1 and IRE1 in regulating the quality control of APP homeostasis and mitigating its pathogenic effects, with implications for the understanding and treatment of AD.
Assuntos
Doença de Alzheimer , Precursor de Proteína beta-Amiloide , Proteínas de Drosophila , Endorribonucleases , Receptores de Quinase C Ativada , Animais , Doença de Alzheimer/metabolismo , Peptídeos beta-Amiloides/metabolismo , Precursor de Proteína beta-Amiloide/genética , Precursor de Proteína beta-Amiloide/metabolismo , Proteínas de Drosophila/genética , Proteínas de Drosophila/metabolismo , Proteínas Serina-Treonina Quinases , Receptores de Quinase C Ativada/genética , Receptores de Quinase C Ativada/metabolismo , Drosophila melanogaster , Modelos Animais de Doenças , Endorribonucleases/genética , Endorribonucleases/metabolismoRESUMO
Experimental studies in flies, mice, and humans suggest a significant role of impaired axonal transport in the pathogenesis of Alzheimer's disease (AD). The mechanisms underlying these impairments in axonal transport, however, remain poorly understood. Here we report that the Swedish familial AD mutation causes a standstill of the amyloid precursor protein (APP) in the axons at the expense of its reduced anterograde transport. The standstill reflects the perturbed directionality of the axonal transport of APP, which spends significantly more time traveling in the retrograde direction. This ineffective movement is accompanied by an enhanced association of dynactin-1 with APP, which suggests that reduced anterograde transport of APP is the result of enhanced activation of the retrograde molecular motor dynein by dynactin-1. The impact of the Swedish mutation on axonal transport is not limited to the APP vesicles since it also reverses the directionality of a subset of early endosomes, which become enlarged and aberrantly accumulate in distal locations. In addition, it also reduces the trafficking of lysosomes due to their less effective retrograde movement. Altogether, our experiments suggest a pivotal involvement of retrograde molecular motors and transport in the mechanisms underlying impaired axonal transport in AD and reveal significantly more widespread derangement of axonal transport pathways in the pathogenesis of AD.
Assuntos
Doença de Alzheimer , Precursor de Proteína beta-Amiloide , Transporte Axonal , Animais , Humanos , Camundongos , Doença de Alzheimer/metabolismo , Doença de Alzheimer/genética , Doença de Alzheimer/patologia , Precursor de Proteína beta-Amiloide/genética , Precursor de Proteína beta-Amiloide/metabolismo , Transporte Axonal/genética , Axônios/metabolismo , Axônios/patologia , Complexo Dinactina/metabolismo , Complexo Dinactina/genética , Dineínas/metabolismo , Endossomos/metabolismo , Endossomos/genética , Lisossomos/metabolismo , Mutação , Variação GenéticaRESUMO
The amyloid precursor protein (APP) is a key protein in Alzheimer's disease synthesized in the endoplasmic reticulum (ER) and translocated to the plasma membrane where it undergoes proteolytic cleavages by several proteases. Conversely, to other known proteases, we previously elucidated rhomboid protease RHBDL4 as a novel APP processing enzyme where several cleavages likely occur already in the ER. Interestingly, the pattern of RHBDL4-derived large APP C-terminal fragments resembles those generated by the η-secretase or MT5-MMP, which was described to generate so-called Aη fragments. The similarity in large APP C-terminal fragments between both proteases raised the question of whether RHBDL4 may contribute to η-secretase activity and Aη-like fragments. Here, we identified two cleavage sites of RHBDL4 in APP by mass spectrometry, which, intriguingly, lie in close proximity to the MT5-MMP cleavage sites. Indeed, we observed that RHBDL4 generates Aη-like fragments in vitro without contributions of α-, ß-, or γ-secretases. Such Aη-like fragments are likely generated in the ER since RHBDL4-derived APP-C-terminal fragments do not reach the cell surface. Inherited, familial APP mutations appear to not affect this processing pathway. In RHBDL4 knockout mice, we observed increased cerebral full-length APP in comparison to wild type (WT) in support of RHBDL4 being a physiologically relevant protease for APP. Furthermore, we found secreted Aη fragments in dissociated mixed cortical cultures from WT mice, however significantly fewer Aη fragments in RHBDL4 knockout cultures. Our data underscores that RHBDL4 contributes to the η-secretease-like processing of APP and that RHBDL4 is a physiologically relevant protease for APP.
RESUMO
The key role of APP for Alzheimer pathogenesis is well established. However, perinatal lethality of germline knockout mice lacking the entire APP family has so far precluded the analysis of its physiological functions for the developing and adult brain. Here, we generated conditional APP/APLP1/APLP2 triple KO (cTKO) mice lacking the APP family in excitatory forebrain neurons from embryonic day 11.5 onwards. NexCre cTKO mice showed altered brain morphology with agenesis of the corpus callosum and disrupted hippocampal lamination. Further, NexCre cTKOs revealed reduced basal synaptic transmission and drastically reduced long-term potentiation that was associated with reduced dendritic length and reduced spine density of pyramidal cells. With regard to behavior, lack of the APP family leads not only to severe impairments in a panel of tests for learning and memory, but also to an autism-like phenotype including repetitive rearing and climbing, impaired social communication, and deficits in social interaction. Together, our study identifies essential functions of the APP family during development, for normal hippocampal function and circuits important for learning and social behavior.
Assuntos
Precursor de Proteína beta-Amiloide/genética , Transtorno Autístico/genética , Animais , Transtorno Autístico/fisiopatologia , Comportamento Animal , Região CA1 Hipocampal/fisiologia , Feminino , Aprendizagem , Potenciação de Longa Duração , Masculino , Camundongos Knockout , Neurônios/fisiologia , Fenótipo , Prosencéfalo/citologia , Comportamento Social , Sinapses/fisiologia , Transmissão SinápticaRESUMO
APP gene dosage is strongly associated with Alzheimer's disease (AD) pathogenesis. Genomic duplication of the APP locus leads to autosomal dominant early-onset AD. Individuals with Down syndrome (trisomy of chromosome 21) harbour three copies of the APP gene and invariably develop progressive AD with highly characteristic neuropathological features. Restoring expression of APP to the equivalent of that of two gene copies, or lower, is a rational therapeutic strategy, as it would restore physiological levels of neuronal APP protein without the potentially deleterious consequences of inadvertently inducing loss of APP function. Here we find that antisense oligonucleotides (ASOs) targeting APP are an effective approach to reduce APP protein levels and rescue endolysosome and autophagy dysfunction in APP duplication and Trisomy 21 human induced pluripotent stem cell (hiPSC)-derived cortical neurons. Importantly, using ultrasensitive single-aggregate imaging techniques, we show that APP targeting ASOs significantly reduce both intracellular and extracellular amyloid-ß-containing aggregates. Our results highlight the potential of APP ASOs as a therapeutic approach for forms of AD caused by duplication of the APP gene, including monogenic AD and AD related to Down syndrome.
Assuntos
Doença de Alzheimer , Peptídeos beta-Amiloides , Precursor de Proteína beta-Amiloide , Síndrome de Down , Células-Tronco Pluripotentes Induzidas , Lisossomos , Oligonucleotídeos Antissenso , Humanos , Doença de Alzheimer/metabolismo , Doença de Alzheimer/genética , Doença de Alzheimer/patologia , Precursor de Proteína beta-Amiloide/genética , Precursor de Proteína beta-Amiloide/metabolismo , Peptídeos beta-Amiloides/metabolismo , Oligonucleotídeos Antissenso/farmacologia , Lisossomos/metabolismo , Lisossomos/efeitos dos fármacos , Células-Tronco Pluripotentes Induzidas/metabolismo , Células-Tronco Pluripotentes Induzidas/efeitos dos fármacos , Síndrome de Down/genética , Síndrome de Down/metabolismo , Síndrome de Down/patologia , Neurônios/metabolismo , Neurônios/efeitos dos fármacos , Endossomos/metabolismo , Endossomos/efeitos dos fármacos , Células CultivadasRESUMO
Autophagy is a highly conserved catabolic mechanism by which unnecessary or dysfunctional cellular components are removed. The dysregulation of autophagy has been implicated in various neurodegenerative diseases, including Alzheimer's disease (AD). Understanding the molecular mechanism(s)/molecules that influence autophagy may provide important insights into developing therapeutic strategies against AD and other neurodegenerative disorders. Engulfment adaptor phosphotyrosine-binding domain-containing protein 1 (GULP1) is an adaptor that interacts with amyloid precursor protein (APP) to promote amyloid-ß peptide production via an unidentified mechanism. Emerging evidence suggests that GULP1 has a role in autophagy. Here, we show that GULP1 is involved in autophagy through an interaction with autophagy-related 14 (ATG14), which is a regulator of autophagosome formation. GULP1 potentiated the stimulatory effect of ATG14 on autophagy by modulating class III phosphatidylinositol 3-kinase complex 1 (PI3KC3-C1) activity. The effect of GULP1 is attenuated by a GULP1 mutation (GULP1m) that disrupts the GULP1-ATG14 interaction. Conversely, PI3KC3-C1 activity is enhanced in cells expressing APP but not in those expressing an APP mutant that does not bind GULP1, which suggests a role of GULP1-APP in regulating PI3KC3-C1 activity. Notably, GULP1 facilitates the targeting of ATG14 to the endoplasmic reticulum (ER). Moreover, the levels of both ATG14 and APP are elevated in the autophagic vacuoles (AVs) of cells expressing GULP1, but not in those expressing GULP1m. APP processing is markedly enhanced in cells co-expressing GULP1 and ATG14. Hence, GULP1 alters APP processing by promoting the entry of APP into AVs. In summary, we unveil a novel role of GULP1 in enhancing the targeting of ATG14 to the ER to stimulate autophagy and, consequently, APP processing.
Assuntos
Proteínas Adaptadoras de Transdução de Sinal , Precursor de Proteína beta-Amiloide , Proteínas Relacionadas à Autofagia , Autofagia , Humanos , Precursor de Proteína beta-Amiloide/metabolismo , Precursor de Proteína beta-Amiloide/genética , Proteínas Relacionadas à Autofagia/metabolismo , Proteínas Relacionadas à Autofagia/genética , Proteínas Adaptadoras de Transdução de Sinal/metabolismo , Proteínas Adaptadoras de Transdução de Sinal/genética , Proteínas Adaptadoras de Transporte Vesicular/metabolismo , Proteínas Adaptadoras de Transporte Vesicular/genética , Células HEK293 , Ligação Proteica , Doença de Alzheimer/metabolismo , Doença de Alzheimer/genética , Doença de Alzheimer/patologia , Classe III de Fosfatidilinositol 3-Quinases/metabolismo , Classe III de Fosfatidilinositol 3-Quinases/genética , Proteínas de Transporte Vesicular/metabolismo , Proteínas de Transporte Vesicular/genéticaRESUMO
Proteins delivered by endocytosis or autophagy to lysosomes are degraded by exo- and endoproteases. In humans 15 lysosomal cathepsins (CTS) act as important physiological regulators. The cysteine proteases CTSB and CTSL and the aspartic protease CTSD are the most abundant and functional important lysosomal proteinases. Whereas their general functions in proteolysis in the lysosome, their individual substrate, cleavage specificity, and their possible sequential action on substrate proteins have been previously studied, their functional redundancy is still poorly understood. To address a possible common role of highly expressed and functional important CTS proteases, we generated CTSB-, CTSD-, CTSL-, and CTSBDL-triple deficient (KO) human neuroblastoma-derived SH-SY5Y cells and CTSB-, CTSD-, CTSL-, CTSZ and CTSBDLZ-quadruple deficient (KO) HeLa cells. These cells with a combined cathepsin deficiency exhibited enlarged lysosomes and accumulated lipofuscin-like storage material. The lack of the three (SH-SY5Y) or four (HeLa) major CTSs caused an impaired autophagic flux and reduced degradation of endocytosed albumin. Proteome analyses of parental and CTS-depleted cells revealed an enrichment of cleaved peptides, lysosome/autophagy-associated proteins, and potentially endocytosed membrane proteins like the amyloid precursor protein (APP), which can be subject to endocytic degradation. Amino- and carboxyterminal APP fragments accumulated in the multiple CTS-deficient cells, suggesting that multiple CTS-mediated cleavage events regularly process APP. In summary, our analyses support the idea that different lysosomal cathepsins act in concert, have at least partially and functionally redundant substrates, regulate protein degradation in autophagy, and control cellular proteostasis, as exemplified by their involvement in the degradation of APP fragments.
Assuntos
Autofagia , Catepsinas , Lisossomos , Proteólise , Humanos , Lisossomos/metabolismo , Catepsinas/metabolismo , Catepsinas/genética , Células HeLa , Endocitose , Catepsina L/metabolismo , Catepsina L/genética , Linhagem Celular Tumoral , Precursor de Proteína beta-Amiloide/metabolismo , Precursor de Proteína beta-Amiloide/genéticaRESUMO
Balanced synaptic inhibition, controlled by multiple synaptic adhesion proteins, is critical for proper brain function. MDGA1 (meprin, A-5 protein, and receptor protein-tyrosine phosphatase mu [MAM] domain-containing glycosylphosphatidylinositol anchor protein 1) suppresses synaptic inhibition in mammalian neurons, yet the molecular mechanisms underlying MDGA1-mediated negative regulation of GABAergic synapses remain unresolved. Here, we show that the MDGA1 MAM domain directly interacts with the extension domain of amyloid precursor protein (APP). Strikingly, MDGA1-mediated synaptic disinhibition requires the MDGA1 MAM domain and is prominent at distal dendrites of hippocampal CA1 pyramidal neurons. Down-regulation of APP in presynaptic GABAergic interneurons specifically suppressed GABAergic, but not glutamatergic, synaptic transmission strength and inputs onto both the somatic and dendritic compartments of hippocampal CA1 pyramidal neurons. Moreover, APP deletion manifested differential effects in somatostatin- and parvalbumin-positive interneurons in the hippocampal CA1, resulting in distinct alterations in inhibitory synapse numbers, transmission, and excitability. The infusion of MDGA1 MAM protein mimicked postsynaptic MDGA1 gain-of-function phenotypes that involve the presence of presynaptic APP. The overexpression of MDGA1 wild type or MAM, but not MAM-deleted MDGA1, in the hippocampal CA1 impaired novel object-recognition memory in mice. Thus, our results establish unique roles of APP-MDGA1 complexes in hippocampal neural circuits, providing unprecedented insight into trans-synaptic mechanisms underlying differential tuning of neuronal compartment-specific synaptic inhibition.
Assuntos
Precursor de Proteína beta-Amiloide/metabolismo , Hipocampo/metabolismo , Hipocampo/fisiopatologia , Moléculas de Adesão de Célula Nervosa/genética , Inibição Neural , Sinapses/metabolismo , Precursor de Proteína beta-Amiloide/genética , Região CA1 Hipocampal , Proteínas de Transporte , Dendritos/metabolismo , Neurônios GABAérgicos/metabolismo , Interneurônios , Modelos Biológicos , Moléculas de Adesão de Célula Nervosa/química , Moléculas de Adesão de Célula Nervosa/metabolismo , Inibição Neural/genética , Ligação Proteica , Domínios e Motivos de Interação entre Proteínas , Células Piramidais/metabolismo , Receptores de GABA-B/metabolismo , Transmissão SinápticaRESUMO
Aberrant cleavage of amyloid precursor protein (APP) by γ-secretase is closely associated with Alzheimer's disease (AD). γ-secretase activating protein (GSAP) specifically promotes γ-secretasemediated cleavage of APP. However, the underlying mechanism remains enigmatic. Here, we demonstrate that the 16-kDa C-terminal fragment of GSAP (GSAP-16K) undergoes phase separation in vitro and forms puncta-like condensates in cells. GSAP-16K exerts dual modulation on γ-secretase cleavage; GSAP-16K in dilute phase increases APPC-terminal 99-residue fragment (C99) cleavage toward preferred production of ß-amyloid peptide 42 (Aß42), but GSAP-16K condensates reduce APP-C99 cleavage through substrate sequestration. Notably, the Aß42/Aß40 ratio is markedly elevated with increasing concentrations of GSAP-16K. GSAP-16K stably associates with APP-C99 through specific sequence elements. These findings mechanistically explain GSAP-mediated modulation of γ-secretase activity that may have ramifications on the development of potential therapeutics.
Assuntos
Doença de Alzheimer , Secretases da Proteína Precursora do Amiloide , Doença de Alzheimer/metabolismo , Secretases da Proteína Precursora do Amiloide/metabolismo , Peptídeos beta-Amiloides/metabolismo , Precursor de Proteína beta-Amiloide/metabolismo , Humanos , Fragmentos de Peptídeos/metabolismoRESUMO
The intracellular trafficking of ß-site amyloid precursor protein (APP) cleaving enzyme (BACE1) and APP regulates amyloid-ß production. Our previous work demonstrated that newly synthesized BACE1 and APP are segregated into distinct trafficking pathways from the trans-Golgi network (TGN), and that alterations in their trafficking lead to an increase in Aß production in non-neuronal and neuronal cells. However, it is not known whether BACE1 and APP are transported through the Golgi stacks together and sorted at the TGN or segregated prior to arrival at the TGN. To address this question, we have used high-resolution Airyscan technology followed by Huygens deconvolution to quantify the overlap of BACE1 and APP in Golgi subcompartments in HeLa cells and primary neurons. Here, we show that APP and BACE1 are segregated, on exit from the endoplasmic reticulum and in the cis-Golgi and throughout the Golgi stack. In contrast, the transferrin receptor, which exits the TGN in AP-1 mediated transport carriers as for BACE1, colocalizes with BACE1, but not APP, throughout the Golgi stack. The segregation of APP and BACE1 is independent of the Golgi ribbon structure and the cytoplasmic domain of the cargo. Overall, our findings reveal the segregation of different membrane cargoes early in the secretory pathway, a finding relevant to the regulation of APP processing events.
Assuntos
Doença de Alzheimer , Precursor de Proteína beta-Amiloide , Doença de Alzheimer/metabolismo , Secretases da Proteína Precursora do Amiloide/metabolismo , Peptídeos beta-Amiloides/metabolismo , Precursor de Proteína beta-Amiloide/metabolismo , Ácido Aspártico Endopeptidases/metabolismo , Complexo de Golgi/metabolismo , Células HeLa , Humanos , Transporte Proteico/fisiologiaRESUMO
The perforant path provides the primary cortical excitatory input to the hippocampus. Because of its important role in information processing and coding, entorhinal projections to the dentate gyrus have been studied in considerable detail. Nevertheless, synaptic transmission between individual connected pairs of entorhinal stellate cells and dentate granule cells remains to be characterized. Here, we have used mouse organotypic entorhino-hippocampal tissue cultures of either sex, in which the entorhinal cortex (EC) to dentate granule cell (GC; EC-GC) projection is present, and EC-GC pairs can be studied using whole-cell patch-clamp recordings. By using cultures of wild-type mice, the properties of EC-GC synapses formed by afferents from the lateral and medial entorhinal cortex were compared, and differences in short-term plasticity were identified. As the perforant path is severely affected in Alzheimer's disease, we used tissue cultures of amyloid precursor protein (APP)-deficient mice to examine the role of APP at this synapse. APP deficiency altered excitatory neurotransmission at medial perforant path synapses, which was accompanied by transcriptomic and ultrastructural changes. Moreover, presynaptic but not postsynaptic APP deletion through the local injection of Cre-expressing adeno-associated viruses in conditional APPflox/flox tissue cultures increased the neurotransmission efficacy at perforant path synapses. In summary, these data suggest a physiological role for presynaptic APP at medial perforant path synapses that may be adversely affected under altered APP processing conditions.SIGNIFICANCE STATEMENT The hippocampus receives input from the entorhinal cortex via the perforant path. These projections to hippocampal dentate granule cells are of utmost importance for learning and memory formation. Although there is detailed knowledge about perforant path projections, the functional synaptic properties at the level of individual connected pairs of neurons are not well understood. In this study, we investigated the role of APP in mediating functional properties and transmission rules in individually connected neurons using paired whole-cell patch-clamp recordings and genetic tools in organotypic tissue cultures. Our results show that presynaptic APP expression limits excitatory neurotransmission via the perforant path, which could be compromised in pathologic conditions such as Alzheimer's disease.