RESUMEN
Accumulation of cytoplasmic inclusions containing fused in sarcoma (FUS), an RNA/DNA-binding protein, is a common hallmark of frontotemporal lobar degeneration and amyotrophic lateral sclerosis neuropathology. We have previously shown that DNA damage can trigger the cytoplasmic accumulation of N-terminally phosphorylated FUS. However, the functional consequences of N-terminal FUS phosphorylation are unknown. To gain insight into this question, we utilized proximity-dependent biotin labeling via ascorbate peroxidase 2 aired with mass spectrometry to investigate whether N-terminal phosphorylation alters the FUS protein-protein interaction network (interactome), and subsequently, FUS function. We report the first analysis comparing the interactomes of three FUS variants: homeostatic wildtype FUS (FUS WT), phosphomimetic FUS (FUS PM; a proxy for N-terminally phosphorylated FUS), and the toxic FUS proline 525 to leucine mutant (FUS P525L) that causes juvenile amyotrophic lateral sclerosis. We found that the phosphomimetic FUS interactome is uniquely enriched for a group of cytoplasmic proteins that mediate mRNA metabolism and translation, as well as nuclear proteins involved in the spliceosome and DNA repair functions. Furthermore, we identified and validated the RNA-induced silencing complex RNA helicase MOV10 as a novel interacting partner of FUS. Finally, we provide functional evidence that N-terminally phosphorylated FUS may disrupt homeostatic translation and steady-state levels of specific mRNA transcripts. Taken together, these results highlight phosphorylation as a unique modulator of the interactome and function of FUS.
Asunto(s)
Esclerosis Amiotrófica Lateral , Daño del ADN , Proteína FUS de Unión a ARN , Esclerosis Amiotrófica Lateral/metabolismo , Humanos , Mutación , Fosforilación , ARN Helicasas/metabolismo , ARN Mensajero/metabolismo , Proteína FUS de Unión a ARN/genética , Proteína FUS de Unión a ARN/metabolismo , Proteínas de Unión al ARN/metabolismoRESUMEN
Frontotemporal dementia (FTD) and amyotrophic lateral sclerosis (ALS) are fatal neurodegenerative disorders that are thought to exist on a clinical and pathological spectrum. FTD and ALS are linked by shared genetic causes (e.g. C9orf72 hexanucleotide repeat expansions) and neuropathology, such as inclusions of ubiquitinated, misfolded proteins (e.g. TAR DNA-binding protein 43; TDP-43) in the CNS. Furthermore, some genes that cause FTD or ALS when mutated encode proteins that localize to the lysosome or modulate endosome-lysosome function, including lysosomal fusion, cargo trafficking, lysosomal acidification, autophagy, or TFEB activity. In this review, we summarize evidence that lysosomal dysfunction, caused by genetic mutations (e.g. C9orf72, GRN, MAPT, TMEM106B) or toxic-gain of function (e.g. aggregation of TDP-43 or tau), is an important pathogenic disease mechanism in FTD and ALS. Further studies into the normal function of many of these proteins are required and will help uncover the mechanisms that cause lysosomal dysfunction in FTD and ALS. Mutations or polymorphisms in genes that encode proteins important for endosome-lysosome function also occur in other age-dependent neurodegenerative diseases, including Alzheimer's (e.g. APOE, PSEN1, APP) and Parkinson's (e.g. GBA, LRRK2, ATP13A2) disease. A more complete understanding of the common and unique features of lysosome dysfunction across the spectrum of neurodegeneration will help guide the development of therapies for these devastating diseases.
Asunto(s)
Esclerosis Amiotrófica Lateral/metabolismo , Esclerosis Amiotrófica Lateral/patología , Demencia Frontotemporal/metabolismo , Demencia Frontotemporal/patología , Lisosomas/metabolismo , Lisosomas/patología , Esclerosis Amiotrófica Lateral/genética , Animales , Autofagia/fisiología , Demencia Frontotemporal/genética , Humanos , Lisosomas/genética , Proteínas del Tejido Nervioso/genética , Proteínas del Tejido Nervioso/metabolismo , Enfermedades Neurodegenerativas/genética , Enfermedades Neurodegenerativas/metabolismo , Enfermedades Neurodegenerativas/patologíaRESUMEN
It has been more than a decade since heterozygous loss-of-function mutations in the progranulin gene (GRN) were first identified as an important genetic cause of frontotemporal lobar degeneration (FTLD). Due to the highly diverse biological functions of the progranulin (PGRN) protein, encoded by GRN, multiple possible disease mechanisms have been proposed. Early work focused on the neurotrophic properties of PGRN and its role in the inflammatory response. However, since the discovery of homozygous GRN mutations in patients with a lysosomal storage disorder, investigation into the possible roles of PGRN and its proteolytic cleavage products granulins, in lysosomal function and dysfunction, has taken center stage. In this chapter, we summarize the GRN mutational spectrum and its associated phenotypes followed by an in-depth discussion on the possible disease mechanisms implicated in FTLD-GRN. We conclude with key outstanding questions which urgently require answers to ensure safe and successful therapy development for GRN mutation carriers.
Asunto(s)
Degeneración Lobar Frontotemporal , Péptidos y Proteínas de Señalización Intercelular , Biología , Degeneración Lobar Frontotemporal/genética , Humanos , Péptidos y Proteínas de Señalización Intercelular/genética , Lisosomas/genética , Mutación , Progranulinas/genéticaRESUMEN
Fused in sarcoma (FUS) is a RNA/DNA protein involved in multiple nuclear and cytoplasmic functions including transcription, splicing, mRNA trafficking, and stress granule formation. To accomplish these many functions, FUS must shuttle between cellular compartments in a highly regulated manner. When shuttling is disrupted, FUS abnormally accumulates into cytoplasmic inclusions that can be toxic. Disrupted shuttling of FUS into the nucleus is a hallmark of ~10% of frontotemporal lobar degeneration (FTLD) cases, the neuropathology that underlies frontotemporal dementia (FTD). Multiple pathways are known to disrupt nuclear/cytoplasmic shuttling of FUS. In earlier work, we discovered that double-strand DNA breaks (DSBs) trigger DNA-dependent protein kinase (DNA-PK) to phosphorylate FUS (p-FUS) at N-terminal residues leading to the cytoplasmic accumulation of FUS. Therefore, DNA damage may contribute to the development of FTLD pathology with FUS inclusions. In the present study, we examined how DSBs effect FUS phosphorylation in various primate and mouse cellular models. All cell lines derived from human and non-human primates exhibit N-terminal FUS phosphorylation following calicheamicin γ1 (CLM) induced DSBs. In contrast, we were unable to detect FUS phosphorylation in mouse-derived primary neurons or immortalized cell lines regardless of CLM treatment, duration, or concentration. Despite DNA damage induced by CLM treatment, we find that mouse cells do not phosphorylate FUS, likely due to reduced levels and activity of DNA-PK compared to human cells. Taken together, our work reveals that mouse-derived cellular models regulate FUS in an anomalous manner compared to primate cells. This raises the possibility that mouse models may not fully recapitulate the pathogenic cascades that lead to FTLD with FUS pathology.
Asunto(s)
Encéfalo/metabolismo , Daño del ADN/fisiología , ADN/metabolismo , Degeneración Lobar Frontotemporal/metabolismo , Proteína FUS de Unión a ARN/genética , Animales , Degeneración Lobar Frontotemporal/genética , Humanos , Cuerpos de Inclusión/genética , Cuerpos de Inclusión/metabolismo , Ratones , Mutación/genética , Neuronas/metabolismo , Fosforilación , Factores Asociados con la Proteína de Unión a TATA/genéticaRESUMEN
Abnormal cytoplasmic accumulation of Fused in Sarcoma (FUS) in neurons defines subtypes of amyotrophic lateral sclerosis (ALS) and frontotemporal lobar degeneration (FTLD). FUS is a member of the FET protein family that includes Ewing's sarcoma (EWS) and TATA-binding protein-associated factor 2N (TAF15). FET proteins are predominantly localized to the nucleus, where they bind RNA and DNA to modulate transcription, mRNA splicing, and DNA repair. In ALS cases with FUS inclusions (ALS-FUS), mutations in the FUS gene cause disease, whereas FTLD cases with FUS inclusions (FTLD-FUS) do not harbor FUS mutations. Notably, in FTLD-FUS, all FET proteins accumulate with their nuclear import receptor Transportin 1 (TRN1), in contrast ALS-FUS inclusions are exclusively positive for FUS. In the present study, we show that induction of DNA damage replicates several pathologic hallmarks of FTLD-FUS in immortalized human cells and primary human neurons and astrocytes. Treatment with the antibiotic calicheamicin γ1, which causes DNA double-strand breaks, leads to the cytoplasmic accumulation of FUS, TAF15, EWS, and TRN1. Moreover, cytoplasmic translocation of FUS is mediated by phosphorylation of its N terminus by the DNA-dependent protein kinase. Finally, we observed elevated levels of phospho-H2AX in FTLD-FUS brains, indicating that DNA damage occurs in patients. Together, our data reveal a novel regulatory mechanism for FUS localization in cells and suggest that DNA damage may contribute to the accumulation of FET proteins observed in human FTLD-FUS cases, but not in ALS-FUS.
Asunto(s)
Citoplasma/metabolismo , Daño del ADN/fisiología , Proteína Quinasa Activada por ADN/metabolismo , Degeneración Lobar Frontotemporal/patología , Proteína FUS de Unión a ARN/metabolismo , Aminoglicósidos/farmacología , Antibióticos Antineoplásicos/farmacología , Astrocitos/efectos de los fármacos , Astrocitos/metabolismo , Células Cultivadas , Citoplasma/efectos de los fármacos , Daño del ADN/efectos de los fármacos , Enediinos/farmacología , Degeneración Lobar Frontotemporal/metabolismo , Humanos , Inmunoprecipitación , Mutágenos/farmacología , Mutación/genética , Neuronas , Proteínas Nucleares/metabolismo , Fosforilación/efectos de los fármacos , Proteína EWS de Unión a ARN/metabolismo , Receptores Citoplasmáticos y Nucleares/metabolismo , Factores Asociados con la Proteína de Unión a TATA/metabolismoRESUMEN
Progranulin (PGRN) is a secreted glycoprotein expressed in neurons and glia that is implicated in neuronal survival on the basis that mutations in the GRN gene causing haploinsufficiency result in a familial form of frontotemporal dementia (FTD). Recently, a direct interaction between PGRN and tumor necrosis factor receptors (TNFR I/II) was reported and proposed to be a mechanism by which PGRN exerts anti-inflammatory activity, raising the possibility that aberrant PGRN-TNFR interactions underlie the molecular basis for neuroinflammation in frontotemporal lobar degeneration pathogenesis. Here, we report that we find no evidence for a direct physical or functional interaction between PGRN and TNFRs. Using coimmunoprecipitation and surface plasmon resonance (SPR) we replicated the interaction between PGRN and sortilin and that between TNF and TNFRI/II, but not the interaction between PGRN and TNFRs. Recombinant PGRN or transfection of a cDNA encoding PGRN did not antagonize TNF-dependent NFκB, Akt, and Erk1/2 pathway activation; inflammatory gene expression; or secretion of inflammatory factors in BV2 microglia and bone marrow-derived macrophages (BMDMs). Moreover, PGRN did not antagonize TNF-induced cytotoxicity on dopaminergic neuroblastoma cells. Last, co-addition or pre-incubation with various N- or C-terminal-tagged recombinant PGRNs did not alter lipopolysaccharide-induced inflammatory gene expression or cytokine secretion in any cell type examined, including BMDMs from Grn+/- or Grn-/- mice. Therefore, the neuroinflammatory phenotype associated with PGRN deficiency in the CNS is not a direct consequence of the loss of TNF antagonism by PGRN, but may be a secondary response by glia to disrupted interactions between PGRN and Sortilin and/or other binding partners yet to be identified.
Asunto(s)
Citocinas/metabolismo , Regulación de la Expresión Génica/inmunología , Péptidos y Proteínas de Señalización Intercelular/metabolismo , Receptores del Factor de Necrosis Tumoral/metabolismo , Transducción de Señal/fisiología , Proteínas Adaptadoras del Transporte Vesicular/metabolismo , Análisis de Varianza , Animales , Línea Celular , Proliferación Celular/efectos de los fármacos , Medios de Cultivo Condicionados/farmacología , Regulación de la Expresión Génica/efectos de los fármacos , Regulación de la Expresión Génica/genética , Granulinas , Humanos , Inmunoprecipitación , Péptidos y Proteínas de Señalización Intercelular/deficiencia , Péptidos y Proteínas de Señalización Intercelular/genética , Isoquinolinas/metabolismo , Lipopolisacáridos/farmacología , Sistema de Señalización de MAP Quinasas/efectos de los fármacos , Sistema de Señalización de MAP Quinasas/inmunología , Macrófagos/efectos de los fármacos , Macrófagos/inmunología , Masculino , Ratones , Ratones Endogámicos C57BL , Ratones Noqueados , Microglía/efectos de los fármacos , Microglía/metabolismo , FN-kappa B/metabolismo , Progranulinas , Unión Proteica/genética , Receptores del Factor de Necrosis Tumoral/genética , Proteínas Recombinantes/farmacología , Transducción de Señal/efectos de los fármacos , Resonancia por Plasmón de Superficie , TransfecciónRESUMEN
γ-Secretase catalyzes the final cleavage of the amyloid precursor protein (APP), resulting in the production of amyloid-ß (Aß) peptides with different carboxyl termini. Presenilin (PSEN) and amyloid precursor protein (APP) mutations linked to early onset familial Alzheimer's disease modify the profile of Aß isoforms generated, by altering both the initial γ-secretase cleavage site and subsequent processivity in a manner that leads to increased levels of the more amyloidogenic Aß42 and in some circumstances Aß43. Compounds termed γ-secretase modulators (GSMs) and inverse GSMs (iGSMs) can decrease and increase levels of Aß42, respectively. As GSMs lower the level of production of pathogenic forms of long Aß isoforms, they are of great interest as potential Alzheimer's disease therapeutics. The factors that regulate GSM modulation are not fully understood; however, there is a growing body of evidence that supports the hypothesis that GSM activity is influenced by the amino acid sequence of the γ-secretase substrate. We have evaluated whether mutations near the luminal border of the transmembrane domain (TMD) of APP alter the ability of both acidic, nonsteroidal anti-inflammatory drug-derived carboxylate and nonacidic, phenylimidazole-derived classes of GSMs and iGSMs to modulate γ-secretase cleavage. Our data show that point mutations can dramatically reduce the sensitivity to modulation of cleavage by GSMs but have weaker effects on iGSM activity. These studies support the concept that the effect of GSMs may be substrate selective; for APP, it is dependent on the amino acid sequence of the substrate near the junction of the extracellular domain and luminal segment of the TMD.
Asunto(s)
Secretasas de la Proteína Precursora del Amiloide/química , Secretasas de la Proteína Precursora del Amiloide/metabolismo , Procesamiento Proteico-Postraduccional/genética , Secuencia de Aminoácidos , Secretasas de la Proteína Precursora del Amiloide/genética , Péptidos beta-Amiloides/química , Animales , Células CHO , Cricetinae , Cricetulus , Regulación hacia Abajo/genética , Datos de Secuencia Molecular , Mutagénesis Sitio-Dirigida , Mutación Puntual/genética , Especificidad por Sustrato/genética , Regulación hacia Arriba/genéticaRESUMEN
Aggregation and accumulation of Aß42 play an initiating role in Alzheimer's disease (AD); thus, selective lowering of Aß42 by γ-secretase modulators (GSMs) remains a promising approach to AD therapy. Based on evidence suggesting that steroids may influence Aß production, we screened 170 steroids at 10 µM for effects on Aß42 secreted from human APP-overexpressing Chinese hamster ovary cells. Many acidic steroids lowered Aß42, whereas many nonacidic steroids actually raised Aß42. Studies on the more potent compounds showed that Aß42-lowering steroids were bonafide GSMs and Aß42-raising steroids were inverse GSMs. The most potent steroid GSM identified was 5ß-cholanic acid (EC50=5.7 µM; its endogenous analog lithocholic acid was virtually equipotent), and the most potent inverse GSM identified was 4-androsten-3-one-17ß-carboxylic acid ethyl ester (EC50=6.25 µM). In addition, we found that both estrogen and progesterone are weak inverse GSMs with further complex effects on APP processing. These data suggest that certain endogenous steroids may have the potential to act as GSMs and add to the evidence that cholesterol, cholesterol metabolites, and other steroids may play a role in modulating Aß production and thus risk for AD. They also indicate that acidic steroids might serve as potential therapeutic leads for drug optimization/development.
Asunto(s)
Secretasas de la Proteína Precursora del Amiloide/efectos de los fármacos , Secretasas de la Proteína Precursora del Amiloide/metabolismo , Precursor de Proteína beta-Amiloide/metabolismo , Esteroides/química , Esteroides/farmacología , Animales , Células CHO , Línea Celular , Colesterol/farmacología , Cricetinae , Cricetulus , Ensayo de Inmunoadsorción Enzimática , Estrógenos/farmacología , Humanos , Espectrometría de Masas , Progesterona/farmacologíaRESUMEN
Selective lowering of Abeta42 levels (the 42-residue isoform of the amyloid-beta peptide) with small-molecule gamma-secretase modulators (GSMs), such as some non-steroidal anti-inflammatory drugs, is a promising therapeutic approach for Alzheimer's disease. To identify the target of these agents we developed biotinylated photoactivatable GSMs. GSM photoprobes did not label the core proteins of the gamma-secretase complex, but instead labelled the beta-amyloid precursor protein (APP), APP carboxy-terminal fragments and amyloid-beta peptide in human neuroglioma H4 cells. Substrate labelling was competed by other GSMs, and labelling of an APP gamma-secretase substrate was more efficient than a Notch substrate. GSM interaction was localized to residues 28-36 of amyloid-beta, a region critical for aggregation. We also demonstrate that compounds known to interact with this region of amyloid-beta act as GSMs, and some GSMs alter the production of cell-derived amyloid-beta oligomers. Furthermore, mutation of the GSM binding site in the APP alters the sensitivity of the substrate to GSMs. These findings indicate that substrate targeting by GSMs mechanistically links two therapeutic actions: alteration in Abeta42 production and inhibition of amyloid-beta aggregation, which may synergistically reduce amyloid-beta deposition in Alzheimer's disease. These data also demonstrate the existence and feasibility of 'substrate targeting' by small-molecule effectors of proteolytic enzymes, which if generally applicable may significantly broaden the current notion of 'druggable' targets.
Asunto(s)
Secretasas de la Proteína Precursora del Amiloide/antagonistas & inhibidores , Secretasas de la Proteína Precursora del Amiloide/metabolismo , Precursor de Proteína beta-Amiloide/química , Precursor de Proteína beta-Amiloide/metabolismo , Antiinflamatorios no Esteroideos/metabolismo , Antiinflamatorios no Esteroideos/farmacología , Enfermedad de Alzheimer/tratamiento farmacológico , Enfermedad de Alzheimer/enzimología , Enfermedad de Alzheimer/metabolismo , Precursor de Proteína beta-Amiloide/antagonistas & inhibidores , Precursor de Proteína beta-Amiloide/genética , Animales , Antiinflamatorios no Esteroideos/química , Sitios de Unión/efectos de los fármacos , Células CHO , Línea Celular Tumoral , Cricetinae , Cricetulus , Femenino , Humanos , Ratones , Unión Proteica/efectos de los fármacos , Receptores Notch/genética , Receptores Notch/metabolismo , Especificidad por Sustrato/efectos de los fármacosRESUMEN
TFEB (transcription factor EB) regulates multiple genes involved in the process of macroautophagy/autophagy and plays a critical role in lifespan determination. However, the detailed mechanisms that regulate TFEB activity are not fully clear. In this study, we identified a role for HSP90AA1 in modulating TFEB. HSP90AA1 was phosphorylated by CDK5 at Ser 595 under basal condition. This phosphorylation inhibited HSP90AA1, disrupted its binding to TFEB, and impeded TFEB's nuclear localization and subsequent autophagy induction. Pro-autophagy signaling attenuated CDK5 activity and enhanced TFEB function in an HSP90AA1-dependent manner. Inhibition of HSP90AA1 function or decrease in its expression significantly attenuated TFEB's nuclear localization and transcriptional function following autophagy induction. HSP90AA1-mediated regulation of a TFEB ortholog was involved in the extended lifespan of Caenorhabditis elegans in the absence of its food source bacteria. Collectively, these findings reveal that this regulatory process plays an important role in modulation of TFEB, autophagy, and longevity.Abbreviations : AL: autolysosome; AP: autophagosome; ATG: autophagy related; BafA1: bafilomycin A1; CDK5: cyclin-dependent kinase 5; CDK5R1: cyclin dependent kinase 5 regulatory subunit 1; CR: calorie restriction; FUDR: 5-fluorodeoxyuridine; HSP90AA1: heat shock protein 90 alpha family class A member 1; MAP1LC3: microtubule associated protein 1 light chain 3; NB: novobiocin sodium; SQSTM1: sequestosome 1; TFEB: transcription factor EB; WT: wild type.
Asunto(s)
Autofagia , Longevidad , Animales , Autofagia/genética , Núcleo Celular/metabolismo , Factores de Transcripción Básicos con Cremalleras de Leucinas y Motivos Hélice-Asa-Hélice/metabolismo , Autofagosomas/metabolismo , Transducción de Señal/genética , Chaperonas Moleculares/metabolismo , Caenorhabditis elegans/metabolismo , Lisosomas/metabolismoRESUMEN
Progranulin (PGRN) deficiency is linked to neurodegenerative diseases including frontotemporal dementia, Alzheimer's disease, Parkinson's disease, and neuronal ceroid lipofuscinosis. Proper PGRN levels are critical to maintain brain health and neuronal survival, however the function of PGRN is not well understood. PGRN is composed of 7.5 tandem repeat domains, called granulins, and is proteolytically processed into individual granulins inside the lysosome. The neuroprotective effects of full-length PGRN are well-documented, but the role of granulins is still unclear. Here we report, for the first time, that expression of single granulins is sufficient to rescue the full spectrum of disease pathology in mice with complete PGRN deficiency (Grn-/-). Specifically, rAAV delivery of either human granulin-2 or granulin-4 to Grn-/- mouse brain ameliorates lysosome dysfunction, lipid dysregulation, microgliosis, and lipofuscinosis similar to full-length PGRN. These findings support the idea that individual granulins are the functional units of PGRN, likely mediate neuroprotection within the lysosome, and highlight their importance for developing therapeutics to treat FTD-GRN and other neurodegenerative diseases.
RESUMEN
A subset of non-steroidal anti-inflammatory drugs modulates the γ cleavage site in the amyloid precursor protein (APP) to selectively reduce production of Aß42. It is unclear precisely how these γ-secretase modulators (GSMs) act to preferentially spare Aß40 production as well as Notch processing and signaling. In an effort to determine the substrate requirements in NSAID/GSM activity, we determined the effects of sulindac sulfide and flurbiprofen on γ-cleavage of artificial constructs containing several γ-secretase substrates. Using FLAG-tagged constructs that expressed extracellularly truncated APP, Notch-1, or CD44, we found that these substrates have different sensitivities to sulindac sulfide. γ-Secretase cleavage of APP was altered by sulindac sulfide, but CD44 and Notch-1 were either insensitive or only minimally altered by this compound. Using chimeric APP constructs, we observed that the transmembrane domain (TMD) of APP played a pivotal role in determining drug sensitivity. Substituting the APP TMD with that of APLP2 retained the sensitivity to γ-cleavage modulation, but replacing TMDs from Notch-1 or ErbB4 rendered the resultant molecules insensitive to drug treatment. Specifically, the GXXXG motif within APP appeared to be critical to GSM activity. Consequently, the modulatory effects on γ-cleavage appears to be substrate-dependent. We hypothesize that the substrate present in the γ-secretase complex influences the conformation of the complex so that the binding site of GSMs is either stabilized or less favorable to influence the cleavage of the respective substrates.
Asunto(s)
Secretasas de la Proteína Precursora del Amiloide/metabolismo , Precursor de Proteína beta-Amiloide/metabolismo , Receptores de Hialuranos/metabolismo , Receptor Notch1/metabolismo , Secuencias de Aminoácidos , Secretasas de la Proteína Precursora del Amiloide/genética , Precursor de Proteína beta-Amiloide/genética , Antiinflamatorios no Esteroideos/farmacología , Células HEK293 , Humanos , Receptores de Hialuranos/genética , Estructura Terciaria de Proteína , Receptor Notch1/genética , Proteínas Recombinantes de Fusión/genética , Proteínas Recombinantes de Fusión/metabolismo , Sulindac/análogos & derivados , Sulindac/farmacologíaRESUMEN
γ-Secretase is a multiprotein intramembrane cleaving aspartyl protease (I-CLiP) that catalyzes the final cleavage of the amyloid ß precursor protein (APP) to release the amyloid ß peptide (Aß). Aß is the primary component of senile plaques in Alzheimer's disease (AD), and its mechanism of production has been studied intensely. γ-Secretase executes multiple cleavages within the transmembrane domain of APP, with cleavages producing Aß and the APP intracellular domain (AICD), referred to as γ and ε, respectively. The heterogeneous nature of the γ cleavage that produces various Aß peptides is highly relevant to AD, as increased production of Aß 1-42 is genetically and biochemically linked to the development of AD. We have identified an amino acid in the juxtamembrane region of APP, lysine 624, on the basis of APP695 numbering (position 28 relative to Aß) that plays a critical role in determining the final length of Aß peptides released by γ-secretase. Mutation of this lysine to alanine (K28A) shifts the primary site of γ-secretase cleavage from 1-40 to 1-33 without significant changes to ε cleavage. These results further support a model where ε cleavage occurs first, followed by sequential proteolysis of the remaining transmembrane fragment, but extend these observations by demonstrating that charged residues at the luminal boundary of the APP transmembrane domain limit processivity of γ-secretase.
Asunto(s)
Secretasas de la Proteína Precursora del Amiloide/metabolismo , Precursor de Proteína beta-Amiloide/metabolismo , Membrana Celular/metabolismo , Lisina/metabolismo , Proteolisis , Secretasas de la Proteína Precursora del Amiloide/genética , Precursor de Proteína beta-Amiloide/genética , Línea Celular Tumoral , Membrana Celular/genética , Células HEK293 , Humanos , Lisina/genética , Estructura Terciaria de ProteínaRESUMEN
Increased Abeta42 production has been linked to the development of Alzheimer disease. We now identify a number of compounds that raise Abeta42. Among the more potent Abeta42-raising agents identified are fenofibrate, an antilipidemic agent, and celecoxib, a COX-2-selective NSAID. Many COX-2-selective NSAIDs tested raised Abeta42, including multiple COX-2-selective derivatives of two Abeta42-lowering NSAIDs. Compounds devoid of COX activity and the endogenous isoprenoids FPP and GGPP also raised Abeta42. These compounds seem to target the gamma-secretase complex, increasing gamma-secretase-catalyzed production of Abeta42 in vitro. Short-term in vivo studies show that two Abeta42-raising compounds increase Abeta42 levels in the brains of mice. The elevations in Abeta42 by these compounds are comparable to the increases in Abeta42 induced by Alzheimer disease-causing mutations in the genes encoding amyloid beta protein precursor and presenilins, raising the possibility that exogenous compounds or naturally occurring isoprenoids might increase Abeta42 production in humans.
Asunto(s)
Enfermedad de Alzheimer/metabolismo , Péptidos beta-Amiloides/biosíntesis , Encéfalo/metabolismo , Endopeptidasas/metabolismo , Secretasas de la Proteína Precursora del Amiloide , Antiinflamatorios no Esteroideos/química , Antiinflamatorios no Esteroideos/farmacología , Ácido Aspártico Endopeptidasas , Celecoxib , Línea Celular , Inhibidores de la Ciclooxigenasa/química , Inhibidores de la Ciclooxigenasa/farmacología , Activación Enzimática/efectos de los fármacos , Ensayo de Inmunoadsorción Enzimática , Femenino , Fenofibrato/química , Fenofibrato/farmacología , Humanos , Hipolipemiantes/química , Hipolipemiantes/farmacología , Inmunoprecipitación , Péptidos y Proteínas de Señalización Intracelular , Espectrometría de Masas , Proteínas Serina-Treonina Quinasas/metabolismo , Pirazoles/química , Pirazoles/farmacología , Sulfonamidas/química , Sulfonamidas/farmacología , Transfección , Quinasas Asociadas a rho , Proteína de Unión al GTP rhoA/metabolismoRESUMEN
Heterozygous, loss-of-function mutations in the granulin gene (GRN) encoding progranulin (PGRN) are a common cause of frontotemporal dementia (FTD). Homozygous GRN mutations cause neuronal ceroid lipofuscinosis-11 (CLN11), a lysosome storage disease. PGRN is a secreted glycoprotein that can be proteolytically cleaved into seven bioactive 6 kDa granulins. However, it is unclear how deficiency of PGRN and granulins causes neurodegeneration. To gain insight into the mechanisms of FTD pathogenesis, we utilized Tandem Mass Tag isobaric labeling mass spectrometry to perform an unbiased quantitative proteomic analysis of whole-brain tissue from wild type (Grn+/+) and Grn knockout (Grn-/-) mice at 3- and 19-months of age. At 3-months lysosomal proteins (i.e. Gns, Scarb2, Hexb) are selectively increased indicating lysosomal dysfunction is an early consequence of PGRN deficiency. Additionally, proteins involved in lipid metabolism (Acly, Apoc3, Asah1, Gpld1, Ppt1, and Naaa) are decreased; suggesting lysosomal degradation of lipids may be impaired in the Grn-/- brain. Systems biology using weighted correlation network analysis (WGCNA) of the Grn-/- brain proteome identified 26 modules of highly co-expressed proteins. Three modules strongly correlated to Grn deficiency and were enriched with lysosomal proteins (Gpnmb, CtsD, CtsZ, and Tpp1) and inflammatory proteins (Lgals3, GFAP, CD44, S100a, and C1qa). We find that lysosomal dysregulation is exacerbated with age in the Grn-/- mouse brain leading to neuroinflammation, synaptic loss, and decreased markers of oligodendrocytes, myelin, and neurons. In particular, GPNMB and LGALS3 (galectin-3) were upregulated by microglia and elevated in FTD-GRN brain samples, indicating common pathogenic pathways are dysregulated in human FTD cases and Grn-/- mice. GPNMB levels were significantly increased in the cerebrospinal fluid of FTD-GRN patients, but not in MAPT or C9orf72 carriers, suggesting GPNMB could be a biomarker specific to FTD-GRN to monitor disease onset, progression, and drug response. Our findings support the idea that insufficiency of PGRN and granulins in humans causes neurodegeneration through lysosomal dysfunction, defects in autophagy, and neuroinflammation, which could be targeted to develop effective therapies.
Asunto(s)
Demencia Frontotemporal/genética , Demencia Frontotemporal/metabolismo , Progranulinas/genética , Anciano , Animales , Autofagia/fisiología , Encéfalo/metabolismo , Encéfalo/patología , Femenino , Demencia Frontotemporal/patología , Humanos , Inflamación/metabolismo , Inflamación/patología , Lisosomas/metabolismo , Lisosomas/patología , Masculino , Ratones , Ratones Noqueados , Persona de Mediana Edad , Mutación , Proteoma , Tripeptidil Peptidasa 1RESUMEN
Alpha-synuclein (αSynAgg) are pathological hallmarks of Parkinson's disease (PD) and other synucleinopathies that induce microglial activation and immune-mediated neurotoxicity, but the molecular mechanisms of αSynAgg-induced immune activation are poorly defined. We performed quantitative proteomics by mass spectrometry coupled with PCR, immunohistochemical and functional validations studies to define the molecular characteristics of alpha synuclein mediated microglial activation. In mouse microglia, αSynAgg induced robust pro-inflammatory activation (increased expression of 864 genes including Irg1, Ifit1, and Pyhin) and increased nuclear proteins involved in RNA synthesis, splicing, and anti-viral defense mechanisms. Conversely, αSynAgg decreased expression several proteins (including Cdc123, Sod1, and Grn), which were predominantly cytosolic and involved in metabolic, proteasomal and lysosomal mechanisms. Pathway analyses and confirmatory in vitro studies suggested that αSynAgg partly mediates its effects via Stat3 activation. As predicted by our proteomic findings, we verified that αSynAgg induces mitochondrial dysfunction in microglia. Twenty-six proteins differentially expressed by αSynAgg were also identified as PD risk genes in genome-wide association studies (upregulated: Brd2, Clk1, Siglec1; down-regulated: Memo1, Arhgap18, Fyn, and Pgrn/Grn). We validated progranulin (PGRN) as a lysosomal PD-associated protein that is downregulated by αSynAgg in microglia in-vivo and is expressed by microglia in post-mortem PD brain, congruent with our in vitro findings. Conclusion: Together, proteomics approach both reveals novel molecular insights into αSyn-mediated neuroinflammation in PD and other synucleinopathies.
Asunto(s)
Microglía/efectos de los fármacos , Microglía/metabolismo , Progranulinas/metabolismo , Agregado de Proteínas , Proteoma , alfa-Sinucleína/farmacología , Animales , Encéfalo/metabolismo , Línea Celular , Regulación hacia Abajo/efectos de los fármacos , Estudio de Asociación del Genoma Completo , Humanos , Inflamación/inducido químicamente , Inflamación/metabolismo , Lisosomas/metabolismo , Ratones , Ratones Endogámicos C57BL , Enfermedad de Parkinson/metabolismo , Enfermedad de Parkinson/patología , Progranulinas/inmunología , Proteómica/métodos , Proteínas Recombinantes/farmacologíaRESUMEN
GGGGCC hexanucleotide repeat expansions (HREs) in C9orf72 cause amyotrophic lateral sclerosis (ALS) and frontotemporal dementia (FTD) and lead to the production of aggregating dipeptide repeat proteins (DPRs) via repeat associated non-AUG (RAN) translation. Here, we show the similar intronic GGCCTG HREs that causes spinocerebellar ataxia type 36 (SCA36) is also translated into DPRs, including poly(GP) and poly(PR). We demonstrate that poly(GP) is more abundant in SCA36 compared to c9ALS/FTD patient tissue due to canonical AUG-mediated translation from intron-retained GGCCTG repeat RNAs. However, the frequency of the antisense RAN translation product poly(PR) is comparable between c9ALS/FTD and SCA36 patient samples. Interestingly, in SCA36 patient tissue, poly(GP) exists as a soluble species, and no TDP-43 pathology is present. We show that aggregate-prone chimeric DPR (cDPR) species underlie the divergent DPR pathology between c9ALS/FTD and SCA36. These findings reveal key differences in translation, solubility, and protein aggregation of DPRs between c9ALS/FTD and SCA36.
Asunto(s)
Esclerosis Amiotrófica Lateral/genética , Proteína C9orf72/genética , Dipéptidos/genética , Demencia Frontotemporal/genética , Proteínas Mutantes Quiméricas/genética , Ataxias Espinocerebelosas/genética , Secuencia de Aminoácidos , Animales , Animales Recién Nacidos , Elementos sin Sentido (Genética)/genética , Expansión de las Repeticiones de ADN , Femenino , Humanos , Intrones/genética , Ratones , Ratones Endogámicos C57BL , Embarazo , Secuencias Repetitivas de Ácidos NucleicosRESUMEN
Inhibition of gamma-secretase cleavage of the amyloid precursor protein (APP) is a prime target for the development of therapeutics for treating Alzheimer's disease; however, complete inhibition of this activity would also impair the processing of many other proteins, including the APP homologues, amyloid precursor-like protein (APLP) 1 and 2. To prevent unwanted side effects, therapeutically useful gamma-secretase inhibitors should specifically target APP processing while sparing cleavage of other gamma-substrates. Thus, since APLP1 and APLP2 are more similar to APP than any of the other known gamma-secretase substrates and have important physiological roles in their own right, we reasoned that comparison of the effect of gamma-secretase inhibitors on APLP processing should provide a sensitive indicator of the selectivity of putative inhibitors. To address this issue, we have optimized microsome and cell culture assays to monitor the gamma-secretase proteolysis of APP and APLPs. Production of the gamma-secretase-generated intracellular domain (ICD) occurs more rapidly from APLP1 than from either APLP2 or APP, suggesting that APLP1 is a better gamma-substrate and that substrate recognition is not restricted to the highly conserved amino acid sequences surrounding the epsilon-site. As expected, the well-characterized gamma-secretase modulator, fenofibrate, did not inhibit ICD release, whereas a related compound, FT-9, inhibited gamma-secretase both in microsomes and in whole cells. Importantly, FT-9 displayed a preferential effect, inhibiting cleavage of APP much more effectively than cleavage of APLP1. These findings suggest that selective inhibitors can be developed and that screening of compounds against APP and APLPs should assist in this process.
Asunto(s)
Secretasas de la Proteína Precursora del Amiloide/antagonistas & inhibidores , Precursor de Proteína beta-Amiloide/metabolismo , Antiinflamatorios no Esteroideos/farmacología , Fenofibrato/análogos & derivados , Inhibidores de Proteasas/farmacología , Secretasas de la Proteína Precursora del Amiloide/metabolismo , Péptidos beta-Amiloides/metabolismo , Animales , Benzodiazepinonas/farmacología , Biocatálisis/efectos de los fármacos , Células CHO , Cricetinae , Cricetulus , Dipéptidos/farmacología , Fenofibrato/farmacología , Flurbiprofeno/farmacología , Humanos , Cinética , Microsomas/efectos de los fármacos , Microsomas/enzimología , Microsomas/metabolismo , Proteínas del Tejido Nervioso/metabolismo , Fragmentos de Péptidos/metabolismo , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Especificidad por Sustrato/efectos de los fármacos , TransfecciónRESUMEN
Processing of amyloid-ß (Aß) precursor protein (APP) by γ-secretase produces multiple species of Aß: Aß40, short Aß peptides (Aß37-39), and longer Aß peptides (Aß42-43). γ-Secretase modulators, a class of Alzheimer's disease therapeutics, reduce production of the pathogenic Aß42 but increase the relative abundance of short Aß peptides. To evaluate the pathological relevance of these peptides, we expressed Aß36-40 and Aß42-43 in Drosophila melanogaster to evaluate inherent toxicity and potential modulatory effects on Aß42 toxicity. In contrast to Aß42, the short Aß peptides were not toxic and, when coexpressed with Aß42, were protective in a dose-dependent fashion. In parallel, we explored the effects of recombinant adeno-associated virus-mediated expression of Aß38 and Aß40 in mice. When expressed in nontransgenic mice at levels sufficient to drive Aß42 deposition, Aß38 and Aß40 did not deposit or cause behavioral alterations. These studies indicate that treatments that lower Aß42 by raising the levels of short Aß peptides could attenuate the toxic effects of Aß42.
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Péptidos beta-Amiloides/metabolismo , Péptidos beta-Amiloides/toxicidad , Fragmentos de Péptidos/metabolismo , Fragmentos de Péptidos/toxicidad , Amiloide/genética , Amiloide/metabolismo , Péptidos beta-Amiloides/química , Péptidos beta-Amiloides/genética , Precursor de Proteína beta-Amiloide/genética , Animales , Animales Modificados Genéticamente , Encéfalo/metabolismo , Encéfalo/patología , Drosophila melanogaster , Ojo/metabolismo , Ojo/patología , Ojo/ultraestructura , Femenino , Locomoción , Ratones , Ratones Transgénicos , Fragmentos de Péptidos/química , Fragmentos de Péptidos/genética , Fenotipo , Agregado de Proteínas , Agregación Patológica de Proteínas/metabolismoRESUMEN
The cytoplasmic mislocalization and aggregation of TAR DNA-binding protein-43 (TDP-43) is a common histopathological hallmark of the amyotrophic lateral sclerosis and frontotemporal dementia disease spectrum (ALS/FTD). However, the composition of aggregates and their contribution to the disease process remain unknown. Here we used proximity-dependent biotin identification (BioID) to interrogate the interactome of detergent-insoluble TDP-43 aggregates and found them enriched for components of the nuclear pore complex and nucleocytoplasmic transport machinery. Aggregated and disease-linked mutant TDP-43 triggered the sequestration and/or mislocalization of nucleoporins and transport factors, and interfered with nuclear protein import and RNA export in mouse primary cortical neurons, human fibroblasts and induced pluripotent stem cell-derived neurons. Nuclear pore pathology is present in brain tissue in cases of sporadic ALS and those involving genetic mutations in TARDBP and C9orf72. Our data strongly implicate TDP-43-mediated nucleocytoplasmic transport defects as a common disease mechanism in ALS/FTD.