RESUMEN
Alzheimer's disease (AD) is characterized pathologically by amyloid ß (Aß)-containing plaques. Generation of Aß from amyloid precursor protein (APP) by two enzymes, ß- and γ-secretase, has therefore been in the AD research spotlight for decades. Despite this, how the physical interaction of APP with the secretases influences APP processing is not fully understood. Herein, we compared two genetically identical human iPSC-derived neuronal cell types: low Aß-secreting neuroprogenitor cells (NPCs) and high Aß-secreting mature neurons, as models of low versus high Aß production. We investigated levels of substrate, enzymes and products of APP amyloidogenic processing and correlated them with the proximity of APP to ß- and γ-secretase in endo-lysosomal organelles. In mature neurons, increased colocalization of full-length APP with the ß-secretase BACE1 correlated with increased ß-cleavage product sAPPß. Increased flAPP/BACE1 colocalization was mainly found in early endosomes. In the same way, increased colocalization of APP-derived C-terminal fragment (CTF) with presenilin-1 (PSEN1), the catalytic subunit of γ-secretase, was seen in neurons as compared to NPCs. Furthermore, most of the interaction of APP with BACE1 in low Aß-secreting NPCs seemed to derive from CTF, the remaining APP part after BACE1 cleavage, indicating a possible novel product-enzyme inhibition. In conclusion, our results suggest that interaction of APP and APP cleavage products with their secretases can regulate Aß production both positively and negatively. ß- and γ-Secretases are difficult targets for AD treatment due to their ubiquitous nature and wide range of substrates. Therefore, targeting APP-secretase interactions could be a novel treatment strategy for AD. Colocalization of APP species with BACE1 in a novel model of low- versus high-Aß secretion-Two genetically identical human iPSC-derived neuronal cell types: low Aß-secreting neuroprogenitor cells (NPCs) and high Aß secreting mature neurons, were compared. Increased full-length APP (flAPP)/BACE1 colocalization in early endosomes was seen in neurons, while APP-CTF/BACE1 colocalization was much higher than flAPP/BACE1 colocalization in NPCs, although the cellular location was not determined.
Asunto(s)
Enfermedad de Alzheimer , Células Madre Pluripotentes Inducidas , Humanos , Precursor de Proteína beta-Amiloide , Péptidos beta-Amiloides , Secretasas de la Proteína Precursora del Amiloide , Ácido Aspártico Endopeptidasas , NeuronasRESUMEN
γ-Secretase inhibitors have been considered promising drug candidates against Alzheimer's disease (AD) due to their ability to reduce amyloid-ß (Aß) production. However, clinical trials have been halted due to lack of clinical efficacy and/or side effects. Recent in vitro studies suggest that low doses of γ-secretase inhibitors may instead increase Aß production. Using a stem cell-derived human model of cortical neurons and low doses of the γ-secretase inhibitor DAPT, the effects on a variety of Aß peptides were studied using mass spectrometry. One major focus was to develop a novel method for specific detection of oligomeric Aß (oAß), and this was used to study the effects of low-dose γ-secretase inhibitor treatment on intracellular oAß accumulation. Low-dose treatment (2 and 20nM) with DAPT increased the secretion of several Aß peptides, especially Aßx-42. Furthermore, using the novel method for oAß detection, we found that 2nM DAPT treatment of cortical neurons resulted in increased oAß accumulation. Thus, low dose-treatment with DAPT causes both increased production of long, aggregation-prone Aß peptides and accumulation of intracellular Aß oligomers, both believed to contribute to AD pathology.
Asunto(s)
Secretasas de la Proteína Precursora del Amiloide/antagonistas & inhibidores , Péptidos beta-Amiloides/metabolismo , Diaminas/farmacología , Neuronas/metabolismo , Tiazoles/farmacología , Línea Celular , Humanos , Neuronas/efectos de los fármacosRESUMEN
Alzheimer's disease (AD) is characterized by accumulation of two misfolded and aggregated proteins, ß-amyloid and hyperphosphorylated tau. Both cellular systems responsible for clearance of misfolded and aggregated proteins, the lysosomal and the proteasomal, have been shown to be malfunctioning in the aged brain and more so in patients with neurodegenerative diseases, including AD. This malfunction could be contributing to ß-amyloid and tau accumulation, eventually aggregating in plaques and tangles. We have investigated the impact of decreased proteasome activity on tau phosphorylation as well as on microtubule stability and transport. To do this, we used our recently developed neuronal model where human SH-SY5Y cells obtain neuronal morphology and function through differentiation. We found that exposure to low doses of the proteasome inhibitor MG-115 caused tau phosphorylation, microtubule destabilization and disturbed neuritic transport. Furthermore, reduced proteasome activity activated several proteins implicated in tau phosphorylation and AD pathology, including c-Jun N-terminal kinase, c-Jun and extracellular signal-regulated protein kinase (ERK) 1/2. Restoration of the microtubule transport was achieved by inhibiting ERK 1/2 activation, and simultaneous inhibition of both ERK 1/2 and c-Jun reversed the proteasome inhibition-induced tau phosphorylation. Taken together, this study suggests that a decrease in proteasome activity can, through activation of c-Jun and ERK 1/2, result in several events related to neurodegenerative diseases. Restoration of proteasome activity or modulation of ERK 1/2 and c-Jun function can open new treatment possibilities against neurodegenerative diseases such as AD.
Asunto(s)
Transporte Axonal/efectos de los fármacos , Leupeptinas/farmacología , MAP Quinasa Quinasa 4/metabolismo , Inhibidores de Proteasoma/farmacología , Enfermedad de Alzheimer/metabolismo , Línea Celular Tumoral , Humanos , Microtúbulos/efectos de los fármacos , Microtúbulos/metabolismo , Proteína Quinasa 1 Activada por Mitógenos/metabolismo , Proteína Quinasa 3 Activada por Mitógenos/metabolismo , Neuritas/efectos de los fármacos , Neuritas/metabolismo , Fosforilación , Proteínas tau/metabolismoRESUMEN
The spreading of pathology through neuronal pathways is likely to be the cause of the progressive cognitive loss observed in Alzheimer's disease (AD) and other neurodegenerative diseases. We have recently shown the propagation of AD pathology via cell-to-cell transfer of oligomeric amyloid beta (Aß) residues 1-42 (oAß1-42) using our donor-acceptor 3-D co-culture model. We now show that different Aß-isoforms (fluorescently labeled 1-42, 3(pE)-40, 1-40 and 11-42 oligomers) can transfer from one cell to another. Thus, transfer is not restricted to a specific Aß-isoform. Although different Aß isoforms can transfer, differences in the capacity to clear and/or degrade these aggregated isoforms result in vast differences in the net amounts ending up in the receiving cells and the net remaining Aß can cause seeding and pathology in the receiving cells. This insufficient clearance and/or degradation by cells creates sizable intracellular accumulations of the aggregation-prone Aß1-42 isoform, which further promotes cell-to-cell transfer; thus, oAß1-42 is a potentially toxic isoform. Furthermore, cell-to-cell transfer is shown to be an early event that is seemingly independent of later appearances of cellular toxicity. This phenomenon could explain how seeds for the AD pathology could pass on to new brain areas and gradually induce AD pathology, even before the first cell starts to deteriorate, and how cell-to-cell transfer can act together with the factors that influence cellular clearance and/or degradation in the development of AD.
Asunto(s)
Péptidos beta-Amiloides/metabolismo , Comunicación Celular/fisiología , Neuritas/metabolismo , Fragmentos de Péptidos/metabolismo , Péptidos beta-Amiloides/ultraestructura , Factor Neurotrófico Derivado del Encéfalo/farmacología , Diferenciación Celular/efectos de los fármacos , Línea Celular Transformada , Técnicas de Cocultivo , Matriz Extracelular/fisiología , Proteínas Fluorescentes Verdes/genética , Proteínas Fluorescentes Verdes/metabolismo , Humanos , Lisosomas/metabolismo , Lisosomas/ultraestructura , Potencial de la Membrana Mitocondrial/efectos de los fármacos , Factor de Crecimiento Nervioso/farmacología , Neurregulina-1/farmacología , Neuritas/ultraestructura , Neuroblastoma/patología , Fragmentos de Péptidos/ultraestructura , Isoformas de Proteínas , Factores de Tiempo , Tretinoina/farmacologíaRESUMEN
Studies on genetic robustness recently revealed transcriptional adaptation (TA) as a mechanism by which an organism can compensate for genetic mutations through activation of homologous genes. Here, we discovered that genetic mutations, introducing a premature termination codon (PTC) in the amyloid precursor protein-b (appb) gene, activated TA of two other app family members, appa and amyloid precursor-like protein-2 (aplp2), in zebrafish. The observed transcriptional response of appa and aplp2 required degradation of mutant mRNA and did not depend on Appb protein level. Furthermore, TA between amyloid precursor protein (APP) family members was observed in human neuronal progenitor cells; however, compensation was only present during early neuronal differentiation and could not be detected in a more differentiated neuronal stage or adult zebrafish brain. Using knockdown and chemical inhibition, we showed that nonsense-mediated mRNA decay (NMD) is involved in degradation of mutant mRNA and that Upf1 and Upf2, key proteins in the NMD pathway, regulate the endogenous transcript levels of appa, appb, aplp1, and aplp2 In conclusion, our results suggest that the expression level of App family members is regulated by the NMD pathway and that mutations destabilizing app/APP mRNA can induce genetic compensation by other family members through TA in both zebrafish and human neuronal progenitors.
Asunto(s)
Precursor de Proteína beta-Amiloide , Degradación de ARNm Mediada por Codón sin Sentido , Pez Cebra , Animales , Precursor de Proteína beta-Amiloide/genética , Precursor de Proteína beta-Amiloide/metabolismo , Humanos , Proteínas de Pez Cebra/metabolismo , Proteínas de Pez Cebra/genética , ARN Mensajero/metabolismo , Células-Madre Neurales/metabolismo , Mutación , Animales Modificados GenéticamenteRESUMEN
Alzheimer's disease (AD) is the major cause of dementia. During the development of AD, neurofibrillary tangles progress in a fixed pattern, starting in the transentorhinal cortex followed by the hippocampus and cortical areas. In contrast, the deposition of ß-amyloid (Aß) plaques, which are the other histological hallmark of AD, does not follow the same strict spatiotemporal pattern, and it correlates poorly with cognitive decline. Instead, soluble Aß oligomers have received increasing attention as probable inducers of pathogenesis. In this study, we use microinjections into electrophysiologically defined primary hippocampal rat neurons to demonstrate the direct neuron-to-neuron transfer of soluble oligomeric Aß. Additional studies conducted in a human donor-acceptor cell model show that this Aß transfer depends on direct cellular connections. As the transferred oligomers accumulate, acceptor cells gradually show beading of tubulin, a sign of neurite damage, and gradual endosomal leakage, a sign of cytotoxicity. These observations support that intracellular Aß oligomers play a role in neurodegeneration, and they explain the manner in which Aß can drive disease progression, even if the extracellular plaque load is poorly correlated with the degree of cognitive decline. Understanding this phenomenon sheds light on the pathophysiological mechanism of AD progression. Additional elucidation will help uncover the detailed mechanisms responsible for the manner in which AD progresses via anatomical connections and will facilitate the development of new strategies for stopping the progression of this incapacitating disease.
Asunto(s)
Péptidos beta-Amiloides/metabolismo , Péptidos beta-Amiloides/toxicidad , Comunicación Celular/fisiología , Degeneración Nerviosa/inducido químicamente , Degeneración Nerviosa/patología , Neuronas/metabolismo , Fragmentos de Péptidos/metabolismo , Fragmentos de Péptidos/toxicidad , Transmisión Sináptica/fisiología , Animales , Animales Recién Nacidos , Comunicación Celular/efectos de los fármacos , Diferenciación Celular/efectos de los fármacos , Células Cultivadas , Técnicas de Cocultivo , Dendritas/metabolismo , Relación Dosis-Respuesta a Droga , Endocitosis/efectos de los fármacos , Endocitosis/fisiología , Exocitosis/efectos de los fármacos , Exocitosis/fisiología , Femenino , Proteínas Fluorescentes Verdes/genética , Proteínas Fluorescentes Verdes/metabolismo , Compuestos Heterocíclicos con 3 Anillos/administración & dosificación , Compuestos Heterocíclicos con 3 Anillos/metabolismo , Hipocampo/citología , Humanos , Proteína 2 de la Membrana Asociada a los Lisosomas/metabolismo , Masculino , Glicoproteínas de Membrana/metabolismo , Microinyecciones , Microscopía Electrónica de Transmisión , Neocórtex/citología , Proteínas del Tejido Nervioso/metabolismo , Neuroblastoma/patología , Neuroglía/efectos de los fármacos , Neuroglía/metabolismo , Neuronas/efectos de los fármacos , Neuronas/ultraestructura , Técnicas de Placa-Clamp , Ratas , Ratas Sprague-Dawley , Rodaminas , Transmisión Sináptica/efectos de los fármacos , Sales de Tetrazolio , Tiazoles , Factores de Tiempo , Transfección , Proteínas de Unión al GTP rab5/metabolismoRESUMEN
The Niemann-Pick type C1 (NPC1) protein mediates the trafficking of cholesterol from lysosomes to other organelles. Mutations in the NPC1 gene lead to the retention of cholesterol and other lipids in the lysosomal compartment, and such defects are the basis of NPC disease. Several parallels exist between NPC disease and Alzheimer's disease (AD), including altered cholesterol homeostasis, changes in the lysosomal system, neurofibrillary tangles, and increased amyloid-beta generation. How the expression of NPC1 in the human brain is affected in AD has not been investigated so far. In the present study, we measured NPC1 mRNA and protein expression in three distinct regions of the human brain, and we revealed that NPC1 expression is upregulated at both mRNA and protein levels in the hippocampus and frontal cortex of AD patients compared to control individuals. In the cerebellum, a brain region that is relatively spared in AD, no difference in NPC1 expression was detected. Similarly, murine NPC1 mRNA levels were increased in the hippocampus of 12-month-old transgenic mice expressing a familial AD form of human amyloid-beta precursor protein (APP) and presenilin-1 (APP/PS1tg) compared to 12-month-old wild type mice, whereas no change in NPC1 was detected in mouse cerebellum. Immunohistochemical analysis of human hippocampus indicated that NPC1 expression was strongest in neurons. However, in vitro studies revealed that NPC1 expression was not induced by transfecting SK-N-SH neurons with human APP or by treating them with oligomeric amyloid-beta peptide. Total cholesterol levels were reduced in hippocampus from AD patients compared to control individuals, and it is therefore possible that the increased expression of NPC1 is linked to perturbed cholesterol homeostasis in AD.
Asunto(s)
Enfermedad de Alzheimer/genética , Proteínas/genética , Anciano , Anciano de 80 o más Años , Enfermedad de Alzheimer/sangre , Enfermedad de Alzheimer/metabolismo , Péptidos beta-Amiloides/genética , Animales , Células CHO , Estudios de Casos y Controles , Células Cultivadas , Colesterol/sangre , Colesterol/metabolismo , Cricetinae , Cricetulus , Femenino , Humanos , Péptidos y Proteínas de Señalización Intracelular , Masculino , Ratones , Ratones Transgénicos , Proteína Niemann-Pick C1 , Presenilina-1/genética , Proteínas/metabolismo , Regulación hacia ArribaRESUMEN
Herpes simplex virus 1 (HSV-1) and 2 (HSV-2) can infect the central nervous system (CNS) with dire consequences; in children and adults, HSV-1 may cause focal encephalitis, while HSV-2 causes meningitis. In neonates, both viruses can cause severe, disseminated CNS infections with high mortality rates. Here, we differentiated human induced pluripotent stem cells (iPSCs) towards cortical neurons for infection with clinical CNS strains of HSV-1 or HSV-2. Progenies from both viruses were produced at equal quantities in iPSCs, neuroprogenitors and cortical neurons. HSV-1 and HSV-2 decreased viability of neuroprogenitors by 36.0% and 57.6% (p < 0.0001), respectively, 48 h post-infection, while cortical neurons were resilient to infection by both viruses. However, in these functional neurons, both HSV-1 and HSV-2 decreased gene expression of two markers of synaptic activity, CAMK2B and ARC, and affected synaptic activity negatively in multielectrode array experiments. However, unaltered secretion levels of the neurodegeneration markers tau and NfL suggested intact axonal integrity. Viral replication of both viruses was found after six days, coinciding with 6-fold and 22-fold increase in gene expression of cellular RNA polymerase II by HSV-1 and HSV-2, respectively. Our results suggest a resilience of human cortical neurons relative to the replication of HSV-1 and HSV-2.
Asunto(s)
Diferenciación Celular , Herpes Simple/virología , Herpesvirus Humano 1 , Herpesvirus Humano 2 , Neuronas/virología , Diferenciación Celular/genética , Supervivencia Celular , Sistema Nervioso Central , Regulación de la Expresión Génica , Herpes Simple/patología , Humanos , Células Madre Pluripotentes Inducidas , Neuronas/patología , Replicación Viral/fisiologíaRESUMEN
Lysosomal storage diseases, including mucopolysaccharidoses, result from genetic defects that impair lysosomal catabolism. Here, we describe two patients from two independent families presenting with progressive psychomotor regression, delayed myelination, brain atrophy, neutropenia, skeletal abnormalities, and mucopolysaccharidosis-like dysmorphic features. Both patients were homozygous for the same intronic variant in VPS16, a gene encoding a subunit of the HOPS and CORVET complexes. The variant impaired normal mRNA splicing and led to an ~85% reduction in VPS16 protein levels in patient-derived fibroblasts. Levels of other HOPS/CORVET subunits, including VPS33A, were similarly reduced, but restored upon re-expression of VPS16. Patient-derived fibroblasts showed defects in the uptake and endosomal trafficking of transferrin as well as accumulation of autophagosomes and lysosomal compartments. Re-expression of VPS16 rescued the cellular phenotypes. Zebrafish with disrupted vps16 expression showed impaired development, reduced myelination, and a similar accumulation of lysosomes and autophagosomes in the brain, particularly in glia cells. This disorder resembles previously reported patients with mutations in VPS33A, thus expanding the family of mucopolysaccharidosis-like diseases that result from mutations in HOPS/CORVET subunits.
Asunto(s)
Mucopolisacaridosis , Pez Cebra , Animales , Endosomas , Humanos , Lisosomas , Proteínas de Transporte Vesicular/genéticaRESUMEN
Protection of the brain from viral infections involves the type I IFN (IFN-I) system, defects in which render humans susceptible to herpes simplex encephalitis (HSE). However, excessive cerebral IFN-I levels lead to pathologies, suggesting the need for tight regulation of responses. Based on data from mouse models, human HSE cases, and primary cell culture systems, we showed that microglia and other immune cells undergo apoptosis in the HSV-1-infected brain through a mechanism dependent on the cyclic GMP-AMP synthase/stimulator of interferon genes (cGAS/STING) pathway, but independent of IFN-I. HSV-1 infection of microglia induced cGAS-dependent apoptosis at high viral doses, whereas lower viral doses led to IFN-I responses. Importantly, inhibition of caspase activity prevented microglial cell death and augmented IFN-I responses. Accordingly, HSV-1-infected organotypic brain slices or mice treated with a caspase inhibitor exhibited lower viral load and an improved infection outcome. Collectively, we identify an activation-induced apoptosis program in brain immune cells that downmodulates local immune responses.
Asunto(s)
Encéfalo/inmunología , Herpes Simple/inmunología , Herpesvirus Humano 1/inmunología , Interferón Tipo I/inmunología , Proteínas de la Membrana/inmunología , Nucleotidiltransferasas/inmunología , Animales , Apoptosis/genética , Apoptosis/inmunología , Encéfalo/virología , Herpes Simple/genética , Humanos , Interferón Tipo I/genética , Masculino , Proteínas de la Membrana/genética , Ratones , Ratones Noqueados , Microglía/inmunología , Microglía/virología , Nucleotidiltransferasas/genéticaRESUMEN
BACKGROUND: Alzheimer's disease (AD) is the most common form of age-related neurodegenerative diseases. Cerebral deposition of Aß peptides, especially Aß42, is considered the major neuropathological hallmark of AD and the putative cause of AD-related neurotoxicity. Aß peptides are produced by sequential proteolytic processing of APP, with ß-secretase (BACE) being the initiating enzyme. Therefore, BACE has been considered an attractive therapeutic target in AD research and several BACE inhibitors have been tested in clinical trials, but so far, all have had negative outcomes or even led to worsening of cognitive function. AD can be triggered by Aß years before the first symptoms appear and one reason for the failures could be that the clinical trials were initiated too late in the disease process. Another possible explanation could be that BACE inhibition alters physiological APP processing in a manner that impairs synaptic function, causing cognitive deterioration. METHODS: The aim of this study was to investigate if partial BACE inhibition, mimicking the putative protective effect of the Icelandic mutation in the APP gene, could reduce Aß generation without affecting synaptic transmission. To investigate this, we used an optical electrophysiology platform, in which effects of compounds on synaptic transmission in cultured neurons can be monitored. We employed this method on primary cortical rat neuronal cultures treated with three different BACE inhibitors (BACE inhibitor IV, LY2886721, and lanabecestat) and monitored Aß secretion into the cell media. RESULTS: We found that all three BACE inhibitors tested decreased synaptic transmission at concentrations leading to significantly reduced Aß secretion. However, low-dose BACE inhibition, resulting in less than a 50% decrease in Aß secretion, did not affect synaptic transmission for any of the inhibitors tested. CONCLUSION: Our results indicate that Aß production can be reduced by up to 50%, a level of reduction of relevance to the protective effect of the Icelandic mutation, without causing synaptic dysfunction. We therefore suggest that future clinical trials aimed at prevention of Aß build-up in the brain should aim for a moderate CNS exposure of BACE inhibitors to avoid side effects on synaptic function.
Asunto(s)
Enfermedad de Alzheimer , Secretasas de la Proteína Precursora del Amiloide , Secretasas de la Proteína Precursora del Amiloide/metabolismo , Péptidos beta-Amiloides , Precursor de Proteína beta-Amiloide , Animales , Ácido Aspártico Endopeptidasas/metabolismo , Ratas , Transmisión SinápticaRESUMEN
An amendment to this paper has been published and can be accessed via a link at the top of the paper.
RESUMEN
One of the neuropathological hallmarks of Alzheimer's disease (AD) is cerebral deposition of amyloid plaques composed of amyloid ß (Aß) peptides and the cerebrospinal fluid concentrations of those peptides are used as a biomarker for AD. Mature induced pluripotent stem cell (iPSC)-derived cortical neurons secrete Aß peptides in ratios comparable to those secreted to cerebrospinal fluid in human, however the protocol to achieve mature neurons is time consuming. In this study, we investigated if differentiation of neuroprogenitor cells (NPCs) in BrainPhys medium, previously reported to enhance synaptic function of neurons in culture, would accelerate neuronal maturation and, thus increase Aß secretion as compared to the conventional neural maintenance medium. We found that NPCs cultured in BrainPhys displayed increased expression of markers for cortical deep-layer neurons, increased synaptic maturation and number of astroglial cells. This accelerated neuronal maturation was accompanied by increased APP processing, resulting in increased secretion of Aß peptides and an increased Aß38 to Aß40 and Aß42 ratio. However, during long-term culturing in BrainPhys, non-neuronal cells appeared and eventually took over the cultures. Taken together, BrainPhys culturing accelerated neuronal maturation and increased Aß secretion from iPSC-derived cortical neurons, but changed the cellular composition of the cultures.
Asunto(s)
Péptidos beta-Amiloides/metabolismo , Medios de Cultivo/química , Células Madre Pluripotentes Inducidas/citología , Neuronas/citología , Biomarcadores/metabolismo , Técnicas de Cultivo de Célula , Diferenciación Celular , Células Cultivadas , Sinapsis Eléctricas/metabolismo , Perfilación de la Expresión Génica , Regulación de la Expresión Génica , Humanos , Células Madre Pluripotentes Inducidas/metabolismo , Neuronas/metabolismoRESUMEN
BACKGROUND: Tau aggregation in neurons and glial cells characterizes tauopathies as Alzheimer's disease (AD), progressive supranuclear palsy (PSP) and corticobasal degeneration (CBD). Tau proteolysis has been proposed as a trigger for tau aggregation and tau fragments have been observed in brain and cerebrospinal fluid (CSF). Our group identified a major tau cleavage at amino acid (aa) 224 in CSF; N-terminal tau fragments ending at aa 224 (N-224) were significantly increased in AD and lacked correlation to total tau (t-tau) and phosphorylated tau (p-tau) in PSP and CBD. OBJECTIVE: Previous studies have shown cleavage from calpain proteases at sites adjacent to aa 224. Our aim was to investigate if calpain-1 or -2 could be responsible for cleavage at aa 224. METHODS: Proteolytic activity of calpain-1, calpain-2, and brain protein extract was assessed on a custom tau peptide (aa 220-228), engineered with fluorescence resonance energy transfer (FRET) technology. Findings were confirmed with in-gel trypsination and mass spectrometry (MS) analysis of brain-derived bands with proteolytic activity on the FRET substrate. Finally, knock-down of the calpain-2 catalytic subunit gene (CAPN2) was performed in a neuroblastoma cell line (SH-SY5Y). RESULTS: Calpain-2 and brain protein extract, but not calpain-1, showed proteolytic activity on the FRET substrate. MS analysis of active gel bands revealed presence of calpain-2 subunits, but not calpain-1. Calpain-2 depletion and chemical inhibition suppressed proteolysis of the FRET substrate. CAPN2 knock-down caused a 76.4% reduction of N-224 tau in the cell-conditioned media. CONCLUSIONS: Further investigation of the calpain-2 pathway in the pathogenesis of tauopathies is encouraged.
Asunto(s)
Calpaína/metabolismo , Tauopatías/metabolismo , Proteínas tau/metabolismo , Western Blotting , Encéfalo/metabolismo , Línea Celular Tumoral , Electroforesis en Gel de Agar , Femenino , Transferencia Resonante de Energía de Fluorescencia , Humanos , Inmunoprecipitación , Masculino , Espectrometría de Masas , Persona de Mediana Edad , Fragmentos de Péptidos/metabolismo , Tauopatías/etiologíaRESUMEN
Herpes simplex virus (HSV) is the main cause of viral encephalitis in the Western world, and the type I interferon (IFN) system is important for antiviral control in the brain. Here, we have compared Ifnb induction in mixed murine brain cell cultures by a panel of HSV1 mutants, each devoid of one mechanism to counteract the IFN-stimulating cGAS-STING pathway. We found that a mutant lacking the deubiquitinase (DUB) activity of the VP1-2 protein induced particularly strong expression of Ifnb and IFN-stimulated genes. HSV1 ΔDUB also induced elevated IFN expression in murine and human microglia and exhibited reduced viral replication in the brain. This was associated with increased ubiquitination of STING and elevated phosphorylation of STING, TBK1, and IRF3. VP1-2 associated directly with STING, leading to its deubiquitination. Recruitment of VP1-2 to STING was dependent on K150 of STING, which was ubiquitinated by TRIM32. Thus, the DUB activity of HSV1 VP1-2 is a major viral immune-evasion mechanism in the brain.
Asunto(s)
Encéfalo/virología , Enzimas Desubicuitinizantes/metabolismo , Herpesvirus Humano 1/metabolismo , Interferón Tipo I/metabolismo , Proteínas de la Membrana/metabolismo , Proteínas Virales/metabolismo , Animales , Encéfalo/patología , Células Cultivadas , Citoplasma/metabolismo , ADN Viral/metabolismo , Células HEK293 , Humanos , Lisina/metabolismo , Ratones Endogámicos C57BL , Microglía/metabolismo , Mutación/genética , Nucleotidiltransferasas/metabolismo , Proteínas Serina-Treonina Quinasas/metabolismo , Transducción de Señal , Ubiquitina/metabolismo , Ubiquitinación , Replicación Viral/fisiologíaRESUMEN
Synaptic function and neurotransmitter release are regulated by specific proteins. Cortical neuronal differentiation of human induced pluripotent stem cells (hiPSC) provides an experimental model to obtain more information about synaptic development and physiology in vitro. In this study, expression and secretion of the synaptic proteins, neurogranin (NRGN), growth-associated protein-43 (GAP-43), synaptosomal-associated protein-25 (SNAP-25) and synaptotagmin-1 (SYT-1) were analyzed during cortical neuronal differentiation. Protein levels were measured in cells, modeling fetal cortical development and in cell-conditioned media which was used as a model of cerebrospinal fluid (CSF), respectively. Human iPSC-derived cortical neurons were maintained over a period of at least 150 days, which encompasses the different stages of neuronal development. The differentiation was divided into the following stages: hiPSC, neuro-progenitors, immature and mature cortical neurons. We show that NRGN was first expressed and secreted by neuro-progenitors while the maximum was reached in mature cortical neurons. GAP-43 was expressed and secreted first by neuro-progenitors and its expression increased markedly in immature cortical neurons. SYT-1 was expressed and secreted already by hiPSC but its expression and secretion peaked in mature neurons. SNAP-25 was first detected in neuro-progenitors and the expression and secretion increased gradually during neuronal stages reaching a maximum in mature neurons. The sensitive analytical techniques used to monitor the secretion of these synaptic proteins during cortical development make these data unique, since the secretion of these synaptic proteins has not been investigated before in such experimental models. The secretory profile of synaptic proteins, together with low release of intracellular content, implies that mature neurons actively secrete these synaptic proteins that previously have been associated with neurodegenerative disorders, including Alzheimer's disease. These data support further studies of human neuronal and synaptic development in vitro, and would potentially shed light on the mechanisms underlying altered concentrations of the proteins in bio-fluids in neurodegenerative diseases.
Asunto(s)
Diferenciación Celular/fisiología , Corteza Cerebral/metabolismo , Proteínas de la Membrana/biosíntesis , Células-Madre Neurales/metabolismo , Neuronas/metabolismo , Sinapsis/metabolismo , Línea Celular , Células Cultivadas , Corteza Cerebral/citología , Expresión Génica , Humanos , Proteínas de la Membrana/genética , Neurogranina/biosíntesis , Neurogranina/genética , Proteína 25 Asociada a Sinaptosomas/biosíntesis , Proteína 25 Asociada a Sinaptosomas/genética , Sinaptotagmina I/biosíntesis , Sinaptotagmina I/genéticaRESUMEN
Parkinson's disease and other alpha-synucleinopathies are progressive neurodegenerative diseases characterized by aggregates of misfolded alpha-synuclein spreading throughout the brain. Recent evidence suggests that the pathological progression is likely due to neuron-to-neuron transfer of these aggregates between neuroanatomically connected areas of the brain. As the impact of this pathological spreading mechanism is currently debated, we aimed to investigate the transfer and subcellular location of alpha-synuclein species in a novel 3D co-culture human cell model based on highly differentiated SH-SY5Y cells. Fluorescently-labeled monomeric, oligomeric and fibrillar species of alpha-synuclein were introduced into a donor cell population and co-cultured with an EGFP-expressing acceptor-cell population of differentiated neuron-like cells. Subsequent transfer and colocalization of the different species were determined with confocal microscopy. We could confirm cell-to-cell transfer of all three alpha-synuclein species investigated. Interestingly the level of transferred oligomers and fibrils and oligomers were significantly higher than monomers, which could affect the probability of seeding and pathology in the recipient cells. Most alpha-synuclein colocalized with the lysosomal/endosomal system, both pre- and postsynaptically, suggesting its importance in the processing and spreading of alpha-synuclein.
Asunto(s)
Lisosomas/metabolismo , Neuronas/citología , Agregado de Proteínas , alfa-Sinucleína/química , alfa-Sinucleína/metabolismo , Diferenciación Celular/efectos de los fármacos , Línea Celular Tumoral , Técnicas de Cocultivo , Endosomas/efectos de los fármacos , Endosomas/metabolismo , Humanos , Lisosomas/efectos de los fármacos , Multimerización de Proteína , Estructura Cuaternaria de Proteína , Transporte de Proteínas , Sinapsis/efectos de los fármacos , Sinapsis/metabolismo , alfa-Sinucleína/toxicidadRESUMEN
Amyloid precursor protein (APP) and its cleavage product amyloid ß (Aß) have been thoroughly studied in Alzheimer's disease. However, APP also appears to be important for neuronal development. Differentiation of induced pluripotent stem cells (iPSCs) towards cortical neurons enables in vitro mechanistic studies on human neuronal development. Here, we investigated expression and proteolytic processing of APP during differentiation of human iPSCs towards cortical neurons over a 100-day period. APP expression remained stable during neuronal differentiation, whereas APP processing changed. α-Cleaved soluble APP (sAPPα) was secreted early during differentiation, from neuronal progenitors, while ß-cleaved soluble APP (sAPPß) was first secreted after deep-layer neurons had formed. Short Aß peptides, including Aß1-15/16, peaked during the progenitor stage, while processing shifted towards longer peptides, such as Aß1-40/42, when post-mitotic neurons appeared. This indicates that APP processing is regulated throughout differentiation of cortical neurons and that amyloidogenic APP processing, as reflected by Aß1-40/42, is associated with mature neuronal phenotypes.
Asunto(s)
Precursor de Proteína beta-Amiloide/metabolismo , Diferenciación Celular , Corteza Cerebral/patología , Neuronas/patología , Procesamiento Proteico-Postraduccional , Potenciales de Acción , Secretasas de la Proteína Precursora del Amiloide/metabolismo , Precursor de Proteína beta-Amiloide/genética , Regulación de la Expresión Génica , Humanos , Células Madre Pluripotentes Inducidas/metabolismo , Neuronas/metabolismo , Fragmentos de Péptidos/metabolismo , ARN Mensajero/genética , ARN Mensajero/metabolismo , SolubilidadRESUMEN
The sporadic, late onset form of Alzheimer's disease (AD) shares pathological hallmarks with the familial form; however, no clear reason for increased ß-amyloid (Aß) generation has been found in the former. It has long been speculated that the late onset form of AD is caused by reduced degradation and/or clearance of Aß. Indeed, both intracellular degradation systems, the proteasomal and lysosomal systems, have been shown to be defective in AD. Reduced proteasome activity increases levels of intracellular and secreted Aß. Furthermore, accumulation of improperly degraded Aß in the lysosomes causes lysosomal disruption and cell death. We recently showed that oligomeric Aß can be transmitted from one neuron to another, which causes neurotoxicity. In both the donating and receiving cells, Aß accumulates in the endo-lysosomal compartment. It is possible that ineffective degradation of Aß causes its transfer to neighboring neurons, thereby spreading AD pathology. This review summarizes the data underlying the idea of reduced Aß clearance and subsequent Aß spread in AD, and also suggests new therapeutic methods, which are aimed at targeting the degradation systems and synaptic transfer. By enhancing degradation of intracellular accumulated Aß, it can be possible to remove it and avoid Aß-induced neurodegeneration without disturbing the endogenously important pool of secreted Aß. Additionally, drugs targeted to inhibit the spread of intracellular toxic Aß aggregates may also be useful in stopping the progression of pathology, without affecting the level of Aß that normally occurs in the brain.
Asunto(s)
Enfermedad de Alzheimer/metabolismo , Péptidos beta-Amiloides/metabolismo , Enfermedad de Alzheimer/tratamiento farmacológico , Péptidos beta-Amiloides/antagonistas & inhibidores , Animales , Autofagia , Encéfalo/metabolismo , Humanos , Lisosomas/metabolismo , Neuronas/efectos de los fármacos , Neuronas/metabolismo , Complejo de la Endopetidasa Proteasomal/fisiología , Receptores de LDL/fisiologíaRESUMEN
The success of future intervention strategies for Alzheimer's disease (AD) will likely rely on the development of treatments starting early in the disease course, before irreversible brain damage occurs. The pre-symptomatic stage of AD occurs at least one decade before the clinical onset, highlighting the need for validated biomarkers that reflect this early period. Reliable biomarkers for AD are also needed in research and clinics for diagnosis, patient stratification, clinical trials, monitoring of disease progression and the development of new treatments. Changes in the lysosomal network, i.e., the endosomal, lysosomal and autophagy systems, are among the first alterations observed in an AD brain. In this study, we performed a targeted search for lysosomal network proteins in human cerebrospinal fluid (CSF). Thirty-four proteins were investigated, and six of them, early endosomal antigen 1 (EEA1), lysosomal-associated membrane proteins 1 and 2 (LAMP-1, LAMP-2), microtubule-associated protein 1 light chain 3 (LC3), Rab3 and Rab7, were significantly increased in the CSF from AD patients compared with neurological controls. These results were confirmed in a validation cohort of CSF samples, and patients with no neurochemical evidence of AD, apart from increased total-tau, were found to have EEA1 levels corresponding to the increased total-tau levels. These findings indicate that increased levels of LAMP-1, LAMP-2, LC3, Rab3 and Rab7 in the CSF might be specific for AD, and increased EEA1 levels may be a sign of general neurodegeneration. These six lysosomal network proteins are potential AD biomarkers and may be used to investigate lysosomal involvement in AD pathogenesis.