RESUMEN
Frontotemporal dementia (FTD) is a heterogeneous clinical disorder characterized by progressive abnormalities in behavior, executive functions, personality, language and/or motricity. A neuropathological subtype of FTD, frontotemporal lobar degeneration (FTLD)-FET, is characterized by protein aggregates consisting of the RNA-binding protein fused in sarcoma (FUS). The cause of FTLD-FET is not well understood and there is a lack of genetic evidence to aid in the investigation of mechanisms of the disease. The goal of this study was to identify genetic variants contributing to FTLD-FET and to investigate their effects on FUS pathology. We performed whole-exome sequencing on a 50-year-old FTLD patient with ubiquitin and FUS-positive neuronal inclusions and unaffected parents, and identified a de novo postzygotic nonsense variant in the NCDN gene encoding Neurochondrin (NCDN), NM_014284.3:c.1206G > A, p.(Trp402*). The variant was associated with a ~ 31% reduction in full-length protein levels in the patient's brain, suggesting that this mutation leads to NCDN haploinsufficiency. We examined the effects of NCDN haploinsufficiency on FUS and found that depleting primary cortical neurons of NCDN causes a reduction in the total number of FUS-positive cytoplasmic granules. Moreover, we found that these granules were significantly larger and more highly enriched with FUS. We then examined the effects of a loss of FUS function on NCDN in neurons and found that depleting cells of FUS leads to a decrease in NCDN protein and mRNA levels. Our study identifies the NCDN protein as a likely contributor of FTLD-FET pathophysiology. Moreover, we provide evidence for a negative feedback loop of toxicity between NCDN and FUS, where loss of NCDN alters FUS cytoplasmic dynamics, which in turn has an impact on NCDN expression.
Asunto(s)
Encéfalo/patología , Demencia Frontotemporal/genética , Proteínas del Tejido Nervioso/genética , Neuronas/patología , Proteína FUS de Unión a ARN/metabolismo , Codón sin Sentido , Femenino , Demencia Frontotemporal/patología , Haploinsuficiencia , Humanos , Persona de Mediana EdadRESUMEN
The SorLA protein, encoded by the SORL1 gene, is a major player in Alzheimer's disease (AD) pathophysiology. Functional and genetic studies demonstrated that SorLA deficiency results in increased production of Aß peptides, and thus a higher risk of AD. A large number of SORL1 missense variants have been identified in AD patients, but their functional consequences remain largely undefined. Here, we identified a new pathophysiological mechanism, by which rare SORL1 missense variants identified in AD patients result in altered maturation and trafficking of the SorLA protein. An initial screening, based on the overexpression of 70 SorLA variants in HEK293 cells, revealed that 15 of them (S114R, R332W, G543E, S564G, S577P, R654W, R729W, D806N, Y934C, D1535N, D1545E, P1654L, Y1816C, W1862C, P1914S) induced a maturation and trafficking-deficient phenotype. Three of these variants (R332W, S577P, and R654W) and two maturation-competent variants (S124R and N371T) were further studied in details in CRISPR/Cas9-modified hiPSCs. When expressed at endogenous levels, the R332W, S577P, and R654W SorLA variants also showed a maturation defective profile. We further demonstrated that these variants were largely retained in the endoplasmic reticulum, resulting in a reduction in the delivery of SorLA mature protein to the plasma membrane and to the endosomal system. Importantly, expression of the R332W and R654W variants in hiPSCs was associated with a clear increase of Aß secretion, demonstrating a loss-of-function effect of these SorLA variants regarding this ultimate readout, and a direct link with AD pathophysiology. Furthermore, structural analysis of the impact of missense variants on SorLA protein suggested that impaired cellular trafficking of SorLA protein could be due to subtle variations of the protein 3D structure resulting from changes in the interatomic interactions.
Asunto(s)
Enfermedad de Alzheimer/genética , Enfermedad de Alzheimer/metabolismo , Proteínas Relacionadas con Receptor de LDL/genética , Proteínas Relacionadas con Receptor de LDL/metabolismo , Proteínas de Transporte de Membrana/genética , Proteínas de Transporte de Membrana/metabolismo , Células HEK293 , Humanos , Mutación MissenseRESUMEN
Tauopathies are neurodegenerative diseases characterized by the presence of aggregates of abnormally phosphorylated Tau. Deciphering the pathophysiological mechanisms that lead from the alteration of Tau biology to neuronal death depends on the identification of Tau cellular partners. Combining genetic and transcriptomic analyses in Drosophila, we identified 77 new modulators of human Tau-induced toxicity, bringing to 301 the number of Tau genetic interactors identified so far in flies. Network analysis showed that 229 of these genetic modulators constitute a connected network. The addition of 77 new genes strengthened the network structure, increased the intergenic connectivity and brought up key hubs with high connectivities, namely Src64B/FYN, Src42A/FRK, kuz/ADAM10, heph/PTBP1, scrib/SCRIB, and Cam/CALM3. Interestingly, we established for the first time a genetic link between Tau-induced toxicity and ADAM10, a recognized Alzheimer Disease protective factor. In addition, our data support the importance of the presynaptic compartment in mediating Tau toxicity.
RESUMEN
The DNA- and RNA-binding protein fused in sarcoma (FUS) has been pathologically and genetically linked to amyotrophic lateral sclerosis (ALS) or frontotemporal lobar degeneration (FTLD). Cytoplasmic FUS-positive inclusions were identified in the brain and spinal cord of a subset of patients suffering with ALS/FTLD. An increasing number of reports suggest that FUS protein can behave in a prion-like manner. However, no neuropathological studies or experimental data were available regarding cell-to-cell spread of these pathological protein assemblies. In the present report, we investigated the ability of wild-type and mutant forms of FUS to transfer between neuronal cells. We combined the use of Drosophila models for FUS proteinopathies with that of the primary neuronal cultures to address neuron-to-neuron transfer of FUS proteins. Using conditional co-culture models and an optimized flow cytometry-based methodology, we demonstrated that ALS-mutant forms of FUS proteins can transfer between well-differentiated mature Drosophila neurons. These new observations support that a propagating mechanism could be applicable to FUS, leading to the sequential dissemination of pathological proteins over years.
Asunto(s)
Mutación , Neuronas/metabolismo , Proteína FUS de Unión a ARN/metabolismo , Esclerosis Amiotrófica Lateral/genética , Animales , Células Cultivadas , Drosophila , Proteína FUS de Unión a ARN/genética , Proteína FUS de Unión a ARN/toxicidadRESUMEN
Late onset neurodegenerative diseases represent a major public health concern as the population in many countries ages. Both frequent diseases such as Alzheimer disease (AD, 14% incidence for 80-84 year-old Europeans) or Parkinson disease (PD, 1.4% prevalence for >55 years old) share, with other low-incidence neurodegenerative pathologies such as spinocerebellar ataxias (SCAs, 0.01% prevalence) and frontotemporal lobar degeneration (FTLD, 0.02% prevalence), a lack of efficient treatment in spite of important research efforts. Besides significant progress, studies with animal models have revealed unexpected complexities in the degenerative process, emphasizing a need to better understand the underlying pathological mechanisms. Recently, microRNAs (miRNAs), a class of small regulatory non-coding RNAs, have been implicated in some neurodegenerative diseases. The current data supporting a role of miRNAs in PD, tauopathies, dominant ataxias, and FTLD will first be discussed to emphasize the different levels of the pathological processes which may be affected by miRNAs. To investigate a potential involvement of miRNA dysregulation in the early stages of these neurodegenerative diseases we have used Drosophila models for seven diseases (PD, 3 FTLD, 3 dominant ataxias) that recapitulate many features of the human diseases. We performed deep sequencing of head small RNAs after 3 days of pathological protein expression in the fly head neurons. We found no evidence for a statistically significant difference in miRNA expression in this early stage of the pathological process. In addition, we could not identify small non-coding CAG repeat RNAs (sCAG) in polyQ disease models. Thus our data suggest that transcriptional deregulation of miRNAs or sCAG is unlikely to play a significant role in the initial stages of neurodegenerative diseases.
RESUMEN
Recently, the fused in sarcoma/translated in liposarcoma (FUS) protein has been identified as a major constituent of nuclear and/or cytoplasmic ubiquitin-positive inclusions in patients with frontotemporal lobar degeneration or amyotrophic lateral sclerosis. The molecular mechanisms underlying FUS toxicity are currently not understood. To address aspects of FUS pathogenesis in vivo, we have generated new Drosophila transgenic models expressing a full-length wild-type isoform of human FUS protein. We found that when expressed in retinal cells, FUS proteins are mainly recovered as soluble forms, and their overexpression results in a mild eye phenotype, with malformed interommatidial bristles and the appearance of ectopic extensions. On the other hand, when FUS proteins are specifically targeted to adult differentiated neurons, they are mainly recovered as insoluble forms, and their overexpression drastically reduces fly life span. Importantly, FUS neurotoxicity occurs regardless of inclusion formation. Lastly, we showed that molecular chaperones reduce FUS toxicity by modulating protein solubility. Altogether, our data indicate that accumulation of insoluble non-aggregated FUS forms might represent the primary toxic species in human FUS proteinopathies.
Asunto(s)
Drosophila melanogaster/metabolismo , Enfermedades Neurodegenerativas/metabolismo , Neuronas/metabolismo , Proteína FUS de Unión a ARN/metabolismo , Animales , Animales Modificados Genéticamente , Encefalopatías Metabólicas/genética , Encefalopatías Metabólicas/metabolismo , Encefalopatías Metabólicas/patología , Modelos Animales de Enfermedad , Drosophila melanogaster/genética , Femenino , Humanos , Masculino , Enfermedades Neurodegenerativas/genética , Enfermedades Neurodegenerativas/patología , Neuronas/patología , Proteína FUS de Unión a ARN/genética , SolubilidadRESUMEN
The microtubule-associated protein Tau is found in large amount in axons of neurons and is involved in human neurodegenerative diseases called tauopathies, which include Alzheimer's disease. In these diseases, the Tau protein is abnormally hyperphosphorylated and one therapeutic strategy currently under consideration consists in inhibiting Tau phosphorylation. However, the consequences of an excess of hypophosphorylated Tau onto neuronal physiology have not been investigated in vivo. Here we studied how important is Tau phosphorylation for axonal transport and neurohormone release in vivo, using the Drosophila model. Surprisingly, our results demonstrate a stronger toxicity of hypophosphorylated Tau for neuronal function, when compared with normal or pseudophosphorylated Tau. This reveals a potential limit of the current therapeutic strategy aimed at inhibiting Tau phosphorylation.
Asunto(s)
Drosophila/metabolismo , Neuronas/metabolismo , Tauopatías/metabolismo , Proteínas tau/metabolismo , Animales , Animales Modificados Genéticamente , Transporte Axonal , Modelos Animales de Enfermedad , Drosophila/genética , Femenino , Humanos , Masculino , Microtúbulos/metabolismo , Neuronas/efectos de los fármacos , Fosforilación , Isoformas de Proteínas/genética , Isoformas de Proteínas/metabolismo , Tauopatías/genética , Proteínas tau/genética , Proteínas tau/toxicidadRESUMEN
Tauopathies, including Alzheimer's disease (AD), fronto-temporal dementia with parkinsonism linked to chromosome 17 (FTDP-17), Pick's disease and progressive supranuclear palsy, are neurodegenerative disorders neuropathologically characterized by the presence of intraneuronal fibrillary inclusions composed of abnormally phosphorylated-Tau. Tau protein is a neuronal microtubule-associated protein (MAP) involved in microtubules polymerization and stabilization. So far, the molecular mechanisms underlying Tau-mediated cellular toxicity remain elusive. To address the determinants of Tau neurotoxicity, we previously performed a misexpression screening in a Drosophila tauopathy model to identify genetic modifiers of the human Tau-induced neurodegeneration. We identified several components of the actin network as modifiers of Tau V337M-induced neurodegeneration, i.e. Filamin-A, Myosin VI, Paxillin and Transgelin-3. The aim of this study was to assess whether these genetic interactions were associated with a colocalization of the proteins (i) in the brains of patients with Tau pathologies, and (ii) in the brain of transgenic mice overexpressing human mutant Tau. We found that Filamin-A and Myosin VI indeed colocalize with fibrillary Tau protein in AD and FTDP-17 and in Thy-Tau22 transgenic mice.
Asunto(s)
Enfermedad de Alzheimer/metabolismo , Encéfalo/metabolismo , Proteínas Contráctiles/metabolismo , Demencia Frontotemporal/metabolismo , Proteínas de Microfilamentos/metabolismo , Cadenas Pesadas de Miosina/metabolismo , Proteínas tau/metabolismo , Anciano , Animales , Cromosomas Humanos Par 17 , Femenino , Filaminas , Demencia Frontotemporal/genética , Humanos , Masculino , Ratones , Ratones Transgénicos , Persona de Mediana Edad , Ovillos Neurofibrilares/metabolismo , Enfermedad de Pick/metabolismo , Parálisis Supranuclear Progresiva/metabolismo , Tauopatías/metabolismo , Proteínas tau/genéticaRESUMEN
Tau is a neuronal microtubule-associated protein involved in microtubules assembly and stabilization. Tauopathies, including Alzheimer's disease and fronto-temporal dementia with parkinsonism linked to chromosome 17, are a group of neurodegenerative disorders characterized by the presence of intraneuronal filamentous inclusions of abnormally and hyperphosphorylated Tau. Currently, the molecular mechanisms underlying Tau-mediated cellular toxicity remain elusive. To address the determinants of Tau neurotoxicity, we used Drosophila models of human tauopathies to study the microtubule-binding properties of human Tau proteins in vivo. We showed that, in contrast to endogenous Drosophila Tau, human Tau proteins bind very poorly to microtubules in Drosophila, and are mostly recovered as soluble cytosolic hyperphosphorylated species. This weak binding of human Tau to microtubules is neither because of microtubules saturation nor competition with endogenous Drosophila Tau, but clearly depends on its phosphorylation degree. We also reported that accumulation of cytosolic hyperphosphorylated forms of human Tau proteins correlates with human Tau-mediated neurodegeneration in flies, supporting the key role of soluble cytosolic hyperphosphorylated Tau proteins as toxic species in vivo.
Asunto(s)
Drosophila melanogaster/metabolismo , Sistema Nervioso/metabolismo , Neuronas/metabolismo , Tauopatías/metabolismo , Proteínas tau/metabolismo , Animales , Animales Modificados Genéticamente , Citosol/metabolismo , Modelos Animales de Enfermedad , Drosophila melanogaster/genética , Femenino , Humanos , Cuerpos de Inclusión/metabolismo , Cuerpos de Inclusión/patología , Masculino , Microscopía Electrónica , Microtúbulos/metabolismo , Microtúbulos/patología , Sistema Nervioso/patología , Sistema Nervioso/fisiopatología , Neuronas/patología , Fosforilación/efectos de los fármacos , Unión Proteica/fisiología , Solubilidad , Tauopatías/patología , Tauopatías/fisiopatología , Proteínas tau/genética , Proteínas tau/toxicidadRESUMEN
Tauopathies, including Alzheimer's disease and fronto-temporal dementia and parkinsonism linked to chromosome 17 (FTDP-17), are a group of neurodegenerative disorders characterized by the presence of intraneuronal filamentous inclusions of aberrantly phosphorylated-tau. Tau is a neuronal microtubule-associated protein involved in microtubule assembly and stabilization. Currently, the molecular mechanisms underlying tau-mediated cellular toxicity remain elusive. To address the determinants of tau neurotoxicity, we first characterized the cellular alterations resulting from the over-expression of a mutant form of human tau associated with FTDP-17 (tau V337M) in Drosophila. We found that the over-expression of tau V337M, in Drosophila larval motor neurons, induced disruption of the microtubular network at presynaptic nerve terminals and changes in neuromuscular junctions morphological features. Secondly, we performed a misexpression screen to identify genetic modifiers of the tau V337M-mediated rough eye phenotype. The screening of 1250 mutant Drosophila lines allowed us to identify several components of the cytoskeleton, and particularly from the actin network, as specific modifiers of tau V337M-induced neurodegeneration. Furthermore, we found that numerous tau modulators identified in our screen were involved in the maintenance of synaptic function. Taken together, these findings suggest that disruption of the microtubule network in presynaptic nerve terminals could constitute early events in the pathological process leading to synaptic dysfunction in tau V337M pathology.
Asunto(s)
Proteínas del Citoesqueleto/metabolismo , Drosophila melanogaster/efectos de los fármacos , Drosophila melanogaster/metabolismo , Proteínas Mutantes/toxicidad , Degeneración Nerviosa/patología , Proteínas tau/genética , Proteínas tau/toxicidad , Animales , Citoesqueleto/efectos de los fármacos , Citoesqueleto/metabolismo , Drosophila melanogaster/ultraestructura , Ojo/patología , Ojo/ultraestructura , Femenino , Humanos , Larva/efectos de los fármacos , Larva/metabolismo , Masculino , Metionina/genética , Chaperonas Moleculares/metabolismo , Proteínas Mutantes/metabolismo , Degeneración Nerviosa/inducido químicamente , Unión Neuromuscular/efectos de los fármacos , Unión Neuromuscular/patología , Péptidos/toxicidad , Fenotipo , Fosforilación/efectos de los fármacos , Valina/genética , Proteínas tau/metabolismoRESUMEN
We report duplication of the APP locus on chromosome 21 in five families with autosomal dominant early-onset Alzheimer disease (ADEOAD) and cerebral amyloid angiopathy (CAA). Among these families, the duplicated segments had a minimal size ranging from 0.58 to 6.37 Mb. Brains from individuals with APP duplication showed abundant parenchymal and vascular deposits of amyloid-beta peptides. Duplication of the APP locus, resulting in accumulation of amyloid-beta peptides, causes ADEOAD with CAA.
Asunto(s)
Enfermedad de Alzheimer/genética , Péptidos beta-Amiloides/genética , Encéfalo/patología , Angiopatía Amiloide Cerebral/genética , Duplicación de Gen , Edad de Inicio , Enfermedad de Alzheimer/epidemiología , Enfermedad de Alzheimer/patología , Péptidos beta-Amiloides/análisis , Estudios de Casos y Controles , Angiopatía Amiloide Cerebral/epidemiología , Angiopatía Amiloide Cerebral/patología , Femenino , Genes Dominantes , Humanos , Masculino , Repeticiones de Microsatélite , Reacción en Cadena de la Polimerasa/métodosRESUMEN
Tau-positive inclusions in neurons are consistent neuropathologic features of the most common causes of dementias such Alzheimer's disease and frontotemporal dementia. Ubiquitinated tau-positive inclusions have been reported in brains of Alzheimer's disease patients, but involvement of the ubiquitin-dependent proteasomal system in tau degradation remains controversial. Before considering the tau degradation in pathologic conditions, it is important to determine whether or not endogenous tau is normally degraded by the proteasome pathway. We therefore investigated this question using two complementary approaches in vitro and in vivo. Firstly, SH-SY5Y human neuroblastoma cells were treated with different proteasome inhibitors, MG132, lactacystin, and epoxomicin. Under these conditions, neither total nor phosphorylated endogenous tau protein levels were increased. Instead, an unexpected decrease of tau protein was observed. Secondly, we took advantage of a temperature-sensitive mutant allele of the 20S proteasome in Drosophila. Genetic inactivation of the proteasome also resulted in a decrease of tau levels in Drosophila. These results obtained in vitro and in vivo demonstrate that endogenous tau is not normally degraded by the proteasome.