RESUMEN
More than 50% of patients with primary familial brain calcification (PFBC), a rare neurological disorder, remain genetically unexplained. While some causative genes are yet to be identified, variants in non-coding regions of known genes may represent a source of missed diagnoses. We hypothesized that 5'-Untranslated Region (UTR) variants introducing an AUG codon may initiate mRNA translation and result in a loss of function in some of the PFBC genes. After reannotation of exome sequencing data of 113 unrelated PFBC probands, we identified two upstream AUG-introducing variants in the 5'UTR of PDGFB. One, NM_002608.4:c.-373C>G, segregated with PFBC in the family. It was predicted to create an upstream open reading frame (ORF). The other one, NM_002608.4:c.-318C>T, was found in a simplex case. It was predicted to result in an ORF overlapping the natural ORF with a frameshift. In a GFP reporter assay, both variants were associated with a dramatic decrease in GFP levels, and, after restoring the reading frame with the GFP sequence, the c.-318C>T variant was associated with a strong initiation of translation as measured by western blotting. Overall, we found upstream AUG-introducing variants in the 5'UTR of PDGFB in 2/113 (1.7%) undiagnosed PFBC cases. Such variants thus represent a source of putative pathogenic variants.
Asunto(s)
Regiones no Traducidas 5' , Calcinosis , Sistemas de Lectura Abierta , Humanos , Calcinosis/genética , Calcinosis/patología , Femenino , Masculino , Encefalopatías/genética , Encefalopatías/patología , Proteínas Proto-Oncogénicas c-sis/genética , Linaje , Adulto , Persona de Mediana Edad , Codón Iniciador/genética , Mutación del Sistema de LecturaRESUMEN
SORL1 loss of function is associated with Alzheimer's disease (AD) risk through increased Aß peptide secretion. We expressed 10 maturation-defective rare missense SORL1 variants in HEK cells and showed that decreasing growing temperature led to a significant increase in the maturation of the encoded protein SorLA for 6/10. In edited hiPSC carrying two of these variants, maturation of the protein was restored partially by decreasing the culture temperature and was associated with concomitant decrease in Aß secretion. Correcting SorLA maturation in the context of maturation-defective missense variants could thus be a relevant strategy to improve SorLA protective function against AD.
Asunto(s)
Enfermedad de Alzheimer , Humanos , Enfermedad de Alzheimer/genética , Enfermedad de Alzheimer/metabolismo , Proteínas Relacionadas con Receptor de LDL/genética , Proteínas Relacionadas con Receptor de LDL/metabolismo , Mutación Missense , Predisposición Genética a la Enfermedad , Proteínas de Transporte de MembranaRESUMEN
Cornelia de Lange syndrome (CdLS) is a clinically-recognizable rare developmental disorder. About 70% of patients carry a missense or loss-of-function pathogenic variant in the NIPBL gene. We hypothesized that some variants in the 5'-untranslated region (UTR) of NIPBL may create an upstream open reading frame (uORF), putatively leading to a loss of function. We searched for NIPBL 5'-UTR variants potentially introducing uORF by (i) reannotating NGS data of 102 unsolved CdLS patients and (ii) literature and variant databases search. We set up a green fluorescent protein (GFP) reporter assay and studied NIPBL expression in a lymphoblastoid cell line (LCL). We identified two variants introducing a novel ATG codon sequence in the 5'-UTR of NIPBL, both predicted to introduce uORF: a novel c.-457_-456delinsAT de novo mutation in a 15-year-old male with classic CdLS, and a c.-94C>T variant in a published family. Our reporter assay showed a significant decrease of GFP levels in both mutant contexts, with similar levels of messenger RNA (mRNA) as compared to wt constructs. Assessment of LCL of one patient showed consistent results with decreased NIPBL protein and unchanged mRNA levels. 5'-UTR uORF-introducing NIPBL variants may represent a rare source of pathogenic variants in unsolved CdLS patients.
Asunto(s)
Síndrome de Cornelia de Lange , Regiones no Traducidas 5' , Adolescente , Proteínas de Ciclo Celular/genética , Síndrome de Cornelia de Lange/diagnóstico , Síndrome de Cornelia de Lange/genética , Humanos , Masculino , Sistemas de Lectura Abierta/genética , Fenotipo , ARN Mensajero/genética , ARN Mensajero/metabolismoRESUMEN
Tau proteins belong to the microtubule associated protein family and are mainly expressed in neurons. Tau accumulates in patients' brain in several neurodegenerative diseases, including Fronto-temporal dementia and Alzheimer's disease. Recently, we described a 17q21.31 duplication in patients presenting different cognitive or motor symptoms and characterized by the accumulation of different Tau isoforms. This duplication involves four genes, including the MAPT gene that encodes the Tau protein. The main pathophysiological consequence associated with this duplication was a 1.6-1.9-fold increase in the MAPT messenger RNA as measured in blood samples of duplication carriers. However, the pathophysiological consequences of this duplication in a cell type relevant for neurodegenerative diseases have never been explored so far. In this study, we developed the first model of primary tauopathy linked to a 17q21.31 duplication in iPSC-induced neurons from 2 unrelated carriers. As in patients' blood, we demonstrated that this duplication was associated with an increase in MAPT mRNA resulting in elevated Tau protein levels in iPSC-derived cortical neurons. We believe that these iPSC lines will be a pertinent tool to elucidate how a same genetic cause could lead to distinct types of tauopathies and the pathophysiological mechanisms associated with Tau-mediated neurodegeneration in the 17q21.31 duplication context.
Asunto(s)
Células Madre Pluripotentes Inducidas , Enfermedades Neurodegenerativas , Tauopatías , Humanos , Células Madre Pluripotentes Inducidas/metabolismo , Enfermedades Neurodegenerativas/metabolismo , Neuronas/metabolismo , Tauopatías/genética , Tauopatías/metabolismo , Proteínas tau/genética , Proteínas tau/metabolismoRESUMEN
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
Microduplications of the 17q21.31 chromosomal region encompassing the MAPT gene, which encodes the Tau protein, were identified in patients with a progressive disorder initially characterized by severe memory impairment with or without behavioral changes that can clinically mimic Alzheimer disease. The unique neuropathological report showed a primary tauopathy, which could not be unanimously classified in a given known subtype, showing both 4R- and 3R-tau inclusions, mainly within temporal cortical subregions and basal ganglia, without amyloid deposits. Recently, two subjects harboring the same duplication were reported with an atypical extrapyramidal syndrome and gait disorder. To decipher the phenotypic spectrum associated with MAPT duplications, we studied ten carriers from nine families, including two novel unrelated probands, gathering clinical (n = 10), cerebrospinal fluid (n = 6), MRI (n = 8), dopamine transporter scan (n = 4), functional (n = 5), amyloid (n = 3) and Tau-tracer (n = 2) PET imaging data as well as neuropathological examination (n = 4). Ages at onset ranged from 37 to 57 years, with prominent episodic memory impairment in 8/10 patients, associated with behavioral changes in four, while two patients showed atypical extrapyramidal syndrome with gait disorder at presentation, including one with associated cognitive deficits. Amyloid imaging was negative but Tau imaging showed significant deposits mainly in both mesiotemporal cortex. Dopaminergic denervation was found in 4/4 patients, including three without extrapyramidal symptoms. Neuropathological examination exclusively showed Tau-immunoreactive lesions. Distribution, aspect and 4R/3R tau aggregates composition suggested a spectrum from predominantly 3R, mainly cortical deposits well correlating with cognitive and behavioral changes, to predominantly 4R deposits, mainly in the basal ganglia and midbrain, in patients with prominent extrapyramidal syndrome. Finally, we performed in vitro seeding experiments in HEK-biosensor cells. Morphological features of aggregates induced by homogenates of three MAPT duplication carriers showed dense/granular ratios graduating between those induced by homogenates of a Pick disease and a progressive supranuclear palsy cases. These results suggest that MAPT duplication causes a primary tauopathy associated with diverse clinical and neuropathological features.
Asunto(s)
Encéfalo/patología , Tauopatías/patología , Proteínas tau/metabolismo , Adulto , Edad de Inicio , Enfermedad de Alzheimer/metabolismo , Enfermedad de Alzheimer/patología , Disfunción Cognitiva/metabolismo , Disfunción Cognitiva/patología , Femenino , Heterocigoto , Humanos , Cuerpos de Inclusión/patología , Masculino , Persona de Mediana Edad , Tauopatías/metabolismo , Proteínas tau/genéticaRESUMEN
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
Alzheimer's disease (AD) is neuropathologically defined by two key hallmarks: extracellular senile plaques composed primarily of amyloid-ß (Aß) peptide and intraneuronal neurofibrillary tangles, containing abnormally hyperphosphorylated tau protein. The tau protein is encoded by the MAPT gene. Recently, the H1 and H2 haplotypes of the MAPT gene were associated with AD risk. The minor MAPT H2 haplotype has been linked with a decreased risk of developing late-onset AD (LOAD). MAPT haplotypes show different levels of MAPT/Tau expression with H1 being â¼1.5-fold more expressed than H2, suggesting that MAPT expression level could be related to LOAD risk. In this study, we investigated whether this moderate difference in MAPT/Tau expression could influence Aß-induced toxicity in vivo. We show that modest overexpression of tau protein in Drosophila exacerbates neuronal phenotypes in AßPP/BACE1 flies. The exacerbation of neuronal defects correlates with the accumulation of insoluble dTau oligomers, suggesting that the moderate difference in level of tau expression observed between H1 and H2 haplotypes could influence Aß toxicity through the production of oligomeric tau insoluble species.
Asunto(s)
Péptidos beta-Amiloides/toxicidad , Neuronas/metabolismo , Fenotipo , Proteínas tau/biosíntesis , Animales , Animales Modificados Genéticamente , Drosophila , Expresión Génica , Neuronas/efectos de los fármacos , Proteínas tau/genéticaRESUMEN
Tauopathies are a class of neurodegenerative diseases characterized by the presence of pathological intracellular deposits of Tau proteins. Six isoforms of Tau are expressed in the adult human brain, resulting from alternative splicing of the MAPT gene. Tau splicing is developmentally regulated such that only the smallest Tau isoform is expressed in fetal brain, contrary to the adult brain showing the expression of all 6 isoforms. Induced Pluripotent Stem Cell (iPSC) technology has opened up new perspectives in human disease modeling, including tauopathies. However, a major challenge to in vitro recapitulation of Tau pathology in iPSC-derived neurons is their relative immaturity. In this study, we examined the switch in Tau splicing from fetal-only to all adult Tau isoforms during the differentiation of iPSC-derived neurons in a new 3D culture system. First, we showed that iPSC-induced neurons inside Matrigel-coated alginate capsules were able to differentiate into cortical neurons. Then, using a new assay that allowed both the qualitative and the quantitative analysis of all adult MAPT mRNA isoforms individually, we demonstrated that BrainPhys-maintained neurons expressed the 6 adult MAPT mRNA transcripts from 25â¯weeks of maturation, making this model highly suitable for modeling Tau pathology and therapeutic purposes.
Asunto(s)
Técnicas de Cultivo de Célula/métodos , Neuronas/metabolismo , Proteínas tau/metabolismo , Alginatos/química , Línea Celular , Linaje de la Célula , Corteza Cerebral/metabolismo , Colágeno/química , Combinación de Medicamentos , Humanos , Células Madre Pluripotentes Inducidas/citología , Células Madre Pluripotentes Inducidas/metabolismo , Laminina/química , Neuronas/citología , Isoformas de Proteínas/genética , Isoformas de Proteínas/metabolismo , Proteoglicanos/química , Proteínas tau/genéticaRESUMEN
TAR DNA-binding protein-43 (TDP-43) is a ubiquitously expressed DNA-/RNA-binding protein that has been linked to numerous aspects of the mRNA life cycle. Similar to many RNA-binding proteins, TDP-43 expression is tightly regulated through an autoregulatory negative feedback loop. Cell function and survival depend on the strict control of TDP-43 protein levels. TDP-43 has been identified as the major constituent of ubiquitin-positive inclusions in patients with Amyotrophic Lateral Sclerosis (ALS) and Frontotemporal Lobar Degeneration (FTLD). Several observations argue for a pathogenic role of elevated TDP-43 levels in these disorders. Modulation of the cycle of TDP-43 production might therefore provide a new therapeutic strategy. Using a Drosophila model mimicking key features of the TDP-43 autoregulatory feedback loop, we identified CG42724 as a genetic modulator of TDP-43 production in vivo. We found that CG42724 protein influences qualitatively and quantitatively the TDP-43 mRNA transcript pattern. CG42724 overexpression promotes the production of transcripts that can be efficiently released into the cytoplasm for protein translation. Importantly, we showed that TCERG1, the human homolog of the Drosophila CG42724 protein, also caused an increase of TDP-43 protein steady-state levels in mammalian cells. Therefore, our data suggest the possibility that targeting TCERG1 could be therapeutic in TDP-43 proteinopathies.
Asunto(s)
Proteínas de Unión al ADN/metabolismo , Proteínas de Drosophila/metabolismo , Factores de Elongación Transcripcional/metabolismo , Adhesinas de Escherichia coli , Animales , Animales Modificados Genéticamente , Células Cultivadas , Proteínas de Unión al ADN/genética , Modelos Animales de Enfermedad , Drosophila , Proteínas de Drosophila/genética , Regulación de la Expresión Génica/efectos de los fármacos , Proteínas Fluorescentes Verdes/genética , Proteínas Fluorescentes Verdes/metabolismo , Ribonucleoproteína Heterogénea-Nuclear Grupo F-H/metabolismo , Humanos , Microscopía Electrónica , ARN Mensajero/metabolismo , Transfección/métodosRESUMEN
TDP-43 is a major disease-causing protein in amyotrophic lateral sclerosis (ALS) and Frontotemporal Lobar Degeneration (FTLD). Today, >50 missense mutations in the TARDBP/TDP-43 gene have been described in patients with FTLD/ALS. However, the functional consequences of FTLD/ALS-linked TDP-43 mutations are not fully elucidated. In the physiological state, TDP-43 expression is tightly regulated through an autoregulatory negative feedback loop. Maintaining normal TDP-43 protein levels is critical for proper physiological functions of the cells. In the present study, we investigated whether the FTLD/ALS-associated mutations could interfere with TDP-43 protein's capacity to modulate its own protein levels using Drosophila as an experimental model. Our data show that FTLD/ALS-associated mutant proteins regulate TDP-43 production with the same efficiency as the wild-type form of the protein. Thus, FTLD/ALS-linked TDP-43 mutations do not alter TDP-43's ability to self-regulate its expression and consequently of the homeostasis of TDP-43 protein levels.
Asunto(s)
Proteínas de Unión al ADN/metabolismo , Drosophila melanogaster/metabolismo , Mutación , Animales , Proteínas de Unión al ADN/genética , Homeostasis/fisiología , Humanos , Cuerpos de Inclusión/metabolismo , Mutación/genéticaRESUMEN
TDP-43 is a critical RNA-binding factor associated with RNA metabolism. In the physiological state, maintaining normal TDP-43 protein levels is critical for proper physiological functions of the cells. As such, TDP-43 expression is tightly regulated through an autoregulatory negative feedback loop. TDP-43 is a major disease-causing protein in Amyotrophic Lateral Sclerosis (ALS) and Frontotemporal Lobar Degeneration (FTLD). Several studies argue for a pathogenic role of elevated TDP-43 levels in these disorders. Modulating the cycle of TDP-43 production might therefore provide a new therapeutic strategy. In this study, we developed a new transgenic Drosophila model mimicking the TDP-43 autoregulatory feedback loop in order to identify genetic modulators of TDP-43 protein steady-state levels in vivo. First, we showed that our TDP-43_TDPBR Drosophila model recapitulates key features of the TDP-43 autoregulatory processes previously described in mammalian and cellular models, namely alternative splicing events, differential usage of polyadenylation sites, nuclear retention of the transcript and a decrease in steady-state mRNA levels. Using this new Drosophila model, we identified several splicing factors, including SF2, Rbp1 and Sf3b1, as genetic modulators of TDP-43 production. Interestingly, our data indicate that these three RNA-binding proteins regulate TDP-43 protein production, at least in part, by controlling mRNA steady-state levels.
Asunto(s)
Proteínas de Unión al ADN/genética , Proteínas de Unión al ADN/metabolismo , Factores de Empalme de ARN/metabolismo , Empalme Alternativo , Esclerosis Amiotrófica Lateral/genética , Animales , Animales Modificados Genéticamente , Modelos Animales de Enfermedad , Drosophila/genética , Proteínas de Drosophila/genética , Proteínas de Drosophila/metabolismo , Degeneración Lobar Frontotemporal/genética , Regulación de la Expresión Génica , Redes Reguladoras de Genes , Humanos , Factores de Empalme de ARN/genética , ARN Mensajero/genética , Proteínas de Unión al ARN/metabolismoRESUMEN
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
Transactive response DNA-binding protein 43 kDa (TDP-43) is considered a major pathological protein in amyotrophic lateral sclerosis and frontotemporal lobar degeneration. The precise mechanisms by which TDP-43 dysregulation leads to toxicity in neurons are not fully understood. Using TDP-43-expressing Drosophila, we examined whether mitochondrial dysfunction is a central determinant in TDP-43 pathogenesis. Expression of human wild-type TDP-43 in Drosophila neurons results in abnormally small mitochondria. The mitochondrial fragmentation is correlated with a specific decrease in the mRNA and protein levels of the Drosophila profusion gene mitofusin/marf. Importantly, overexpression of Marf ameliorates defects in spontaneous walking activity and startle-induced climbing response of TDP-43-expressing flies. Partial inactivation of the mitochondrial profission factor, dynamin-related protein 1, also mitigates TDP-43-induced locomotor deficits. Expression of TDP-43 impairs neuromuscular junction transmission upon repetitive stimulation of the giant fiber circuit that controls flight muscles, which is also ameliorated by Marf overexpression. We show here for the first time that enhancing the profusion gene mitofusin/marf is beneficial in an in vivo model of TDP-43 proteinopathies, serving as a potential therapeutic target.
Asunto(s)
Proteínas de Unión al ADN/genética , Proteínas de Unión al ADN/metabolismo , Proteínas de Drosophila/genética , Proteínas de Drosophila/metabolismo , Expresión Génica/genética , Proteínas de la Membrana/genética , Proteínas de la Membrana/metabolismo , Mitocondrias/genética , Mitocondrias/patología , Dinámicas Mitocondriales/genética , Dinámicas Mitocondriales/fisiología , Unión Neuromuscular/fisiopatología , Proteinopatías TDP-43/genética , Proteinopatías TDP-43/terapia , Animales , Modelos Animales de Enfermedad , Drosophila , Proteínas de Drosophila/fisiología , Dinaminas/fisiología , Locomoción/genética , Locomoción/fisiología , Terapia Molecular Dirigida , Neuronas/metabolismo , ARN Mensajero/metabolismo , Proteinopatías TDP-43/fisiopatologíaRESUMEN
Amyotrophic lateral sclerosis (ALS) is a neurodegenerative disease where motor neurons in cortex, brain stem, and spinal cord die progressively, resulting in muscle wasting, paralysis, and death. Currently, effective therapies for ALS are lacking; however, identification of pathological TAR DNA-binding protein 43 (TDP-43) as the hallmark lesion in sporadic ALS suggests new therapeutic targets for pharmacological intervention. Pathological TDP-43 phosphorylation appears to drive the onset and progression of ALS and may result from upregulation of the protein kinase CK-1 in affected neurons, resulting in postranslational TDP-43 modification. Consequently, brain penetrant specific CK-1 inhibitors may provide a new therapeutic strategy for treating ALS and other TDP-43 proteinopathies. Using a chemical genetic approach, we report the discovery and further optimization of a number of potent CK-1δ inhibitors. Moreover, these small heterocyclic molecules are able to prevent TDP-43 phosphorylation in cell cultures, to increase Drosophila lifespan by reduction of TDP-43 neurotoxicity, and are predicted to cross the blood-brain barrier. Thus, N-(benzothiazolyl)-2-phenyl-acetamides are valuable drug candidates for further studies and may be a new therapeutic approach for ALS and others pathologies in which TDP-43 is involved.
Asunto(s)
Esclerosis Amiotrófica Lateral/tratamiento farmacológico , Quinasa de la Caseína I/antagonistas & inhibidores , Inhibidores de Proteínas Quinasas/síntesis química , Inhibidores de Proteínas Quinasas/uso terapéutico , Animales , Animales Modificados Genéticamente , Benzotiazoles/síntesis química , Benzotiazoles/farmacología , Barrera Hematoencefálica/efectos de los fármacos , Barrera Hematoencefálica/metabolismo , Permeabilidad de la Membrana Celular/efectos de los fármacos , Células Cultivadas , Proteínas de Unión al ADN/metabolismo , Drosophila , Diseño de Fármacos , Descubrimiento de Drogas , Células HEK293 , Compuestos Heterocíclicos/síntesis química , Compuestos Heterocíclicos/farmacología , Ensayos Analíticos de Alto Rendimiento , Humanos , Espectroscopía de Resonancia Magnética , Modelos Moleculares , Conformación Molecular , Neuronas/efectos de los fármacos , Neuronas/enzimología , Síndromes de Neurotoxicidad/tratamiento farmacológico , Fosforilación , Especificidad por SustratoRESUMEN
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
Enlarged early endosomes have been observed in neurons and fibroblasts in Down syndrome (DS). These endosome abnormalities have been implicated in the early development of Alzheimer's disease (AD) pathology in these subjects. Here, we show the presence of enlarged endosomes in blood mononuclear cells and lymphoblastoid cell lines (LCLs) from individuals with DS using immunofluorescence and confocal microscopy. Genotype-phenotype correlations in LCLs carrying partial trisomies 21 revealed that triplication of a 2.56 Mb locus in 21q22.11 is associated with the endosomal abnormalities. This locus contains the gene encoding the phosphoinositide phosphatase synaptojanin 1 (SYNJ1), a key regulator of the signalling phospholipid phosphatidylinositol-4,5-biphosphate that has been shown to regulate clathrin-mediated endocytosis. We found that SYNJ1 transcripts are increased in LCLs from individuals with DS and that overexpression of SYNJ1 in a neuroblastoma cell line as well as in transgenic mice leads to enlarged endosomes. Moreover, the proportion of enlarged endosomes in fibroblasts from an individual with DS was reduced after silencing SYNJ1 expression with RNA interference. In LCLs carrying amyloid precursor protein (APP) microduplications causing autosomal dominant early-onset AD, enlarged endosomes were absent, suggesting that APP overexpression alone is not involved in the modification of early endosomes in this cell type. These findings provide new insights into the contribution of SYNJ1 overexpression to the endosomal changes observed in DS and suggest an attractive new target for rescuing endocytic dysfunction and lipid metabolism in DS and in AD.
Asunto(s)
Síndrome de Down/enzimología , Endosomas/química , Proteínas del Tejido Nervioso/genética , Proteínas del Tejido Nervioso/metabolismo , Monoéster Fosfórico Hidrolasas/genética , Monoéster Fosfórico Hidrolasas/metabolismo , Trisomía , Animales , Línea Celular , Mapeo Cromosómico , Cromosomas Humanos Par 21/enzimología , Cromosomas Humanos Par 21/genética , Síndrome de Down/genética , Endosomas/metabolismo , Humanos , Ratones , Ratones TransgénicosRESUMEN
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.