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1.
Brain ; 146(9): 3770-3782, 2023 09 01.
Artículo en Inglés | MEDLINE | ID: mdl-36883643

RESUMEN

Amyotrophic lateral sclerosis is a fatal and incurable neurodegenerative disease that mainly affects the neurons of the motor system. Despite the increasing understanding of its genetic components, their biological meanings are still poorly understood. Indeed, it is still not clear to which extent the pathological features associated with amyotrophic lateral sclerosis are commonly shared by the different genes causally linked to this disorder. To address this point, we combined multiomics analysis covering the transcriptional, epigenetic and mutational aspects of heterogenous human induced pluripotent stem cell-derived C9orf72-, TARDBP-, SOD1- and FUS-mutant motor neurons as well as datasets from patients' biopsies. We identified a common signature, converging towards increased stress and synaptic abnormalities, which reflects a unifying transcriptional program in amyotrophic lateral sclerosis despite the specific profiles due to the underlying pathogenic gene. In addition, whole genome bisulphite sequencing linked the altered gene expression observed in mutant cells to their methylation profile, highlighting deep epigenetic alterations as part of the abnormal transcriptional signatures linked to amyotrophic lateral sclerosis. We then applied multi-layer deep machine-learning to integrate publicly available blood and spinal cord transcriptomes and found a statistically significant correlation between their top predictor gene sets, which were significantly enriched in toll-like receptor signalling. Notably, the overrepresentation of this biological term also correlated with the transcriptional signature identified in mutant human induced pluripotent stem cell-derived motor neurons, highlighting novel insights into amyotrophic lateral sclerosis marker genes in a tissue-independent manner. Finally, using whole genome sequencing in combination with deep learning, we generated the first mutational signature for amyotrophic lateral sclerosis and defined a specific genomic profile for this disease, which is significantly correlated to ageing signatures, hinting at age as a major player in amyotrophic lateral sclerosis. This work describes innovative methodological approaches for the identification of disease signatures through the combination of multiomics analysis and provides novel knowledge on the pathological convergencies defining amyotrophic lateral sclerosis.


Asunto(s)
Esclerosis Amiotrófica Lateral , Células Madre Pluripotentes Inducidas , Enfermedades Neurodegenerativas , Humanos , Esclerosis Amiotrófica Lateral/metabolismo , Multiómica , Enfermedades Neurodegenerativas/metabolismo , Proteína C9orf72/genética , Superóxido Dismutasa-1/genética , Células Madre Pluripotentes Inducidas/metabolismo , Neuronas Motoras/metabolismo
2.
Cell Mol Life Sci ; 79(3): 189, 2022 Mar 14.
Artículo en Inglés | MEDLINE | ID: mdl-35286466

RESUMEN

Amyotrophic lateral sclerosis (ALS) is a rare neurodegenerative disorder characterized by progressive degeneration of motor neurons (MNs). Most cases are sporadic, whereas 10% are familial. The pathological mechanisms underlying the disease are partially understood, but it is increasingly being recognized that alterations in RNA metabolism and deregulation of microRNA (miRNA) expression occur in ALS. In this study, we performed miRNA expression profile analysis of iPSC-derived MNs and related exosomes from familial patients and healthy subjects. We identified dysregulation of miR-34a, miR-335 and miR-625-3p expression in both MNs and exosomes. These miRNAs regulate genes and pathways which correlate with disease pathogenesis, suggesting that studying miRNAs deregulation can contribute to deeply investigate the molecular mechanisms underlying the disease. We also assayed the expression profile of these miRNAs in the cerebrospinal fluid (CSF) of familial (fALS) and sporadic patients (sALS) and we identified a significant dysregulation of miR-34a-3p and miR-625-3p levels in ALS compared to controls. Taken together, all these findings suggest that miRNA analysis simultaneously performed in different human biological samples could represent a promising molecular tool to understand the etiopathogenesis of ALS and to develop new potential miRNA-based strategies in this new propitious therapeutic era.


Asunto(s)
Esclerosis Amiotrófica Lateral/genética , Exosomas/genética , Células Madre Pluripotentes Inducidas/fisiología , MicroARNs/genética , Neuronas Motoras/fisiología , Esclerosis Amiotrófica Lateral/líquido cefalorraquídeo , Esclerosis Amiotrófica Lateral/patología , Estudios de Casos y Controles , Comunicación Celular/genética , Células Cultivadas , Regulación de la Expresión Génica , Humanos , Células Madre Pluripotentes Inducidas/citología , Neuronas Motoras/patología
3.
Alzheimers Dement ; 19(4): 1245-1259, 2023 04.
Artículo en Inglés | MEDLINE | ID: mdl-35993441

RESUMEN

INTRODUCTION: The most common genetic cause of frontotemporal dementia (FTD) and amyotrophic lateral sclerosis (ALS) are hexanucleotide repeats in chromosome 9 open reading frame 72 (C9orf72). These repeats produce dipeptide repeat proteins with poly(PR) being the most toxic one. METHODS: We performed a kinome-wide CRISPR/Cas9 knock-out screen in human induced pluripotent stem cell (iPSC) -derived cortical neurons to identify modifiers of poly(PR) toxicity, and validated the role of candidate modifiers using in vitro, in vivo, and ex-vivo studies. RESULTS: Knock-down of NIMA-related kinase 6 (NEK6) prevented neuronal toxicity caused by poly(PR). Knock-down of nek6 also ameliorated the poly(PR)-induced axonopathy in zebrafish and NEK6 was aberrantly expressed in C9orf72 patients. Suppression of NEK6 expression and NEK6 activity inhibition rescued axonal transport defects in cortical neurons from C9orf72 patient iPSCs, at least partially by reversing p53-related DNA damage. DISCUSSION: We identified NEK6, which regulates poly(PR)-mediated p53-related DNA damage, as a novel therapeutic target for C9orf72 FTD/ALS.


Asunto(s)
Esclerosis Amiotrófica Lateral , Demencia Frontotemporal , Células Madre Pluripotentes Inducidas , Animales , Humanos , Esclerosis Amiotrófica Lateral/genética , Demencia Frontotemporal/genética , Células Madre Pluripotentes Inducidas/metabolismo , Proteína C9orf72/genética , Proteína p53 Supresora de Tumor/genética , Proteína p53 Supresora de Tumor/metabolismo , Sistemas CRISPR-Cas , Pez Cebra/genética , Pez Cebra/metabolismo , Neuronas/metabolismo , Expansión de las Repeticiones de ADN/genética , Quinasas Relacionadas con NIMA/genética , Quinasas Relacionadas con NIMA/metabolismo
4.
Acta Neuropathol ; 144(3): 465-488, 2022 09.
Artículo en Inglés | MEDLINE | ID: mdl-35895140

RESUMEN

A 'GGGGCC' repeat expansion in the first intron of the C9orf72 gene is the most common genetic cause of amyotrophic lateral sclerosis (ALS) and frontotemporal dementia (FTD). The exact mechanism resulting in these neurodegenerative diseases remains elusive, but C9 repeat RNA toxicity has been implicated as a gain-of-function mechanism. Our aim was to use a zebrafish model for C9orf72 RNA toxicity to identify modifiers of the ALS-linked phenotype. We discovered that the RNA-binding protein heterogeneous nuclear ribonucleoprotein K (HNRNPK) reverses the toxicity of both sense and antisense repeat RNA, which is dependent on its subcellular localization and RNA recognition, and not on C9orf72 repeat RNA binding. We observed HNRNPK cytoplasmic mislocalization in C9orf72 ALS patient fibroblasts, induced pluripotent stem cell (iPSC)-derived motor neurons and post-mortem motor cortex and spinal cord, in line with a disrupted HNRNPK function in C9orf72 ALS. In C9orf72 ALS/FTD patient tissue, we discovered an increased nuclear translocation, but reduced expression of ribonucleotide reductase regulatory subunit M2 (RRM2), a downstream target of HNRNPK involved in the DNA damage response. Last but not least, we showed that increasing the expression of HNRNPK or RRM2 was sufficient to mitigate DNA damage in our C9orf72 RNA toxicity zebrafish model. Overall, our study strengthens the relevance of RNA toxicity as a pathogenic mechanism in C9orf72 ALS and demonstrates its link with an aberrant DNA damage response, opening novel therapeutic avenues for C9orf72 ALS/FTD.


Asunto(s)
Esclerosis Amiotrófica Lateral , Demencia Frontotemporal , Enfermedad de Pick , Esclerosis Amiotrófica Lateral/patología , Animales , Proteína C9orf72/genética , Proteína C9orf72/metabolismo , Daño del ADN , Expansión de las Repeticiones de ADN/genética , Demencia Frontotemporal/patología , Ribonucleoproteína Heterogénea-Nuclear Grupo K/genética , Ribonucleoproteína Heterogénea-Nuclear Grupo K/metabolismo , Enfermedad de Pick/genética , ARN/metabolismo , ARN sin Sentido , Pez Cebra/genética , Pez Cebra/metabolismo
5.
Acta Neuropathol ; 135(3): 427-443, 2018 03.
Artículo en Inglés | MEDLINE | ID: mdl-29302778

RESUMEN

The exact mechanism underlying amyotrophic lateral sclerosis (ALS) and frontotemporal dementia (FTD) associated with the GGGGCC repeat expansion in C9orf72 is still unclear. Two gain-of-function mechanisms are possible: repeat RNA toxicity and dipeptide repeat protein (DPR) toxicity. We here dissected both possibilities using a zebrafish model for ALS. Expression of two DPRs, glycine-arginine and proline-arginine, induced a motor axonopathy. Similarly, expanded sense and antisense repeat RNA also induced a motor axonopathy and formed mainly cytoplasmic RNA foci. However, DPRs were not detected in these conditions. Moreover, stop codon-interrupted repeat RNA still induced a motor axonopathy and a synergistic role of low levels of DPRs was excluded. Altogether, these results show that repeat RNA toxicity is independent of DPR formation. This RNA toxicity, but not the DPR toxicity, was attenuated by the RNA-binding protein Pur-alpha and the autophagy-related protein p62. Our findings demonstrate that RNA toxicity, independent of DPR toxicity, can contribute to the pathogenesis of C9orf72-associated ALS/FTD.


Asunto(s)
Esclerosis Amiotrófica Lateral/metabolismo , Proteína C9orf72/metabolismo , ARN/metabolismo , Esclerosis Amiotrófica Lateral/patología , Animales , Animales Modificados Genéticamente , Axones/metabolismo , Axones/patología , Proteína C9orf72/genética , Expansión de las Repeticiones de ADN , Modelos Animales de Enfermedad , Escherichia coli , Técnicas de Transferencia de Gen , Humanos , Neuronas Motoras/metabolismo , Neuronas Motoras/patología , Pez Cebra
6.
Hum Mol Genet ; 22(9): 1783-90, 2013 May 01.
Artículo en Inglés | MEDLINE | ID: mdl-23364049

RESUMEN

Defects in axonal transport are thought to contribute to the pathogenesis of neurodegenerative disease. Because α-tubulin acetylation facilitates axonal transport, inhibition of the α-tubulin deacetylating enzymes, histone deacetylase 6 (Hdac6) and silent information regulator 2 (Sirt2), is thought to be an interesting therapeutic strategy for these conditions. Amyotrophic lateral sclerosis (ALS) is a one such rapidly progressive and fatal neurodegenerative disorder, in which axonal transport defects have been found in vitro and in vivo. To establish whether the inhibition of Hdac6 or Sirt2 may be of interest for ALS treatment, we investigated whether deleting Hdac6 or Sirt2 from the superoxide dismutase 1, SOD1(G93A) mouse affects the motor neuron degeneration in this ALS model. Deletion of Hdac6 significantly extended the survival of SOD1(G93A) mice without affecting disease onset, and maintained motor axon integrity. This protective effect was associated with increased α-tubulin acetylation. Deletion of Sirt2 failed to affect the disease course, but also did not modify α-tubulin acetylation. These findings show that Hdac6, rather than Sirt2, is a therapeutic target for the treatment of ALS. Moreover, Sirt2 appears not to be a major α-tubulin deacetylase in the nervous system.


Asunto(s)
Esclerosis Amiotrófica Lateral/genética , Eliminación de Gen , Histona Desacetilasas/genética , Sirtuina 2/genética , Acetilación , Esclerosis Amiotrófica Lateral/patología , Animales , Transporte Axonal/genética , Axones/patología , Modelos Animales de Enfermedad , Progresión de la Enfermedad , Femenino , Histona Desacetilasa 6 , Histona Desacetilasas/metabolismo , Masculino , Ratones , Ratones Transgénicos , Sirtuina 2/metabolismo , Superóxido Dismutasa/genética , Superóxido Dismutasa/metabolismo , Tubulina (Proteína)/metabolismo
7.
Brain ; 136(Pt 2): 471-82, 2013 Feb.
Artículo en Inglés | MEDLINE | ID: mdl-23378219

RESUMEN

Oligodendrocytes are well known targets for immune-mediated and infectious diseases, and have been suggested to play a role in neurodegeneration. Here, we report the involvement of oligodendrocytes and their progenitor cells in the ventral grey matter of the spinal cord in amyotrophic lateral sclerosis, a neurodegenerative disease of motor neurons. Degenerative changes in oligodendrocytes were abundantly present in human patients with amyotrophic lateral sclerosis and in an amyotrophic lateral sclerosis mouse model. In the mouse model, morphological changes in grey matter oligodendrocytes became apparent before disease onset, increasingly so during disease progression, and oligodendrocytes ultimately died. This loss was compensated by increased proliferation and differentiation of oligodendrocyte precursor cells. However, these newly differentiated oligodendrocytes were dysfunctional as suggested by their reduced myelin basic protein and monocarboxylate transporter 1 expression. Mutant superoxide dismutase 1 was found to directly affect monocarboxylate transporter 1 protein expression. Our data suggest that oligodendroglial dysfunction may be a contributor to motor neuron degeneration in amyotrophic lateral sclerosis.


Asunto(s)
Esclerosis Amiotrófica Lateral/patología , Esclerosis Amiotrófica Lateral/fisiopatología , Modelos Animales de Enfermedad , Oligodendroglía/patología , Esclerosis Amiotrófica Lateral/enzimología , Animales , Línea Celular Tumoral , Proliferación Celular , Genes Reporteros , Humanos , Ratones , Ratones Endogámicos C57BL , Ratones Transgénicos , Oligodendroglía/enzimología , Superóxido Dismutasa/genética
8.
J Neurol ; 2024 Sep 28.
Artículo en Inglés | MEDLINE | ID: mdl-39340541

RESUMEN

Amyotrophic lateral sclerosis (ALS) is a rare neurodegenerative disorder characterized by relentless and progressive loss of motor neurons. A molecular diagnosis, supported by the identification of specific biomarkers, might promote the definition of multiple biological subtypes of ALS, improving patient stratification and providing prognostic information. Here, we investigated the levels of neurofilament light chain (NfL), chitotriosidase (CHIT1) and microRNA-181b (miR-181b) in the cerebrospinal fluid (CSF) of ALS subjects (N = 210) as well as neurologically healthy and neurological disease controls (N = 218, including N = 74 with other neurodegenerative diseases) from a large European multicentric cohort, evaluating their specific or combined utility as diagnostic and prognostic biomarkers. NfL, CHIT1 and miR-181b all showed significantly higher levels in ALS subjects compared to controls, with NfL showing the most effective diagnostic performance. Importantly, all three biomarkers were increased compared to neurodegenerative disease controls and, specifically, to patients with Alzheimer's disease (AD; N = 44), with NfL and CHIT1 being also higher in ALS than in alpha-synucleinopathies (N = 22). Notably, ALS patients displayed increased CHIT1 levels despite having, compared to controls, a higher prevalence of a polymorphism lowering CHIT1 expression. While no relationship was found between CSF miR-181b and clinical measures in ALS (disease duration, functional disability, and disease progression rate), CSF NfL was the best independent predictor of disease progression and survival. This study deepens our knowledge of ALS biomarkers, highlighting the relative specificity of CHIT1 for ALS among neurodegenerative diseases and appraising the potential diagnostic utility of CSF miR-181b.

9.
Biochem Biophys Res Commun ; 429(3-4): 210-3, 2012 Dec 14.
Artículo en Inglés | MEDLINE | ID: mdl-23131553

RESUMEN

Amyotrophic Lateral Sclerosis (ALS) is a devastating neurodegenerative disease causing progressive paralysis of the patient followed by death on average 3-5 years after diagnosis. Disease pathology is multi-factorial including the process of excitotoxicity that induces cell death by cytosolic Ca(2+) overload. In this study, we increased the neuronal expression of an endoplasmic reticulum (ER) Ca(2+) release channel, inositol 1,4,5-trisphosphate receptor 2 (IP(3)R2), to assess whether increased cytosolic Ca(2+) originating from the ER is detrimental for neurons. Overexpression of IP(3)R2 in N2a cells using a Thy1.2-IP(3)R2 construct increases cytosolic Ca(2+) concentrations evoked by bradykinin. In addition, mice generated from this construct have increased expression of IP(3)R2 in the spinal cord and brain. This overexpression of IP(3)R2 does not affect symptom onset, but decreases disease duration and shortens the lifespan of the ALS mice significantly. These data suggest that ER Ca(2+) released by IP(3) receptors may be detrimental in ALS and that motor neurons are vulnerable to impaired Ca(2+) metabolism.


Asunto(s)
Esclerosis Amiotrófica Lateral/metabolismo , Receptores de Inositol 1,4,5-Trifosfato/biosíntesis , Neuronas/metabolismo , Esclerosis Amiotrófica Lateral/genética , Animales , Modelos Animales de Enfermedad , Retículo Endoplásmico/metabolismo , Humanos , Receptores de Inositol 1,4,5-Trifosfato/genética , Ratones , Ratones Endogámicos C57BL , Ratones Transgénicos , Superóxido Dismutasa/genética , Superóxido Dismutasa-1
10.
Mol Neurobiol ; 59(6): 3414-3430, 2022 Jun.
Artículo en Inglés | MEDLINE | ID: mdl-35320455

RESUMEN

Charcot-Marie-Tooth disease (CMT) is the most common inherited peripheral neuropathy, with currently no effective treatment or cure. CMT1A is caused by a duplication of the PMP22 gene, which leads to Schwann cell differentiation defects and dysmyelination of the peripheral nerves. The epigenetic regulator histone deacetylase 3 (HDAC3) has been shown to negatively regulate myelination as well as its associated signaling pathways, PI3K-AKT and MAPK-ERK. We showed that these signaling pathways are indeed downregulated in the C3-PMP22 mouse model, similar to what has been shown in the CMT1A rat model. We confirmed that early postnatal defects are present in the peripheral nerves of the C3-PMP22 mouse model, which led to a progressive reduction in axon caliber size and myelination. The aim of this study was to investigate whether pharmacological HDAC3 inhibition could be a valuable therapeutic approach for this CMT1A mouse model. We demonstrated that early treatment of CMT1A mice with the selective HDAC3 inhibitor RGFP966 increased myelination and myelin g-ratios, which was associated with improved electrophysiological recordings. However, a high dose of RGFP966 caused a decline in rotarod performance and a decline in overall grip strength. Additionally, macrophage presence in peripheral nerves was increased in RGFP966 treated CMT1A mice. We conclude that HDAC3 does not only play a role in regulating myelination but is also important in the neuroimmune modulation. Overall, our results indicate that correct dosing of HDAC3 inhibitors is of crucial importance if translated to a clinical setting for demyelinating forms of CMT or other neurological disorders.


Asunto(s)
Enfermedad de Charcot-Marie-Tooth , Enfermedades Desmielinizantes , Animales , Enfermedad de Charcot-Marie-Tooth/genética , Enfermedades Desmielinizantes/metabolismo , Modelos Animales de Enfermedad , Histona Desacetilasas/metabolismo , Ratones , Fosfatidilinositol 3-Quinasas/metabolismo , Ratas , Células de Schwann/metabolismo
11.
Mol Ther Methods Clin Dev ; 20: 508-519, 2021 Mar 12.
Artículo en Inglés | MEDLINE | ID: mdl-33614825

RESUMEN

Oligodendrocyte dysfunction has been implicated in the pathophysiology of amyotrophic lateral sclerosis (ALS), a neurodegenerative disorder characterized by progressive motor neuron loss. The failure of trophic support provided by oligodendrocytes is associated with a concomitant reduction in oligodendroglial monocarboxylate transporter 1 (MCT1) expression and is detrimental for the long-term survival of motor neuron axons. Therefore, we established an adeno-associated virus 9 (AAV9)-based platform by which MCT1 was targeted mostly to white matter oligodendrocytes to investigate whether this approach could provide a therapeutic benefit in the SOD1G93A mouse model of ALS. Despite good oligodendrocyte transduction and AAV-mediated MCT1 transgene expression, the disease outcome of SOD1G93A mice was not altered. Our study further increases our current understanding about the complex nature of oligodendrocyte pathology in ALS and provides valuable insights into the future development of therapeutic strategies to efficiently modulate these cells.

12.
Endocrinology ; 149(5): 2313-24, 2008 May.
Artículo en Inglés | MEDLINE | ID: mdl-18202120

RESUMEN

The rat anterior pituitary expresses beta(2)-adrenoceptors (ARs) on somatotrophs, lactotrophs, and corticotrophs. The present study investigates whether beta(1)-ARs exist in the anterior pituitary, in which cell type(s) they are found, and whether they are regulated by glucocorticoids. As determined by quantitative RT-PCR and Western immunoblotting, the rat anterior pituitary expressed beta(1)-AR mRNA and protein. Unlike the beta(2)-AR, expression decreased to very low levels after 5-d aggregate cell culture but was strongly up-regulated in a dose- and time-dependent manner by dexamethasone (DEX). Glucocorticoids attenuated isoproterenol-induced down-regulation of beta(1)-AR mRNA levels. As examined by immunofluorescence confocal microscopy, beta(1)-AR immunoreactivity was detected in a subpopulation of gonadotrophs, but not in somatotrophs, lactotrophs, corticotrophs, thyrotrophs, or folliculo-stellate cells. beta(1)-AR-immunoreactivity cells were often surrounded by cup-shaped lactotrophs. Consistent with these findings, beta(1)-AR mRNA was considerably more abundant in the gonadotrophic alphaT3-1 and LbetaT2 cell lines than in the GHFT, GH3, and TtT/GF cell lines. DEX did not affect expression level in the cell lines. DEX also failed to up-regulate beta(1)-AR mRNA levels in aggregates from a subpopulation enriched in large gonadotrophs obtained by gradient sedimentation. In contrast, excessive DEX-dependent up-regulation of beta(1)-AR mRNA was found in a subpopulation enriched in small nonhormonal cells. The present data indicate that beta(1)-AR is expressed in a subpopulation of gonadotrophs with a topographical relationship to lactotrophs. However, the glucocorticoid-induced up-regulation does not seem to occur directly in the gonadotrophs but within (an)other unidentified cell type(s), or is transduced by that cell type on gonadotrophs.


Asunto(s)
Gonadotrofos/metabolismo , Receptores Adrenérgicos beta 1/genética , Agonistas de Receptores Adrenérgicos beta 1 , Agonistas de Receptores Adrenérgicos beta 2 , Animales , Línea Celular , Dexametasona/farmacología , Regulación de la Expresión Génica/efectos de los fármacos , Glucocorticoides/farmacología , Hormonas Glicoproteicas de Subunidad alfa/metabolismo , Gonadotrofos/efectos de los fármacos , Isoproterenol/farmacología , Hormona Luteinizante de Subunidad beta/metabolismo , Masculino , Ratones , Adenohipófisis/efectos de los fármacos , Adenohipófisis/metabolismo , Adenohipófisis Porción Intermedia/efectos de los fármacos , Adenohipófisis Porción Intermedia/metabolismo , ARN Mensajero/metabolismo , Ratas , Ratas Wistar , Receptores Adrenérgicos beta 1/metabolismo , Receptores Adrenérgicos beta 2/genética , Receptores Adrenérgicos beta 2/metabolismo
13.
Cell Rep ; 24(3): 529-537.e4, 2018 07 17.
Artículo en Inglés | MEDLINE | ID: mdl-30021151

RESUMEN

RNA-binding protein aggregation is a pathological hallmark of several neurodegenerative disorders, including amyotrophic lateral sclerosis (ALS) and frontotemporal lobar degeneration (FTLD). To gain better insight into the molecular interactions underlying this process, we investigated FUS, which is mutated and aggregated in both ALS and FTLD. We generated a Drosophila model of FUS toxicity and identified a previously unrecognized synergistic effect between the N-terminal prion-like domain and the C-terminal arginine-rich domain to mediate toxicity. Although the prion-like domain is generally considered to mediate aggregation of FUS, we find that arginine residues in the C-terminal low-complexity domain are also required for maturation of FUS in cellular stress granules. These data highlight an important role for arginine-rich domains in the pathology of RNA-binding proteins.


Asunto(s)
Proteínas de Drosophila/química , Proteínas de Drosophila/toxicidad , Drosophila melanogaster/metabolismo , Ribonucleoproteína Heterogénea-Nuclear Grupo F-H/química , Ribonucleoproteína Heterogénea-Nuclear Grupo F-H/toxicidad , Secuencia de Aminoácidos , Animales , Arginina/metabolismo , Línea Celular Tumoral , Proteínas de Drosophila/genética , Ribonucleoproteína Heterogénea-Nuclear Grupo F-H/genética , Humanos , Actividad Motora , Neuronas Motoras/patología , Degeneración Nerviosa/patología , Dominios Proteicos , Relación Estructura-Actividad
14.
Hum Mutat ; 28(8): 830, 2007 Aug.
Artículo en Inglés | MEDLINE | ID: mdl-17623484

RESUMEN

The 27 kDa heat shock protein 1 (HSP27) is a member of the ubiquitously expressed small heat shock protein family and has pleiotropic cytoprotective functions. Since HSP27 may act as a motor neuron survival factor, we analyzed the genetic contribution of the human HSPB1 gene (HSPB1) to the etiology of amyotrophic lateral sclerosis (ALS). In a cohort of sporadic ALS patients, we identified three rare genetic variations and one of which (c.-217T>C) targeted a conserved nucleotide of the Heat Shock Element (HSE) in the HSPB1 promoter. Since binding of Heat Shock Factor 1 (HSF1) to this HSE is essential for stress-induced transcription of HSPB1, we examined the effect of the c.-217C allele on transcriptional activity and HSF binding. The basal promoter activity of the HSPB1 c.-217C mutant allele decreased to 50% as compared to the wild-type promoter in neuronal and non-neuronal cells. Following heat shock, the HSE variant attenuated significantly the stress-related increase in transcription. Electrophoretic mobility shift assays demonstrated a dramatically reduced HSF-binding to the c.-217C mutant allele as compared to the c.-217T wild-type allele. In conclusion, our study underscores the importance of the c.-217T nucleotide for HSF binding and heat inducibility of HSPB1. Therefore, our study suggests that the functional HSPB1 variant may represent a genetic modifier in the pathogenesis of motor neuron disease; however, it is necessary to confirm this HSPB1 variant in additional ALS patients.


Asunto(s)
Proteínas de Choque Térmico/genética , Respuesta al Choque Térmico/genética , Mutación/genética , Proteínas de Neoplasias/genética , Animales , Secuencia de Bases , Células COS , Chlorocebus aethiops , Secuencia de Consenso , Análisis Mutacional de ADN , Ensayo de Cambio de Movilidad Electroforética , Femenino , Proteínas de Choque Térmico HSP27 , Humanos , Masculino , Persona de Mediana Edad , Chaperonas Moleculares , Datos de Secuencia Molecular , Regiones Promotoras Genéticas , Unión Proteica , Elementos de Respuesta/genética , Transcripción Genética
15.
Endocrinology ; 146(9): 3985-98, 2005 Sep.
Artículo en Inglés | MEDLINE | ID: mdl-15932930

RESUMEN

A side population (SP) has been identified in a number of tissues, where it typically represents a small population enriched in stem/progenitor cells. In this study we show that the adult mouse anterior pituitary (AP) also contains a characteristic SP displaying verapamil-sensitive Hoechst dye efflux capacity. A majority of the SP cells express stem cell antigen 1 at a high level (Sca1high). Using (semi)quantitative RT-PCR and immunofluorescence, we characterized the Sca1high SP as a population enriched in cells expressing stem/progenitor cell-associated factors and components of the Notch, Wnt, and sonic hedgehog signaling pathways, functional in stem cell homeostasis as well as in early pituitary embryogenesis. Lhx4, a transcription factor pivotal for early embryonic development of the AP, was only detected in the Sca1high SP, whereas Lhx3, in contrast to Lhx4 not down-regulated after AP development, was only found in the main population. The Sca1high SP was depleted from cells expressing phenotypic markers of differentiated AP cells (hormones), but contained a small proportion of folliculo-stellate cells. Stem cells of many tissues can clonally expand to nonadherent spheres in culture. Clonal spheres also developed in AP cell cultures. Spheres showed an expression pattern resembling that of Sca1high SP cells. Moreover, the sphere-initiating cells of the pituitary segregated to the SP and not to the main population. In conclusion, we show that the adult pituitary contains a hitherto undescribed population of cells with SP phenotype and clonal expansion capacity. These cells express (signaling) molecules generally found in stem/progenitor cells and/or operative during pituitary early embryonic development. These characteristics are supportive of a stem/progenitor cell phenotype.


Asunto(s)
Adenohipófisis/citología , Células Madre/citología , Factores de Edad , Animales , Antígenos CD34/genética , Ataxina-1 , Ataxinas , Biomarcadores , Separación Celular , Células Cultivadas , Femenino , Expresión Génica , Proteínas Hedgehog , Proteínas de Homeodominio/genética , Péptidos y Proteínas de Señalización Intercelular/genética , Proteínas con Homeodominio LIM , Masculino , Proteínas de la Membrana/genética , Ratones , Ratones Endogámicos , Proteínas del Tejido Nervioso/genética , Proteínas Nucleares/genética , Fenotipo , Adenohipófisis/embriología , Proteínas Proto-Oncogénicas c-kit/genética , Receptores Notch , Transactivadores/genética , Factores de Transcripción/genética , Proteínas Wnt
16.
Neurobiol Aging ; 31(12): 2185-91, 2010 Dec.
Artículo en Inglés | MEDLINE | ID: mdl-19185395

RESUMEN

Influx of Ca(2+) ions through the α-amino-3-hydroxy-5-methylisoxazole propionic acid (AMPA) receptors is toxic to neurons and contributes to motor neuron degeneration observed in amyotrophic lateral sclerosis (ALS). The Ca(2+) permeability of the AMPA receptor depends on its subunit composition. If the GluR2 subunit is present in the receptor complex, the AMPA receptor is impermeable to Ca(2+). In this study, we identified vascular endothelial growth factor-A (VEGF) as a GluR2 inducing molecule. Cultured motor neurons pretreated with VEGF displayed higher GluR2 levels. This resulted in AMPA receptor currents with a low relative Ca(2+) permeability and in motor neurons that were less vulnerable to AMPA receptor-mediated excitotoxicity. This effect of VEGF was mediated through the VEGFR2 present on the motor neurons and was due to stimulation of GluR2 transcription. Intracerebroventricular treatment with VEGF similarly induced GluR2 expression in the ventral spinal cord of rats and this mechanism contributes to the protective effect of VEGF on motor neurons.


Asunto(s)
Neuronas Motoras/metabolismo , Degeneración Nerviosa/terapia , Neurotoxinas/antagonistas & inhibidores , Receptores AMPA/biosíntesis , Regulación hacia Arriba/fisiología , Factor A de Crecimiento Endotelial Vascular/fisiología , Animales , Señalización del Calcio/fisiología , Células Cultivadas , Técnicas de Cocultivo , Inyecciones Intraventriculares/métodos , Neuronas Motoras/fisiología , Degeneración Nerviosa/metabolismo , Degeneración Nerviosa/fisiopatología , Neurotoxinas/farmacología , Ratas , Ratas Wistar , Receptores AMPA/genética , Receptores AMPA/fisiología , Médula Espinal/citología , Médula Espinal/metabolismo , Médula Espinal/fisiología , Factor A de Crecimiento Endotelial Vascular/administración & dosificación
17.
Hum Mol Genet ; 16(19): 2359-65, 2007 Oct 01.
Artículo en Inglés | MEDLINE | ID: mdl-17636250

RESUMEN

The development of small animal models is of major interest to unravel the pathogenesis and treatment of neurodegenerative diseases, especially because of their potential in large-scale chemical and genetic screening. We have investigated the zebrafish as a model to study amyotrophic lateral sclerosis (ALS), a fatal neurodegenerative disorder characterized by the selective loss of motor neurons, caused by mutations in superoxide dismutase 1 (SOD1) in a subset of patients. Overexpression of mutant human SOD1 in zebrafish embryos induced a motor axonopathy that was specific, dose-dependent and found for all mutations studied. Moreover, using this newly established animal model for ALS, we investigated the role of a known modifier in the disease: vascular endothelial growth factor (VEGF). Lowering VEGF induced a more severe phenotype, whereas upregulating VEGF rescued the mutant SOD1 axonopathy. This novel zebrafish model underscores the potential of VEGF for the treatment of ALS and furthermore will permit large-scale genetic and chemical screening to facilitate the identification of new therapeutic targets in motor neuron disease.


Asunto(s)
Enfermedad de la Neurona Motora/genética , Mutación , Superóxido Dismutasa/genética , Animales , Western Blotting , Humanos , Inmunohistoquímica , Enfermedad de la Neurona Motora/enzimología , Enfermedad de la Neurona Motora/patología , ARN Mensajero/genética , ARN Mensajero/metabolismo , Superóxido Dismutasa/metabolismo , Superóxido Dismutasa-1 , Factor A de Crecimiento Endotelial Vascular/genética , Factor A de Crecimiento Endotelial Vascular/metabolismo , Pez Cebra
18.
Proc Natl Acad Sci U S A ; 104(37): 14825-30, 2007 Sep 11.
Artículo en Inglés | MEDLINE | ID: mdl-17804792

RESUMEN

Influx of Ca(2+) ions through alpha-amino-3-hydroxy-5-methylisoxazole-4-propionic acid (AMPA) receptors contributes to neuronal damage in stroke, epilepsy, and neurodegenerative disorders such as ALS. The Ca(2+) permeability of AMPA receptors is largely determined by the glutamate receptor 2 (GluR2) subunit, receptors lacking GluR2 being permeable to Ca(2+) ions. We identified a difference in GluR2 expression in motor neurons from two rat strains, resulting in a difference in vulnerability to AMPA receptor-mediated excitotoxicity both in vitro and in vivo. Astrocytes from the ventral spinal cord were found to mediate this difference in GluR2 expression in motor neurons. The presence of ALS-causing mutant superoxide dismutase 1 in astrocytes abolished their GluR2-regulating capacity and thus affected motor neuron vulnerability to AMPA receptor-mediated excitotoxicity. These results reveal a mechanism through which astrocytes influence neuronal functioning in health and disease.


Asunto(s)
Astrocitos/fisiología , Neuronas Motoras/efectos de los fármacos , Neuronas Motoras/metabolismo , Receptores AMPA/genética , Receptores AMPA/metabolismo , Esclerosis Amiotrófica Lateral/metabolismo , Animales , Astrocitos/enzimología , Calcio/metabolismo , Células Cultivadas , Técnicas de Cocultivo , Agonistas de Aminoácidos Excitadores/farmacología , Femenino , Expresión Génica/fisiología , Genes Reporteros , Luciferasas/metabolismo , Modelos Biológicos , Neuronas Motoras/fisiología , Mutación , Técnicas de Placa-Clamp , Proteínas/metabolismo , ARN Mensajero/metabolismo , Ratas , Ratas Sprague-Dawley , Ratas Wistar , Médula Espinal/citología , Superóxido Dismutasa/genética , Ácido alfa-Amino-3-hidroxi-5-metil-4-isoxazol Propiónico/farmacología
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