RESUMEN
The dysfunction of TAR DNA-binding protein 43 (TDP-43) is implicated in various neurodegenerative diseases, though the specific contributions of its toxic gain-of-function versus loss-of-function effects remain unclear. This study investigates the impact of TARDBP loss on cellular metabolism and viability using human-induced pluripotent stem cell-derived motor neurons and HeLa cells. TARDBP silencing led to reduced metabolic activity and cell growth, accompanied by neurite degeneration and decreased oxygen consumption rates in both cell types. Notably, TARDBP depletion induced a metabolic shift, impairing ATP production, increasing metabolic inflexibility, and elevating free radical production, indicating a critical role for TDP-43 in maintaining cellular bioenergetics. Furthermore, TARDBP loss triggered non-apoptotic cell death, increased ACSL4 expression, and reprogrammed lipid metabolism towards lipid droplet accumulation, while paradoxically enhancing resilience to ferroptosis inducers. Overall, our findings highlight those essential cellular traits such as ATP production, metabolic activity, oxygen consumption, and cell survival are highly dependent on TARDBP function.
RESUMEN
Spinal Muscular Atrophy (SMA) is a severe genetic neuromuscular disorder that occurs in childhood and is caused by misexpression of the survival motor neuron (SMN) protein. SMN reduction induces spinal cord motoneuron (MN) degeneration, which leads to progressive muscular atrophy and weakness. The link between SMN deficiency and the molecular mechanisms altered in SMA cells remains unclear. Autophagy, deregulation of intracellular survival pathways and ERK hyperphosphorylation may contribute to SMN-reduced MNs collapse, offering a useful strategy to develop new therapies to prevent neurodegeneration in SMA. Using SMA MN in vitro models, the effect of pharmacological inhibition of PI3K/Akt and ERK MAPK pathways on SMN and autophagy markers modulation was studied by western blot analysis and RT-qPCR. Experiments involved primary cultures of mouse SMA spinal cord MNs and differentiated SMA human MNs derived from induced pluripotent stem cells (iPSCs). Inhibition of the PI3K/Akt and the ERK MAPK pathways reduced SMN protein and mRNA levels. Importantly, mTOR phosphorylation, p62, and LC3-II autophagy markers protein level were decreased after ERK MAPK pharmacological inhibition. Furthermore, the intracellular calcium chelator BAPTA prevented ERK hyperphosphorylation in SMA cells. Our results propose a link between intracellular calcium, signaling pathways, and autophagy in SMA MNs, suggesting that ERK hyperphosphorylation may contribute to autophagy deregulation in SMN-reduced MNs.
RESUMEN
Spinal Muscular Atrophy (SMA) is a genetic neuromuscular disorder caused by reduction of the ubiquitously expressed protein Survival Motor Neuron (SMN). Low levels of SMN impact on spinal cord motoneurons (MNs) causing their degeneration and progressive muscle weakness and atrophy. To study the molecular mechanisms leading to cell loss in SMN-reduced MNs, we analyzed the NF-κB intracellular pathway in SMA models. NF-κB pathway activation is required for survival and regulates SMN levels in cultured MNs. Here we describe that NF-κB members, inhibitor of kappa B kinase beta (IKKß), and RelA, were reduced in SMA mouse and human MNs. In addition, we observed that Gemin3 protein level was decreased in SMA MNs, but not in non-neuronal SMA cells. Gemin3 is a core member of the SMN complex responsible for small nuclear ribonucleoprotein biogenesis, and it regulates NF-κB activation through the mitogen-activated protein kinase TAK1. Our experiments showed that Gemin3 knockdown reduced SMN, IKKß, and RelA protein levels, and caused significant neurite degeneration. Overexpression of SMN increased Gemin3 protein in SMA MNs, but did not prevent neurite degeneration in Gemin3 knockdown cells. These data indicated that Gemin3 reduction may contribute to cell degeneration in SMA MNs.
RESUMEN
Spinal muscular atrophy (SMA) is a neuromuscular genetic disease caused by reduced survival motor neuron (SMN) protein. SMN is ubiquitous and deficient levels cause spinal cord motoneurons (MNs) degeneration and muscle atrophy. Nevertheless, the mechanism by which SMN reduction in muscle contributes to SMA disease is not fully understood. Therefore, studies evaluating atrophy mechanisms in SMA muscles will contribute to strengthening current knowledge of the pathology. Here we propose to evaluate autophagy in SMA muscle, a pathway altered in myotube atrophy. We analized autophagy proteins and mTOR in muscle biopsies, fibroblasts, and lymphoblast cell lines from SMA patients and in gastrocnemius muscles from a severe SMA mouse model. Human MNs differentiated from SMA and unaffected control iPSCs were also included in the analysis of the autophagy. Muscle biopsies, fibroblasts, and lymphoblast cell lines from SMA patients showed reduction of the autophagy marker LC3-II. In SMA mouse gastrocnemius, we observed lower levels of LC3-II, Beclin 1, and p62/SQSTM1 proteins at pre-symptomatic stage. mTOR phosphorylation at Ser2448 was decreased in SMA muscle cells. However, in mouse and human cultured SMA MNs mTOR phosphorylation and LC3-II levels were increased. These results suggest a differential regulation in SMA of the autophagy process in muscle cells and MNs. Opposite changes in autophagy proteins and mTOR phosphorylation between muscle cells and neurons were observed. These differences may reflect a specific response to SMN reduction, which could imply diverse tissue-dependent reactions to therapies that should be taken into account when treating SMA patients.
Asunto(s)
Autofagia/fisiología , Neuronas Motoras/patología , Músculo Esquelético/patología , Atrofia Muscular Espinal/patología , Animales , Femenino , Humanos , Masculino , Ratones , Neuronas Motoras/metabolismo , Músculo Esquelético/metabolismo , Atrofia Muscular Espinal/metabolismoRESUMEN
Spinal Muscular Atrophy (SMA) is a severe neuromuscular disorder caused by loss of the Survival Motor Neuron 1 gene (SMN1). Due to this depletion of the survival motor neuron (SMN) protein, the disease is characterized by the degeneration of spinal cord motoneurons (MNs), progressive muscular atrophy, and weakness. Nevertheless, the ultimate cellular and molecular mechanisms leading to cell loss in SMN-reduced MNs are only partially known. We have investigated the activation of apoptotic and neuronal survival pathways in several models of SMA cells. Even though the antiapoptotic proteins FAIM-L and XIAP were increased in SMA MNs, the apoptosis executioner cleaved-caspase-3 was also elevated in these cells, suggesting the activation of the apoptosis process. Analysis of the survival pathway PI3K/Akt showed that Akt phosphorylation was reduced in SMA MNs and pharmacological inhibition of PI3K diminished SMN and Gemin2 at transcriptional level in control MNs. In contrast, ERK phosphorylation was increased in cultured mouse and human SMA MNs. Our observations suggest that apoptosis is activated in SMA MNs and that Akt phosphorylation reduction may control cell degeneration, thereby regulating the transcription of Smn and other genes related to SMN function.
Asunto(s)
Apoptosis/fisiología , Neuronas Motoras/metabolismo , Neuronas Motoras/patología , Atrofia Muscular Espinal/fisiopatología , Transducción de Señal/fisiología , Animales , Supervivencia Celular , Humanos , RatonesRESUMEN
Spinal muscular atrophy (SMA) is a severe neuromuscular disorder caused by loss of the survival motor neuron 1 (SMN1) gene. SMA is characterized by the degeneration of spinal cord motoneurons (MNs), progressive skeletal muscle atrophy, and weakness. The cellular and molecular mechanisms causing MN loss of function are only partially known. Recent advances in SMA research postulate the role of calpain protease regulating survival motor neuron (SMN) protein and the positive effect on SMA phenotype of treatment with calpain inhibitors. We analyzed the level of calpain pathway members in mice and human cellular SMA models. Results indicate an increase of calpain activity in SMN-reduced MNs. Spinal cord analysis of SMA mice treated with calpeptin, a calpain inhibitor, showed an increase of SMN, calpain, and its endogenous inhibitor calpastatin in MNs. Finally, in vitro calpeptin treatment prevented microtubule-associated protein 1A/1B-light chain 3 (LC3) increase in MNs neurites, indicating that calpain inhibition may reduce autophagosome accumulation in neuron prolongations, but not in soma. Thus, our results show that calpain activity is increased in SMA MNs and its inhibition may have a beneficial effect on SMA phenotype through the increase of SMN in spinal cord MNs.
Asunto(s)
Calpaína/metabolismo , Neuronas Motoras/enzimología , Neuronas Motoras/patología , Atrofia Muscular Espinal/enzimología , Atrofia Muscular Espinal/patología , Animales , Proteínas de Unión al Calcio/metabolismo , Proteínas Portadoras/metabolismo , Diferenciación Celular/efectos de los fármacos , Línea Celular , Supervivencia Celular/efectos de los fármacos , Células Cultivadas , Dipéptidos/farmacología , Modelos Animales de Enfermedad , Fibroblastos/efectos de los fármacos , Fibroblastos/patología , Humanos , Células Madre Pluripotentes Inducidas/efectos de los fármacos , Células Madre Pluripotentes Inducidas/metabolismo , Ratones , Ratones Mutantes , Proteínas de Microfilamentos/metabolismo , Proteínas Asociadas a Microtúbulos/metabolismo , Neuronas Motoras/efectos de los fármacos , Proteolisis/efectos de los fármacos , Médula Espinal/embriología , Médula Espinal/patología , Proteína 1 para la Supervivencia de la Neurona Motora/metabolismoRESUMEN
Disruption in membrane excitability contributes to malfunction and differential vulnerability of specific neuronal subpopulations in a number of neurological diseases. The adaptor protein p11, and background potassium channel TASK1, have overlapping distributions in the CNS. Here, we report that the transcription factor Sp1 controls p11 expression, which impacts on excitability by hampering functional expression of TASK1. In the SOD1-G93A mouse model of ALS, Sp1-p11-TASK1 dysregulation contributes to increased excitability and vulnerability of motor neurons. Interference with either Sp1 or p11 is neuroprotective, delaying neuron loss and prolonging lifespan in this model. Nitrosative stress, a potential factor in human neurodegeneration, stimulated Sp1 expression and human p11 promoter activity, at least in part, through a Sp1-binding site. Disruption of Sp1 or p11 also has neuroprotective effects in a traumatic model of motor neuron degeneration. Together our work suggests the Sp1-p11-TASK1 pathway is a potential target for treatment of degeneration of motor neurons.
Asunto(s)
Esclerosis Amiotrófica Lateral/patología , Anexina A2/metabolismo , Neuronas Motoras/patología , Degeneración Nerviosa/patología , Proteínas del Tejido Nervioso/genética , Canales de Potasio de Dominio Poro en Tándem/genética , Proteínas S100/metabolismo , Factor de Transcripción Sp1/metabolismo , Esclerosis Amiotrófica Lateral/etiología , Animales , Membrana Celular/patología , Modelos Animales de Enfermedad , Femenino , Regulación de la Expresión Génica , Técnicas de Silenciamiento del Gen , Células HEK293 , Humanos , Masculino , Potenciales de la Membrana , Ratones , Ratones Transgénicos , Neuronas Motoras/citología , Degeneración Nerviosa/etiología , Proteínas del Tejido Nervioso/metabolismo , Canales de Potasio de Dominio Poro en Tándem/metabolismo , Cultivo Primario de Células , Regiones Promotoras Genéticas , Ratas , Factor de Transcripción Sp1/genética , Médula Espinal/citología , Médula Espinal/patologíaRESUMEN
Spinal muscular atrophy (SMA), a leading genetic cause of infant death, is caused by the loss of survival motor neuron 1 (SMN1) gene. SMA is characterized by the degeneration and loss of spinal cord motoneurons (MNs), muscular atrophy, and weakness. SMN2 is the centromeric duplication of the SMN gene, whose numbers of copies determine the intracellular levels of SMN protein and define the disease onset and severity. It has been demonstrated that elevating SMN levels can be an important strategy in treating SMA and can be achieved by several mechanisms, including promotion of protein stability. SMN protein is a direct target of the calcium-dependent protease calpain and induces its proteolytic cleavage in muscle cells. In this study, we examined the involvement of calpain in SMN regulation on MNs. In vitro experiments showed that calpain activation induces SMN cleavage in CD1 and SMA mouse spinal cord MNs. Additionally, calpain 1 knockdown or inhibition increased SMN level and prevent neurite degeneration in these cells. We examined the effects of calpain inhibition on the phenotype of two severe SMA mouse models. Treatment with the calpain inhibitor, calpeptin, significantly improved the lifespan and motor function of these mice. Our observations show that calpain regulates SMN level in MNs and calpeptin administration improves SMA phenotype demonstrating the potential utility of calpain inhibitors in SMA therapy.
Asunto(s)
Calpaína/antagonistas & inhibidores , Neuronas Motoras/patología , Atrofia Muscular Espinal/patología , Médula Espinal/patología , Proteína 1 para la Supervivencia de la Neurona Motora/metabolismo , Animales , Calpaína/metabolismo , Células Cultivadas , Dipéptidos/administración & dosificación , Dipéptidos/farmacología , Técnicas de Silenciamiento del Gen , Glicoproteínas/farmacología , Potenciales de la Membrana/efectos de los fármacos , Ratones Transgénicos , Actividad Motora/efectos de los fármacos , Neuronas Motoras/efectos de los fármacos , Neuronas Motoras/metabolismo , Atrofia Muscular Espinal/complicaciones , Atrofia Muscular Espinal/fisiopatología , Mutación/genética , Degeneración Nerviosa/complicaciones , Degeneración Nerviosa/patología , Neuritas/efectos de los fármacos , Neuritas/metabolismo , Fenotipo , Potasio/farmacologíaRESUMEN
C-bouton-type cholinergic afferents exert an important function in controlling motoneuron (MN) excitability. During the immunocytochemical analysis of the role of c-Jun in MNs with a monoclonal (clone Y172) antibody against phospho (p)-c-Jun (serine [Ser]63), unexpected labeling was identified in the cell body cytoplasm. As predicted for c-Jun in adult spinal cord, very few, if any MNs exhibited nuclear immunoreactivity with the Y172 antibody; conversely, virtually all MNs displayed strong Y172 immunostaining in cytoplasmic structures scattered throughout the soma and proximal dendrites. The majority of these cytoplasmic Y172-positive profiles was closely associated with VAChT-positive C-boutons, but not with other types of nerve afferents contacting MNs. Ultrastructural analysis revealed that cytoplasmic Y172 immunostaining was selectively located at the subsurface cistern (SSC) of C-boutons and also in the inner areas of the endoplasmic reticulum (ER). We also described changes in cytoplasmic Y172 immunoreactivity in injured and degenerating MNs. Moreover, we noticed that MNs from NRG1 type III-overexpressing transgenic mice, which show abnormally expanded SSCs, exhibited an increase in the density and size of peripherally located Y172-positive profiles. A similar immunocytochemical pattern to that of the Y172 antibody in MNs was found with a polyclonal antibody against p-c-Jun (Ser63) but not with another polyclonal antibody that recognizes c-Jun phosphorylated at a different site. No differential band patterns were found by western blotting with any of the antibodies against c-Jun or p-c-Jun used in our study. In cultured MNs, Y172-positive oval profiles were distributed in the cell body and proximal dendrites. The in vitro lentiviral-based knockdown of c-Jun resulted in a dramatic decrease in nuclear Y172 immunostaining in MNs without any reduction in the density of cytoplasmic Y172-positive profiles, suggesting that the synaptic antigen recognized by the antibody corresponds to a C-bouton-specific protein other than p-c-Jun. Our results lay the foundation for further studies aimed at identifying this protein and determining its role in this particular type of synapse.
RESUMEN
Survival motor neuron (SMN) protein deficiency causes the genetic neuromuscular disorder spinal muscular atrophy (SMA), characterized by spinal cord motoneuron degeneration. Since SMN protein level is critical to disease onset and severity, analysis of the mechanisms involved in SMN stability is one of the central goals of SMA research. Here, we describe the role of several members of the NF-κB pathway in regulating SMN in motoneurons. NF-κB is one of the main regulators of motoneuron survival and pharmacological inhibition of NF-κB pathway activity also induces mouse survival motor neuron (Smn) protein decrease. Using a lentiviral-based shRNA approach to reduce the expression of several members of NF-κB pathway, we observed that IKK and RelA knockdown caused Smn reduction in mouse-cultured motoneurons whereas IKK or RelB knockdown did not. Moreover, isolated motoneurons obtained from the severe SMA mouse model showed reduced protein levels of several NF-κB members and RelA phosphorylation. We describe the alteration of NF-κB pathway in SMA cells. In the context of recent studies suggesting regulation of altered intracellular pathways as a future pharmacological treatment of SMA, we propose the NF-κB pathway as a candidate in this new therapeutic approach.
Asunto(s)
Regulación de la Expresión Génica/fisiología , Neuronas Motoras/metabolismo , FN-kappa B/metabolismo , Transducción de Señal/fisiología , Médula Espinal/metabolismo , Proteína 1 para la Supervivencia de la Neurona Motora/metabolismo , Animales , Células Cultivadas , Modelos Animales de Enfermedad , Femenino , Masculino , Ratones , Ratones Transgénicos , Neuronas Motoras/citología , Fosforilación , Médula Espinal/citología , Proteína 1 para la Supervivencia de la Neurona Motora/genéticaRESUMEN
Spinal muscular atrophy (SMA) is a recessive autosomal neuromuscular disease, due to homozygous mutations or deletions in the telomeric survival motoneuron gene 1 (SMN1). SMA is characterized by motor impairment, muscle atrophy, and premature death following motor neuron (MN) degeneration. Emerging evidence suggests that dysregulation of autophagy contributes to MN degeneration. We here investigated the role of autophagy in the SMNdelta7 mouse model of SMA II (intermediate form of the disease) which leads to motor impairment by postnatal day 5 (P5) and to death by P13. We first showed by immunoblots that Beclin 1 and LC3-II expression levels increased in the lumbar spinal cord of the SMA pups. Electron microscopy and immunofluorescence studies confirmed that autophagic markers were enhanced in the ventral horn of SMA pups. To clarify the role of autophagy, we administered intracerebroventricularly (at P3) either an autophagy inhibitor (3-methyladenine, 3-MA), or an autophagy inducer (rapamycin) in SMA pups. Motor behavior was assessed daily with different tests: tail suspension, righting reflex, and hindlimb suspension tests. 3-MA significantly improved motor performance, extended the lifespan, and delayed MN death in lumbar spinal cord (10372.36 ± 2716 MNs) compared to control-group (5148.38 ± 94 MNs). Inhibition of autophagy by 3-MA suppressed autophagosome formation, reduced the apoptotic activation (cleaved caspase-3 and Bcl2) and the appearance of terminal deoxynucleotidyl transferase dUTP nick end labeling (TUNEL)-positive neurons, underlining that apoptosis and autophagy pathways are intricately intertwined. Therefore, autophagy is likely involved in MN death in SMA II, suggesting that it might represent a promising target for delaying the progression of SMA in humans as well.
Asunto(s)
Autofagia/efectos de los fármacos , Neuronas Motoras/enzimología , Neuronas Motoras/metabolismo , Atrofia Muscular Espinal/tratamiento farmacológico , Atrofia Muscular Espinal/metabolismo , Adenina/análogos & derivados , Adenina/uso terapéutico , Animales , Apoptosis/efectos de los fármacos , Modelos Animales de Enfermedad , Genotipo , Etiquetado Corte-Fin in Situ , Ratones , Sirolimus/farmacologíaRESUMEN
During development, motoneurons experience significant changes in their size and in the number and strength of connections that they receive, which requires adaptive changes in their passive and active electrical properties. Even after reaching maturity, motoneurons continue to adjust their intrinsic excitability and synaptic activity for proper functioning of the sensorimotor circuit in accordance with physiological demands. Likewise, if some elements of the circuit become dysfunctional, the system tries to compensate for the alterations to maintain appropriate function. In Spinal Muscular Atrophy (SMA), a severe motor disease, spinal motoneurons receive less excitation from glutamatergic sensory fibers and interneurons and are electrically hyperexcitable. Currently, the origin and relationship among these alterations are not completely established. In this study, we investigated whether Survival of Motor Neuron (SMN), the ubiquitous protein defective in SMA, regulates the excitability of motoneurons before and after the establishment of the synaptic contacts. To this end, we performed patch-clamp recordings in embryonic spinal motoneurons forming complex synaptic networks in primary cultures, and in differentiated NSC-34 motoneuron-like cells in the absence of synaptic contacts. Our results show that in both conditions, Smn-deficient cells displayed lower action potential threshold, greater action potential amplitudes, and larger density of voltage-dependent sodium currents than cells with normal Smn-levels. These results indicate that Smn participates in the regulation of the cell-autonomous excitability of motoneurons at an early stage of development. This finding may contribute to a better understanding of motoneuron excitability in SMA during the development of the disease.
Asunto(s)
Autofagia/fisiología , Neuronas Motoras/metabolismo , Atrofia Muscular Espinal/patología , Proteínas del Tejido Nervioso/metabolismo , Animales , Animales Recién Nacidos , Autofagia/efectos de los fármacos , Autofagia/genética , Supervivencia Celular/genética , Supervivencia Celular/fisiología , Células Cultivadas , Modelos Animales de Enfermedad , Embrión de Mamíferos , Inhibidores Enzimáticos/farmacología , Regulación del Desarrollo de la Expresión Génica/efectos de los fármacos , Regulación del Desarrollo de la Expresión Génica/genética , Regulación del Desarrollo de la Expresión Génica/fisiología , Macrólidos/farmacología , Ratones , Ratones Transgénicos , Neuronas Motoras/efectos de los fármacos , Neuronas Motoras/ultraestructura , Atrofia Muscular Espinal/genética , Factores de Crecimiento Nervioso/farmacología , Proteínas del Tejido Nervioso/genética , Proteína Sequestosoma-1/metabolismo , Médula Espinal/citología , Médula Espinal/crecimiento & desarrollo , Proteína 1 para la Supervivencia de la Neurona Motora/genética , Proteína 1 para la Supervivencia de la Neurona Motora/metabolismo , Proteína 2 para la Supervivencia de la Neurona Motora/genética , Proteína 2 para la Supervivencia de la Neurona Motora/metabolismoRESUMEN
Amyotrophic Lateral Sclerosis (ALS), a severe neurodegenerative disease, affects the upper and lower motor neurons in the brain and spinal cord. In some studies, ALS disease progression has been associated with an increase in calcium-dependent degeneration processes. Motoneurons are specifically vulnerable to sustained membrane depolarization and excessive elevation of intracellular calcium concentration. The present study analyzed intracellular events in embryonic motoneurons and adult spinal cords of the hSOD1G93A ALS mouse model. We observed activation of calpain, a calcium-dependent cysteine protease that degrades a variety of substrates, and a reduction in calcium-calmodulin dependent protein kinase type IV (CaMKIV) levels in protein extracts from spinal cords obtained at several time-points of hSOD1G93A mice disease progression. However, in cultured embryonic motoneurons these differences between controls and hSOD1G93A mutants are not evident. Our results support the hypothesis that age-dependent changes in calcium homeostasis and resulting events, e.g., calpain activation and CaMKIV processing, are involved in ALS pathogenesis.
Asunto(s)
Esclerosis Amiotrófica Lateral/patología , Proteína Quinasa Tipo 4 Dependiente de Calcio Calmodulina/metabolismo , Calpaína/metabolismo , Regulación de la Expresión Génica/genética , Médula Espinal/metabolismo , Médula Espinal/patología , Factores de Edad , Esclerosis Amiotrófica Lateral/genética , Análisis de Varianza , Animales , Células Cultivadas , Modelos Animales de Enfermedad , Embrión de Mamíferos , Regulación de la Expresión Génica/efectos de los fármacos , Humanos , Péptidos y Proteínas de Señalización Intercelular/farmacología , Ratones , Ratones Endogámicos C57BL , Ratones Transgénicos , Neuronas Motoras/efectos de los fármacos , Neuronas Motoras/fisiología , Proteínas del Tejido Nervioso/metabolismo , Potasio/farmacología , Médula Espinal/efectos de los fármacos , Superóxido Dismutasa/genéticaRESUMEN
Spinal muscular atrophy (SMA) is a neurodegenerative disease produced by low levels of Survival Motor Neuron (SMN) protein that affects alpha motoneurons in the spinal cord. Notch signaling is a cell-cell communication system well known as a master regulator of neural development, but also with important roles in the adult central nervous system. Aberrant Notch function is associated with several developmental neurological disorders; however, the potential implication of the Notch pathway in SMA pathogenesis has not been studied yet. We report here that SMN deficiency, induced in the astroglioma cell line U87MG after lentiviral transduction with a shSMN construct, was associated with an increase in the expression of the main components of Notch signaling pathway, namely its ligands, Jagged1 and Delta1, the Notch receptor and its active intracellular form (NICD). In the SMNΔ7 mouse model of SMA we also found increased astrocyte processes positive for Jagged1 and Delta1 in intimate contact with lumbar spinal cord motoneurons. In these motoneurons an increased Notch signaling was found, as denoted by increased NICD levels and reduced expression of the proneural gene neurogenin 3, whose transcription is negatively regulated by Notch. Together, these findings may be relevant to understand some pathologic attributes of SMA motoneurons.
Asunto(s)
Neuronas Motoras/metabolismo , Neuronas Motoras/patología , Atrofia Muscular Espinal/metabolismo , Atrofia Muscular Espinal/patología , Receptores Notch/metabolismo , Transducción de Señal , Animales , Factores de Transcripción con Motivo Hélice-Asa-Hélice Básico/metabolismo , Proteínas de Unión al Calcio/metabolismo , Línea Celular Tumoral , Gliosis/metabolismo , Gliosis/patología , Humanos , Péptidos y Proteínas de Señalización Intercelular/metabolismo , Péptidos y Proteínas de Señalización Intracelular/metabolismo , Proteína Jagged-1 , Proteínas de la Membrana/metabolismo , Ratones , Proteínas del Tejido Nervioso/metabolismo , Proteínas Serrate-Jagged , Médula Espinal/metabolismo , Médula Espinal/patología , Proteína 1 para la Supervivencia de la Neurona Motora/metabolismoRESUMEN
Intracellular pathways related to cell survival regulate neuronal physiology during development and neurodegenerative disorders. One of the pathways that have recently emerged with an important role in these processes is nuclear factor-κB (NF-κB). The activity of this pathway leads to the nuclear translocation of the NF-κB transcription factors and the regulation of anti-apoptotic gene expression. Different stimuli can activate the pathway through different intracellular cascades (canonical, non-canonical, and atypical), contributing to the translocation of specific dimers of the NF-κB transcription factors, and each of these dimers can regulate the transcription of different genes. Recent studies have shown that the activation of this pathway regulates opposite responses such as cell survival or neuronal degeneration. These apparent contradictory effects depend on conditions such as the pathway stimuli, the origin of the cells, or the cellular context. In the present review, the authors summarize these findings and discuss their significance with respect to survival or death in the nervous system.
Asunto(s)
Supervivencia Celular/fisiología , FN-kappa B/metabolismo , Sistema Nervioso/metabolismo , Neuronas/metabolismo , Transducción de Señal/fisiología , Animales , HumanosRESUMEN
Spinal muscular atrophy (SMA) is a neurodegenerative disease that affects alpha motoneurons in the spinal cord caused by homozygous deletion or specific mutations in the survival motoneuron-1 (SMN1) gene. Cell migration is critical at many stages of nervous system development; to investigate the role of SMN in cell migration, U87MG astroglioma cells were transduced with shSMN lentivectors and about 60% reduction in SMN expression was achieved. In a monolayer wound-healing assay, U87MG SMN-depleted cells exhibit reduced cell migration. In these cells, RhoA was activated and phosphorylated levels of myosin regulatory light chain (MLC), a substrate of the Rho kinase (ROCK), were found increased. The decrease in cell motility was related to activation of RhoA/Rho kinase (ROCK) signaling pathway as treatment with the ROCK inhibitor Y-27632 abrogated both the motility defects and MLC phosphorylation in SMN-depleted cells. As cell migration is regulated by continuous remodeling of the actin cytoskeleton, the actin distribution was studied in SMN-depleted cells. A shift from filamentous to monomeric (globular) actin, involving the disappearance of stress fibers, was observed. In addition, profilin I, an actin-sequestering protein showed an increased expression in SMN-depleted cells. SMN is known to physically interact with profilin, reducing its actin-sequestering activity. The present results suggest that in SMN-depleted cells, the increase in profilin I expression and the reduction in SMN inhibitory action on profilin could lead to reduced filamentous actin polymerization, thus decreasing cell motility. We propose that the alterations reported here in migratory activity in SMN-depleted cells, related to abnormal activation of RhoA/ROCK pathway and increased profilin I expression could have a role in developing nervous system by impairing normal neuron and glial cell migration and thus contributing to disease pathogenesis in SMA.
Asunto(s)
Astrocitoma/metabolismo , Movimiento Celular/fisiología , Profilinas/metabolismo , Proteína 1 para la Supervivencia de la Neurona Motora/metabolismo , Quinasas Asociadas a rho/metabolismo , Proteína de Unión al GTP rhoA/metabolismo , Citoesqueleto de Actina/metabolismo , Actinas/metabolismo , Amidas/farmacología , Astrocitoma/genética , Movimiento Celular/genética , Humanos , Neuronas Motoras/metabolismo , Neuronas/metabolismo , Profilinas/genética , Piridinas/farmacología , Atrofias Musculares Espinales de la Infancia/metabolismo , Proteína 1 para la Supervivencia de la Neurona Motora/genética , Quinasas Asociadas a rho/antagonistas & inhibidores , Quinasas Asociadas a rho/genéticaRESUMEN
In vivo and in vitro motoneuron survival depends on the support of neurotrophic factors. These factors activate signaling pathways related to cell survival or inactivate proteins involved in neuronal death. In the present work, we analyzed the involvement of the nuclear factor-κB (NF-κB) pathway in mediating mouse spinal cord motoneuron survival promoted by neurotrophic factors. This pathway comprises ubiquitously expressed transcription factors that could be activated by two different routes: the canonical pathway, associated with IKKα/IKKß kinase phosphorylation and nuclear translocation RelA (p65)/p50 transcription factors; and the noncanonical pathway, related to IKKα kinase homodimer phosphorylation and RelB/p52 transcription factor activation. In our system, we show that neurotrophic factors treatment induced IKKα and IKKß phosphorylation and RelA nuclear translocation, suggesting NF-κB pathway activation. Protein levels of different members of the canonical or noncanonical pathways were reduced in a primary culture of isolated embryonic motoneurons using an interference RNA approach. Even in the presence of neurotrophic factors, selective reduction of IKKα, IKKß, or RelA proteins induced cell death. In contrast, RelB protein reduction did not have a negative effect on motoneuron survival. Together these results demonstrated that the canonical NF-κB pathway mediates motoneuron survival induced by neurotrophic factors, and the noncanonical pathway is not related to this survival effect. Canonical NF-κB blockade induced an increase of Bim protein level and apoptotic cell death. Bcl-x(L) overexpression or Bax reduction counteracted this apoptotic effect. Finally, RelA knockdown causes changes of CREB and Smn protein levels.
Asunto(s)
Apoptosis/fisiología , Regulación del Desarrollo de la Expresión Génica/fisiología , Neuronas Motoras/fisiología , FN-kappa B/metabolismo , Transducción de Señal/fisiología , Médula Espinal/citología , Análisis de Varianza , Animales , Apoptosis/efectos de los fármacos , Proteínas Reguladoras de la Apoptosis/metabolismo , Proteína 11 Similar a Bcl2 , Proteína de Unión a CREB/metabolismo , Supervivencia Celular , Células Cultivadas , Cromonas/farmacología , Embrión de Mamíferos , Activación Enzimática/efectos de los fármacos , Inhibidores Enzimáticos/farmacología , Femenino , Regulación del Desarrollo de la Expresión Génica/efectos de los fármacos , Proteínas Fluorescentes Verdes/genética , Humanos , Quinasa I-kappa B/antagonistas & inhibidores , Quinasa I-kappa B/genética , Quinasa I-kappa B/metabolismo , Masculino , Proteínas de la Membrana/metabolismo , Ratones , Modelos Biológicos , Morfolinas/farmacología , Neuronas Motoras/efectos de los fármacos , Factores de Crecimiento Nervioso/farmacología , Péptidos/farmacología , Fosforilación/fisiología , Transporte de Proteínas/efectos de los fármacos , Proteínas Proto-Oncogénicas/metabolismo , Interferencia de ARN/fisiología , Transducción de Señal/efectos de los fármacos , Factor de Transcripción ReIA/metabolismo , Transfección/métodos , Proteína bcl-X/metabolismoRESUMEN
Spinal muscular atrophy (SMA) is a motoneuron disorder characterized by deletions or specific mutations in the Survival Motor Neuron gene (SMN). SMN is ubiquitously expressed and has a general role in the assembly of small nuclear ribonucleoprotein (snRNP) and pre-mRNA splicing requirements. However, in motoneuron axons SMN deficiency results in inappropriate levels of certain transcripts in the distal axon, suggesting that the specific susceptibility of motoneurons to SMN deficiency is related to a specialized function in these cells. Although mouse models of SMA have been generated and are useful for in vivo and in vitro studies, the limited number of isolated MNs that could be obtained from them makes it difficult to perform biochemical, genetic and pharmacological approaches. We describe here an in vitro model of isolated embryonic mouse motoneurons in which the cellular levels of endogenous SMN are reduced. These cells show neurite degeneration and cell death after several days of SMN knockdown. We found that the over-expression of the anti-apoptotic protein Bcl-x(L) into motoneurons rescues these cells from the phenotypic changes observed. This result demonstrates that Bcl-x(L) signaling could be a possible pharmacological target of SMA therapeutics.
Asunto(s)
Neuronas Motoras/metabolismo , Atrofia Muscular Espinal/metabolismo , Degeneración Nerviosa/metabolismo , Neuritas/metabolismo , Proteína 1 para la Supervivencia de la Neurona Motora/metabolismo , Proteína bcl-X/metabolismo , Análisis de Varianza , Animales , Western Blotting , Muerte Celular , Células Cultivadas , Modelos Animales de Enfermedad , Ratones , Neuronas Motoras/patología , Atrofia Muscular Espinal/genética , Atrofia Muscular Espinal/patología , Degeneración Nerviosa/genética , Degeneración Nerviosa/patología , Neuritas/patología , Ratas , Proteína 1 para la Supervivencia de la Neurona Motora/genética , Proteína bcl-X/genéticaRESUMEN
FLICE-inhibitory protein (FLIP) is an endogenous inhibitor of the signaling pathway triggered by the activation of death receptors. Here, we reveal a novel biological function for the long form of FLIP (FLIP-L) in neuronal differentiation, which can be dissociated from its antiapoptotic role. We show that FLIP-L is expressed in different regions of the mouse embryonic nervous system. Immunohistochemistry of mouse brain sections at different stages reveals that, in neurons, FLIP is expressed early during the embryonic neuronal development (embryonic day 16) and decreases at later stages (postnatal days 5-15), when its expression is essentially detected in glial cells. FLIP-L overexpression significantly enhances neurotrophin-induced neurite outgrowth in motoneurons, superior cervical ganglion neurons, and PC12 cells. Conversely, the downregulation of FLIP-L protein levels by specific RNA interference significantly reduces neurite outgrowth, even in the presence of the appropriate neurotrophin stimulus. Moreover, NGF-dependent activation of two main intracellular pathways involved in the regulation of neurite outgrowth, extracellular signal-regulated kinases (ERKs) and nuclear factor kappaB (NF-kappaB), is impaired when endogenous FLIP-L is downregulated, although TrkA remains activated. Finally, we demonstrate that FLIP-L interacts with TrkA, and not with p75(NTR), in an NGF-dependent manner, and endogenous FLIP-L interacts with TrkB in whole-brain lysates from embryonic day 15 mice embryos. Altogether, we uncover a new role for FLIP-L as an unexpected critical player in neurotrophin-induced mitogen-activated protein kinase/ERK- and NF-kappaB-mediated control of neurite growth in developing neurons.