RESUMEN
Spinal muscular atrophy (SMA) is a motor neuron disease caused by insufficient levels of the survival motor neuron (SMN) protein. One of the most prominent pathological characteristics of SMA involves defects of the neuromuscular junction (NMJ), such as denervation and reduced clustering of acetylcholine receptors (AChRs). Recent studies suggest that upregulation of agrin, a crucial NMJ organizer promoting AChR clustering, can improve NMJ innervation and reduce muscle atrophy in the delta7 mouse model of SMA. To test whether the muscle-specific kinase (MuSK), part of the agrin receptor complex, also plays a beneficial role in SMA, we treated the delta7 SMA mice with an agonist antibody to MuSK. MuSK agonist antibody #13, which binds to the NMJ, significantly improved innervation and synaptic efficacy in denervation-vulnerable muscles. MuSK agonist antibody #13 also significantly increased the muscle cross-sectional area and myofiber numbers in these denervation-vulnerable muscles but not in denervation-resistant muscles. Although MuSK agonist antibody #13 did not affect the body weight, our study suggests that preservation of NMJ innervation by the activation of MuSK may serve as a complementary therapy to SMN-enhancing drugs to maximize the therapeutic effectiveness for all types of SMA patients.
Asunto(s)
Neuronas Motoras/enzimología , Atrofia Muscular Espinal/enzimología , Unión Neuromuscular/enzimología , Proteínas Tirosina Quinasas Receptoras/metabolismo , Animales , Modelos Animales de Enfermedad , Activación Enzimática , Ratones , Ratones Transgénicos , Neuronas Motoras/patología , Atrofia Muscular Espinal/genética , Atrofia Muscular Espinal/patología , Unión Neuromuscular/genética , Unión Neuromuscular/patología , Proteínas Tirosina Quinasas Receptoras/genética , Proteína 1 para la Supervivencia de la Neurona Motora/genética , Proteína 1 para la Supervivencia de la Neurona Motora/metabolismoRESUMEN
Aminoacyl-RNA synthetases (aaRSs) are among the key enzymes of protein biosynthesis. They are responsible for conducting the first step in the protein biosynthesis, namely attaching amino acids to the corresponding tRNA molecules both in cytoplasm and mitochondria. More and more research demonstrates that mutations in the genes encoding aaRSs lead to the development of various neurodegenerative diseases, such as incurable Charcot-Marie-Tooth disease (CMT) and distal spinal muscular atrophy. Some mutations result in the loss of tRNA aminoacylation activity, while other mutants retain their classical enzyme activity. In the latter case, disease manifestations are associated with additional neuron-specific functions of aaRSs. At present, seven aaRSs (GlyRS, TyrRS, AlaRS, HisRS, TrpRS, MetRS, and LysRS) are known to be involved in the CMT etiology with glycyl-tRNA synthetase (GlyRS) being the most studied of them.
Asunto(s)
Glicina-ARNt Ligasa/genética , Mutación , Enfermedades del Sistema Nervioso/enzimología , Enfermedad de Charcot-Marie-Tooth/enzimología , Enfermedad de Charcot-Marie-Tooth/genética , Enfermedad de Charcot-Marie-Tooth/fisiopatología , Femenino , Humanos , Masculino , Atrofia Muscular Espinal/enzimología , Atrofia Muscular Espinal/genética , Atrofia Muscular Espinal/fisiopatología , Enfermedades del Sistema Nervioso/genética , Enfermedades del Sistema Nervioso/fisiopatología , Neuronas/enzimología , Neuronas/fisiologíaRESUMEN
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
BACKGROUND: Distal motor neuropathies with a genetic origin have a heterogeneous clinical presentation with overlapping features affecting distal nerves and including spinal muscular atrophies and amyotrophic lateral sclerosis. This indicates that their genetic background is heterogeneous. PATIENT AND METHODS: In this work, we have identified and characterized the genetic and molecular base of a patient with a distal sensorimotor neuropathy of unknown origin. For this study, we performed whole-exome sequencing, molecular modelling, cloning and expression of mutant gene, and biochemical and cell biology analysis of the mutant protein. RESULTS: A novel homozygous recessive mutation in the human VRK1 gene, coding for a chromatin kinase, causing a substitution (c.637T > C; p.Tyr213His) in exon 8, was detected in a patient presenting since childhood a progressive distal sensorimotor neuropathy and spinal muscular atrophy syndrome, with normal intellectual development. Molecular modelling predicted this mutant VRK1 has altered the kinase activation loop by disrupting its interaction with the C-terminal regulatory region. The p.Y213H mutant protein has a reduced kinase activity with different substrates, including histones H3 and H2AX, proteins involved in DNA damage responses, such as p53 and 53BP1, and coilin, the scaffold for Cajal bodies. The mutant VRK1(Y213H) protein is unable to rescue the formation of Cajal bodies assembled on coilin, in the absence of wild-type VRK1. CONCLUSION: The VRK1(Y213H) mutant protein alters the activation loop, impairs the kinase activity of VRK1 causing a functional insufficiency that impairs the formation of Cajal bodies assembled on coilin, a protein that regulates SMN1 and Cajal body formation.
Asunto(s)
Cuerpos Enrollados , Péptidos y Proteínas de Señalización Intracelular/genética , Atrofia Muscular Espinal/enzimología , Atrofia Muscular Espinal/genética , Proteínas Serina-Treonina Quinasas/genética , Adulto , Consanguinidad , Humanos , MasculinoRESUMEN
Acid ceramidase (aCDase, ASAH1) hydrolyzes lysosomal membrane ceramide into sphingosine, the backbone of all sphingolipids, to regulate many cellular processes. Abnormal function of aCDase leads to Farber disease, spinal muscular atrophy with progressive myoclonic epilepsy, and is associated with Alzheimer's, diabetes, and cancer. Here, we present crystal structures of mammalian aCDases in both proenzyme and autocleaved forms. In the proenzyme, the catalytic center is buried and protected from solvent. Autocleavage triggers a conformational change exposing a hydrophobic channel leading to the active site. Substrate modeling suggests distinct catalytic mechanisms for substrate hydrolysis versus autocleavage. A hydrophobic surface surrounding the substrate binding channel appears to be a site of membrane attachment where the enzyme accepts substrates facilitated by the accessory protein, saposin-D. Structural mapping of disease mutations reveals that most would destabilize the protein fold. These results will inform the rational design of aCDase inhibitors and recombinant aCDase for disease therapeutics.
Asunto(s)
Ceramidasa Ácida/química , Ceramidasa Ácida/metabolismo , Lipogranulomatosis de Farber/enzimología , Atrofia Muscular Espinal/enzimología , Ceramidasa Ácida/genética , Sitios de Unión , Biocatálisis , Ceramidas/química , Ceramidas/metabolismo , Activación Enzimática , Lipogranulomatosis de Farber/genética , Humanos , Interacciones Hidrofóbicas e Hidrofílicas , Atrofia Muscular Espinal/genética , Mutación , Pliegue de Proteína , Saposinas/genética , Saposinas/metabolismo , Esfingolípidos/química , Esfingolípidos/metabolismoRESUMEN
C5-substituted 2,4-diaminoquinazoline inhibitors of the decapping scavenger enzyme DcpS (DAQ-DcpSi) have been developed for the treatment of spinal muscular atrophy (SMA), which is caused by genetic deficiency in the Survival Motor Neuron (SMN) protein. These compounds are claimed to act as SMN2 transcriptional activators but data underlying that claim are equivocal. In addition it is unclear whether the claimed effects on SMN2 are a direct consequence of DcpS inhibitor or might be a consequence of lysosomotropism, which is known to be neuroprotective. DAQ-DcpSi effects were characterized in cells in vitro utilizing DcpS knockdown and 7-methyl analogues as probes for DcpS vs non-DcpS-mediated effects. We also performed analysis of Smn transcript levels, RNA-Seq analysis of the transcriptome and SMN protein in order to identify affected pathways underlying the therapeutic effect, and studied lysosomotropic and non-lysosomotropic DAQ-DCpSi effects in 2B/- SMA mice. Treatment of cells caused modest and transient SMN2 mRNA increases with either no change or a decrease in SMNΔ7 and no change in SMN1 transcripts or SMN protein. RNA-Seq analysis of DAQ-DcpSi-treated N2a cells revealed significant changes in expression (both up and down) of approximately 2,000 genes across a broad range of pathways. Treatment of 2B/- SMA mice with both lysomotropic and non-lysosomotropic DAQ-DcpSi compounds had similar effects on disease phenotype indicating that the therapeutic mechanism of action is not a consequence of lysosomotropism. In striking contrast to the findings in vitro, Smn transcripts were robustly changed in tissues but there was no increase in SMN protein levels in spinal cord. We conclude that DAQ-DcpSi have reproducible benefit in SMA mice and a broad spectrum of biological effects in vitro and in vivo, but these are complex, context specific, and not the result of simple SMN2 transcriptional activation.
Asunto(s)
Endorribonucleasas/antagonistas & inhibidores , Inhibidores Enzimáticos/farmacología , Atrofia Muscular Espinal/tratamiento farmacológico , Atrofia Muscular Espinal/enzimología , Quinazolinas/farmacología , Animales , Línea Celular , Modelos Animales de Enfermedad , Inhibidores Enzimáticos/química , Femenino , Técnicas de Silenciamiento del Gen , Células HEK293 , Humanos , Masculino , Ratones , Ratones Noqueados , Atrofia Muscular Espinal/genética , Regiones Promotoras Genéticas , Quinazolinas/química , ARN Mensajero/genética , ARN Mensajero/metabolismo , Proteína 2 para la Supervivencia de la Neurona Motora/deficiencia , Proteína 2 para la Supervivencia de la Neurona Motora/genética , Proteína 2 para la Supervivencia de la Neurona Motora/metabolismoRESUMEN
Spinal Muscular Atrophy (SMA) is a motoneuron disease caused by low levels of functional survival of motoneuron protein (SMN). Molecular disease mechanisms downstream of functional SMN loss are still largely unknown. Previous studies suggested an involvement of Rho kinase (ROCK) as well as the extracellular signal-regulated kinases (ERK) pathways in the pathomechanism. Both pathways are bi-directionally linked and inhibit each other. Thus, we hypothesize that both pathways regulate SMA pathophysiology in vivo in a combined manner rather than acting separately. Here, we applied the repurposed drugs, selumetinib, an ERK inhibitor, and the ROCK inhibitor fasudil to severe SMA mice. Thereby, separately applied inhibitors as well as a combination enabled us to explore the impact of the ROCK-ERK signaling network on SMA pathophysiology. ROCK inhibition specifically ameliorated the phenotype of selumetinib-treated SMA mice demonstrating an efficient ROCK to ERK crosstalk relevant for the SMA pathophysiology. However, ERK inhibition alone aggravated the condition of SMA mice and reduced the number of motoneurons indicating a compensatory hyper-activation of ERK in motoneurons. Taken together, we identified a regulatory network acting downstream of SMN depletion and upstream of the SMA pathophysiology thus being a future treatment target in combination with SMN dependent strategies.
Asunto(s)
Quinasas MAP Reguladas por Señal Extracelular/metabolismo , Atrofia Muscular Espinal/enzimología , Quinasas Asociadas a rho/metabolismo , 1-(5-Isoquinolinesulfonil)-2-Metilpiperazina/análogos & derivados , 1-(5-Isoquinolinesulfonil)-2-Metilpiperazina/farmacología , Animales , Bencimidazoles/farmacología , Muerte Celular/efectos de los fármacos , Muerte Celular/fisiología , Línea Celular , Citoplasma/efectos de los fármacos , Citoplasma/enzimología , Citoplasma/patología , Modelos Animales de Enfermedad , Progresión de la Enfermedad , Inhibidores Enzimáticos/farmacología , Quinasas MAP Reguladas por Señal Extracelular/antagonistas & inhibidores , Ratones Transgénicos , Neuronas Motoras/efectos de los fármacos , Neuronas Motoras/enzimología , Neuronas Motoras/patología , Atrofia Muscular Espinal/patología , ARN Interferente Pequeño , Distribución Aleatoria , Índice de Severidad de la Enfermedad , Transducción de Señal/efectos de los fármacos , Médula Espinal/efectos de los fármacos , Médula Espinal/enzimología , Médula Espinal/patología , Regulación hacia Arriba/efectos de los fármacos , Quinasas Asociadas a rho/antagonistas & inhibidoresRESUMEN
Spinal muscular atrophy (SMA) is an autosomal recessive neurodegenerative disease characterized by motor neurons degeneration and muscular atrophy. There is no effective SMA treatment. Loganin is a botanical candidate with anti-inflammatory, anti-oxidant, glucose-lowering and anti-diabetic nephropathy activities. The aim of this study is to investigate the potential protective effects of loganin on SMA using two cellular models, SMN-deficient NSC34 cells and SMA patient fibroblasts, and an animal disease model, SMAΔ7 mice. In SMN-deficient NSC34 cells, loganin increased cell viability, neurite length, and expressions of SMN, Gemin2, SMN-Gemin2 complex, p-Akt, p-GSK-3ß, p-CREB, BDNF and Bcl-2. However, both AG1024 (IGF-1 R antagonist) and IGF-1 R siRNA attenuated the protective effects of loganin on SMN level and cell viability in SMN-deficient NSC34 cells. In SMA patient fibroblasts, loganin up-regulated levels of SMN, FL-SMN2, and Gemins, increased numbers of SMN-containing nuclear gems, modulated splicing factors, and up-regulated p-Akt. Furthermore, in the brain, spinal cord and gastrocnemius muscle of SMAΔ7 mice, loganin up-regulated the expressions of SMN and p-Akt. Results from righting reflex and hind-limb suspension tests indicated loganin improved muscle strength of SMAΔ7 mice; moreover, loganin activated Akt/mTOR signal and inhibited atrogin-1/MuRF-1 signal in gastrocnemius muscle of SMAΔ7 mice. Loganin also increased body weight, but the average lifespan of loganin (20mg/kg/day)-treated SMA mice was 16.80±0.73 days, while saline-treated SMA mice was 10.91±0.96 days. In conclusion, the present results demonstrate that loganin provides benefits to SMA therapeutics via improving SMN restoration, muscle strength and body weight. IGF-1 plays an important role in loganin neuroprotection. Loganin can be therefore a valuable complementary candidate for treatment of neuromuscular diseases via regulation of muscle protein synthesis and neuroprotection.
Asunto(s)
Iridoides/farmacología , Neuronas Motoras/efectos de los fármacos , Atrofia Muscular Espinal/tratamiento farmacológico , Fármacos Neuroprotectores/farmacología , Proteínas Proto-Oncogénicas c-akt/metabolismo , Proteína 1 para la Supervivencia de la Neurona Motora/biosíntesis , Serina-Treonina Quinasas TOR/metabolismo , Animales , Apoptosis/efectos de los fármacos , Línea Celular , Citoprotección , Modelos Animales de Enfermedad , Relación Dosis-Respuesta a Droga , Fibroblastos/efectos de los fármacos , Fibroblastos/enzimología , Fibroblastos/patología , Predisposición Genética a la Enfermedad , Humanos , Factor I del Crecimiento Similar a la Insulina/metabolismo , Ratones , Ratones Transgénicos , Actividad Motora/efectos de los fármacos , Neuronas Motoras/enzimología , Neuronas Motoras/patología , Proteínas Musculares/metabolismo , Fuerza Muscular/efectos de los fármacos , Músculo Esquelético/efectos de los fármacos , Músculo Esquelético/enzimología , Músculo Esquelético/patología , Atrofia Muscular Espinal/enzimología , Atrofia Muscular Espinal/genética , Atrofia Muscular Espinal/fisiopatología , Mutación , Degeneración Nerviosa , Fenotipo , Fosforilación , Biosíntesis de Proteínas , Interferencia de ARN , Proteínas Ligasas SKP Cullina F-box/metabolismo , Transducción de Señal/efectos de los fármacos , Proteína 1 para la Supervivencia de la Neurona Motora/genética , Factores de Tiempo , Transfección , Proteínas de Motivos Tripartitos/metabolismo , Ubiquitina-Proteína Ligasas/metabolismo , Aumento de Peso/efectos de los fármacosRESUMEN
To broaden the scope of existing methods based on (19)F nucleotide labeling, we developed a new method for the synthesis of fluorophosphate (oligo)nucleotide analogues containing an O to F substitution at the terminal position of the (oligo)phosphate moiety and evaluated them as tools for (19)F NMR studies. Using three efficient and comprehensive synthetic approaches based on phosphorimidazolide chemistry and tetra-n-butylammonium fluoride, fluoromonophosphate, or fluorophosphate imidazolide as fluorine sources, we prepared over 30 fluorophosphate-containing nucleotides, varying in nucleobase type (A, G, C, U, m(7)G), phosphate chain length (from mono to tetra), and presence of additional phosphate modifications (thio, borano, imido, methylene). Using fluorophosphate imidazolide as fluorophosphorylating reagent for 5'-phosphorylated oligos we also synthesized oligonucleotide 5'-(2-fluorodiphosphates), which are potentially useful as (19)F NMR hybridization probes. The compounds were characterized by (19)F NMR and evaluated as (19)F NMR molecular probes. We found that fluorophosphate nucleotide analogues can be used to monitor activity of enzymes with various specificities and metal ion requirements, including human DcpS enzyme, a therapeutic target for spinal muscular atrophy. The compounds can also serve as reporter ligands for protein binding studies, as exemplified by studying interaction of fluorophosphate mRNA cap analogues with eukaryotic translation initiation factor (eIF4E).
Asunto(s)
Endorribonucleasas/química , Factor 4E Eucariótico de Iniciación/química , Fluoruros/síntesis química , Radioisótopos de Flúor/química , Nucleótidos/síntesis química , Oligonucleótidos/síntesis química , Fosfatos/síntesis química , Compuestos de Amonio Cuaternario/química , ARN Mensajero/química , Marcadores de Spin/síntesis química , Endorribonucleasas/farmacología , Fluoruros/química , Humanos , Espectroscopía de Resonancia Magnética , Atrofia Muscular Espinal/enzimología , Atrofia Muscular Espinal/metabolismo , Nucleótidos/química , Oligonucleótidos/química , Fosfatos/química , Unión Proteica , ARN Mensajero/síntesis químicaRESUMEN
AIM: Levels of ubiquitin carboxyl-terminal hydrolase L1 (UCHL1) are robustly increased in spinal muscular atrophy (SMA) patient fibroblasts and mouse models. We therefore wanted to establish whether changes in UCHL1 contribute directly to disease pathogenesis, and to assess whether pharmacological inhibition of UCHL1 represents a viable therapeutic option for SMA. METHODS: SMA mice and control littermates received a pharmacological UCHL1 inhibitor (LDN-57444) or DMSO vehicle. Survival and weight were monitored daily, a righting test of motor performance was performed, and motor neurone loss, muscle fibre atrophy and neuromuscular junction pathology were all quantified. Ubiquitin-like modifier activating enzyme 1 (Uba1) was then pharmacologically inhibited in neurones in vitro to examine the relationship between Uba1 levels and UCHL1 in SMA. RESULTS: Pharmacological inhibition of UCHL1 failed to improve survival, motor symptoms or neuromuscular pathology in SMA mice and actually precipitated the onset of weight loss. LDN-57444 treatment significantly decreased spinal cord mono-ubiquitin levels, further exacerbating ubiquitination defects in SMA mice. Pharmacological inhibition of Uba1, levels of which are robustly reduced in SMA, was sufficient to induce accumulation of UCHL1 in primary neuronal cultures. CONCLUSION: Pharmacological inhibition of UCHL1 exacerbates rather than ameliorates disease symptoms in a mouse model of SMA. Thus, pharmacological inhibition of UCHL1 is not a viable therapeutic target for SMA. Moreover, increased levels of UCHL1 in SMA likely represent a downstream consequence of decreased Uba1 levels, indicative of an attempted supportive compensatory response to defects in ubiquitin homeostasis caused by low levels of SMN protein.
Asunto(s)
Indoles/uso terapéutico , Atrofia Muscular Espinal/tratamiento farmacológico , Atrofia Muscular Espinal/enzimología , Oximas/uso terapéutico , Proteína 1 para la Supervivencia de la Neurona Motora/metabolismo , Ubiquitina Tiolesterasa/antagonistas & inhibidores , Ubiquitina Tiolesterasa/metabolismo , Animales , Células Cultivadas , Modelos Animales de Enfermedad , Fibroblastos/efectos de los fármacos , Fibroblastos/enzimología , Homeostasis/efectos de los fármacos , Humanos , Indoles/administración & dosificación , Indoles/efectos adversos , Ratones , Actividad Motora/efectos de los fármacos , Neuronas Motoras/efectos de los fármacos , Neuronas Motoras/metabolismo , Atrofia Muscular Espinal/patología , Oximas/administración & dosificación , Oximas/efectos adversosRESUMEN
Among hereditary diseases, the group of motor neuron diseases (MNDs) includes some of the most devastating and rapidly progressive lethal conditions. Although degeneration of motor neurons is common to all of them, the phenotypic spectrum of MNDs is relatively broad and ranges from perinatal conditions like spinal muscular atrophy (SMA) to adult-onset diseases such as amyotrophic lateral sclerosis (ALS). While the understanding of the pathology of the diseases is constantly growing, the development of therapeutic approaches lags behind. In fact, there is no approved therapy for MNDs available at the moment. Recent findings demonstrated the existence of some patterns that are shared by several MNDs such as transcriptional dysregulation. In addition, conditions like SMA or certain types of Charcot-Marie-Tooth disease provide some defined targets which may be amenable to therapeutic approaches. Consequently, counteracting this dysregulation may be a valuable therapeutic option and ameliorate disease progression in MND patients. The feasibility of such an approach has been proven during the past years by the epigenetic treatment of various neoplastic entities with histone deacetylase inhibitors (HDACi). On these grounds, also epigenetic therapy of MNDs has become a promising option. So far, several HDACi have been tested in vitro and in animal models and some proceeded further and were evaluated in clinical trials. This review will summarize the advances of HDACi in MNDs and will give a perspective where the road will lead us.
Asunto(s)
Histonas/metabolismo , Terapia Molecular Dirigida , Enfermedad de la Neurona Motora/tratamiento farmacológico , Neuronas Motoras/efectos de los fármacos , Proteínas del Tejido Nervioso/antagonistas & inhibidores , Fármacos Neuroprotectores/uso terapéutico , Procesamiento Proteico-Postraduccional/efectos de los fármacos , Acetilación/efectos de los fármacos , Esclerosis Amiotrófica Lateral/tratamiento farmacológico , Esclerosis Amiotrófica Lateral/enzimología , Esclerosis Amiotrófica Lateral/metabolismo , Animales , Atrofia Bulboespinal Ligada al X/tratamiento farmacológico , Atrofia Bulboespinal Ligada al X/enzimología , Atrofia Bulboespinal Ligada al X/metabolismo , Modelos Animales de Enfermedad , Inhibidores de Histona Desacetilasas/uso terapéutico , Humanos , Ratones , Enfermedad de la Neurona Motora/enzimología , Enfermedad de la Neurona Motora/metabolismo , Neuronas Motoras/enzimología , Neuronas Motoras/metabolismo , Atrofia Muscular Espinal/tratamiento farmacológico , Atrofia Muscular Espinal/enzimología , Atrofia Muscular Espinal/metabolismo , Proteínas del Tejido Nervioso/metabolismoRESUMEN
Spinal muscular atrophy (SMA) is an autosomal recessive neuromuscular disease caused by mutations in the survival of motor neuron 1 (SMN1) gene and deficient expression of the ubiquitously expressed SMN protein. Pathologically, SMA is characterized by motor neuron loss and severe muscle atrophy. During muscle atrophy, the E3 ligase atrogenes, atrogin-1 and muscle ring finger 1 (MuRF1), mediate muscle protein breakdown through the ubiquitin proteasome system. Atrogene expression can be induced by various upstream regulators. During acute denervation, they are activated by myogenin, which is in turn regulated by histone deacetylases 4 and 5. Here we show that atrogenes are induced in SMA model mice and in SMA patient muscle in association with increased myogenin and histone deacetylase-4 (HDAC4) expression. This activation during both acute denervation and SMA disease progression is suppressed by treatment with a histone deacetylase inhibitor; however, this treatment has no effect when atrogene induction occurs independently of myogenin. These results indicate that myogenin-dependent atrogene induction is amenable to pharmacological intervention with histone deacetylase inhibitors and help to explain the beneficial effects of these agents on SMA and other denervating diseases.
Asunto(s)
Proteínas Musculares/genética , Atrofia Muscular Espinal/genética , Miogenina/metabolismo , Proteínas Ligasas SKP Cullina F-box/genética , Animales , Células HEK293 , Inhibidores de Histona Desacetilasas/farmacología , Histona Desacetilasas/metabolismo , Humanos , Ratones , Ratones Endogámicos , Proteínas Musculares/metabolismo , Atrofia Muscular Espinal/enzimología , Proteínas Ligasas SKP Cullina F-box/metabolismo , Proteínas de Motivos Tripartitos , Ubiquitina-Proteína Ligasas/genética , Ubiquitina-Proteína Ligasas/metabolismoRESUMEN
Spinal muscular atrophy (SMA) and amyotrophic lateral sclerosis (ALS) are among the most common motor neuron diseases to afflict the human population. A deficiency of the survival of motor neuron (SMN) protein causes SMA and is also reported to be an exacerbating factor in the development of ALS. However, pathways linking the two diseases have yet to be defined and it is not clear precisely how the pathology of ALS is aggravated by reduced SMN or whether mutant proteins underlying familial forms of ALS interfere with SMN-related biochemical pathways to exacerbate the neurodegenerative process. In this study, we show that mutant superoxide dismutase-1 (SOD1), a cause of familial ALS, profoundly alters the sub-cellular localization of the SMN protein, preventing the formation of nuclear 'gems' by disrupting the recruitment of the protein to Cajal bodies. Overexpressing the SMN protein in mutant SOD1 mice, a model of familial ALS, alleviates this phenomenon, most likely in a cell-autonomous manner, and significantly mitigates the loss of motor neurons in the spinal cord and in culture dishes. In the mice, the onset of the neuromuscular phenotype is delayed and motor function enhanced, suggestive of a therapeutic benefit for ALS patients treated with agents that augment the SMN protein. Nevertheless, this finding is tempered by an inability to prolong survival, a limitation most likely imposed by the inexorable denervation that characterizes ALS and eventually disrupts the neuromuscular synapses even in the presence of increased SMN.
Asunto(s)
Esclerosis Amiotrófica Lateral/enzimología , Núcleo Celular/metabolismo , Atrofia Muscular Espinal/enzimología , Atrofia Muscular Espinal/genética , Mutación , Superóxido Dismutasa/genética , Proteína 1 para la Supervivencia de la Neurona Motora/metabolismo , Esclerosis Amiotrófica Lateral/genética , Esclerosis Amiotrófica Lateral/metabolismo , Animales , Modelos Animales de Enfermedad , Ratones , Ratones Noqueados , Ratones Transgénicos , Atrofia Muscular Espinal/metabolismo , Superóxido Dismutasa/metabolismo , Superóxido Dismutasa-1 , Proteína 1 para la Supervivencia de la Neurona Motora/genéticaRESUMEN
Amyotrophic lateral sclerosis (ALS) is a progressive, fatal, neurodegenerative disorder caused by the degeneration of motor neurons in the CNS, which results in complete paralysis of skeletal muscles. Recent experimental studies have suggested that the disease could initiate in skeletal muscle, rather than in the motor neurons. To establish the timeframe of motor neuron degeneration in relation to muscle atrophy in motor neuron disease, we have used MRI to monitor changes throughout disease in brain and skeletal muscle of G93A-SOD1 mice, a purported model of ALS. Longitudinal MRI examination of the same animals indicated that muscle volume in the G93A-SOD1 mice was significantly reduced from as early as week 8 of life, 4 weeks prior to clinical onset. Progressive muscle atrophy from week 8 onwards was confirmed by histological analysis. In contrast, brain MRI indicated that neurodegeneration occurs later in G93A-SOD1 mice, with hyperintensity MRI signals detected only at weeks 10-18. Neurodegenerative changes were observed only in the motor nuclei areas of the brainstem; MRI changes indicative of neurodegeneration were not detected in the motor cortex where first motor neurons originate, even at the late disease stage. This longitudinal MRI study establishes unequivocally that, in the experimental murine model of ALS, muscle degeneration occurs before any evidence of neurodegeneration and clinical signs, supporting the postulate that motor neuron disease can initiate from muscle damage and result from retrograde dying-back of the motor neurons.
Asunto(s)
Esclerosis Amiotrófica Lateral/patología , Encéfalo/patología , Neuronas Motoras/patología , Músculo Esquelético/patología , Degeneración Retrógrada/patología , Alanina/genética , Esclerosis Amiotrófica Lateral/enzimología , Esclerosis Amiotrófica Lateral/genética , Animales , Encéfalo/enzimología , Modelos Animales de Enfermedad , Glicina/genética , Miembro Posterior/patología , Humanos , Estudios Longitudinales , Masculino , Ratones , Ratones Endogámicos , Ratones Transgénicos , Neuronas Motoras/enzimología , Músculo Esquelético/enzimología , Atrofia Muscular Espinal/enzimología , Atrofia Muscular Espinal/genética , Atrofia Muscular Espinal/patología , Degeneración Retrógrada/enzimología , Degeneración Retrógrada/genéticaRESUMEN
Spinal muscular atrophy (SMA) is an inherited disease resulting in the highest mortality of children under the age of two. SMA is caused by mutations or deletions in the survival motor neuron 1 (SMN1) gene, leading to aberrant neuromuscular junction (NMJ) development and the loss of spinal cord alpha-motor neurons. Here, we show that Smn depletion leads to increased activation of RhoA, a major regulator of actin dynamics, in the spinal cord of an intermediate SMA mouse model. Treating these mice with Y-27632, which inhibits ROCK, a direct downstream effector of RhoA, dramatically improves their survival. This lifespan rescue is independent of Smn expression and is accompanied by an improvement in the maturation of the NMJs and an increase in muscle fiber size in the SMA mice. Our study presents evidence linking disruption of actin cytoskeletal dynamics to SMA pathogenesis and, for the first time, identifies RhoA effectors as viable targets for therapeutic intervention in the disease.
Asunto(s)
Atrofia Muscular Espinal/enzimología , Atrofia Muscular Espinal/mortalidad , Proteína 1 para la Supervivencia de la Neurona Motora/genética , Proteína de Unión al GTP rhoA/metabolismo , Amidas/administración & dosificación , Animales , Modelos Animales de Enfermedad , Femenino , Humanos , Ratones , Ratones Endogámicos C57BL , Ratones Noqueados , Atrofia Muscular Espinal/genética , Unión Neuromuscular/genética , Unión Neuromuscular/metabolismo , Inhibidores de Proteínas Quinasas/administración & dosificación , Piridinas/administración & dosificación , Médula Espinal/enzimología , Médula Espinal/metabolismo , Sobrevida , Proteína 1 para la Supervivencia de la Neurona Motora/metabolismo , Proteína de Unión al GTP rhoA/antagonistas & inhibidores , Proteína de Unión al GTP rhoA/genéticaRESUMEN
Histone deacetylases (HDACs) play a key role in homeostasis of protein acetylation in histones and other proteins and in regulating fundamental cellular activities such as transcription. A wide range of brain disorders are associated with imbalances in protein acetylation levels and transcriptional dysfunctions. Treatment with various HDAC inhibitors can correct these deficiencies and has emerged as a promising new strategy for therapeutic intervention in neurodegenerative disease. Here, we review and discuss intriguing recent developments in the use of HDAC inhibitors to combat neurodegenerative conditions in cellular and disease models. HDAC inhibitors have neuroprotective, neurotrophic and anti-inflammatory properties; improvements in neurological performance, learning/memory and other disease phenotypes are frequently seen in these models. We discuss the targets and mechanisms underlying these effects of HDAC inhibition and comment on the potential for some HDAC inhibitors to prove clinically effective in the treatment of neurodegenerative disorders.
Asunto(s)
Inhibidores de Histona Desacetilasas/uso terapéutico , Histona Desacetilasas/metabolismo , Enfermedades Neurodegenerativas/enzimología , Acetilación , Enfermedad de Alzheimer/tratamiento farmacológico , Enfermedad de Alzheimer/enzimología , Enfermedad de Alzheimer/fisiopatología , Esclerosis Amiotrófica Lateral/tratamiento farmacológico , Esclerosis Amiotrófica Lateral/enzimología , Esclerosis Amiotrófica Lateral/fisiopatología , Animales , Modelos Animales de Enfermedad , Histonas/metabolismo , Humanos , Enfermedad de Huntington/tratamiento farmacológico , Enfermedad de Huntington/enzimología , Enfermedad de Huntington/fisiopatología , Atrofia Muscular Espinal/tratamiento farmacológico , Atrofia Muscular Espinal/enzimología , Atrofia Muscular Espinal/fisiopatología , Enfermedades Neurodegenerativas/tratamiento farmacológico , Enfermedades Neurodegenerativas/patología , Enfermedades Neurodegenerativas/fisiopatología , Fármacos Neuroprotectores/clasificación , Fármacos Neuroprotectores/uso terapéutico , Enfermedad de Parkinson/tratamiento farmacológico , Enfermedad de Parkinson/enzimología , Enfermedad de Parkinson/fisiopatología , Ratas , Accidente Cerebrovascular/tratamiento farmacológico , Accidente Cerebrovascular/enzimología , Accidente Cerebrovascular/fisiopatologíaAsunto(s)
Predisposición Genética a la Enfermedad/genética , Atrofia Muscular Espinal/enzimología , Atrofia Muscular Espinal/genética , Proteínas del Tejido Nervioso/genética , Péptido Hidrolasas/genética , Canales de Potasio/genética , Dipeptidil-Peptidasas y Tripeptidil-Peptidasas , Femenino , Variación Genética/genética , Humanos , Masculino , Persona de Mediana Edad , Factores de RiesgoRESUMEN
Distal spinal muscular atrophy type 1 (DSMA1) is an autosomal recessive disease that is clinically characterized by distal limb weakness and respiratory distress. In this disease, the degeneration of alpha-motoneurons is caused by mutations in the immunoglobulin mu-binding protein 2 (IGHMBP2). This protein has been implicated in DNA replication, pre-mRNA splicing and transcription, but its precise function in all these processes has remained elusive. We have purified catalytically active recombinant IGHMBP2, which has enabled us to assess its enzymatic properties and to identify its cellular targets. Our data reveal that IGHMBP2 is an ATP-dependent 5' --> 3' helicase, which unwinds RNA and DNA duplices in vitro. Importantly, this helicase localizes predominantly to the cytoplasm of neuronal and non-neuronal cells and associates with ribosomes. DSMA1-causing amino acid substitutions in IGHMBP2 do not affect ribosome binding yet severely impair ATPase and helicase activity. We propose that IGHMBP2 is functionally linked to translation, and that mutations in its helicase domain interfere with this function in DSMA1 patients.
Asunto(s)
ADN Helicasas/metabolismo , Proteínas de Unión al ADN/metabolismo , Atrofia Muscular Espinal/enzimología , Ribosomas/enzimología , Factores de Transcripción/metabolismo , Adenosina Trifosfatasas/metabolismo , Adenosina Trifosfato/metabolismo , Animales , Extractos Celulares , Línea Celular Tumoral , ADN Helicasas/química , Proteínas de Unión al ADN/química , Activación Enzimática , Humanos , Ratones , Proteínas Mutantes/metabolismo , Unión Proteica , Ribonucleoproteínas/metabolismo , Factores de Transcripción/químicaRESUMEN
Aminoacyl-tRNA synthetases (ARSs) are ubiquitously expressed, essential enzymes responsible for performing the first step of protein synthesis. Specifically, ARSs attach amino acids to their cognate tRNA molecules in the cytoplasm and mitochondria. Recent studies have demonstrated that mutations in genes encoding ARSs can result in neurodegeneration, raising many questions about the role of these enzymes (and protein synthesis in general) in neuronal function. In this review, we summarize the current knowledge of genetic diseases that are associated with mutations in ARS-encoding genes, discuss the potential pathogenic mechanisms underlying these disorders, and point to likely areas of future research that will advance our understanding about the role of ARSs in genetic diseases.
Asunto(s)
Aminoacil-ARNt Sintetasas/genética , Enfermedades Genéticas Congénitas/enzimología , Enfermedades Genéticas Congénitas/genética , Aminoacil-ARNt Sintetasas/inmunología , Aminoacil-ARNt Sintetasas/metabolismo , Animales , Autoanticuerpos/biosíntesis , Ataxia Cerebelosa/enzimología , Ataxia Cerebelosa/genética , Enfermedad de Charcot-Marie-Tooth/enzimología , Enfermedad de Charcot-Marie-Tooth/genética , Modelos Animales de Enfermedad , Genes Mitocondriales , Enfermedades Genéticas Congénitas/fisiopatología , Humanos , Ratones , Atrofia Muscular Espinal/enzimología , Atrofia Muscular Espinal/genética , Mutación , Neuronas/enzimología , Enfermedades del Sistema Nervioso Periférico/enzimología , Enfermedades del Sistema Nervioso Periférico/genéticaRESUMEN
Amyotrophic lateral sclerosis (ALS) is a devastating motor neuron degenerative disease whose etiology and pathogenesis remain poorly understood. Most cases of ALS ( approximately 90%) are sporadic (SALS), occurring in the absence of genetic associations. Approximately 20% of familial ALS (FALS) cases are due to known mutations in the copper, zinc superoxide dismutase (SOD1) gene. Molecular evidence for a common pathogenesis of SALS and FALS has remained elusive. Here we use covalent chemical modification to reveal an attribute of spinal cord SOD1 common to both SOD1-linked FALS and SALS, but not present in normal or disease-affected tissues from other neurodegenerative diseases, including Alzheimer's, Parkinson's, and Huntington's diseases and spinal muscular atrophy, a non-ALS motor neuron disease. Biotinylation reveals a 32-kDa, covalently cross-linked SOD1-containing protein species produced not only in FALS caused by SOD1 mutation, but also in SALS. These studies use chemical modification as a novel tool for the detection of a disease-associated biomarker. Our results identify a shared molecular event involving a known target gene and suggest a common step in the pathogenesis between SALS and FALS.