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1.
Transl Neurosci ; 15(1): 20220330, 2024 Jan 01.
Artículo en Inglés | MEDLINE | ID: mdl-38283997

RESUMEN

Objective: Heterozygous mutations within the voltage-gated sodium channel α subunit (SCN1A) are responsible for the majority of cases of Dravet syndrome (DS), a severe developmental and epileptic encephalopathy. Development of novel therapeutic approaches is mandatory in order to directly target the molecular consequences of the genetic defect. The aim of the present study was to investigate whether cis-acting long non-coding RNAs (lncRNAs) of SCN1A are expressed in brain specimens of children and adolescent with epilepsy as these molecules comprise possible targets for precision-based therapy approaches. Methods: We investigated SCN1A mRNA expression and expression of two SCN1A related antisense RNAs in brain tissues in different age groups of pediatric non-Dravet patients who underwent surgery for drug resistant epilepsy. The effect of different antisense oligonucleotides (ASOs) directed against SCN1A specific antisense RNAs on SCN1A expression was tested. Results: The SCN1A related antisense RNAs SCN1A-dsAS (downstream antisense, RefSeq identifier: NR_110598) and SCN1A-usAS (upstream AS, SCN1A-AS, RefSeq identifier: NR_110260) were widely expressed in the brain of pediatric patients. Expression patterns revealed a negative correlation of SCN1A-dsAS and a positive correlation of lncRNA SCN1A-usAS with SCN1A mRNA expression. Transfection of SK-N-AS cells with an ASO targeted against SCN1A-dsAS was associated with a significant enhancement of SCN1A mRNA expression and reduction in SCN1A-dsAS transcripts. Conclusion: These findings support the role of SCN1A-dsAS in the suppression of SCN1A mRNA generation. Considering the haploinsufficiency in genetic SCN1A related DS, SCN1A-dsAS is an interesting target candidate for the development of ASOs (AntagoNATs) based precision medicine therapeutic approaches aiming to enhance SCN1A expression in DS.

2.
Mol Ther Nucleic Acids ; 34: 102055, 2023 Dec 12.
Artículo en Inglés | MEDLINE | ID: mdl-37928443

RESUMEN

Insulin-like growth factor I (IGF-I) is a growth-promoting anabolic hormone that fosters cell growth and tissue homeostasis. IGF-I deficiency is associated with several diseases, including growth disorders and neurological and musculoskeletal diseases due to impaired regeneration. Despite the vast regenerative potential of IGF-I, its unfavorable pharmacokinetic profile has prevented it from being used therapeutically. In this study, we resolved these challenges by the local administration of IGF-I mRNA, which ensures desirable homeostatic kinetics and non-systemic, local dose-dependent expression of IGF-I protein. Furthermore, IGF-I mRNA constructs were sequence engineered with heterologous signal peptides, which improved in vitro protein secretion (2- to 6-fold) and accelerated in vivo functional regeneration (16-fold) over endogenous IGF-I mRNA. The regenerative potential of engineered IGF-I mRNA was validated in a mouse myotoxic muscle injury and rabbit spinal disc herniation models. Engineered IGF-I mRNA had a half-life of 17-25 h in muscle tissue and showed dose-dependent expression of IGF-I over 2-3 days. Animal models confirm that locally administered IGF-I mRNA remained at the site of injection, contributing to the safety profile of mRNA-based treatment in regenerative medicine. In summary, we demonstrate that engineered IGF-I mRNA holds therapeutic potential with high clinical translatability in different diseases.

3.
Hum Mol Genet ; 31(1): 82-96, 2021 12 17.
Artículo en Inglés | MEDLINE | ID: mdl-34368854

RESUMEN

Spinal muscular atrophy (SMA) is caused by the loss of the survival motor neuron 1 (SMN1) gene function. The related SMN2 gene partially compensates but produces insufficient levels of SMN protein due to alternative splicing of exon 7. Evrysdi™ (risdiplam), recently approved for the treatment of SMA, and related compounds promote exon 7 inclusion to generate full-length SMN2 mRNA and increase SMN protein levels. SMNΔ7 type I SMA mice survive without treatment for ~17 days. SMN2 mRNA splicing modulators increase survival of SMN∆7 mice with treatment initiated at postnatal day 3 (PND3). To define SMN requirements for adult mice, SMNΔ7 mice were dosed with an SMN2 mRNA splicing modifier from PND3 to PND40, then dosing was stopped. Mice not treated after PND40 showed progressive weight loss, necrosis, and muscle atrophy after ~20 days. Male mice presented a more severe phenotype than female mice. Mice dosed continuously did not show disease symptoms. The estimated half-life of SMN protein is 2 days indicating that the SMA phenotype reappeared after SMN protein levels returned to baseline. Although SMN protein levels decreased with age in mice and SMN protein levels were higher in brain than in muscle, our studies suggest that SMN protein is required throughout the life of the mouse and is especially essential in adult peripheral tissues including muscle. These studies indicate that drugs such as risdiplam will be optimally therapeutic when given as early as possible after diagnosis and potentially will be required for the life of an SMA patient.


Asunto(s)
Atrofia Muscular Espinal , Empalme Alternativo , Animales , Modelos Animales de Enfermedad , Progresión de la Enfermedad , Exones , Femenino , Humanos , Masculino , Ratones , Atrofia Muscular Espinal/metabolismo , Empalme del ARN , 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
5.
Cells ; 9(6)2020 06 22.
Artículo en Inglés | MEDLINE | ID: mdl-32580353

RESUMEN

Forced expression of insulin-like growth factor binding proteins (IGFBPs) in transgenic mice has clearly revealed inhibitory effects on somatic growth. However, by this approach, it cannot be solved if or how IGFBPs rule insulin-like growth factor (IGF)-dependent growth under normal conditions. In order to address this question, we have used growth-selected mouse models (obese and lean) and studied IGF-1 and IGFBPs in serum with respect to longitudinal growth activity in males and females compared with unselected controls. In mice of both genders, body weights were recorded and daily weight gains were calculated. Between 2 and 54 weeks of age, serum IGF-1 was determined by ELISA and intact IGFBP-2, -3 and -4 were quantified by Western ligand blotting. The molar ratio of IGF-1 to the sum of IGFBP-2 to -4 was calculated for all groups and plotted against the daily weight gain curve. Growth-selected mice are characterized by higher daily weight gains and extended periods of elevated growth activity if compared to matched unselected controls. Therefore, adult mice from the obese and lean groups can achieve more than twofold increased body weight in both genders (p < 0.001). Between 2 and 11 weeks of age, in obese and lean mice of both genders, serum IGF-1 concentrations are increased more prominently if compared to unselected controls (p < 0.001). Instead, substantial decreases of IGFBPs, particularly of IGFBP-2, are observed in males and females of all groups at the age of 2 to 4 weeks (p < 0.001). Due to the strong increase of IGF-1 but not of IGFBPs between two and four weeks of age, the ratio of IGF-1 to IGFBP-2 to -4 in serum significantly increased in all groups and genders (p < 0.05). Notably, the IGF-1 to IGFBP ratio was higher in male and female obese mice if compared to unselected controls (p < 0.05).


Asunto(s)
Proteínas de Unión a Factor de Crecimiento Similar a la Insulina/sangre , Factor I del Crecimiento Similar a la Insulina/metabolismo , Aumento de Peso/fisiología , Animales , Modelos Animales de Enfermedad , Femenino , Estudios Longitudinales , Masculino , Ratones , Ratones Transgénicos
6.
Sci Rep ; 9(1): 7799, 2019 05 24.
Artículo en Inglés | MEDLINE | ID: mdl-31127156

RESUMEN

Spinal muscular atrophy (SMA) is caused by loss-of-function mutations in the survival of motoneuron gene 1 (SMN1). SMA is characterized by motoneuron death, skeletal muscle denervation and atrophy. Disease severity inversely correlates with copy number of a second gene (SMN2), which harbors a splicing defect that causes the production of inadequate levels of functional SMN protein. Small molecules that modify SMN2 splicing towards increased production of functional SMN significantly ameliorate SMA phenotypes in mouse models of severe SMA. At suboptimal doses, splicing modifiers, such as SMN-C1, have served to generate mice that model milder SMA, referred to as pharmacological SMA mice, which survive into early adulthood. Nerve sprouting at endplates, known as terminal sprouting, is key to normal muscle fiber reinnervation following nerve injury and its promotion might mitigate neuromuscular symptoms in mild SMA. Sprouting has been difficult to study in severe SMA mice due to their short lifespan. Here, we show that pharmacological SMA mice are capable of terminal sprouting following reinnervation that is largely SMN-C1 dose-independent, but that they display a reinnervation delay that is critically SMN-C1 dose-dependent. Data also suggest that SMN-C1 can induce by itself a limited terminal sprouting response in SMA and wild-type normally-innervated endplates.


Asunto(s)
Músculo Esquelético/inervación , Atrofia Muscular Espinal/fisiopatología , Unión Neuromuscular/fisiopatología , Animales , Modelos Animales de Enfermedad , Humanos , Ratones , Fibras Musculares Esqueléticas/metabolismo , Fibras Musculares Esqueléticas/patología , Músculo Esquelético/patología , Músculo Esquelético/fisiopatología , Atrofia Muscular Espinal/inducido químicamente , Atrofia Muscular Espinal/patología , Regeneración Nerviosa , Unión Neuromuscular/patología , Células de Schwann/patología
7.
PLoS One ; 13(10): e0205589, 2018.
Artículo en Inglés | MEDLINE | ID: mdl-30304024

RESUMEN

Spinal muscular atrophy (SMA) is a severe genetic disorder that manifests in progressive neuromuscular degeneration. SMA originates from loss-of-function mutations of the SMN1 (Survival of Motor Neuron 1) gene. Recent evidence has implicated peripheral deficits, especially in skeletal muscle, as key contributors to disease progression in SMA. In this study we generated myogenic cells from two SMA-affected human embryonic stem cell (hESC) lines with deletion of SMN1 bearing two copies of the SMN2 gene and recapitulating the molecular phenotype of Type 1 SMA. We characterized myoblasts and myotubes by comparing them to two unaffected, control hESC lines and demonstrate that SMA myoblasts and myotubes showed altered expression of various myogenic markers, which translated into an impaired in vitro myogenic maturation and development process. Additionally, we provide evidence that these SMN1 deficient cells display functional deficits in cholinergic calcium signaling response, glycolysis and oxidative phosphorylation. Our data describe a novel human myogenic SMA model that might be used for interrogating the effect of SMN depletion during skeletal muscle development, and as model to investigate biological mechanisms targeting myogenic differentiation, mitochondrial respiration and calcium signaling processes in SMA muscle cells.


Asunto(s)
Células Madre Embrionarias Humanas/metabolismo , Desarrollo de Músculos/fisiología , Fibras Musculares Esqueléticas/metabolismo , Atrofia Muscular Espinal/metabolismo , Mioblastos/metabolismo , Adenosina Trifosfato/metabolismo , Calcio/metabolismo , Cationes Bivalentes/metabolismo , Línea Celular , Expresión Génica , Células Madre Embrionarias Humanas/patología , Humanos , Fibras Musculares Esqueléticas/patología , Atrofia Muscular Espinal/genética , Atrofia Muscular Espinal/patología , Mioblastos/patología , Receptores Colinérgicos/metabolismo , 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/metabolismo
8.
J Med Chem ; 61(15): 6501-6517, 2018 Aug 09.
Artículo en Inglés | MEDLINE | ID: mdl-30044619

RESUMEN

SMA is an inherited disease that leads to loss of motor function and ambulation and a reduced life expectancy. We have been working to develop orally administrated, systemically distributed small molecules to increase levels of functional SMN protein. Compound 2 was the first SMN2 splicing modifier tested in clinical trials in healthy volunteers and SMA patients. It was safe and well tolerated and increased SMN protein levels up to 2-fold in patients. Nevertheless, its development was stopped as a precautionary measure because retinal toxicity was observed in cynomolgus monkeys after chronic daily oral dosing (39 weeks) at exposures in excess of those investigated in patients. Herein, we describe the discovery of 1 (risdiplam, RG7916, RO7034067) that focused on thorough pharmacology, DMPK and safety characterization and optimization. This compound is undergoing pivotal clinical trials and is a promising medicine for the treatment of patients in all ages and stages with SMA.


Asunto(s)
Compuestos Azo/farmacología , Descubrimiento de Drogas , Atrofia Muscular Espinal/tratamiento farmacológico , Atrofia Muscular Espinal/genética , Pirimidinas/farmacología , Empalme del ARN/efectos de los fármacos , Proteína 2 para la Supervivencia de la Neurona Motora/genética , Animales , Compuestos Azo/efectos adversos , Compuestos Azo/uso terapéutico , Humanos , Pirimidinas/efectos adversos , Pirimidinas/uso terapéutico , Seguridad
9.
Restor Neurol Neurosci ; 36(4): 559-569, 2018.
Artículo en Inglés | MEDLINE | ID: mdl-29889090

RESUMEN

BACKGROUND: Traumatic brain injury can result in lasting cognitive dysfunction due to degeneration of mature hippocampal neurons as well as the loss of immature neurons within the dentate gyrus. While endogenous neurogenesis affords a partial recovery of the immature neuron population, hippocampal neurogenesis may be enhanced through therapeutic intervention. Insulin-like growth factor-1 (IGF-1) has the potential to improve cognitive function and promote neurogenesis after TBI, but its short half-life in the systemic circulation makes it difficult to maintain a therapeutic concentration. IGF-1 modified with a polyethylene glycol moiety (PEG-IGF-1) exhibits improved stability and half-life while retaining its ability to enter the brain from the periphery, increasing its viability as a translational approach. OBJECTIVE: The goal of this study was to evaluate the ability of systemic PEG-IGF-1 administration to attenuate acute neuronal loss and stimulate the recovery of hippocampal immature neurons in brain-injured mice. METHODS: In a series of studies utilizing a well-established contusion brain injury model, PEG-IGF-1 was administered subcutaneously after injury. Serum levels of PEG were verified using ELISA and histological staining was used to investigate numbers of degenerating neurons and cortical contusion size at 24 h after injury. Immunofluorescent staining was used to evaluate numbers of immature neurons at 10 d after injury. RESULTS: Although subcutaneous injections of PEG-IGF-1 increased serum IGF-1 levels in a dose-dependent manner, no effects were observed on cortical contusion size, neurodegeneration within the dentate gyrus, or recovery of hippocampal immature neuron numbers. CONCLUSIONS: In contrast to its efficacy in rodent models of neurodegenerative diseases, PEG- IGF-1 was not effective in ameliorating early neuronal loss after contusion brain trauma.


Asunto(s)
Lesiones Traumáticas del Encéfalo/tratamiento farmacológico , Factor I del Crecimiento Similar a la Insulina/administración & dosificación , Fármacos Neuroprotectores/administración & dosificación , Polietilenglicoles/uso terapéutico , Análisis de Varianza , Animales , Lesiones Traumáticas del Encéfalo/patología , Modelos Animales de Enfermedad , Relación Dosis-Respuesta a Droga , Proteínas de Dominio Doblecortina , Fluoresceínas/farmacocinética , Lateralidad Funcional , Hipocampo/efectos de los fármacos , Hipocampo/metabolismo , Hipocampo/patología , Factor I del Crecimiento Similar a la Insulina/metabolismo , Masculino , Ratones , Ratones Endogámicos C57BL , Proteínas Asociadas a Microtúbulos/metabolismo , Neuronas/efectos de los fármacos , Neuronas/metabolismo , Neuropéptidos/metabolismo
10.
Nat Commun ; 9(1): 2032, 2018 05 23.
Artículo en Inglés | MEDLINE | ID: mdl-29795225

RESUMEN

Modification of SMN2 exon 7 (E7) splicing is a validated therapeutic strategy against spinal muscular atrophy (SMA). However, a target-based approach to identify small-molecule E7 splicing modifiers has not been attempted, which could reveal novel therapies with improved mechanistic insight. Here, we chose as a target the stem-loop RNA structure TSL2, which overlaps with the 5' splicing site of E7. A small-molecule TSL2-binding compound, homocarbonyltopsentin (PK4C9), was identified that increases E7 splicing to therapeutic levels and rescues downstream molecular alterations in SMA cells. High-resolution NMR combined with molecular modelling revealed that PK4C9 binds to pentaloop conformations of TSL2 and promotes a shift to triloop conformations that display enhanced E7 splicing. Collectively, our study validates TSL2 as a target for small-molecule drug discovery in SMA, identifies a novel mechanism of action for an E7 splicing modifier, and sets a precedent for other splicing-mediated diseases where RNA structure could be similarly targeted.


Asunto(s)
Imidazoles/farmacología , Indoles/farmacología , Atrofia Muscular Espinal/tratamiento farmacológico , ARN Mensajero/metabolismo , Empalme Alternativo , Animales , Animales Modificados Genéticamente , Drosophila , Evaluación Preclínica de Medicamentos , Exones/genética , Células HeLa , Humanos , Imidazoles/química , Imidazoles/uso terapéutico , Indoles/química , Indoles/uso terapéutico , Terapia Molecular Dirigida/métodos , Atrofia Muscular Espinal/genética , Fenotipo , Sitios de Empalme de ARN , ARN Mensajero/química , ARN Mensajero/genética , Elementos Reguladores de la Transcripción/efectos de los fármacos , Proteína 2 para la Supervivencia de la Neurona Motora/genética
11.
Nat Commun ; 8(1): 1476, 2017 11 14.
Artículo en Inglés | MEDLINE | ID: mdl-29133793

RESUMEN

Small molecule splicing modifiers have been previously described that target the general splicing machinery and thus have low specificity for individual genes. Several potent molecules correcting the splicing deficit of the SMN2 (survival of motor neuron 2) gene have been identified and these molecules are moving towards a potential therapy for spinal muscular atrophy (SMA). Here by using a combination of RNA splicing, transcription, and protein chemistry techniques, we show that these molecules directly bind to two distinct sites of the SMN2 pre-mRNA, thereby stabilizing a yet unidentified ribonucleoprotein (RNP) complex that is critical to the specificity of these small molecules for SMN2 over other genes. In addition to the therapeutic potential of these molecules for treatment of SMA, our work has wide-ranging implications in understanding how small molecules can interact with specific quaternary RNA structures.


Asunto(s)
Atrofia Muscular Espinal/tratamiento farmacológico , Piperazinas/farmacología , Precursores del ARN/metabolismo , Empalme del ARN/efectos de los fármacos , ARN Mensajero/metabolismo , Proteínas de Unión al ARN/metabolismo , Biflavonoides/farmacología , Sistema Libre de Células , Biología Computacional , Compuestos Epoxi/farmacología , Exones/genética , Fibroblastos , Células HEK293 , Células HeLa , Humanos , Ligandos , Macrólidos/farmacología , Atrofia Muscular Espinal/genética , Piperazinas/síntesis química , Unión Proteica , Estructura Cuaternaria de Proteína , Proteómica/métodos , Precursores del ARN/genética , ARN Mensajero/genética , Empalmosomas/efectos de los fármacos , Empalmosomas/metabolismo , Proteína 1 para la Supervivencia de la Neurona Motora/genética , Proteína 2 para la Supervivencia de la Neurona Motora/genética
12.
J Med Chem ; 60(10): 4444-4457, 2017 05 25.
Artículo en Inglés | MEDLINE | ID: mdl-28441483

RESUMEN

Spinal muscular atrophy (SMA) is caused by mutation or deletion of the survival motor neuron 1 (SMN1) gene, resulting in low levels of functional SMN protein. We have reported recently the identification of small molecules (coumarins, iso-coumarins and pyrido-pyrimidinones) that modify the alternative splicing of SMN2, a paralogous gene to SMN1, restoring the survival motor neuron (SMN) protein level in mouse models of SMA. Herein, we report our efforts to identify a novel chemotype as one strategy to potentially circumvent safety concerns from earlier derivatives such as in vitro phototoxicity and in vitro mutagenicity associated with compounds 1 and 2 or the in vivo retinal findings observed in a long-term chronic tox study with 3 at high exposures only. Optimized representative compounds modify the alternative splicing of SMN2, increase the production of full length SMN2 mRNA, and therefore levels of full length SMN protein upon oral administration in two mouse models of SMA.


Asunto(s)
Benzamidas/química , Benzamidas/farmacología , Atrofia Muscular Espinal/genética , Empalme del ARN/efectos de los fármacos , ARN Mensajero/genética , Proteína 2 para la Supervivencia de la Neurona Motora/genética , Animales , Benzamidas/farmacocinética , Diseño de Fármacos , Ratones , Modelos Moleculares , Atrofia Muscular Espinal/tratamiento farmacológico
13.
Sci Rep ; 7(1): 241, 2017 03 21.
Artículo en Inglés | MEDLINE | ID: mdl-28325900

RESUMEN

Insulin-like growth factor-I (IGF-I) is involved in the maturation and maintenance of neurons, and impaired IGF-I signaling has been shown to play a role in various neurological diseases including stroke. The aim of the present study was to investigate the efficacy of an optimized IGF-I variant by adding a 40 kDa polyethylene glycol (PEG) chain to IGF-I to form PEG-IGF-I. We show that PEG-IGF-I has a slower clearance which allows for twice-weekly dosing to maintain steady-state serum levels in mice. Using a photothrombotic model of focal stroke, dosing from 3 hrs post-stroke dose-dependently (0.3-1 mg/kg) decreases the volume of infarction and improves motor behavioural function in both young 3-month and aged 22-24 month old mice. Further, PEG-IGF-I treatment increases GFAP expression when given early (3 hrs post-stroke), increases Synaptophysin expression and increases neurogenesis in young and aged. Finally, neurons (P5-6) cultured in vitro on reactive astrocytes in the presence of PEG-IGF-I showed an increase in neurite length, indicating that PEG-IGF-I can aid in sprouting of new connections. This data suggests a modulatory role of IGF-I in both protective and regenerative processes, and indicates that therapeutic approaches using PEG-IGF-I should be given early and where the endogenous regenerative potential is still high.


Asunto(s)
Factor I del Crecimiento Similar a la Insulina/administración & dosificación , Isquemia/terapia , Accidente Cerebrovascular/terapia , Animales , Modelos Animales de Enfermedad , Factor I del Crecimiento Similar a la Insulina/farmacocinética , Locomoción , Ratones , Suero/química , Resultado del Tratamiento
14.
Growth Horm IGF Res ; 30-31: 1-10, 2016.
Artículo en Inglés | MEDLINE | ID: mdl-27544574

RESUMEN

OBJECTIVE: The insulin-like growth factor binding proteins (IGFBPs) are thought to modulate cell size and homeostasis via IGF-I-dependent and -independent pathways. There is a considerable dearth of information regarding the function of IGFBPs in skeletal muscle, particularly their role in the pathophysiology of Duchenne muscular dystrophy (DMD). In this study we tested the hypothesis that intramuscular IGFBP-2 overexpression would ameliorate the pathology in mdx dystrophic mice. DESIGN: 4week old male C57Bl/10 and mdx mice received a single intramuscular injection of AAV6-empty or AAV6-IGFBP-2 vector into the tibialis anterior muscle. At 8weeks post-injection the effect of IGFBP-2 overexpression on the structure and function of the injected muscle was assessed. RESULTS: AAV6-mediated IGFBP-2 overexpression in the tibialis anterior (TA) muscles of 4-week-old C57BL/10 and mdx mice reduced the mass of injected muscle after 8weeks, inducing a slower muscle phenotype in C57BL/10 but not mdx mice. Analysis of inflammatory and fibrotic gene expression revealed no changes between control and IGFBP-2 injected muscles in dystrophic (mdx) mice. CONCLUSIONS: Together these results indicate that the IGFBP-2-induced promotion of a slower muscle phenotype is impaired in muscles of dystrophin-deficient mdx mice, which contributes to the inability of IGFBP-2 to ameliorate the dystrophic pathology. The findings implicate the dystrophin-glycoprotein complex (DGC) in the signaling required for this adaptation.


Asunto(s)
Proteína 2 de Unión a Factor de Crecimiento Similar a la Insulina/genética , Músculo Esquelético/metabolismo , Animales , Modelos Animales de Enfermedad , Fibrosis/genética , Inflamación/genética , Proteína 2 de Unión a Factor de Crecimiento Similar a la Insulina/metabolismo , Ratones , Ratones Endogámicos mdx , Músculo Esquelético/inmunología , Músculo Esquelético/patología , Distrofia Muscular de Duchenne , Fenotipo , Transcriptoma
15.
J Med Chem ; 59(13): 6086-100, 2016 07 14.
Artículo en Inglés | MEDLINE | ID: mdl-27299419

RESUMEN

Spinal muscular atrophy (SMA) is the leading genetic cause of infant and toddler mortality, and there is currently no approved therapy available. SMA is caused by mutation or deletion of the survival motor neuron 1 (SMN1) gene. These mutations or deletions result in low levels of functional SMN protein. SMN2, a paralogous gene to SMN1, undergoes alternative splicing and exclusion of exon 7, producing an unstable, truncated SMNΔ7 protein. Herein, we report the identification of a pyridopyrimidinone series of small molecules that modify the alternative splicing of SMN2, increasing the production of full-length SMN2 mRNA. Upon oral administration of our small molecules, the levels of full-length SMN protein were restored in two mouse models of SMA. In-depth lead optimization in the pyridopyrimidinone series culminated in the selection of compound 3 (RG7800), the first small molecule SMN2 splicing modifier to enter human clinical trials.


Asunto(s)
Empalme Alternativo/efectos de los fármacos , Atrofia Muscular Espinal/tratamiento farmacológico , Pirimidinonas/química , Pirimidinonas/farmacología , ARN Mensajero/genética , Proteína 2 para la Supervivencia de la Neurona Motora/genética , Animales , Exones/efectos de los fármacos , Humanos , Ratones , Atrofia Muscular Espinal/genética , Pirimidinonas/farmacocinética , Pirimidinonas/uso terapéutico
16.
Hum Mol Genet ; 25(10): 1885-1899, 2016 05 15.
Artículo en Inglés | MEDLINE | ID: mdl-26931466

RESUMEN

Spinal muscular atrophy (SMA) is caused by the loss or mutation of both copies of the survival motor neuron 1 (SMN1) gene. The related SMN2 gene is retained, but due to alternative splicing of exon 7, produces insufficient levels of the SMN protein. Here, we systematically characterize the pharmacokinetic and pharmacodynamics properties of the SMN splicing modifier SMN-C1. SMN-C1 is a low-molecular weight compound that promotes the inclusion of exon 7 and increases production of SMN protein in human cells and in two transgenic mouse models of SMA. Furthermore, increases in SMN protein levels in peripheral blood mononuclear cells and skin correlate with those in the central nervous system (CNS), indicating that a change of these levels in blood or skin can be used as a non-invasive surrogate to monitor increases of SMN protein levels in the CNS. Consistent with restored SMN function, SMN-C1 treatment increases the levels of spliceosomal and U7 small-nuclear RNAs and corrects RNA processing defects induced by SMN deficiency in the spinal cord of SMNΔ7 SMA mice. A 100% or greater increase in SMN protein in the CNS of SMNΔ7 SMA mice robustly improves the phenotype. Importantly, a ∼50% increase in SMN leads to long-term survival, but the SMA phenotype is only partially corrected, indicating that certain SMA disease manifestations may respond to treatment at lower doses. Overall, we provide important insights for the translation of pre-clinical data to the clinic and further therapeutic development of this series of molecules for SMA treatment.


Asunto(s)
Isocumarinas/administración & dosificación , Atrofia Muscular Espinal/tratamiento farmacológico , Atrofia Muscular Espinal/genética , Piperazinas/administración & dosificación , Bibliotecas de Moléculas Pequeñas/farmacocinética , Proteína 2 para la Supervivencia de la Neurona Motora/genética , Empalme Alternativo/efectos de los fármacos , Empalme Alternativo/genética , Animales , Sistema Nervioso Central/metabolismo , Modelos Animales de Enfermedad , Relación Dosis-Respuesta a Droga , Exones/genética , Humanos , Leucocitos Mononucleares/efectos de los fármacos , Ratones , Ratones Transgénicos , Atrofia Muscular Espinal/sangre , Atrofia Muscular Espinal/patología , Empalme del ARN/efectos de los fármacos , Empalme del ARN/genética , Piel/metabolismo , Bibliotecas de Moléculas Pequeñas/administración & dosificación , Proteína 2 para la Supervivencia de la Neurona Motora/sangre
17.
Hum Mol Genet ; 25(5): 964-75, 2016 Mar 01.
Artículo en Inglés | MEDLINE | ID: mdl-26758873

RESUMEN

Spinal muscular atrophy (SMA) is a genetic disease characterized by atrophy of muscle and loss of spinal motor neurons. SMA is caused by deletion or mutation of the survival motor neuron 1 (SMN1) gene, and the nearly identical SMN2 gene fails to generate adequate levels of functional SMN protein due to a splicing defect. Currently, several therapeutics targeted to increase SMN protein are in clinical trials. An outstanding issue in the field is whether initiating treatment in symptomatic older patients would confer a therapeutic benefit, an important consideration as the majority of patients with milder forms of SMA are diagnosed at an older age. An SMA mouse model that recapitulates the disease phenotype observed in adolescent and adult SMA patients is needed to address this important question. We demonstrate here that Δ7 mice, a model of severe SMA, treated with a suboptimal dose of an SMN2 splicing modifier show increased SMN protein, survive into adulthood and display SMA disease-relevant pathologies. Increasing the dose of the splicing modifier after the disease symptoms are apparent further mitigates SMA histopathological features in suboptimally dosed adult Δ7 mice. In addition, inhibiting myostatin using intramuscular injection of AAV1-follistatin ameliorates muscle atrophy in suboptimally dosed Δ7 mice. Taken together, we have developed a new murine model of symptomatic SMA in adolescents and adult mice that is induced pharmacologically from a more severe model and demonstrated efficacy of both SMN2 splicing modifiers and a myostatin inhibitor in mice at later disease stages.


Asunto(s)
Folistatina/farmacología , Factores Inmunológicos/farmacología , Atrofia Muscular Espinal/tratamiento farmacológico , Empalme del ARN/efectos de los fármacos , Proteína 1 para la Supervivencia de la Neurona Motora/genética , Proteína 2 para la Supervivencia de la Neurona Motora/agonistas , Adolescente , Adulto , Edad de Inicio , Animales , Dependovirus/genética , Dependovirus/metabolismo , Modelos Animales de Enfermedad , Eliminación de Gen , Vectores Genéticos/química , Vectores Genéticos/metabolismo , Humanos , Ratones , Neuronas Motoras/efectos de los fármacos , Neuronas Motoras/metabolismo , Neuronas Motoras/patología , Atrofia Muscular Espinal/genética , Atrofia Muscular Espinal/metabolismo , Atrofia Muscular Espinal/patología , Miostatina/antagonistas & inhibidores , Miostatina/genética , Miostatina/metabolismo , Fenotipo , 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/metabolismo
18.
Aging Cell ; 15(1): 111-7, 2016 Feb.
Artículo en Inglés | MEDLINE | ID: mdl-26507795

RESUMEN

Impaired growth is often associated with an extension of lifespan. However, the negative correlation between somatic growth and life expectancy is only true within, but not between, species. This can be observed because smaller species have, as a rule, a shorter lifespan than larger species. In insects and worms, reduced reproductive development and increased fat storage are associated with prolonged lifespan. However, in mammals the relationship between the dynamics of reproductive development, fat metabolism, growth rate, and lifespan are less clear. To address this point, female transgenic mice that were overexpressing similar levels of either intact (D-mice) or mutant insulin-like growth factor-binding protein-2 (IGFBP-2) lacking the Arg-Gly-Asp (RGD) motif (E- mice) were investigated. Both lines of transgenic mice exhibited a similar degree of growth impairment (-9% and -10%) in comparison with wild-type controls (C-mice). While in D-mice, sexual maturation was found to be delayed and life expectancy was significantly increased in comparison with C-mice, these parameters were unaltered in E-mice in spite of their reduced growth rate. These observations indicate that the RGD-domain has a major influence on the pleiotropic effects of IGFBP-2 and suggest that somatic growth and time of sexual maturity or somatic growth and life expectancy are less closely related than thought previously.


Asunto(s)
Peso Corporal/fisiología , Proteína 2 de Unión a Factor de Crecimiento Similar a la Insulina/metabolismo , Esperanza de Vida , Metabolismo de los Lípidos/fisiología , Tamaño de los Órganos/fisiología , Animales , Peso Corporal/genética , Femenino , Proteína 2 de Unión a Factor de Crecimiento Similar a la Insulina/genética , Metabolismo de los Lípidos/genética , Ratones Endogámicos C57BL , Ratones Transgénicos , Tamaño de los Órganos/genética , Factores de Tiempo
19.
Growth Horm IGF Res ; 26: 42-9, 2016 02.
Artículo en Inglés | MEDLINE | ID: mdl-26597140

RESUMEN

The insulin-like growth factor binding proteins (IGFBPs) are determinants of local IGF-effects and thus have an impact on growth and metabolism in vertebrate species. In farm animals, IGFBPs are associated with traits such as growth rate, body composition, milk production, or fertility. It may be assumed, that selective breeding and characteristic phenotypes of breeds are related to differential expression of IGFBPs. Therefore, the aim of the present study was to investigate the effects of selective breeding on blood IGFBP concentrations of farm animals. Breeds of the sheep, goat, and cattle species were investigated. IGFBP-3, -2, and -4 were analyzed with quantitative Western ligand blotting (qWLB), enabling comprehensive monitoring of intact IGFBPs with IGF-binding capacity. We show that in sera of all species and breeds investigated, IGFBP-3, -2, and -4 were simultaneously detectable by qWLB analysis. IGFBP-3 and the total amount of IGFBPs were significantly increased (P<0.05) in Cameroon sheep, if compared to 3 of 4 other sheep breeds, as well as in Dwarf goats versus Toggenburg and Boer goats (P<0.01). IGFBP-2 was elevated in Cameroon sheep and Boer goats, if compared to other breeds of these species (P<0.01), respectively. Holstein Friesian dairy cows had higher levels of IGFBP-4 (P<0.05), if compared to conventional crossbreeds of beef cattle. In Dwarf goats the ratio of IGFBP-3/IGFBP-2 was about 3-fold higher than in other goat breeds (P<0.001). The total IGFBP amount of Toggenburg goats was reduced (P<0.05), compared to the other goat breeds. In conclusion, our data indicate that common and specific features of IGFBP fingerprints are found in different ruminant species and breeds. Our findings may introduce quantitative Western ligand blotting as an attractive tool for biomarker development and molecular phenotyping in farm animal breeds.


Asunto(s)
Proteínas de Unión a Factor de Crecimiento Similar a la Insulina/metabolismo , Rumiantes/metabolismo , Animales , Animales Domésticos , Western Blotting , Bovinos , Proteína 2 de Unión a Factor de Crecimiento Similar a la Insulina/metabolismo , Proteína 3 de Unión a Factor de Crecimiento Similar a la Insulina/metabolismo , Proteína 4 de Unión a Factor de Crecimiento Similar a la Insulina/metabolismo , Factor I del Crecimiento Similar a la Insulina/metabolismo , Factor II del Crecimiento Similar a la Insulina/metabolismo , Ovinos , Especificidad de la Especie
20.
Cell Metab ; 20(4): 662-9, 2014 Oct 07.
Artículo en Inglés | MEDLINE | ID: mdl-25176146

RESUMEN

Most mitochondrial proteins possess N-terminal presequences that are required for targeting and import into the organelle. Upon import, presequences are cleaved off by matrix processing peptidases and subsequently degraded by the peptidasome Cym1/PreP, which also degrades Amyloid-beta peptides (Aß). Here we find that impaired turnover of presequence peptides results in feedback inhibition of presequence processing enzymes. Moreover, Aß inhibits degradation of presequence peptides by PreP, resulting in accumulation of mitochondrial preproteins and processing intermediates. Dysfunctional preprotein maturation leads to rapid protein degradation and an imbalanced organellar proteome. Our findings reveal a general mechanism by which Aß peptide can induce the multiple diverse mitochondrial dysfunctions accompanying Alzheimer's disease.


Asunto(s)
Péptidos beta-Amiloides/metabolismo , Metaloproteasas/metabolismo , Mitocondrias/metabolismo , Proteínas Mitocondriales/metabolismo , Proteínas de Saccharomyces cerevisiae/metabolismo , Serina Endopeptidasas/metabolismo , Enfermedad de Alzheimer/metabolismo , Enfermedad de Alzheimer/patología , Animales , Encéfalo/metabolismo , Humanos , Metaloproteasas/antagonistas & inhibidores , Metaloproteasas/genética , Ratones , Ratones Endogámicos C57BL , Proteínas Mitocondriales/antagonistas & inhibidores , Mutación , Proto-Oncogenes Mas , Proteínas Proto-Oncogénicas/metabolismo , Especies Reactivas de Oxígeno/metabolismo , Receptores Acoplados a Proteínas G/metabolismo , Saccharomyces cerevisiae/metabolismo , Proteínas de Saccharomyces cerevisiae/antagonistas & inhibidores , Proteínas de Saccharomyces cerevisiae/genética , Superóxido Dismutasa/metabolismo
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