RESUMEN
Phase 3 randomized controlled trials (RCTs), while the gold standard for treatment efficacy and safety, are not always feasible, are expensive, can be prolonged and can be limited in generalizability. Other under-recognized sources of evidence can also help advance drug development. Basic science, proof-of-concept studies and early-phase RCTs can provide evidence regarding the potential for clinical benefit. Real-world evidence generated from registries or observational datasets can provide insights into the treatment of rare diseases that often pose a challenge for trial recruitment. Pragmatic trials embedded in healthcare systems can assess the treatment effects in clinical settings among patient populations sometimes excluded from trials. This Perspective discusses potential sources of evidence that may be used to complement explanatory phase 3 RCTs and to speed the development of new cardiovascular medications. Content is derived from the 19th Global Cardiovascular Clinical Trialists meeting (December 2022), involving clinical trialists, patients, clinicians, regulators, funders and industry representatives.
Asunto(s)
Desarrollo de Medicamentos , Humanos , Desarrollo de Medicamentos/métodos , Fármacos Cardiovasculares/uso terapéutico , Fármacos Cardiovasculares/efectos adversos , Ensayos Clínicos Fase III como Asunto , Ensayos Clínicos Pragmáticos como Asunto/métodos , Proyectos de Investigación/normas , Enfermedades Cardiovasculares/tratamiento farmacológico , Medicina Basada en la Evidencia/métodos , Resultado del Tratamiento , Ensayos Clínicos Controlados Aleatorios como Asunto/métodos , Estudios Observacionales como Asunto/métodosRESUMEN
Dysfunction of proteasomal protein degradation is involved in neurodegeneration in Parkinson's disease (PD). Recently we identified the regulatory proteasomal subunit S6 ATPase as a novel interactor of synphilin-1, which is a substrate of the ubiquitin-ligase Parkin (PARK2) and an interacting protein of alpha-synuclein (PARK1). To further investigate a potential role in the pathogenesis of PD, we performed a detailed mutation analysis of the S6 ATPase gene in a large sample of 486 German sporadic and familial PD patients. Direct sequencing revealed two novel intronic variants. An insertion/deletion variant in intron 5 of the S6 ATPase gene was more frequent in patients compared to controls. Moreover, this variant was significantly more frequent in early-onset compared to late-onset PD patients. The identification of a genetic link between a regulatory proteasomal subunit and PD further underscores the relevance of disturbed protein degradation in PD.
Asunto(s)
Adenosina Trifosfatasas/genética , Enfermedad de Parkinson/epidemiología , Enfermedad de Parkinson/genética , Complejo de la Endopetidasa Proteasomal/genética , ATPasas Asociadas con Actividades Celulares Diversas , Anciano , Alelos , Proteínas Portadoras/genética , Cromatografía Líquida de Alta Presión , Análisis Mutacional de ADN , ADN Complementario/biosíntesis , ADN Complementario/genética , Femenino , Genotipo , Alemania/epidemiología , Humanos , Intrones/genética , Masculino , Persona de Mediana Edad , Proteínas del Tejido Nervioso/genética , Polimorfismo de Longitud del Fragmento de Restricción , Desnaturalización Proteica , ARN Mensajero/biosíntesis , ARN Mensajero/genética , Reacción en Cadena de la Polimerasa de Transcriptasa InversaRESUMEN
Synphilin-1 is linked to Parkinson's disease (PD), based on its role as an alpha-synuclein (PARK1)-interacting protein and substrate of the ubiquitin E3 ligase Parkin (PARK2) and because of its presence in Lewy bodies (LB) in brains of PD patients. We found that overexpression of synphilin-1 in cells leads to the formation of ubiquitinated cytoplasmic inclusions supporting a derangement of the ubiquitin-proteasome system in PD. We report here a novel specific interaction of synphilin-1 with the regulatory proteasomal protein S6 ATPase (tbp7). Functional characterization of this interaction on a cellular level revealed colocalization of S6 and synphilin-1 in aggresome-like intracytoplasmic inclusions. Overexpression of synphilin-1 and S6 in cells caused reduced proteasomal activity associated with a significant increase in inclusion formation compared to cells expressing synphilin-1 alone. Steady-state levels of synphilin-1 in cells were not altered after cotransfection of S6 and colocalization of synphilin-1-positive inclusions with lysosomal markers suggests the presence of an alternative lysosomal degradation pathway. Subsequent immunohistochemical studies in brains of PD patients identified S6 ATPase as a component of LB. This is the first study investigating the physiological role of synphilin-1 in the ubiquitin proteasome system. Our data suggest a direct interaction of synphilin-1 with the regulatory complex of the proteasome modulating proteasomal function.
Asunto(s)
Proteínas Portadoras/metabolismo , Proteínas del Tejido Nervioso/metabolismo , Enfermedad de Parkinson/etiología , Complejo de la Endopetidasa Proteasomal/metabolismo , ATPasas Asociadas con Actividades Celulares Diversas , Encéfalo/patología , Humanos , Cuerpos de Inclusión/metabolismo , Cuerpos de Lewy , Lisosomas/metabolismo , Complejo de la Endopetidasa Proteasomal/fisiologíaRESUMEN
Recently targeted disruption of Omi/HtrA2 has been found to cause neurodegeneration and a parkinsonian phenotype in mice. Using a candidate gene approach, we performed a mutation screening of the Omi/HtrA2 gene in German Parkinson's disease (PD) patients. In four patients, we identified a novel heterozygous G399S mutation, which was absent in healthy controls. Moreover, we identified a novel A141S polymorphism that was associated with PD (P<0.05). Both mutations resulted in defective activation of the protease activity of Omi/HtrA2. Immunohistochemistry and functional analysis in stably transfected cells revealed that S399 mutant Omi/HtrA2 and to a lesser extent, the risk allele of the A141S polymorphism induced mitochondrial dysfunction associated with altered mitochondrial morphology. Cells overexpressing S399 mutant Omi/HtrA2 were more susceptible to stress-induced cell death than wild-type. On the basis of functional genomics, our results provide a novel link between mitochondrial dysfunction and neurodegeneration in PD.
Asunto(s)
Enfermedad de Parkinson/genética , Secuencia de Aminoácidos , Animales , Secuencia de Bases , Sitios de Unión , Encéfalo/metabolismo , Muerte Celular , Células Cultivadas , Análisis Mutacional de ADN , Femenino , Regulación de la Expresión Génica , Serina Peptidasa A2 que Requiere Temperaturas Altas , Humanos , Masculino , Potenciales de la Membrana , Ratones , Persona de Mediana Edad , Mitocondrias/metabolismo , Mitocondrias/ultraestructura , Proteínas Mitocondriales , Modelos Moleculares , Datos de Secuencia Molecular , Enfermedad de Parkinson/metabolismo , Enfermedad de Parkinson/patología , Mutación Puntual , Homología de Secuencia de Aminoácido , Serina Endopeptidasas/genética , Serina Endopeptidasas/metabolismo , TransfecciónRESUMEN
Mutations in the parkin gene have been identified as a common cause of autosomal recessive inherited Parkinson disease (PD) associated with early disease manifestation. However, based on linkage data, mutations in other genes contribute to the genetic heterogeneity of early-onset PD (EOPD). Recently, two mutations in the DJ1 gene were described as a second cause of autosomal recessive EOPD (PARK7). Analyzing the PARK7/DJ1 gene in 104 EOPD patients, we identified a third mutation, c.192G>C (p.E64D), associated with EOPD in a patient of Turkish ancestry and characterized the functional significance of this amino acid substitution. In the patient, a substantial reduction of dopamine uptake transporter (DAT) binding was found in the striatum using [(18)F]FP-CIT and PET, indicating a serious loss of presynaptic dopaminergic afferents. His sister, homozygous for E64D, was clinically unaffected but showed reduced dopamine uptake when compared with a clinically unaffected brother, who is heterozygous for E64D. We demonstrate by crystallography that the E64D mutation does not alter the structure of the DJ1 protein, however we observe a tendency towards decreased levels of the mutant protein when overexpressed in HEK293 or COS7 cells. Using immunocytochemistry in contrast to the homogenous nuclear and cytoplasmic staining in HEK293 cells overexpressing wild-type DJ1, about 5% of the cells expressing E64D and up to 80% of the cells expressing the recently described L166P mutation displayed a predominant nuclear localization of the mutant DJ1 protein.
Asunto(s)
Sustitución de Aminoácidos , Mutación Missense , Proteínas Oncogénicas/genética , Enfermedad de Parkinson/genética , Mutación Puntual , Adulto , Edad de Inicio , Animales , Células COS , Línea Celular/metabolismo , Núcleo Celular/química , Chlorocebus aethiops , Cuerpo Estriado/diagnóstico por imagen , Cuerpo Estriado/metabolismo , Cristalografía por Rayos X , Análisis Mutacional de ADN , Dopamina/metabolismo , Proteínas de Transporte de Dopamina a través de la Membrana Plasmática , Femenino , Heterogeneidad Genética , Pruebas Genéticas , Genotipo , Alemania/epidemiología , Humanos , Péptidos y Proteínas de Señalización Intracelular , Riñón , Masculino , Glicoproteínas de Membrana/metabolismo , Proteínas de Transporte de Membrana/metabolismo , Persona de Mediana Edad , Modelos Moleculares , Proteínas del Tejido Nervioso/metabolismo , Proteínas Oncogénicas/análisis , Proteínas Oncogénicas/biosíntesis , Proteínas Oncogénicas/química , Enfermedad de Parkinson/diagnóstico por imagen , Enfermedad de Parkinson/epidemiología , Linaje , Tomografía de Emisión de Positrones , Conformación Proteica , Proteína Desglicasa DJ-1 , Proteínas Recombinantes de Fusión/análisis , Proteínas Recombinantes de Fusión/biosíntesis , Turquía/etnologíaRESUMEN
Neurofilament M, a major component of Lewy bodies, represents an interesting candidate in the pathogenesis of Parkinson's disease (PD). We performed detailed mutation analyses of the NF-M gene in 322 familial and sporadic PD patients. Two polymorphisms (Ala475Thr and Gly697Arg) occurred at similar frequencies in PD patients and controls. A Pro725Gln substitution and a deletion of valine in position 829 were identified in two PD patients. These substitutions affect residues of the NF-M protein that are highly conserved among different species. None of our patients carried the Gly336Ser substitution, which has been described in familial PD. Our results argue against a major role of NF-M in PD. However, rare variants of the NF-M gene may act as susceptibility factors for PD and functional analyses of the identified variations are warranted to decipher possible mechanisms in neurodegeneration.
Asunto(s)
Predisposición Genética a la Enfermedad/genética , Proteínas de Neurofilamentos/genética , Enfermedad de Parkinson/genética , Mutación Puntual/genética , Adulto , Sustitución de Aminoácidos/genética , Secuencia de Bases/genética , Encéfalo/metabolismo , Encéfalo/patología , Encéfalo/fisiopatología , Análisis Mutacional de ADN , Femenino , Frecuencia de los Genes/genética , Pruebas Genéticas , Humanos , Cuerpos de Lewy/genética , Cuerpos de Lewy/metabolismo , Masculino , Persona de Mediana Edad , Datos de Secuencia Molecular , Proteínas de Neurofilamentos/deficiencia , Neuronas/metabolismo , Neuronas/patología , Enfermedad de Parkinson/metabolismo , Linaje , Polimorfismo Genético/genética , Homología de Secuencia de AminoácidoRESUMEN
Synphilin-1 is linked to the pathogenesis of Parkinson's disease (PD) based on its identification as an alpha-synuclein (PARK1) and parkin (PARK2) interacting protein. Moreover, synphilin-1 is a component of Lewy bodies (LB) in brains of sporadic PD patients. Therefore, we performed a detailed mutation analysis of the synphilin-1 gene in 328 German familial and sporadic PD patients. In two apparently sporadic PD patients we deciphered a novel C to T transition in position 1861 of the coding sequence leading to an amino acid substitution from arginine to cysteine in position 621 (R621C). This mutation was absent in a total of 702 chromosomes of healthy German controls. To define a possible role of mutant synphilin-1 in the pathogenesis of PD we performed functional analyses in SH-SY5Y cells. We found synphilin-1 capable of producing cytoplasmic inclusions in transfected cells. Moreover we observed a significantly reduced number of inclusions in cells expressing C621 synphilin-1 compared with cells expressing wild-type (wt) synphilin-1, when subjected to proteasomal inhibition. C621 synphilin-1 transfected cells were more susceptible to staurosporine-induced cell death than cells expressing wt synphilin-1. Our findings argue in favour of a causative role of the R621C mutation in the synphilin-1 gene in PD and suggest that the formation of intracellular inclusions may be beneficial to cells and that a mutation in synphilin-1 that reduces this ability may sensitize neurons to cellular stress.