RESUMEN
Mutations in PINK1 (PARK6), a serine/threonine kinase involved in mitochondrial homeostasis, are associated with early onset Parkinson's disease. Fibroblasts from Parkinson's disease patients with compound heterozygous mutations in exon 7 (c.1488 + 1G > A; c.1252_1488del) showed no apparent signs of mitochondrial impairment. To elucidate changes primarily caused by lack of functional PINK1, we over-expressed wild-type PINK1, which induced a significant downregulation of LRRK2 (PARK8). Indeed, we found that LRRK2 protein basal levels were significantly higher in the mutant PINK1 fibroblasts. To examine the interaction between the two PARK genes in a disease-relevant cell context, we generated induced pluripotent stem cell (iPSC) lines from mutant, carrier and control fibroblasts by lentiviral-mediated re-programming. Efficiency of neural induction and dopamine differentiation using a floor-plate induction protocol was similar in all genotypes. As observed in fibroblasts, PINK1 mutant neurons showed increased LRRK2 expression both at the RNA and protein level and transient over-expression of wild-type PINK1 efficiently downregulated LRRK2 levels. Additionally, we confirmed a dysregulation of LRRK2 expression in fibroblasts from patients with a different homozygous mutation in PINK1 exon 4, c.926G > A (G309D). Thus, our results identify a novel role of PINK1 modulating the levels of LRRK2 in Parkinson's disease fibroblasts and neurons, suggest a convergent pathway for these PARK genes, and broaden the role of LRRK2 in the pathogenesis of Parkinson's disease.
Asunto(s)
Fibroblastos/metabolismo , Proteína 2 Quinasa Serina-Treonina Rica en Repeticiones de Leucina/genética , Mutación/genética , Neuronas/metabolismo , Enfermedad de Parkinson/genética , Enfermedad de Parkinson/patología , Proteínas Quinasas/genética , Regulación hacia Abajo , Femenino , Fibroblastos/patología , Humanos , Células Madre Pluripotentes Inducidas/metabolismo , Proteína 2 Quinasa Serina-Treonina Rica en Repeticiones de Leucina/metabolismo , Masculino , Persona de Mediana Edad , Neuronas/patologíaRESUMEN
Gerstmann-Sträussler-Scheinker (GSS) syndrome is a fatal autosomal dominant neurodegenerative prionopathy clinically characterized by ataxia, spastic paraparesis, extrapyramidal signs and dementia. In some GSS familiar cases carrying point mutations in the PRNP gene, patients also showed comorbid tauopathy leading to mixed pathologies. In this study we developed an induced pluripotent stem (iPS) cell model derived from fibroblasts of a GSS patient harboring the Y218N PRNP mutation, as well as an age-matched healthy control. This particular PRNP mutation is unique with very few described cases. One of the cases presented neurofibrillary degeneration with relevant Tau hyperphosphorylation. Y218N iPS-derived cultures showed relevant astrogliosis, increased phospho-Tau, altered microtubule-associated transport and cell death. However, they failed to generate proteinase K-resistant prion. In this study we set out to test, for the first time, whether iPS cell-derived neurons could be used to investigate the appearance of disease-related phenotypes (i.e, tauopathy) identified in the GSS patient.
Asunto(s)
Enfermedad de Gerstmann-Straussler-Scheinker/genética , Enfermedad de Gerstmann-Straussler-Scheinker/patología , Células Madre Pluripotentes Inducidas/patología , Mutación/genética , Proteínas Priónicas/genética , Proteínas tau/metabolismo , Astrocitos/metabolismo , Astrocitos/patología , Secuencia de Bases , Encéfalo/patología , Diferenciación Celular , Células Cultivadas , Femenino , Gliosis/patología , Humanos , Células Madre Pluripotentes Inducidas/metabolismo , Persona de Mediana Edad , Mitocondrias/metabolismo , Neuronas/metabolismo , Neuronas/patología , FosforilaciónRESUMEN
BACKGROUND: Mutations in leucine-rich repeat kinase 2 (LRRK2) contribute to both familial and idiopathic forms of Parkinson's disease (PD). Neuroinflammation is a key event in neurodegeneration and aging, and there is mounting evidence of LRRK2 involvement in inflammatory pathways. In a previous study, we described an alteration of the inflammatory response in dermal fibroblasts from PD patients expressing the G2019S and R1441G mutations in LRRK2. METHODS: Taking advantage of cellular reprogramming, we generated induced pluripotent stem cell (iPSC) lines and neurons thereafter, harboring LRRK2G2019S and LRRK2R1441G mutations. We used gene silencing and functional reporter assays to characterize the effect of the mutations. We examined the temporal profile of TNFα-induced changes in proteins of the NF-κB pathway and optimized western blot analysis to capture α-synuclein dynamics. The effects of the mutations and interventions were analyzed by two-way ANOVA tests with respect to corresponding controls. RESULTS: LRRK2 silencing decreased α-synuclein protein levels in mutated neurons and modified NF-κB transcriptional targets, such as PTGS2 (COX-2) and TNFAIP3 (A20). We next tested whether NF-κB and α-synuclein pathways converged and found that TNFα modulated α-synuclein levels, although we could not detect an effect of LRRK2 mutations, partly because of the individual variability. Nevertheless, we confirmed NF-κB dysregulation in mutated neurons, as shown by a protracted recovery of IκBα and a clear impairment in p65 nuclear translocation in the LRRK2 mutants. CONCLUSIONS: Altogether, our results show that LRRK2 mutations affect α-synuclein regulation and impair NF-κB canonical signaling in iPSC-derived neurons. TNFα modulated α-synuclein proteostasis but was not modified by the LRRK2 mutations in this paradigm. These results strengthen the link between LRRK2 and the innate immunity system underscoring the involvement of inflammatory pathways in the neurodegenerative process in PD.
Asunto(s)
Proteína 2 Quinasa Serina-Treonina Rica en Repeticiones de Leucina/genética , Mutación/genética , FN-kappa B/metabolismo , Neuronas/metabolismo , Células Madre Pluripotentes/fisiología , Diferenciación Celular/genética , Células Cultivadas , Citocinas/metabolismo , Análisis Mutacional de ADN , Dopamina/metabolismo , Fibroblastos , Regulación de la Expresión Génica/genética , Proteína Ácida Fibrilar de la Glía/metabolismo , Humanos , Proteína 2 Quinasa Serina-Treonina Rica en Repeticiones de Leucina/metabolismo , Enfermedad de Parkinson/genética , Enfermedad de Parkinson/patología , Transducción de Señal/genética , Transducción de Señal/fisiología , Antígenos Embrionarios Específico de Estadio/metabolismo , Transfección , Tubulina (Proteína)/metabolismo , alfa-Sinucleína/metabolismoRESUMEN
Levodopa-induced dyskinesias (LID) are a frequent complication of Parkinson's disease pharmacotherapy that causes significant disability and narrows the therapeutic window. Pharmacological management of LID is challenging partly because the precise molecular mechanisms are not completely understood. Here, our aim was to determine molecular changes that could unveil targetable mechanisms underlying this drug complication. We examined the expression and downstream activity of dopamine receptors (DR) in the striatum of 1-methyl-4-phenyl-1,2,3,6 tetrahydropiridine (MPTP)-lesioned monkeys with and without L-DOPA treatment. Four monkeys were made dyskinetic and other four received a shorter course of L-DOPA and did not develop LID. Our results show that L-DOPA treatment induces an increase in DRD2 and DRD3 expression in the postcommissural putamen, but only DRD3 is correlated with the severity of LID. Dyskinetic monkeys show a hyperactivation of the canonical DRD1-signaling pathway, measured by an increased phosphorylation of protein kinase A (PKA) and its substrates, particularly DARPP32. In contrast, activation of the DRD2-signaling pathway, visible in the levels of Akt phosphorylated on Thr308 and GSK3ß on Ser9, is associated with L-DOPA treatment, independently of the presence of dyskinesias. Our data clearly demonstrate that dyskinetic monkeys present a dysregulation of the DRD3 receptor and the DRD1 pathway with a sustained increase of PKA activity in the postcommissural putamen. Importantly, we found that all signaling changes related to long-term L-DOPA administration are exquisitely restricted to the postcommissural putamen, which may be related to the recurrent failure of pharmacological approaches.
Asunto(s)
Proteínas Quinasas Dependientes de AMP Cíclico/metabolismo , Trastornos Parkinsonianos/metabolismo , Putamen/metabolismo , Animales , Cuerpo Estriado/efectos de los fármacos , Cuerpo Estriado/metabolismo , Levodopa/farmacología , Levodopa/uso terapéutico , Macaca fascicularis , Masculino , Trastornos Parkinsonianos/tratamiento farmacológico , Fosforilación/efectos de los fármacos , Putamen/efectos de los fármacos , Receptores Dopaminérgicos/metabolismo , Transducción de Señal/efectos de los fármacos , Transducción de Señal/fisiologíaRESUMEN
Inflammatory mechanisms are activated in aging and late-onset neurodegenerative diseases, such as Parkinson's disease (PD). Mutations in leucine-rich repeat kinase 2 (LRRK2) contribute to both idiopathic and familial forms of PD. Here, we investigated the involvement of LRRK2 in inflammatory pathways using primary dermal fibroblasts from patients with 2 common mutations in LRRK2 (G2019S and R1441G), idiopathic PD and age-matched healthy individuals. Basal cyclooxygenase (COX)-2 RNA levels were very high in the fibroblasts of all patients. Remarkably, LRRK2 silencing experiments significantly reduced basal COX-2 levels and COX-2 induction after a pro-inflammatory stimulus. Additionally, in samples from patients with the R1441G mutation and with idiopathic PD, we found a prominent cytoplasmic re-distribution of human antigen R, a protein that, among others, stabilizes COX-2 RNA. Furthermore, the response to lipopolysaccharide was defective in these 2 groups, which showed weak induction of pro-inflammatory cytokines and reduced NFκB transcriptional activation. In summary, we describe multiple defects in inflammatory pathways in which LRRK2 appears to be critically involved. Further studies are required to establish the therapeutic implications of inflammatory dysregulation in the pathophysiology of Parkinson's disease.