Project description:We have generated human induced Pluripotent Stem cells (hiPSc) from Parkinson's Disease patients, using retrovirus-mediated delivery of reprogramming factors. hiPSc lines have been screened using SNP array to assess chromosomal stability (alongside the fibroblast lines from which they derived), and validation of the pluripotency of the hiPSc lines is provided by Pluritest assessment of transcriptome datasets, prior to differentiation to dopaminergic neuronal clutures and downstream functional assays. Fernandes H.J.R., Hartfield E.M., Badger J., Christian H. C., Emmanoulidou E., Vowles J., Evetts S., Vekrellis K., Talbot K., Hu M.T., James W., Cowley S.A., and Wade-Martins, R. Heterozygous glucocerebrosidase mutations in Parkinson's increase autophagic demand, but decrease capacity, in induced pluripotent stem cell-derived dopaminergic neuronal cultures. submitted for publication human iPSc lines were derived from human dermal fibroblasts from 2 Parkinson's Disease patients with heterozygous glucocerebrosidase mutations (GBA N370S) mutations, and 2 idiopathic Parkinson's Disease patients. SNP datasets from the 2 control individuals used in this study have been published previously [PMID 23951090; A mature physiological cellular model of human dopaminergic neurons Hartfield E.M., Yamasaki-Mann M., Fernandes H.J., Vowles., James W.S., Cowley S.A, and Wade-Martins R. In revision]
Project description:We have generated human induced Pluripotent Stem cells (hiPSc) from Parkinson's Disease patients, using retrovirus-mediated delivery of reprogramming factors. hiPSc lines have been screened using SNP array to assess chromosomal stability (alongside the fibroblast lines from which they derived), and validation of the pluripotency of the hiPSc lines is provided by Pluritest assessment of transcriptome datasets, prior to differentiation to dopaminergic neuronal clutures and downstream functional assays. human iPSc lines were derived from human dermal fibroblasts from 2 Parkinson's Disease patients with heterozygous glucocerebrosidase mutations (GBA N370S) mutations, and 2 idiopathic Parkinson's Disease patients. SNP datasets from the 2 control individuals used in this study have been published previously [PMID 23951090; A mature physiological cellular model of human dopaminergic neurons Hartfield E.M., Yamasaki-Mann M., Fernandes H.J., Vowles., James W.S., Cowley S.A, and Wade-Martins R. In revision]
Project description:Induced pluripotent stem cells (iPSCs) derived from somatic cells of patients represent a powerful tool for biomedical research and may provide a source for replacement therapies. However, the use of viruses encoding the reprogramming factors represents a major limitation of the current technology since even low vector expression may alter the differentiation potential of the iPSCs or induce malignant transformation. Here, we show that fibroblasts from five patients with idiopathic Parkinson's disease can be efficiently reprogrammed and subsequently differentiated into dopaminergic neurons. Moreover, we derived hiPSCs free of reprogramming factors using Cre-recombinase excisable viruses. Factor-free hiPSCs maintain a pluripotent state and show a global gene expression profile, more closely related to hESCs than to hiPSCs carrying the transgenes. Our results indicate that residual transgene expression in virus-carrying hiPSCs can affect their molecular characteristics and that factor-free hiPSCs therefore represent a more suitable source of cells for modeling of human disease.
Project description:Parkinson's disease (PD) is a multifactorial neurodegenerative disorder. The pathogenesis of this disease is associated with gene and environmental factors. Mutations in leucine-rich repeat kinase 2 (LRRK2) are the most frequent genetic cause of familial and sporadic PD. Moreover, posttranslational modifications, including protein acetylation, are involved in the molecular mechanism of PD. Acetylation of lysine proteins is a dynamic process that is modulated in PD. In this descriptive study, we characterized the acetylated proteins and peptides in primary fibroblasts from idiopathic PD (IPD) and genetic PD harboring G2019S or R1441G LRRK2 mutations. Identified acetylated peptides are modulated between individuals' groups. Although acetylated nuclear proteins are the most represented in cells, they are hypoacetylated in IPD. Results display that the level of hyperacetylated and hypoacetylated peptides are, respectively, enhanced in genetic PD and in IPD cells.
Project description:In this study, we evaluate the hypothesis that transcriptome pattern of a cell model of Parkinson’s disease (PD) could be altered compared to healthy individuals. We cultured fibroblasts from Parkin-associated PD (PRKN-PD; n=4) and healthy controls (HC; n=4) and analysed the transcriptome profile by using whole RNA sequencing. We found 343 differentially expressed transcripts (DET) between patients and controls being 206 up-regulated and 137 down-regulated. These genes are related with the gene ontology terms cell adhesion, cell growth, aminoacid biosynthesis and folate metabolism amongst others. Our findings indicate that PRKN mutations are associated with global gene expression changes as observed in fibroblasts, and support an emerging view of PD as a systemic disease affecting also peripheral non-neural tissues such as the skin. Overall design: Evaluation of transcript expression by RNA sequencing of fibroblasts from Parkin-associated Parkinson's Disease patients versus fibroblasts from healthy controls
Project description:A major barrier to research on Parkinson's disease is inaccessibility of diseased tissue for study. One solution is to derive induced pluripotent stem cells from patients and differentiate them into neurons affected by disease. Triplication of SNCA, encoding ?-synuclein, causes a fully penetrant, aggressive form of Parkinson's disease with dementia. ?-Synuclein dysfunction is the critical pathogenic event in Parkinson's disease, multiple system atrophy and dementia with Lewy bodies. Here we produce multiple induced pluripotent stem cell lines from an SNCA triplication patient and an unaffected first-degree relative. When these cells are differentiated into midbrain dopaminergic neurons, those from the patient produce double the amount of ?-synuclein protein as neurons from the unaffected relative, precisely recapitulating the cause of Parkinson's disease in these individuals. This model represents a new experimental system to identify compounds that reduce levels of ?-synuclein, and to investigate the mechanistic basis of neurodegeneration caused by ?-synuclein dysfunction.
Project description:The long-term goal of nuclear transfer or alternative reprogramming approaches is to create patient-specific donor cells for transplantation therapy, avoiding immunorejection, a major complication in current transplantation medicine. It was recently shown that the four transcription factors Oct4, Sox2, Klf4, and c-Myc induce pluripotency in mouse fibroblasts. However, the therapeutic potential of induced pluripotent stem (iPS) cells for neural cell replacement strategies remained unexplored. Here, we show that iPS cells can be efficiently differentiated into neural precursor cells, giving rise to neuronal and glial cell types in culture. Upon transplantation into the fetal mouse brain, the cells migrate into various brain regions and differentiate into glia and neurons, including glutamatergic, GABAergic, and catecholaminergic subtypes. Electrophysiological recordings and morphological analysis demonstrated that the grafted neurons had mature neuronal activity and were functionally integrated in the host brain. Furthermore, iPS cells were induced to differentiate into dopamine neurons of midbrain character and were able to improve behavior in a rat model of Parkinson's disease upon transplantation into the adult brain. We minimized the risk of tumor formation from the grafted cells by separating contaminating pluripotent cells and committed neural cells using fluorescence-activated cell sorting. Our results demonstrate the therapeutic potential of directly reprogrammed fibroblasts for neuronal cell replacement in the animal model.
Project description: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.
Project description:Data from both toxin-based and gene-based models suggest that dysfunction of the microtubule system contributes to the pathogenesis of Parkinson's disease, even if, at present, no evidence of alterations of microtubules in vivo or in patients is available. Here we analyze cytoskeleton organization in primary fibroblasts deriving from patients with idiopathic or genetic Parkinson's disease, focusing on mutations in parkin and leucine-rich repeat kinase 2. Our analyses reveal that genetic and likely idiopathic pathology affects cytoskeletal organization and stability, without any activation of autophagy or apoptosis. All parkinsonian fibroblasts have a reduced microtubule mass, represented by a higher fraction of unpolymerized tubulin in respect to control cells, and display significant changes in microtubule stability-related signaling pathways. Furthermore, we show that the reduction of microtubule mass is so closely related to the alteration of cell morphology and behavior that both pharmacological treatment with microtubule-targeted drugs, and genetic approaches, by transfecting the wild type parkin or leucine-rich repeat kinase 2, restore the proper microtubule stability and are able to rescue cell architecture. Taken together, our results suggest that microtubule destabilization is a point of convergence of genetic and idiopathic forms of parkinsonism and highlight, for the first time, that microtubule dysfunction occurs in patients and not only in experimental models of Parkinson's disease. Therefore, these data contribute to the knowledge on molecular and cellular events underlying Parkinson's disease and, revealing that correction of microtubule defects restores control phenotype, may offer a new therapeutic target for the management of the disease.
Project description:Identification of early Parkinson's disease events by developing methodology that utilizes recent innovations in human pluripotent stem cells and chemical sensors of HSP90-incorporating chaperome networks.