ABSTRACT: Derivation, characterization, and neuronal differentiation of integration-free induced pluripotent stem cell lines from Parkinsons disease patients carrying SNCA, LRRK2, PARK2, and GBA mutations
Project description:Analysis of whole genome expression in control and patients with Parkinsons disease iPSC lines. The hypothesis tested in present study was that deficient GBA, SNCA, LRRK2 and PARKIN expression can be used for disease modeling by their affects on multiple pathways. These results provide information on relationship between PD genes and mitochondria related gene expression.
Project description:Analysis of whole genome expression in control and patients with Parkinsons disease iPSC lines. The hypothesis tested in present study was that deficient GBA, SNCA, LRRK2 and PARKIN expression can be used for disease modeling by their affects on multiple pathways. These results provide information on relationship between PD genes and mitochondria related gene expression. Total RNA obtained from patient iPSC line compare to control iPSC line
Project description:Mutation of the gene PARK2 is the most common cause of early-onset Parkinson's Disease (PD)1,2. PARK2 encodes a gene product with E3 ubiquitin ligase activity3. In a search for multisite tumor suppressors, we identified PARK2 as a frequently targeted gene on chromosome 6q25.2-q27 in cancer. Here, we describe inactivating somatic mutations and frequent intragenic deletions of PARK2 in human malignancies. The PARK2 mutations in cancer occur in the same domains, and sometimes, at the same residues as the germline mutations causing familial PD. Cancer-specific mutations abrogate the growth suppressive effects of PARK2. PARK2 mutations in cancer decrease the gene product's E3 ligase activity, compromising its ability to ubiquitinate cyclin E and resulting in mitotic instability. These data strongly point to PARK2 as a tumor suppressor on 6q25.2-q27. PARK2, a gene that causes neuronal dysfunction when mutated in the germline, may instead contribute to oncogenesis when altered in non-neuronal somatic cells. Human colorectal samples were profiled on Agilent 244K aCGH arrays per manufacturer's instructions. Pooled reference normal DNA was used as the reference.
Project description:Coding sequences and putative regulatory sequences (retrieved from Ensembl and including up to 2500 bp of promoter sequence upstream the transcription start site) were re-sequenced to a mean depth of 49X on 38 genes associated to Parkinson's disease (RAB25, NUCKS1, RAB7L1, GBA, SYT11, ACMSD, STK39, MCCC1, STBD1, GAK, DGKQ, BST1, SCARB2, HLA-DRB5, GPNMB, FGF20, ITGA8, HIP1R, STX1B, SETD1A, SREBF1, MED13, RAI1, MAPT, RIT2, GIGYF2, HTRA2, EIF4G1, SNCA, LRRK2, VPS35, PINK1, DJ1, ATP13A2, UCHL1, PARK2, FBXO7, and PLA2G6). Sequencing was performed on 249 Parkinson's idiopathic cases and 145 unrelated controls of Spanish origin. All sequencing data was generated with an Illumina Hiseq2000 instrument after enrichment with a custom NimbleGen array. Raw reads were mapped to human reference genome hg19/GRCh37 using BWA aligner.
Project description:We compared transcriptome of monocyte-derived macrophages of 5 patients with GBA-PD (4 L444P/N, 1 N370S/N) and 4 asymptomatic GBA mutation carriers (GBA-carriers) (3 L444P/N, 1 N370S/N) and 4 controls. We also conducted comparative transcriptome analysis for L444P/N only GBA-PD patients and GBA-carriers. Revealed deregulated genes in GBA-PD independently of GBA mutations (L444P or N370S) were involved in immune response, neuronal function. We found upregulated pathway associated with zinc metabolism in L444P/N GBA-PD patients. The potential important role of DUSP1 in the pathogenesis of GBA-PD was suggested.
Project description:The discovery of cell-free micro-RNAs in body fluids has made them a promising biomarker target in the field of neurodegenerative diseases. Although they have been reported to be differentially expressed in biofluids and tissue from sporadic Parkinson’s disease patients, it remains unclear whether similar observations can be made in patients with genetic forms of the disease. Since induced pluripotent stem cell derived neurons represent a widely used research model for both sporadic and familial Parkinson’s disease, we sought to assess the usability of this model for the identification of differentially expressed cell-free micro-RNAs in the context of the Parkinson’s disease related LRRK2 G2019S mutation in a proof-of-concept study. We isolated extracellular vesicles carrying cell-free RNA from patient-derived induced pluripotent stem cell lines carrying the LRRK2 G2019S mutation and their gene corrected isogenic controls. After generation of small-RNA libraries and differential expression analysis, we validated fourteen micro-RNAs in an independent batch of cell-free and cellular RNA via RT-qPCR. Finally, we selected eleven differentially expressed micro-RNAs from our cell culture experiments and quantified their expression levels in cerebrospinal fluid derived from two LRRK2 G2019S patients and two healthy controls.
Project description:Mutation of the gene PARK2 is the most common cause of early-onset Parkinson's Disease (PD)1,2. PARK2 encodes a gene product with E3 ubiquitin ligase activity3. In a search for multisite tumor suppressors, we identified PARK2 as a frequently targeted gene on chromosome 6q25.2-q27 in cancer. Here, we describe inactivating somatic mutations and frequent intragenic deletions of PARK2 in human malignancies. The PARK2 mutations in cancer occur in the same domains, and sometimes, at the same residues as the germline mutations causing familial PD. Cancer-specific mutations abrogate the growth suppressive effects of PARK2. PARK2 mutations in cancer decrease the gene product's E3 ligase activity, compromising its ability to ubiquitinate cyclin E and resulting in mitotic instability. These data strongly point to PARK2 as a tumor suppressor on 6q25.2-q27. PARK2, a gene that causes neuronal dysfunction when mutated in the germline, may instead contribute to oncogenesis when altered in non-neuronal somatic cells.
Project description:We compared transcriptome of monocyte-derived macrophages of 5 patients with GBA-PD (4 L444P/N, 1 N370S/N) and 4 asymptomatic GBA mutation carriers (GBA-carriers) (3 L444P/N, 1 N370S/N) and 4 controls. We also conducted comparative transcriptome analysis for L444P/N only GBA-PD patients and GBA-carriers. Revealed deregulated genes in GBA-PD independently of GBA mutations (L444P or N370S) were involved in immune response, neuronal function. We found upregulated pathway associated with zinc metabolism in L444P/N GBA-PD patients. The potential important role of DUSP1 in the pathogenesis of GBA-PD was suggested.
Project description:We have generated isogenic induced pluripotent stem cell lines by reprogramming human fibroblasts from patients carrying the LRRK2 G2019S mutation with subsequent zinc finger nuclease - mediated targeted correction of the diseased allele. These iPS cell lines were differentiated for 30 days using a direct differentiation protocol towards midbrain dopaminergic neurons (mDANs). Isogenic human iPS cells carrying the LRRK2 WT and G2019S locus were differentiated to dopaminergic neurons to detect gene expression changes associated with mutated LRRK2.
Project description:Wildtype and LRRK2-deficient mouse embryonic stem (ES) cells were used to investigate the effects of LRRK2 on neuronal differentiation.
