scholarly journals Cellular alterations identified in pluripotent stem cell-derived midbrain spheroids generated from a female patient with progressive external ophthalmoplegia and parkinsonism who carries a novel variation (p.Q811R) in the POLG1 gene

2019 ◽  
Vol 7 (1) ◽  
Author(s):  
Margarita Chumarina ◽  
Kaspar Russ ◽  
Carla Azevedo ◽  
Andreas Heuer ◽  
Maria Pihl ◽  
...  
Keyword(s):  
Mitochondrial Dna ◽  
Female Patient ◽  
Dopaminergic Neurons ◽  
Alpha Synuclein ◽  
Genetic Identification ◽  
Progressive External Ophthalmoplegia ◽  
Gene Encoding ◽  
Mitochondrial Dna Copy Number ◽  
And Control ◽  
Cellular Alterations

AbstractVariations in the POLG1 gene encoding the catalytic subunit of the mitochondrial DNA polymerase gamma, have recently been associated with Parkinson’s disease (PD), especially in patients diagnosed with progressive external ophthalmoplegia (PEO). However, the majority of the studies reporting this association mainly focused on the genetic identification of the variation in POLG1 in PD patient primary cells, and determination of mitochondrial DNA copy number, providing little information about the cellular alterations existing in patient brain cells, in particular dopaminergic neurons. Therefore, through the use of induced pluripotent stem cells (iPSCs), we assessed cellular alterations in novel p.Q811R POLG1 (POLG1Q811R) variant midbrain dopaminergic neuron-containing spheroids (MDNS) from a female patient who developed early-onset PD, and compared them to cultures derived from a healthy control of the same gender. Both POLG1 variant and control MDNS contained functional midbrain regionalized TH/FOXA2-positive dopaminergic neurons, capable of releasing dopamine. Western blot analysis identified the presence of high molecular weight oligomeric alpha-synuclein in POLG1Q811R MDNS compared to control cultures. In order to assess POLG1Q811R-related cellular alterations within the MDNS, we applied mass-spectrometry based quantitative proteomic analysis. In total, 6749 proteins were identified, with 61 significantly differentially expressed between POLG1Q811R and control samples. Pro- and anti-inflammatory signaling and pathways involved in energy metabolism were altered. Notably, increased glycolysis in POLG1Q811R MDNS was suggested by the increase in PFKM and LDHA levels and confirmed using functional analysis of glycolytic rate and oxygen consumption levels. Our results validate the use of iPSCs to assess cellular alterations in relation to PD pathogenesis, in a unique PD patient carrying a novel p.Q811R variation in POLG1, and identify several altered pathways that may be relevant to PD pathogenesis.

scholarly journals Activators of Alpha Synuclein Expression Identified by Reporter Cell Line-based High Throughput Drug Screen

2021 ◽  
Author(s):  
Fabian Stahl ◽  
Philip Denner ◽  
Dominik Piston ◽  
Bernd Evert ◽  
Ina Schmitt ◽  
...  
Keyword(s):  
Dopaminergic Neurons ◽  
Control Cell ◽  
Alpha Synuclein ◽  
Drug Screen ◽  
Reporter Cell Line ◽  
Reporter Cell ◽  
Protein Levels ◽  
Approved Drugs ◽  
And Control ◽  
Fda Approved Drugs

Abstract Multiplications, mutations and dysregulation of the alpha synuclein gene (SNCA) are associated with the demise of dopaminergic neurons and are considered to play important roles in the pathogenesis of familial and sporadic forms of Parkinson’s disease. Regulation of SNCA expression might thus be an appropriate target for treatment. We aimed to identify specific modulators of SNCA transcription, generated CRISPR/Cas9 modified SNCA-GFP-luciferase (LUC) genomic fusion- and control cell lines and screened a library of 1649 bioactive compounds, including the FDA approved drugs. We found no inhibitors but three selective activators which increased SNCA mRNA and protein levels.

scholarly journals Alpha-synuclein induces epigenomic dysregulation of glutamate signaling and locomotor pathways

2021 ◽  
Author(s):  
Samantha Lynn Schaffner ◽  
Zinah Wassouf ◽  
Diana Lazaro ◽  
Mary Xylaki ◽  
Nicole Gladish ◽  
...  
Keyword(s):  
Dna Methylation ◽  
Dopaminergic Neurons ◽  
Alpha Synuclein ◽  
Wild Type ◽  
Gene Encoding ◽  
Dna Hydroxymethylation ◽  
Glutamate Signaling ◽  
Body Regions ◽  
Transcriptional Changes ◽  
Or Gene

Background: Mutations and multiplications in the gene encoding for alpha-synuclein are associated with Parkinson's disease (PD). However, not all individuals with alpha-synuclein variants develop PD, suggesting that additional factors are involved. We hypothesized that increased alpha-synuclein might alter epigenetic regulation of PD pathways. Objectives: To identify genome-wide DNA methylation and hydroxymethylation changes induced by overexpression of two alpha-synuclein variants in human dopaminergic neurons, and to relate these to the corresponding transcriptome. Methods: We assessed DNA methylation and hydroxymethylation at >850,000 CpGs using the EPIC BeadChip in LUHMES cells differentiated to dopaminergic neurons. Control LUHMES neurons, LUHMES neurons overexpressing wild type alpha-synuclein, and LUHMES neurons overexpressing A30P alpha-synuclein were compared. We used SMITE network analysis to identify functionally related genes with altered DNA methylation, DNA hydroxymethylation, and/or gene expression, incorporating LUHMES H3K4me1 ChIP-seq to delineate enhancers in addition to the default promoter and gene body regions. Results: Using stringent statistical thresholds, we found that increased expression of wild type or A30P mutant alpha-synuclein induced DNA methylation changes at thousands of CpGs and DNA hydroxymethylation changes at hundreds of CpGs. Differentially methylated sites in both genotypes were enriched for several processes including movement-associated pathways and glutamate signaling. For glutamate and other signaling pathways (i.e. PDGF, insulin), this differential DNA methylation was also associated with transcriptional changes. Conclusions: Our results indicated that alpha-synuclein altered the DNA methylome of dopaminergic neurons, influencing regulation of pathways involved in development, signaling, and metabolism. This supports a role for alpha-synuclein in the epigenetic etiology of PD.

scholarly journals Mitochondrial DNA copy number in cervical exfoliated cells and risk of cervical cancer among HPV-positive women

2019 ◽  
Author(s):  
Wei Sun ◽  
Xueyun Qin ◽  
Jing Zhou ◽  
Mingjing Xu ◽  
Zhangyan Lyu ◽  
...  
Keyword(s):  
Cervical Cancer ◽  
Mitochondrial Dna ◽  
Copy Number ◽  
Hpv Infection ◽  
Dna Copy Number ◽  
Mitochondrial Dna Copy Number ◽  
Exfoliated Cells ◽  
Hpv Types ◽  
Dose Response Effect ◽  
And Control

Abstract Background: Although human papillomavirus (HPV) infection has been recognized as the major cause of cervical cancer, only a minority of HPV-infected women develop this malignancy. An increasing amount of evidence suggests that alterations of mitochondrial DNA copy number (mtCN) may contribute to carcinogenesis. However, the relationship between mtCN and cervical cancer remains unknown. Methods: In this case-control study, we included 591 cervical cancer cases and 373 cancer-free controls, all of whom were infected with high-risk HPV. Relative mtCN in cervical cancer exfoliated cells was measured by qRT-PCR assays, and logistic regression analysis was performed to calculate odds ratios (ORs) and 95% confidence intervals (CIs). Results: HPV16, 18, 52, and 58 were the most common types in both case and control groups. Median mtCN in cases was significantly higher than that in controls ( P = 0.03). After adjustment for age and HPV types, the highest quartile of mtCN was associated with increased odds of having cervical cancer (OR = 1.77, 95% CI = 1.19, 2.62; P < 0.01), as compared to the lowest quartile. A dose-response effect of mtCN on cervical cancer was also observed ( P trend < 0.001). The interaction between mtCN and HPV types was statistically nonsignificant. Conclusions: Increased mtCN in cervical exfoliated cells is associated with cervical cancer after HPV infection. Our study provides a basis for future studies to determine the potential of mtCN as a novel biomarker in cervical cancer screening.

scholarly journals Activators of alpha synuclein expression identified by reporter cell line-based high throughput drug screen

2021 ◽  
Vol 11 (1) ◽  
Author(s):  
Fabian Stahl ◽  
Philip Denner ◽  
Dominik Piston ◽  
Bernd O. Evert ◽  
Laura de Boni ◽  
...  
Keyword(s):  
Dopaminergic Neurons ◽  
Control Cell ◽  
Alpha Synuclein ◽  
Drug Screen ◽  
Reporter Cell Line ◽  
Reporter Cell ◽  
Protein Levels ◽  
Approved Drugs ◽  
And Control ◽  
Fda Approved Drugs

AbstractMultiplications, mutations and dysregulation of the alpha synuclein gene (SNCA) are associated with the demise of dopaminergic neurons and are considered to play important roles in the pathogenesis of familial and sporadic forms of Parkinson’s disease. Regulation of SNCA expression might thus be an appropriate target for treatment. We aimed to identify specific modulators of SNCA transcription, generated CRISPR/Cas9 modified SNCA-GFP-luciferase (LUC) genomic fusion- and control cell lines and screened a library of 1649 bioactive compounds, including the FDA approved drugs. We found no inhibitors but three selective activators which increased SNCA mRNA and protein levels.

scholarly journals Imaging mass cytometry reveals generalised deficiency in OXPHOS complexes in Parkinson’s disease

2021 ◽  
Vol 7 (1) ◽  
Author(s):  
Chun Chen ◽  
David McDonald ◽  
Alasdair Blain ◽  
Ashwin Sachdeva ◽  
Laura Bone ◽  
...  
Keyword(s):  
Parkinson’S Disease ◽  
Parkinson's Disease ◽  
Oxidative Phosphorylation ◽  
Mitochondrial Dysfunction ◽  
Mitochondrial Disease ◽  
Dopaminergic Neurons ◽  
Neuronal Response ◽  
Proteomic Profiling ◽  
Mass Cytometry ◽  
And Control

AbstractHere we report the application of a mass spectrometry-based technology, imaging mass cytometry, to perform in-depth proteomic profiling of mitochondrial complexes in single neurons, using metal-conjugated antibodies to label post-mortem human midbrain sections. Mitochondrial dysfunction, particularly deficiency in complex I has previously been associated with the degeneration of dopaminergic neurons in Parkinson’s disease. To further our understanding of the nature of this dysfunction, and to identify Parkinson’s disease specific changes, we validated a panel of antibodies targeting subunits of all five mitochondrial oxidative phosphorylation complexes in dopaminergic neurons from Parkinson’s disease, mitochondrial disease, and control cases. Detailed analysis of the expression profile of these proteins, highlighted heterogeneity between individuals. There is a widespread decrease in expression of all complexes in Parkinson’s neurons, although more severe in mitochondrial disease neurons, however, the combination of affected complexes varies between the two groups. We also provide evidence of a potential neuronal response to mitochondrial dysfunction through a compensatory increase in mitochondrial mass. This study highlights the use of imaging mass cytometry in the assessment and analysis of expression of oxidative phosphorylation proteins, revealing the complexity of deficiencies of these proteins within individual neurons which may contribute to and drive neurodegeneration in Parkinson’s disease.

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