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See detailDiscordant Monozygotic Parkinson Disease Twins: Role of Mitochondrial Integrity
Dulovic-Mahlow, Marija; König, Inke R.; Trinh, Joanne et al

in Annals of Neurology (2020)

Objective Even though genetic predisposition has proven to be an important element in Parkinson's disease (PD) etiology, monozygotic (MZ) twins with PD displayed a concordance rate of only about 20 ... [more ▼]

Objective Even though genetic predisposition has proven to be an important element in Parkinson's disease (PD) etiology, monozygotic (MZ) twins with PD displayed a concordance rate of only about 20% despite their shared identical genetic background. Methods We recruited 5 pairs of MZ twins discordant for idiopathic PD and established skin fibroblast cultures to investigate mitochondrial phenotypes in these cellular models against the background of a presumably identical genome. To test for genetic differences, we performed whole genome sequencing, deep mitochondrial DNA (mtDNA) sequencing, and tested for mitochondrial deletions by multiplex real‐time polymerase chain reaction (PCR) in the fibroblast cultures. Further, the fibroblast cultures were tested for mitochondrial integrity by immunocytochemistry, immunoblotting, flow cytometry, and real‐time PCR to quantify gene expression. Results Genome sequencing did not identify any genetic difference. We found decreased mitochondrial functionality with reduced cellular adenosine triphosphate (ATP) levels, altered mitochondrial morphology, elevated protein levels of superoxide dismutase 2 (SOD2), and increased levels of peroxisome proliferator‐activated receptor‐gamma coactivator‐α (PPARGC1A) messenger RNA (mRNA) in skin fibroblast cultures from the affected compared to the unaffected twins. Further, there was a tendency for a higher number of somatic mtDNA variants among the affected twins. Interpretation We demonstrate disease‐related differences in mitochondrial integrity in the genetically identical twins. Of note, the clinical expression matches functional alterations of the mitochondria [less ▲]

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See detailMitochondrial damage-associated inflammation highlights biomarkers in PRKN/PINK1 parkinsonism
Borsche, Max; Koenig, Inke; Delcambre, Sylvie UL et al

in Brain: a Journal of Neurology (2020)

There is increasing evidence for a role of inflammation in Parkinson’s disease. Recent research in murine models suggests that parkin and PINK1 deficiency leads to impaired mitophagy, which causes the ... [more ▼]

There is increasing evidence for a role of inflammation in Parkinson’s disease. Recent research in murine models suggests that parkin and PINK1 deficiency leads to impaired mitophagy, which causes the release of mitochondrial DNA (mtDNA), thereby triggering inflammation. Specifically, the CGAS (cyclic GMP-AMP synthase)-STING (stimulator of interferon genes) pathway mitigates activation of the innate immune system, quantifiable as increased interleukin-6 (IL6) levels. However, the role of IL6 and circulating cell-free mtDNA in unaffected and affected individuals harbouring mutations in PRKN/PINK1 and idiopathic Parkinson’s disease patients remain elusive. We investigated IL6, C-reactive protein, and circulating cell-free mtDNA in serum of 245 participants in two cohorts from tertiary movement disorder centres. We performed a hypothesis-driven rank-based statistical approach adjusting for multiple testing. We detected (i) elevated IL6 levels in patients with biallelic PRKN/PINK1 mutations compared to healthy control subjects in a German cohort, supporting the concept of a role for inflammation in PRKN/PINK1-linked Parkinson’s disease. In addition, the comparison of patients with biallelic and heterozygous mutations in PRKN/PINK1 suggests a gene dosage effect. The differences in IL6 levels were validated in a second independent Italian cohort; (ii) a correlation between IL6 levels and disease duration in carriers of PRKN/PINK1 mutations, while no such association was observed for idiopathic Parkinson’s disease patients. These results highlight the potential of IL6 as progression marker in Parkinson’s disease due to PRKN/PINK1 mutations; (iii) increased circulating cell-free mtDNA serum levels in both patients with biallelic or with heterozygous PRKN/PINK1 mutations compared to idiopathic Parkinson’s disease, which is in line with previous findings in murine models. By contrast, circulating cell-free mtDNA concentrations in unaffected heterozygous carriers of PRKN/PINK1 mutations were comparable to control levels; and (iv) that circulating cell-free mtDNA levels have good predictive potential to discriminate between idiopathic Parkinson’s disease and Parkinson’s disease linked to heterozygous PRKN/PINK1 mutations, providing functional evidence for a role of heterozygous mutations in PRKN or PINK1 as Parkinson’s disease risk factor. Taken together, our study further implicates inflammation due to impaired mitophagy and subsequent mtDNA release in the pathogenesis of PRKN/PINK1-linked Parkinson’s disease. In individuals carrying mutations in PRKN/PINK1, IL6 and circulating cell-free mtDNA levels may serve as markers of Parkinson’s disease state and progression, respectively. Finally, our study suggests that targeting the immune system with anti-inflammatory medication holds the potential to influence the disease course of Parkinson’s disease, at least in this subset of patients. [less ▲]

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See detailHaploinsufficiency due to a novel ACO2 deletion causes mitochondrial dysfunction in fibroblasts from a patient with dominant optic nerve atrophy
Neumann, Marie Anne-Catherine UL; Grossmann, Dajana UL; Schimpf-Linzenbold, Simone et al

in Scientific Reports (2020)

ACO2 is a mitochondrial protein, which is critically involved in the function of the tricarboxylic acid cycle (TCA), the maintenance of iron homeostasis, oxidative stress defense and the integrity of ... [more ▼]

ACO2 is a mitochondrial protein, which is critically involved in the function of the tricarboxylic acid cycle (TCA), the maintenance of iron homeostasis, oxidative stress defense and the integrity of mitochondrial DNA (mtDNA). Mutations in the ACO2 gene were identified in patients suffering from a broad range of symptoms, including optic nerve atrophy, cortical atrophy, cerebellar atrophy, hypotonia, seizures and intellectual disabilities. In the present study, we identified a heterozygous 51 bp deletion (c.1699_1749del51) in ACO2 in a family with autosomal dominant inherited isolated optic atrophy. A complementation assay using aco1-deficient yeast revealed a growth defect for the mutant ACO2 variant substantiating a pathogenic effect of the deletion. We used patient-derived fibroblasts to characterize cellular phenotypes and found a decrease of ACO2 protein levels, while ACO2 enzyme activity was not affected compared to two age- and gender-matched control lines. Several parameters of mitochondrial function, including mitochondrial morphology, mitochondrial membrane potential or mitochondrial superoxide production, were not changed under baseline conditions. However, basal respiration, maximal respiration, and spare respiratory capacity were reduced in mutant cells. Furthermore, we observed a reduction of mtDNA copy number and reduced mtDNA transcription levels in ACO2-mutant fibroblasts. Inducing oxidative stress led to an increased susceptibility for cell death in ACO2-mutant fibroblasts compared to controls. Our study reveals that a monoallelic mutation in ACO2 is sufficient to promote mitochondrial dysfunction and increased vulnerability to oxidative stress as main drivers of cell death related to optic nerve atrophy. [less ▲]

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See detailMitochondrial Mechanisms of LRRK2 G2019S Penetrance
Delcambre, Sylvie UL; Ghelfi, Jenny UL; Ouzren, Nassima et al

in Frontiers in Neurology (2020)

Several mutations in leucine-rich repeat kinase-2 (LRRK2) have been associated with Parkinson’s disease (PD). The most common substitution, G2019S, interferes with LRRK2 kinase activity, which is ... [more ▼]

Several mutations in leucine-rich repeat kinase-2 (LRRK2) have been associated with Parkinson’s disease (PD). The most common substitution, G2019S, interferes with LRRK2 kinase activity, which is regulated by autophosphorylation. Yet, the penetrance of this gain-of-function mutation is incomplete, and thus far, few factors have been correlated with disease status in carriers. This includes (i) LRRK2 autophosphorylation in urinary exosomes, (ii) serum levels of the antioxidant urate, and (iii) abundance of mitochondrial DNA (mtDNA) transcription-associated 7S DNA. In light of a mechanistic link between LRRK2 kinase activity and mtDNA lesion formation, we previously investigated mtDNA integrity in fibroblasts from manifesting (LRRK2+/PD+) and non-manifesting carriers (LRRK2+/PD−) of the G2019S mutation as well as from aged-matched controls. In our published study, mtDNA major arc deletions correlated with PD status, with manifesting carriers presenting the highest levels. In keeping with these findings, we now further explored mitochondrial features in fibroblasts derived from LRRK2+/PD+ (n = 10), LRRK2+/PD− (n = 21), and control (n = 10) individuals. In agreement with an accumulation of mtDNA major arc deletions, we also detected reduced NADH dehydrogenase activity in the LRRK2+/PD+ group. Moreover, in affected G2019S carriers, we observed elevated mitochondrial mass and mtDNA copy numbers as well as increased expression of the transcription factor nuclear factor erythroid 2-related factor 2 (Nrf2), which regulates antioxidant signaling. Taken together, these results implicate mtDNA dyshomeostasis—possibly as a consequence of impaired mitophagy—in the penetrance of LRRK2-associated PD. Our findings are a step forward in the pursuit of unveiling markers that will allow monitoring of disease progression of LRRK2 mutation carriers [less ▲]

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See detailMtDNA deletions discriminate affected from unaffected LRRK2 mutation carriers
Ouzren, Nassima UL; Delcambre, Sylvie UL; Ghelfi, Jenny UL et al

in Annals of Neurology (2019), 86(2), 324-326

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See detailIntegration of VDR genome wide binding and GWAS genetic variation data reveals co-occurrence of VDR and NF-κB binding that is linked to immune phenotypes
Singh, Prashant K.; van den Berg, Patrick R.; Long, Mark D. et al

in BMC Genomics (2017)

Background The nuclear hormone receptor superfamily acts as a genomic sensor of diverse signals. Their actions are often intertwined with other transcription factors. Nuclear hormone receptors are targets ... [more ▼]

Background The nuclear hormone receptor superfamily acts as a genomic sensor of diverse signals. Their actions are often intertwined with other transcription factors. Nuclear hormone receptors are targets for many therapeutic drugs, and include the vitamin D receptor (VDR). VDR signaling is pleotropic, being implicated in calcaemic function, antibacterial actions, growth control, immunomodulation and anti-cancer actions. Specifically, we hypothesized that the biologically significant relationships between the VDR transcriptome and phenotype-associated biology could be discovered by integrating the known VDR transcription factor binding sites and all published trait- and disease-associated SNPs. By integrating VDR genome-wide binding data (ChIP-seq) with the National Human Genome Research Institute (NHGRI) GWAS catalog of SNPs we would see where and which target gene interactions and pathways are impacted by inherited genetic variation in VDR binding sites, indicating which of VDR’s multiple functions are most biologically significant. Results To examine how genetic variation impacts VDR function we overlapped 23,409 VDR genomic binding peaks from six VDR ChIP-seq datasets with 191,482 SNPs, derived from GWAS-significant SNPs (Lead SNPs) and their correlated variants (r 2 > 0.8) from HapMap3 and the 1000 genomes project. In total, 574 SNPs (71 Lead and 503 SNPs in linkage disequilibrium with Lead SNPs) were present at VDR binding loci and associated with 211 phenotypes. For each phenotype a hypergeometric test was used to determine if SNPs were enriched at VDR binding sites. Bonferroni correction for multiple testing across the 211 phenotypes yielded 42 SNPs that were either disease- or phenotype-associated with seven predominately immune related including self-reported allergy; esophageal cancer was the only cancer phenotype. Motif analyses revealed that only two of these 42 SNPs reside within a canonical VDR binding site (DR3 motif), and that 1/3 of the 42 SNPs significantly impacted binding and gene regulation by other transcription factors, including NF-κB. This suggests a plausible link for the potential cross-talk between VDR and NF-κB. Conclusions These analyses showed that VDR peaks are enriched for SNPs associated with immune phenotypes suggesting that VDR immunomodulatory functions are amongst its most important actions. The enrichment of genetic variation in non-DR3 motifs suggests a significant role for the VDR to bind in multimeric complexes containing other transcription factors that are the primary DNA binding component. Our work provides a framework for the combination of ChIP-seq and GWAS findings to provide insight into the underlying phenotype-associated biology of a given transcription factor. [less ▲]

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See detailIn vitro Metabolic Studies of Dopamine Synthesis and the Toxicity of L-DOPA in Human Cells
Delcambre, Sylvie UL

Doctoral thesis (2016)

This work is divided in two parts. In the first, I investigated the effects of 2,3- dihydroxy-L-phenylalanine (L-DOPA) on the metabolism of human tyrosine hydroxylase (TH)-positive neuronal LUHMES cells ... [more ▼]

This work is divided in two parts. In the first, I investigated the effects of 2,3- dihydroxy-L-phenylalanine (L-DOPA) on the metabolism of human tyrosine hydroxylase (TH)-positive neuronal LUHMES cells. L-DOPA is the gold standard treatment for Parkinson’s disease (PD) and its effects on cellular metabolism are controversial. It induced a re-routing of intracellular carbon supplies. While glutamine contribution to tricarboxylic acid (TCA) cycle intermediates increased, glucose contribution to the same metabolites decreased. Carbon contribution from glucose was decreased in lactate and was compensated by an increased pyruvate contribution. Pyruvate reacted with hydrogen peroxide generated during the auto-oxidation of L-DOPA and lead to an increase of acetate in the medium. In the presence of L-DOPA, this acetate was taken up by the cells. In combination with an increased glutamate secretion, all these results seem to point towards a mitochondrial complex II inhibition. In the second part of this work, I studied and compared dopamine (DA)-producing in vitro systems. First, I compared gene and protein expression of catecholamine (CA)- related genes. Then, I performed molecular engineering to increase TH expression in LUHMES and SH-SY5Y cells. This was sufficient to induce DA production in SH-SY5Y, but not in LUHMES cells, indicating that TH expression is not sufficient to characterize dopaminergic neurons. Therefore I used SH-SY5Y cells overexpressing TH to study substrates for DA production. Upon overexpression of aromatic amino acid decarboxylase (AADC), LUHMES cells produced DA after L-DOPA supplementation. This model was useful to study L-DOPA uptake in LUHMES cells and I showed that L-DOPA is imported via large amino acid transporter (LAT). In conclusion, the expression of TH is not sufficient to obtain a DA-producing cell system and this work opened many and answered some questions about DA metabolism. [less ▲]

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See detailStable Isotope-Assisted Evaluation of Different Extraction Solvents for Untargeted Metabolomics of Plants
Doppler, Maria; Kluger, Bernhard; Bueschl, Christoph et al

in International Journal of Molecular Sciences (2016)

The evaluation of extraction protocols for untargeted metabolomics approaches is still difficult. We have applied a novel stable isotope-assisted workflow for untargeted LC-HRMS-based plant metabolomics ... [more ▼]

The evaluation of extraction protocols for untargeted metabolomics approaches is still difficult. We have applied a novel stable isotope-assisted workflow for untargeted LC-HRMS-based plant metabolomics , which allows for the first time every detected feature to be considered for method evaluation. The efficiency and complementarity of commonly used extraction solvents, namely 1 + 3 (v/v) mixtures of water and selected organic solvents (methanol, acetonitrile or methanol/acetonitrile 1 + 1 (v/v)), with and without the addition of 0.1% (v/v) formic acid were compared. Four different wheat organs were sampled, extracted and analysed by LC-HRMS. Data evaluation was performed with the in-house-developed MetExtract II software and R. With all tested solvents a total of 871 metabolites were extracted in ear, 785 in stem, 733 in leaf and 517 in root samples, respectively. Between 48% (stem) and 57% (ear) of the metabolites detected in a particular organ were found with all extraction mixtures, and 127 of 996 metabolites were consistently shared between all extraction agent/organ combinations. In aqueous methanol, acidification with formic acid led to pronounced pH dependency regarding the precision of metabolite abundance and the number of detectable metabolites, whereas extracts of acetonitrile-containing mixtures were less affected. Moreover, methanol and acetonitrile have been found to be complementary with respect to extraction efficiency. Interestingly, the beneficial properties of both solvents can be combined by the use of a water-methanol-acetonitrile mixture for global metabolite extraction instead of aqueous methanol or aqueous acetonitrile alone. [less ▲]

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See detailLoss of DJ-1 impairs antioxidant response by altered glutamine and serine metabolism
Meiser, Johannes UL; Delcambre, Sylvie UL; Wegner, André UL et al

in Neurobiology of disease (2016), 89

The oncogene DJ-1 has been originally identified as a suppressor of PTEN. Further on, loss-of-function mutations have been described as a causative factor in Parkinson's disease (PD). DJ-1 has an ... [more ▼]

The oncogene DJ-1 has been originally identified as a suppressor of PTEN. Further on, loss-of-function mutations have been described as a causative factor in Parkinson's disease (PD). DJ-1 has an important function in cellular antioxidant responses, but its role in central metabolism of neurons is still elusive. We applied stable isotope assisted metabolic profiling to investigate the effect of a functional loss of DJ-1 and show that DJ-1 deficient neuronal cells exhibit decreased glutamine influx and reduced serine biosynthesis. By providing precursors for GSH synthesis, these two metabolic pathways are important contributors to cellular antioxidant response. Down-regulation of these pathways, as a result of loss of DJ-1 leads to an impaired antioxidant response. Furthermore, DJ-1 deficient mouse microglia showed a weak but constitutive pro-inflammatory activation. The combined effects of altered central metabolism and constitutive activation of glia cells raise the susceptibility of dopaminergic neurons towards degeneration in patients harboring mutated DJ-1. Our work reveals metabolic alterations leading to increased cellular instability and identifies potential new intervention points that can further be studied in the light of novel translational medicine approaches. [less ▲]

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See detailDopamine Metabolism and Reactive Oxygen Species Production
Delcambre, Sylvie UL

in Buhlman, Lori (Ed.) Mitochondrial Mechanisms of Degeneration and Repair in Parkinson’s Disease (2016)

Due to their chemical nature, dopamine and its metabolites are easily oxidized—a process often accompanied by the production of reactive oxygen species. While the MAO-mediated degradation of dopamine ... [more ▼]

Due to their chemical nature, dopamine and its metabolites are easily oxidized—a process often accompanied by the production of reactive oxygen species. While the MAO-mediated degradation of dopamine leads to the formation of hydrogen peroxide, the autoxidation of many intermediates of dopamine metabolism produces highly reactive quinones. These quinones can bind to cysteinyl residues of reduced GSH or proteins, leading to their inactivation if this residue is located in the active site of a protein. One way to overcome this problem is to increase production or uptake of antioxidants such as GSH or ascorbate. Interaction with astrocytes is an important fact for dopaminergic neuron survival: astrocytes provide the neurons with the GSH-precursor glutamine and are able to degrade excessive dopamine released by neurons. In dopaminergic neurons, excessive amounts of catecholamines can also be inactivated by their polymerization to neuromelanin. This polymer pigment itself can, however, have both a protective or deleterious effect, depending on the cellular context. It should be noted that although l-DOPA still represents the state-of-the-art treatment for Parkinson’s disease, it also represents a catecholamine that can contribute to oxidative stress in already damaged dopaminergic neurons. Oxidative damage outside the brain induced by longtime l-DOPA treatment (e.g., in melanocytes) has often been discussed; however, there is little clinical evidence for that theory. Finally, dopamine metabolism can be altered by genetic factors such as tyrosine hydroxylase deficiency or genetic variants of the dopamine transporter, as well as environmental factors such as pesticides or drugs. [less ▲]

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