Polygenic risk scores validated in patient-derived cells stratify for mitochondrial subtypes of Parkinson\textquoterights disease 2023.05.12.23289877

E-print/Working paper (2023)

Background Parkinson's disease (PD) is the fastest growing neurodegenerative disorder, with affected individuals expected to double during the next 20 years. This raises the urgent need to better ... [more ▼]

Background Parkinson's disease (PD) is the fastest growing neurodegenerative disorder, with affected individuals expected to double during the next 20 years. This raises the urgent need to better understand the genetic architecture and downstream cellular alterations underlying PD pathogenesis, in order to identify more focused therapeutic targets. While only ~10\% of PD cases can be clearly attributed to monogenic causes, there is mounting evidence that additional genetic factors could play a role in idiopathic PD (iPD). In particular, common variants with low to moderate effect size in multiple genes regulating key neuroprotective activities may act as risk factors for PD. In light of the well-established involvement of mitochondrial dysfunction in PD, we hypothesized that a fraction of iPD cases may harbour a pathogenic combination of common variants in nuclear-encoded mitochondrial genes, ultimately resulting in neurodegeneration.Methods: To capture this mitochondria-related 'missing heritability', we leveraged on existing data from previous genome-wide association studies (GWAS) i.e., the large PD GWAS from Nalls and colleagues. We then used computational approaches based on mitochondria-specific polygenic risk scores (mitoPRSs) for imputing the genotype data obtained from different iPD case-control datasets worldwide, including the Luxembourg Parkinson\textquoterights Study (412 iPD patients and 576 healthy controls) and the COURAGE-PD cohorts (7270 iPD cases and 6819 healthy controls).Results: Applying this approach to gene sets controlling mitochondrial pathways potentially relevant for neurodegeneration in PD, we demonstrated that common variants in genes regulating Oxidative Phosphorylation (OXPHOS-PRS) were significantly associated with a higher PD risk both in the Luxembourg Parkinson\textquoterights Study (odds ratio, OR=1.31[1.14-1.50], p=5.4e-04) and in COURAGE-PD (OR=1.23[1.18-1.27], p=1.5e-29). Functional analyses in primary skin fibroblasts and in the corresponding induced pluripotent stem cells-derived neuronal progenitor cells from Luxembourg Parkinson's Study iPD patients stratified according to the OXPHOS-PRS, revealed significant differences in mitochondrial respiration between high and low risk groups (p < 0.05). Finally, we also demonstrated that iPD patients with high OXPHOS-PRS have a significantly earlier age at disease onset compared to low-risk patients.Conclusions: Our findings suggest that OXPHOS-PRS may represent a promising strategy to stratify iPD patients into pathogenic subgroups in which the underlying neurodegeneration is due to a genetically defined mitochondrial burden potentially eligible for future, more tailored mitochondrially targeted treatments. [less ▲]

From methylation to myelination: epigenomic and transcriptomic profiling of chronic inactive demyelinated multiple sclerosis lesions.

in Acta neuropathologica (2023)

In the progressive phase of multiple sclerosis (MS), the hampered differentiation capacity of oligodendrocyte precursor cells (OPCs) eventually results in remyelination failure. We have previously shown ... [more ▼]

In the progressive phase of multiple sclerosis (MS), the hampered differentiation capacity of oligodendrocyte precursor cells (OPCs) eventually results in remyelination failure. We have previously shown that DNA methylation of Id2/Id4 is highly involved in OPC differentiation and remyelination. In this study, we took an unbiased approach by determining genome-wide DNA methylation patterns within chronically demyelinated MS lesions and investigated how certain epigenetic signatures relate to OPC differentiation capacity. We compared genome-wide DNA methylation and transcriptional profiles between chronically demyelinated MS lesions and matched normal-appearing white matter (NAWM), making use of post-mortem brain tissue (n = 9/group). DNA methylation differences that inversely correlated with mRNA expression of their corresponding genes were validated for their cell-type specificity in laser-captured OPCs using pyrosequencing. The CRISPR-dCas9-DNMT3a/TET1 system was used to epigenetically edit human-iPSC-derived oligodendrocytes to assess the effect on cellular differentiation. Our data show hypermethylation of CpGs within genes that cluster in gene ontologies related to myelination and axon ensheathment. Cell type-specific validation indicates a region-dependent hypermethylation of MBP, encoding for myelin basic protein, in OPCs obtained from white matter lesions compared to NAWM-derived OPCs. By altering the DNA methylation state of specific CpGs within the promotor region of MBP, using epigenetic editing, we show that cellular differentiation and myelination can be bidirectionally manipulated using the CRISPR-dCas9-DNMT3a/TET1 system in vitro. Our data indicate that OPCs within chronically demyelinated MS lesions acquire an inhibitory phenotype, which translates into hypermethylation of crucial myelination-related genes. Altering the epigenetic status of MBP can restore the differentiation capacity of OPCs and possibly boost (re)myelination. [less ▲]

Molecular phenotypes of mitochondrial dysfunction in clinically non-manifesting heterozygous PRKN variant carriers

in NPJ Parkinson's Disease (2023)

Homozygous or compound heterozygous (biallelic) variants in PRKN are causal for PD with highly penetrant symptom expression, while the much more common heterozygous variants may predispose to PD with ... [more ▼]

Homozygous or compound heterozygous (biallelic) variants in PRKN are causal for PD with highly penetrant symptom expression, while the much more common heterozygous variants may predispose to PD with highly reduced penetrance, through altered mitochondrial function. In the presence of pathogenic heterozygous variants, it is therefore important to test for mitochondrial alteration in cells derived from variant carriers to establish potential presymptomatic molecular markers. We generated lymphoblasts (LCLs) and human induced pluripotent stem cell (hiPSC)-derived neurons from non-manifesting heterozygous PRKN variant carriers and tested them for mitochondrial functionality. In LCLs, we detected hyperactive mitochondrial respiration, and, although milder compared to a biallelic PRKN-PD patient, hiPSC-derived neurons of non-manifesting heterozygous variant carriers also displayed several phenotypes of altered mitochondrial function. Overall, we identified molecular phenotypes that might be used to monitor heterozygous PRKN variant carriers during the prodromal phase. Such markers might also be useful to identify individuals at greater risk of eventual disease development and for testing potential mitochondrial function-based neuroprotective therapies before neurodegeneration advances [less ▲]

Generation of two induced pluripotent stem cell lines and the corresponding isogenic controls from Parkinson’s disease patients carrying the heterozygous mutations c.815G>A (p.R272Q) or c.1348C>T (p.R450C) in the RHOT1 gene encoding Miro1

in Stem Cell Research (2023)

Fibroblasts from two Parkinson’s disease (PD) patients carrying either the heterozygous mutation c.815G>A (Miro1 p.R272Q) or c.1348C>T (Miro1 p.R450C) in the RHOT1 gene, were converted into induced ... [more ▼]

Fibroblasts from two Parkinson’s disease (PD) patients carrying either the heterozygous mutation c.815G>A (Miro1 p.R272Q) or c.1348C>T (Miro1 p.R450C) in the RHOT1 gene, were converted into induced pluripotent stem cells (iPSCs) using RNA-based and episomal reprogramming, respectively. The corresponding isogenic gene-corrected lines have been generated using CRISPR/Cas9 technology. These two isogenic pairs will be used to study Miro1-related molecular mechanisms underlying neurodegeneration in relevant iPSC-derived neuronal models (e.g., midbrain dopaminergic neurons and astrocytes). [less ▲]

Generation of two induced pluripotent stem cell lines and the corresponding isogenic controls from Parkinson's disease patients carrying the heterozygous mutations c.1290A > G (p.T351A) or c.2067A > G (p.T610A) in the RHOT1 gene encoding Miro1.

in Stem cell research (2023), 69

Primary skin fibroblasts from two Parkinson's disease (PD) patients carrying distinct heterozygous mutations in the RHOT1 gene encoding Miro1, namely c.1290A > G (Miro1 p.T351A) and c.2067A > G (Miro1 p ... [more ▼]

Primary skin fibroblasts from two Parkinson's disease (PD) patients carrying distinct heterozygous mutations in the RHOT1 gene encoding Miro1, namely c.1290A > G (Miro1 p.T351A) and c.2067A > G (Miro1 p.T610A), were converted into induced pluripotent stem cells (iPSCs) by episomal reprogramming. The corresponding isogenic gene-corrected lines have been generated using CRISPR/Cas9 technology. Here, we provide a comprehensive characterization and quality assurance of both isogenic pairs, which will be used to study Miro1-related molecular mechanisms underlying neurodegeneration in iPSC-derived neuronal models (e.g., midbrain dopaminergic neurons and astrocytes). [less ▲]

Mitochondrial DNA heteroplasmy distinguishes disease manifestation in PINK1- and PRKN-linked Parkinson's disease 2022.05.17.22275087

E-print/Working paper (2022)

Biallelic mutations in PINK1 and PRKN cause recessively inherited Parkinson's disease (PD). Though some studies suggest that PINK1/PRKN monoallelic mutations may not contribute to risk, deep phenotyping ... [more ▼]

Biallelic mutations in PINK1 and PRKN cause recessively inherited Parkinson's disease (PD). Though some studies suggest that PINK1/PRKN monoallelic mutations may not contribute to risk, deep phenotyping assessment showed that PINK1 or PRKN monoallelic pathogenic variants were at a significantly higher rate in PD compared to controls. Given the established role of PINK1 and Parkin in regulating mitochondrial dynamics, we explored mitochondrial DNA (mtDNA) integrity and inflammation as potential disease modifiers in carriers of mutations in these genes. MtDNA integrity, global gene expression and serum cytokine levels were investigated in a large collection of biallelic (n=84) and monoallelic (n=170) carriers of PINK1/PRKN mutations, iPD patients (n=67) and controls (n=90). Affected and unaffected PINK1/PRKN monoallelic mutation carriers can be distinguished by heteroplasmic mtDNA variant load (AUC=0.83, CI:0.74-0.93). Biallelic PINK1/PRKN mutation carriers harbor more heteroplasmic mtDNA variants in blood (p=0.0006, Z=3.63) compared to monoallelic mutation carriers. This enrichment was confirmed in iPSC-derived and postmortem midbrain neurons from biallelic PRKN-PD patients. Lastly, the heteroplasmic mtDNA variant load was found to correlate with IL6 levels in PINK1/PRKN mutation carriers (r=0.57, p=0.0074). PINK1/PRKN mutations predispose individuals to mtDNA variant accumulation in a dose- and disease-dependent manner. MtDNA variant load over time is a potential marker of disease manifestation in PINK1/PRKN mutation carriers.Competing Interest StatementThe authors have declared no competing interest.Funding StatementThe authors wish to thank the many patients and their families who volunteered, and the efforts of the many clinical teams involved. Funding has been obtained from the German Research Foundation (ProtectMove; FOR 2488, GR 3731/5-1; SE 2608/2-1; KO 2250/7-1), the Luxembourg National Research Fund in the ATTRACT (Model-IPD, FNR9631103), NCER-PD (FNR11264123) and INTER programmes (ProtectMove, FNR11250962; MiRisk-PD, C17/BM/11676395, NB 4328/2-1), the BMBF (MitoPD), the Hermann and Lilly Schilling Foundation, the European Community (SysMedPD), the Canadian Institutes of Health Research (CIHR), Peter and Traudl Engelhorn Foundation. Initial studies in Tunisia on familial parkinsonism were in collaboration with Lefkos Middleton, Rachel Gibson, and the GlaxoSmithKline PD Programme Team (2002-2005). We would like to thank Dr Helen Tuppen from the Welcome Trust Centre for Mitochondrial Research, Newcastle University, UK for providing us with the plasmid p7D1. Moreover, this project was supported by the high throughput/high content screening platform and HPC facility at the Luxembourg Centre for Systems Biomedicine, and the University of Luxembourg.Author DeclarationsI confirm all relevant ethical guidelines have been followed, and any necessary IRB and/or ethics committee approvals have been obtained.YesThe details of the IRB/oversight body that provided approval or exemption for the research described are given below:University of Lubeck Ethics CommitteeI confirm that all necessary patient/participant consent has been obtained and the appropriate institutional forms have been archived, and that any patient/participant/sample identifiers included were not known to anyone (e.g., hospital staff, patients or participants themselves) outside the research group so cannot be used to identify individuals.YesI understand that all clinical trials and any other prospective interventional studies must be registered with an ICMJE-approved registry, such as ClinicalTrials.gov. I confirm that any such study reported in the manuscript has been registered and the trial registration ID is provided (note: if posting a prospective study registered retrospectively, please provide a statement in the trial ID field explaining why the study was not registered in advance).Yes I have followed all appropriate research reporting guidelines and uploaded the relevant EQUATOR Network research reporting checklist(s) and other pertinent material as supplementary files, if applicable.YesAll data produced in the present study are available upon reasonable request to the authors [less ▲]

Single-cell sequencing of human midbrain reveals glial activation and a Parkinson-specific neuronal state.

in Brain : a journal of neurology (2022), 145(3), 964-978

Idiopathic Parkinson's disease is characterized by a progressive loss of dopaminergic neurons, but the exact disease etiology remains largely unknown. To date, Parkinson's disease research has mainly ... [more ▼]

Idiopathic Parkinson's disease is characterized by a progressive loss of dopaminergic neurons, but the exact disease etiology remains largely unknown. To date, Parkinson's disease research has mainly focused on nigral dopaminergic neurons, although recent studies suggest disease-related changes also in non-neuronal cells and in midbrain regions beyond the substantia nigra. While there is some evidence for glial involvement in Parkinson's disease, the molecular mechanisms remain poorly understood. The aim of this study was to characterize the contribution of all cell types of the midbrain to Parkinson's disease pathology by single-nuclei RNA sequencing and to assess the cell type-specific risk for Parkinson's disease employing the latest genome-wide association study. We profiled >41 000 single-nuclei transcriptomes of postmortem midbrain from six idiopathic Parkinson's disease patients and five age-/sex-matched controls. To validate our findings in a spatial context, we utilized immunolabeling of the same tissues. Moreover, we analyzed Parkinson's disease-associated risk enrichment in genes with cell type-specific expression patterns. We discovered a neuronal cell cluster characterized by CADPS2 overexpression and low TH levels, which was exclusively present in IPD midbrains. Validation analyses in laser-microdissected neurons suggest that this cluster represents dysfunctional dopaminergic neurons. With regard to glial cells, we observed an increase in nigral microglia in Parkinson's disease patients. Moreover, nigral idiopathic Parkinson's disease microglia were more amoeboid, indicating an activated state. We also discovered a reduction in idiopathic Parkinson's disease oligodendrocyte numbers with the remaining cells being characterized by a stress-induced upregulation of S100B. Parkinson's disease risk variants were associated with glia- and neuron-specific gene expression patterns in idiopathic Parkinson's disease cases. Furthermore, astrocytes and microglia presented idiopathic Parkinson's disease-specific cell proliferation and dysregulation of genes related to unfolded protein response and cytokine signaling. While reactive patient astrocytes showed CD44 overexpression, idiopathic Parkinson's disease-microglia revealed a pro-inflammatory trajectory characterized by elevated levels of IL1B, GPNMB, and HSP90AA1. Taken together, we generated the first single-nuclei RNA sequencing dataset from the idiopathic Parkinson's disease midbrain, which highlights a disease-specific neuronal cell cluster as well as 'pan-glial' activation as a central mechanism in the pathology of the movement disorder. This finding warrants further research into inflammatory signaling and immunomodulatory treatments in Parkinson's disease. [less ▲]

Neurodegeneration and Neuroinflammation in Parkinson’s Disease: a Self-Sustained Loop

in Current Neurology and Neuroscience Reports (2022), 22(8), 427440

Purpose of Review: Neuroinflammation plays a significant role in Parkinson’s disease (PD) etiology along with mitochondrial dysfunction and impaired proteostasis. In this context, mechanisms related to ... [more ▼]

Purpose of Review: Neuroinflammation plays a significant role in Parkinson’s disease (PD) etiology along with mitochondrial dysfunction and impaired proteostasis. In this context, mechanisms related to immune response can act as modifiers at different steps of the neurodegenerative process and justify the growing interest in anti-inflammatory agents as potential disease-modifying treatments in PD. The discovery of inherited gene mutations in PD has allowed researchers to develop cellular and animal models to study the mechanisms of the underlying biology, but the original cause of neuroinflammation in PD is still debated to date. Recent Findings: Cell autonomous alterations in neuronal cells, including mitochondrial damage and protein aggregation, could play a role, but recent findings also highlighted the importance of intercellular communication at both local and systemic level. This has given rise to debate about the role of non-neuronal cells in PD and reignited intense research into the gut-brain axis and other non-neuronal interactions in the development of the disease. Whatever the original trigger of neuroinflammation in PD, what appears quite clear is that the aberrant activation of glial cells and other components of the immune system creates a vicious circle in which neurodegeneration and neuroinflammation nourish each other. Summary: In this review, we will provide an up-to-date summary of the main cellular alterations underlying neuroinflammation in PD, including those induced by environmental factors (e.g. the gut microbiome) and those related to the genetic background of affected patients. Starting from the lesson provided by familial forms of PD, we will discuss pathophysiological mechanisms linked to inflammation that could also play a role in idiopathic forms. Finally, we will comment on the potential clinical translatability of immunobiomarkers identified in PD patient cohorts and provide an update on current therapeutic strategies aimed at overcoming or preventing inflammation in PD. © 2022, The Author(s). [less ▲]

A genome on shaky ground: exploring the impact of mitochondrial DNA integrity on Parkinson's disease by highlighting the use of cybrid models.

in Cellular and molecular life sciences : CMLS (2022), 79(5), 283

Mitochondria play important roles in the regulation of key cellular processes, including energy metabolism, oxidative stress response, and signaling towards cell death or survival, and are distinguished ... [more ▼]

Mitochondria play important roles in the regulation of key cellular processes, including energy metabolism, oxidative stress response, and signaling towards cell death or survival, and are distinguished by carrying their own genome (mtDNA). Mitochondrial dysfunction has emerged as a prominent cellular mechanism involved in neurodegeneration, including Parkinson's disease (PD), a neurodegenerative movement disorder, characterized by progressive loss of dopaminergic neurons and the occurrence of proteinaceous Lewy body inclusions. The contribution of mtDNA variants to PD pathogenesis has long been debated and is still not clearly answered. Cytoplasmic hybrid (cybrid) cell models provided evidence for a contribution of mtDNA variants to the PD phenotype. However, conclusive evidence of mtDNA mutations as genetic cause of PD is still lacking. Several models have shown a role of somatic, rather than inherited mtDNA variants in the impairment of mitochondrial function and neurodegeneration. Accordingly, several nuclear genes driving inherited forms of PD are linked to mtDNA quality control mechanisms, and idiopathic as well as familial PD tissues present increased mtDNA damage. In this review, we highlight the use of cybrids in this PD research field and summarize various aspects of how and to what extent mtDNA variants may contribute to the etiology of PD. [less ▲]

Coffee, smoking and aspirin are associated with age at onset in idiopathic Parkinson's disease.

in Journal of neurology (2022)

Parkinson's disease (PD) is a progressive neurodegenerative disorder. Genetic modifiers, environmental factors and gene-environment interactions have been found to modify PD risk and disease progression ... [more ▼]

Parkinson's disease (PD) is a progressive neurodegenerative disorder. Genetic modifiers, environmental factors and gene-environment interactions have been found to modify PD risk and disease progression. The objective of this study was to evaluate the association of smoking, caffeine and anti-inflammatory drugs with age at onset (AAO) in a large PD cohort. A total of 35,963 American patients with idiopathic PD (iPD) from the Fox Insight Study responded to health and lifestyle questionnaires. We compared the median AAO between different groups using the non-parametric Mann-Whitney U test. Non-parametric Spearman's correlation was used for correlation assessments and regression analysis was used to assess interaction between variables. We found that smoking (p < 0.0001), coffee drinking (p < 0.0001) and aspirin intake (p < 0.0001) show an exploratory association with AAO in PD, that was further supported by multivariate regression models. The association of aspirin with PD AAO was replicated in another cohort (EPIPARK) (n = 237 patients with PD). [less ▲]