Polygenic Risk Scores Validated in Patient-Derived Cells Stratify for Mitochondrial Subtypes of Parkinson's Disease.

OBJECTIVE: The aim of our study is to better understand the genetic architecture and pathological mechanisms underlying neurodegeneration in idiopathic Parkinson's disease (iPD). We hypothesized that a fraction of iPD patients may harbor a combination of common variants in nuclear-encoded mitochondrial genes ultimately resulting in neurodegeneration. METHODS: We used mitochondria-specific polygenic risk scores (mitoPRSs) and created pathway-specific mitoPRSs using genotype data from different iPD case-control datasets worldwide, including the Luxembourg Parkinson's Study (412 iPD patients and 576 healthy controls) and COURAGE-PD cohorts (7,270 iPD cases and 6,819 healthy controls). Cellular models from individuals stratified according to the most significant mitoPRS were subsequently used to characterize different aspects of mitochondrial function. RESULTS: Common variants in genes regulating Oxidative Phosphorylation (OXPHOS-PRS) were significantly associated with a higher PD risk in independent cohorts (Luxembourg Parkinson's Study odds ratio, OR = 1.31[1.14-1.50], p-value = 5.4e-04; COURAGE-PD OR = 1.23[1.18-1.27], p-value = 1.5e-29). Functional analyses in fibroblasts and induced pluripotent stem cells-derived neuronal progenitors revealed significant differences in mitochondrial respiration between iPD patients with high or low OXPHOS-PRS (p-values < 0.05). Clinically, iPD patients with high OXPHOS-PRS have a significantly earlier age at disease onset compared to low-risk patients (false discovery rate [FDR]-adj p-value = 0.015), similar to prototypic monogenic forms of PD. Finally, iPD patients with high OXPHOS-PRS responded more effectively to treatment with mitochondrially active ursodeoxycholic acid. INTERPRETATION: OXPHOS-PRS may provide a precision medicine tool to stratify iPD patients into a pathogenic subgroup genetically defined by specific mitochondrial impairment, making these individuals eligible for future intelligent clinical trial designs. ANN NEUROL 2024;96:133-149.

Parkin Deficiency Impairs Mitochondrial DNA Dynamics and Propagates Inflammation.

BACKGROUND: Mutations in the E3 ubiquitin ligase parkin cause autosomal recessive Parkinson's disease (PD). Together with PTEN-induced kinase 1 (PINK1), parkin regulates the clearance of dysfunctional mitochondria. New mitochondria are generated through an interplay of nuclear- and mitochondrial-encoded proteins, and recent studies suggest that parkin influences this process at both levels. In addition, parkin was shown to prevent mitochondrial membrane permeability, impeding mitochondrial DNA (mtDNA) escape and subsequent neuroinflammation. However, parkin's regulatory roles independent of mitophagy are not well described in patient-derived neurons. OBJECTIVES: We sought to investigate parkin's role in preventing neuronal mtDNA dyshomeostasis, release, and glial activation at the endogenous level. METHODS: We generated induced pluripotent stem cell (iPSC)-derived midbrain neurons from PD patients with parkin (PRKN) mutations and healthy controls. Live-cell imaging, proteomic, mtDNA integrity, and gene expression analyses were employed to investigate mitochondrial biogenesis and genome maintenance. To assess neuroinflammation, we performed single-nuclei RNA sequencing in postmortem tissue and quantified interleukin expression in mtDNA/lipopolysaccharides (LPS)-treated iPSC-derived neuron-microglia co-cultures. RESULTS: Neurons from patients with PRKN mutations revealed deficits in the mitochondrial biogenesis pathway, resulting in mtDNA dyshomeostasis. Moreover, the energy sensor sirtuin 1, which controls mitochondrial biogenesis and clearance, was downregulated in parkin-deficient cells. Linking mtDNA disintegration to neuroinflammation, in postmortem midbrain with PRKN mutations, we confirmed mtDNA dyshomeostasis and detected an upregulation of microglia overexpressing proinflammatory cytokines. Finally, parkin-deficient neuron-microglia co-cultures elicited an enhanced immune response when exposed to mtDNA/LPS. CONCLUSIONS: Our findings suggest that parkin coregulates mitophagy, mitochondrial biogenesis, and mtDNA maintenance pathways, thereby protecting midbrain neurons from neuroinflammation and degeneration. © 2022 The Authors. Movement Disorders published by Wiley Periodicals LLC on behalf of International Parkinson and Movement Disorder Society.

Parkinson's Disease Phenotypes in Patient Neuronal Cultures and Brain Organoids Improved by 2-Hydroxypropyl-ß-Cyclodextrin Treatment.

BACKGROUND: The etiology of Parkinson's disease (PD) is only partially understood despite the fact that environmental causes, risk factors, and specific gene mutations are contributors to the disease. Biallelic mutations in the phosphatase and tensin homolog (PTEN)-induced putative kinase 1 (PINK1) gene involved in mitochondrial homeostasis, vesicle trafficking, and autophagy are sufficient to cause PD. OBJECTIVES: We sought to evaluate the difference between controls' and PINK1 patients' derived neurons in their transition from neuroepithelial stem cells to neurons, allowing us to identify potential pathways to target with repurposed compounds. METHODS: Using two-dimensional and three-dimensional models of patients' derived neurons we recapitulated PD-related phenotypes. We introduced the usage of midbrain organoids for testing compounds. Using Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR)/CRISPR-associated protein 9 (Cas9), we corrected the point mutations of three patients' derived cells. We evaluated the effect of the selected compound in a mouse model. RESULTS: PD patient-derived cells presented differences in their energetic profile, imbalanced proliferation, apoptosis, mitophagy, and a reduced differentiation efficiency to tyrosine hydroxylase positive (TH+) neurons compared to controls' cells. Correction of a patient's point mutation ameliorated the metabolic properties and neuronal firing rates as well as reversing the differentiation phenotype, and reducing the increased astrocytic levels. Treatment with 2-hydroxypropyl-ß-cyclodextrin increased the autophagy and mitophagy capacity of neurons concomitant with an improved dopaminergic differentiation of patient-specific neurons in midbrain organoids and ameliorated neurotoxicity in a mouse model. CONCLUSION: We show that treatment with a repurposed compound is sufficient for restoring the impaired dopaminergic differentiation of PD patient-derived cells. © 2021 The Authors. Movement Disorders published by Wiley Periodicals LLC on behalf of International Parkinson and Movement Disorder Society.

Generation and characterization of induced pluripotent stem cells from a Parkinson's disease patient carrying the digenic LRRK2 p.G2019S and GBA1 p.N409S mutations.

We describe an induced pluripotent stem cell (iPSC) line that was derived from fibroblasts obtained from a Parkinson's disease (PD) patient carrying the p.G2019S mutation in the LRRK2 gene and the p.N409S mutation in the GBA1 gene. iPSCs were generated via Sendai virus transduction of Yamanaka factors. The presence of GBA1 p.N409S and LRRK2 p.G2019S was confirmed by Sanger sequencing. The iPSCs express pluripotency markers, are capable of in vitro differentiation into the three germ layers and have a normal karyotype. The newly generated line will be used for in vitro PD modeling by investigating the role of each mutation in iPSC-derived dopaminergic neurons.

Wild-type measles virus interferes with short-term engraftment of human CD34+ hematopoietic progenitor cells.

Transient lymphopenia is a hallmark of measles virus (MV)-induced immunosuppression. To address to what extent replenishment of the peripheral lymphocyte compartment from bone marrow (BM) progenitor/stem cells might be affected, we analyzed the interaction of wild-type MV with hematopoietic stem and progenitor cells (HS/PCs) and stroma cells in vitro. Infection of human CD34(+) HS/PCs or stroma cells with wild-type MV is highly inefficient yet noncytolytic. It occurs independently of CD150 in stroma cells but also in HS/PCs, where infection is established in CD34(+) CD150(-) and CD34(+) CD150(+) (in humans representing HS/PC oligopotent precursors) subsets. Stroma cells and HS/PCs can mutually transmit MV and may thereby create a possible niche for continuous viral exchange in the BM. Infected lymphocytes homing to this compartment may serve as sources for HS/PC or stroma cell infection, as reflected by highly efficient transmission of MV from both populations in cocultures with MV-infected B or T cells. Though MV exposure does not detectably affect the viability, expansion, and colony-forming activity of either CD150(+) or CD150(-) HS/PCs in vitro, it efficiently interferes with short- but not long-term hematopoietic reconstitution in NOD/SCID mice. Altogether, these findings support the hypothesis that MV accession of the BM compartment by infected lymphocytes may contribute to peripheral blood mononuclear cell lymphopenia at the level of BM suppression.

Generation and characterization of a genetic Parkinson's disease-patient derived iPSC line DJ-1-delP (LCSBi008-A).

Here, we describe an induced pluripotent stem cell (iPSC) line that was derived from fibroblasts obtained from a monogenic Parkinson's disease (PD) patient. The disease was caused by a c.634-636delGCC mutation in the PARK7 gene leading to p.158P deletion in the protein DJ-1. iPSCs were generated via electroporation using three episomal plasmids encoding human Oct3/4, Sox2, Klf4, Lin28, L-Myc combined with a short hairpin RNA for p53. The presence of the c.471_473delGCC mutation in exon 7 of PARK7 was confirmed by Sanger sequencing. The iPSCs express pluripotency markers, are capable of in vitro differentiation into the three germ layers and obtain karyotypic integrity.

Generation of isogenic control DJ-1-delP GC13 for the genetic Parkinson's disease-patient derived iPSC line DJ-1-delP (LCSBi008-A-1).

We describe the generation of an isogenic control cell line DJ-1-delP GC13 from an induced pluripotent stem cell (iPSC) line DJ-1-delP LCSBi008-A that was derived from fibroblasts obtained from a Parkinson's disease (PD) patient. Using CRISPR/Cas9 technology, we corrected the disease causing c.471_473delGCC homozygous mutation in the PARK7 gene leading to p.158P deletion in the encoded protein DJ-1. The generated isogenic pair will be used for phenotypic analysis of PD-patient derived neurons and astrocytes.

Alpha synuclein determines ferroptosis sensitivity in dopaminergic neurons via modulation of ether-phospholipid membrane composition.

There is a continued unmet need for treatments that can slow Parkinson's disease progression due to the lack of understanding behind the molecular mechanisms underlying neurodegeneration. Since its discovery, ferroptosis has been implicated in several diseases and represents a therapeutic target in Parkinson's disease. Here, we use two highly relevant human dopaminergic neuronal models to show that endogenous levels of α-synuclein can determine the sensitivity of dopaminergic neurons to ferroptosis. We show that reducing α-synuclein expression in dopaminergic neurons leads to ferroptosis evasion, while elevated α-synuclein expression in patients' small-molecule-derived neuronal precursor cells with SNCA triplication causes an increased vulnerability to lipid peroxidation and ferroptosis. Lipid profiling reveals that ferroptosis resistance is due to a reduction in ether-linked phospholipids, required for ferroptosis, in neurons depleted of α-synuclein (α-syn). These results provide a molecular mechanism linking α-syn levels to the sensitivity of dopaminergic neurons to ferroptosis, suggesting potential therapeutic relevance.

GDAP1 loss of function inhibits the mitochondrial pyruvate dehydrogenase complex by altering the actin cytoskeleton.

Charcot-Marie-Tooth (CMT) disease 4A is an autosomal-recessive polyneuropathy caused by mutations of ganglioside-induced differentiation-associated protein 1 (GDAP1), a putative glutathione transferase, which affects mitochondrial shape and alters cellular Ca2+ homeostasis. Here, we identify the underlying mechanism. We found that patient-derived motoneurons and GDAP1 knockdown SH-SY5Y cells display two phenotypes: more tubular mitochondria and a metabolism characterized by glutamine dependence and fewer cytosolic lipid droplets. GDAP1 interacts with the actin-depolymerizing protein Cofilin-1 and beta-tubulin in a redox-dependent manner, suggesting a role for actin signaling. Consistently, GDAP1 loss causes less F-actin close to mitochondria, which restricts mitochondrial localization of the fission factor dynamin-related protein 1, instigating tubularity. GDAP1 silencing also disrupts mitochondria-ER contact sites. These changes result in lower mitochondrial Ca2+ levels and inhibition of the pyruvate dehydrogenase complex, explaining the metabolic changes upon GDAP1 loss of function. Together, our findings reconcile GDAP1-associated phenotypes and implicate disrupted actin signaling in CMT4A pathophysiology.

The Role of DJ-1 in Cellular Metabolism and Pathophysiological Implications for Parkinson's Disease.

DJ-1 is a multifunctional protein associated with pathomechanisms implicated in different chronic diseases including neurodegeneration, cancer and diabetes. Several of the physiological functions of DJ-1 are not yet fully understood; however, in the last years, there has been increasing evidence for a potential role of DJ-1 in the regulation of cellular metabolism. Here, we summarize the current knowledge on specific functions of DJ-1 relevant to cellular metabolism and their role in modulating metabolic pathways. Further, we illustrate pathophysiological implications of the metabolic effects of DJ-1 in the context of neurodegeneration in Parkinson´s disease.

Integrated, automated maintenance, expansion and differentiation of 2D and 3D patient-derived cellular models for high throughput drug screening.

Patient-derived cellular models become an increasingly powerful tool to model human diseases for precision medicine approaches. The identification of robust cellular disease phenotypes in these models paved the way towards high throughput screenings (HTS) including the implementation of laboratory advanced automation. However, maintenance and expansion of cells for HTS remains largely manual work. Here, we describe an integrated, complex automated platform for HTS in a translational research setting also designed for maintenance and expansion of different cell types. The comprehensive design allows automation of all cultivation steps and is flexible for development of methods for variable cell types. We demonstrate protocols for controlled cell seeding, splitting and expansion of human fibroblasts, induced pluripotent stem cells (iPSC), and neural progenitor cells (NPC) that allow for subsequent differentiation into different cell types and image-based multiparametric screening. Furthermore, we provide automated protocols for neuronal differentiation of NPC in 2D culture and 3D midbrain organoids for HTS. The flexibility of this multitask platform makes it an ideal solution for translational research settings involving experiments on different patient-derived cellular models for precision medicine.

PINK1 deficiency impairs adult neurogenesis of dopaminergic neurons.

Recent evidence suggests neurogenesis is on-going throughout life but the relevance of these findings for neurodegenerative disorders such as Parkinson's disease (PD) is poorly understood. Biallelic PINK1 mutations cause early onset, Mendelian inherited PD. We studied the effect of PINK1 deficiency on adult neurogenesis of dopaminergic (DA) neurons in two complementary model systems. Zebrafish are a widely-used model to study neurogenesis in development and through adulthood. Using EdU analyses and lineage-tracing studies, we first demonstrate that a subset of ascending DA neurons and adjacent local-projecting DA neurons are each generated into adulthood in wild type zebrafish at a rate that decreases with age. Pink1-deficiency impedes DA neurogenesis in these populations, most significantly in early adult life. Pink1 already exerts an early effect on Th1+ progenitor cells rather than on differentiated DA neurons only. In addition, we investigate the effect of PINK1 deficiency in a human isogenic organoid model. Global neuronal differentiation in PINK1-deficient organoids and isogenic controls is similar, but PINK1-deficient organoids display impeded DA neurogenesis. The observation of impaired adult dopaminergic neurogenesis in Pink1 deficiency in two complementing model systems may have significant consequences for future therapeutic approaches in human PD patients with biallelic PINK1 mutations.

Bidirectional Relation Between Parkinson's Disease and Glioblastoma Multiforme.

Cancer and Parkinson's disease (PD) define two disease entities that include opposite concepts. Indeed, the involved mechanisms are at different ends of a spectrum related to cell survival - one due to enhanced cellular proliferation and the other due to premature cell death. There is increasing evidence indicating that patients with neurodegenerative diseases like PD have a reduced incidence for most cancers. In support, epidemiological studies demonstrate an inverse association between PD and cancer. Both conditions apparently can involve the same set of genes, however, in affected tissues the expression was inversely regulated: genes that are down-regulated in PD were found to be up-regulated in cancer and vice versa, for example p53 or PARK7. When comparing glioblastoma multiforme (GBM), a malignant brain tumor with poor overall survival, with PD, astrocytes are dysregulated in both diseases in opposite ways. In addition, common genes, that are involved in both diseases and share common key pathways of cell proliferation and metabolism, were shown to be oppositely deregulated in PD and GBM. Here, we provide an overview of the involvement of PD- and GBM-associated genes in common pathways that are dysregulated in both conditions. Moreover, we illustrate why the simultaneous study of PD and GBM regarding the role of common pathways may lead to a deeper understanding of these still incurable conditions. Eventually, considering the inverse regulation of certain genes in PD and GBM will help to understand their mechanistic basis, and thus to define novel target-based strategies for causative treatments.

A patient-based model of RNA mis-splicing uncovers treatment targets in Parkinson's disease.

Parkinson's disease (PD) is a heterogeneous neurodegenerative disorder with monogenic forms representing prototypes of the underlying molecular pathology and reproducing to variable degrees the sporadic forms of the disease. Using a patient-based in vitro model of PARK7-linked PD, we identified a U1-dependent splicing defect causing a drastic reduction in DJ-1 protein and, consequently, mitochondrial dysfunction. Targeting defective exon skipping with genetically engineered U1-snRNA recovered DJ-1 protein expression in neuronal precursor cells and differentiated neurons. After prioritization of candidate drugs, we identified and validated a combinatorial treatment with the small-molecule compounds rectifier of aberrant splicing (RECTAS) and phenylbutyric acid, which restored DJ-1 protein and mitochondrial dysfunction in patient-derived fibroblasts as well as dopaminergic neuronal cell loss in mutant midbrain organoids. Our analysis of a large number of exomes revealed that U1 splice-site mutations were enriched in sporadic PD patients. Therefore, our study suggests an alternative strategy to restore cellular abnormalities in in vitro models of PD and provides a proof of concept for neuroprotection based on precision medicine strategies in PD.

Detection of adenoviruses and rotaviruses in drinking water sources used in rural areas of Benin, West Africa.

Diseases associated with viruses also found in environmental samples cause major health problems in developing countries. Little is known about the frequency and pattern of viral contamination of drinking water sources in these resource-poor settings. We established a method to analyze 10 liters of water from drinking water sources in a rural area of Benin for the presence of adenoviruses and rotaviruses. Overall, 541 samples from 287 drinking water sources were tested. A total of 12.9% of the sources were positive for adenoviruses and 2.1% of the sources were positive for rotaviruses at least once. Due to the temporary nature of viral contamination in drinking water sources, the probability of virus detection increased with the number of samples taken at one test site over time. No seasonal pattern for viral contaminations was found after samples obtained during the dry and wet seasons were compared. Overall, 3 of 15 surface water samples (20%) and 35 of 247 wells (14.2%) but also 2 of 25 pumps (8%) tested positive for adenoviruses or rotaviruses. The presence of latrines within a radius of 50 m in the vicinity of pumps or wells was identified as being a risk factor for virus detection. In summary, viral contamination was correlated with the presence of latrines in the vicinity of drinking water sources, indicating the importance of appropriate decision support systems in these socioeconomic prospering regions.

Quality Control Strategy for CRISPR-Cas9-Based Gene Editing Complicated by a Pseudogene.

CRISPR-Cas9 mediated gene editing in induced pluripotent stem cells became an efficient tool to investigate biological mechanisms underlying genetic-driven diseases while accounting for the respective genetic background. This technique relies on the targeting of a specific nucleotide sequence present in the gene of interest. Therefore, the gene editing of some genes can be complicated by non-coding pseudogenes presenting a high homology of sequence with their respective genes. Among them, GBA is raising special interest because of its implication as the most common genetic risk factor for Parkinson's disease. In this study, we present an easy-to-use CRISPR-Cas9 gene editing strategy allowing for specific editing of point mutations in a gene without genetic alteration of its pseudogene exemplified by the correction or insertion of the common N370S mutation in GBA. A quality control strategy by combined fluorescence and PCR-based screening allows the early identification of correctly edited clones with unambiguous identification of the status of its pseudogene, GBAP1. Successful gene editing was confirmed by functional validation. Our work presents the first CRISPR-Cas9 based editing of a point mutation in GBA and paves the way for technically demanding gene engineering due to the presence of pseudogenes.

Derivation of Human Midbrain-Specific Organoids from Neuroepithelial Stem Cells.

Research on human brain development and neurological diseases is limited by the lack of advanced experimental in vitro models that truly recapitulate the complexity of the human brain. Here, we describe a robust human brain organoid system that is highly specific to the midbrain derived from regionally patterned neuroepithelial stem cells. These human midbrain organoids contain spatially organized groups of dopaminergic neurons, which make them an attractive model for the study of Parkinson's disease. Midbrain organoids are characterized in detail for neuronal, astroglial, and oligodendrocyte differentiation. Furthermore, we show the presence of synaptic connections and electrophysiological activity. The complexity of this model is further highlighted by the myelination of neurites. The present midbrain organoid system has the potential to be used for advanced in vitro disease modeling and therapy development.

Stable coreceptor usage of HIV in patients with ongoing treatment failure on HAART.

BACKGROUND: Disease progression in HIV infection has been associated with switch of viral coreceptor usage from CCR5 to CXCR4. OBJECTIVES: To investigate the relationship between HIV-coreceptor tropism and clinical and virological outcome in 40 heavily pretreated patients over time. METHODS: Coreceptor phenotype was predicted after sequencing the V3 loop of the HIV glycoprotein 120. RESULTS: Coreceptor use was stable during observation time in 87% of patients, and CCR5 tropism was predominant. Viral mutations in the pol gene and clinical parameters were not predictive for coreceptor switching. CONCLUSIONS: Even in patients with repeated HAART failure, CCR5 antagonists might be a valuable treatment option.

Functional analysis of a chromosomal deletion associated with myelodysplastic syndromes using isogenic human induced pluripotent stem cells.

Chromosomal deletions associated with human diseases, such as cancer, are common, but synteny issues complicate modeling of these deletions in mice. We use cellular reprogramming and genome engineering to functionally dissect the loss of chromosome 7q (del(7q)), a somatic cytogenetic abnormality present in myelodysplastic syndromes (MDS). We derive del(7q)- and isogenic karyotypically normal induced pluripotent stem cells (iPSCs) from hematopoietic cells of MDS patients and show that the del(7q) iPSCs recapitulate disease-associated phenotypes, including impaired hematopoietic differentiation. These disease phenotypes are rescued by spontaneous dosage correction and can be reproduced in karyotypically normal cells by engineering hemizygosity of defined chr7q segments in a 20-Mb region. We use a phenotype-rescue screen to identify candidate haploinsufficient genes that might mediate the del(7q)- hematopoietic defect. Our approach highlights the utility of human iPSCs both for functional mapping of disease-associated large-scale chromosomal deletions and for discovery of haploinsufficient genes.