Genetic Parkinson disease (PD) has been associated with mutations in PINK1, a gene encoding a mitochondrial kinase implicated in the regulation of mitochondrial degradation. While the studies so far ...examined PINK1 function in non-neuronal systems or through PINK1 knockdown approaches, there is an imperative to examine the role of endogenous PINK1 in appropriate human-derived and biologically relevant cell models. Here we report the generation of induced pluripotent stem (iPS) cells from skin fibroblasts taken from three PD patients with nonsense (c.1366C>T; p.Q456X) or missense (c.509T>G; p.V170G) mutations in the PINK1 gene. These cells were differentiated into dopaminergic neurons that upon mitochondrial depolarization showed impaired recruitment of lentivirally expressed Parkin to mitochondria, increased mitochondrial copy number, and upregulation of PGC-1α, an important regulator of mitochondrial biogenesis. Importantly, these alterations were corrected by lentiviral expression of wild-type PINK1 in mutant iPS cell-derived PINK1 neurons. In conclusion, our studies suggest that fibroblasts from genetic PD can be reprogrammed and differentiated into neurons. These neurons exhibit distinct phenotypes that should be amenable to further mechanistic studies in this relevant biological context.
Parkinson’s disease patients report disturbed sleep patterns long before motor dysfunction. Here, in parkin and pink1 models, we identify circadian rhythm and sleep pattern defects and map these to ...specific neuropeptidergic neurons in fly models and in hypothalamic neurons differentiated from patient induced pluripotent stem cells (iPSCs). Parkin and Pink1 control the clearance of mitochondria by protein ubiquitination. Although we do not observe major defects in mitochondria of mutant neuropeptidergic neurons, we do find an excess of endoplasmic reticulum-mitochondrial contacts. These excessive contact sites cause abnormal lipid trafficking that depletes phosphatidylserine from the endoplasmic reticulum (ER) and disrupts the production of neuropeptide-containing vesicles. Feeding mutant animals phosphatidylserine rescues neuropeptidergic vesicle production and acutely restores normal sleep patterns in mutant animals. Hence, sleep patterns and circadian disturbances in Parkinson’s disease models are explained by excessive ER-mitochondrial contacts, and blocking their formation or increasing phosphatidylserine levels rescues the defects in vivo.
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•Parkinson’s disease (PD) models (parkin/pink1 loss) have circadian and sleep defects•Increased ER-mitochondria contacts cause neuropeptide accumulation in ER in PD models•Depletion of phosphatidylserine from ER causes sleep pattern phenotypes in PD models•Phosphatidylserine supplementation rescues circadian and sleep defects of PD models
Valadas et al. show that ER lipid imbalance causes sleep pattern defects in Parkinson’s disease by preventing the formation of secretory vesicles required for the release of the neuropeptides. Restoring the ER lipid balance by supplementation with phosphatidylserine rescues the cellular and behavioral defects.
Under resting conditions, Pink1 knockout cells and cells derived from patients with PINK1 mutations display a loss of mitochondrial complex I reductive activity, causing a decrease in the ...mitochondrial membrane potential. Analyzing the phosphoproteome of complex I in liver and brain from Pink1–/– mice, we found specific loss of phosphorylation of serine-250 in complex I subunit NdufA10. Phosphorylation of serine-250 was needed for ubiquinone reduction by complex I. Phosphomimetic NdufA10 reversed Pink1 deficits in mouse knockout cells and rescued mitochondrial depolarization and synaptic transmission defects in pinkB9-null mutant Drosophila. Complex I deficits and adenosine triphosphate synthesis were also rescued in cells derived from PINK1 patients. Thus, this evolutionary conserved pathway may contribute to the pathogenic cascade that eventually leads to Parkinson's disease in patients with PINK1 mutations.
Mutations in WDR45 cause neurodegeneration with brain iron accumulation. Seibler et al. report that loss of WDR45 function in cells from patients results in increased cellular iron levels and ...oxidative stress, accompanied by mitochondrial and lysosomal dysfunction. Activation of autophagy may serve as a potential therapeutic approach.
Abstract
Beta-propeller protein-associated neurodegeneration is a subtype of monogenic neurodegeneration with brain iron accumulation caused by de novo mutations in WDR45. The WDR45 protein functions as a beta-propeller scaffold and plays a putative role in autophagy through its interaction with phospholipids and autophagy-related proteins. Loss of WDR45 function due to disease-causing mutations has been linked to defects in autophagic flux in patient and animal cells. However, the role of WDR45 in iron homeostasis remains elusive. Here we studied patient-specific WDR45 mutant fibroblasts and induced pluripotent stem cell-derived midbrain neurons. Our data demonstrated that loss of WDR45 increased cellular iron levels and oxidative stress, accompanied by mitochondrial abnormalities, autophagic defects, and diminished lysosomal function. Restoring WDR45 levels partially rescued oxidative stress and the susceptibility to iron treatment, and activation of autophagy reduced the observed iron overload in WDR45 mutant cells. Our data suggest that iron-containing macromolecules and organelles cannot effectively be degraded through the lysosomal pathway due to loss of WDR45 function.
Aside from the degeneration of dopaminergic neurons, inflammation is a key component in the movement disorder Parkinson's disease (PD). Microglia activation as well as elevated cytokine levels were ...observed in the brains of PD patients, but the specific role of microglia in the disease process is unknown. Here, we generate human cellular models by differentiating iPSCs into dopaminergic neurons and microglia. We combine these cells in co-culture to perform cytokine profiling, representing the final functional outcome of various signaling pathways. For this, we used unstimulated conditions and treatment with inflammatory stressors. Importantly, only co-cultures but not the monocultures responded to IL-1β treatment suggesting co-culture-related crosstalk. Moreover, we identified the main types of released cytokines and chemokines in this model system and found a preference for the activation of the chemotaxis pathway in response to all treatments, which informs future studies on the cell-type-specific reaction to inflammatory stimulation. Finally, we detected protein level changes in PD risk factor GPNMB upon stress in microglia, further confirming the link between PD-associated genes and inflammation in human-derived cellular models.
Individualised cellular models of disease are a key tool for precision medicine to recapitulate chronic inflammatory processes. Organoid models can be derived from induced pluripotent stem cells ...(iPSCs) or from primary stem cells
. These models have been emerging over the past decade and have been used to reconstruct the respective organ-specific physiology and pathology, at an unsurpassed depth. In cancer research, patient-derived cancer organoids opened new perspectives in predicting therapy response and provided novel insights into tumour biology. In precision medicine of chronic inflammatory disorders, stem-cell based organoid models are currently being evaluated in pre-clinical pharmacodynamic studies (clinical studies in a dish) and are employed in clinical studies, e.g., by re-transplanting autologous epithelial organoids to re-establish intestinal barrier integrity. A particularly exciting feature of iPSC systems is their ability to provide insights into organ systems and inflammatory disease processes, which cannot be monitored with clinical biopsies, such as immune reactions in neurodegenerative disorders. Refinement of differentiation protocols, and next-generation co-culturing methods, aimed at generating self-organised, complex tissues
, will be the next logical steps. In this mini-review, we critically discuss the current state-of-the-art stem cell and organoid technologies, as well as their future impact, potential and promises in combating immune-mediated chronic diseases.
Myoclonus-dystonia (DYT-SGCE, formerly DYT11) is characterized by alcohol-sensitive, myoclonic-like appearance of fast dystonic movements. It is caused by mutations in the
gene encoding ε-sarcoglycan ...leading to a dysfunction of this transmembrane protein, alterations in the cerebello-thalamic pathway and impaired striatal plasticity. To elucidate underlying pathogenic mechanisms, we investigated induced pluripotent stem cell (iPSC)-derived striatal medium spiny neurons (MSNs) from two myoclonus-dystonia patients carrying a heterozygous mutation in the
gene (c.298T>G and c.304C>T with protein changes W100G and R102X) in comparison to two matched healthy control lines. Calcium imaging showed significantly elevated basal intracellular Ca
content and lower frequency of spontaneous Ca
signals in SGCE MSNs. Blocking of voltage-gated Ca
channels by verapamil was less efficient in suppressing KCl-induced Ca
peaks of SGCE MSNs. Ca
amplitudes upon glycine and acetylcholine applications were increased in SGCE MSNs, but not after GABA or glutamate applications. Expression of voltage-gated Ca
channels and most ionotropic receptor subunits was not altered. SGCE MSNs showed significantly reduced GABAergic synaptic density. Whole-cell patch-clamp recordings displayed elevated amplitudes of miniature postsynaptic currents and action potentials in SGCE MSNs. Our data contribute to a better understanding of the pathophysiology and the development of novel therapeutic strategies for myoclonus-dystonia.
iPS models of Parkin and PINK1 Rakovic, Aleksandar; Seibler, Philip; Klein, Christine
Biochemical Society transactions
43, Številka:
2
Journal Article
Recenzirano
Parkinson disease (PD) is a degenerative disorder of the central nervous system resulting from depletion of dopaminergic neurons and currently remains incurable despite enormous international ...research efforts. The development of induced pluripotent stem cell (iPSC) technology opened up the unique possibility of studying disease mechanisms in human tissue that was otherwise not accessible, such as the brain. Of particular interest are the monogenetic forms of PD as they closely resemble the more common 'idiopathic' PD and, through the mutated protein, provide a clear research target in iPSC-derived neurons. Recessively inherited Parkin and PTEN-induced putative kinase 1 (PINK1) mutations have been investigated in this context and the present review describes the first insights gained from studies in iPSC-derived dopaminergic neurons, which comprise abnormalities in mitochondrial and dopamine homoeostasis, microtubular stability and axonal outgrowth. These new models of PD have a high translational potential that includes the identification of druggable targets, testing of known and novel therapeutic agents in the disease-relevant tissue using well-defined read-outs and potential regenerative approaches.
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).
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