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The two-pore potassium channel TRESK is a potential drug target in pain and migraine. Pettingill et al. show that the F139WfsX2 mutation causes TRESK loss of function and hyperexcitability in nociceptors derived from iPSCs of patients with migraine. Cloxyquin, a TRESK activator, reverses migraine-relevant phenotypes in vitro and in vivo., The two-pore potassium channel, TRESK has been implicated in nociception and pain disorders. We have for the first time investigated TRESK function in human nociceptive neurons using induced pluripotent stem cell-based models. Nociceptors from migraine patients with the F139WfsX2 mutation show loss of functional TRESK at the membrane, with a corresponding significant increase in neuronal excitability. Furthermore, using CRISPR-Cas9 engineering to correct the F139WfsX2 mutation, we show a reversal of the heightened neuronal excitability, linking the phenotype to the mutation. In contrast we find no change in excitability in induced pluripotent stem cell derived nociceptors with the C110R mutation and preserved TRESK current; thereby confirming that only the frameshift mutation is associated with loss of function and a migraine relevant cellular phenotype. We then demonstrate the importance of TRESK to pain states by showing that the TRESK activator, cloxyquin, can reduce the spontaneous firing of nociceptors in an in vitro human pain model. Using the chronic nitroglycerine rodent migraine model, we demonstrate that mice lacking TRESK develop exaggerated nitroglycerine-induced mechanical and thermal hyperalgesia, and furthermore, show that cloxyquin conversely is able to prevent sensitization. Collectively, our findings provide evidence for a role of TRESK in migraine pathogenesis and its suitability as a therapeutic target.

Summary Reproducibility in molecular and cellular studies is fundamental to scientific discovery. To establish the reproducibility of a well-defined long-term neuronal differentiation protocol, we repeated the cellular and molecular comparison of the same two iPSC lines across five distinct laboratories. Despite uncovering acceptable variability within individual laboratories, we detect poor cross-site reproducibility of the differential gene expression signature between these two lines. Factor analysis identifies the laboratory as the largest source of variation along with several variation-inflating confounders such as passaging effects and progenitor storage. Single-cell transcriptomics shows substantial cellular heterogeneity underlying inter-laboratory variability and being responsible for biases in differential gene expression inference. Factor analysis-based normalization of the combined dataset can remove the nuisance technical effects, enabling the execution of robust hypothesis-generating studies. Our study shows that multi-center collaborations can expose systematic biases and identify critical factors to be standardized when publishing novel protocols, contributing to increased cross-site reproducibility., Highlights • Cross-site reproducibility in iPSC-based molecular experiments is poor • Factor analysis-based normalization can be used to analyze nuisance variation • External validation of iPSC experimental molecular data is critical for reproducibility • Collaborative studies are needed to reveal systematic biases to improve reproducibility, In this article, Lakics and colleagues show that, while individual laboratories are able to identify consistent molecular and seemingly statistically robust differences between iPSC neuronal models, cross-site reproducibility is poor. Their findings support multi-center collaborations to expose systematic biases and identify critical factors to be standardized to improve reproducibility in iPSC-based molecular experiments.

Friedreich's ataxia (FRDA) is a neurodegenerative disorder associated with cardiomyopathy and diabetes. Effective therapies for FRDA are an urgent unmet need; there are currently no options to prevent or treat this orphan disease. FRDA is caused by reduced expression of the mitochondrial protein frataxin. We have previously demonstrated that pancreatic β-cell dysfunction and death cause diabetes in FRDA. This is secondary to mitochondrial dysfunction and apoptosis but the underlying molecular mechanisms are not known. Here we show that β-cell demise in frataxin deficiency is the consequence of oxidative stress-mediated activation of the intrinsic pathway of apoptosis. The pro-apoptotic Bcl-2 family members Bad, DP5 and Bim are the key mediators of frataxin deficiency-induced β-cell death. Importantly, the intrinsic pathway of apoptosis is also activated in FRDA patients' induced pluripotent stem cell-derived neurons. Interestingly, cAMP induction normalizes mitochondrial oxidative status and fully prevents activation of the intrinsic pathway of apoptosis in frataxin-deficient β-cells and neurons. This preclinical study suggests that incretin analogs hold potential to prevent/delay both diabetes and neurodegeneration in FRDA., info:eu-repo/semantics/published

Friedreich's ataxia (FRDA) is a recessive neurodegenerative disorder commonly associated with hypertrophic cardiomyopathy. FRDA is due to expanded GAA repeats within the first intron of the gene encoding frataxin, a conserved mitochondrial protein involved in iron-sulphur cluster biosynthesis. This mutation leads to partial gene silencing and substantial reduction of the frataxin level. To overcome limitations of current cellular models of FRDA, we derived induced pluripotent stem cells (iPSCs) from two FRDA patients and successfully differentiated them into neurons and cardiomyocytes, two affected cell types in FRDA. All FRDA iPSC lines displayed expanded GAA alleles prone to high instability and decreased levels of frataxin, but no biochemical phenotype was observed. Interestingly, both FRDA iPSC-derived neurons and cardiomyocytes exhibited signs of impaired mitochondrial function, with decreased mitochondrial membrane potential and progressive mitochondrial degeneration, respectively. Our data show for the first time that FRDA iPSCs and their neuronal and cardiac derivatives represent promising models for the study of mitochondrial damage and GAA expansion instability in FRDA., Journal Article, SCOPUS: ar.j, info:eu-repo/semantics/published

Objective: The development of cellular models of neurological diseases to study pathogenesis and test potential therapeutics has been hindered by the inaccessibility of relevant cell types. Induced pluripotent stem (iPS) cells are generated from easily accessible cells and provide a long term source of self-renewing embryonic stem (ES)-like cells that can be differentiated into the cell types of interest. Background Friedreich9s ataxia (FRDA) is an autosomal recessive disease caused by relative deficiency of the mitochondrial protein frataxin. Most patients are homozygous for the hyperexpansion of a GAA repeat in the first intron of the frataxin gene, which causes its downregulation by epigenetic mechanisms. Frataxin deficiency impairs iron-sulfur cluster biogenesis in mitochondria, resulting in multiple enzyme deficiencies, energy deficit, oxidative damage and altered iron metabolism. However, though the defect is in every cell, only specific neuronal populations, cardiomyocytes an beta cells degenerate. Design/Methods: We generated iPS cells from FRDA patients and controls by lentiviral transduction of a specific set of transcription factors (Oct4, Sox2, Lin28 and Nanog) and validated them for pluripotency and genetic integrity. We obtained neurons and cardiomyocytes from these iPS cells and morphologically and functionally characterized them. Results: FRDA-derived iPS cells are frataxin deficient and show GAA repeat instability. They can generate neural precursors (neurospheres), neurons and cardiomyocytes. GAA repeats stabilize during neurogenesis, but remain unstable during cardiomyocyte differentiation. FRDA iPS-derived neurons have the same cell body size as controls, but extend less processes and tend to mature more slowly. After 5-6 weeks of differentiation, significantly more control than FRDA neurons express sodium currents and generate action potentials. FRDA cardiomyocytes progressively develop mitochondrial abnormalities. Studies on calcium signaling, iron metabolism, mitochondrial function and biogenesis are ongoing. Conclusions: iPS cells derived from FRDA patients recapitulate genetic, epigenetic and pathogenic mechanisms occurring in the disease. Supported by: European Community 7th Framework Programme (EFACTS collaborative project). Disclosure: Dr. Pandolfo has received personal compensation for activities with Santhera.Dr. Pandolfo has received (royalty or license fee or contractual rights)payments from Athena Diagnostic. Dr. Pandolfo has received research support from Repligen. Dr. Chintawar has nothing to disclose. Dr. Wattenhofer-Donze has nothing to disclose. Dr. Puccio has nothing to disclose.