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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.

Friedreich ataxia is an autosomal recessive neurodegenerative disease associated with a high diabetes prevalence. No treatment is available to prevent or delay disease progression. Friedreich ataxia is caused by intronic GAA trinucleotide repeat expansions in the frataxin-encoding FXN gene that reduce frataxin expression, impair iron-sulfur cluster biogenesis, cause oxidative stress, and result in mitochondrial dysfunction and apoptosis. Here we examined the metabolic, neuroprotective and frataxin-inducing effects of glucagon-like-peptide 1 (GLP-1) analogs in in vivo and in vitro models and in Friedreich ataxia patients. The GLP-1 analog exenatide improved glucose homeostasis of frataxin-deficient mice through enhanced insulin content and secretion in pancreatic β-cells. Exenatide induced frataxin and iron-sulfur cluster-containing proteins in β-cells and brain, and was protective to sensory neurons in dorsal root ganglia. GLP-1 analogs also induced frataxin expression, reduced oxidative stress and improved mitochondrial function in Friedreich ataxia patients' induced pluripotent stem cell-derived β-cells and sensory neurons. The frataxin-inducing effect of exenatide was confirmed in a pilot trial in Friedreich ataxia patients, showing modest frataxin induction in platelets over a 5-week treatment course. Taken together, GLP-1 analogs improve mitochondrial function in frataxin-deficient cells and induce frataxin expression. Our findings identify incretin receptors as a therapeutic target in Friedreich ataxia., info:eu-repo/semantics/published

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.

The majority of epilepsies are focal in origin, with seizures emanating from one brain region. Although focal epilepsies often arise from structural brain lesions, many affected individuals have normal brain imaging. The etiology is unknown in the majority of individuals, although genetic factors are increasingly recognized. Autosomal dominant familial focal epilepsy with variable foci (FFEVF) is notable because family members have seizures originating from different cortical regions. Using exome sequencing, we detected DEPDC5 mutations in two affected families. We subsequently identified mutations in five of six additional published large families with FFEVF. Study of families with focal epilepsy that were too small for conventional clinical diagnosis with FFEVF identified DEPDC5 mutations in approximately 12% of families (10/82). This high frequency establishes DEPDC5 mutations as a common cause of familial focal epilepsies. Shared homology with G protein signaling molecules and localization in human neurons suggest a role of DEPDC5 in neuronal signal transduction. Refereed/Peer-reviewed

Estimation of cerebellar Purkinje cell number is an important evaluation parameter for the study of the development of the cerebellum as well as for investigating the changes occurring in various experimental and physiological conditions. The introduction of stereological techniques in biological and biomedical research has made quantification of Purkinje cells not only more reliable, because independent from morphology-related sources of bias, but also easier and less time consuming. In this paper, we will briefly overview the stereological techniques that have been used so far for estimation of Purkinje cell number in different animal species and we will describe a simple method (the confocal physical disector) for Purkinje cell quantification on confocal microscope optical slices, based on a variation of the physical disector technique that can be adopted without the need of any dedicated stereological workstation. NeuroQuantology | March 2012 | Volume 10 | Issue 1 | Page 125-131