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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 cellular prion protein (PrPC) is a key neuronal receptor for β-amyloid oligomers (AβO), mediating their neurotoxicity, which contributes to the neurodegeneration in Alzheimer's disease (AD). Similarly to the amyloid precursor protein (APP), PrPC is proteolytically cleaved from the cell surface by a disintegrin and metalloprotease, ADAM10. We hypothesized that ADAM10-modulated PrPC shedding would alter the cellular binding and cytotoxicity of AβO. Here, we found that in human neuroblastoma cells, activation of ADAM10 with the muscarinic agonist carbachol promotes PrPC shedding and reduces the binding of AβO to the cell surface, which could be blocked with an ADAM10 inhibitor. Conversely, siRNA-mediated ADAM10 knockdown reduced PrPC shedding and increased AβO binding, which was blocked by the PrPC-specific antibody 6D11. The retinoic acid receptor analog acitretin, which up-regulates ADAM10, also promoted PrPC shedding and decreased AβO binding in the neuroblastoma cells and in human induced pluripotent stem cell (iPSC)-derived cortical neurons. Pretreatment with acitretin abolished activation of Fyn kinase and prevented an increase in reactive oxygen species caused by AβO binding to PrPC. Besides blocking AβO binding and toxicity, acitretin also increased the nonamyloidogenic processing of APP. However, in the iPSC-derived neurons, Aβ and other amyloidogenic processing products did not exhibit a reciprocal decrease upon acitretin treatment. These results indicate that by promoting the shedding of PrPC in human neurons, ADAM10 activation prevents the binding and cytotoxicity of AβO, revealing a potential therapeutic benefit of ADAM10 activation in AD.

From the earliest stages of development, when cerebral angiogenesis and neurogenesis are entwined, to the end of life, the interplay between vascular and neural systems of the brain is critical in health and disease. Cerebral microvascular endothelial cells constitute the blood–brain barrier and in concert with pericytes or smooth muscle cells, glia and neurons, integrate into a functional neurovascular unit (NVU). This multicellular NVU maintains homoeostasis of the brain’s microenvironment by restricting the entry of systemic pathogens and neurotoxins as well as meeting the metabolic demands of neural activity. Recent evidence of cerebral microvascular pathologies in vascular diseases and dementia, including Alzheimer’s disease, has challenged the notion that vascular events are merely the consequence of neuronal pathology. This review focuses on molecular mechanisms of neurovascular dysfunction in dementia and outlines currently employed in vitro models to decode such mechanisms. Deciphering neurovascular crosstalk is likely to be more important in understanding the molecular mechanisms of disease than previously anticipated and may offer novel therapeutic opportunities for dementia and related conditions.

Sterol regulatory element-binding proteins (SREBPs) are transcription factors governing transcription of genes related to cholesterol and fatty acid metabolism. To become active, SREBPs must undergo a proteolytic cleavage to allow an active NH(2)-terminal segment to translocate into the nucleus. SKI-1/S1P is the first protease in the proteolytic activation cascade of SREBPs. SREBP inhibition may be useful, for example, in the treatment of liver steatosis caused by homocysteine-induced lipid synthesis. Accordingly, we overexpressed inhibitory prodomains (proSKI) of SKI-1/S1P in HepG2 cells to block SREBP activation to evaluate the potential of SKI-1/S1P in controlling cellular cholesterol synthesis. SKI-1/S1P inhibition resulted in reduced cholesterol synthesis and mRNA levels of the rate-limiting enzymes, HMG-CoA reductase and squalene epoxidase, in the cholesterol synthetic pathway. The inhibitory effect was maintained in the presence of homocysteine-induced endoplasmic reticulum stress. A gene set enrichment analysis was performed to elucidate other metabolic effects caused by SKI-1/S1P inhibition. SKI-1/S1P inhibition was observed to affect a number of other metabolic pathways, including glycolysis and citric acid cycle. These results demonstrate that inhibition of SREBPs decreases cholesterol synthesis in HepG2 cells both in the absence and presence of homocysteine. SKI-1/S1P inhibition may cause widespread changes in other key metabolic pathways.

Induced pluripotent stem cell (iPSC)-derived cortical neurons potentially present a powerful new model to understand corticogenesis and neurological disease. Previous work has established that differentiation protocols can produce cortical neurons, but little has been done to characterize these at cellular resolution. In particular, it is unclear to what extent in vitro two-dimensional, relatively disordered culture conditions recapitulate the development of in vivo cortical layer identity. Single-cell multiplex reverse transcriptase-quantitative polymerase chain reaction (RT-qPCR) was used to interrogate the expression of genes previously implicated in cortical layer or phenotypic identity in individual cells. Totally, 93.6% of single cells derived from iPSCs expressed genes indicative of neuronal identity. High proportions of single neurons derived from iPSCs expressed glutamatergic receptors and synaptic genes. And, 68.4% of iPSC-derived neurons expressing at least one layer marker could be assigned to a laminar identity using canonical cortical layer marker genes. We compared single-cell RNA-seq of our iPSC-derived neurons to available single-cell RNA-seq data from human fetal and adult brain and found that iPSC-derived cortical neurons closely resembled primary fetal brain cells. Unexpectedly, a subpopulation of iPSC-derived neurons co-expressed canonical fetal deep and upper cortical layer markers. However, this appeared to be concordant with data from primary cells. Our results therefore provide reassurance that iPSC-derived cortical neurons are highly similar to primary cortical neurons at the level of single cells but suggest that current layer markers, although effective, may not be able to disambiguate cortical layer identity in all cells.

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

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

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

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

How grafted neural stem cells (NSCs) and their progeny integrate into recipient brain tissue and functionally interact with host cells is as yet unanswered. We report that, in organotypic slice cultures analyzed by ratiometric time-lapse calcium imaging, current-clamp recordings, and dye-coupling methods, an early and essential way in which grafted murine or human NSCs integrate functionally into host neural circuitry and affect host cells is via gap-junctional coupling, even before electrophysiologically mature neuronal differentiation. The gap junctions, which are established rapidly, permit exogenous NSCs to influence directly host network activity, including synchronized calcium transients with host cells in fluctuating networks. The exogenous NSCs also protect host neurons from death and reduce such signs of secondary injury as reactive astrogliosis. To determine whether gap junctions between NSCs and host cells may also mediate neuroprotection in vivo, we examined NSC transplantation in two murine models characterized by degeneration of the same cell type (Purkinje neurons) from different etiologies, namely, the nervous and SCA1 mutants. In both, gap junctions (containing connexin 43) formed between NSCs and host cells at risk, and were associated with rescue of neurons and behavior (when implantation was performed before overt neuron loss). Both in vitro and in vivo beneficial NSC effects were abrogated when gap junction formation or function was suppressed by pharmacologic and/or RNA-inhibition strategies, supporting the pivotal mediation by gap-junctional coupling of some modulatory, homeostatic, and protective actions on host systems as well as establishing a template for the subsequent development of electrochemical synaptic intercellular communication.

The B05 transgenic SCA1 mice, expressing human ataxin-1 with an expanded polyglutamine tract in cerebellar Purkinje cells (PCs), recapitulate many pathological and behavioral characteristics of the neurodegenerative disease spinocerebellar ataxia type 1 (SCA1), including progressive ataxia and PC loss. We transplanted neural precursor cells (NPCs) derived from the subventricular zone of GFP-expressing adult mice into the cerebellar white matter of SCA1 mice when they showed absent (5 weeks), initial (13 weeks), and significant (24 weeks) PC loss. Only in mice with significant cell loss, grafted NPCs migrated into the cerebellar cortex. These animals showed improved motor skills compared with sham-treated controls. No grafted cell adopted the morphological and immunohistochemical characteristics of PCs, but the cerebellar cortex in NPC-grafted SCA1 mice had a significantly thicker molecular layer and more surviving PCs. Perforated patch-clamp recordings revealed a normalization of the PC basal membrane potential, which was abnormally depolarized in sham-treated animals. No significant increase in levels of several neurotrophic factors was observed, suggesting, along with morphological observation, that the neuroprotective effect of grafted NPCs was mediated by direct contact with the host PCs. We postulate that a similar neuroprotective effect of NPCs may be applicable to other cerebellar degenerative diseases.

In the stem cell niche, neural stem cells (NSCs) are in close contact with the specialized blood–brain barrier (BBB) endothelial cells (ECs) that modulate their proliferation and differentiation behavior. NSCs are also an attractive source for cell transplantation and neural tissue repair after central nervous system injury. After systemic grafting, they are confronted with the BBB before they can enter the brain parenchyma. We investigated the interactions of human fetal neural precursor cells (hfNPCs) with human brain ECs in an in vitro model using primary cultures. We demonstrated that hfNPCs efficiently differentiate to neurons, astrocytes, and oligodendrocytes and move to the subendothelial space of human BBB endothelium, but not to pulmonary artery ECs. Effective differentiation was found to be dependent on the chemokine CCL2/MCP-1, but not on CXCL8/IL-8. Our findings suggest that neural precursor cells specifically interact with the BBB endothelium and differentiate in the subendothelial niche into astrocytes, neurons, and oligodendrocytes, under the influence of the chemokine CCL2/MCP-1. Disclosure of potential conflicts of interest is found at the end of this article.

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.