Progress in Understanding and Treating SCN2A-Mediated Disorders

Advances in gene discovery for neurodevelopmental disorders have identified SCN2A dysfunction as a leading cause of infantile seizures, autism spectrum disorder, and intellectual disability. SCN2A encodes the neuronal sodium channel NaV1.2. Functional assays demonstrate strong correlation between genotype and phenotype. This insight can help guide therapeutic decisions and raises the possibility that ligands that selectively enhance or diminish channel function may improve symptoms. The well-defined function of sodium channels makes SCN2A an important test case for investigating the neurobiology of neurodevelopmental disorders more generally. Here, we discuss the progress made, through the concerted efforts of a diverse group of academic and industry scientists as well as policy advocates, in understanding and treating SCN2A-related disorders

Neurotrophic requirements of human motor neurons defined using amplified and purified stem-cell derived cultures

Neurotrophic requirements of human motor neurons defined using amplified and purified stem-cell derived culturesHuman motor neurons derived from embryonic and induced pluripotent stem cells (hESCs and hiPSCs) are a potentially important tool for studying motor neuron survival and pathological cell death. However, their basic survival requirements remain poorly characterized. Here, we sought to optimize a robust survival assay and characterize their response to different neurotrophic factors. First, to increase motor neuron yield, we screened a small-molecule collection and found that the Rho-associated kinase (ROCK) inhibitor Y-27632 enhances motor neuron progenitor proliferation up to 4-fold in hESC and hiPSC cultures. Next, we FACS-purified motor neurons expressing the Hb9::GFP reporter from Y-27632-amplified embryoid bodies and cultured them in the presence of mitotic inhibitors to eliminate dividing progenitors. Survival of these purified motor neurons in the absence of any other cell type was strongly dependent on neurotrophic support. GDNF, BDNF and CNTF all showed potent survival effects (EC(50) 1-2 pM). The number of surviving motor neurons was further enhanced in the presence of forskolin and IBMX, agents that increase endogenous cAMP levels. As a demonstration of the ability of the assay to detect novel neurotrophic agents, Y-27632 itself was found to support human motor neuron survival. Thus, purified human stem cell-derived motor neurons show survival requirements similar to those of primary rodent motor neurons and can be used for rigorous cell-based screening.This work was funded by Project A.L.S., P2ALS and NYSTEM grant number CO24415. The work of N.J.L. was supported by the Portuguese Foundation for Science and Technology SFRH/BD/33421/2008 and the Luso-American Development Foundation. B.J.-K. was supported by the National Institute of Neurological Disorders and Stroke (NINDS). L.R. was supported by the Swedish Brain Foundation/Hjarnfonden. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript

A C. elegans model of C9orf72-associated ALS/FTD uncovers a conserved role for eIF2D in RAN translation

A hexanucleotide repeat expansion GGGGCC in the non-coding region of C9orf72 is the most common cause of inherited amyotrophic lateral sclerosis (ALS) and frontotemporal dementia (FTD). Toxic dipeptide repeats (DPRs) are synthesized from GGGGCC via repeat-associated non-AUG (RAN) translation. Here, we develop C. elegans models that express, either ubiquitously or exclusively in neurons, 75 GGGGCC repeats flanked by intronic C9orf72 sequence. The worms generate DPRs (poly-glycine-alanine [poly-GA], poly-glycine-proline [poly-GP]) and poly-glycine-arginine [poly-GR]), display neurodegeneration, and exhibit locomotor and lifespan defects. Mutation of a non-canonical translation-initiating codon (CUG) upstream of the repeats selectively reduces poly-GA steady-state levels and ameliorates disease, suggesting poly-GA is pathogenic. Importantly, loss-of-function mutations in the eukaryotic translation initiation factor 2D (eif-2D/eIF2D) reduce poly-GA and poly-GP levels, and increase lifespan in both C. elegans models. Our in vitro studies in mammalian cells yield similar results. Here, we show a conserved role for eif-2D/eIF2D in DPR expression

Role of Ucp1 enhancer methylation and chromatin remodelling in the control of Ucp1 expression in murine adipose tissue

Aims/hypothesis Increasing the expression of the brown adipose tissue-specific gene uncoupling protein-1 (Ucp1) is a potential target for treating obesity. We investigated the role of DNA methylation and histone modification in Ucp1 expression in adipose cell lines and ex vivo murine adipose tissues. Methods Methylation state of the Ucp1 enhancer was studied using bisulphite mapping in murine adipose cell lines, and tissue taken from cold-stressed mice, coupled with functional assays of the effects of methylation and demethylation of the Ucp1 promoter on gene expression and nuclear protein binding. Results We show that demethylation of the Ucp1 promoter by 5-aza-deoxycytidine increases Ucp1 expression while methylation of Ucp1 promoter–reporter constructs decreases expression. Brown adipose tissue-specific Ucp1 expression is associated with decreased CpG dinucleotide methylation of the Ucp1 enhancer. The lowest CpG dinucleotide methylation state was found in two cyclic AMP response elements (CRE3, CRE2) in the Ucp1 promoter and methylation of the CpG in CRE2, but not CRE3 decreased nuclear protein binding. Chromatin immunoprecipitation assays revealed the presence of the silencing DiMethH3K9 modification on the Ucp1 enhancer in white adipose tissue and the appearance of the active TriMethH3K4 mark at the Ucp1 promoter in brown adipose tissue in response to a cold environment. Conclusions/interpretation The results demonstrate that CpG dinucleotide methylation of the Ucp1 enhancer exhibits tissue-specific patterns in murine tissue and cell lines and suggest that adipose tissue-specific Ucp1 expression involves demethylation of CpG dinucleotides found in regulatory CREs in the Ucp1 enhancer, as well as modification of histone tails

Inherent Plasticity of Brown Adipogenesis in White Fat of Mice Allows for Recovery from Effects of Post-Natal Malnutrition

Interscapular brown adipose tissue (iBAT) is formed during fetal development and stable for the life span of the mouse. In addition, brown adipocytes also appear in white fat depots (wBAT) between 10 and 21 days of age in mice maintained at a room temperature of 23°C. However, this expression is transient. By 60 days of age the brown adipocytes have disappeared, but they can re-emerge if the adult mouse is exposed to the cold (5°C) or treated with β3-adrenergic agonists. Since the number of brown adipocytes that can be induced in white fat influences the capacity of the mouse to resist the obese state, we determined the effects of the nutritional conditions on post-natal development (birth to 21 days) of wBAT and its long-term effects on diet-induced obesity (DIO). Under-nutrition caused essentially complete suppression of wBAT in inguinal fat at 21 days of age, as indicated by expression of Ucp1 and genes of mitochondrial structure and function based upon microarray and qRT-PCR analysis, whereas over-nutrition had no discernible effects on wBAT induction. Surprisingly, the suppression of wBAT at 21 days of age did not affect DIO in adult mice maintained at 23°C, nor did it affect the reduction in obesity or cold tolerance when DIO mice were exposed to the cold at 5°C for one week. Gene expression analysis indicated that mice raised under conditions that suppressed wBAT at 21 days of age were able to normally induce wBAT as adults. Therefore, neither severe hypoleptinemia nor hypoinsulinemia during suckling permanently impaired brown adipogenesis in white fat. In addition, energy balance studies of DIO mice exposed to cold indicates that mice with reduced adipose stores preferentially increased food intake, whereas those with larger adipose tissue depots preferred to utilize energy from their adipose stores

TonEBP/NFAT5 promotes obesity and insulin resistance by epigenetic suppression of white adipose tissue beiging

Tonicity-responsive enhancer binding protein (TonEBP or NFAT5) is a regulator of cellular adaptation to hypertonicity, macrophage activation and T-cell development. Here we report that TonEBP is an epigenetic regulator of thermogenesis and obesity. In mouse subcutaneous adipocytes, TonEBP expression increases > 50-fold in response to high-fat diet (HFD) feeding. Mice with TonEBP haplo-deficiency or adipocyte-specific TonEBP deficiency are resistant to HFD-induced obesity and metabolic defects (hyperglycemia, hyperlipidemia, and hyperinsulinemia). They also display increased oxygen consumption, resistance to hypothermia, and beiging of subcutaneous fat tissues. TonEBP suppresses the promoter of beta 3-adrenoreceptor gene, a critical regulator of lipolysis and thermogenesis, in ex vivo and cultured adipocytes. This involves recruitment of DNMT1 DNA methylase and methylation of the promoter. In human subcutaneous adipocytes TonEBP expression displays a correlation with body mass index but an inverse correlation with beta 3-adrenoreceptor expression. Thus, TonEBP is an attractive therapeutic target for obesity, insulin resistance, and hyperlipidemia

Somatic coding mutations in human induced pluripotent stem cells

Defined transcription factors can induce epigenetic reprogramming of adult mammalian cells into induced pluripotent stem cells. Although DNA factors are integrated during some reprogramming methods, it is unknown whether the genome remains unchanged at the single nucleotide level. Here we show that 22 human induced pluripotent stem (hiPS) cell lines reprogrammed using five different methods each contained an average of five protein-coding point mutations in the regions sampled (an estimated six protein coding point mutations per exome). The majority of these mutations were non-synonymous, nonsense, or splice variants, and were enriched in genes mutated or having causative effects in cancers. At least half of these reprogramming-associated mutations pre-existed in fibroblast progenitors at low frequencies, while the rest were newly occurring during or after reprogramming. Thus, hiPS cells acquire genetic modifications in addition to epigenetic modifications. Extensive genetic screening should become a standard procedure to ensure hiPS safety before clinical use

Ontogeny and thermogenic role for sternal fat in female sheep

Brown adipose tissue acting through a unique uncoupling protein (UCP1) has a critical role in preventing hypothermia in new-born sheep but is then considered to rapidly disappear during postnatal life. The extent to which the anatomical location of fat influences postnatal development and thermogenic function, particularly following feeding, in adulthood, are not known and were both examined in our study. Changes in gene expression of functionally important pathways (i.e. thermogenesis, development, adipogenesis and metabolism) were compared between sternal and retroperitoneal fat depots together with a representative skeletal muscle over the first month of postnatal life, coincident with the loss of brown fat and accumulation of white fat. In adult sheep, implanted temperature probes were used to characterise the thermogenic response of fat and muscle to feeding and the effects of reduced or increased adiposity. UCP1 was more abundant within sternal than retroperitoneal fat and was only retained in the sternal depot of adults. Distinct differences in the abundance of gene pathway markers were apparent between tissues, with sternal fat exhibiting some similarities with muscle that were not apparent in the retroperitoneal depot. In adults, the post-prandial rise in temperature was greater and more prolonged in sternal than retroperitoneal fat and muscle, a difference that was maintained with altered adiposity. In conclusion, sternal adipose tissue retains UCP1 into adulthood when it shows a greater thermogenic response to feeding than muscle and retroperitoneal fat. Sternal fat may be more amenable to targeted interventions that promote thermogenesis in large mammals

Absence of RIP140 Reveals a Pathway Regulating glut4-Dependent Glucose Uptake in Oxidative Skeletal Muscle through UCP1-Mediated Activation of AMPK

Skeletal muscle constitutes the major site of glucose uptake leading to increased removal of glucose from the circulation in response to insulin. Type 2 diabetes and obesity are often associated with insulin resistance that can be counteracted by exercise or the use of drugs increasing the relative proportion of oxidative fibers. RIP140 is a transcriptional coregulator with a central role in metabolic tissues and we tested the effect of modulating its level of expression on muscle glucose and lipid metabolism in two mice models. Here, we show that although RIP140 protein is expressed at the same level in both oxidative and glycolytic muscles, it inhibits both fatty acid and glucose utilization in a fiber-type dependent manner. In RIP140-null mice, fatty acid utilization increases in the extensor digitorum longus and this is associated with elevated expression of genes implicated in fatty acid binding and transport. In the RIP140-null soleus, depletion of RIP140 leads to increased GLUT4 trafficking and glucose uptake with no change in Akt activity. AMPK phosphorylation/activity is inhibited in the soleus of RIP140 transgenic mice and increased in RIP140-null soleus. This is associated with increased UCP1 expression and mitochondrial uncoupling revealing the existence of a signaling pathway controlling insulin-independent glucose uptake in the soleus of RIP140-null mice. In conclusion, our findings reinforce the participation of RIP140 in the maintenance of energy homeostasis by acting as an inhibitor of energy production and particularly point to RIP140 as a promising therapeutic target in the treatment of insulin resistance