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

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, whereas the rest occurred 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 cell safety before clinical use

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

CLIP-Seq analysis enables the design of protective ribosomal RNA bait oligonucleotides against C9ORF72 ALS/FTD poly-GR pathophysiology

Amyotrophic lateral sclerosis and frontotemporal dementia patients with a hexanucleotide repeat expansion in C9ORF72 (C9-HRE) accumulate poly-GR and poly-PR aggregates. The pathogenicity of these arginine-rich dipeptide repeats (R-DPRs) is thought to be driven by their propensity to bind low-complexity domains of multivalent proteins. However, the ability of R-DPRs to bind native RNA and the significance of this interaction remain unclear. Here, we used computational and experimental approaches to characterize the physicochemical properties of R-DPRs and their interaction with RNA. We find that poly-GR predominantly binds ribosomal RNA (rRNA) in cells and exhibits an interaction that is predicted to be energetically stronger than that for associated ribosomal proteins. Critically, modified rRNA “bait” oligonucleotides restore poly-GR–associated ribosomal deficits and ameliorate poly-GR toxicity in patient neurons and Drosophila models. Our work strengthens the hypothesis that ribosomal function is impaired by R-DPRs, highlights a role for direct rRNA binding in mediating ribosomal dysfunction, and presents a strategy for protecting against C9-HRE pathophysiological mechanisms.This work was supported by the U.S. National Institutes of Health (NIH) National Institute of Neurological Disorders and Stroke (NINDS) and National Institute of Aging (NIA) grant R01NS104219 (E.K.); NIH/NINDS grant R21NS107761 (E.K.); AFM-Telethon French Muscular Dystrophy Association Trampoline Grant #23648 (J.A.O.); AFM-Telethon postdoctoral fellowship (J.A.O.); Ramon y Cajal fellowships RYC2019-026980-I (J.A.O.) and RYC2021-033294-I (I.R.S.); Gipuzkoa Foru Aldundia 2019-FELL-000017-01 (I.R.S.); Maria de Maeztu Units of Excellence CEX2021-001159-M (J.A.O.) and MDM-2017-0720 (I.R.S.); NINDS grants R01NS097850 and R01NS131409 (J.K.I.); Department of Defense grants PR211919 and W81XWH2110131 (J.K.I.); John Douglas French Alzheimer’s Foundation (J.K.I.); Center for Regenerative Nanomedicine at the Simpson Querrey Institute (S.I.S. and T.D.C.); Intramural Research Program, NIH, National Cancer Institute (NCI), Center for Cancer Research (M.B. and S.L.W.); Les Turner ALS Foundation (E.K.); and New York Stem Cell Foundation (J.K.I. and E.K.).With funding from the Spanish government through the "Severo Ochoa Centre of Excellence" accreditation (CEX2021-001159-M (J.A.O.)).Peer reviewe

Supplementary Materials for CLIP-Seq analysis enables the design of protective ribosomal RNA bait oligonucleotides against C9ORF72 ALS/FTD poly-GR pathophysiology

This PDF file includes: Figs. S1 to S10 Legends for tables S1 to S6Other Supplementary Material includes the following: Tables S1 to S6Peer reviewe

A novel platform to enable the high-throughput derivation and characterization of feeder-free human iPSCs

Human induced pluripotent stem cells (hiPSCs) hold enormous potential, however several obstacles impede their translation to industrial and clinical applications. Here we describe a platform to efficiently generate, characterize and maintain single cell and feeder-free (FF) cultured hiPSCs by means of a small molecule cocktail media additive. Using this strategy we have developed an effective multiplex sorting and high-throughput selection platform where individual clonal hiPSC lines are readily obtained from a pool of candidate clones, expanded and thoroughly characterized. By promoting survival and self-renewal, the selected hiPSC clones can be rapidly expanded over multiple FF, single-cell passages while maintaining their pluripotency and genomic stability as demonstrated by trilineage differentiation, karyotype and copy number variation analysis. This study provides a robust platform that increases efficiency, throughput, scale and quality of hiPSC generation and facilitates the industrial and clinical use of iPSC technology

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

Generation of Healthy Mice from Gene-Corrected Disease-Specific Induced Pluripotent Stem Cells

Using the murine model of tyrosinemia type 1 (fumarylacetoacetate hydrolase [FAH] deficiency; FAH−/− mice) as a paradigm for orphan disorders, such as hereditary metabolic liver diseases, we evaluated fibroblast-derived FAH−/−-induced pluripotent stem cells (iPS cells) as targets for gene correction in combination with the tetraploid embryo complementation method. First, after characterizing the FAH−/− iPS cell lines, we aggregated FAH−/−-iPS cells with tetraploid embryos and obtained entirely FAH−/−-iPS cell–derived mice that were viable and exhibited the phenotype of the founding FAH−/− mice. Then, we transduced FAH cDNA into the FAH−/−-iPS cells using a third-generation lentiviral vector to generate gene-corrected iPS cells. We could not detect any chromosomal alterations in these cells by high-resolution array CGH analysis, and after their aggregation with tetraploid embryos, we obtained fully iPS cell–derived healthy mice with an astonishing high efficiency for full-term development of up to 63.3%. The gene correction was validated functionally by the long-term survival and expansion of FAH-positive cells of these mice after withdrawal of the rescuing drug NTBC (2-(2-nitro-4-fluoromethylbenzoyl)-1,3-cyclohexanedione). Furthermore, our results demonstrate that both a liver-specific promoter (transthyretin, TTR)-driven FAH transgene and a strong viral promoter (from spleen focus-forming virus, SFFV)-driven FAH transgene rescued the FAH-deficiency phenotypes in the mice derived from the respective gene-corrected iPS cells. In conclusion, our data demonstrate that a lentiviral gene repair strategy does not abrogate the full pluripotent potential of fibroblast-derived iPS cells, and genetic manipulation of iPS cells in combination with tetraploid embryo aggregation provides a practical and rapid approach to evaluate the efficacy of gene correction of human diseases in mouse models

Nuclear poly(ADP-ribose) activity is a therapeutic target in amyotrophic lateral sclerosis

Abstract Amyotrophic lateral sclerosis (ALS) is a devastating and fatal motor neuron disease. Diagnosis typically occurs in the fifth decade of life and the disease progresses rapidly leading to death within ~ 2–5 years of symptomatic onset. There is no cure, and the few available treatments offer only a modest extension in patient survival. A protein central to ALS is the nuclear RNA/DNA-binding protein, TDP-43. In > 95% of ALS patients, TDP-43 is cleared from the nucleus and forms phosphorylated protein aggregates in the cytoplasm of affected neurons and glia. We recently defined that poly(ADP-ribose) (PAR) activity regulates TDP-43-associated toxicity. PAR is a posttranslational modification that is attached to target proteins by PAR polymerases (PARPs). PARP-1 and PARP-2 are the major enzymes that are active in the nucleus. Here, we uncovered that the motor neurons of the ALS spinal cord were associated with elevated nuclear PAR, suggesting elevated PARP activity. Veliparib, a small-molecule inhibitor of nuclear PARP-1/2, mitigated the formation of cytoplasmic TDP-43 aggregates in mammalian cells. In primary spinal-cord cultures from rat, Veliparib also inhibited TDP-43-associated neuronal death. These studies uncover that PAR activity is misregulated in the ALS spinal cord, and a small-molecular inhibitor of PARP-1/2 activity may have therapeutic potential in the treatment of ALS and related disorders associated with abnormal TDP-43 homeostasis