Traumatic injury causes selective degeneration and TDP-43 mislocalization in human iPSC-derived C9orf72-associated ALS/FTD motor neurons.

A hexanucleotide repeat expansion (HRE) in C9orf72 is the most common genetic cause of amyotrophic lateral sclerosis (ALS) and frontotemporal dementia (FTD). However, patients with the HRE exhibit a wide disparity in clinical presentation and age of symptom onset suggesting an interplay between genetic background and environmental stressors. Neurotrauma as a result of traumatic brain or spinal cord injury has been shown to increase the risk of ALS/FTD in epidemiological studies. Here, we combine patient-specific induced pluripotent stem cells (iPSCs) with a custom-built device to deliver biofidelic stretch trauma to C9orf72 patient and isogenic control motor neurons (MNs) in vitro. We find that mutant but not control MNs exhibit selective degeneration after a single incident of severe trauma, which can be partially rescued by pretreatment with a C9orf72 antisense oligonucleotide. A single incident of mild trauma does not cause degeneration but leads to cytoplasmic accumulation of TDP-43 in C9orf72 MNs. This mislocalization, which only occurs briefly in isogenic controls, is eventually restored in C9orf72 MNs after 6 days. Lastly, repeated mild trauma ablates the ability of patient MNs to recover. These findings highlight alterations in TDP-43 dynamics in C9orf72 ALS/FTD patient MNs following traumatic injury and demonstrate that neurotrauma compounds neuropathology in C9orf72 ALS/FTD. More broadly, our work establishes an in vitro platform that can be used to interrogate the mechanistic interactions between ALS/FTD and neurotrauma.

Integrative analysis of epilepsy-associated genes reveals expression-phenotype correlations.

Epilepsy is a highly prevalent neurological disorder characterized by recurrent seizures. Patients exhibit broad genetic, molecular, and clinical diversity involving mild to severe comorbidities. The factors that contribute to this phenotypic diversity remain unclear. Here we used publicly available datasets to systematically interrogate the expression pattern of 230 epilepsy-associated genes across human tissues, developmental stages, and central nervous system (CNS) cellular subtypes. We grouped genes based on their curated phenotypes into 3 broad classes: core epilepsy genes (CEG), where seizures are the dominant phenotype, developmental and epileptic encephalopathy genes (DEEG) that are associated with developmental and epileptic encephalopathy, and seizure-related genes (SRG), which are characterized by the presence of seizures and gross brain malformations. We find that compared to the other two groups of genes, DEEGs are highly expressed within the adult CNS, exhibit the highest and most dynamic expression in various brain regions across development, and are significantly enriched in GABAergic neurons. Our analysis provides an overview of the expression pattern of epilepsy-associated genes with spatiotemporal resolution and establishes a broad expression-phenotype correlation in epilepsy.

ALS-Associated TDP-43 Dysfunction Compromises UPF1-Dependent mRNA Metabolism Pathways Including Alternative Polyadenylation and 3'UTR Length.

UPF1-mediated decay entails several mRNA surveillance pathways that play a crucial role in cellular homeostasis. However, the precise role of UPF1 in postmitotic neurons remains unresolved, as does its activity in amyotrophic lateral sclerosis (ALS), a devastating neurodegenerative disease characterized by TDP-43 pathology and disrupted mRNA metabolism. Here, we used human iPSC-derived spinal motor neurons (MNs) to identify mRNAs subject to UPF1 degradation by integrating RNA-seq before and after UPF1 knockdown with RIP-seq to identify RNAs that co-immunoprecipitate with the active form of phosphorylated UPF1. We define a stringent set of bona fide UPF1 targets in MNs that are functionally enriched for autophagy and structurally enriched for GC-rich and long 3' UTRs but not for premature termination codon (PTC)-containing transcripts. TDP-43 depletion in iPSC-derived MNs reduces UPF1 phosphorylation and consequently post-transcriptional upregulation of UPF1 targets, suggesting that TDP-43 dysfunction compromises UPF1-mediated mRNA surveillance. Intriguingly, our datasets reveal that UPF1 and TDP-43 regulate alternative polyadenylation and 3'UTR length of mRNAs associated with synaptic and axonal function, a process that we find to be compromised in ALS models in vitro and ALS patient tissue. Our study provides a comprehensive description of UPF1-mediated mRNA decay activity in neurons, reveals overlapping roles between UPF1 and TDP-43 in regulating 3'UTR length, and offers novel insight into the intricate interplay between RNA metabolism and neurodegeneration in ALS.

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.

Polyurethane Culture Substrates Enable Long-Term Neuron Monoculture in a Human in vitro Model of Neurotrauma.

Human induced pluripotent stem cell (hiPSC)-derived cells can reproduce human-specific pathophysiology, patient-specific vulnerability, and gene-environment interactions in neurological disease. Human in vitro models of neurotrauma therefore have great potential to advance the field. However, this potential cannot be realized until important biomaterials challenges are addressed. Status quo stretch injury models of neurotrauma culture cells on sheets of polydimethylsiloxane (PDMS) that are incompatible with long-term monoculture of hiPSC-derived neurons. Here, we overcame this challenge in an established human in vitro neurotrauma model by replacing PDMS with a highly biocompatible form of polyurethane (PU). This substitution allowed long-term monoculture of hiPSC-derived neurons. It also changed the biomechanics of stretch injury. We quantified these changes experimentally using high-speed videography and digital image correlation. We used finite element modeling to quantify the influence of the culture substrate's thickness, stiffness, and coefficient of friction on membrane stretch and concluded that the coefficient of friction explained most of the observed biomechanical changes. Despite these changes, we demonstrated that the modified model produced a robust, dose-dependent trauma phenotype in hiPSC-derived neuron monocultures. In summary, the introduction of this PU film makes it possible to maintain hiPSC-derived neurons in monoculture for long periods in a human in vitro neurotrauma model. In doing so, it opens new horizons in the field of neurotrauma by enabling the unique experimental paradigms (e.g., isogenic models) associated with hiPSC-derived neurons.

Enhanced Neuron Growth and Electrical Activity by a Supramolecular Netrin-1 Mimetic Nanofiber.

Neurotrophic factors are essential not only for guiding the organization of the developing nervous system but also for supporting the survival and growth of neurons after traumatic injury. In the central nervous system (CNS), inhibitory factors and the formation of a glial scar after injury hinder the functional recovery of neurons, requiring exogenous therapies to promote regeneration. Netrin-1, a neurotrophic factor, can initiate axon guidance, outgrowth, and branching, as well as synaptogenesis, through activation of deleted in colorectal cancer (DCC) receptors. We report here the development of a nanofiber-shaped supramolecular mimetic of netrin-1 with monomers that incorporate a cyclic peptide sequence as the bioactive component. The mimetic structure was found to activate the DCC receptor in primary cortical neurons using low molar ratios of the bioactive comonomer. The supramolecular nanofibers enhanced neurite outgrowth and upregulated maturation as well as pre- and postsynaptic markers over time, resulting in differences in electrical activity similar to neurons treated with the recombinant netrin-1 protein. The results suggest the possibility of using the supramolecular structure as a therapeutic to promote regenerative bioactivity in CNS injuries.

Analysis of proteome-wide degradation dynamics in ALS SOD1 iPSC-derived patient neurons reveals disrupted VCP homeostasis.

Mutations in SOD1 cause amyotrophic lateral sclerosis (ALS) through gain-of-function effects, yet the mechanisms by which misfolded mutant SOD1 (mutSOD1) protein impairs human motor neurons (MNs) remain unclear. Here, we use induced-pluripotent-stem-cell-derived MNs coupled to metabolic stable isotope labeling and mass spectrometry to investigate proteome-wide degradation dynamics. We find several proteins, including the ALS-causal valosin-containing protein (VCP), which predominantly acts in proteasome degradation and autophagy, that degrade slower in mutSOD1 relative to isogenic control MNs. The interactome of VCP is altered in mutSOD1 MNs in vitro, while VCP selectively accumulates in the affected motor cortex of ALS-SOD1 patients. Overexpression of VCP rescues mutSOD1 toxicity in MNs in vitro and in a C. elegans model in vivo, in part due to its ability to modulate the degradation of insoluble mutSOD1. Our results demonstrate that VCP contributes to mutSOD1-dependent degeneration, link two distinct ALS-causal genes, and highlight selective protein degradation impairment in ALS pathophysiology.

Loss of function of the ALS-associated NEK1 kinase disrupts microtubule homeostasis and nuclear import.

Loss-of-function variants in NIMA-related kinase 1 (NEK1) constitute a major genetic cause of amyotrophic lateral sclerosis (ALS), accounting for 2 to 3% of all cases. However, how NEK1 mutations cause motor neuron (MN) dysfunction is unknown. Using mass spectrometry analyses for NEK1 interactors and NEK1-dependent expression changes, we find functional enrichment for proteins involved in the microtubule cytoskeleton and nucleocytoplasmic transport. We show that α-tubulin and importin-ß1, two key proteins involved in these processes, are phosphorylated by NEK1 in vitro. NEK1 is essential for motor control and survival in Drosophila models in vivo, while using several induced pluripotent stem cell (iPSC)-MN models, including NEK1 knockdown, kinase inhibition, and a patient mutation, we find evidence for disruptions in microtubule homeostasis and nuclear import. Notably, stabilizing microtubules with two distinct classes of drugs restored NEK1-dependent deficits in both pathways. The capacity of NEK1 to modulate these processes that are critically involved in ALS pathophysiology renders this kinase a formidable therapeutic candidate.

Integrative Analysis of Epilepsy-Associated Genes Reveals Expression-Phenotype Correlations.

Epilepsy is a highly prevalent neurological disorder characterized by recurrent seizures. Patients exhibit broad genetic, molecular, and clinical diversity involving mild to severe comorbidities. The factors that contribute to this phenotypic diversity remain unclear. We used publicly available datasets to systematically interrogate the expression pattern of 247 epilepsy-associated genes across human tissues, developmental stages, and central nervous system (CNS) cellular subtypes. We grouped genes based on their curated phenotypes into 3 broad classes: core epilepsy genes (CEG), where seizures are the core syndrome, developmental and epileptic encephalopathy genes (DEEG) that are associated with developmental delay, and seizure-related genes (SRG), which are characterized by developmental delay and gross brain malformations. We find that DEEGs are highly expressed within the CNS, while SRGs are most abundant in non-CNS tissues. DEEGs and CEGs exhibit highly dynamic expression in various brain regions across development, spiking during the prenatal to infancy transition. Lastly, the abundance of CEGs and SRGs is comparable within cellular subtypes in the brain, while the average expression level of DEEGs is significantly higher in GABAergic neurons and non-neuronal cells. Our analysis provides an overview of the expression pattern of epilepsy-associated genes with spatiotemporal resolution and establishes a broad expression-phenotype correlation in epilepsy.

Artificial extracellular matrix scaffolds of mobile molecules enhance maturation of human stem cell-derived neurons.

Human induced pluripotent stem cell (hiPSC) technologies offer a unique resource for modeling neurological diseases. However, iPSC models are fraught with technical limitations including abnormal aggregation and inefficient maturation of differentiated neurons. These problems are in part due to the absence of synergistic cues of the native extracellular matrix (ECM). We report on the use of three artificial ECMs based on peptide amphiphile (PA) supramolecular nanofibers. All nanofibers display the laminin-derived IKVAV signal on their surface but differ in the nature of their non-bioactive domains. We find that nanofibers with greater intensity of internal supramolecular motion have enhanced bioactivity toward hiPSC-derived motor and cortical neurons. Proteomic, biochemical, and functional assays reveal that highly mobile PA scaffolds caused enhanced ß1-integrin pathway activation, reduced aggregation, increased arborization, and matured electrophysiological activity of neurons. Our work highlights the importance of designing biomimetic ECMs to study the development, function, and dysfunction of human neurons.

Excitatory Dysfunction Drives Network and Calcium Handling Deficits in 16p11.2 Duplication Schizophrenia Induced Pluripotent Stem Cell-Derived Neurons.

BACKGROUND: Schizophrenia (SCZ) is a debilitating psychiatric disorder with a large genetic contribution; however, its neurodevelopmental substrates remain largely unknown. Modeling pathogenic processes in SCZ using human induced pluripotent stem cell-derived neurons (iNs) has emerged as a promising strategy. Copy number variants confer high genetic risk for SCZ, with duplication of the 16p11.2 locus increasing the risk 14.5-fold. METHODS: To dissect the contribution of induced excitatory neurons (iENs) versus GABAergic (gamma-aminobutyric acidergic) neurons (iGNs) to SCZ pathophysiology, we induced iNs from CRISPR (clustered regularly interspaced short palindromic repeats)-Cas9 isogenic and SCZ patient-derived induced pluripotent stem cells and analyzed SCZ-related phenotypes in iEN monocultures and iEN/iGN cocultures. RESULTS: In iEN/iGN cocultures, neuronal firing and synchrony were reduced at later, but not earlier, stages of in vitro development. These were fully recapitulated in iEN monocultures, indicating a primary role for iENs. Moreover, isogenic iENs showed reduced dendrite length and deficits in calcium handling. iENs from 16p11.2 duplication-carrying patients with SCZ displayed overlapping deficits in network synchrony, dendrite outgrowth, and calcium handling. Transcriptomic analysis of both iEN cohorts revealed molecular markers of disease related to the glutamatergic synapse, neuroarchitecture, and calcium regulation. CONCLUSIONS: Our results indicate the presence of 16p11.2 duplication-dependent alterations in SCZ patient-derived iENs. Transcriptomics and cellular phenotyping reveal overlap between isogenic and patient-derived iENs, suggesting a central role of glutamatergic, morphological, and calcium dysregulation in 16p11.2 duplication-mediated pathogenesis. Moreover, excitatory dysfunction during early neurodevelopment is implicated as the basis of SCZ pathogenesis in 16p11.2 duplication carriers. Our results support network synchrony and calcium handling as outcomes directly linked to this genetic risk variant.

NUP62 localizes to ALS/FTLD pathological assemblies and contributes to TDP-43 insolubility.

A G4C2 hexanucleotide repeat expansion in the C9orf72 gene is the most common genetic cause of ALS and FTLD (C9-ALS/FTLD) with cytoplasmic TDP-43 inclusions observed in regions of neurodegeneration. The accumulation of repetitive RNAs and dipeptide repeat protein (DPR) are two proposed mechanisms of toxicity in C9-ALS/FTLD and linked to impaired nucleocytoplasmic transport. Nucleocytoplasmic transport is regulated by the phenylalanine-glycine nucleoporins (FG nups) that comprise the nuclear pore complex (NPC) permeability barrier. However, the relationship between FG nups and TDP-43 pathology remains elusive. Our studies show that nuclear depletion and cytoplasmic mislocalization of one FG nup, NUP62, is linked to TDP-43 mislocalization in C9-ALS/FTLD iPSC neurons. Poly-glycine arginine (GR) DPR accumulation initiates the formation of cytoplasmic RNA granules that recruit NUP62 and TDP-43. Cytoplasmic NUP62 and TDP-43 interactions promotes their insolubility and NUP62:TDP-43 inclusions are frequently found in C9orf72 ALS/FTLD as well as sporadic ALS/FTLD postmortem CNS tissue. Our findings indicate NUP62 cytoplasmic mislocalization contributes to TDP-43 proteinopathy in ALS/FTLD.

'Channeling' therapeutic discovery for epileptic encephalopathy through iPSC technologies.

Induced pluripotent stem cell (iPSC) and gene editing technologies have revolutionized the field of in vitro disease modeling, granting us access to disease-pertinent human cells of the central nervous system. These technologies are particularly well suited for the study of diseases with strong monogenic etiologies. Epilepsy is one of the most common neurological disorders in children, with approximately half of all genetic cases caused by mutations in ion channel genes. These channelopathy-associated epilepsies are clinically diverse, mechanistically complex, and hard to treat. Here, we review the genetic links to epilepsy, the opportunities and challenges of iPSC-based approaches for developing in vitro models of channelopathy-associated disorders, the available tools for effective phenotyping of iPSC-derived neurons, and discuss the potential therapeutic approaches for these devastating diseases.

Homozygous might be hemizygous: CRISPR/Cas9 editing in iPSCs results in detrimental on-target defects that escape standard quality controls.

The ability to precisely edit the genome of human induced pluripotent stem cell (iPSC) lines using CRISPR/Cas9 has enabled the development of cellular models that can address genotype to phenotype relationships. While genome editing is becoming an essential tool in iPSC-based disease modeling studies, there is no established quality control workflow for edited cells. Moreover, large on-target deletions and insertions that occur through DNA repair mechanisms have recently been uncovered in CRISPR/Cas9-edited loci. Yet the frequency of these events in human iPSCs remains unclear, as they can be difficult to detect. We examined 27 iPSC clones generated after targeting 9 loci and found that 33% had acquired large, on-target genomic defects, including insertions and loss of heterozygosity. Critically, all defects had escaped standard PCR and Sanger sequencing analysis. We describe a cost-efficient quality control strategy that successfully identified all edited clones with detrimental on-target events and could facilitate the integrity of iPSC-based studies.

Biofidelic dynamic compression of human cortical spheroids reproduces neurotrauma phenotypes.

Fundamental questions about patient heterogeneity and human-specific pathophysiology currently obstruct progress towards a therapy for traumatic brain injury (TBI). Human in vitro models have the potential to address these questions. Three-dimensional spheroidal cell culture protocols for human-origin neural cells have several important advantages over their two-dimensional monolayer counterparts. Three-dimensional spheroidal cultures may mature more quickly, develop more biofidelic electrophysiological activity and/or reproduce some aspects of brain architecture. Here, we present the first human in vitro model of non-penetrating TBI employing three-dimensional spheroidal cultures. We used a custom-built device to traumatize these spheroids in a quantifiable, repeatable and biofidelic manner, and correlated the heterogeneous mechanical strain field with the injury phenotype. Trauma reduced cell viability, mitochondrial membrane potential and spontaneous synchronous electrophysiological activity in the spheroids. Electrophysiological deficits emerged at lower injury severities than changes in cell viability. Also, traumatized spheroids secreted lactate dehydrogenase, a marker of cell damage, and neurofilament light chain, a promising clinical biomarker of neurotrauma. These results demonstrate that three-dimensional human in vitro models can reproduce important phenotypes of neurotrauma in vitro.

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.

Dyshomeostatic modulation of Ca2+-activated K+ channels in a human neuronal model of KCNQ2 encephalopathy.

Mutations in KCNQ2, which encodes a pore-forming K+ channel subunit responsible for neuronal M-current, cause neonatal epileptic encephalopathy, a complex disorder presenting with severe early-onset seizures and impaired neurodevelopment. The condition is exceptionally difficult to treat, partially because the effects of KCNQ2 mutations on the development and function of human neurons are unknown. Here, we used induced pluripotent stem cells (iPSCs) and gene editing to establish a disease model and measured the functional properties of differentiated excitatory neurons. We find that patient iPSC-derived neurons exhibit faster action potential repolarization, larger post-burst afterhyperpolarization and a functional enhancement of Ca2+-activated K+ channels. These properties, which can be recapitulated by chronic inhibition of M-current in control neurons, facilitate a burst-suppression firing pattern that is reminiscent of the interictal electroencephalography pattern in patients. Our findings suggest that dyshomeostatic mechanisms compound KCNQ2 loss-of-function leading to alterations in the neurodevelopmental trajectory of patient iPSC-derived neurons.

Effect of Ezogabine on Cortical and Spinal Motor Neuron Excitability in Amyotrophic Lateral Sclerosis: A Randomized Clinical Trial.

Importance: Amyotrophic lateral sclerosis (ALS) is a progressive neurodegenerative disease of the motor nervous system. Clinical studies have demonstrated cortical and spinal motor neuron hyperexcitability using transcranial magnetic stimulation and threshold tracking nerve conduction studies, respectively, although metrics of excitability have not been used as pharmacodynamic biomarkers in multi-site clinical trials. Objective: To ascertain whether ezogabine decreases cortical and spinal motor neuron excitability in ALS. Design, Setting, and Participants: This double-blind, placebo-controlled phase 2 randomized clinical trial sought consent from eligible participants from November 3, 2015, to November 9, 2017, and was conducted at 12 US sites within the Northeast ALS Consortium. Participants were randomized in equal numbers to a higher or lower dose of ezogabine or to an identical matched placebo, and they completed in-person visits at screening, baseline, week 6, and week 8 for clinical assessment and neurophysiological measurements. Interventions: Participants were randomized to receive 600 mg/d or 900 mg/d of ezogabine or a matched placebo for 10 weeks. Main Outcomes and Measures: The primary outcome was change in short-interval intracortical inhibition (SICI; SICI-1 was used in analysis to reflect stronger inhibition from an increase in amplitude) from pretreatment mean at screening and baseline to the full-dose treatment mean at weeks 6 and 8. The secondary outcomes included levels of cortical motor neuron excitability (including resting motor threshold) measured by transcranial magnetic stimulation and spinal motor neuron excitability (including strength-duration time constant) measured by threshold tracking nerve conduction studies. Results: A total of 65 participants were randomized to placebo (23), 600 mg/d of ezogabine (23), and 900 mg/d of ezogabine (19 participants); 45 were men (69.2%) and the mean (SD) age was 58.3 (8.8) years. The SICI-1 increased by 53% (mean ratio, 1.53; 95% CI, 1.12-2.09; P = .009) in the 900-mg/d ezogabine group vs placebo group. The SICI-1 did not change in the 600-mg/d ezogabine group vs placebo group (mean ratio, 1.15; 95% CI, 0.87-1.52; P = .31). The resting motor threshold increased in the 600-mg/d ezogabine group vs placebo group (mean ratio, 4.61; 95% CI, 0.21-9.01; P = .04) but not in the 900-mg/d ezogabine group vs placebo group (mean ratio, 1.95; 95% CI, -2.64 to 6.54; P = .40). Ezogabine caused a dose-dependent decrease in excitability by several other metrics, including strength-duration time constant in the 900-mg/d ezogabine group vs placebo group (mean ratio, 0.73; 95% CI, 0.60 to 0.87; P < .001). Conclusions and Relevance: Ezogabine decreased cortical and spinal motor neuron excitability in participants with ALS, suggesting that such neurophysiological metrics may be used as pharmacodynamic biomarkers in multisite clinical trials. Trial Registration: ClinicalTrials.gov Identifier: NCT02450552.

RNA dependent suppression of C9orf72 ALS/FTD associated neurodegeneration by Matrin-3.

The most common genetic cause of amyotrophic lateral sclerosis (ALS) is a GGGGCC (G4C2) hexanucleotide repeat expansions in first intron of the C9orf72 gene. The accumulation of repetitive RNA sequences can mediate toxicity potentially through the formation of intranuclear RNA foci that sequester key RNA-binding proteins (RBPs), and non-ATG mediated translation into toxic dipeptide protein repeats. However, the contribution of RBP sequestration to the mechanisms underlying RNA-mediated toxicity remain unknown. Here we show that the ALS-associated RNA-binding protein, Matrin-3 (MATR3), colocalizes with G4C2 RNA foci in patient tissues as well as iPSC-derived motor neurons harboring the C9orf72 mutation. Hyperexpansion of C9 repeats perturbed subcellular distribution and levels of endogenous MATR3 in C9-ALS patient-derived motor neurons. Interestingly, we observed that ectopic expression of human MATR3 strongly mitigates G4C2-mediated neurodegeneration in vivo. MATR3-mediated suppression of C9 toxicity was dependent on the RNA-binding domain of MATR3. Importantly, we found that expression of MATR3 reduced the levels of RAN-translation products in mammalian cells in an RNA-dependent manner. Finally, we have shown that knocking down endogenous MATR3 in C9-ALS patient-derived iPSC neurons decreased the presence of G4C2 RNA foci in the nucleus. Overall, these studies suggest that MATR3 genetically modifies the neuropathological and the pathobiology of C9orf72 ALS through modulating the RNA foci and RAN translation.

TETs compete with DNMT3 activity in pluripotent cells at thousands of methylated somatic enhancers.

Mammalian cells stably maintain high levels of DNA methylation despite expressing both positive (DNMT3A/B) and negative (TET1-3) regulators. Here, we analyzed the independent and combined effects of these regulators on the DNA methylation landscape using a panel of knockout human embryonic stem cell (ESC) lines. The greatest impact on global methylation levels was observed in DNMT3-deficient cells, including reproducible focal demethylation at thousands of normally methylated loci. Demethylation depends on TET expression and occurs only when both DNMT3s are absent. Dynamic loci are enriched for hydroxymethylcytosine and overlap with subsets of putative somatic enhancers that are methylated in ESCs and can be activated upon differentiation. We observe similar dynamics in mouse ESCs that were less frequent in epiblast stem cells (EpiSCs) and scarce in somatic tissues, suggesting a conserved pluripotency-linked mechanism. Taken together, our data reveal tightly regulated competition between DNMT3s and TETs at thousands of somatic regulatory sequences within pluripotent cells.