Glucose hypometabolism prompts RAN translation and exacerbates C9orf72-related ALS/FTD phenotypes.

The most prevalent genetic cause of both amyotrophic lateral sclerosis and frontotemporal dementia is a (GGGGCC)n nucleotide repeat expansion (NRE) occurring in the first intron of the C9orf72 gene (C9). Brain glucose hypometabolism is consistently observed in C9-NRE carriers, even at pre-symptomatic stages, but its role in disease pathogenesis is unknown. Here, we show alterations in glucose metabolic pathways and ATP levels in the brains of asymptomatic C9-BAC mice. We find that, through activation of the GCN2 kinase, glucose hypometabolism drives the production of dipeptide repeat proteins (DPRs), impairs the survival of C9 patient-derived neurons, and triggers motor dysfunction in C9-BAC mice. We also show that one of the arginine-rich DPRs (PR) could directly contribute to glucose metabolism and metabolic stress by inhibiting glucose uptake in neurons. Our findings provide a potential mechanistic link between energy imbalances and C9-ALS/FTD pathogenesis and suggest a feedforward loop model with potential opportunities for therapeutic intervention.

Differential response of C9orf72 transcripts following neuronal depolarization.

The (G4C2)n nucleotide repeat expansion (NRE) mutation in C9orf72 is the most common genetic cause of ALS and FTD. The biological functions of C9orf72 are becoming understood, but it is unclear if this gene is regulated in a neural-specific manner. Neuronal activity is a crucial modifier of biological processes in health and neurodegenerative disease contexts. Here, we show that prolonged membrane depolarization in healthy human iPSC-cortical neurons leads to a significant downregulation of a transcript variant 3 (V3) of C9orf72, with a concomitant increase in variant 2 (V2), which leads to total C9orf72 RNA transcript levels remaining unchanged. However, the same response is not observed in cortical neurons derived from patients with the C9-NRE mutation. These findings reveal the impact of depolarization on C9orf72 transcripts, and how this response diverges in C9-NRE-carriers, which may have important implications in the underlying unique clinical associations of C9-NRE transcripts and disease pathogenesis.

Breakdown of the central synapses in C9orf72-linked ALS/FTD.

Amyotrophic lateral sclerosis (ALS) is a progressive, fatal neurodegenerative disease that leads to the death of motor and cortical neurons. The clinical manifestations of ALS are heterogenous, and efficacious treatments to significantly slow the progression of the disease are lacking. Cortical hyper-excitability is observed pre-symptomatically across disease-causative genetic variants, as well as in the early stages of sporadic ALS, and typically precedes motor neuron involvement and overt neurodegeneration. The causes of cortical hyper-excitability are not yet fully understood but is mainly agreed to be an early event. The identification of the nucleotide repeat expansion (GGGGCC)n in the C9ORF72 gene has provided evidence that ALS and another neurodegenerative disease, frontotemporal dementia (FTD), are part of a disease spectrum with common genetic origins. ALS and FTD are diseases in which synaptic dysfunction is reported throughout disease onset and stages of progression. It has become apparent that ALS/FTD-causative genes, such as C9ORF72, may have roles in maintaining the normal physiology of the synapse, as mutations in these genes often manifest in synaptic dysfunction. Here we review the dysfunctions of the central nervous system synapses associated with the nucleotide repeat expansion in C9ORF72 observed in patients, organismal, and cellular models of ALS and FTD.

A mouse model with widespread expression of the C9orf72-linked glycine-arginine dipeptide displays non-lethal ALS/FTD-like phenotypes.

Translation of the hexanucleotide G4C2 expansion associated with C9orf72 amyotrophic lateral sclerosis and frontotemporal dementia (ALS/FTD) produces five different dipeptide repeat protein (DPR) species that can confer toxicity. There is yet much to learn about the contribution of a single DPR to disease pathogenesis. We show here that a short repeat length is sufficient for the DPR poly-GR to confer neurotoxicity in vitro, a phenomenon previously unobserved. This toxicity is also reported in vivo in our novel knock-in mouse model characterized by widespread central nervous system (CNS) expression of the short-length poly-GR. We observe sex-specific chronic ALS/FTD-like phenotypes in these mice, including mild motor neuron loss, but no TDP-43 mis-localization, as well as motor and cognitive impairments. We suggest that this model can serve as the foundation for phenotypic exacerbation through second-hit forms of stress.

Targeting TNFα produced by astrocytes expressing amyotrophic lateral sclerosis-linked mutant fused in sarcoma prevents neurodegeneration and motor dysfunction in mice.

Genetic mutations that cause amyotrophic lateral sclerosis (ALS), a progressively lethal motor neuron disease, are commonly found in ubiquitously expressed genes. In addition to direct defects within motor neurons, growing evidence suggests that dysfunction of non-neuronal cells is also an important driver of disease. Previously, we demonstrated that mutations in DNA/RNA binding protein fused in sarcoma (FUS) induce neurotoxic phenotypes in astrocytes in vitro, via activation of the NF-κB pathway and release of pro-inflammatory cytokine TNFα. Here, we developed an intraspinal cord injection model to test whether astrocyte-specific expression of ALS-causative FUSR521G variant (mtFUS) causes neuronal damage in vivo. We show that restricted expression of mtFUS in astrocytes is sufficient to induce death of spinal motor neurons leading to motor deficits through upregulation of TNFα. We further demonstrate that TNFα is a key toxic molecule as expression of mtFUS in TNFα knockout animals does not induce pathogenic changes. Accordingly, in mtFUS-transduced animals, administration of TNFα neutralizing antibodies prevents neurodegeneration and motor dysfunction. Together, these studies strengthen evidence that astrocytes contribute to disease in ALS and establish, for the first time, that FUS-ALS astrocytes induce pathogenic changes to motor neurons in vivo. Our work identifies TNFα as the critical driver of mtFUS-astrocytic toxicity and demonstrates therapeutic success of targeting TNFα to attenuate motor neuron dysfunction and death. Ultimately, through defining and subsequently targeting this toxic mechanism, we provide a viable FUS-ALS specific therapeutic strategy, which may also be applicable to sporadic ALS where FUS activity and cellular localization are frequently perturbed.

The effects of molecular crowding and CpG hypermethylation on DNA G-quadruplexes formed by the C9orf72 nucleotide repeat expansion.

A nucleotide repeat expansion (NRE), (G4C2)n, located in a classically noncoding region of C9orf72 (C9), is the most common genetic mutation associated with ALS/FTD. There is increasing evidence that nucleic acid structures formed by the C9-NRE may both contribute to ALS/FTD, and serve as therapeutic targets, but there is limited characterization of these nucleic acid structures under physiologically and disease relevant conditions. Here we show in vitro that the C9-NRE DNA can form both parallel and antiparallel DNA G-quadruplex (GQ) topological structures and that the structural preference of these DNA GQs can be dependent on the molecular crowding conditions. Additionally, 5-methylcytosine DNA hypermethylation, which is observed in the C9-NRE locus in some patients, has minimal effects on GQ topological preferences. Finally, molecular dynamic simulations of methylated and nonmethylated GQ structures support in vitro data showing that DNA GQ structures formed by the C9-NRE DNA are stable, with structural fluctuations limited to the cytosine-containing loop regions. These findings provide new insight into the structural polymorphic preferences and stability of DNA GQs formed by the C9-NRE in both the methylated and nonmethylated states, as well as reveal important features to guide the development of upstream therapeutic approaches to potentially attenuate C9-NRE-linked diseases.

G-quadruplexes offer a conserved structural motif for NONO recruitment to NEAT1 architectural lncRNA.

The long non-coding RNA NEAT1 serves as a scaffold for the assembly of paraspeckles, membraneless nuclear organelles involved in gene regulation. Paraspeckle assembly requires NEAT1 recruitment of the RNA-binding protein NONO, however the NEAT1 elements responsible for recruitment are unknown. Herein we present evidence that previously unrecognized structural features of NEAT1 serve an important role in these interactions. Led by the initial observation that NONO preferentially binds the G-quadruplex conformation of G-rich C9orf72 repeat RNA, we find that G-quadruplex motifs are abundant and conserved features of NEAT1. Furthermore, we determine that NONO binds NEAT1 G-quadruplexes with structural specificity and provide evidence that G-quadruplex motifs mediate NONO-NEAT1 association, with NONO binding sites on NEAT1 corresponding largely to G-quadruplex motifs, and treatment with a G-quadruplex-disrupting small molecule causing dissociation of native NONO-NEAT1 complexes. Together, these findings position G-quadruplexes as a primary candidate for the NONO-recruiting elements of NEAT1 and provide a framework for further investigation into the role of G-quadruplexes in paraspeckle formation and function.

Synaptic dysfunction induced by glycine-alanine dipeptides in C9orf72-ALS/FTD is rescued by SV2 replenishment.

The most common cause of amyotrophic lateral sclerosis (ALS) and frontotemporal dementia (FTD) is an intronic hexanucleotide repeat expansion in the C9orf72 gene. In disease, RNA transcripts containing this expanded region undergo repeat-associated non-AUG translation to produce dipeptide repeat proteins (DPRs), which are detected in brain and spinal cord of patients and are neurotoxic both in vitro and in vivo paradigms. We reveal here a novel pathogenic mechanism for the most abundantly detected DPR in ALS/FTD autopsy tissues, poly-glycine-alanine (GA). Previously, we showed motor dysfunction in a GA mouse model without loss of motor neurons. Here, we demonstrate that mobile GA aggregates are present within neurites, evoke a reduction in synaptic vesicle-associated protein 2 (SV2), and alter Ca2+ influx and synaptic vesicle release. These phenotypes could be corrected by restoring SV2 levels. In GA mice, loss of SV2 was observed without reduction of motor neuron number. Notably, reduction in SV2 was seen in cortical and motor neurons derived from patient induced pluripotent stem cell lines, suggesting synaptic alterations also occur in patients.

Repeat-associated non-AUG translation in C9orf72-ALS/FTD is driven by neuronal excitation and stress.

Nucleotide repeat expansions (NREs) are prevalent mutations in a multitude of neurodegenerative diseases. Repeat-associated non-AUG (RAN) translation of these repeat regions produces mono or dipeptides that contribute to the pathogenesis of these diseases. However, the mechanisms and drivers of RAN translation are not well understood. Here we analyzed whether different cellular stressors promote RAN translation of dipeptide repeats (DPRs) associated with the G4C2 hexanucleotide expansions in C9orf72, the most common genetic cause of amyotrophic lateral sclerosis (ALS) and frontotemporal dementia (FTD). We found that activating glutamate receptors or optogenetically increasing neuronal activity by repetitive trains of depolarization induced DPR formation in primary cortical neurons and patient derived spinal motor neurons. Increases in the integrated stress response (ISR) were concomitant with increased RAN translation of DPRs, both in neurons and different cell lines. Targeting phosphorylated-PERK and the phosphorylated-eif2α complex reduces DPR levels revealing a potential therapeutic strategy to attenuate DPR-dependent disease pathogenesis in NRE-linked diseases.

The expanding biology of the C9orf72 nucleotide repeat expansion in neurodegenerative disease.

A nucleotide repeat expansion (NRE) within the chromosome 9 open reading frame 72 (C9orf72) gene was the first of this type of mutation to be linked to multiple neurological conditions, including amyotrophic lateral sclerosis and frontotemporal dementia. The pathogenic mechanisms through which the C9orf72 NRE contributes to these disorders include loss of C9orf72 function and gain-of-function mechanisms of C9orf72 driven by toxic RNA and protein species encoded by the NRE. These mechanisms have been linked to several cellular defects - including nucleocytoplasmic trafficking deficits and nuclear stress - that have been observed in both patients and animal models.

The C9orf72 repeat expansion disrupts nucleocytoplasmic transport.

The hexanucleotide repeat expansion (HRE) GGGGCC (G4C2) in C9orf72 is the most common cause of amyotrophic lateral sclerosis (ALS) and frontotemporal dementia (FTD). Recent studies support an HRE RNA gain-of-function mechanism of neurotoxicity, and we previously identified protein interactors for the G4C2 RNA including RanGAP1. A candidate-based genetic screen in Drosophila expressing 30 G4C2 repeats identified RanGAP (Drosophila orthologue of human RanGAP1), a key regulator of nucleocytoplasmic transport, as a potent suppressor of neurodegeneration. Enhancing nuclear import or suppressing nuclear export of proteins also suppresses neurodegeneration. RanGAP physically interacts with HRE RNA and is mislocalized in HRE-expressing flies, neurons from C9orf72 ALS patient-derived induced pluripotent stem cells (iPSC-derived neurons), and in C9orf72 ALS patient brain tissue. Nuclear import is impaired as a result of HRE expression in the fly model and in C9orf72 iPSC-derived neurons, and these deficits are rescued by small molecules and antisense oligonucleotides targeting the HRE G-quadruplexes. Nucleocytoplasmic transport defects may be a fundamental pathway for ALS and FTD that is amenable to pharmacotherapeutic intervention.

Emerging role of RNA•DNA hybrids in C9orf72-linked neurodegeneration.

RNA plays an active role in structural polymorphism of the genome through the formation of stable RNA•DNA hybrids (R-loops). R-loops can modulate normal physiological processes and are also associated with pathological conditions, such as those related to nucleotide repeat expansions. A guanine-rich hexanucleotide repeat expansion in chromosome 9 open reading frame 72 (C9orf72) has been linked to a spectrum of neurological conditions including amyotrophic lateral sclerosis (ALS) and frontotemporal dementia (FTD). Here we discuss the possible roles, both locally and genome-wide, of R-loops that may arise from the C9orf72 hexanucleotide repeat. R-loops have the potential to influence the pathological processes identified in many repeat expansion diseases, such as repeat instability, transcriptional dysregulation, epigenetic modification, and antisense-mediated gene regulation. We propose that, given the wide-ranging consequences of R-loops in the cell, these structures could underlie multiple pathological processes in C9orf72-linked neurodegeneration.

C9orf72 nucleotide repeat structures initiate molecular cascades of disease.

A hexanucleotide repeat expansion (HRE), (GGGGCC)n, in C9orf72 is the most common genetic cause of the neurodegenerative diseases amyotrophic lateral sclerosis (ALS) and frontotemporal dementia (FTD). Here we identify a molecular mechanism by which structural polymorphism of the HRE leads to ALS/FTD pathology and defects. The HRE forms DNA and RNA G-quadruplexes with distinct structures and promotes RNA•DNA hybrids (R-loops). The structural polymorphism causes a repeat-length-dependent accumulation of transcripts aborted in the HRE region. These transcribed repeats bind to ribonucleoproteins in a conformation-dependent manner. Specifically, nucleolin, an essential nucleolar protein, preferentially binds the HRE G-quadruplex, and patient cells show evidence of nucleolar stress. Our results demonstrate that distinct C9orf72 HRE structural polymorphism at both DNA and RNA levels initiates molecular cascades leading to ALS/FTD pathologies, and provide the basis for a mechanistic model for repeat-associated neurodegenerative diseases.

RNA toxicity from the ALS/FTD C9ORF72 expansion is mitigated by antisense intervention.

A hexanucleotide GGGGCC repeat expansion in the noncoding region of the C9ORF72 gene is the most common genetic abnormality in familial and sporadic amyotrophic lateral sclerosis (ALS) and frontotemporal dementia (FTD). The function of the C9ORF72 protein is unknown, as is the mechanism by which the repeat expansion could cause disease. Induced pluripotent stem cell (iPSC)-differentiated neurons from C9ORF72 ALS patients revealed disease-specific (1) intranuclear GGGGCCexp RNA foci, (2) dysregulated gene expression, (3) sequestration of GGGGCCexp RNA binding protein ADARB2, and (4) susceptibility to excitotoxicity. These pathological and pathogenic characteristics were confirmed in ALS brain and were mitigated with antisense oligonucleotide (ASO) therapeutics to the C9ORF72 transcript or repeat expansion despite the presence of repeat-associated non-ATG translation (RAN) products. These data indicate a toxic RNA gain-of-function mechanism as a cause of C9ORF72 ALS and provide candidate antisense therapeutics and candidate human pharmacodynamic markers for therapy.

LacI-DNA-IPTG loops: equilibria among conformations by single-molecule FRET.

The E. coli Lac repressor (LacI) tetramer binds simultaneously to a promoter-proximal DNA binding site (operator) and an auxiliary operator, resulting in a DNA loop, which increases repression efficiency. Induction of the lac operon by allolactose reduces the affinity of LacI for DNA, but induction does not completely prevent looping in vivo. Our previous work on the conformations of LacI loops used a hyperstable model DNA construct, 9C14, that contains a sequence directed bend flanked by operators. Single-molecule fluorescence resonance energy transfer (SM-FRET) on a dual fluorophore-labeled LacI-9C14 loop showed that it adopts a single, stable, high-FRET V-shaped LacI conformation. Ligand-induced changes in loop geometry can affect loop stability, and the current work assesses loop population distributions for LacI-9C14 complexes containing the synthetic inducer IPTG. SM-FRET confirms that the high-FRET LacI-9C14 loop is only partially destabilized by saturating IPTG. LacI titration experiments and FRET fluctuation analysis suggest that the addition of IPTG induces loop conformational dynamics and re-equilibration between loop population distributions that include a mixture of looped states that do not exhibit high-efficiency FRET. The results show that repression by looping even at saturating IPTG should be considered in models for regulation of the operon. We propose that persistent DNA loops near the operator function biologically to accelerate rerepression upon exhaustion of inducer.

FRET studies of a landscape of Lac repressor-mediated DNA loops.

DNA looping mediated by the Lac repressor is an archetypal test case for modeling protein and DNA flexibility. Understanding looping is fundamental to quantitative descriptions of gene expression. Systematic analysis of LacI•DNA looping was carried out using a landscape of DNA constructs with lac operators bracketing an A-tract bend, produced by varying helical phasings between operators and the bend. Fluorophores positioned on either side of both operators allowed direct Förster resonance energy transfer (FRET) detection of parallel (P1) and antiparallel (A1, A2) DNA looping topologies anchored by V-shaped LacI. Combining fluorophore position variant landscapes allows calculation of the P1, A1 and A2 populations from FRET efficiencies and also reveals extended low-FRET loops proposed to form via LacI opening. The addition of isopropyl-ß-D-thio-galactoside (IPTG) destabilizes but does not eliminate the loops, and IPTG does not redistribute loops among high-FRET topologies. In some cases, subsequent addition of excess LacI does not reduce FRET further, suggesting that IPTG stabilizes extended or other low-FRET loops. The data align well with rod mechanics models for the energetics of DNA looping topologies. At the peaks of the predicted energy landscape for V-shaped loops, the proposed extended loops are more stable and are observed instead, showing that future models must consider protein flexibility.