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) 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.

The nuclear import receptor Kapβ2 modifies neurotoxicity mediated by poly(GR) in C9orf72-linked ALS/FTD.

Expanded intronic GC repeats in the C9ORF72 gene cause amyotrophic lateral sclerosis (ALS) and frontotemporal dementia (FTD). These intronic repeats are translated through a non-AUG-dependent mechanism into five different dipeptide repeat proteins (DPRs), including poly-glycine-arginine (GR), which is aggregation-prone and neurotoxic. Here, we report that Kapβ2 and GR interact, co-aggregating, in cultured neurons in-vitro and CNS tissue in-vivo.

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) nucleotide repeat expansion (NRE) occurring in the first intron of the gene (C9). Brain glucose hypometabolism is consistently observed in C9-NRE carriers, even at pre-symptomatic stages, although its potential role in disease pathogenesis is unknown. Here, we identified alterations in glucose metabolic pathways and ATP levels in the brain of asymptomatic C9-BAC mice.

Differential response of C9orf72 transcripts following neuronal depolarization.

The (GC) 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.

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.

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.

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.

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

A nucleotide repeat expansion (NRE), (GC), 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.

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.

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).

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.

In situ investigation of production processes in a large chamber scanning electron microscope.

A large-chamber scanning electron microscope (LC-SEM) provides an ideal platform for the installation of large-scale in situ experiments. Our LC-SEM has internal chamber dimensions of 1,2 × 1,3 × 1,4 m (W × H × D) (Fig.1) and makes it possible to incorporate novel in situ experimental devices, which are reported on here.

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.

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).

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).

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.

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.

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.

Pretreatment with aldosterone or corticosterone blocks the memory-enhancing effects of nimodipine, captopril, CGP 37,849, and strychnine in mice.

Oral pretreatment with aldosterone or corticosterone blocked the memory-enhancing effects of the calcium antagonist nimodipine, the ACE inhibitor captopril, the NMDA blocker CGP 37,849, and the glycine antagonist strychnine in a passive-avoidance test in mice. The memory-disturbing effects of phenobarbitone, diazepam, CGP 37,849 and scopolamine were not influenced by the hormonal pretreatment. These findings could indicate the involvement of a steroid-sensitive mechanism in drug-induced improvement of memory.

Chronic methamphetamine and its withdrawal modify behavioral and neuroendocrine circadian rhythms.

Chronic methamphetamine (MA) administration via the drinking water not only induced hyperactivity, but phase delayed the rat rest-activity cycle under entrained conditions. The minimum and/or maximum of the rhythms in eating, drinking, body weight, core body temperature and plasma/urine corticosterone were delayed. The different phase shifts of peak and trough values can also be a result of modulation in the wave form of the rhythms.