Molecular conformations and dynamics of nucleotide repeats associated with neurodegenerative diseases: double helices and CAG hairpin loops.

Pathogenic DNA secondary structures have been identified as a common and causative factor for expansion in trinucleotide, hexanucleotide, and other simple sequence repeats. These expansions underlie about fifty neurological and neuromuscular disorders known as "anticipation diseases". Cell toxicity and death have been linked to the pathogenic conformations and functional changes of the RNA transcripts, of DNA itself and, when trinucleotides are present in exons, of the translated proteins. We review some of our results for the conformations and dynamics of pathogenic structures for both RNA and DNA, which include mismatched homoduplexes formed by trinucleotide repeats CAG and GAC; CCG and CGG; CTG(CUG) and GTC(GUC); the dynamics of DNA CAG hairpins; mismatched homoduplexes formed by hexanucleotide repeats (GGGGCC) and (GGCCCC); and G-quadruplexes formed by (GGGGCC) and (GGGCCT). We also discuss the dynamics of strand slippage in DNA hairpins formed by CAG repeats as observed with single-molecule Fluorescence Resonance Energy Transfer. This review focuses on the rich behavior exhibited by the mismatches associated with these simple sequence repeat noncanonical structures.

Enhanced insulin signalling ameliorates C9orf72 hexanucleotide repeat expansion toxicity in Drosophila.

G4C2 repeat expansions within the C9orf72 gene are the most common genetic cause of amyotrophic lateral sclerosis (ALS) and frontotemporal dementia (FTD). The repeats undergo repeat-associated non-ATG translation to generate toxic dipeptide repeat proteins. Here, we show that insulin/IGF signalling is reduced in fly models of C9orf72 repeat expansion using RNA sequencing of adult brain. We further demonstrate that activation of insulin/IGF signalling can mitigate multiple neurodegenerative phenotypes in flies expressing either expanded G4C2 repeats or the toxic dipeptide repeat protein poly-GR. Levels of poly-GR are reduced when components of the insulin/IGF signalling pathway are genetically activated in the diseased flies, suggesting a mechanism of rescue. Modulating insulin signalling in mammalian cells also lowers poly-GR levels. Remarkably, systemic injection of insulin improves the survival of flies expressing G4C2 repeats. Overall, our data suggest that modulation of insulin/IGF signalling could be an effective therapeutic approach against C9orf72 ALS/FTD.

Prevalence of C9orf72 hexanucleotide repeat expansion in Greek patients with sporadic ALS.

A total of 178 consecutive patients with definite sALS without frontotemporal dementia (FTD) were enrolled in this study, after complete clinical evaluation. A Repeat-Primed Polymerase Chain Reaction (RP-PCR) protocol was applied to detect the G4C2 repeats expansions. In the studied sALS patients, 5.06% (n = 9) carried the C9orf72 mutation. Among carriers, 2/3 of them were females and spinal onset accounted for 78% and bulbar for 22%, while the mean age of onset was about 60 years. Our study showed that the prevalence of C9orf72 repeat expansion in Greek sALS patients is similar to the overall frequency of the mutation in European populations. The pathogenic mutation remains a promising biomarker for genetic testing and targeted treatment.

1,3-Di(quinolin-2-yl)guanidine binds to GGCCCC hexanucleotide repeat DNA in C9ORF72.

Aberrant expansion of GGGGCC (G4C2) hexanucleotide repeat (HNR) in the first intron of C9ORF72 has been found in frontotemporal dementia and amyotrophic lateral sclerosis (FTD/ALD). The non-canonical DNA structures of the expanded repeats are causative to repeat instability leading to contraction and expansion. We demonstrate that 1,3-di(quinolin-2-yl)guanidine (DQG) binds to GGCCCC/GGCCCC (G2C4/G2C4) motif in double stranded DNA and also antisense G2C4 HNR DNA in C9ORF72. Large increase in the melting temperature of dsDNA containing the G2C4/G2C4 motif was confirmed by the binding of DQG. The marked CD spectral changes indicated structural transition of d(G2C4)9 from i-motifs and/or hairpins to DQG-stabilized d(G2C4)9 structures.

Structural and Dynamical Characterization of DNA and RNA Quadruplexes Obtained from the GGGGCC and GGGCCT Hexanucleotide Repeats Associated with C9FTD/ALS and SCA36 Diseases.

A (GGGGCC) hexanucleotide repeat (HR) expansion in the C9ORF72 gene has been considered the major cause behind both frontotemporal dementia and amyotrophic lateral sclerosis, while a (GGGCCT) is associated with spinocerebellar ataxia 36. Recent experiments involving NMR, CD, optical melting and 1D 1H NMR spectroscopy, suggest that the r(GGGGCC) HR can adopt a hairpin structure with G-G mismatches in equilibrium with a G-quadruplex structure. G-Quadruplexes have also been identified for d(GGGGCC). As these experiments lack molecular resolution, we have used molecular dynamics microsecond simulations to obtain a structural characterization of the G-quadruplexes associated with both HRs. All DNA G-quadruplexes, parallel or antiparallel, with or without loops are stable, while only parallel and one antiparallel (stabilized by diagonal loops) RNA G-quadruplexes are stable. It is known that antiparallel G-quadruplexes require alternating guanines to be in a syn conformation that is hindered by the C3'-endo pucker preferred by RNA. Initial RNA antiparallel quadruplexes built with C2'-endo sugars evolve such that the transition (C2'-endo)-to-(C3'-endo) triggers unwinding and buckling of the flat G-tetrads, resulting in the unfolding of the RNA antiparallel quadruplex. Finally, a parallel G-quadruplex stabilizes an adjacent C-tetrad in both DNA and RNA (thus effectively becoming a mixed quadruplex of 5 layers). The C-tetrad is stabilized by the stacking interactions with the preceding G-tetrad, by cyclical hydrogen bonds C(N4)-(O2), and by an ion between the G-tetrad and the C-tetrad. In addition, antiparallel DNA G-quadruplexes also stabilize flat C-layers at the ends of the quadruplexes.

A PCR-based protocol to accurately size C9orf72 intermediate-length alleles.

Although large expansions of the non-coding GGGGCC repeat in C9orf72 gene are clearly defined as pathogenic for Amyotrophic Lateral Sclerosis (ALS) and Frontotemporal Lobar Degeneration (FTLD), intermediate-length expansions have also been associated with those and other neurodegenerative diseases. Intermediate-length allele sizing is complicated by intrinsic properties of current PCR-based methodologies, in that somatic mosaicism could be suspected. We designed a protocol that allows the exact sizing of intermediate-length alleles, as well as the identification of large expansions.

Characterization of DNA G-quadruplex species forming from C9ORF72 G4C2-expanded repeats associated with amyotrophic lateral sclerosis and frontotemporal lobar degeneration.

The G4C2 hexanucleotide repeat expansion, located in the first intron of the C9ORF72 gene, represents a major genetic hallmark of amyotrophic lateral sclerosis and frontotemporal lobar degeneration. Several hypotheses have been proposed on how the transcribed repeat RNA leads to the development of amyotrophic lateral sclerosis and frontotemporal lobar degeneration. However, despite their importance, factors affecting the transcription of expanded-repeat RNA are not well known. As transcription is dependent on the DNA containing the expanded repeats, it is crucial to understand its structure. G-quadruplexes are known to affect expression on the level of DNA, therefore whether they form on the expanded-repeat DNA constitutes an important biological question. Using nuclear magnetic resonance and circular dichroism spectroscopy we show that DNA G4C2 with varying number of repeats d(G4C2)n form planar guanine quartets characteristic of G-quadruplexes. Additionally, we show DNA G-quadruplexes can form inter- and intra-molecularly in either parallel or anti-parallel orientation, based on d(G4C2) sequence length. This potential structural heterogeneity of longer disease-relevant repeats should therefore be taken into account when studying their role in disease pathogenesis.

Identification of C9orf72 repeat expansions in patients with amyotrophic lateral sclerosis and frontotemporal dementia in mainland China.

The GGGGCC repeat expansion in the C9orf72 gene was recently identified as a major cause of amyotrophic lateral sclerosis (ALS) and frontotemporal dementia (FTD) in white populations. To estimate the frequency of hexanucleotide repeats in patients with ALS and FTD from mainland China, we screened for C9orf72 in a cohort of 128 patients and 150 control subjects using the repeat-primed polymerase chain reaction method. We observed pathogenic repeat expansions in a family with ALS-FTD and in a patient with sporadic FTD. In the family with ALS-FTD, the proband and the 2 asymptomatic siblings exhibited C9orf72 repeat expansions, and the clinical feature of the proband was characterized by pure motor syndrome with no cognitive impairment. The patient with sporadic FTD presented primarily with deteriorating behavior and mental status. Genotype analysis revealed that the proband shared the previously reported 20-single nucleotide polymorphism risk haplotype, whereas the patient with sporadic FTD carried all single nucleotide polymorphisms except rs2814707-A. To our knowledge, this study is the first to report 2 C9orf72 mutation patients in mainland China, and they shared the similar risk haplotype identified in white populations, suggesting that ALS and FTD associated with C9orf72 mutation was probably derived from a single founder.