Roberto Simone

G-quadruplexes: Emerging roles in neurodegenerative diseases and the non-coding transcriptome

G‐rich sequences in DNA and RNA have a propensity to fold into stable secondary structures termed G‐quadruplexes. G‐quadruplex forming sequences are widespread throughout the human genome, within both, protein coding and non‐coding genes, and regulatory regions. G‐quadruplexes have been implicated in multiple cellular functions including chromatin epigenetic regulation, DNA recombination, transcriptional regulation of gene promoters and enhancers, and translation. Here we will review the evidence for the occurrence of G‐quadruplexes both in vitro and in vivo; their role in neurological diseases including G‐quadruplex‐forming repeat expansions in the C9orf72 gene in frontotemporal dementia and amyotrophic lateral sclerosis and loss of the G‐quadruplex binding protein FMRP in the intellectual disability fragile X syndrome. We also review mounting evidence that supports a role for G‐quadruplexes in regulating the processing or function of a range of non‐coding RNAs. Finally we will highlight current perspectives for therapeutic interventions that target G‐quadruplexes.

The MAPT p.A152T variant is a risk factor associated with tauopathies with atypical clinical and neuropathological features.

Microtubule-associated protein tau (MAPT) mutations have been shown to underlie frontotemporal dementia and a variety of additional sporadic tauopathies. We identified a rare p.A152T variant in MAPT exon 7 in two (of eight) patients with clinical presentation of parkinsonism and postmortem finding of neurofibrillary tangle pathology. Two siblings of one patient also carried the p.A152T variant, and both have progressive cognitive impairment. Further screening identified the variant in two other cases: one with pathologically confirmed corticobasal degeneration and another with the diagnosis of Parkinsons disease with dementia. The balance of evidence suggests this variant is associated with disease, but the very varied phenotype of the cases with the mutation is not consistent with it being a fully penetrant pathogenic mutation. Interestingly, this variation results in the creation of a new phosphorylation site that could cause reduced microtubule binding. We suggest that the A152T variant is a risk factor associated with the development of atypical neurodegenerative conditions with abnormal tau accumulation.

In vitro prion-like behaviour of TDP-43 in ALS

Amyotrophic lateral sclerosis (ALS) is the most common form of motor neuron disease (MND), and > 95% of familial and sporadic cases involve the deposition of insoluble aggregated, phosphorylated and cleaved TDP-43 protein. Accumulating clinical and biological evidence now indicates that ALS bears a number of similarities to the prion diseases, with TDP-43 acting as a misfolded ‘prion-like’ protein demonstrating similar underlying pathobiology. Here we systematically address the hypothesis that ALS is a prion-like disorder. First we demonstrate that TDP-43 demonstrates seeded polymerisation in vitro directly from both ALS brain and spinal cord. We next show that the seeding of TDP-43 results in the formation of characteristic insoluble, aggregated, and phosphorylated TDP-43 pathology that directly recapitulates the morphological diversity of TDP-43 inclusions detected in ALS patient CNS tissue. We next demonstrate that this reaction can be serially propagated to produce increasing amounts of phosphorylated TDP-43 pathology, and that aggregates can spread from cell to cell in an analogous fashion to that seen in the prion diseases. Finally, we reproduced our findings in a murine motor neuron-like cell line (NSC-34), where the seeding of TDP-43 induces the formation of TDP-43 oligomers and reduced cell viability. These findings may guide therapeutic strategies in this rapidly progressive and invariably fatal disease.

Widespread RNA metabolism impairment in sporadic inclusion body myositis TDP43-proteinopathy.

TDP43 protein mislocalization is a hallmark of the neurodegenerative diseases amyotrophic lateral sclerosis and frontotemporal dementia, and mutations in the gene encoding TDP43 cause both disorders, further highlighting its role in disease pathogenesis. TDP43 is a heterogenous ribonucleoprotein, therefore suggesting that alterations in RNA metabolism play a role in these disorders, although direct evidence in patients is lacking. Sporadic inclusion body myositis (sIBM) is the most common acquired myopathy occurring in adults aged older than 50 years and abnormal cytoplasmic accumulations of TDP43 have been consistently described in sIBM myofibers. Here, we exploit high quality RNA from frozen sIBM muscle biopsies for transcriptomic studies on TDP43-proteinopathy patient tissue. Surprisingly, we found widespread sIBM-specific changes in the RNA metabolism pathways themselves. Consistent with this finding, we describe novel RNA binding proteins to mislocalize in the cytoplasm of sIBM myofibers and splicing changes in MAPT, a gene previously shown to play a role in sIBM. Our data indicate widespread alterations of RNA metabolism are a novel aspect of disease pathogenesis in sIBM. These findings also document an association, in TDP43-proteinopathy patients, between heterogenous ribonucleoprotein pathology and RNA metabolism alterations and carry importance for neurodegenerative diseases, such as amyotrophic lateral sclerosis and frontotemporal dementia.

G‐quadruplex‐binding small molecules ameliorate C9orf72 FTD/ALS pathology in vitro and in vivo

Intronic GGGGCC repeat expansions in C9orf72 are the most common known cause of frontotemporal dementia (FTD) and amyotrophic lateral sclerosis (ALS), which are characterised by degeneration of cortical and motor neurons, respectively. Repeat expansions have been proposed to cause disease by both the repeat RNA forming foci that sequester RNA‐binding proteins and through toxic dipeptide repeat proteins generated by repeat‐associated non‐ATG translation. GGGGCC repeat RNA folds into a G‐quadruplex secondary structure, and we investigated whether targeting this structure is a potential therapeutic strategy. We performed a screen that identified three structurally related small molecules that specifically stabilise GGGGCC repeat G‐quadruplex RNA. We investigated their effect in C9orf72 patient iPSC‐derived motor and cortical neurons and show that they significantly reduce RNA foci burden and the levels of dipeptide repeat proteins. Furthermore, they also reduce dipeptide repeat proteins and improve survival in vivo, in GGGGCC repeat‐expressing Drosophila. Therefore, small molecules that target GGGGCC repeat G‐quadruplexes can ameliorate the two key pathologies associated with C9orf72 FTD/ALS. These data provide proof of principle that targeting GGGGCC repeat G‐quadruplexes has therapeutic potential.

Assessment of common variability and expression quantitative trait loci for genome-wide associations for progressive supranuclear palsy

Progressive supranuclear palsy is a rare parkinsonian disorder with characteristic neurofibrillary pathology consisting of hyperphosphorylated tau protein. Common variation defining the microtubule associated protein tau gene (MAPT) H1 haplotype strongly contributes to disease risk. A recent genome-wide association study (GWAS) revealed 3 novel risk loci on chromosomes 1, 2, and 3 that primarily implicate STX6, EIF2AK3, and MOBP, respectively. Genetic associations, however, rarely lead to direct identification of the relevant functional allele. More often, they are in linkage disequilibrium with the causative polymorphism(s) that could be a coding change or affect gene expression regulatory motifs. To identify any such changes, we sequenced all coding exons of those genes directly implicated by the associations in progressive supranuclear palsy cases and analyzed regional gene expression data from control brains to identify expression quantitative trait loci within 1 Mb of the risk loci. Although we did not find any coding variants underlying the associations, GWAS-associated single-nucleotide polymorphisms at these loci are in complete linkage disequilibrium with haplotypes that completely overlap with the respective genes. Although implication of EIF2AK3 and MOBP could not be fully assessed, we show that the GWAS single-nucleotide polymorphism rs1411478 (STX6) is a strong expression quantitative trait locus with significantly lower expression of STX6 in white matter in carriers of the risk allele.

Corrigendum to “Assessment of common variability and expression quantitative trait loci for genome-wide associations for progressive supranuclear palsy.” [Neurobiol. Aging 35 (2014) 1514.e1–1514.e12]

Corrigendum Corrigendum to “Assessment of common variability and expression quantitative trait loci for genome-wide associations for progressive supranuclear palsy.” [Neurobiol. Aging 35 (2014) 1514.e1e1514.e12] Raffaele Ferrari, Mina Ryten, Roberto Simone, Daniah Trabzuni, Nayia Nicolaou, Geshanthi Hondhamuni, Adaikalavan Ramasamy, JanaVandrovcova, UK Brain Expression Consortium, Michael E. Weale, Andrew J. Lees, Parastoo Momeni, John Hardy, Rohan de Silva

Foamy Virus Vectors Transduce Visceral Organs and Hippocampal Structures following In Vivo Delivery to Neonatal Mice

Viral vectors are rapidly being developed for a range of applications in research and gene therapy. Prototype foamy virus (PFV) vectors have been described for gene therapy, although their use has mainly been restricted to ex vivo stem cell modification. Here we report direct in vivo transgene delivery with PFV vectors carrying reporter gene constructs. In our investigations, systemic PFV vector delivery to neonatal mice gave transgene expression in the heart, xiphisternum, liver, pancreas, and gut, whereas intracranial administration produced brain expression until animals were euthanized 49 days post-transduction. Immunostaining and confocal microscopy analysis of injected brains showed that transgene expression was highly localized to hippocampal architecture despite vector delivery being administered to the lateral ventricle. This was compared with intracranial biodistribution of lentiviral vectors and adeno-associated virus vectors, which gave a broad, non-specific spread through the neonatal mouse brain without regional localization, even when administered at lower copy numbers. Our work demonstrates that PFV can be used for neonatal gene delivery with an intracranial expression profile that localizes to hippocampal neurons, potentially because of the mitotic status of the targeted cells, which could be of use for research applications and gene therapy of neurological disorders.

Multiple roles for TDP-43 in tau gene expression regulation

post hoc test applied when appropriate. Data are expressed as mean 6 SD and p< 0.05 was considered statistically significant.Results:Dex produced significant increases in hippocampal p-tau levels at the AT8, CP13, and PHF-1 phosphoepitopes 30 min (0.5h) following treatment (Fig. 1). Interestingly, at 2h post-Dex, a significant increase in p-tau was observed at all three phosphoepitopes despite recovery from anesthesia. No significant changes in total-tau levels were observed following Dex administration. Kinase activation could not explain the increase in p-tau levels; however, significant decreases in demethylation levels of the catalytic subunit of protein phosphastase 2A (PP2A-C) and the A subunit of PP2A (PP2A-A) paralleled the p-tau increase. Conclusions: These data indicate that Dex directly induces tau hyperphosphorylation in the mouse hippocampus. This effect persists after the recovery from anesthesia and may involve the deregulation of PP2A. The impact of this anesthetic exposure on neurofibrillary pathology warrants further investigation.