We were introduced to the topic by a very insightful plenary lecture by Laura Ranum, Professor of Molecular Genetics and Microbiology at the University of Florida. Professor Ranum started out by describing how the central dogma of molecular biology, namely that DNA makes RNA makes protein, had been modified during the last decade using examples from her ground-breaking research on myotonic dystrophy and SCA8.
She introduced the ideas of RNA toxicity in repeat disorders as well as the fascinating phenomenon of repeat associated non-ATG (RAN) translation. The second half of the lecture focussed on C9orf72, where she reviewed the specific evidence for RAN translation. Professor Ranum showed an interesting panel of antibodies against dipeptides which showed that they are translated beyond the hexanucleotide repeat, and suggested that these ‘tails’ of the dipeptide proteins may be of pathologic significance. Finally, Professor Ranum showed data from two new transgenic mice generated in her laboratory: the first was carried a monoallellic expansion of around 500 repeats, the second carried a biallellic expansion of 36 repeats in one allele and 500 in the other. Both mice formed sense and antisense foci in the cortex as well as dipeptide protein. Analysis of the phenotype and motor neuron pathology is currently under way.
The next talk was presented by MRC/MNDA Lady Edith Wolfson Clinical Research Fellow Dr Cooper-Knock from Sheffield. He presented very interesting data from pathological specimens from ALS patients with the C9orf72 expansion. He showed that sense foci were more present in granule cells of the cerebellum, whereas antisense foci were more abundant in cerebellar purkinje cells and motor neurons. Only antisense and not sense foci were associated with loss of TDP-43 from the nucleus, potentially pointing towards different pathophysiological roles of the two different groups of RNA foci.
Louis de Muynck from the University of Leuven, Belgium presented expression data for the three C9orf72 mRNA isoforms using TaqMan assays. RNA was extracted from blood, frontal cortex and occipital cortex of a large cohort of 34 expansion carriers, 77 controls and 137 sporadic ALS patients. Repeat expansion carriers did not have significantly differences in total C9orf72 mRNA expression but isoform V2 was expressed at lower levels whereas V3 was expressed at higher levels in C9orf72 carriers in both frontal cortex and occipital cortex. Louis de Muynck also showed very interesting data on correlations of C9orf72 expression with age: while in controls C9orf72 mRNA expression decreased with age, c9orf72 carriers had increased c9orf72 mRNA expression with age, whereas it was raised independently of age in sporadic ALS patients, suggesting a role for C9orf72 in sporadic ALS.
The final talk in the session was given by Michael Niblock from King’s College London. He analysed the possibility of intron retention in lymphoblast cells, iPS derived neurons and neuropathological tissue from frontal cortex of C9orf72 patients and controls. All introns were spliced out correctly in C9orf72 expansion carriers except intron 1. There was clear intron retention in lymphoblast cells of C9orf72 expansion carriers compared to controls and also some more subltle retention seen in the frontal cortex. When nuclear and cytoplasmic fractionation was carried out, most RNA with the retained intron was found in the nucleus (mature mRNAs were selected using a probe against the polyA tail), which is counterintuitive given the fact that dipeptide protein must be translated in the cytoplasm.
The four talks demonstrated the impressive depth and breadth of research into RNA dysregulation that is currently being done in the C9orf72 field. The presentations stimulated a lot of discussion and demonstrated how fast the field is moving ahead to determine the contribution of RNA foci and RNA dysregulation to the pathophysiology of the disease.