RNA Processing at the MNDA Symposium on ALS/MND

Around the time I first started working in Sheffield in 2008 it was discovered that an RNA processing protein called TDP-43 is a key component of brain pathology in the majority of motor neuron disease (MND) / amyotrophic lateral sclerosis (ALS).  Following that finding the importance of RNA processing to our understanding of the disease pathogenesis has grown and grown.  This has only increased with the discovery of an intronic GGGGCC-repeat expansion in C9orf72 in 10% of all ALS patients; it appears that this mutation is intimately tied up with basic RNA biology in the cell.  So to me it seemed that the RNA processing session at the MNDA Symposium was likely to be very exciting and right at the cutting edge of our disease understanding.  I was right.

The first talk in the RNA Processing session came from Laura Ranum from the University of Florida.  Professor Ranum has been at the forefront of research into neuromuscular disease resulting from repeat expansion mutations for a number of years and hence in recent times she has turned her attention to C9orf72.  Her group was key to discoveries in spinocerebellar ataxia 8 and myotonic dystrophy linking these disorders directly to defects in RNA splicing.  They defined several novel disease mechanisms including bidirectional transcription of the disease linked repeat expansion and the translation of the repeat-RNA into protein despite the absence of a traditional ATG initiation site.  A series of seminal experiments demonstrated that translation occurs in all possible reading frames in a manner dependent on repeat length and that the key to initiation of translation was hairpin secondary structure in the RNA-repeat molecules.  Turning her attention to C9orf72 mutations, Professor Ranum described the detection of sense and antisense transcripts derived from the C9orf72 repeat expansion and the subsequent translation of both of these transcripts into five different dipeptide repeat proteins.  In discussing the relative toxicity of the RNA and protein molecules Professor Ranum highlighted recent work which has shown the proteins are toxic in cell models independent of the RNA, but she urged caution, highlighting the fact that thus far this has only been shown in overexpression models which may not be representative of the in-vivo biology.  Finally Professor Ranum described early work on characterisation of a mouse model of C9orf72 disease.  Mice have been generated using a bacterial artificial chromosome (BAC) containing a mutated human gene.  Lines have been generated to express the entire human C9orf72 gene including an expansion of approximately 500 GGGGCC-repeats.  This is significantly shorter than the >2000 repeats found in many patients but it appears that the expansion tends to collapse during the manipulation necessary to generate the BAC model.  Despite this the mice exhibit a number of pathological features associated with C9orf72-disease including the bidirectional transcription and repeat-associated non-ATG translation described above.  As yet the mice only have limited symptoms but this research is progressing and will potentially yield key insights as we seek to treat patients with and without C9orf72 mutations.

Dr Johnathan Cooper-Knock, MND Association/MRC Lady Edith Wolfson Clinical Fellow blogs about the symposium via our peer-to-peer blog

Next I talked about work in Sheffield comparing and contrasting RNA foci derived from sense and antisense transcription of the C9orf72 repeat expansion in patient tissue.  We have shown that in motor neurons, the primary cell of ALS, antisense (but not sense) RNA foci are significantly associated with nuclear loss of TDP-43, a key step in the vast majority of ALS.  As a result we investigated the difference between these species of RNA foci and showed that they actually had very similar binding partners including many proteins key to RNA processing such as SC-35 and ALYREF.  Given that their interactions appeared similar we took a step backwards and asked whether the two RNA species might be produced and preserved differently in different cells.  This was indeed the case and we demonstrated that motor neurons exhibit antisense RNA foci at a significantly higher frequency than sense RNA foci whereas other neuronal populations in the cerebellum do the opposite.  Following this we showed that dipeptide repeat proteins derived from antisense RNA transcripts are also present at a much higher rate in motor neurons than sense RNA derived proteins.  It appears the antisense RNA molecules and their downstream consequences are likely to be key to future translational research in C9orf72-ALS.

The third talk came from Louis De Muynck from Leuven who talked about very careful quantification of C9orf72 mRNA transcripts in blood and CNS tissue from ALS patients with and without C9orf72 disease.  Louis’s approach is to be commended and his combination of standard qPCR techniques with cutting edge digital PCR technology will hopefully bring clarity to an area which has been confused with varying reports from different labs across the world.  In blood samples Louis showed that expression of C9orf72 mRNA reduces with age in normal controls but increases with age in C9orf72-patients; fascinatingly he showed that expression is elevated independent of age in ALS patients without C9orf72 mutations.  Moreover he showed that levels of C9orf72 mRNA or its derivative transcripts, were inversely correlated with survival in both C9orf72 and non-C9orf72 patients.  This is very interesting and supports a role for the C9orf72 protein in disease even when it is not mutated.  The discussion following this talk was very informative and a number of audience members highlighted the difficulty in quantifying transcripts when transcription initiation may be occurring at numerous abnormal start sites; the effect of the repeat expansion on the mix of C9orf72 mRNAs is likely to be complex as was already highlighted by Professor Ranum’s talk earlier in the session.

The final talk of the session came from Michael Niblock of King’s College London who had also been using quantitative PCR to examine the processing of C9orf72 mRNAs.  He demonstrated elevated levels of transcripts with a retained intron 1 (the intron containing the GGGGCC-repeat) in lymphoblastoid cell line, neural differentiated induced pluripotent stem cells (iPSCs), and CNS tissue derived from patients with expansions of C9orf72.  Thus he suggested that the intron is not being correctly processed and degraded in these patients. Furthermore he performed nuclear-cytoplasmic separation and suggested that the intron-containing transcripts were being maintained in the nucleus and not exported into the cytoplasm.  This has implications for other areas of C9orf72 research: thus far it has been presumed that the repeat-RNA must be exported to the cytoplasm to gain access to the translation machinery and produce dipeptide repeat proteins.  Michael was not excluding this but he is suggesting that exported mRNA may form a small proportion of the total.  The alternative is nuclear translation, perhaps a new area for ALS research?