Much of the symposium this year involved discussions on the disease associated proteins of MND. This session specifically focussed on the cellular biology and resultant pathology that arises as a consequence of the aberrant functioning of these proteins.The session started with an interesting talk by Dr Baralle who has uncovered the key sites on the TDP-43 protein required for RNA interactions, RM1 and RM2, and the regions responsible for aggregate formation, a Q/N rich region at the c-terminus. Additionally the group showed that TDP-43 is capable of regulating its own protein levels via a negative feedback loop through binding to the 3’UTR to trigger RNA degradation. Staying with the theme of TDP-43 interactions the second talk, given by Dr Darnescu identified futsch, a microtubule stabilising protein, as an RNA binding partner of TDP-43. The protein is also post-translationally regulated by TDP-43. Following interaction between the two proteins futsch is retained in the cell body losing its normal localisation at the neuromuscular junction which results in architectural damage. Overexpression of futsch in drosophila overcame locomotor defects and TDP-43 pathology.
In the third talk Janice Robertson gave an insightful presentation on the localisation of the two isoforms of the disease associated protein c9orf72, short (s) and long (l). Interestingly in patients with c9orf72 ALS there is a decrease in the long isoform but an increase in the short isoform in the temporal lobe and frontal cortex compared with sporadic ALS cases. Cellular localisation of the two isoforms also differed with the short isoform completely labelling the nuclear membrane and the long isoform showing a speckled labelling in both the cell body and neuronal projections in sporadic ALS. In contrast in c9+ ALS patients there is complete loss of membrane staining of the short isoform and loss to a varied degree of the long isoform staining.
Continuing the exploration into c9orf72 Steven Boeynaems presented data to show that c9orf72 knockdown in zebrafish brings on a phenotype similar to that seen with SOD1 and TDP-43 mutations. Injection of high repeat numbers of c9orf72 sense RNA into zebrafish also induces a toxic phenotype, in contrast to the antisense RNA which wasn’t toxic. Further investigation uncovered the ability of the sense RNA to form a quadruplex secondary structure, whereas the antisense forms a hairpin like structure, therefore it was hypothesised that the quadruplex secondary structure of c9orf72 sense RNA may be essential in c9 toxicity.
Professor Vladimir Buchaman presented data examining the propensity of FUS variants to form inclusions and the cellular conditions in which this occurs. The mutations in FUS appear most often in the nuclear localisation sequence resulting in cytoplasmic confinement of the protein which increases the likelihood of pathological aggregation. A novel RNA dependent aggregation of FUS into granules that structurally mimic yet physiologically differ from regular stress granules has been observed. Through the inhibition of transcription and translation, i.e. in the absence of newly synthesised RNA, these granules fall apart. However this leaves a localised high concentration of FUS protein which can in turn form secondary aggregates.
In the final talk of this session, by Dr Bento-Abreu, we were introduced to ELP3, a member of the Elongator complex involved in regulation of transcription. The disease-associated protein is capable of preventing the pathogenic phenotype of the SOD1 A4V zebrafish, which exhibit decreased axonal length and branching. Overexpression of the protein is also capable of delaying the onset and increasing survival in SOD1 G93A mice when delivered directly to the lumbar spinal cord and when general expression of the protein was induced postnatally. However SOD1G93A x ELP3+/- mice become symptomatic earlier than the SOD1G93A mice and ELP3 knockdown in zebrafish induces axonopathy.
From this session it is clear that understanding the normal and aberrant functioning of disease associated proteins and their pathogenic mechanisms is vital for identifying potential therapeutic targets in ALS to modify disease processes.