Protein structure: better to stay in shape!

The 24th International Symposium has just ended and I’m starting to rearrange all my notes to write a report regarding the last scientific session of the meeting (10A) entitled Protein Processing and Degradation.

Like the rest of the meeting, this session was very busy and international, with speakers coming from many parts of the World, such as Europe and USA, Canada and Australia.

But let’s start talking about the main characters of the session: proteins.

As we all know, proteins are involved in many functions inside the cells. But to work well, it is essential that they have a well-defined structure. If their conformation is altered (i.e. they are misfolded) this could lead to problems in their functionality that could translate into a pathology.

This session was devoted to the study of alterations in different proteins (like SOD1, TDP43 and c9orf72) whose mutated forms have been linked to ALS. The speakers showed how misfolded proteins and aggregate formation are a hallmark of this pathology.

Let’s start from the beginning.

The “prionic hypothesis” of ALS spreading characterized many of the talks of this session. This mechanism was beautifully introduced and explained by the first speaker, Dr. Aguzzi (University Hospital Zurich, Switzerland).  Prion diseases are a class of pathologies like Creutzfeldt-Jakob and Scrapie where a misfolded protein alters the conformation of its normal counterpart in a domino reaction, leading to the formation of a huge amount of altered proteins that ultimately form aggregates.

As in ALS different brain and spinal cord regions are progressively affected, the question is whether the spreading of this disease could be explained by a prion-like mechanism.

Dr. Cashman’s (University of British Columbia, Canada) presentation provided evidence that mutant (and misfolded) SOD1 could alter the structure of its wild type counterpart. Even though the clinical cases of ALS due to mutations in the SOD1 gene are few, an interesting emerging finding is that aggregated SOD1 is also observed in the spinal cord of sporadic cases. Therefore, investigating the alteration of this protein can have broad implications for the pathogenesis of both familial and sporadic disease. In his experiments, Dr. Cashman injected the mutant form G127X in the primary motor cortex of mice overexpressing normal SOD1 and found that the wild type protein became misfolded, too. Moreover, he demonstrated that a single aminoacide residue (the tryptophan in position 32) was necessary for this conversion, acting really as a “prion domain”.

Dr. Tokuda’s (Umea University, Sweden) talk was also focused on the interaction between mutant and wild type SOD1 in the spinal cord of transgenic mice co-expressing the two isoforms. In this particular model, he showed an exacerbation of the disease in comparison to mice overexpressing only the G127X. The wild type SOD1 is aggregated to an extent similar to that of the mutant protein, and the same observation was obtained also in spinal cord of patients carrying the same mutation.

Now, let’s take a step ahead: are there any experimental evidences that the progression of ALS could be due to a prion-like spreading of misfolded proteins in human patients?

Dr. Brettschneider’s (University of Pennsylvania, USA) observations on the widespread distribution of phosphorylated and aggregated TDP-43 in ALS autoptic samples suggest that this may be indeed the case. What he saw was a progressive accumulation of the inclusions in different brain regions, according to which he classified different stages of TDP-43 pathology: 1- motor cortex, 2- initial involvement of prefrontal cortex, brainstem reticular formation, precerebellar nuclei and red nucleus, 3- prefrontal and postcentral neocortex and striatum, 4- portion of the temporal lobe, hippocampus. Beside that, he pointed our attention to the apparent frontal propagation of the aggregates, thus establishing a possible link with cognitive symptoms observed in ALS-FTD pathology. Based on the observations that TDP pathology was present in regions highly connected by axonal projections and that instead some areas close to inclusions were aggregates-free, he also suggested that the dissemination travels through axonal transport rather than cell-cell propagation.

From large to small, Dr. Ng (University of Cambridge, UK) showed us what happens to TDP-43 inside a single cell. To visualize it, she tagged both the full-length protein and the C-terminal fragment, previously demonstrated to be prone to aggregation, with fluorescent arsenical dyes. Once transfected into the cell, at first both proteins localize correctly into the nucleus, but after 48-72 hours, they translocate and aggregate into the cytoplasm. This model could prove useful to study the dynamics of the interaction of TDP-43 with other cellular proteins.

When talking about protein processing and degradation, a topic that can’t be overlooked is protein stability. In fact, instability and consequent misfolding can be induced by both mutations and/or posttranslational modifications. Dr. Wright (University of Liverpool, UK) described an example of both these processes. First, he analyzed mutations in the RRM (RNA-recognition motif) region of TDP-43 that decrease the turnover of the protein, increase its stability and make it resistant to aggregation. Second, he described how a mutation in hCCS (human Copper Chaperon for SOD1) recapitulates the SOD1 pathology. This is a chaperon protein involved in the maturation and posttranslational modification of SOD1. The mutation R163W of hCCS makes it more prone to aggregation, unstable and unable to bind SOD1.  This data points to an indirect mechanism through which the functionality of SOD1 is affected.

The last talk of the session was from Dr. Farg (La Trobe University, Australia), who brought our attention on c9orf72. Only two years ago a 6-nucleotide expansion on this gene has been identified as a main cause of both sporadic and familial ALS. Different pathogenic mechanisms have been proposed, including toxic RNA production from the expanded region and aberrant protein production and aggregation, but so far its function in cells has not been cleared. The work discussed by Dr. Farg shows evidences that c9orf72 is involved in endocytosis and autophagy-related endosomal trafficking. These were not the only findings: c9orf72 also interacts with ubiquilin2 and the ribonuclear proteins hnRNPA1 and hnRNPA2/B1, which have also been linked to ALS, therefore suggesting its possible role in protein degradation and RNA processing.

In conclusion, what is the take home message of this session?

The structure of a protein is crucial not only for its own functionality, but also for the well being of the others surrounding it. Having a correct and healthy protein set is essential for cellular homeostasis.  Moreover, the theory of the prionic spreading of ALS that dominated this session is becoming a more and more relevant hypothesis in the pathogenesis of this disease.