Although the underlying cause of sALS remains a mystery, there is a rapidly growing list of genes in which mutations have been implicated in ALS (fALS; accounting for 5-10% of all ALS cases). A subset of these genes have been associated with either protein aggregates found in motor neurones (e.g. SOD1, TDP-43, FUS, VAPB), protein degradation machinery (e.g. UBQLN2, VCP, SQSTM1) or both. Although we cannot yet definitively say whether protein aggregation is a cause or consequence of ALS pathology, their presence provides strong indications that the topic of this session protein processing and degradation – otherwise known as protein homeostasis – is disrupted.
The term protein homeostasis or proteostasis refers to the maintenance of the proteome as a set of individual proteins in a conformation, concentration and in a location that is required for their correct function. Regardless of the energetically expensive attempts of the cell to maintain proteostasis, burdens such as mutations that cause mis-splicing, inhibition of protein transport, or blockages to degradation machinery may overwhelm the cells proteostasis machinery and allow protein accumulation and aggregation to proceed. Work presented in this session by Manal Farg, La Trobe University Australia, suggests that c9orf72 protein contributes to protein degradation pathways, specifically autophagy related endosomal trafficking. In addition, knock down of c9orf72 protein, as occurs in c9orf72 ALS, impaired autophagy. An impairment in pathways such as these may lead to a diverse range of consequences from increased degradation of a protein(s), thus loss of function at one end of the scale, to overproduction or stifled degradation resulting in protein aggregation and inclusion formation at the other.
Another mechanism underpinning dysfunctional protein homeostasis is the direct effect of mutation on protein stability. Work presented by Dr Gareth Wright, University of Liverpool UK, suggests that mutations in ALS associated proteins such as SOD1 and TDP-43 affect their half-life. Dr Wright reported that without proper post-translational modifications, such as those promoted by interactions with its copper chaperone hCCS, SOD1 is structurally compromised. In support of this idea Dr Eiichi Tokuda presented work to show that SOD1 mutation promotes instability and may shorten half-life – certainly the while expression was similar mutant SOD1 concentration is much lower than that of the overexpressed human wtSOD1. On the other hand Dr Wright showed that mutations in TDP-43 increase the stability of the molecule, thus extending its half-life.
One significant consequence of protein misfolding is that it can promote further protein misfolding and aggregation by acting as a seed. This concept of seeding and propagation of protein misfolding is what is thought to drive transmission of prion protein in Creutzfeldt-Jakob disease and other prion related disorders as related by Prof Adriano Aguzzi, University hospital Zurich, Switzerland, in the plenary seminar. There is a growing list of neurodegenerative diseases where evidence of cell-to-cell transmission of protein misfolding is emerging. Indeed, progression of ALS pathology, beginning focally and its contiguous spread is consistent with a prion like transmission. Evidence for TDP-43 pathological gradation was presented by Dr Johannes BrettSchneider, University of Ulm, Germany. Data was presented that suggested that there may be a sequential and progressive pattern of pathology in humans that moves through i) motor neurons ii) brainstem reticular formation, precerebellar nuclei and the red nucleus, iii) prefrontal, postcentral neocortex and striatum, and iv) temporal lobe, including the hippocampus. The observed spread is consistent with propagation along axonal pathways more than a non-specific cell-to-cell transmission.
Evidence that SOD1 can act in a prion-like manner, or as a “propagon” a term introduced in Prof Aguzzi’s talk, comes from work presented by Dr Eiichi Tokuda from Umea University, Sweden. Dr Tokuda presented work demonstrating that overexpression of wtSOD1 can hasten disease in G127X mutant SOD1 mice. The expression of wtSOD1 accelerated SOD1 aggregate formation, and wtSOD1 was found in aggregates at similar levels to the mutant. These results are likely due to both an increased burden on the protein homeostasis machinery in the cell and the propagation of misfolding from G127X to wtSOD1. Dr Tokuda also presented work to show wtSOD1 can be found aggregated in human post mortem tissue of G127X mutation carriers. Further evidence that misfolded SOD1 comes from the work of Dr Neil Cashman from the University of British Columbia in Canada. Prof Cashman presented in vitro experiments clarifying the role of wtSOD1 in a prion-like propagation of misfolding. In addition, when recombinantly G127X SOD1 or the propagation resistant double mutant G127X/W32S was injected in to the motor cortex of human wtSOD1 expressing mice misfolded SOD1 was measured after injection of G127X but not the control cases. This provides evidence that misfolded SOD1 can enter cells and promote further misfolding in the recipient cells in vivo.
While the data presented above is evidence that propagation of misfolding can occur in human ALS, it is possible that propagation of misfolding may be uncoupled from cell death. Prof Aguzzi presented data to suggest just that. While all molecules that stop propagation of misfolding stopped toxicity in vitro in slice cultures. Data presented suggests that inhibition of calpain or prevention of ROS formation can prevent cell death but allow prion propagation, suggesting the two can be uncoupled. Prof Aguzzi then presented work that demonstrated that antibody binding to Prion protein on the surface of cells is toxic – he proposed that the binding of the antibody, and by extension the PrPSc, alters the PrPC conformation such that the flexible tail inserts in to the membrane triggering toxicity in the neuron.
In summary, it is becoming clear that protein misfolding is a major part of ALS pathogenesis. Indeed, while dysfunction of mRNA metabolism and/or protein degradation may lead to misfolding, accumulation and aggregation of various proteins within a cell, protein misfolding clearly may be transmitted to either nearby neurons, non-neuronal cells, or those cells in the axon pathways. One may conclude then that therapeutic strategies such as misfolded protein antibodies may be useful in slowing or even stopping ALS. To do so we will need new models, such as those presented by Janice Ng, University of Cambridge UK, to understand the precise molecular mechanism(s) of propagation of misfolding inside cells.