The interplay between the “rna/protein quality control system” and “exosomes” as a spreading mechanism in amyotrophic lateral sclerosis (EX_ALS)

Amyotrophic lateral sclerosis (ALS) is a fatal motoneuron disease characterized by loss of upper and lower motoneurons, which includes clinically indistinguishable sporadic (sALS) and familial (fALS) forms. Affected motoneurons and their neighbouring cells contain protein aggregates, which may interfere with several cell functions. Intriguingly, most of the proteins encoded by ALS genes (SOD1, TDP-43 and FUS) may aberrantly behave in their wild type forms also in sALS, indicating that common toxic mechanisms exist. Indeed, in most sALS, a nucleus to cytoplasm TDP-43 mislocalization occurs, leading to the formation of TDP-43 aggregates that sequester other ALS-proteins (e.g. SQSTM1/p62, TIA-1, optineurin, ubiquilin 2). When mutated in fALS, these proteins misfold becoming highly aggregation prone, including ALS-linked C9orf72 dipeptide repeats (DPRs). Aggregating ALS proteins and DPRs induce proteotoxic and oxidative stress. In addition, excitotoxic phenomena and hypoxia may enhance ALS-protein aggregation, perturbing neuronal homeostasis. Besides misfolding, also deregulation of RNA homeostasis is found in ALS. The ALS-proteins FUS, TIA-1 and TDP-43 regulate RNA metabolism and the dynamic of liquid-like stress granules (SGs). The ALS-proteins promote an aberrant transition of SGs into aggregates, which can co-aggregate with misfolded proteins that escape the protein quality control (PQC) system. Altered SG dynamics and RNA interactions perturb RNA engagement into polysomes, as well as translation rate. Recent findings support that loss of RNA and protein homeostasis are interconnected, and represent key events in ALS progression. ALS affects not only motoneurons, but also other neuronal, glial and muscle cells, whose dysfunctions all contribute to disease. The rapid disease progression strongly suggests that cell-to-cell spreading of noxious factors could take place in ALS pathogenesis. Extracellular vesicles (EVs; exosomes, EXOs and microvesicles, MVs) could potentially spread the disease. EVs contain misfolded ALS-proteins, PQC proteins, and RNA species, which might be carried to recipient cells, conveying cytotoxic components and causing a stress response in targeted cells. So far, no direct evidence exists showing that EVs released from ALS cells subjected to stressful conditions transfer potentially toxic products to recipient cells, ultimately altering their function and viability. Here, we will test the hypotheses that: 1) environmental, proteotoxic and excitotoxic stress influence the amount and composition of EVs released by ALS cells; 2) once uptaken by recipient cells, the proteins and RNA species, transferred by EVs, can elicit a stressful response, leading to an imbalance of protein and RNA homeostasis. We will take advantage of the complementary expertise and long-standing successful collaborations between consortium units to: 1: study how altered intracellular RNA and protein handling impacts on the degradative pathways and modulates EV production and disease spreading. This will be achieved by quantifying the effects of different cell stressors, including excitotoxicity on the RNA-binding of ALS-proteins, RNA/RBP redistribution, misfolded protein dynamics, autophagy and proteasome modulation and how these ultimately affect EV release. These data will be validated with animal and human derived EV preparations. 2: study the physiopathological role of EVs, focusing on the characterization of the content of EVs according to their cell source and type of stress. The isolation of pure and polydisperse EVs will allow RNA content analysis and correlation of changes of RNA, ALS-proteins and PQC system components in EVs. We will measure the energy metabolism associated to EVs and evaluate the potential chaperone effect of proteins and RNAs isolated from EVs at the single-molecule level, with optical tweezers. 3: test if released EVs mediate cytotoxicity and induce aberrant RNA/protein aggregat