ALS-RELATED FUS PROTEIN IS MISLOCALIZED TO CYTOPLASM AND RECRUITED INTO STRESS GRANULES IN FIBROBLASTS OF ASYMPTOMATIC FUS P525L MUTATION CARRIERS

Abstract

Symptoms onset in Amyotrophic Lateral Sclerosis (ALS) occur when over 70% of motor neurons is already lost, suggesting a relatively long pre-symptomatic phase. The description of several genes linked to ALS (e.g., SOD1, FUS, TARDP, C9orf72) has now allowed identification of pre-symptomatic carriers. These pre-symptomatic (or even preclinical) carriers can be followed up with the aim to identify the very early clinical disease-related changes or a valuable biomarker. These efforts seem at present the best approach for the implementation of an early symptomatic therapy or for the disease prevention. In this work, we studied the expression of FUS protein in cultured skin fibroblasts from pre-symptomatic FUS P525L mutation carriers. We cultured skin fibroblasts from two sisters belonging to an ALS family with FUS P525L mutation and carrying the same mutation, one healthy control and two patients with sporadic ALS (sALS), with no identified gene mutations. The two carriers were clinically asymptomatic, while the two patients fulfilled the El-Escorial and Awaji criteria for definite ALS. Western blot and immunocytochemistry were performed with specific antibodies to study the expression and subcellular localization of FUS protein in the skin fibroblasts. In sporadic ALS, FUS protein showed an almost exclusive nuclear localization. In the healthy control, FUS was also mostly nuclear, with some cells showing a faint cytoplasmic expression. In the FUS P525L mutation carriers, the protein was strongly expressed in both nucleus and cytoplasm in most cells, with a relatively high proportion of cells showing an exclusive cytoplasmic FUS localization. Furthermore, either heat-shock or dithiothreitol were applied to assess the effect of stress on FUS expression and cytoplasmic redistribution. Stress induced cytoplasmic granules formation in all subjects, and FUS was recruited into them. After a single stress exposure, we observed in all subjects a time-dependent decrease of cells containing granules. However, granules persisted longer in fibroblasts from the two FUS P525L carriers, where the number of granules per cell was found to be higher than HC and sALS, suggesting that these granules may accumulate during chronic stress and thus be the precursors of the pathological FUS inclusions. These data might represent an early molecular change occurring before ALS onset, suggesting a transient pre-aggregative state