Alterazioni del trasporto nucleocitoplasmatico delle UsnRNPs nella patogenesi della Sclerosi Laterale Amiotrofica familiare

Amyotrophic lateral sclerosis (ALS) is a late-onset neurodegenerative disease characterizedby the selective death of both upper motor neurons in the motor cortex, and lower motorneurons in the brainstem and spinal cord (Azuma et al., 2018). Loss of these neurons causesgeneralized muscle weakness, spasticity and muscle atrophy, leading to progressiveparalysis until death caused by respiratory failure (Cozzolino et al., 2012; Taylor et al.,2016). About 90% of ALS cases are considered sporadic (sSLA), while the remaining 10%are familial (fSLA), although they present the same clinical phenotypes andneuropathological features.ALS is a multifactorial disease, determined by the alteration of several key processes forcell viability. Although the contribution of each of them to ALS pathogenesis has not beenfully clarified, the regulation of the integrated stress response (ISR), the nucleo-cytosolictransport of RNAs and proteins, as well as the regulation of alternative splicing seem to playa particularly relevant role. Recently, it has been shown that the activation of the ISRinduces the accumulation into stress granules (SGs) of important factors that are mediatorsof the molecular traffic between the nucleus and the cytosol, such as importin-?1, importin-?2 and exportin-1 (Zhang et al., 2018). This suggests the existence of a functionalconnection between the activation of the integrated stress response and the regulation ofnucleo-cytosolic trafficking of RNAs and proteins. Recently, we have observed in our labthat pharmacological activation of ISR in HeLa cells by sodium arsenite (NaAs) orthapsigargin (TG) treatment induces a strong decrease in the nuclear distribution of CajalBodies (CBs) and Gems. These are membrane-less organelles that assist the maturation andstorage of UsnRNPs (U-rich small nuclear ribonucleoproteins), the key constituents of thespliceosome machinery, and SMN complex, essential in the cytosolic assembly ofUsnRNPs. Overall, these data indicate that the altered localization and the reducedavailability of nucleo-cytosolic mediators may disturb the transport of UsnRNPs betweenthe nucleus and the cytosol, and that the modulation of this process may represent amechanism of regulation of gene expression under stress conditions. However, themechanisms regulating this process have not been investigated so far, nor it is clear whetherdys-regulation of such process might be involved in ALS pathogenesis.On this basis, the first aim of this study was to define the mechanism by which thepharmacological activation of the ISR regulates the dynamics of Cajal Bodies, and to verifywhether this has an effect on the regulation of alternative splicing. Further, the second aim of this study was to investigate whether the activationof the ISR induced by the expression of ALS proteins is functionally related to the observedreduction of nuclear CBs, and to verify whether the previously defined mechanism ofregulation of UsnRNPs trafficking via the importin-?1/SPN1 import machinery in responseto stress is impaired in ALS conditions.Results from our study indicate that:i. the activation of the ISR in response to stress induces changes in the regulation ofalternative splicing by modulating the nucleo-cytosolic trafficking of UsnRNPs that ismediated by the snurportin1/importin-?1 machinery;ii. in cells expressing mutated proteins associated with ALS, this regulatory mechanism isimpaired and mutant ALS proteins directly affect the nucleo-cytoplasmic transport ofUsnRNPs, independently from or downstream ISR activation.