The maintenance of protein homeostasis is vital for all cells, but it is of utmost importance in post-mitotic cells, such as neurons that cannot dilute aggregates by cell division. Dysregulation of the proteostasis network can lead to neurodegenerative disorders such as Parkinson’s disease (PD), Alzheimer’s disease, Huntington’s disease, Amyotrophic Lateral Sclerosis (ALS), and prion diseases. The small molecule Sephin1 is a promising lead against proteostasis disruption, but its mechanism of action is uncertain. We assessed the therapeutic efficacy of Sephin1 in an established PD mouse model. Our laboratory has recently characterized a mouse expressing via bacterial artificial chromosome (BAC) the human LRRK2 G2019S protein, a variant linked to PD. Our data show that Sephin1 treatment rescues the motor deficit observed in BAC human-G2019S mice. Our experimental evidence shows that Sephin1 binds the monomeric globular actin (G-actin) in cell-free assays. By combining PAL chemistry to MS/MS analysis we identified the putative Sephin1 binding site on actin. In vitro, Sephin1 drives actin misfolding, and eventually, its precipitation. Upon Sephin1 treatment in HeLa cells, we visualized actin clusters localized to the lysosomes. This event at the lysosome impairs the normal autophagic flux. At the same time, Sephin1 induces the inactivation of the mammalian target of rapamycin (mTORC1), thus allowing the nuclear translocation of the transcription Factor EB (TFEB) and the expression of TFEB-direct target genes, on the longer term. In parallel, Sephin1 elicits the phosphorylation of the α subunit of the Eukaryotic Initiation Factor 2 (eIF2) and the ER-stress independent expression of the C/EBP homologous protein (CHOP). CHOP is a transcription factor that contributes to the integrated stress response as well as to autophagy. As such, Sephin1 triggers the activation of two main players in the autophagic response, TFEB and CHOP. Accordingly, we reported that, after the initial impairment, Sephin1 stimulates autophagy. Taken together, our results reveal a novel Sephin1 molecular mechanism in which lysosomal stress may regulate autophagy via mTORC1-TFEB complemented with the eIF2α signalling pathway. Although several questions remain to be answered, Sephin1, which successfully completed the phase I clinical trial for ALS and Charcot–Marie–Tooth disease, represents a promising therapeutic strategy that targets autophagy to regulate the homeostatic balance of proteins in neurodegenerative diseases.
| Identifer | oai:union.ndltd.org:unitn.it/oai:iris.unitn.it:11572/363904 |
| Date | 17 January 2023 |
| Creators | Frapporti, Giulia |
| Contributors | Frapporti, Giulia, Piccoli, Giovanni |
| Publisher | Università degli studi di Trento, place:TRENTO |
| Source Sets | Università di Trento |
| Language | English |
| Detected Language | English |
| Type | info:eu-repo/semantics/doctoralThesis |
| Rights | info:eu-repo/semantics/embargoedAccess |
| Relation | firstpage:1, lastpage:89, numberofpages:89 |
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