The role of ALS-related proteins in different cellular contexts

Author(s)

Amanda Faria Assoni

• Document type: Doctoral Thesis
• Press: São Paulo.
• Institution: Universidade de São Paulo (USP). Instituto de Biociências (IBIOC/SB)
• Defense date: 2022-05-19

Examining board members

Oswaldo Keith Okamoto; Miguel Mitne Neto; Ellen Nollen; Mayana Zatz

• Advisor: Oswaldo Keith Okamoto

Abstract

Amyotrophic Lateral Sclerosis (ALS) is a neurodegenerative disease for which there is currently no effective treatment. Therefore, it is of great importance to further investigate the mechanisms leading to motor neuron death to find new potential therapeutic targets. In this thesis, we deepen our understanding of 3 proteins involved in the pathogenesis of ALS (Fused in Sarcoma -FUS, Vesicle-associated Membrane Protein B -VAPB, and the ephrin receptor A4-EPHA4) by analyzing their effects when mutated, absent, or when their activity is inhibited. In addition, there is evidence for overlapping mechanisms in neurodegeneration and cell transformation. Therefore, we further investigated these ALS -related proteins in different cellular contexts (neurodegeneration and cancer). To this end, we used the cellular models of iPSCs from ALS type 6 patients as well as a central nervous system tumor, medulloblastoma. Herein, we describe that protein translation is one of the first and most important pathways affected by ALS -related mutations. Indeed, all mechanisms that are disrupted in ALS -cells ultimately lead to downregulation of protein synthesis rates. Consistent with the literature, our results show that protein synthesis rates are decreased in iPSC-derived motor neurons from ALS6 patients, and this correlates with the presence of the generally nuclear localized FUS protein in the cytoplasm. We also show that interferon-gamma (IFN-y) - a multifunctional cytokine that, among other things, aids in antiviral response - upregulates translation-associated genes specifically in ALS MNs when these iPSC-derived motor neurons from ALS patients are treated in the presence of oxidative stress, and that the cytoplasmic localization of FUS is reduced in ALS6 MNs after IFN-y treatment. This treatment prevents apoptosis of ALS6 MNs. Therefore, decreased protein synthesis may be a hallmark of ALS pathogenesis, and increasing translation with IFN-y is a potential treatment for these patients. Regarding the function of ALS associated proteins in medulloblastoma, we reveal novel roles for VAPB and EPHA4 in tumor progression. We find that VAPB - which has lower mRNA levels in the cerebrospinal fluid of sporadic ALS cases - correlates with lower overall patient survival when expressed at higher levels in medulloblastoma. Moreover, VAPB knockout arrests cells in G1/0 and alters transcript levels of many WNT-related proteins, including CTNNB1. We also show that downregulation of EPHA4 appears to be beneficial for cell proliferation in medulloblastoma. Furthermore, we found that VAPB binds to EPHA4 in nontransformed neuronal tissues but not in medulloblastoma cells. However, removal of VAPB in medulloblastoma increased EPHA4 phosphorylation, whereas inhibition of EPHA4 phosphorylation increased the cycling of VAPB-KO cells, highlighting the interplay between different signaling pathways. In summary, here we provide evidence to support the hypothesis that neurodegeneration and tumorigenesis are the result of the same deregulated signaling pathways, albeit in different directions. Therefore, it is of utmost importance to connect knowledge of cancer research and neurodegenerative diseases. This will not only lead to a better understanding of these devastating diseases, but could generate new intervention strategies to improve the lives of patients in the future. (AU)

• FAPESP's process: 16/09707-8 - Interaction between the protein VAPB and the development of embryonic tumors on the central nervous system
• Grantee: Amanda Faria Assoni
• Support Opportunities: Scholarships in Brazil - Doctorate