Determination of readthrough susceptibility among different Schwann cell lines carrying nonsense mutations

Authors

• Trushali Vansadia, University of Alabama at Birmingham

Advisor(s)

David Bedwell
David Bedwell

Committee Member(s)

Deeann Wallis
Kim Keeling

School

Joint Health Sciences (Interdisciplinary)

Document Type

Thesis

Department (new version)

Joint Health Sciences

Date of Award

9-9-2024

Abstract

Neurofibromatosis Type 1 (NF1) is a genetic disorder that affects the nervous system through the formation of tumors or neurofibromas. Encoded by the NF1 gene, neurofibromin functions as a tumor suppressor by converting active GTP-Ras to inactive GDP-Ras. 20% of NF1 patients carry a genomic nonsense mutation, which generates a premature termination codon (PTC) in the NF1 mRNA, leading to the formation of a truncated neurofibromin protein. Termination at PTCs can be suppressed by the incorporation of an amino acid at the site of the PTC, also known as readthrough. Readthrough results in continued translation elongation downstream of the PTC in the correct ribosomal reading frame, allowing the rescue of a full-length protein with full or partial function. To restore neurofibromin function, nonsense suppression therapy, which uses small molecules that target translation, can promote readthrough at in-frame PTCs. While readthrough occurs naturally at low levels (0.1%), it can be greatly amplified using small molecules such as aminoglycosides and other compounds that lead to depletion of the termination factor eRF1. This approach is yet to be explored in NF1 patients carrying these mutations. We hypothesize that readthrough compounds such as the eRF1 degrader SRI-41315, can suppress PTCs in NF1 patient-derived Schwann cell lines and rescue neurofibromin expression and function. The purpose of this study is to 1) characterize different NF1 patient-derived Schwann cells; 2) investigate the effect of small molecule eRF1 degrader SRI-41315 on PTC readthrough in different patient-derived Schwann cell lines. In these patient-derived Schwann cell lines, we anticipate a restoration of neurofibromin expression, which will be assessed by western blotting. We also anticipate the rescue of neurofibromin function, which will be assessed by phosphorylated p-ERK/ERK levels using western blotting. Rescue of neurofibromin function is expected to decrease phosphorylated ERK (p-ERK), which is a downstream effector of the Ras pathway that is negatively controlled by neurofibromin.

ProQuest ID

https://proquest.com/docview/3101841804