A Screening Assay for Myelin Protein Zero Mutants: Unfolded Protein Response Activation and Its Significant Reduction by Curcumin Treatment (IN1-1.001)

Abstract

Objective: To develop an in vitro assay to identify CMT mutations causing ER stress and activation of the unfolded protein response (UPR). Background Multiple mutations in Myelin Protein Zero (MPZ) and Peripheral Myelin Protein 22 (PMP22) result in protein misfolding and retention of the mutant protein in the endoplasmic reticulum (ER). The overload of misfolded proteins activates the Unfolded Protein Response (UPR) that contributes to the neuropathy. Medications exist that can reduce UPR activation. Design/Methods: We utilized transient transfection assays with mutant MPZ constructs and a GFP fused XBP1 construct in which XBP1 splicing shifts the frame and leads to the emission of green fluorescence. Cos7 cells were transiently cotransfected with wild type (wt) or mutant human MPZ and XBP1-GFP. Only CMT1B causing mutations that activated the UPR emitted green fluorescence in their nuclei. Results: Wt MPZ reached the cells surface and activated XBP1 splicing only to a minimal extent (10%) due to protein overexpression. Mutants known to activate the UPR in mice (R98C p=0,004, S63del p=0,026) activated XBP1 splicing to a significantly higher extent (about 25%) than wt MPZ. XBP-1 activation by these mutants was reduced significantly (8%) with curcumin treatment. However, the protein remained retained in the ER. Two or 10 µM curcumin treatment had an equally beneficial effect. Other mutants (S63C, R227S) reached the cell surface and did not activate XBP1 splicing to a higher extent than wt. Though the T124M mutant reached the cell surface it activated the UPR significantly (25%, p Conclusions: UPR activation and cell surface expression are not mutually exclusive This cell culture assay may prove to be a fast and simple screening method for identifying mutations that may activate the UPR and thus selecting patients who might benefit from curcumin or similar treatments. Supported by: Muscular Dystrophy Association. NINDS: R01 NS41319A. Disclosure: Dr. Patzko has nothing to disclose. Dr. Kamholz has nothing to disclose. Dr. Sohi has nothing to disclose. Dr. Tinelli has nothing to disclose. Dr. saveri has nothing to disclose. Dr. Vizzuso has nothing to disclose. Dr. Feltri has nothing to disclose. Dr. Wrabetz has nothing to disclose. Dr. Shy has nothing to disclose.