Effect on cell survival and cytoophidium assembly of the adRP-10-related IMPDH1 missense mutation Asp226Asn

Abstract

Introduction: Inosine monophosphate dehydrogenase 1 (IMPDH1) is a critical enzyme in the retina, essential for the correct functioning of photoreceptor cells. Mutations in IMPDH1 have been linked to autosomal dominant retinitis pigmentosa subtype 10 (adRP-10), a genetic eye disorder. Some of these mutations such as the Asp226Asn (D226N) lead to the assembly of large filamentous structures termed cytoophidia. D226N also gives IMPDH1 resistance to feedback inhibition by GDP/GTP. This study aims to emulate the adRP-10 condition with a long-term expression of IMPDH1-D226N in vitro and explore cytoophidium assembly and cell survival. We also assessed whether the introduction of an additional mutation (Y12C) to disrupt the cytoophidium has an attenuating effect on the toxicity caused by the D226N mutation.Results: Expression of IMPDH1-D226N in HEp-2 cells resulted in cytoophidium assembly in ∼70% of the cells, but the presence of the Y12C mutation disrupted the filaments. Long-term cell survival was significantly affected by the presence of the D226N mutation, with a decrease of ∼40% in the cells expressing IMPDH1-D226N when compared to IMPDH1-WT; however, survival was significantly recovered in IMPDH1-Y12C/D226N, with only a ∼10% decrease when compared to IMPDH1-WT. On the other hand, the IMPDH1 expression level in the D226N-positive cells was <30% of that of the IMPDH1-WT-positive cells and only slightly higher in the Y12C/D226N, suggesting that although cell survival in Y12C/D226N was recovered, higher expression levels of the mutated IMPDH1 were not tolerated by the cells in the long term.Conclusion: The IMPDH1-D226N effect on photoreceptor cell survival may be the result of a sum of problems: nucleotide unbalance plus a toxic long-life cytoophidium, supported by the observation that by introducing Y12C in IMPDH1 the cytoophidium was disrupted and cell survival significantly recovered, but not the sensibility to GDP/GTP regulation since higher expression levels of IMPDH1-D226N were not tolerated.

Light-sensitive phosphorylation regulates enzyme activity and filament assembly of human IMPDH1 retinal splice variants

“…4 ). Since overexpression alone also causes IMPDH1 filament assembly (Gunter et al, 2008; Keppeke et al, 2018; Keppeke et al, 2023), we performed the same experiment without ribavirin treatment and observed similar results ( Supp. Fig.…”

“…These data suggest two important points: (1) human IMPDH1 and IMPDH2 co-assemble into the same filament (Keppeke et al, 2023) and (2) because IMPDH1(546)-S477D forms filaments in vitro , its inability to form filaments in cells suggests that IMPDH filaments in living cells predominantly assemble using the large interface. Together, these points imply that phosphorylation at S477 is a molecular mechanism for regulating flux through de novo GTP synthesis by controlling higher-order assembly of the pathway’s rate-limiting enzyme.…”

IMPDH2 filaments protect from neurodegeneration in AMPD2 deficiency

“…Some of these mutations impair the disassembly of IMPDH1 filaments upon guanine nucleotide supplementation in vitro. Consequently, these mutant IMDPH1 variants constitutively polymerize when overexpressed in cells (Fernandez-Justel et al, 2019;Keppeke et al, 2023). Furthermore, recent data shows that the long-term overexpression of a constitutively polymerizing IMPDH1 disease variant induces cell death in HEp-2 cells line.…”

Cytoophidia Influence Cell Cycle and Size in Schizosaccharomyces pombe

“…In Drosophila , cytoophidia are involved in optic lobe development and oogenesis; recent studies showed that cytoophidia take part in adipose architecture and metabolism, and loss of cytoophidia reduced adipocyte expansion and inhibition of lipogenesis [ 15 , 17 , 18 , 19 ]. In human cells, cytoophidia stabilize the CTPS protein by extending its half-life [ 20 , 21 ] and abnormal cytoophidia affects cell survival [ 22 ]. Cytoophidia participate in ovarian stress response in Drosophila [ 23 ] and respond to changes in nutrition, pH, temperature, and osmolality for stress adaptation in yeast [ 24 , 25 ].…”