Disease associated mutations in inosine monophosphate dehydrogenase 1 (IMPDH1) cause aberrant steady state metabolism in zebrafish retina

Abstract

Purpose : It is unclear why dominant mutations in IMPDH1 cause photoreceptor degeneration. IMPDH1 is a rate limiting enzyme in the de novo purine biosynthesis pathway and is expressed abundantly in rod and cone photoreceptors. In vitro, some IMPDH1 mutants are hyperactive, suggesting that increased IMPDH1 activity in the retina could lead to photoreceptor degeneration.

Methods : We generated transgenic zebrafish lines with either wild type (WT) or mutated myc-tagged IMPDH1 expressed in cones (gnat2:IMPDH1a-X1-K238E, gnat2:IMPDH1a-X1-D226N, and gnat2:IMPDH1-X1). IMPDH1 expression levels were quantified with a custom-made IMPDH1 antibody and normalized to actin. To track cone degeneration, transgenic lines were crossed with a cone marker line (gnat2:EGFP) and nuclei were visualized with Hoechst. To visualize IMPDH1 filaments, we performed immunocytochemistry and/or expansion microscopy using IMPDH1 antibodies. For metabolomics, whole eyes or isolated retinas were collected prior to degeneration. Extracted metabolites were analyzed by liquid chromatography/mass spectrometry.

Results : Transgenic IMPDH1 expression ranged from 1.5 – 3.0x higher than endogenous WT IMPDH1. The K238E mutant shows signs of degeneration (~50%) by 2.5 months whereas the D226N mutant shows early signs of degeneration by 2 years. Zebrafish with mutated IMPDH1 have abnormally large filaments concentrated in cone synapses (and nucleus for D226N mutant) whereas in WT zebrafish smaller IMPDH1 filaments are distributed throughout the cell body and synapse. There are distinct differences in steady state metabolites for both mutants. There is significantly less adenosine and inosine with the K238E mutation as compared to WT (Adenosine: 51.94 ng/mg WT vs. 21.55 ng/mg K238E p = 0.03; Inosine: 1104 ng/mg WT vs. 712.1 ng/mg K238E p =0.03). D226N has increased cytosine (1.71:1 mutant:wild type p=0.02). cGMP levels were not significantly different for either mutant. Transgenically expressed WT IMPDH1 shows no abnormalities in purine or pyrimidine biosynthesis. Metabolic flux experiments are in progress.

Conclusions : Mutations in IMPDH1 cause cone degeneration in zebrafish. Mutated IMPDH1 filaments are larger and mislocalized as compared to WT filaments. IMPDH1 filaments may be interacting with other metabolic proteins as mutants have decreased purine and/or pyrimidine steady state metabolites.

This abstract was presented at the 2024 ARVO Annual Meeting, held in Seattle, WA, May 5-9, 2024.