Zinc-metallopeptidase Lmln deficiency induces subretinal microglia activation and retinal neurodegeneration

Abstract

Purpose : Our goal is to identify and study genes essential to retinal development and homeostasis using an unbiased forward genetics approach. Here we characterize a novel association involving a gene coding for zinc-metallopeptidase Leishmanolysin Like Peptidase (Lmln).

Methods : We used a forward genetics screening pipeline (N-ethyl-N- nitrosourea mutagenesis in C57BL/6J mice) to identify in an unbiased manner a non-redundant gene leading to changes in retinal optical coherence tomography (OCT). Targeting this gene using CRISPR/Cas9 technology is allowing us to further characterize the resulting phenotype.

Results : The nemeth phenotype was identified in 3 mice homozygous for a mutation in zinc-metallopeptidase Lmln. All 3 mice showed approximately 20% thinning of the outer nuclear layer (ONL; p = 8.1 x 10^-12). A CRISPR-generated Lmln knockout (KO) mouse line was generated, and Western blot confirmed absence of the Lmln protein product. ONL loss was recapitulated in the CRISPR KO line with approximately 21% thinning (p = 7 x 10^-22) at 6 months of age. This was accompanied by accumulation of retinal fundus spots in an age-dependent manner. Furthermore, RPE and retina flat mount staining showed accumulation of Iba1⁺/Gal3⁺ activated subretinal microglia. Electrophysiology demonstrated retinal functional deterioration. We are now doing electron microscopy studies to better understand specific ultrastructural aspects of the neurodegeneration we are observing. Moreover, in order to further characterize the mechanisms by which Lmln deficiency drives retinal disease, we are currently conducting cytokine array and bulk RNA-sequencing experiments. We hope to better understand early molecular changes triggered by the absence of this zinc-metallopeptidase and how they may result in neurodegeneration.

Conclusions : Screening using ONL measurements allowed us to identify a novel non-redundant gene-retinal phenotype association involving a gene coding for zinc-metallopeptidase Lmln. Metallopeptidases participate in many major physiological processes, and their dysregulation is involved in multiple diseases. Given the presence of a variety of similar enzymes in the retina, it is interesting to see that other molecules cannot compensate for the absence of Lmln. Our studies will help characterize the specific role of this particular metallopeptidase in retinal neurodegeneration.

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