Nonredundant Role of Leishmanolysin-Like (Lmln) Zinc-Metallopeptidase in Retinal Homeostasis

Abstract

To determine if Lmln, a Zn-metallopeptidase, is important for retinal homeostasis. Combining an unbiased N-ethyl-N-nitrosourea mutagenesis pipeline in mice with optical coherence tomography (OCT) screening and automated meiotic mapping, we identified an allele (nemeth) that seemed to be associated with outer nuclear layer (ONL) thinning. Since nemeth was predicted to lead to a nonsense mutation of the Lmln gene, we targeted Lmln using CRISPR/Cas-9 technology and characterized the impact on retinal anatomy and function. OCT imaging demonstrated an outer retinal degeneration in Lmln-/- mice (P = 7.3 × 10-9 for ONL at 2 m) that progressed over the first 6 months of life and then stabilized. Light microscopy showed loss of ONL nuclei (P ranged between .00033 and .0097 for posterior measurements), and a TUNEL assay revealed a small but significant increase in apoptosis (P = .034). Lmln-/- mice accumulated fundus spots (P = .0030 by 2 m of age) and activated subretinal microglia (p ranged from .0007 to 8 × 10-13 for Gal3+ cells). Scotopic electroretinography demonstrated a decrease in retinal function in Lmln-/- mice both at 6 m (only a-wave, P < .01 for all stimuli) and at 10 m of age (P < .01 for both a-wave and b-wave with all stimuli). Our work revealed a previously unknown essential requirement for Lmln in maintaining retinal anatomy and function. Further studies using this new model will be aimed at determining the cellular expression of Lmln and its mechanisms of action within the retina.