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

PURPOSE: To determine if Lmln, a Zinc-metallopeptidase, is important for retinal homeostasis. DESIGN: Basic research in mouse models of retinal degeneration. METHODS: 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. RESULTS: 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). CONCLUSIONS: Our work revealed a previously unknown essential role 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.

E3 ubiquitin ligase Herc3 deficiency leads to accumulation of subretinal microglia and retinal neurodegeneration.

Activated microglia have been implicated in the pathogenesis of age-related macular degeneration (AMD), diabetic retinopathy, and other neurodegenerative and neuroinflammatory disorders, but our understanding of the mechanisms behind their activation is in infant stages. With the goal of identifying novel genes associated with microglial activation in the retina, we applied a semiquantitative fundus spot scoring scale to an unbiased, state-of-the-science mouse forward genetics pipeline. A mutation in the gene encoding the E3 ubiquitin ligase Herc3 led to prominent accumulation of fundus spots. CRISPR mutagenesis was used to generate Herc3-/- mice, which developed prominent accumulation of fundus spots and corresponding activated Iba1 + /CD16 + subretinal microglia, retinal thinning on OCT and histology, and functional deficits by Optomotory and electrophysiology. Bulk RNA sequencing identified activation of inflammatory pathways and differentially expressed genes involved in the modulation of microglial activation. Thus, despite the known expression of multiple E3 ubiquitin ligases in the retina, we identified a non-redundant role for Herc3 in retinal homeostasis. Our findings are significant given that a dysregulated ubiquitin-proteasome system (UPS) is important in prevalent retinal diseases, in which activated microglia appear to play a role. This association between Herc3 deficiency, retinal microglial activation and retinal degeneration merits further study.