**Background:** The ubiquitin-proteasome system (UPS) is critical for retinal homeostasis, and its dysregulation is implicated in diseases like age-related macular degeneration (AMD), retinitis pigmentosa, and diabetic retinopathy. E3 ubiquitin ligases, which target specific proteins for ubiquitination, are numerous, but their individual roles in the retina are poorly understood. Activated microglia accumulate in the subretinal space in various retinal degenerations, but the genetic control of this process is unclear. This study used an unbiased forward genetics approach to identify novel genes modulating retinal immune cell activation, focusing on the E3 ligase Herc3.
**Methods:** A forward genetics pipeline using ENU mutagenesis in C57BL/6J mice was combined with a semiquantitative fundus spot scoring scale to screen for mutations causing yellow fundus spot accumulation, a surrogate for subretinal microglia. Over 5,900 G3 mice were screened, and a nonsense mutation (Q137*) in Herc3 (allele 'aegean') was identified. Two independent Herc3 knockout mouse lines were generated using CRISPR/Cas9 (1 bp insertions in exon 14 causing frameshift and premature stop). Retinal phenotype was assessed by fundus photography (scored 0-8), optical coherence tomography (OCT) measuring total retinal thickness (TRT), outer nuclear layer (ONL), outermost neural retina thickness (ONRT), and ganglion cell complex (GCC), and histology (H&E, TUNEL, cone arrestin). Subretinal microglia were quantified on RPE/choroid flat mounts stained for Iba1, F4/80, TMEM119, and CCR2. Visual function was tested by optomotor response (spatial frequency threshold) and full-field scotopic electroretinography (ERG) at 10 and 16-18 months. Single-cell RNA sequencing (scRNA-seq) of 4-month-old C57BL/6J retinas and bulk RNA-seq of 7-8-month-old Herc3-/- vs. wild-type retinas were performed. RNAscope in situ hybridization confirmed Herc3 expression.
**Key Results:** The ENU screen identified a strong association between the Herc3 aegean allele and fundus spots (p=1.3×10⁻¹¹). CRISPR Herc3-/- mice showed progressive fundus spot accumulation, with significant differences at 3-6 months (p=0.0028), 6-9 months (p=0.00049), and 9-15 months (p=2.2×10⁻⁵). Subretinal Iba1+ cells were significantly increased in Herc3-/- mice in central (p=0.0011), paracentral (p=6.5×10⁻⁵), mid-peripheral (p=0.0047), and peripheral (p=0.023) regions. These cells were F4/80+/TMEM119+/CCR2-, confirming microglial origin. OCT revealed progressive outer retinal thinning: ONL thinning was significant from 3-5 months (p<0.01), while TRT and ONRT became significant at 6-7 months (p<0.01). GCC showed no difference. Linear regression confirmed age-dependent thinning of TRT (R²=0.2, p=0.0079) and ONL (R²=0.374, p=0.00012). Histology confirmed ONL thinning and reduced nuclear counts. TUNEL showed no difference in apoptosis. Cone density was unchanged. Optomotor testing at 1 year showed significantly reduced spatial frequency threshold in Herc3-/- mice (p=0.002). ERG at 10 months showed reduced scotopic a-wave (p=9.0×10⁻⁵) and b-wave (p=7.4×10⁻⁵) at 1 log cd.s.m⁻², with greater deficits at 16-18 months. scRNA-seq showed Herc3 is the only small Herc family member significantly expressed in rod photoreceptors, with moderate expression in multiple retinal cell types. Bulk RNA-seq identified 192 upregulated and 65 downregulated genes (p<0.01, fold change>1.5). Pathway analysis (IPA) revealed activation of inflammatory pathways including IL-4, IL-10, IL-12 signaling, complement system, and multiple sclerosis signaling. Differentially expressed genes included complement components (C1ra, C3, C4b) and microglia-modulating genes (Edn2, Neat1, Meg3, Npy6r). Sex-stratified post-hoc analysis suggested female Herc3-/- mice may have more fundus spots and ONL thinning than males, but the study was not powered for this comparison.
**Clinical Implications:** This study demonstrates that Herc3, an E3 ubiquitin ligase, has a non-redundant and essential role in maintaining retinal homeostasis, particularly in the outer retina. Herc3 deficiency leads to progressive retinal neurodegeneration with subretinal microglial accumulation and activation of inflammatory pathways, mimicking features of human retinal degenerations like AMD. The findings suggest that Herc3 may be a novel therapeutic target for retinal diseases involving UPS dysfunction or microglial activation. The Herc3-/- mouse model provides a valuable tool to study the interplay between ubiquitination, microglial activation, and photoreceptor degeneration. Further research is needed to identify Herc3's specific substrates in the retina and to determine whether microglial activation is a primary or secondary event.