The temporal progression of retinal degeneration and early-stage idebenone treatment in the Pde6b rd1/rd1 mouse model of retinal dystrophy

**Background:** Retinitis pigmentosa (RP) is a leading cause of irreversible vision loss, driven by genetic mutations in photoreceptor or RPE cells. The rd1 mouse model, carrying a nonsense mutation in the Pde6b gene, exhibits rapid rod photoreceptor degeneration within two weeks post-birth. While later stages of degeneration and remodeling are well-studied, early pathological events preceding cell death remain poorly understood. This study aimed to characterize the molecular and cellular changes in the rd1 retina before P14 and to evaluate early idebenone treatment as a potential intervention targeting mitochondrial dysfunction.

**Methods:** Pde6b rd1/rd1 mice (rd1) and C57BL/6J wild-type (WT) controls were used. Retinal morphology was assessed by hematoxylin and eosin staining at P1, P3, P7, P10, and P14. Ultrastructural analysis via transmission electron microscopy (TEM) was performed at P1, P3, P7, P10, and P14 to evaluate mitochondria, Golgi apparatus, and synaptic ribbons. Pde6b mRNA expression was quantified by qRT-PCR at P1, P3, P7, P10, and P14. Immunohistochemistry and immunofluorescence for PKCα (rod bipolar cells), Calbindin D28K (horizontal cells), and CtBP2 (presynaptic ribbons) were conducted at P7, P10, and P14. For the treatment study, lactating mother mice received oral idebenone (200 mg/kg/day) from P0 to P21. Offspring were analyzed at P14 and P21 via ERG, F-VEP, histology, and immunofluorescence. Statistical analyses used independent two-sample t-tests and ANOVA.

**Key Results:** Pde6b mRNA expression in rd1 mice was significantly lower than WT from P3 onward (rd1: 2.63±0.21 vs WT: 4.09±0.53 at P3; rd1: 0.14±0.01 vs WT: 87.91±8.52 at P14). Histologically, the ONL of rd1 mice thinned to 2–4 rows by P14, while the INL remained intact. TEM revealed mitochondrial cristae defects in rd1 rods as early as P3, with swollen, vesicular mitochondria increasing by P7 and P10. Golgi apparatus were scarce in rd1 from P7 onward, whereas WT showed abundant Golgi at P7 and P14. At P14, rd1 OPL lacked normal synaptic ribbons, with terminals containing vesicles but no invaginations. Immunofluorescence showed that CtBP2-positive puncta were sparse and flattened in rd1 from P10, preceding photoreceptor degeneration. Horizontal cell dendrites decreased from P10, and cell bodies declined at P14. Idebenone treatment at P14 significantly increased ONL thickness and CtBP2-positive puncta compared to untreated rd1, and F-VEP amplitudes were significantly improved (P<0.05). However, at P21, these effects were not significant, and ERG b-wave amplitudes showed only a non-significant trend toward improvement. No differences in horizontal cell dendrites were observed between treated and untreated rd1 at either time point.

**Clinical Implications:** This study demonstrates that mitochondrial dysfunction is an early and critical event in rd1 retinal degeneration, occurring before photoreceptor cell death and before significant cGMP elevation. The findings suggest that therapeutic interventions targeting mitochondrial health may need to be initiated very early—potentially before P10—to be effective. Idebenone, already approved for LHON, showed only transient benefit in this model, indicating that monotherapy may be insufficient for RP. The observed abnormal synaptogenesis in second-order neurons (rod bipolar cells and horizontal cells) before photoreceptor loss underscores the importance of early intervention to preserve retinal circuitry. These results support the concept of a 'metabolic ecosystem' in the retina and highlight the need for combinatorial or multi-targeted approaches to address the complex pathophysiology of RP. Future studies should explore optimal dosing, timing, and combination therapies to achieve sustained photoreceptor rescue.