Neural Regeneration Research · 10 authors, 2 centres
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Homer1a overexpression protects retinal tissue and Müller cells from ischemia/reperfusion injury by inhibiting the caspase-8/NF-κB/NLRP3 inflammasome pathway, reducing inflammatory cytokine release and apoptosis. Homer1a knockdown exacerbates damage, while combined treatment with Homer1a protein and the NF-κB inhibitor JSH-23 provides additive protection. These findings identify Homer1a as a potential therapeutic target for retinal ischemic diseases.
**Background:** Retinal ischemia/reperfusion (I/R) injury underlies many blinding diseases such as retinal vascular occlusion, diabetic retinopathy, and ocular trauma. The inflammatory cascade, particularly the NLRP3 inflammasome, is a major driver of damage. Homer scaffold protein 1 (Homer1a), a short form of Homer1, is upregulated by oxygen-glucose deprivation and protects neurons in cerebral I/R models, but its role in retinal I/R injury was unknown. This study investigated whether Homer1a modulates the inflammatory response via the NLRP3 and caspase-8 inflammasomes in retinal tissue and Müller cells.
**Methods:** Retinal I/R injury was induced in male C57BL/6J mice (8 weeks old, 25±2 g) by elevating intraocular pressure to 85 mmHg for 90 minutes. In vitro, mouse Müller cells (>90% purity) were subjected to 60 minutes of oxygen-glucose deprivation followed by reperfusion. Homer1a was overexpressed (OE) or knocked down (KD) using adeno-associated virus (AAV) in vivo or lentivirus in vitro. Additional groups received intravitreal injections of NF-κB inhibitor JSH-23 (20 μM), caspase-8 inhibitor Z-IETD-fmk (20 μM), or Homer1a protein (1 μg) 6 hours before I/R. Homer1^flox/−/Homer1a^+/−/Nestin-Cre^+/− conditional knockout mice were also used. Outcomes included retinal thickness (H&E), apoptosis (TUNEL), Müller cell viability (CCK-8), expression of 200 inflammatory factors (cytokine array), NLRP3/caspase-8/ASC inflammasome proteins (western blot, co-immunoprecipitation), IL-1β and IL-18 levels (ELISA, western blot, RT-PCR), and NF-κB P65 phosphorylation and nuclear translocation (western blot). Statistical analysis used one-way ANOVA with Tukey's test; P<0.05 was significant.
**Key Results:** Retinal thickness decreased to 60.40±7.44% of sham at 24 hours (vs. 104.40±5.94%, P<0.0001), and Müller cell viability fell to 45.67±5.51% of control (vs. 100.70±8.02%, P<0.0001). Homer1a mRNA and protein peaked at 24 hours post-reperfusion both in vivo and in vitro. Homer1a OE significantly increased retinal thickness to 78.40±3.98% (vs. I/R 56.20±6.02%, P=0.0004) and reduced apoptosis (TUNEL+ rate 0.23±0.04 vs. 0.56±0.08, P<0.0001). Homer1a KD worsened these parameters (thickness 34.40±3.21%, P=0.0005; apoptosis 0.88±0.07, P<0.0001). Cytokine array revealed that Homer1a OE suppressed bFGF, KC/CXCL1, M-CSF, TNFR1, and TNFR2 (all P<0.0001 vs. I/R), while Homer1a KD increased them. Homer1a OE reduced caspase-8, NLRP3, ASC, and cleaved caspase-1/caspase-1 ratio (e.g., NLRP3: 1.38±0.14 vs. 1.91±0.31, P=0.0186; cleaved caspase-1/caspase-1: 1.50±0.12 vs. 2.27±0.15, P<0.0001), and Homer1a KD increased them (all P<0.01). Co-IP showed Homer1a OE inhibited ASC binding to both caspase-8 and NLRP3, while Homer1a KD enhanced binding. Homer1a OE reduced IL-1β and IL-18 processing (cleaved IL-1β/IL-1β: 1.68±0.13 vs. 2.10±0.21, P=0.0118; cleaved IL-18/IL-18: 1.34±0.11 vs. 1.67±0.21, P=0.0231), and Homer1a KD increased them (P<0.0001). NF-κB P65 phosphorylation and nuclear translocation were reduced by Homer1a OE (nuclear P65: 1.39±0.24 vs. 2.98±0.15, P<0.0001) and increased by Homer1a KD (4.93±0.26, P<0.0001). JSH-23 (NF-κB inhibitor) decreased NLRP3 (1.41±0.10 vs. 1.81±0.12, P=0.0005) and cleaved IL-1β/IL-1β (1.16±0.16 vs. 1.49±0.15, P=0.0195) but did not affect caspase-8 (1.46±0.28 vs. 1.48±0.19, P=0.9878). Caspase-8 inhibitor Z-IETD-fmk reduced NF-κB P65 phosphorylation (P<0.001). In conditional knockout mice, combined Homer1a protein and JSH-23 treatment reduced apoptosis (TUNEL+ rate 0.15±0.04 vs. I/R 0.61±0.05, P<0.0001) and preserved retinal thickness (90.60±4.93% vs. I/R 46.40±4.04%, P<0.0001) more effectively than either agent alone.
**Clinical Implications:** This study demonstrates that Homer1a is a critical endogenous regulator of the inflammatory response in retinal I/R injury, acting through the caspase-8/NF-κB/NLRP3 signaling axis. Homer1a overexpression or exogenous protein administration reduces inflammasome activation, cytokine release, and cell death, while knockdown exacerbates damage. The additive benefit of combining Homer1a with an NF-κB inhibitor suggests a potential multi-target therapeutic strategy. These findings provide a mechanistic foundation for developing Homer1a-based therapies—such as exosome-delivered Homer1a protein—for retinal ischemic diseases including diabetic retinopathy, retinal vein occlusion, and glaucoma. Limitations include the use of a single I/R model and the need for cell-type-specific knockout studies to identify the precise cellular targets of Homer1a in the retina.