[Regulation of SARM1 on SNPH expression and its participation in glaucomatous optic neuropathy]

Objective: To investigate the molecular mechanism of toll-like receptor adaptor protein SARM1 in glaucomatous optic neuropathy. Methods: The experimental study was conducted from February 2024 to October 2025. A chronic ocular hypertension glaucoma model was established by injecting micro-magnetic beads into the anterior chamber of 8- to 10-week-old male Wistar rats. At 3 days, 1 week, and 2 weeks post-modeling, retinal and optic nerve tissues from 6 eyes of 6 rats were collected as the chronic ocular hypertension glaucoma model group, and 6 eyes from 6 wild-type rats that received an equal volume of saline via anterior chamber injection served as the control group. Intraocular pressure was measured using a TonoLab tonometer. Retinal whole-mounts were prepared and POU domain class 4 transcription factor 1 (POU4F1 or Brn3A) immunofluorescence staining was used to detect retinal ganglion cell loss. Western blotting was performed to detect the expression levels of SARM1 and SNPH in the rat retina and optic nerve. Immunofluorescence staining was used to examine their distribution in these tissues. Furthermore, CRISPR/Cas9 technology was used to knock down the expression of SARM1 and SNPH in mouse 661W retinal ganglion cells, respectively. Cells were collected 48 hours after transfection, and Western blotting was performed to detect the expression levels of SARM1 and SNPH. Normally distributed continuous data are presented as mean±SEM. Comparisons between two groups were performed using the Student's t-test, while comparisons among multiple groups were assessed by the one-way analysis of variance followed by the Tukey's multiple comparisons test. Results: Western blot analysis revealed that in the glaucoma model group, the relative expression level of SARM1 protein in the optic nerve at one week post-modeling (1.22±0.06) was significantly higher than that in the control group (1.03±0.01; P=0.027, q=4.38). In contrast, the expression level of SARM1 in the retina at three days post-modeling (0.79±0.02) was significantly lower than that in the control group (1.04±0.03; P<0.001, q=6.86). Concurrently, the expression level of SNPH at three days post-modeling (0.74±0.01) was lower than that in the control group (1.03±0.04; P=0.040, q=0.58), and its expression at one week post-modeling (1.19±0.10; P=0.002, q=4.36) was significantly higher than that at three days (0.74±0.01). Consistent with the Western blot results, immunofluorescence staining results showed that in the optic nerve of the glaucoma model group, the expression of SARM1 was significantly higher than that in the control group at one week post-modeling, while the expression of SNPH was lower than that in the control group at three days post-modeling. Both proteins partially co-localized with the neuronal marker β3-tubulin. Immunofluorescence staining also revealed co-localization of these two proteins within axons. Additionally, SARM1 co-localized with the mitochondrial marker protein TOM20. Western blot results from 661W cells showed that the knockdown of SARM1 expression (0.54±0.04) significantly reduced SNPH expression (0.54±0.05; P=0.003, q=7.98), whereas the knockdown of SNPH expression (0.39±0.06) did not markedly affect SARM1 levels (0.75±0.05; P=0.010, q=6.39). Conclusion: The elevated expression of SARM1 protein in the axons of the rat glaucoma model can promote retinal ganglion cell axonal pathology by localizing to axonal mitochondria and regulating SNPH expression.