Fig. 6.
Autophagy inhibition by HCQ treatment in vivo increased photoreceptor survival and rescued retinal function in GCP4 heterozygotes. a Western blot analysis showed that LC3B-II and SQSTM1 was accumulated in heterozygous retinas after 2 months of HCQ treatment via the drinking water. GAPDH was used as an internal control. b Quantification of LC3B-II and SQSTM1 expression levels in retinas in panel a. c Real-time quantitative PCR of Sqstm1 in retinas in (a). β-actin was used as an internal control. The results were representative of 3 independent experiments and represented as means ± SD. The data were analyzed using Student’s t-test. *p < 0.05; **p < 0.01. d Histologic analysis of heterozygous retinas with or without HCQ treatment at age of 8 month. RPE, retinal pigment epithelium; OS, outer segment; IS, inner segment; ONL, outer nuclear layer; OPL, outer plexiform layer; INL, inner nuclear layer; IPL, inner plexiform layer; GCL, ganglion cell layer. Scale bar: 20 µm. e Statistical analysis of ONL thickness in Tubgcp4+/− mice with or without HCQ treatment (n = 3). The ONL thickness was measured along the vertical meridian at each defined distance from the optic nerve head. Data were represented as mean ± SD. Two-way ANOVA followed by Bonferroni posttest was used for statistical analysis. *p < 0.05; **p < 0.01. f, h, j Saturating ERG responses of the retinas of the mice as indicated to 510 nm flashes at an intensity of −35 log scot. cd. s/m2 under scotopic (f, h) and photopic (j) conditions. Each trace is the average of individual records from five mice. g, i, k Statistical analysis of the saturating amplitude of a- and b-waves of the mice as indicated under scotopic (g, i) and photopic (k) conditions. Amplitude levels of a- and b-waves were significantly rescued in the HCQ-treated Tubgcp4+/− mice. The mice were 8-month-old littermates. Data were represented as means ± SD of five mice. p-values were calculated by two-tailed t-test, *p < 0.05; **p < 0.01