Fig. 4. Increased proteasome activity in the P23H retina decreases autophagy flux.

a Chymotrypsin-like activities measured in the presence of ATP (7 µM) in retinal lysates from P23H mice treated with rolipram or vehicle-only as control (CTL) for 2 weeks, normalized to age-matched C57 (wild-type) mice. Representative western blots from retinas of untreated C57 and P23H mice treated with rolipram and vehicle-only (CTL) for 2 weeks, probed and quantified for b ubiquitin, c 20S proteasome and autophagy markers d, e Beclin1, LC3, f, g phosphorylated SQSTM1/p62 and total SQSTM1/p62. To allow for determination of autophagy flux, animals received an intraperitoneal injection of leupeptin (40 mg/kg body weight) 4 h before tissue harvest. Statistical analyses with one-way ANOVA. h Representative fluorescence micrographs of retinas from of 1-month-old P23H GFP-LC3 mice treated with rolipram or vehicle-only (CTL) for 2 weeks. GFP-LC3 puncta localizes primarily to the photoreceptor inner segment (IS), with none localizing to the outer segments (OS). Nuclei of the photoreceptors were stained with DAPI (blue). Scale bar: 25 µm. i Quantification of the number of GFP-LC3 puncta per counting unit indicated in (H) as a red rectangle. Results are shown as individual symbols representing data values along with mean and standard deviation (SD). Statistical analyses with unpaired t-test. *p ≤ 0.05. **p ≤ 0.01. ***p ≤ 0.001. ****p ≤ 0.0001