Figure 3

Modification of the UPS induced by intrahippocampal LPS injection. (A) Profile of the mRNA expression of the E2 ubiquitin-conjugating enzyme UB2L6 at different times post-LPS injection. Data are presented as the mean ± SD relative to saline-injected animals. Data were obtained from triplicate assays. (B) Profile of the mRNA expression of the β5i subunit (black line) in the same animals as in A. (C) At the top is shown a representative western blot of the β5i subunit (mature form) in samples from rats injected with saline and LPS, sacrificed at times indicated. Densitometric quantification of western blot experiments (n = 3) is shown in B (red line). At the bottom is shown a representative western blot of POMP in the same animals as shown before. The experiment was repeated three times with similar results. Note the strong time-dependent increase in the content of this protein involved in proteasome assembly, indicating an active process of de novo proteasome biogenesis. (D) Immunofluorescence analysis of the cellular distribution of ubiquitinated proteins and the β5i in hippocampal slices from LPS-injected rats. Images correspond to animals sacrificed 3 days after LPS injection. The β5i immunostaining was mainly located in neuronal fibers in the stratum oriens and stratum radiatum. Some scattered cellular bodies were also observed in the stratum lucidum. Arrows indicate colocalization of ubiquitin and β5i immunostaining. Bar scale 75 μm. (E) Chymotrypsin-like activity was analyzed in saline- and LPS-injected rats at different times post-injection (n = 4 per time point). LPS injection significantly decreased the chymotrypsin activity of hippocampal proteasomes. *P <0.05. LPS: lipopolysaccharide; POMP: proteasome maturation protein; sl: stratum lucidum; so: stratum oriens; sp: stratum pyramidale; sr: stratum radiatum.