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. 2011 Oct 12;31(41):14508–14520. doi: 10.1523/JNEUROSCI.1560-11.2011

Figure 3.

Figure 3.

UPS inhibition using the specific proteasome inhibitor CLBL results in significantly elevated α-synuclein levels in vivo independent of the preexisting α-synuclein in burden. A, C, Immunoblot levels of α-synuclein in homogenates of recovered cortical tissue of nontransgenic, human α-synuclein transgenic, and α-synuclein-GFP transgenic mice after treatment with proteasome inhibitor CLBL (20 μm; 24 h), MG132 (100 μm; 24 h), or vehicle. Non-tg: 0.43 ± 0.03, n = 17 (vehicle), 0.58 ± 0.04, n = 14 (CLBL); ANOVA, p < 0.0001; Dunnett's posttest, p < 0.05; (h)αSyn tg: 0.42 ± 0.05, n = 15 (vehicle), 0.85 ± 0.11, n = 14 (CLBL), 1.14 ± 0.09, n = 2 (MG132); ANOVA, p = 0.0001; Dunnett's posttest, p < 0.001; αSyn-GFP tg: αSyn-GFP: 0.25 ± 0.04, n = 11 (vehicle), 0.72 ± 0.07, n = 11 (CLBL); ANOVA, p < 0.0001; Dunnett's posttest, p < 0.001; endogenous α-synuclein: 0.25 ± 0.05, n = 11 (vehicle), 1.15 ± 0.18, n = 11 (CLBL); ANOVA, p = 0.0001; Dunnett's posttest, p < 0.001. B, D, Representative immunoblots of α-synuclein levels. E, CLBL treatment in human α-synuclein transgenic mice produces a detectable increase in human α-synuclein in the first 300 μm of subsurface, the exact region where proteasome inhibition is the strongest [1.52-fold the level of vehicle-treated littermates within the first 300 μm in this set of experiments; n = 3 (vehicle); n = 3 (CLBL)]. F, Comparison of immunoblot levels of α-synuclein in different age groups in human α-synuclein transgenic mice after proteasome inhibition with CLBL shows a linear increase with age. Aged 3–6 months: 0.30 ± 0.05, n = 5 (vehicle), 0.48 ± 0.03, n = 4 (CLBL); aged 6–9 months: 0.39 ± 0.09, n = 5 (vehicle), 0.67 ± 0.06 n = 4 (CLBL); aged >9 months: 0.56 ± 0.10, n = 5 (vehicle), 1.22 ± 0.14, n = 6 (CLBL); CLBL: ANOVA, p = 0.0017; posttest for linear trend: p = 0.0008; vehicle: ANOVA, p = 0.13; posttest for linear trend, p > 0.05. G, H, Immunoblot levels of the autophagosome marker LC3 in different age groups in human α-synuclein transgenic mice treated with CLBL show no change. Aged 3–6 months: 0.80 ± 0.11, n = 3; aged 6–9 months: 0.72 ± 0.06, n = 3; aged >9 months: 0.76 ± 0.08, n = 3; ANOVA, p > 0.05; posttest for linear trend, p > 0.05. *p < 0.05, **p < 0.01, ***p < 0.001 compared with vehicle-treated littermates. Error bars indicate SEM.