Figure 5
From: Lysosomal enzyme cathepsin D protects against alpha-synuclein aggregation and toxicity
CD reduces α-syn toxicity. a. Enhanced CD expression protects against α-syn overexpression-induced cell death. GFP was visualized under the fluorescence microscope and demonstrated more survival cells after co-transfection of GFP-α-syn and CD compared to transfection with GFP-α-syn alone. Viable cells were counted by trypan blue exclusion method. b. mRNA of α-syn is unchanged by CD transfection. SHSY5Y cells were transfected with vector alone, CD, α-syn, or α-syn+CD. Paired Student t-tests were conducted on RQ values for each group to determine significance. c. Western blot analyses indicate that CD transfection results in truncation of α-syn-GFP (the appearance of a band below the full-length 37 kDa band), and a reduction of endogenous 17 kDa α-syn monomers. CD is synthesized as a prepropeptide (53 kDa); the signal peptide is cleaved upon CD insertion into the endoplasmic reticulum (47 kDa). The CD zymogen is then activated in the acidic lysosomal environment to produce the 32 and 14 kDa products [13, 47]. d. Enhanced CD expression reduces A53T and A30P mutant α-syn-induced cell death, but does not reduce Y125A mutant α-syn-, 10 μM chloroquine-, or 2 μM staurosporine-induced cell death. For a-d, *p < 0.05 compared to control (CTL); †p < 0.05 compared to otherwise identical transfection except without CD. n = 3 transfection for each experimental conditions. Student t-test was used. e. Increased protein levels correlate with transfection of respective cDNAs. SHSY5Y cells were transfected with control vector, or respective cDNA in each lane. The respective antibodies used for the immunoblots are at the left side of the gel images. Mutated α-syn-GFP were produced. CD transfection did not produce significant truncation intermediate products on the mutated α-syn-GFP, suggesting that the protection against A53T and A30P may be via alteration of their intracellular targeting rather than direct cleavage. CD 53 kDa and 47 kDa precursors and the 32 kDa mature product are shown. Actin immunoblot was used as a loading control.