α-Synuclein overexpression represses 14-3-3θ transcription

Abstract

Previous gene microarray studies have shown that expression of 14-3-3θ is significantly decreased in an α-synuclein transgenic mouse model. In this study, we tested whether α-synuclein can regulate 14-3-3θ transcription. We demonstrate that the 14-3-3θ mRNA level is decreased in SH-SY5Y cells overexpressing α-synuclein. Luciferase activity under the control of the 14-3-3θ promoter is reduced both in stable SH-SY5Y cells constitutively overexpressing α-synuclein and in doxycycline-inducible SH-SY5Y cells upon α-synuclein induction, suggesting that the regulation of 14-3-3θ by α-synuclein occurs at the transcriptional level. Knockdown of α-synuclein by RNA interference does not increase the 14-3-3θ mRNA level. These findings suggest that α-synuclein represses 14-3-3θ transcription under pathologic conditions, but that regulation of 14-3-3θ expression is not a function of endogenous α-synuclein at baseline.

Figure 1. 14-3-3θ mRNA levels were reduced in stable SH-SY5Y cells overexpressing α-syn

(A) Immunoblot of protein lysates from control and three different SH-SY5Y stable cell lines that overexpress α-syn (Syn-1, Syn-2 and Syn-5). (B, C, D) RNA was extracted from control and α-syn stable cell lines and reverse transcribed into cDNA. Quantitative PCR was performed using primers specific for 14-3-3θ, ε, or γ. Compared to control cells, 14-3-3θ mRNA levels were significantly reduced in all three α-syn stable lines. 14-3-3ε mRNA levels were reduced in two out of the three α-syn stable lines, whereas 14-3-3γ mRNA levels were not altered. Results were expressed as percentage of control. Experiments were performed three times with two or three replicates per experiment. **p<0.01, ***p<0.001 compared to control (two-way ANOVA followed by Tukey’s multiple comparison test). Error bars reflect S.E.M.

Figure 2. 14-3-3θ mRNA levels were reduced by rotenone treatment but not mutant huntingtin overexpression

(A) SH-SY5Y cells were treated for 24 hrs with 0.2 μM rotenone. RNA was extracted and quantitative PCR was performed using primers specific for 14-3-3θ. Treatment with rotenone caused significant reduction in 14-3-3θ mRNA level. Results were expressed as percentage of the untreated group. Experiments were performed three times with three replicates per experiment. ***p<0.001 compared to untreated (t-test). (B) RNA was extracted from brains of wildtype mice (WT) or BAC transgenic mice expressing human mutant huntingtin (BACHD). 14-3-3θ mRNA levels were analyzed by quantitative PCR and normalized to HPRT. There was no significant difference between the WT (n=5) and BACHD mice (n=7). Results were expressed as percentage of wildtype. n.s. = not significant (t-test). Error bars reflect S.E.M.

Figure 3. 14-3-3θ promoter activity was repressed in stable SH-SY5Y cells overexpressing α-syn

(A) Schematic of luciferase reporter constructs (F2, F9, F12, F15 and F17) containing varying lengths of putative 14-3-3θ promoter region. The nucleotide numbers are relative to the translation start site of 14-3-3θ. (B) Expression of luciferase reporter constructs in control and α-syn stable SH-SY5Y cells. Control and α-syn stable cell line Syn-1 were co-transfected with Renilla luciferase control vector and the control pGL4 vector or one of the five 14-3-3θ luciferase reporter constructs. Luciferase activities were measured by dual luciferase reporter assay and expressed as percentage of the shortest construct F2 in control cells. In control condition, the shorter constructs F2, F9, F12 and F15 drove luciferase expression at a comparable level, whereas the longest construct F17 showed significantly lower basal luciferase activity (white bars, construct effect p<0.001 by three-way ANOVA). Luciferase activities of all of the tested reporter constructs were significantly reduced in α-syn stable cells compared to their respective controls (black bars, ***p<0.001, t-test). Experiments were performed three times with two replicates per experiment. Three-way ANOVA was first done to analyze the overall effect of each grouping factor (cell line, construct and experimental run). T-tests were then used to compare luciferase activities in control cells and α-syn stable cells for each reporter construct. Error bars reflect S.E.M.

Figure 4. 14-3-3θ promoter activity was repressed in doxycycline-inducible SH-SY5Y cells overexpressing α-syn

(A) SH-SY5Y cells were transduced with a doxycycline-inducible α-syn lentivirus and selected with geneticin for stably-transduced cells. Immunoblotting showed increased α-syn expression after induction with 1 μg/ml doxycycline for 48 hrs. (B) Luciferase activities of reporter constructs were significantly reduced in α-syn inducible cells. 14-3-3θ luciferase reporter construct F9 and Renilla luciferase control vector were co-transfected into uninduced cells or cells that had been induced for 1 or 4 days, and then luciferase activity was measured 2 days after transfection. Therefore, cells were induced for a total of 3 or 6 days, respectively. Luciferase activities were expressed as percentage of uninduced cells. Experiments were performed three times with two or three replicates per experiment. ***p<0.001 compared to uninduced (two-way ANOVA followed by Tukey’s multiple comparison test). Error bars reflect S.E.M.

Figure 5. Knockdown of α-syn had no effect on 14-3-3θ mRNA level and did not reverse the effect of HDAC inhibition on 14-3-3θ level

(A) SH-SY5Y cells were transduced with control pLKO.1 or α-syn shRNA lentiviruses and selected with puromycin for stably-transduced cells. Immunoblotting showed decrease in α-syn expression after lentiviral transduction. (B) SH-SY5Y cells stably transduced with the pLKO.1 control or with α-syn shRNA lentiviruses were untreated or treated with 2 mM SB for 24hrs. RNA was extracted and quantitative PCR was performed using primers specific for 14-3-3θ. In untreated cells, 14-3-3θ mRNA level was not changed by the knockdown of α-syn when compared to pLKO.1 control (black bars). Treatment with SB caused significant reduction in 14-3-3θ expression in both pLKO.1 and α-syn knockdown cells (black vs. white bars). 14-3-3θ mRNA level in SB-treated cells transduced with α-syn shRNA lentiviruses was slightly lower than those transduced with pLKO.1 control (white bars). Results were expressed as percentage of untreated pLKO.1. Experiments were performed three times with three replicates per experiment. Three-way ANOVA was first done to analyze the overall effect of each grouping factor (cell line, treatment and experimental run). Two-way ANOVA was then used to compare pairs of experimental groups. *p<0.05, ***p<0.001, n.s. = not significant. Error bars reflect S.E.M.

Figure 6. Treatment of HDAC inhibitors caused a decrease in 14-3-3θ mRNA level and an increase in α-syn protein level

(A, B, C) SH-SY5Y cells were treated for 24 hrs with varying doses of HDAC inhibitors sodium butyrate (SB), trichostatin A (TSA), and MS-275. RNA was extracted and quantitative PCR was performed using primers specific for 14-3-3θ. All three HDAC inhibitors caused decreases in 14-3-3θ mRNA levels. Results were expressed as percentage of the untreated group. Experiments were performed three times with three replicates per experiment. ***p<0.001 compared to untreated (two-way ANOVA followed by Tukey’s multiple comparison test). (D, E) SH-SY5Y cells were treated with 2 mM SB or 200 nM TSA for 24 hrs. Proteins were collected and immunoblotted with antibody against α-syn or actin. Blots were quantified by densitometry. The levels of α-syn were normalized to the levels of actin. Treatment with SB and TSA increased α-syn expression by 2.4 and 2.8-fold, respectively. Densitometric quantification included three independent experiments. *p<0.05, **p<0.01 compared to untreated (one-way ANOVA followed by Dunnett’s multiple comparison test).