PMC

14-3-3 Expression in transgenic α-syn mice and wild-type littermates. 14-3-3θ and γ mRNA levels were significantly reduced in α-syn transgenic mice compared with wild-type mice. RNA was extracted from the cortex of 3-month-old transgenic mice and wild-type littermates. Primers specific to each 14-3-3 isoform were used for quantitative PCR to determine the amount of each 14-3-3 transcript. Results were normalized to GAPDH. n=8 per group. ^*P<0.05 (least-squares means test). Error bar reflects S.E.M.

Overexpression of 14-3-3ɛ, -γ, and -θ protects against MPP⁺ toxicity. Cell lines stably transfected with either a 14-3-3 isoform or an empty vector were treated with varying concentrations of MPP⁺ for 24 h. Cell death was assayed by LDH release into the culture media. LDH release into media was normalized to total LDH release for each well. Lines overexpressing 14-3-3θ (a), 14-3-3ɛ (b), or 14-3-3γ (c) were more resistant to MPP⁺ compared to control stable cells. In contrast, overexpression of 14-3-3β (d), 14-3-3ζ (e), 14-3-3η (f), or 14-3-3σ (g) did not protect against MPP⁺. Results reflect 2–3 independent experiments with at least two replicates per experiment. ^**P<0.01, ^***P<0.001 (Bonferroni's multiple comparison test). Error bars reflect S.E.M.

Subcellular distribution of V5-tagged 14-3-3 isoforms and α-syn in stable cell lines was similar to endogenous expression in control M17 cells. (a–c) Naive M17 cells (a), control stable cells (b), and 14-3-3θ-overexpressing cells (c) were immunostained using a primary monoclonal antibody against 14-3-3θ and a cy-3-conjugated goat anti-mouse secondary antibody to determine the cellular distribution of endogenous and overexpressed 14-3-3θ. Cells were also stained with Sytox Green to localize nuclei. 14-3-3θ Expression in all cells was seen predominantly in the cytoplasm. (d–f) These cell lines were also stained with a primary monoclonal antibody against V5 and a cy3-conjugated goat anti-mouse secondary antibody to determine the cellular distribution of exogenous V5-tagged 14-3-3θ. V5 staining showed similar cellular distribution as 14-3-3θ in 14-3-3θ stable cells (f). No V5 staining was apparent in M17 (d) or control stable cells (e). Cells were stained with Sytox Green to visualize nuclei. (g–j) Control cells (g, i) and 14-3-3θ stable cells (h, j) were stained with an antibody against α-syn to determine subcellular localization of α-syn. Staining was apparent in both nuclear and cytoplasmic regions for both control (g) and 14-3-3θ (h) cells. Sytox Green was used to stain nuclei (i, j). Scale bar=30 μM

Stable M17 cells created to overexpress each 14-3-3 isoform. (a) 14-3-3θ was subcloned into the pcDNA3.1/V5-His vector, and SK-N-BE(2)-M17 cells were transfected with V5/His-tagged 14-3-3θ construct. Cells stably transfected with 14-3-3θ were selected in the presence of G418. A total of 13 different clones were analyzed for their expression of 14-3-3θ. Protein lysates of these clones were blotted with a monoclonal antibody against V5 to detect exogenous 14-3-3θ (top blot) or with a monoclonal antibody against 14-3-3θ to detect total 14-3-3θ levels (exogenous or endogenous; bottom blot). Two clones (clones 4 and 5) with high levels of 14-3-3θ overexpression were used for the LDH experiments described below. (b) 15–20 stable clones for each of the other 14-3-3 isoforms were similarly created and analyzed for 14-3-3 overexpression by western blot against the V5 epitope tag. Two to three clones for each 14-3-3 isoform were selected for the LDH experiments based on high expression levels. Western blots of these selected clones are shown using an antibody against V5. (c) Immunoblotting against 14-3-3θ reveals higher expression level of 14-3-3θ in 14-3-3θ stable cell line clone 5 (Q5) as compared to untransfected naive M17 cells (M17) and two control stable lines (C1, C2). Tubulin was used as a loading control for all blots

Overexpression of other 14-3-3 isoforms also protects against rotenone toxicity, whereas inhibition of 14-3-3s promotes toxicity. (a–f) Cell lines stably transfected with either a 14-3-3 isoform or empty vector were treated with varying concentrations of rotenone for 48 h. Cell death was assayed by LDH release into the culture media. LDH release into media was normalized to total LDH release for each well. Lines overexpressing 14-3-3ɛ (a), 14-3-3γ (b), 14-3-3β (c), or 14-3-3ζ (d) were more resistant to rotenone compared to control stable cells. In contrast, overexpression of 14-3-3η (e) and 14-3-3σ (f) did not protect against rotenone and showed increased toxicity at baseline. Results reflect 2–3 independent experiments with at least two replicates per experiment. ^*P<0.05, ^**P<0.01, ^***P<0.001 (Bonferroni's multiple comparison test). Error bars reflect S.E.M. (g) M17 cells were infected with either tetracycline-inducible difopein-EYFP lentivirus or scrambled difopein-EYFP lentivirus. After 2 days of induction by doxycycline, cells were plated and treated with increasing doses of rotenone for 30 h, and cell death was assayed by LDH release. Difopein-expressing cells were more sensitive to rotenone compared to control stable cells. ^*P<0.05 (least-squares means test). Results reflect three independent experiments with at least two replicates per experiment. Error bars reflect S.E.M.

14-3-3ɛ, -γ, and -θ reduce α-syn inclusions in H4 neuroglioma cells. (a–d) Immunocytochemistry of H4 cells transfected with synT, synphilin, and empty vector (a, b) or 14-3-3θ (c, d) with an antibody against α-syn and an Alexa 488-conjugated goat anti-mouse secondary antibody (green; a, c) or a pan-14-3-3 antibody and a cy-3-conjugated goat anti-rabbit secondary antibody (red; b, d). Fewer cells transfected with 14-3-3θ show α-syn inclusions as compared to control cells. Arrows point to cells with α-syn inclusions. Scale bar=25 μM. (e) H4 cells were co-transfected with either empty vector or a 14-3-3 isoform along with synT and synphilin. At 24 h after transfection, cells were fixed and stained with a monoclonal antibody against α-syn. Cells that stained for α-syn were scored as positive or negative for α-syn inclusions, with the rater masked to experimental condition. Results reflect three independent experiments with four replicates per experiment. ^***P<0.001 (Bonferroni's multiple comparison test). Error bars reflect S.E.M. (f) Expression levels of synT are not affected in H4 cells transfected with 14-3-3θ. Representative western blot of total lysates from empty vector and 14-3-3θ transfected H4 cells incubated with an α-syn antibody is shown. Results were confirmed in three independent experiments. Arrow points to the ∼32 kDa α-syn/GFP fusion protein (synT). Tubulin was used as a loading control

Overexpression of 14-3-3θ protects against dopaminergic cell loss in the α-syn transgenic C. elegans worm model. Expression plasmids, P_dat−1∷ human 14-3-3θ and P_unc−54∷mCherry, were constructed and microinjected into the gonads of C. elegans strain that already expresses α-syn and GFP (P_dat−1∷α-syn; P_dat−1∷GFP) and exhibits age-dependent α-syn-induced degeneration in dopaminergic neurons. Similar double transgenic worms were created to overexpress human 14-3-3ɛ, human 14-3-3γ, or the worm 14-3-3 homologue ftt-2. In addition, a mutant ftt-2 knockout that overexpressed α-syn was also created. (a) α-Syn worm shows loss of dopaminergic neurons (only two CEP neurons and one ADE neuron remain) at day 7. Arrowheads show intact dopaminergic neuron cell bodies. Lined arrows indicate areas where dopaminergic neurons have degenerated. (b) Overexpression of 14-3-3θ in the α-syn-overexpressing line protects dopaminergic neurons from α-syn-induced cell death. Arrowheads show six intact dopaminergic neuron cell bodies. (c, d) 14-3-3θ reduced dopaminergic cell loss, but 14-3-3ɛ and -γ did not. The numbers of α-syn transgenic worms and α-syn/14-3-3 double transgenic worms that had the full complement of six anterior dopaminergic neurons were scored at day 10 (c). The percentage of intact dopaminergic neurons per worm was also scored at day 10 for α-syn and α-syn/14-3-3θ transgenic worms (d). (e, f) Overexpression (OE) of the worm 14-3-3 homologue ftt-2 also reduced dopaminergic cell death, but ftt-2 knockout did not enhance neurodegeneration. The percentage of worms that had the full complement of dopaminergic neurons (e) and the percentage of intact dopaminergic neurons per worm (f) were scored at day 10. For each experiment, three independent transgenic lines were scored, with 30 worms per line analyzed in triplicate experimental trials. Plotted data reflect the average of these three separate lines. ^***P<0.001 (Bonferroni's multiple comparison test). Error bars reflect standard deviation

Overexpression of 14-3-3θ protects M17 cells from rotenone toxicity. (a–c) Cell lines stably transfected with either 14-3-3θ or empty vector were treated with varying concentrations of rotenone for 24 h (a), 30 h (b), or 48 h (c). Cell death was assayed by LDH release into the culture media. LDH release into media was normalized to total LDH release for each well. The 14-3-3θ-overexpressing line was more resistant to rotenone at several different concentrations compared to control stable cells at all time points tested. (d) At the 48 h time point, a second 14-3-3θ-overexpressing stable clone was tested to verify these results. Results reflect 2–3 independent experiments with at least two replicates per experiment. ^*P<0.05, ^**P<0.01, ^***P<0.001 (Bonferroni's multiple comparison test). (e) To confirm these findings using an alternative cell death assay, we transiently transfected with V5/His-tagged 14-3-3θ construct plasmid into naive M17 cells. Control cells were transfected with GFP. At 24 h after transfection, cells were treated with rotenone at 0, 0.2, or 1 μM for 24 h. Afterwards, cells were fixed and immunostained with an antibody against V5 or GFP, followed by nuclear staining with Hoechst 33342. Nuclei of transfected cells were scored as normal or showing apoptotic changes. Rater was blind to experimental condition. n=8 for each experimental condition. ^*P<0.05, ^***P<0.001 (Bonferroni's multiple comparison test). (f) Amount of insoluble α-syn was reduced in 14-3-3θ cells treated with 5 μM rotenone for 24 h compared to control cells. After rotenone treatment, cell lysates were separated into Triton X-100 soluble and insoluble fractions and blotted with an antibody against α-syn. Representative western blot of insoluble fractions is shown. Densitometric quantification of the multiple α-syn bands includes four independent experiments; we quantified the region between ∼30 and ∼70 kDa where all major bands were found. ^**P<0.01 (Tukey's multiple comparison test). Error bars reflect S.E.M.