14-3-3ζ Contributes to Tyrosine Hydroxylase Activity in MN9D Cells

Abstract

The 14-3-3 proteins stimulate the activation of tyrosine hydroxylase (TH), the rate-limiting catecholamine biosynthetic enzyme. To explore if particular endogenous 14-3-3 isoforms specifically affected TH activity and dopamine synthesis, we utilized rodent nigrostriatal tissues and midbrain-derived MN9D dopaminergic cells. Extracts from ventral midbrain and MN9D cells contained similar pools of 14-3-3 mRNAs, with 14-3-3ζ being relatively abundant in both. Protein levels of 14-3-3ζ were also abundant. [³²P]Orthophosphate labeling of MN9D cells, followed by co-immunoprecipitation with pan-TH and pan-14-3-3 antibodies brought down similar amounts of phosphorylated TH in each, confirming that 14-3-3-bound phosphorylated TH in our cells. Co-immunoprecipitation of striatal tissues with a pan-TH antibody precipitated 14-3-3ζ but not another potential TH regulatory isoform, 14-3-3η. In whole cell extracts from MN9D cells after 14-3-3 small interfering RNA treatments, we found that 14-3-3ζ knockdown significantly reduced TH activity and dopamine synthesis whereas knockdown of 14-3-3η had no effect. 14-3-3ζ was found co-localized on mitochondria with TH, and its knockdown by small interfering RNA reduced TH phosphorylation and TH activity in the mitochondrial pool. Together the data support a role for 14-3-3ζ as an endogenous activator of TH in midbrain dopaminergic neurons and furthermore, identify mitochondria as a potential novel site for dopamine synthesis, with implications for Parkinson disease.

Parkinson disease (PD)3 is a progressive neurodegenerative disorder with widespread central nervous system pathology (1). PD motor symptoms emerge after a significant loss of midbrain dopaminergic neurons (2), which reduces striatal dopamine (DA) levels (3). Successful pharmacological treatments for PD often act to restore DA levels, but are unable to halt disease progression (4). To explore alternative strategies that may restore and also be protective, we are working to identify endogenous physiological regulators of DA that may serve as novel therapeutic targets for treating the DA deficit of PD.

One possible therapeutic target is a highly conserved family of multifunctional proteins, 14-3-3, which have long been acknowledged to stimulate the activity of tyrosine hydroxylase (TH; EC 1.14.16.2) (5), the rate-limiting enzyme in catecholamine biosynthesis (6). In mammals, there are seven major 14-3-3 isoforms (β, γ, η, ε, ζ, σ, and τ/θ) (7) with the first five being particularly abundant in brain (8), comprising ∼1% of soluble brain protein (9). With regard to 14-3-3 function, the proteins dimerize (10, 11), after which they can bind to their targets. In doing so, 14-3-3 proteins contribute significantly to several pathways including cell cycle progression, transcriptional activation, signal transduction, and intracellular trafficking (12). Binding of 14-3-3 is often controlled by serine phosphorylation of the target protein (13).

Regarding 14-3-3 and TH, in vitro assays demonstrate that 14-3-3 proteins form a complex with TH after TH serine 19 (Ser-19) phosphorylation, with TH activity increasing ∼2.5-fold after 14-3-3 binding (14). TH Ser-19 phosphorylation is mediated by calcium calmodulin-dependent protein kinase II (CaMKII) (14), and also by other kinases (15, 16). CaMKII phosphorylation increases TH activity only in the presence of 14-3-3 (14, 16, 17). Three 14-3-3 isoforms, β, η, and ζ, have been shown to interact with TH that is phosphorylated on Ser-19 (14, 16). Mutation of Ser-19 to glutamate mimics TH Ser-19 phosphorylation, which enhances TH activity and TH stability (18); whereas cell models reveal that phosphorylation of Ser-19 stimulates a 3-fold increase in the rate of TH Ser-40 phosphorylation, which enhances TH activity (19), an effect also noted in vitro (16). Such phosphorylation promotes TH activation, which in turn contributes to DA synthesis (20). Although a role for 14-3-3 in TH regulation is fairly well known, no one has demonstrated binding between endogenous TH and endogenous 14-3-3 proteins, or evaluated if a particular 14-3-3 isoform physiologically influences TH activity or DA synthesis in dopaminergic cells.

Dopaminergic neurons of the midbrain substantia nigra pars compacta are the source of the axonal fibers that terminate in the striatal neuropil (21, 22), such that evaluating nigral cell bodies in midbrain as well as their axon terminals in striatum promises to provide fundamental clues concerning TH regulation. Immunohistochemical staining of mouse brain with isoform-specific 14-3-3 antibodies by others reveals that 14-3-3ζ protein is particularly enriched in the striatal neuropil, whereas other 14-3-3 isoforms are much less abundant there, or even absent (8). This observation prompted us to explore whether 14-3-3ζ might play a role in TH regulation. Another 14-3-3 isoform that we evaluated was 14-3-3η, the isoform studied in seminal in vitro analyses of 14-3-3-mediated effects on TH (5, 14). Furthermore, 14-3-3η is abundant in neurons (23), including those of substantia nigra (24), although it is less plentiful in striatal neuropil than 14-3-3ζ (8), and η mRNA is most abundant in cerebellum, hippocampus, and olfactory bulb (25). We considered other candidates including 14-3-3β, because the cDNA for this isoform, when myc-tagged and overexpressed in PC12 cells, binds TH as measured by immunoprecipitation with a Myc antibody, a method that also brings down Myc-tagged 14-3-3η overexpressed in PC12 cells (14). However, 14-3-3β immunoreactivity although present on postsynaptic striatal neurons (26), has limited distribution in striatal neuropil, similar to 14-3-3γ (27), making these less likely candidates for TH regulation. A final candidate, 14-3-3τ/θ, can activate TH in vitro (14), however, that isoform is entirely absent from striatum (8). Therefore, we chose the most likely 14-3-3 isoforms, η and ζ, to measure their potential contributions to TH and DA regulation of the nigrostriatal pathway.

Using mouse ventral midbrain and MN9D cells we measured the specific isoforms of 14-3-3 mRNA present in dopaminergic neuronal cells and found that these correlated well with immunohistochemistry by others (8) and also with our own immunoblot data. TH and 14-3-3 interactions were then assessed by co-immunoprecipitation in MN9D cells and striatum. To measure isoform-specific effects of 14-3-3 on TH activity and DA synthesis, we used 14-3-3η or 14-3-3ζ siRNA. Having previously demonstrated that TH localizes to mitochondria (28), we used immunoelectron microscopy and subcellular fractionation and found that mitochondria contained a significant pool of TH that appears to be sensitive to modulation by 14-3-3. These data identify a particular isoform, 14-3-3ζ, as well as a novel subcellular site involved in DA physiology. This knowledge may help point the way to potential restorative treatments for PD.

EXPERIMENTAL PROCEDURES

Cell Culture—MN9D cells (kind gift of Drs. A. Heller and L. Won, University of Chicago) express abundant TH, synthesize DA, and also quantally release DA (28–30). Low-passage cells were grown on TPP plates (LPS, Rochester, NY) in Dulbecco's modified Eagle's medium (D5648; Sigma) and 10% fetal bovine serum (Hyclone, Logan, UT) at 37 °C as previously described (28, 31, 32).

Phosphorylation of Cells and Autoradiography—MN9D phosphorylation was as previously described (28). Briefly, media were replaced with Hepes-buffered saline (150 mm NaCl, 5 mm glucose, 0.1 mm K₂HPO₄, 2.0 mm MgCl, 1.0 mm CaCl₂, and 25 mm Hepes; pH 7.1) supplemented with 0.5 mCi/ml ³²P (NEX-011, disodium ³²PO_4, PerkinElmer Life Sciences) to label endogenous ATP during 1.5-h incubations at 37 °C. Buffers were removed; cells were washed and equilibrated in 200 μl/well Hepes-buffered saline for 20 min at 37 °C and then depolarized 5 min with 250 μm KCl to stimulate CaMKII activity and enhance TH phosphorylation. Co-immunoprecipitation was performed as described below, using rabbit anti-14-3-3 (06-511, Upstate, Lake Placid, NY) or mouse anti-TH MAB318 (Chemicon, Temecula, CA). Proteins were separated by 12–15% SDS-PAGE, gels were dried and exposed to a phosphor screen for five days and analyzed by Storm PhosphorImaging (Molecular Dynamics).

Co-immunoprecipitation (Co-IP) of TH and 14-3-3s—Co-IP was performed as before on adult rat striata (28). Briefly, tissues were homogenized, supernatants collected, and aliquots were separated for protein determination and immunoblots. Mouse anti-TH (5 μg of MAB318, Chemicon, Temecula, CA) was coupled to SiezeX beads following the kit parameters (Pierce). Proteins were eluted, separated on 12–15% SDS-PAGE, and then transferred to nitrocellulose for immunoblots.

Dopamine and Related Catechols—Perchloric acid (90 μl of 1% 0.1 n) was added to each well of MN9D cells on a 6-well plate. The plate was immediately placed on dry ice so that cells were frozen and then thawed. After collection and sonication, samples were centrifuged at 18,000 × g at 4 °C for 10 min and supernatants were transferred to Microspin columns (Millipore, Ultrafree-MC Durapore PVDF 0.22 um) to clear particulates by 12,000 × g with 2.5-min spins. Samples were analyzed immediately or stored at –80 °C until HPLC analysis for dihydroxyphenylalanine (DOPA), DA, dihydroxyphenylacetic acid (DOPAC), and homovanillic acid as previously described (33–35). Briefly, 15–30-μl samples were injected onto C18 columns, 3-μm particle size, 2.0 × 150-mm (ESA Inc., Chelmsford, MA) using a mobile phase consisting of 50 mm H₂NaO₄P·H₂O, 0.72 mm sodium octyl sulfate, 0.075 mm Na₂EDTA, and 16% acetonitrile (v/v), at pH 2.7. The mobile phase was pumped through the system at 0.3 ml/min using an ESA 580 pump (ESA Inc., Chelmsford, MA). Analytes were detected coulometrically using an ESA Coulochem model 5100A detector, an ESA model 5010 conditioning cell, and an ESA model 5014B microdialysis cell (ESA, Inc., Chelmsford, MA). The settings for detection were E₁ = –75 mV, E₂ = +220 mV, and guard cell = +350 mV. The limits of detection for DA were in the femtomole range. Detection for DOPA, DA, DOPAC, and homovanillic acid were identified relative to the retention times set to known standards.

Immunoblotting—Lysates were prepared in 1% Nonidet P-40 containing protease inhibitors and Halt Phosphatase Inhibitor Mixture (Pierce) at 4 °C. Samples were sonicated for 5 s and particulates were eliminated by centrifugation at 14,000 × g for 10 min at 4 °C. Protein concentrations were determined as described above. Samples (20 μg of protein per lane) were separated on 12–15% SDS-PAGE and transferred to nitrocellulose membranes. Loading was assessed by Ponceau S staining before blocking blots in 10% nonfat milk, Tris-buffered saline. Immunoblots were incubated in primary antibodies at 4 °C overnight. Antibodies included: 14-3-3β (gift of Alastair Aitken, University of Edinburgh), 14-3-3γ (sc-731, Santa Cruz), 14-3-3ε (sc-1020, Santa Cruz), 14-3-3ζ (sc-1019, Santa Cruz), 14-3-3η (sc-17287, Santa Cruz, or 18-783-78341, GenWay), β-actin (A5441, Sigma), total-TH (MAB318 or AB151, Chemicon); TH Ser(P)-19 (AB5425, Chemicon); TH Ser(P)-40 (AB5935, Chemicon); and cytochrome c oxidase (COX4, 4844, Cell Signaling). After Tris-buffered saline or phosphate-buffered saline washes, secondary antibodies, tagged with peroxidase or infrared label, were applied for 1 h with signals visualized on BioMax-MR film (Kodak, Rochester, NY) for chemiluminescence (PerkinElmer Life Sciences) or by infrared imaging (LICOR, Odyssey, Lincoln, NB). β-Actin was the loading control for the cytosolic fractions, whereas COX4 was the loading control for the mitochondrial fractions. Data were quantified using ImageQuant (Amersham Biosciences) with normalizing to β-actin or COX4.

Immunoelectron Microscopy—Transmission immunoelectron microscopy was performed as previously described (28). Primary antibodies for 14-3-3ζ (rabbit sc-1019, Santa Cruz) and TH (mouse MAB318, Chemicon) were diluted 1:100. Secondary antibodies were goat anti-rabbit conjugated to 10-nm gold particles and goat anti-mouse conjugated to 5-nm gold particles (Amersham Biosciences) used at a dilution of 1:25.

Isolation of Mitochondria—We utilized a Mitochondrial Isolation Kit for Cultured Cells (Pierce) exactly following the manufacturer's protocol. Mitochondria were isolated from low passage MN9D cells grown to 80–90% confluence on 100-mm plates. Final pellets were resuspended in 1% Nonidet P-40 containing 140 mm NaCl, 3 mm KCl, 25 mm Tris Base plus leupeptin, aprotinin, 4-(2-aminoethyl)benzenesulfonyl fluoride, and Halt Phosphatase Inhibitor Mixture (Pierce) at 4 °C (100 μl). For TH assays, samples were suspended in TH assay buffer (see below). Protein concentrations were determined by Bradford assay (Bio-Rad).

mRNA Levels—Total RNA was extracted from MN9D cells with an RNeasy Mini Kit (Qiagen) and reverse-transcribed to single-stranded cDNA with the SuperScript® First-Strand Synthesis System for RT-PCR (Invitrogen) as per the manufacturer's instructions. Primer sequences for mouse 14-3-3 isoforms (Table 1) were designed using GenBank™ and previously published data (36, 37). Preliminary experiments were used to determine optimal PCR cycles within a linear range of amplification. Based on these results, each PCR was performed using 20 pmol of sense and antisense primers in 50-μl reactions for 18 cycles at 94 °C 45 s, 56 °C 45 s, 72 °C 45 s, and a final extension at 72 °C for 5 min. The amplified products were evaluated on 1.5% agarose gels, producing data that were scanned and quantified with ImageQuant and normalized to β-actin.

TABLE 1.

Primers used for RT-PCR of 14-3-3 and β-actin to evaluate the relative abundance of the various 14-3-3 mRNA isoforms in ventral midbrain and MN9D dopaminergic neuronal cells, both of which are of mouse origin

Molecule	Orientation	T (anneal)	Size	Sequence
		°C	bp
14-3-3β	Sense	64.4	179	5′-ctc ttc ctg gcg tgt cat ct-3′
	Antisense	64.9		5′-act ttg ctt tct gcc tgg gt-3′
14-3-3γ	Sense	63.0	296	5′-gtt ggt ctg gct ctt cat cat-3′
	Antisense	64.4		5′-agg tgc aga gta gac ttg ggt g-3′
14-3-3ε	Sense	63.1	195	5′-ccc cat tcg ttt agg tct tg-3′
	Antisense	63.9		5′-ggt cca cag cgt cag gtt at-3′
14-3-3ζ	Sense	65.1	365	5′-TGC TGG TGA TGA CAA GAA AGG-3′
	Antisense	64.9		5′-GAG GCA GAC AAA GGT TGG AAG-3′
14-3-3η	Sense	66.0	294	5′-atg ggc att tgc tgg act g-3′
	Antisense	64.1		5′-aag gaa tga gtt gtc gct gtg-3′
β-Actin	Sense	62.2	626	5′-CAC TGT GTT GGC ATA GAG GTC-3′
	Antisense	63.9		5′-TTC TAC AAT GAG CTG CGT GTG-3′

siRNA Treatments—A day before transfection, cells were plated and grown to ∼50% confluence. The 14-3-3ζ siRNA (mouse, sc-29585, Santa Cruz Biotechnology) and 14-3-3η siRNA (sc-43582, Santa Cruz) were pools of three target-specific 20–25-nucleotide siRNAs prepared by the manufacturer (Table 2). We utilized Lipofectamine™ 2000 (Invitrogen) for transfection, with 50–100 nm 14-3-3ζ or 14-3-3η siRNA. Scrambled 14-3-3 siRNAs (Santa Cruz) were applied to parallel cultures as negative controls. After 72 h, cells were collected for RT-PCR, HPLC, or mitochondrial isolations.

TABLE 2.

Sequences of the siRNA used for knockdown of 14-3-3ζ and 14-3-3η in MN9D cells

siRNA	Duplex	Sense strand	Antisense strand
14-3-3ζ	1	5′-CUGCUGGUGAUGACAAGAATT-3′	5′-UUCUUGUCAUCACCAGCAGTT-3′
	2	5′-CCAUGUCUAAGCAAAGAAATT-3′	5′-UUUCUUUGCUUAGACAUGGTT-3′
	3	5′-CCUCAGUACUUUACAGAAATT-3′	5′-UUUCUGUAAAGUACUGAGGTT-3′
14-3-3η	1	5-GGAGACAGUUUGCAAUGAUTT-3′	5′-AUCAUUGCAAACUGUCUCCTT-3′
	2	5′-CUGGACUGAUGGUUGCUUUTT-3′	5′-AAAGCAACCAUCAGUCCAGTT-3′
	3	5′-GUAACUCUUUGGCUAUUGUTT-3′	5′-ACAAUAGCCAAAGAGUUACTT-3′

TH Activity—DOPA, formed by the hydroxylation of tyrosine by active TH, was measured in a timed colorimetric assay using established methods (38) with minor modifications. Briefly, samples were sonicated in 8 volumes of 50 mm Hepes buffer containing 10% glycerol, 5 μg/ml leupeptin, 5 μg/ml aprotinin, and 1 mm 4-(2-aminoethyl)benzenesulfonyl fluoride, and then spun 5 min at 4 °C to eliminate particulates. Supernatants were aliquotted separately for protein determination by Bradford and for TH assays. Supernatants (2 μl) were added to 298 μl of H₂O plus 200 μl of a TH assay mixture (50 mm Hepes, 2.0 mm tyrosine, 2 mg/ml catalase, 1 mm FeNH₄SO₄, and 100 mm dithiothreitol) in borosilicate tubes. Samples were then supplemented with tetrahydrobiopterin (PTH₄, 20 mm) and reacted for exactly 2 min at 30 °C. Reactions were halted with 4.8 m HCl and 12.5% sodium nitrite/sodium molybdate. After 20 min at room temperature, samples were supplemented with 2.1 m NaOH (100 μl) and absorbance was immediately read on a diode array spectrophotometer (Agilent Technologies, Santa Clara, CA) at 490 and 800 nm. All samples were assayed in triplicate. DOPA levels were determined relative to freshly prepared standards for each experiment.

Statistical Analyses—Experiments were repeated 2–5 times on independent occasions using duplicate or triplicate samples for the various treatment conditions. Independent sample t tests or ANOVA were performed using Prism 4.0 or Instat software (GraphPad, San Diego, CA). A p value of 0.05 or less was considered significant. Data represent the mean (±S.E.) for each condition.

RESULTS

Multiple 14-3-3 Isoforms Are Present in Similar Relative Amounts in Brain and Midbrain-derived MN9D Neuronal Cells—To quantitatively measure the expression potential of 14-3-3 isoforms in mouse ventral midbrain and MN9D cells, we first used RT-PCR within a linear range of amplification. Quantification of 14-3-3 mRNAs from ventral midbrain (Fig. 1A) and MN9D cells (Fig. 1B) confirmed that 14-3-3β levels were lower than all other isoforms, with ε and γ mRNA being present at similar levels. In ventral midbrain, we noted that levels of 14-3-3η and 14-3-3ζ were also similar (Fig. 1A; p < 0.001, ANOVA). In MN9D cells, the ε, η, and γ mRNAs were significantly less abundant than 14-3-3ζ mRNA, which was the most abundant 14-3-3 isoform (Fig. 1B; p < 0.0001, ANOVA). Thus, it appeared that the relative abundance of 14-3-3 mRNA in both midbrain and MN9D cells was ζ > η > γ ≥ ε > β. These findings are consistent with mouse brain immunohistochemistry, performed by others, using isoform-specific 14-3-3 antibodies in which labeling of the striatal neuropil, a site that is enriched in the terminal processes of ventral midbrain neurons, is strongest for 14-3-3ζ (8).

FIGURE 1.

Ventral midbrain and midbrain-derived MN9D cells contain similar relative amounts of mRNA for the five major brain isoforms of 14-3-3. RT-PCR was performed within a linear range of amplification, using 18 cycles of PCR. A, a representative image of an ethidium bromide-stained agarose gel shows 14-3-3 mRNA levels in mouse ventral midbrain, with a histogram of data from repeated RT-PCR experiments shown below. B, data from a representative ethidium bromide-stained agarose gel of 14-3-3 mRNA from midbrain-derived MN9D cells revealing similar relative levels of the various 14-3-3 isoforms, with a histogram of data from repeated RT-PCR experiments shown below. The 14-3-3ζ signal was set at 100% as a control for comparison to other 14-3-3 mRNAs. The relative sizes of mRNAs in kilobases (Kb) were determined from prestained standards, shown on the left. *, p < 0.05; **, p < 0.01; ***, p < 0.001, ANOVA. Data represent the mean ± S.E. relative to 14-3-3ζ levels.

To evaluate if our mRNA data accurately reflected the expression potential of 14-3-3 proteins in our cells, we next prepared immunoblots with equal amounts of MN9D extracts using isoform-specific 14-3-3 antibodies. The relative protein levels of 14-3-3 were similar, but not identical to RT-PCR data. Notably, 14-3-3ζ appeared to have the highest protein levels: with β, η, and γ protein levels being somewhat lower than ζ, and very similar to each other; whereas 14-3-3ε protein levels appeared lowest overall (Fig. 2). Quantification of data from five independent experiments confirmed the relative levels of 14-3-3 isoforms (Fig. 2, p < 0.05, ANOVA), with post hoc comparisons revealing that only the ε isoform was present at significantly lower levels than 14-3-3ζ protein in MN9D cells (Fig. 2). These results are not unlike published immunoblot data from mouse brain, except for 14-3-3ε, which was much less abundant in MN9D cells than in brain (8). Assuming that all antibody affinities are similar, these data suggest that 14-3-3 protein levels in MN9D cells are as follows: ζ ≥ η = γ = β > ε. As described above, because ζ and η isoforms have been the most extensively studied isoforms with regard to TH regulation and also have similar relative abundance in our cells, we chose those isoforms to further examine for effects on TH.

FIGURE 2.

Immunoblots of 14-3-3 isoform levels in MN9D cells. Equal total protein was separated by SDS-PAGE and transferred to nitrocellulose. A representative immunoblot is shown, which was reacted with 14-3-3 isoform-specific antibodies, revealing that 14-3-3β, η, γ, and ζ isoforms had strong chemiluminescent signals, however, 14-3-3ε had much weaker signal. β-Actin, as a loading control, confirms equivalent protein loading. Histogram of quantitative data for 14-3-3 normalized to β-actin levels, reveals that 14-3-3ζ tended to be the most abundant, but the levels were not significantly greater than β, η, and γ isoforms of 14-3-3; whereas 14-3-3ε levels were significantly lower only when compared with 14-3-3ζ levels. Molecular weights (M_r), determined from prestained standards, are shown on the right. Only the η antibody produced a band at ∼90 kDa that may be nonspecific. *, p < 0.05. Data represent the mean ± S.E. relative to 14-3-3ζ levels.

Co-immunoprecipitation of 14-3-3 Brings Down Phosphorylated TH in MN9D Cells—To confirm the binding of endogenous 14-3-3 to phosphorylated TH in MN9D cells, we labeled cells with [³²P]orthophosphate for 1.5 h after which we stimulated the cells with KCl followed by co-IP of equal amounts of cell extracts using pan-14-3-3 or pan-TH antibodies. After co-IPs, proteins were separated by SDS-PAGE and visualized by autoradiography. We noted equivalent signals at ∼60 kDa in MN9D extracts after TH co-IP or 14-3-3 co-IP, revealing that phosphorylated TH was immunoprecipitated with both antibodies (Fig. 3), and further revealing that endogenous 14-3-3 associates with phosphorylated TH in MN9D cells, although which isoform of 14-3-3 was not yet known.

FIGURE 3.

Co-immunoprecipitation of phosphorylated TH with 14-3-3 in MN9D cells. Duplicate cultures of cells were ³²P-labeled and briefly depolarized with KCl for 5 min to stimulate TH phosphorylation. Equal aliquots of cell extracts were then used for co-IP for TH using a pan-TH antibody or for 14-3-3 using a pan-14-3-3 antibody. A representative autoradiographic image confirms ³²P signal of TH at 60 kDa in the TH co-IP and also in the parallel 14-3-3 co-IP, confirming that 14-3-3 binds to phosphorylated TH in midbrain-derived MN9D neuronal cells. Molecular weights (M_r), determined from prestained standards, are shown on the left. P-TH, phosphorylated TH bands.

Endogenous 14-3-3ζ Interacts with Endogenous TH in Rat Striatum—To evaluate binding interactions between endogenous 14-3-3η or 14-3-3ζ and TH, we performed co-IP from rat striatum using a pan-TH antibody as previously described (28). To demonstrate the efficiency and specificity of the co-IP, we assessed aliquots of input and after co-IP samples as well as negative control co-IPs with preadsorbed TH antibody (Fig. 4). Parallel blots were probed with specific antibodies for 14-3-3η and 14-3-3ζ. We noted measurable levels of 14-3-3η in the input and after co-IP samples, but no 14-3-3η in the TH co-IP (Fig. 4A). In sharp contrast, the 14-3-3ζ signal was apparent in the input, after co-IP, and TH co-IP samples (Fig. 4B). Probing the blots for total TH verified that the co-IP efficiently reduced the level of total TH in the after the co-IP sample, as evidenced by the TH signal in the TH co-IP lanes (Fig. 4, A and B). As expected, preadsorbed TH did not immunoprecipitate TH or 14-3-3 (Fig. 4, A and B). Because 14-3-3 binds to TH that is phosphorylated on Ser-19, we stripped the blot shown in Fig. 4B, and reprobed it using a specific antibody for Ser(P)-19 (39), which revealed Ser(P)-19 TH in input, after co-IP, and TH co-IP samples (Fig. 4C), confirming an association between endogenous 14-3-3ζ and Ser(P)-19 TH in nigral axon terminals of striatum. This result is also consistent with prior immunostaining data showing high levels of 14-3-3ζ, but not 14-3-3η, in striatal neuropil (8).

FIGURE 4.

Co-immunoprecipitation of 14-3-3ζ with TH from rat striatum. A, representative immunoblot from a TH co-IP of striatal homogenates reveals 14-3-3η present in the input and in the final homogenate collected after co-IP, but no signal for 14-3-3η in the TH co-IP lane, revealing that 14-3-3η did not bind striatal TH. Likewise, no signal was noted in the negative control co-IP performed using preadsorbed TH antibody. B, a parallel blot from the same co-IP was reacted with antibodies specific for 14-3-3ζ, revealing 14-3-3ζ in the input and in the final homogenate collected after co-IP, and also in the TH co-IP. No signal was apparent in the negative control co-IP performed with preadsorbed TH antibody. Weak bands at ∼50 and 25 kDa in the negative control lane appear to be IgG bands. C, reprobe of the blot in B with an antibody specific for TH phosphorylated at Ser-19 (PSer19, 1:500) revealed Ser(P)-19 TH in the input, after co-IP, and TH co-IP samples, but not in the preadsorbed control co-IP. The relative molecular weights (M_r), determined from prestained standards, are shown on the right. Input, beginning homogenate; Preads, preabsorbed TH co-IP; Ser(P)-19, TH phosphorylated on serine 19.

14-3-3 siRNA Knockdown Is Efficient and Specific in MN9D Cells—There was no change in cell viability noted between cells treated with the negative control scrambled siRNA or with 14-3-3-specific siRNAs. RT-PCR of RNA extracted from MN9D cells treated with scrambled 14-3-3ζ siRNA did not alter expression of any 14-3-3 isoform (Fig. 5A, left), whereas 14-3-3ζ-specific siRNA efficiently knocked down only 14-3-3ζ mRNA levels (Fig. 5A, right, at arrow). Quantification of data from multiple RT-PCR experiments verified efficient and specific 14-3-3ζ mRNA knockdown in MN9D cells (50.2 ± 3.3%; p < 0.05, ANOVA). Analysis of 14-3-3ζ protein levels in whole cell lysates revealed that the 14-3-3ζ protein was significantly reduced below control levels noted in scrambled siRNA-treated cells (Fig. 5B, 37.1 ± 8.6% p < 0.01, t test). We saw a similar reduction in 14-3-3η protein levels after 14-3-3η-specific siRNA treatment compared with scrambled siRNA-treated control cells (Fig. 5C, 35.7 ± 10.2; p < 0.01, t test), revealing significant and equivalent knockdown of both 14-3-3s after specific siRNA treatments of MN9D cells.

FIGURE 5.

Specificity and efficiency of 14-3-3 knockdown in MN9D cells. A, baseline levels of all five 14-3-3 isoforms are apparent for MN9D cells treated with scrambled siRNA for 72 h. The baseline level of 14-3-3ζ mRNA is apparent at the arrow in the left panel. After 14-3-3ζ-specific siRNA only 14-3-3ζ mRNA levels were reduced at 72 h post-transfection (at arrow, right panel). B, corresponding knockdown of 14-3-3ζ protein levels was evident at 72 h post-transfection with 14-3-3ζ-specific siRNA as measured by immunoblot on duplicate samples, with β-actin loading control demonstrating equivalent protein loading. C, levels of 14-3-3η protein were also diminished on immunoblot of cell extracts 72 h after transfection with 14-3-3η-specific siRNA from duplicate samples. β-Actin, as a loading control, confirms equivalent protein loading. Histogram represents the data from three independent experiments. Scr, scrambled; siRNA, ζ-or η-specific siRNA. **, p < 0.01. Data represent the mean ± S.E. relative to scrambled siRNA.

DOPA and DA Levels Are Reduced in MN9D Cells after 14-3-3ζ siRNA but Not after 14-3-3η siRNA—To assess the impact of 14-3-3ζ and 14-3-3η knockdown on DA synthesis we measured DOPA and DA content of siRNA-treated MN9D cells using HPLC-EC. DOPA is formed by hydroxylation of a tyrosine substrate by active TH. DOPA levels in MN9D cells were significantly reduced to 57.6 ± 13.6% of control levels after 14-3-3ζ siRNA (p < 0.01, t test), but remained similar to scrambled control levels for cells treated with 14-3-3η siRNA (115.0 ± 30.2%, p > 0.05, t test) (Fig. 6A). Correspondingly, DA levels in MN9D cells were also significantly reduced by 14-3-3ζ siRNA (42.6 ± 16.9%; p < 0.05, t test) compared with controls, but not by 14-3-3η siRNA (93.2 ± 19.9%, p > 0.05, t test) (Fig. 6B). Thus, knockdown of 14-3-3ζ but not 14-3-3η reduced the levels of DOPA and DA in MN9D cells, suggesting the possibility that an interaction between 14-3-3ζ and TH physiologically contributes to DA synthesis in midbrain dopaminergic neuronal cells.

FIGURE 6.

Only 14-3-3ζ siRNA reduced DOPA and DA levels in MN9D cells, yet it had no effect on phosphorylated TH levels in whole cell extracts as measured by immunoblot. A, data from siRNA-treated MN9D cells at 72 h post-transfection reveal that DOPA levels, as measured by HPLC, were significantly reduced in cells treated with 14-3-3ζ siRNA (left side), but not after 14-3-3η siRNA treatment (right side). B, similar decreases in DA levels were noted only for cells treated with 14-3-3ζ siRNA, whereas 14-3-3η siRNA produced no change in DA as measured by HPLC of cells 72 h post-transfection. C, immunoblot data from three independent experiments of 14-3-3ζ siRNA-treated MN9D cells revealed no significant difference in TH Ser(P)-19 (left side) or TH Ser(P)-40 (right side) levels in whole cell extracts prepared from 14-3-3ζ siRNA-treated cells. **, p < 0.01. Data represent the mean ± S.E. relative to scrambled siRNA.

14-3-3ζ siRNA Significantly Reduced Phosphorylation of Mitochondrial TH—TH with high levels of phosphorylated serine appear to have higher TH activity, sustained at least in part by interaction with 14-3-3 (40–42). To assess the impact of 14-3-3ζ knockdown on TH phosphorylation, we measured total TH, TH Ser(P)-19, and TH Ser(P)-40 levels by immunoblot from whole cell extracts of MN9D cells following 14-3-3ζ siRNA. We were somewhat surprised to see that the relative levels of Ser(P)-19 and Ser(P)-40 were not reduced in whole cell lysates at 72 h after 14-3-3ζ siRNA (Fig. 6C), even though DOPA and DA levels were significantly reduced in MN9D cells at this time point (Fig. 6, A and B). To assess how DA levels could have been significantly reduced while TH phosphorylation levels remained similar to control levels in whole cell lysates, we further evaluated MN9D cells to establish the source of the changes.

We have previously demonstrated that TH localizes to near mitochondria in MN9D cells along with α-synuclein, another TH regulatory protein that, in contrast to 14-3-3, acts to inhibit TH activity (28). To determine whether 14-3-3ζ localized to mitochondria in MN9D cells, we used transmission electron microscopy after immunolabeling using a 14-3-3ζ-specific antibody. Gold particle labeling revealed that 14-3-3ζ was localized to MN9D cell mitochondria (Fig. 7A), suggesting that this organelle may be a site for physiological interactions between 14-3-3ζ and TH that are associated with DA synthesis. Using double labeling immunoelectron microscopy we further confirmed that TH and 14-3-3ζ co-localized to mitochondria in MN9D cells, as noted by small and large gold particles in close proximity to each other (Fig. 7B). This prompted us to collect mitochondrial fractions from MN9D cells to measure the impact of 14-3-3ζ knockdown on TH and other mitochondrial associated proteins. We first confirmed the purity of the cytosolic and mitochondrial fractions using compartment-specific antibodies, in which immunoblots revealed abundant α-tubulin, a cytoskeletal protein, in the cytosol but absent from the mitochondrial fraction; whereas COX4, a mitochondrial protein was present only in the mitochondrial sample (Fig. 7C). We also measured TH levels, by immunoblot, in each fraction from two independent experiments and found significant levels of TH localized to mitochondria in MN9D cells (40.7 ± 3.1%, p < 0.01, t test). We then assessed other mitochondrially localized proteins by immunoblot in cells treated with scrambled siRNA or 14-3-3ζ siRNA, using COX4 as a loading control (Fig. 7D). We noted that mitochondrial 14-3-3ζ levels were significantly reduced by 14-3-3ζ siRNA compared to control cells treated with scrambled siRNA (Fig. 7D) as confirmed in three independent experiments (48.3 ± 8.2%, p < 0.05, t test). We further noted that total TH levels were unchanged by siRNA treatments, but that 14-3-3ζ siRNA significantly reduced the levels of TH phosphorylated at Ser(P)-19 (74.1 ± 13.9%, p < 0.05, t test) and at Ser(P)-40 (48.7 ± 14.0%, p < 0.05, t test) in the mitochondrial fraction (Fig. 7D). These findings suggested that the reduction in DOPA and DA levels noted in MN9D cells after 14-3-3ζ siRNA may have been largely associated with reduced phosphorylation of TH localized to mitochondria. To further test for this, we measured the impact of 14-3-3ζ knockdown on TH activity in the mitochondrial pool using a well characterized TH activity assay. We noted significant reduction in TH activity in the mitochondrial fraction of 14-3-3ζ siRNA-treated MN9D cells compared to control mitochondria from cells treated with scrambled siRNA in three independent experiments (Fig. 7E, p < 0.001, t test). These findings raise the possibility that 14-3-3ζ may normally interact with TH that is localized to mitochondria in a manner to stimulate TH activity.

FIGURE 7.

Mitochondrially localized 14-3-3ζ contributes to TH phosphorylation and TH activity in MN9D cells. A, transmission immunoelectron microscopy of MN9D cells confirmed the localization of 14-3-3ζ to mitochondria, as evidenced by gold particles (at arrows), which decorate a mitochondrial surface. B, two representative high magnification images of double labeling for 14-3-3ζ (large particles) with TH (small particles) on mitochondria in MN9D cells using immunogold-tagged secondary antibodies to demonstrate co-localization of the proteins. C, a representative immunoblot demonstrates the purity of the isolated mitochondria, where α-tubulin was the marker for the cytosolic fraction and COX4 was the marker for the mitochondrial fraction. D, a representative immunoblot showing reduced 14-3-3ζ protein levels in the mitochondrial pool from MN9D cells treated for 72 h with 14-3-3ζ siRNA (right side) compared with mitochondria from cells treated with scrambled siRNA as a negative control (left side). Total TH levels were unchanged by either treatment; however, phosphorylated TH levels in the mitochondrial pool were reduced after 14-3-3ζ-specific siRNA (right side) as measured using well characterized antibodies specific for TH Ser(P)-19 and Ser(P)-40. COX4 levels were equivalent in both mitochondrial preparations, confirming equal protein loading. E, TH that was localized to mitochondria in MN9D cells was significantly less active after cells were treated with 14-3-3ζ-specific siRNA for 72 h as compared with TH on mitochondria from control cells treated with scrambled siRNA as measured by the ability of TH to convert tyrosine to DOPA in a timed reaction. Histogram shows the mean ± S.E. from three independent experiments from triplicate samples. Cyto, cytosolic fraction; Mito, mitochondrial fraction; Ser(P)-19, TH phosphorylated at serine 19; Ser(P)-40, TH phosphorylated at serine 40. ***, p < 0.001. Size bar in A, 25 nm; and B, 10 nm.

DISCUSSION

TH was the first enzyme shown to bind 14-3-3 proteins (5, 43), an interaction that is modulated by TH phosphorylation (44). The binding of 14-3-3 to TH occurs at Ser(P)-19, an interaction that can stimulate TH activity (16, 41), perhaps by reducing dephosphorylation of TH or by sustaining an active TH conformation (42). Importantly, both η and ζ were the most abundant isoforms of 14-3-3 mRNA in ventral midbrain and in MN9D cells as measured using RT-PCR (Fig. 1), and were also plentiful proteins in MN9D cells (Fig. 2). Furthermore, published immunohistochemistry data (8, 23, 24) made it reasonable for us to test these isoforms for potential contributions to dopaminergic function, especially because recombinant 14-3-3η binds to and stimulates TH activity (43).

We confirmed that endogenous 14-3-3 bound to phosphorylated TH in our cells using co-IPs from ³²P-labeled MN9D cells (Fig. 3). Our use of a pan-14-3-3 antibody for that co-IP necessitated further analysis to determine which isoform(s) of 14-3-3 may have interacted with TH in our cells. Using a pan-TH antibody for co-IP of striatal tissue we discovered that 14-3-3η did not interact with TH, whereas 14-3-3ζ did (Fig. 4). These are among the first data to support a potential functional role for 14-3-3ζ in TH regulation with implications for DA synthesis. They also raise the possibility that co-localization of specific isoforms of 14-3-3 to the same cellular site with TH may significantly contribute to which particular 14-3-3 isoform functionally interacts with and modulates the activity of TH in dopaminergic cells.

To further assess potential contributions of the η and ζ isoforms of 14-3-3 to TH regulation, we performed immunoelectron microscopy and used control and specific 14-3-3η and 14-3-3ζ siRNAs in MN9D cells. Others previously identified 14-3-3ζ, but not 14-3-3η, on mitochondria in rat and human hippocampal neurons (45), and here we demonstrated 14-3-3ζ in close association with TH on mitochondria in MN9D cells. We saw no immunolabeling of mitochondria using 14-3-3η-specific antibodies (not shown). The pool of TH localized to mitochondria in MN9D cells had significantly reduced levels of phosphorylation and activity after knockdown with 14-3-3ζ siRNA, implying that DA synthesis may be modulated, at least in part, in association with these organelles, a hypothesis worthy of further investigation. We previously discovered that α-synuclein, another TH regulatory protein with limited resemblance to 14-3-3 proteins (46) also co-localizes with TH to mitochondria (28). Additional proteins that contribute to DA metabolism also localize to mitochondria, including the mitogen-activated kinase extracellular signal-regulated kinase 2 (ERK2) (47), monoamine oxidase B (48), and protein phosphatase 2A (PP2A) (49, 50), which we also have also verified (not shown). Because all of these proteins can modulate DA synthesis, mitochondria may serve as a site for assembling a macromolecular protein complex that subserves DA synthesis, although this awaits verification. Our findings, along with data from other laboratories, with regard to mitochondria and PD may help explain why these organelles become so compromised in nigral DA neurons during pathogenesis (51), as oxidative species resulting from DA metabolism may contribute significantly to mitochondrial damage.

We noted that following 14-3-3ζ, but not 14-3-3η siRNA treatment, the phosphorylation state of TH was significantly reduced, but only in the mitochondrial pool. This suggested that in MN9D cells, 14-3-3ζ localized to mitochondria may normally bind to Ser(P)-19 on TH in a manner to block TH dephosphorylation, whereas 14-3-3η does not. Furthermore, knockdown of 14-3-3ζ decreased DA synthesis in MN9D cells, as would be anticipated for TH having diminished phosphorylation and reduced enzymatic activity (52). These observations are further supported by data suggesting that 14-3-3 may play a role in DA synthesis by binding to and stimulating TH activity (16), perhaps by holding TH in a conformation that enhances Ser-40 phosphorylation (53), by diminishing TH proteolysis (54), or by acting as a “molecular anvil” to stabilize TH in a more active conformation (55). Together, these findings support a role for 14-3-3ζ as a physiological effector of TH.

With regard to PD, only certain 14-3-3 isoforms accumulate in Lewy bodies (LBs), the intraneuronal inclusions so abundant in nigral neurons during PD pathogenesis (28, 56, 57). The 14-3-3ζ isoform is relatively abundant in LBs, although neither 14-3-3η nor 14-3-3β are LB constituents (24). TH is also present in LBs (58–60), supporting the notion that a functional interaction between these proteins, as described here, may well contribute to their co-accumulation, along with α-synuclein, an abundant LB protein (61). Interestingly, association studies of intragenic polymorphisms rule out 14-3-3η as being causative of PD (57). Collectively, these findings support a distinctive role for 14-3-3ζ in TH regulation, namely, one associated closely with DA synthesis. Intriguingly, the ζ isoform of 14-3-3 has been shown to dissolve aggregated proteins (62) and eliminate misfolded proteins (63), raising the possibility that its accumulation in LBs may be particularly detrimental to DA neurons. We are currently exploring viral gene regulation of 14-3-3ζ in mouse substantia nigra as a model for modulating DA levels in vivo, a strategy that may ultimately prove to be beneficial for the treatment of PD.