Aging Reveals a Role for Nigral Tyrosine Hydroxylase ser31 Phosphorylation in Locomotor Activity Generation

BACKGROUND:Tyrosine hydroxylase (TH) regulates dopamine (DA) bioavailability. Its product, L-DOPA, is an established treatment for Parkinson's disease (PD), suggesting that TH regulation influences locomotion. Site-specific phosphorylation of TH at ser31 and ser40 regulates activity. No direct evidence shows that ser40 phosphorylation is the dominating mechanism of regulating TH activity in vivo, and physiologically-relevant stimuli increase L-DOPA biosynthesis independent of ser40 phosphorylation. Significant loss of locomotor activity occurs in aging as in PD, despite less loss of striatal DA or TH in aging compared to the loss associated with symptomatic PD. However, in the substantia nigra (SN), there is equivalent loss of DA or TH in aging and at the onset of PD symptoms. Growth factors increase locomotor activity in both PD and aging models and increase DA bioavailability and ser31 TH phosphorylation in SN, suggesting that ser31 TH phosphorylation status in the SN, not striatum, regulates DA bioavailability necessary for locomotor activity. METHODOLOGY AND PRINCIPAL FINDINGS:We longitudinally characterized locomotor activity in young and older Brown-Norway Fischer 344 F(1) hybrid rats (18 months apart in age) at two time periods, eight months apart. The aged group served as an intact and pharmacologically-naïve source of deficient locomotor activity. Following locomotor testing, we analyzed DA tissue content, TH protein, and TH phosphorylation in striatum, SN, nucleus accumbens, and VTA. Levels of TH protein combined with ser31 phosphorylation alone reflected inherent differences in DA levels among the four regions. Measures strictly pertaining to locomotor activity initiation significantly correlated to DA content only in the SN. Nigral TH protein and ser31 phosphorylation together significantly correlated to test subject's maximum movement number, horizontal activity, and duration. CONCLUSIONS/SIGNIFICANCE:Together, these results show ser31 TH phosphorylation regulates DA bioavailability in intact neuropil, its status in the SN may regulate locomotor activity generation, and it may represent an accurate target for treating locomotor deficiency. They also show that neurotransmitter regulation in cell body regions can mediate behavioral outcomes and that ser31 TH phosphorylation plays a role in behaviors dependent upon catecholamines, such as dopamine

Dichotomy of Tyrosine Hydroxylase and Dopamine Regulation between Somatodendritic and Terminal Field Areas of Nigrostriatal and Mesoaccumbens Pathways

Measures of dopamine-regulating proteins in somatodendritic regions are often used only as static indicators of neuron viability, overlooking the possible impact of somatodendritic dopamine (DA) signaling on behavior and the potential autonomy of DA regulation between somatodendritic and terminal field compartments. DA reuptake capacity is less in somatodendritic regions, possibly placing a greater burden on de novo DA biosynthesis within this compartment to maintain DA signaling. Therefore, regulation of tyrosine hydroxylase (TH) activity may be particularly critical for somatodendritic DA signaling. Phosphorylation of TH at ser31 or ser40 can increase activity, but their impact on L-DOPA biosynthesis in vivo is unknown. Thus, determining their relationship with L-DOPA tissue content could reveal a mechanism by which DA signaling is normally maintained. In Brown-Norway Fischer 344 F1 hybrid rats, we quantified TH phosphorylation versus L-DOPA accumulation. After inhibition of aromatic acid decarboxylase, L-DOPA tissue content per recovered TH protein was greatest in NAc, matched by differences in ser31, but not ser40, phosphorylation. The L-DOPA per catecholamine and DA turnover ratios were significantly greater in SN and VTA, suggesting greater reliance on de novo DA biosynthesis therein. These compartmental differences reflected an overall autonomy of DA regulation, as seen by decreased DA content in SN and VTA, but not in striatum or NAc, following short-term DA biosynthesis inhibition from local infusion of the TH inhibitor α-methyl-p-tyrosine, as well as in the long-term process of aging. Such data suggest ser31 phosphorylation plays a significant role in regulating TH activity in vivo, particularly in somatodendritic regions, which may have a greater reliance on de novo DA biosynthesis. Thus, to the extent that somatodendritic DA release affects behavior, TH regulation in the midbrain may be critical for DA bioavailability to influence behavior

Relationship of dopamine tissue content to tyrosine hydroxylase protein and phosphorylation <i>in vivo</i>.

<p>DA tissue content and its relationship to recovered TH protein and phosphorylation shows differences in terminal field and somatodendritic TH activity in the four DA regions (<i>n</i> = 20 for all regions except SN (19)) in BNF rats. Values for pmols DA per mg protein were striatum = 842±34, SN = 39±2, NAc = 472±18, and VTA = 125±8. Values for ng TH per mg protein were striatum = 343±16, SN = 96±5, NAc = 256±16, and VTA = 179±16. <b>A. pmols DA per ng TH protein in dissected DA neuropil.</b> DA per TH was calculated by dividing the pmols DA per mg protein by the ng TH per mg protein for each sample. DA per TH was significantly greater in terminal field regions compared to somatodendritic regions. Repeated measures ANOVA, <i>p</i><0.0001,<i>F</i> = 60.2, <i>post-hoc:</i> (***str vs. SN, t = 11.6, <i>p</i><0.001; str vs. NAc, t = 2.74, <b>ns</b>; ***str vs. VTA, t = 9.57, <i>p</i><0.001; ***SN vs. NAc, t = 8.83, <i>p</i><0.001; SN vs. VTA, t = 2.00, <b>ns</b>; ***NAc v. VTA, t = 6.83, <i>p</i><0.001). The pmols DA per ng TH values were str = 2.52±0.12, SN = 0.42±0.02, NAc = 2.02±0.20, VTA = 0.78±0.07. <b>B. ser31 phosphorylation stoichiometry.</b> TH phosphorylation stoichiometry at ser31 was significantly higher in the striatum than all other regions while ser31 phosphorylation stoichiometry was significantly higher in terminal field regions compared to somatodendritic regions (ANOVA, <i>p</i><0.0001, <i>F</i> = 53.1, <i>post-hoc</i> (***str v. SN, t = 11.7, <i>p</i><0.001; ***str v. NAc, t = 5.80, <i>p</i><0.001; ***str v. VTA, t = 9.75; <i>p</i><0.001; ***SN v. NAc, t = 5.87, p<0.001; ***NAc v. VTA, t = 3.95, <i>p</i><0.001)). Stoichiometry values were str = 0.290±0.017, SN = 0.069±0.007, NAc = 0.183±0.019, VTA = 0.104±0.007. These differences reflect the differences in DA per TH observed in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0029867#pone-0029867-g004" target="_blank">Fig <b>4A</b></a>. <b>C. ser40 phosphorylation stoichiometry.</b> TH phosphorylation stoichiometry at ser40 was significantly different between all brain regions except between the striatum and the SN with ser40 phosphorylation being greater in the mesoaccumbens pathway than the nigrostriatal pathway (ANOVA, <i>p</i><0.0001, <i>F</i> = 31.8, <i>post-hoc</i> (***str v. NAc, t = 7.52, <i>p</i><0.001; **Str v. VTA, t = 3.89, <i>p</i><0.01, ***SN v. NAc, t = 8.81, <i>p</i><0.001; ***SN v. VTA, t = 5.19, <i>p</i><0.001; **NAc v. VTA, t = 3.62, <i>p</i><0.01). Stoichiometry values were str = 0.025±0.002, SN = 0.016±0.002, NAc = 0.070±0.006, VTA = 0.051±0.004.</p

Relationship of L-DOPA accumulation to tyrosine hydroxylase phosphorylation <i>in vivo</i>.

<p>Comparisons of L-DOPA per TH and TH phosphorylation stoichiometries between dopaminergic brain regions dissected one hour after 50 mg/kg i.p. administration of NSD-1015 to BNF rat (n = 4). <b>A. pmols L-DOPA per ng TH in dissected DA neuropil.</b> There was significantly greater L-DOPA per TH in NAc (3.18±0.34) compared to all other regions (Striatum, 0.43±0.02; SN, 0.50±0.11; VTA 0.71±0.10). Repeated measures ANOVA, <i>p</i><0.0001,<i>F</i> = 48.7, <i>post-hoc:</i> (str vs. SN, t = 0.27, <b>ns</b>; ***str vs. NAc, t = 10.3, <i>p</i><0.001; str vs. VTA, t = 1.06, <b>ns</b>; ***SN vs. NAc, t = 10.0, <i>p</i><0.001; SN vs. VTA, t = 0.79, <b>ns</b>; ***NAc v. VTA, t = 9.21, <i>p</i><0.001). <b>B. ser19 phosphorylation stoichiombetry</b>; TH phosphorylation stoichiometry at ser19 was significantly higher in the SN than all other regions except the VTA (ANOVA, <i>p</i> = 0.0005, <i>F</i> = 16.5, <i>post-hoc</i> (str v. SN, t = 6.52, <i>p</i><0.001; str v. VTA, t = 3.56, <i>p</i><0.05; **SN v. NAc, t = 5.31, <i>p</i><0.01)). Only significant differences between the NAc and the other brain regions are depicted in the figure in order to better draw a comparison to the significant differences in L-DOPA per TH as observed in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0029867#pone-0029867-g002" target="_blank">Fig 2A</a>. Stoichiometry values were str = 0.098±0.005, SN = 0.340±0.024, NAc = 0.143±0.010, VTA = 0.230±0.047. <b>C. ser31 phosphorylation stoichiometry</b>; NAc had significantly greater ser31 phosphorylation stoichiometry than any other region, SN had significantly less than all other regions, and terminal field regions had significantly greater ser31 phosphorylation stoichiometry than their cognate somatodendritic regions (ANOVA, <i>p</i><0.0001, <i>F</i> = 71.3, <i>post-hoc</i> (str v. SN, t = 9.90, <i>p</i><0.001; *str v. NAc, t = 4.11, <i>p</i><0.05; str v. VTA, t = 4.00, <i>p</i><0.05; ***SN v. NAc, t = 14.0, <i>p</i><0.001; SN v. VTA, t = 5.89, p<0.01; ***NAc v. VTA, t = 8.12 <i>p</i><0.001)). Only significant differences between the NAc and the other brain regions are depicted in the figure in order to better draw a comparison to the significant differences in L-DOPA per TH as observed in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0029867#pone-0029867-g002" target="_blank">Fig 2A</a>. Stoichiometry values were str = 0.305±0.025, SN = 0.083±0.011, NAc = 0.398±0.024, VTA = 0.215±0.025. <b>D. ser40 phosphorylation stoichiometry</b>; TH phosphorylation stoichiometry at ser40 was significantly higher in SN than in all other regions (ANOVA, <i>p</i> = 0.0005, <i>F</i> = 16.9, <i>post-hoc</i> (str v. SN, t = 4.01, <i>p</i><0.05; **SN v. NAc, t = 5.19, <i>p</i><0.01; SN v. VTA, t = 6.82, <i>p</i><0.001)). Only significant differences between the NAc and the other brain regions are depicted in the figure in order to better draw a comparison to the significant differences in L-DOPA per TH as observed in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0029867#pone-0029867-g002" target="_blank">Fig 2A</a>. Stoichiometry values were str = 0.085±0.005, SN = 0.118±0.008, NAc = 0.075±0.006, VTA = 0.062±0.004.</p

Relative TH protein recoveries and TH phosphorylation stoichiometries <i>in vivo</i>.

<p><b>A. Relative TH protein per total protein.</b> Standard curve of TH protein ranging from 0.5 to 6.0 ng TH protein used to demonstrate the relative recovery of TH protein in 6 µg total protein from each of the four regions examined. <b>B. Relative ser¹⁹ TH phosphorylation stoichiometry.</b> Standard curve of calibrated ser¹⁹ phosphorylation standard, expressed as total ng phosphorylated ser¹⁹, ranging from 0.5 to 6.0 ng, used to demonstrate the relative phosphorylation stoichiometry at ser¹⁹ among the four regions examined. The somatodendritic compartments of SN and VTA had significantly greater phosphorylation at this site compared to the cognate terminal field regions in Str and NAc. <b>C. Relative ser³¹ TH phosphorylation stoichiometry.</b> Standard curve of calibrated ser³¹ phosphorylation standard, expressed as total ng phosphorylated ser³¹, ranging from 0.5 to 7.0 ng, used to demonstrate the relative phosphorylation stoichiometry at ser³¹ among the four regions examined. The somatodendritic compartments of SN and VTA had significantly less phosphorylation at this site compared to the cognate terminal field regions in Str and NAc. <b>D. Relative ser⁴⁰ TH phosphorylation stoichiometry.</b> Standard curve of calibrated ser⁴⁰ phosphorylation standard, expressed as total ng phosphorylated ser⁴⁰, ranging from 0.3 to 2.0 ng, used to demonstrate the relative phosphorylation stoichiometry at ser⁴⁰ among the four regions examined.</p

Total TH protein as a function of aging.

<p><b>Striatum:</b> (ANOVA, <i>p</i><0.01, <i>F</i> = 9.4, <i>post-hoc:</i> 6 months v 18 months, *<i>p</i><0.05; 6 months v 24 months, ns; 18 months v 24 months, *<i>p</i><0.01). <b>Substantia nigra:</b> (ANOVA, *<i>p</i><0.05, <i>F</i> = 5.8, <i>post-hoc:</i> 6 months v. 18 months, ns; 6 months vs. 24 months, ns; 18 months vs. 24 months, *<i>p</i><0.05). <b>Nucleus Accumbens:</b> (ANOVA, <i>p</i> = 0.14, ns). <b>Ventral tegmental area:</b> (ANOVA, <i>p</i> = 0.15, ns).</p

Increased reliance of cell body regions upon <i>de novo</i> catecholamine biosynthesis.

<p>The ratios of pmols L-DOPA per pmol catecholamine (DA+NE) and pmols DOPAC per pmol DA (a measure of DA turnover) were calculated from HPLC analysis of dopaminergic brain regions dissected from BNF rats (n = 4) one hour following i.p. administration of NSD-1015 (50 mg/kg). <b>A. pmols L-DOPA per pmol catecholamine,</b> the pmols of L-DOPA recovered from each sample were divided by the pmols catecholamines recovered within the same sample. The measurement of catecholamines refers to the total pmols DA combined with pmols NE recovered within a given sample. As L-DOPA represents a newly synthesized catecholamine precursor, the ratio of pmols L-DOPA per pmol catecholamine serves as a measure of a region's reliance upon <i>de novo</i> catecholamine biosynthesis to maintain adequate stores. VTA had the greatest L-DOPA per catecholamine ratio (1.1±0.12) followed by the SN (0.75±0.044) with the striatum (0.17±0.003) and NAc (0.42±0.006) having similarly low ratios. Repeated measures ANOVA, p<0.0001, <i>F</i> = 55.4, <i>post-hoc:</i> (***str vs. SN, t = 7.35, <i>p</i><0.001; str vs. NAc, t = 3.24, <b>ns</b>; ***str vs. VTA, t = 12.2, <i>p</i><0.001; *SN vs. NAc, t = 4.12, <i>p</i><0.05; **SN vs. VTA, t = 4.81, <i>p</i><0.01; ***NAc vs. VTA, t = 8.93, <i>p</i><0.001). <b>B. pmols DOPAC per pmol DA,</b> the pmols DOPAC (a primary rat metabolite of DA) from each sample were divided by the pmols DA recovered within the same sample. This ratio represents a measure of DA turnover. DA turnover was highest in the SN (0.20±0.016) and VTA (0.18±0.032) and significantly less in the NAc (0.063±0.0010) and striatum (0.016±0.0003). Repeated measures ANOVA, p<0.0001, <i>F</i> = 30.8, <i>post-hoc:</i> (***str vs. SN, t = 8.20, <i>p</i><0.001; str vs. NAc, t = 2.03, <b>ns</b>; ***str vs. VTA, t = 6.99, <i>p</i><0.001; ***SN vs. NAc, t = 6.18, <i>p</i><0.001; SN vs. VTA, t = 1.22, <b>ns</b>; **NAc vs. VTA, t = 4.96, <i>p</i><0.01).</p

Recovery of L-DOPA and TH from dopaminergic tissues following i.p. NSD-1015 infusion.

<p>Recovery of L-DOPA and TH from dopaminergic brain regions dissected one hour after 50 mg/kg i.p. administration of NSD-1015 to BNF rat (n = 4). <b>A.</b> pmols L-DOPA per mg protein in dissected DA neuropil. L-DOPA per protein was significantly different in each of the four regions being greatest in the NAc (298±12) followed by the striatum (269±4), the VTA (172±9), and the SN (40±3). Repeated measures ANOVA, p<0.0001, <i>F</i> = 396.0, <i>post-hoc:</i> (***str vs. SN, t = 27.7, <i>p</i><0.001; *str vs. NAc, t = 3.49, <i>p</i><0.05; ***str vs. VTA, t = 11.8, <i>p</i><0.001; ***SN vs. NAc, t = 31.2, <i>p</i><0.001; ***SN vs. VTA, t = 15.9, <i>p</i><0.001; ***NAc vs. VTA, t = 15.3, <i>p</i><0.001). <b>B.</b> ng TH per mg protein in dissected DA neuropil. TH per protein was significantly greater in the striatum (635±37) than the remaining three regions, while TH per protein was also greater in the VTA (260±47) than in either the SN (87±10) or the NAc (96±8) which were not significantly different from one another. Repeated measures ANOVA, p<0.0001, <i>F</i> = 97.5, <i>post-hoc:</i> (***str vs. SN, t = 14.9, <i>p</i><0.001; ***str vs. NAc, t = 14.7, <i>p</i><0.001; ***str vs. VTA, t = 10.2, <i>p</i><0.001; SN vs. NAc, t = 0.23, <b>ns</b>; **SN vs. VTA, t = 4.71, <i>p</i><0.01; **NAc vs. VTA, t = 4.48, <i>p</i><0.01).</p

Relationship of ser19 phosphorylation to ser31 and ser40 phosphorylation <i>in vivo</i>.

<p><b>A. ser19 and ser40</b> Pearson correlation analysis shows a significant positive correlation of inherent ser19 versus ser40 phosphorylation stoichiometries in striatum (Pearson r = 0.54; <i>p</i> = 0.014, <i>n</i> = 20). Line represents linear regression of data (y = 0.494x+0.006; r² = 0.29). All other regions did not have a significant correlation between ser19 and ser40 phosphorylation. <b>B. ser19 and ser31, Midbrain. </b><b>SN:</b> Pearson correlation analysis shows a significant positive correlation of inherent ser19 versus ser31 phosphorylation stoichiometries in SN (Pearson r = 0.55; <i>p</i> = 0.019, <i>n</i> = 18). <b>VTA:</b> Pearson correlation analysis shows a significant positive correlation of inherent ser19 versus ser31 phosphorylation stoichiometries in VTA (Pearson r = 0.55; <i>p</i> = 0.013, <i>n</i> = 20). <b>Combined:</b> Pearson correlation analysis shows a significant positive correlation of inherent ser19 versus ser31 phosphorylation stoichiometries in SN and VTA (Pearson r = 0.67; p<0.0001, n = 38). Line represents linear regression of data (y = 0.17x+0.03; r² = 0.45). No significant relationship of ser19 with either ser31 or ser40 was observed in the nucleus accumbens.</p