Age-related changes in dopamine transporters and accumulation of 3-nitrotyrosine in rhesus monkey midbrain dopamine neurons: relevance in selective neuronal vulnerability to degeneration

Abstract

Aging is the strongest risk factor for developing Parkinson's disease (PD). There is a preferential loss of dopamine (DA) neurons in the ventral tier of the substantia nigra (vtSN) compared to the dorsal tier and ventral tegmental area (VTA) in PD. Examining age-related and region-specific differences in DA neurons represents a means of identifying factors potentially involved in vulnerability or resistance to degeneration. Nitrative stress is among the factors potentially underlying DA neuron degeneration. We studied the relationship between 3-nitrotyrosine (3NT; a marker of nitrative damage) and DA transporters [DA transporter (DAT) and vesicular monoamine transporter-2 (VMAT)] during aging in DA subregions of rhesus monkeys. The percentage of DA neurons containing 3NT increased significantly only in the vtSN with advancing age, and the vtSN had a greater percentage of 3NT-positive neurons when compared to the VTA. The relationship between 3NT and DA transporters was determined by measuring fluorescence intensity of 3NT, DAT and VMAT staining. 3NT intensity increased with advancing age in the vtSN. Increased DAT, VMAT and DAT/VMAT ratios were associated with increased 3NT in individual DA neurons. These results suggest nitrative damage accumulates in midbrain DA neurons with advancing age, an effect exacerbated in the vulnerable vtSN. The capacity of a DA neuron to accumulate more cytosolic DA, as inferred from DA transporter expression, is related to accumulation of nitrative damage. These findings are consistent with a role for aging-related accrual of nitrative damage in the selective vulnerability of vtSN neurons to degeneration in PD.

Fig. 1

Advancing chronological age was associated with increases in the percentage of DA neurons containing 3NT, and the vtSN had more DA neurons containing 3NT than did the VTA. (A) 3NT⁺ neuron density was significantly greater in the vtSN than in the VTA of old animals (*P < 0.05). The number of 3NT⁺ neurons did not change with advancing age in the vtSN, dtSN or VTA. (B) The percentage of TH⁺ neurons containing 3NT was significantly increased in old animals compared to young animals (^#P < 0.05). In young, middle-aged and old animals the proportion of vtSN TH⁺ neurons containing 3NT was significantly greater than the proportion in the VTA (*P < 0.05).

Fig. 2

Comparisons between 3NT and TH immunohistochemistry demonstrate that 3NT⁺ neurons in the midbrain were mostly DA neurons, and there was an age-related increase in the percentage of DA neurons containing 3NT. (A vs. B, C vs. D, E vs. F and G vs. H) Visually comparing 3NT staining to TH staining clearly illustrates the similarities between neurons labeled with these markers. This suggests the vast majority of 3NT⁺ neurons in the vtSN, dtSN and VTA were DA neurons. In the vtSN (A and B compared to E and F) and VTA (C and D compared to G and H), the proportion of 3NT⁺ TH neurons was significantly greater in the old animals (E and F, and G and H) than in the young animals (A and B, and C and D). In young (A and B compared to C and D) and old animals (E and F compared to G and H), the proportion of 3NT⁺ TH neurons was significantly greater in the vtSN (A and B, and E and F) than in the VTA (C and D, and G and H). Scale bar, 50 µm in H (applies to A–H).

Fig. 3

Immunofluorescence intensity for 3NT, DAT, VMAT and DAT/VMAT in midbrain DA neurons. (A) Only in the vtSN was there a significant increase in 3NT fluorescence intensity in old animals compared to young animals (^#P < 0.05). In young animals, all three DA subregions had similar levels of 3NT immunofluorescence. By contrast, VTA neurons in middle-aged animals and vtSN neurons in old animals had significantly greater levels of 3NT immunofluorescence than dtSN neurons (*P < 0.05 vs. dtSN). (B) In the vtSN and VTA, DAT did not change with age; however, DAT intensity was greater in middle-aged animals than in old animals (^#P < 0.05). In all three age groups, DAT intensity was greatest in vtSN DA neurons, reaching statistical significance when compared to the dtSN (*P < 0.05 vs. dtSN). (C) No age-related changes were detected in VMAT intensity. In young animals, the DA neurons in all three midbrain subregions had similar VMAT intensity. In middle-aged and old animals, the level of VMAT intensity was greatest in vtSN DA neurons, reaching statistical significance compared to the dtSN (*P < 0.05 vs. dtSN). (D) In the vtSN and VTA, the DAT/VMAT ratio did not change with age; however, the DAT/VMAT ratio was greater in middle-aged animals than in old animals (^#P < 0.05). In young and middle-aged animals, the DAT/VMAT ratio was similar between DAergic subregions; however, in aged animals the DAT/VMAT ratio was significantly greater in vtSN DA neurons than in the dtSN (*P < 0.05 vs. dtSN).

Fig. 4

Aging was associated with increased intensity of 3NT immunofluorescence in vtSN DA neurons, and the intensity in vtSN DA neurons was greater than the intensity of dtSN DA neurons. (A, B, C and D) In the vtSN, the intensity of 3NT (green; A and C) in DA neurons (VMAT⁺ neurons: red in B, and D) of (C) aged animals was significantly greater than those in (A) young animals. (E, F, G and H) In the dtSN, there was not a significant change in the intensity of 3NT immunofluorescence with advancing age (E, young; G, old). In aged animals, the intensity of 3NT in vtSN neurons was greater than the intensity of neurons in the dtSN (compare C and G). In contrast, the intensity of 3NT was statistically similar in all three DAergic subregions of young animals [compare (A) vtSN to (E) dtSN]. (B, D, F and H) The co-localization (yellow) of VMAT (red) with 3NT (green) is shown to confirm that 3NT⁺ neurons were DAergic. Scale bar, 50 µm in H (applies to A–H).

Fig. 5

In midbrain DA neurons, DAT intensity decreased with age in the dtSN, VMAT intensity remained unchanged with age, and the intensity of both transporters was greater in the vtSN than in the dtSN. (A, B, C and D) In the vtSN, DAT intensity was similar in (A) young and (C) old DA neurons. In the dtSN, increasing age was significantly correlated with reductions in the level of DAT immunofluorescence [(B) young and (D) old]. Regional comparisons show that the level of DAT was greater in vtSN DA neurons than in dtSN DA neurons in all age groups (compare A to B and C to D). (A′, B′, C′ and D′) The level of VMAT immunofluorescence was unchanged in all DAergic subregions during normal aging (compare A′ to C′ for vtSN and B′ to D′ for dtSN). The level of VMAT immunofluorescence was similar between DAergic subregions of young animals (compare A′ for vtSN with B′ for dtSN). However, VMAT intensity was greater in (C′) vtSN neurons than in (D′) dtSN neurons in old animals. (A″, B″, C″ and D″) Merged images of DAT (green) and VMAT (red) immunofluorescence confirm the co-localization (yellow) of the two markers in DAergic neurons of the midbrain. Scale bar, 50 µm in D″ (applies to A–D″).

Fig. 6

Higher levels of DAT, VMAT and DAT/VMAT ratios were associated with higher 3NT levels in midbrain DA neurons. (A–D) The co-localization of 3NT (red, B) with DAT (green, A) and VMAT (blue, C) was confirmed using confocal microscopy. Analysis through the z-plane in a confocal z-stack clearly illustrates the co-localization of 3NT in midbrain DA neurons (A–D). (E–H) Linear regression models demonstrate that DAT (green, E), VMAT (blue, G) and DAT/VMAT levels were significant predictors for the level of 3NT (red, F) in individual DA neurons. In addition, the level of DAT, VMAT and DAT/VMAT ratios was positively correlated with 3NT levels. The relationship between DA transporter intensity and 3NT intensity are illustrated in the representative DA neurons in E–H. Neuron 1: DAT = 616, VMAT = 1019, DAT/VMAT ratio = 0.60, 3NT = 1580; Neuron 2: DAT = 636, VMAT = 1111, DAT/VMAT ratio = 0.57, 3NT = 1344; Neuron 3: DAT = 447, VMAT = 869, DAT/VMAT ratio = 0.51, 3NT = 742. All images are from the vtSN of an aged animal. Scale bars, 25 µm in D (applies to A–D) and H (applies to E–H).