Reduced vesicular storage of dopamine causes progressive nigrostriatal neurodegeneration

Abstract

The vesicular monoamine transporter 2 (VMAT2; SLC18A2) is responsible for packaging dopamine into vesicles for subsequent release and has been suggested to serve a neuroprotective role in the dopamine system. Here, we show that mice that express approximately 5% of normal VMAT2 (VMAT2 LO) display age-associated nigrostriatal dopamine dysfunction that ultimately results in neurodegeneration. Elevated cysteinyl adducts to L-DOPA and DOPAC are seen early and are followed by increased striatal protein carbonyl and 3-nitrotyrosine formation. These changes were associated with decreased striatal dopamine and decreased expression of the dopamine transporter and tyrosine hydroxylase. Furthermore, we observed an increase in alpha-synuclein immunoreactivity and accumulation and neurodegeneration in the substantia nigra pars compacta in aged VMAT2 LO mice. Thus, VMAT2 LO animals display nigrostriatal degeneration that begins in the terminal fields and progresses to eventual loss of the cell bodies, alpha-synuclein accumulation, and an L-DOPA responsive behavioral deficit, replicating many of the key aspects of Parkinson's disease. These data suggest that mishandling of dopamine via reduced VMAT2 expression is, in and of itself, sufficient to cause dopamine-mediated toxicity and neurodegeneration in the nigrostriatal dopamine system. In addition, the altered dopamine homeostasis resulting from reduced VMAT2 function may be conducive to pathogenic mechanisms induced by genetic or environmental factors thought to be involved in Parkinson's disease.

Figure 1.

Characterization of VMAT2 LO mice. A, Southern blot analysis of mouse genomic DNA. B, Representative Western blot analysis of striatal synaptosomes from 2-month-old VMAT2 WT, HT, and LO mice. C, [³H] Dopamine uptake in purified vesicles from 2-month-old VMAT2 WT and LO whole brain. V _MAX: WT, 35.6; LO, 9.2; K _M: WT, 0.377; LO, 0.482. Results are presented as the mean V _MAX (pmol/mg/min) ± SEM for three animals per genotype. D, HPLC analysis of striatal dopamine levels of 2-month-old VMAT2 WT, HT, and LO mice. E, HPLC analysis of striatal dopamine levels in aged VMAT2 LO mice. F, Analysis of tyrosine hydroxylase synthesis rate in VMAT2 WT and LO mice after injection of 100 mg/kg of the aromatic acid decarboxylase inhibitor NSD 1015. HPLC results are presented as the mean raw values (ng/mg tissue) ± SEM for four animals per age group. **p < 0.01; ***p < 0.001.

Figure 2.

Ratio of DOPAC/DA and HVA/DA in the striatum of VMAT2 WT and LO mice at 2, 6, and 12 months of age. A, B, Results represent mean ratios of raw values (ng/mg tissue) ± SEM for four animals per genotype at each age. *p < 0.05; **p < 0.01; ***p < 0.001.

Figure 3.

Locomotor activity of VMAT2 WT and LO mice. A, General locomotor activity of VMAT2 WT and LO mice was measured at 2, 6, 12, and 18 months of age. B, Locomotor activity was recorded in 18-month-old VMAT2 WT and LO mice for 2 h before and after administration of 15 mg/kg l-DOPA plus benserazide. Locomotor activity results are presented as the mean distance traveled (cm) ± SEM for 8–10 mice per genotype. *p < 0.05; ***p < 0.001.

Figure 4.

Evidence of oxidative stress in aged VMAT2 LO mice. A, HPLC-EC analysis of cysteinyl-DOPA adducts in aged VMAT2 mice. Striatal samples from VMAT2 WT and LO mice at 2 and 12 months of age were used for HPLC determination of cysteinyl-DOPA adducts. A significant increase in cysteinyl-DOPA adducts was seen in VMAT2 LO mice at 2 and 12 months of age compared with their age-matched controls. Results represent the mean raw values (pg/mg tissue) ± SEM for four animals per genotype at each age. ***p < 0.001. B, HPLC-EC analysis of cysteinyl-DOPAC adducts in aged VMAT2 mice. Striatal samples from VMAT2 WT and LO mice at 2 and 12 months of age were used for HPLC determination of cysteinyl-DOPAC adducts. A significant increase in cysteinyl-DOPAC adducts was seen in VMAT2 LO mice at 2 and 12 months of age compared with their age-matched controls. Results represent the mean raw values (pg/mg tissue) ± SEM for four animals per genotype at each age. ***p < 0.001.

Figure 5.

A, Dot blot analysis of striatal protein carbonyls. Striatal samples from VMAT2 WT and LO mice at 2, 6, 12, and 18 months were analyzed for protein carbonyl levels. No change was seen in carbonyl levels in VMAT2 WT mice at any time point. A significant increase in carbonyls was observed in the VMAT2 LO mice at 12 and 18 months, compared with WT. Columns represent the percentage change from control. Results represent the mean ± SEM for four animals per genotype at each age. ***p < 0.001. B, Western blot analysis of striatal samples for 3-NT. Striatal samples from VMAT2 WT and LO mice at 2, 6, 12, and 18 months were analyzed for 3-nitrotyrosine levels. No change was seen in 3-NT levels in VMAT2 WT mice at any time point. A significant increase in 3-NT was observed in the VMAT2 LO mice at 12 and 18 months, compared with WT. C, Representative dot blots corresponding to each age and genotype. Columns represent the percentage change from control. Results represent the mean ± SEM for four animals per genotype at each age. ***p < 0.001.

Figure 6.

Analysis of glutathione and MnSOD in aged VMAT2 mice. A, HPLC-UV analysis of GSH redox potentials in aged VMAT2 mice. Striatal samples from VMAT2 WT and LO mice at 2 and 18 months of age were used for determination of GSH redox potentials. No significant change was seen in redox states in VMAT2 LO at 2 and 18 months of age compared with their age-matched controls. Results represent the mean raw values of E _h (mV) ± SEM for four animals per genotype at each age. B, HPLC-UV analysis of GSH concentrations in aged VMAT2 mice. Striatal samples from VMAT2 WT and LO mice at 2 and 18 months of age were used for determination of GSH concentrations. A significant decrease in GSH was seen in the VMAT2 WT and LO animals at 18 months of age compared with WT and LO animals at 2 months of age. Results represent the mean raw values (μm) ± SEM for four animals per genotype at each age. *p < 0.05. C, Western blot analysis of striatal samples for MnSOD. Striatal samples from VMAT2 WT and LO mice at 2 and 18 months were analyzed for MnSOD levels. A significant decrease was seen in the VMAT2 LO animals at 18 months of age compared with their age-matched controls. No change was seen in MnSOD levels in VMAT2 WT mice. Columns represent the percentage change from control. Results represent the mean ± SEM for four animals per genotype at each age. *p < 0.05.

Figure 7.

Immunohistochemical analysis of DAT and TH in striatum of aged mice. A, DAT immunoreactivity was reduced in VMAT2 LO mice at 6, 12, 18, and 22 months of age, whereas TH immunoreactivity (B) was reduced at 18 and 22 months of age. An analysis was performed on three to four animals per genotype at each age. Representative sections are shown. Scale bars, 300 μm.

Figure 8.

High-power magnification of DAT fiber density in the striatum of aged mice. High-power magnification (40×) of DAT innervation in the striatum of aged VMAT2 mice shows a reduction in DAT immunoreactivity at 6 and 12 months, with an additional reduction in fiber density seen at 18 and 22 months. An analysis was performed on three to four animals per genotype at each age. Representative sections were taken from the middle region of the caudate-putamen and correspond to sections in Figure 7. Scale bar, 100 μm.

Figure 9.

High-power magnification of TH fiber density in the striatum of aged mice. High-power magnification (40×) of TH immunoreactivity in the striatum of aged VMAT2 mice shows a reduction of TH fiber density at 18 and 22 months of age compared with WT. Analysis performed on three to four animals per genotype at each age. Representative sections were taken from the middle region of the caudate-putamen and correspond to sections in Figure 7. Scale bar, 100 μm.

Figure 10.

Immunoblotting analysis of DAT and TH in striatum of aged mice. A, Western blot analysis of striatal synaptosomes from VMAT2 WT and LO mice show an age-dependent reduction in DAT expression in LO mice beginning at 6 months of age. B, Western blot analysis of striatal synaptosomes from VMAT2 WT and LO mice show a reduced expression of TH in VMAT2 LO mice at 18 months of age. Columns represent percentage change from control. Results represent the mean ± SEM for four animals per genotype at each age. **p < 0.01; ***p < 0.001.

Figure 11.

TH+ and Nissl cell counts in the substantia nigra of 2, 18, and 24 month VMAT2 WT and LO mice. A significant reduction in TH+ cells was observed in the 18-month-old VMAT2 LO mice compared with WT (VMAT2 WT, 6193 ± 88.1; VMAT2 LO, 5291 ± 164.0; p < 0.002). In addition, a greater reduction in TH+ cells was seen in 24-month-old VMAT2 LO mice compared with WT (VMAT2 WT, 5951 ± 205.2; VMAT2 LO, 4468 ± 185.7; p < 0.002). Furthermore, a significant reduction in Nissl cells was seen at 18 months (VMAT2 WT, 7307 ± 111; VMAT2 LO, 6312 ± 166; p < 0.005) and 24 months (VMAT2 WT, 6746 ± 135.4; VMAT2 LO, 5500 ± 210.1; p < 0.005) between VMAT2 WT and LO mice. Results represent the mean ± SEM for three animals per genotype at each age. *p < 0.05; ** p <0.01; ***p < 0.001.

Figure 12.

Determination of neuronal degeneration in aged VMAT2 LO mice. A, Fluoro-Jade B staining in 22-month-old VMAT2 WT mice. B, Fluoro-Jade B-positive neurons in the SNpc of 22-month-old VMAT2 LO mice. C, Corresponding TH staining. Note the lack of Fluoro-Jade immunoreactivity in A. D, Corresponding TH-immunoreactive neurons. Arrows denote neurons that are positive for Fluoro-Jade (B, D). Accumulation of silver deposits in SNpc of 22-month-old VMAT2 WT (E) and LO (F) mice. Arrows indicate silver grain deposition in degenerating neurons. Analysis was performed on thee to four animals per genotype. Representative sections are shown. Scale bars, 100 μm.

Figure 13.

Synuclein neuropathology in aged VMAT2 LO mice. A, B, An increase in α-synuclein immunoreactivity in the SNpc was seen in the VMAT2 LO mice compared with WT at 18 months of age. C, D, An increased accumulation of α-synuclein was seen in 22-month-old VMAT2 LO SNpc compared with WT. Analysis was performed on three to four animals per genotype. Representative sections are shown. Scale bars: 300 μm; inset, 20 μm.