EE ameliorates hyperactivity and anxiety, but not motor function, in hA53T α-Syn transgenic mice
Eight-month-old WT or hA53T α-Syn mice were randomly allocated to either the EE group or SC group (n = 15–25 per each group) for two months (Fig. 1a and b). Tests of motor function were performed to determine whether there were motor symptoms in hA53T α-Syn mice at 10 months of age. Grip strength test results showed did not differ significantly between WT (n = 5, 120.20 ± 2.13 g × force), PD-SC (n = 10, 117.50 ± 3.45 g × force), and PD-EE (n = 10, 119.50 ± 1.55 g × force) mice (Fig. 1c). The hanging wire test endurance time did not differ significantly between WT (n = 5, 14.00 ± 4.46 seconds), PD-SC (n = 10, 12.17 ± 2.50 seconds), and PD-EE (n = 10, 13.77 ± 1.78 seconds) mice (Fig. 1d). The cylinder test count did not significantly differ between WT (n = 10, 15.60 ± 2.37), PD-SC (n = 10, 21.60 ± 2.32), and PD-EE (n = 16.80 ± 2.21) mice (Fig. 1e).
The open field test is commonly used to estimate locomotor activity and spontaneous exploration in a new environment (31, 32). During the 25 minutes, total distance traveled significantly increased in PD-SC (n = 15, 38,313 ± 1,614) compared to WT (n = 12, 14,377 ± 512 cm) mice (Fig. 1f). However, the total distance significantly decreased in PD-EE (n = 12, 21,910 ± 2,274 cm) compared to PD-SC mice, and increased in PD-EE (n = 12, 21,910 ± 2,274 cm) compared to WT mice.
To determine whether EE exposure affected anxiety, the percentage of spending time in the inner zone compared to the outer zone (Fig. 1g). The percentage was significantly decreased in PD-SC (n = 12, 7.3 ± 0.8%) compared to WT (n = 10, 20.2 ± 2.7%) mice. However, the percentage significantly increased in PD-EE (n = 11, 13.1 ± 2.4%) compared to PD-SC mice, and decreased in PD-EE (n = 11, 13.1 ± 2.4%) compared to WT mice.
EE reduces degeneration of dopaminergic nerve terminals in NAc of hA53T α-Syn mice
To investigate whether dopaminergic neurons in the substantia nigra pars compacta (SNpc) and ventral tegmental area (VTA) were degenerated at 10 months of age, immunostaining was performed for analysis of TH-positive cells (Fig. 2a). The immunostaining results showed that TH-positive cell bodies (% of WT) in SNpc did not change significantly among the three groups: WT (n = 8, 100.00% ± 8.05), PD-SC (n = 7, 95.19% ± 6.41), and PD-EE (n = 3, 95.05% ± 3.73). Also, TH-positive cell bodies (% of WT) in the VTA did not differ significantly between the three groups: WT (n = 5, 100.00% ± 13.26), PD-SC (n = 3, 104.74% ± 5.07), and PD-EE mice (n = 3, 96.54% ± 5.07) (Fig. 2b).
Immunostaining was performed for analysis of TH density to investigate dopaminergic nerve terminals in the striatum and NAc were degenerated at 10 months of age and the effects of EE exposure on dopaminergic nerve terminals. Immunostaining results showed that dopaminergic nerve terminals in the striatum decreased significantly in PD-SC (n = 5, 57.11% ± 4.40) and PD-EE (n = 5, 95.38% ± 6.82) compared to WT (n = 5, 100.00% ± 5.31) mice (Fig. 2c).
EE increases SNARE expression and alters the expression of dopamine transporter and dopamine receptor in NAc of hA53T α-Syn mice
qRT-PCR and western blot showed the effects of EE on the expression of SNARE genes and associated proteins SNAP-25, Syntaxin1, and VAMP2 in the striatum and NAc of 10-month-old hA53T α-Syn mice. The qRT-PCR results showed that the decreased level of SNAP-25, Syntaxin1, and VAMP2 mRNA expression levels in PD-SC (SNAP-25; n = 4, 0.74 ± 0.04, Syntaxin1; n = 4, 0.39 ± 0.03, VAMP-2; n = 3, 0.64 ± 0.07) tended to restored in PD-EE (SNAP-25; n = 4, 0.93 ± 0.04, Syntaxin1; n = 3, 0.56 ± 0.02, VAMP2; n = 3, 0.89 ± 0.04) compared to WT (SNAP-25; n = 4, 1.00 ± 0.06, Syntaxin1; n = 4, 1.00 ± 0.05, VAMP2; n = 4 1.00 ± 0.06) mice (Fig. 3a). Western blot results indicated that relative protein levels of SNARE proteins significantly increased in PD-EE (n = 5, 0.84 ± 0.01, 0.74 ± 0.01, 1.01 ± 0.01) compared to PD-SC (n = 5, 0.77 ± 0.01, 0.61 ± 0.01, 0.67 ± 0.03) (Fig. 3b).
Next, we investigated the effects of EE on expression of dopamine receptors and dopamine transporters such as DRD1, DRD2, and DAT in the striatum and NAc of 10-month-old hA53T α-Syn mice. The RT-qPCR results showed that the relative mRNA expression of DRD1 was significantly decreased in PD-EE (n = 4, 1.17 ± 0.08) compared to PD-SC (n = 4, 1.53 ± 0.12) mice, but the expression of DAT significantly increased in PD-EE (n = 4, 0.69 ± 0.09) compared to PD-SC (n = 4, 0.28 ± 0.03) mice (Fig. 3c). The expression of DRD2 in PD-SC (n = 3, 1.24 ± 0.10) and PD-EE (n = 4, 1.08 ± 0.07) mice did not differ significantly. Western blot results showed that the relative protein expression of DRD1 was significantly decreased in PD-EE (n = 5, 0.72 ± 0.03) compared to PD-SC (n = 5, 0.72 ± 0.03) mice (Fig. 3d). The relative protein expression of DAT was significantly increased in PD-EE (n = 5, 0.77 ± 0.05) compared to PD-SC (n = 5, 0.6 ± 0.05) mice. Expression of DRD2 in PD-SC (n = 5, 0.87 ± 0.2) and A53T EE (n = 5, 0.77 ± 0.05) mice did not differ significantly.
EE reduces aggregated α-Syn and the interaction between α-Syn to VAMP-2 in NAc of hA53T α-Syn mice
Next, we examined if EE can block α-Syn aggregation since the A53T mutation of α-Syn is known to increase its tendency to aggregate (33). The intensity of α-Syn monomer band (14-kDa) was increased in PD-SC and PD-EE then WT (Fig. 4a). Monomeric and α-Syn aggregates slightly decreased in PD-EE(n = 5, 59.59 ± 21.91, 2.24 ± 0.91) then PD-SC (n = 5, 222.70 ± 50.2, 22.46 ± 8.36). Interestingly, these tendencies that α-Syn monomer in PD-EE was higher than PD-SC was consistent with the result exercise reduced α-Syn oligomer but not monomer in human α-Syn expressing transgenic mice (34).
IHC was performed to investigate the effects of EE on aggregation of α-Syn in striatum and NAc in 10-month-old mice. Both of IHC results showed that pSer129 α-Syn counts tended to decrease in PD-EE (striatum; n = 6, 42.85 ± 7.48, NAc; n = 5, 48.80 ± 22.82, pSer129 α-Syn count / mm3) contrast to PD-SC (striatum; n = 8, 78.50 ± 15.98, NAc; n = 6, 162.67 ± 34.02, pSer129 α-Syn count / mm3) mice (Fig. 4b).
Previous studies showed that pSer129 α-Syn directly bound to the SNARE-protein VAMP2 (35), and α-Syn overexpressed mice showed inhibited intersynaptic vesicle mobility and trafficking (36). To confirm that EE can affect to the interaction between pSer129 α-Syn and VAMP2, in situ PLA assay was conducted in striatum and NAc of PD-SC or PD-EE groups. Red puncta showed the complexes formed between pSer129 α-Syn and VAMP2. Abundant signal was observed in PD-SC of NAc (n = 4, 0.27 ± 0.06), but not in PD-EE group (n = 3, 0.11 ± 0.03) (Fig. 4c). There were no differences between PD-SC (n = 4, 0.19 ± 0.01) and PD-EE (n = 4, 0.13 ± 0.03) in striatum of the hA53T α-Syn mice. We confirm that EE can reduce the interaction of VAMP2 and pSer129 α-Syn in NAc by in situ PLA assay.