Reduced Interaction of VAMP2 and Aggregated α-synuclein by Environmental Enrichment Alleviates Hyperactivity and Anxiety in a Model of Parkinson’s Disease.

and and The therapeutic effects of on of in a mouse model of PD the effects of on non-motor symptoms in human A53T α-Syn overexpressing transgenic (hA53T α-Syn) mice have yet to be determined. In this study, we revealed the on changes in plasticity in the and

presents non-motor symptoms, involving loss of olfactory senses, cognitive decline, sleep disorders, gastrointestinal disorders, sensory disorders, depression, and anxiety, which often appear in the early stages including the pre-motor phase (7).
The pathogenesis of PD is associated with the dysfunction of various brain regions such as substantia nigra, striatum, and cortex with the increased degree of alpha-synuclein pathology (8). Among those brain regions, the nucleus accumbens (NAc), a part of ventral striatum, is the brain region that is mainly responsible for reward and emotional processes and has been implicated in psychiatric and neurodegenerative diseases such as PD (9)(10)(11). Previous studies have shown that the manifestation of non-motor symptoms is related to neurochemical changes and the brain reward circuit including the NAc in PD (9,12). Given the increasing interest in the NAc, various treatments and interventions to this brain region have been conducted to alleviate the non-motor symptoms of PD (13)(14)(15).
Environmental enrichment (EE) is a way of breeding animals in an enormous cage containing running wheels, novel objects, and providing social interactions as a form of a complex stimuli mixture of physical, cognitive, and social experiences (16). In clinical studies, EE is used as a rehabilitation therapy for human patients (17,18). Epidemiological studies supported a link between hard exercise and lessened risk for PD (19,20). Additionally, many studies about the effect of exercise on normal aging or PD supported the advantages of exercise, physical activity, and EE (21)(22)(23). The therapeutic effects of EE in behavioral recuperation of motor function on a mouse model of PD pathology induced by the administration of 1-methyl-4-phenyl-1,2,3,6-tetrahydrophyridine (MPTP) have been reported (24,25).
However, there are no studies on the effects of EE on non-motor symptoms during the pre-motor phase in mice overexpressing human A53T α-Syn (hA53T α-Syn). Through this transgenic mouse model of PD, our study showed the in uence of EE on the induction of synaptic plasticity in the striatum and NAc in the initial phase of PD.
The transgenic mice bred heterozygous progeny that overexpressed one copy of hA53T α -Syn. All animals were raised in a facility quali ed by the Association for Assessment and Accreditation of Laboratory Animal Care (AAALAC) and provided food and water ad libitum with 12-hour light/dark cycles, according to animal protection regulations. The experimental procedures were permitted by the Yonsei University Health System Institutional Animal Care and Use Committee (YUHS-IACUC approval number; 2017-0039).

Genotyping
Genotyping of mice was done based on a manufacturer's protocol from Jackson Laboratories. Genomic DNA was obtained from a 2-mm piece of each mouse tail using the prepGEM Tissue Kit (ZyGEM, New Zealand). The mouse tail tissue was incubated with 1 µL of prepGEM, 10 µL of Buffer Gold, and 89 µL of   autoclaved 3' distilled water at 75 °C for 15 minutes and 95 °C for 5 minutes. The following primers were  used for polymerase chain reaction (PCR): transgene forward, 5'-TCA TGA AAG GAC TTT CAA AGG C-3';  transgene reverse, 5'-CCT CCC CCA GCC TAG ACC-3' (transgene = ~  Housing conditions At  Diego, CA), which consist of a push-pull strain gauge. Each animal grasped a triangular metal wire 2 mm in diameter with its forepaws, then pulled its tail until the animal lose the bar. The machine automatically recorded peak force in gram-force.
Hanging wire test Mice held by their forepaws from a horizontal rod (5 × 5 mm 2 area, 35 cm long, between two poles 50 cm high) tend to assist themselves with their hind limbs to prevent themselves from falling and to aid their progression along the rod. For this test, suspension latencies were recorded for ve minutes (26).

Cylinder test
When a mouse is placed in the cylinder, it will spontaneously rear and use its forepaws for support. For this test, the number of each forelimb touched the cylinder wall (Jeung Do B&P, Seoul, Korea) while the animal was rearing was counted over a period of ve minutes (27).

Open eld test
An open eld test was performed for 25 minutes when mice were 10 months of age to determine whether EE exposure in uenced locomotor activity. Activity was recorded in a square area measuring 30 × 30 × 30 cm 3 . The total distance traveled by mouse was recorded during 25 minutes as an index of hyperactivity (28). The area's oor was composed of 16 sectors. The four inner sectors represented the center, the 12 outer sectors were described as the periphery. Total time spent in four inner sections was recorded as an sign of anxiety (29,30). Mice were individually put into the periphery of the area and let to explore spontaneously for 25 minutes while being recorded with a video camera. The resulting data were examined using the Smart Vision 2.5.21 (Panlab, Barcelona, Spain) video tracking system.

RNA extraction
Mice were sacri ced perfused with cold 1x PBS at 10 months after birth. Total RNA was extracted from the striatum and NAc of mice brains using TRIzol reagent (Invitrogen Life Technologies, Carlsbad, CA, USA). Extracted RNA purity was assessed using the Nanodrop-2000 spectrophotometer (Thermo Fisher Scienti c, Waltham, MA, USA). Puri ed total RNA (1 µg) was used as a template to produce the complementary DNA (cDNA) using The ReverTra Ace qPCR RT master mix with gDNA remover (TOYOBO, Osaka, Japan).
Quantitative real time polymerase chain reaction (qRT-PCR) The following reaction used 1 µL of cDNA in a total volume of 20 µL. qRT-PCR was done in triplicate on a LightCycler 480 (Roche Applied Science, Mannheim, Germany) using the LightCycler 480 SYBR Green master mix (Roche Applied Science, Mannheim, Germany), and the thermocycler conditions were as follows: ampli cations were started with a 5-minute template preincubation step at 95 °C, followed by 40 cycles at 95 °C for 20 seconds, 62 °C for 20 seconds, and 72 °C for 15 seconds. Melting curve analysis initiated at 95 °C for ve seconds, followed by one minute at 60 °C. The speci city of the product was con rmed by melting curve analysis, which showed a distinctive single sharp peak with the expected Tm for all samples. The glyceraldehyde-3-phosphate dehydrogenase (GAPDH) gene was used as the internal control. The expression level of each gene of interest was acquired using the 2 −ΔΔCt method.

Immunohistochemistry (IHC)
Animals were euthanized and perfused with 4% paraformaldehyde (PFA) in 0.1 M phosphate buffer, pH 7.4. Brains were removed and post-xed for 1 hour, followed by cryoprotection in 30% sucrose in TBS containing 0.02% sodium azide. Harvested brain tissue was cryo-sectioned with a slice thickness of 16 µm along the sagittal or coronal plane and IHC was performed on four sections. For immuno uorescence double labeling, sections were stained with the following antibodies: In situ proximity ligation assay (In situ PLA) Animals were euthanized and perfused with 4% paraformaldehyde (PFA) in 0.1 M phosphate buffer, pH 7.4. Brains were removed and post-xed for 1 hour, followed by cryoprotection in 30% sucrose in TBS containing 0.02% sodium azide. Harvested brain tissue was cryo-sectioned with a slice thickness of 16 µm along the sagittal or coronal plane. The sections were stained with VAMP2 (1:400, Abcam, Cambridge, UK) 1:1,000, Synaptic systems) and human pSer129 α-Syn (1:1,000, FUJIFILM Wako Pure Chemical Corporation, Tokyo, Japan) primary antibodies overnight at 4 °C to detect interacting VAMP2 and human pSer129 α-Syn proteins. After rinsing, the sections were simmered with the secondary oligonucleotidelinked antibodies (The Duolink® kit, Olink Bioscience, Uppsala, Sweden) provided in the kit. The oligonucleotides attached to the antibodies were detected using a uorescent probe (Detection Kit 563). The specks were detected by confocal imaging (LSM700, Zeiss, Oberkochen, Germany).

Statistical analysis
Statistical analysis was conducted using Statistical Package for Social Sciences (SPSS) software IBM Corp., released 2017. IBM SPSS Statistics for Windows (Version 25.0. Armonk, NY: IBM Corp.). Data are expressed as the mean ± standard error of the mean (SEM). The results of behavioral tests, qRT-PCR, western blot, and immunohistochemistry were analyzed by the one-way ANOVA followed by a post-hoc Bonferroni to adjust the variance for multiple testing effects (WT, PD-SC, and PD-EE). A P-value < 0.05 was considered statistically signi cant.
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 signi cantly 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).
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 tra cking (36). To con rm 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 con rm that EE can reduce the interaction of VAMP2 and pSer129 α-Syn in NAc by in situ PLA assay.

Discussion
Motor impairments in PD patients are primarily due to a 50 to 70% loss of dopamine neurons in the SNpc (37). The mouse model used in this study, which expresses hA53T α-Syn under the mouse Prnp promoter, does not show degeneration in the SNpc even at the age of 13-to 14-months (38). Dopaminergic neurons in the striatum of 10-month-old hA53T α-Syn mice showed only mild degeneration (~ 20%) compared to WT mice.
The pathogenesis of PD is associated with the increased degree of alpha-synuclein pathology (8). Previous studies have shown that the manifestation of non-motor symptoms like depression and anxiety is related to neurochemical changes and the brain reward circuit including the NAc in PD (9,12). Anxiety is frequent and can be the starting signals of the disease before the onset of motor defects in PD (39,40).
The dysregulation of dopamine levels in NAc related with hyperactivity disorder (41).
The abnormal accumulation of Lewy bodies is the characteristic of Parkinson's disease and is associated with many neurodegenerative diseases. The recent study has revealed that raised the level of impulsive compulsive behaviors in PD is associated with the increased expression of α-Syn proteins and excessive stimulation of dopamine receptors in the NAc of PD patients (42). However, the psychological roles of α-Syn have yet to be fully identi ed. Consistent with the previous study, the present study showed that the mice did not show motor de cits in grip strength test, hanging wire test or cylinder test, but non-motor symptoms, like anxiety and hyperactivity were revealed in the mice as shown by the open eld test results (Fig. 1).
There are numerous lines of evidence have been linked with α-Syn in the control center of neurotransmitter delivery through regulating the formation of the SNARE complex and the size of the synaptic vesicle pool (35,36,43,44). The recent studies showed pSer129 α-Syn proteins can attach to SNARE proteins and interrupt their functions (35,45). In this study, exposure to EE improved the expression of SNAP-25, Syntaxin1, and VAMP2, decreased pathological α-Syn simultaneously (Fig. 3), contrary to hA53T α-Syn mice at 10 months of age ( Fig. 4a and b). Then, we con rmed that EE can ameliorate the close link of VAMP2 and pSer129 α-Syn by in situ PLA assay (Fig. 4c).
The upregulation of dopamine transporter is compensatory responses associated with de cient dopamine signaling (46,47). Kurz et al. reported that the increased striatal dopamine levels in young (8 months) and old (18 months) hA53T α-Syn mice and revealed elevated striatal DRD1 and DRD2 levels in the absence of neurodegeneration (46). Furthermore, as a compensatory effect, the expression of DRD1 could be increased to receive more synaptic signals.
In this study, we explained progressively increasing striatal dopamine levels as an early effect of hA53T α-Syn overexpression prior to neurodegeneration. Our work suggests that EE exerts therapeutic effects on the early symptoms of PD, which includes hyperactivity and anxiety, mainly responsible for the expression of synaptic proteins, dopamine transporters, and dopamine receptors (Fig. 5).

Conclusion
The original functions of v-and t-SNARE proteins help synaptic vesicle to dock and fuse for exocytosis. This event can be interrupted by the aggregated α-Syn proteins in striatum and NAc of PD-SC group. On the other hand, EE can reduce the aggregated α-Syn and enhance the expression level of SNARE proteins, recovering the behavioral functions.  EE reduces aggregated α-Syn and the interaction between α-Syn to VAMP-2 in NAc. (a) Western blot results indicated the relative expressions of α-Syn monomer and oligomer. The expression of α-Syn monomer tends to increase but aggregated α-Syn is decreased in PD-EE than PD-SC mice. (b) Representative images of pSer129 α-Syn immunohistochemistry in striatum and NAc of the three groups.
The density of pSer129 α-Syn tended to decrease in PD-EE compared to PD-SC mice in both region (scale bar = 10 μm). (c)The proximity of VAMP2 and pSer129 α-Syn in the striatum and NAc assessed by proximity ligation assay shows the decreased PLA signals in PD-EE relative to PD-SC mice (scale bar = 10 μm). *P < 0.05, **P < 0.01, and *P < 0.001 versus WT. Data in all panels represent mean ± SEM. α-Syn: αsynuclein, pSer129 α-Syn: phosphorylated serine 129 α-synuclein, PLA: proximity ligation assay, NAc: nucleus accumbens Figure 5 Synopsis of aberrant synaptic signaling in hA53T α-Syn mice recovered by EE. This schematic illustration summarizes the progressive pathology of dopaminergic nerve terminals in the striatum and NAc in PD. Originally, the functions of SNAP-25, Syntaxin1 and VAMP2 help synaptic vesicles to dock and fusion to permit exocytosis. These functions were interrupted by aggregated -Syn proteins; therefore, dopamine release was decreased in the PD-SC group. As a compensatory effect, DRD1s were increased to receive more synaptic signals. EE simultaneously reduced aggregated α-Syn and induced the expression of SNARE proteins like syntaxin1 and VAMP2. SNARE proteins may recover their functions. After all, synaptic vesicles were re-circulated and normalized, so cytosolic dopamine levels may be normalized in NAc. Since dopamine transporters and/or dopamine receptors were normalized, pre-motor symptoms were ameliorated, and the progress of the disease can be delayed.