Fig 2: Adeno‐associated virus (AAV)‐mediated overexpression of SYNGR3 in striatum increased synaptosomal dopamine (DA) uptake. (a) Young 3‐month‐old wild‐type (WT) and LRRK2 mutant mice received intracranial injection of AAV7 (1 μl; injection rate: 0.1 μl/min) at one side of the brain hemisphere to induce SYNGR3 overexpression driven by Synapsin‐I promotor. WT and LRRK2 mutant mice were injected with AAV7‐SYNGR3 for 3 months before sacrifice. Diagram illustrates the design of the viral expression construct and the site of injection. (b) Overexpression of SYNGR3 on the injected side of the brain was confirmed by immunohistochemistry of SYNGR3 (red) compared to the opposite hemisphere of the same mouse brain. (c) Representative Western blots of two WT and two LRRK2R1441G mutant mice showing overexpression of SYNGR3 in synaptosomal lysates extracted from injected mouse striatum 3 months after AAV injection. (d) Overexpression of SYNGR3 significantly increased striatal synaptosomal [3H]‐DA uptake, compared to the opposite non‐injected side of the striatum (paired t‐test). There was no significant difference in synaptosomal DA uptake at non‐injected side of WT and LRRK2 mutant striatum. *p < .05 represents statistical significance between designated groups by paired t‐test. Abbreviation: ns, not significant. R1441G, LRRK2R1441G knockin mice

Fig 3: Effects of SYNGR3 expression on innate marble burying behavior. (a) Young (3 months old) wild‐type (WT) and LRRK2R1441G mutant receiving stereotaxic injection of either adeno‐associated virus‐7–green fluorescent protein (AAV7–GFP) or AAV7‐SYNGR3 were subjected to marble burying test at 3 months after AAV injection. A total of 15 marbles were evenly distributed on the bedding inside the cage in form of a 5 × 3 matrix. (b) The number of marbles which have been covered two‐thirds by bedding were counted at 15 and 30 min. Total number of marbles buried (at 30 min) by LRRK2 mutant mice were significantly increased after overexpressing SYNGR3 in the striatum. Data are expressed as means ± SEM. (N = 5). *p < .05 and **p < .01 represent statistical significance between the two designated groups by unpaired, Student's t‐test.

Fig 4: SYNGR3 co‐localizes and interacts with dopamine uptake transporter (DAT) in striatum. (a) Immunohistochemistry showed co‐localization of SYNGR3 (red) and DAT (green) in dorsal striatal region of mouse brain. Area of SYNGR3‐DAT colocalization was identified using Adobe Photoshop™, which were shown as white puncta in the right panel. (b and c) Immunogold staining under transmission electron microscopy (TEM) demonstrated spatial proximity between SYNGR3 and DAT in striatum. Multiple snapshots show SYNGR3‐DAT co‐localization in striatal pre‐synaptic termini; magnification: 1:245,000. (d) Immunoprecipitation of DAT using anti‐DAT antibody resulted in co‐precipitation of SYNGR3 from wild‐type (WT) (i) and LRRK2R1441G mutant (ii) mouse brain striatal lysates, and (ii, iv) vice versa. Target bands are highlighted by the rectangular boxes.

Fig 5: Overexpression of SYNGR3 increased cellular dopamine (DA) uptake in human SH‐SY5Y neuroblastoma cells. (a) Immunocytochemistry showing that SYNGR3 tagged with green fluorescent protein (GFP) (green) was overexpressed and co‐localized with synaptophysin (SYP; synaptic vesicle marker protein; red) in human SH‐SY5Y cells; magnification: 1:630. The lower panels showed magnified views of transfected cells in the dashed box. (b) Western blot analysis of total cell lysates after transient overexpression of SYNGR3 compared to empty‐vector controls. (c) Overexpression of SYNGR3 in SH‐SY5Y cells caused significant increase in total cellular [3H]‐DA uptake activity compared with empty‐vector controls. (d) Flow cytometry assay after whole cell immunostaining of cell surface DAT. Mean cellular fluorescent intensity was calculated based on measurements of 20,000 cells in each group. SYNGR3 overexpression did not affect levels of cell surface DAT. *p < .05, and ***p < .001 represent statistical significance between groups by unpaired Student's t‐test.