Fig 2: Overexpression of C2B-truncated Syt11 in the SNpc fails to mediate deficits in DA release and motor behavior. a Schematic of unilateral overexpression of C2B-truncated Syt11 (?C2B) in the SNpc with an AAV9-expressing system. b Representative western blots showing the expression of parkin and Syt11 in the bilateral SNpc from mice with unilateral overexpression of ?C2B as in a. Western blots were performed 1 month after virus injection. c, d Statistics of Syt11 (P = 0.002) and parkin (P = 0.196) expression in the SNpc from mice as in b. e, f Representative amperometric currents (Iamp) and statistics showing the intact DA release from ?C2B-overexpressing dopaminergic terminals in the striatum (P = 0.332, n = 9 pairs in slices from five mice). g METH-induced asymmetric rotations (in 90 min) of control (Ctrl), full-length Syt11 (WT), and ?C2B-Syt11 overexpressing mice (P < 0.001). h–k Footprint data showing that unilateral overexpression of ?C2B in the SNpc fails to induce a motor defect in the contralateral limbs (SD, P = 0.900; range, P = 0.516; overlap, P = 0.917, n = 90 Contra and 85 Ipsi steps from 7 mice). Dashed red lines show the 5 and 95% percentiles of stride length in h. Data are shown as mean ± s.e.m for c, d, f, g, i, j. Paired Student’s t-test for d, f, i, j; one-way ANOVA for c, g; Mann–Whitney test for k, box and whisker plots show medians (central line in the box), ranges between 25th and 75th percentiles (box) and minimum–maximum ranges (whiskers). *P < 0.05, **P < 0.01, ***P < 0.001

Fig 3: Effect of Parkin dysfunction on Syt11 accumulation. a, b Co-immunoprecipitation of Syt11 with parkin. YFP-parkin (or GFP) and Myc-Syt11 was co-expressed in HEK293 cells, and the lysates were immunoprecipitated (IP) with anti-GFP or anti-Myc antibodies, followed by immunoblotting (IB) with antibodies as indicated. Whole-cell lysates were also immunoblotted with the indicated antibodies as controls. c Parkin catalyzes the ubiquitination of Syt11. Ubiquitination of Syt11 in HEK293 cells expressing combinations of Myc-Syt11, YFP-parkin, and Flag-ubiquitin (Flag-Ub) as indicated. Cell lysates were immunoprecipitated with anti-Myc, followed by immunoblotting with anti-Flag antibody. d Parkin mediates proteasome-dependent degradation of Syt11. Immunoblotting for Syt11 in HEK293 cells expressing YFP-parkin or parkin mutants (R42P disrupting the ubiquitin-like domain, or R275W disrupting the RING1 domain of parkin), with or without 12 h treatment with MG132 (10 µM). Data were collected from three independent experiments (P = 0.041 for ANOVA, P < 0.001 for further LSD test between Myc-Syt11/YFP-parkin and Myc-Syt11). e Cartoon of virus injection for unilateral parkin knockdown (KD) and parkin/syt11 double-KD in the SNpc. f Representative western blots showing the expression of parkin, Syt11, and TH in the bilateral SNpc from parkin KD and parkin/syt11 double-KD mice 1 month after virus injection as in e. g–j Statistics of parkin and Syt11 expression in the SNpc from mice as indicated (P = 0.000, 0.040, 0.047, and 0.022; number in parentheses represents number of mice/biological repeats). Data are shown as mean ± s.e.m. One-way ANOVA for d and paired Student’s t-test for g–j, *P < 0.05, ***P < 0.001

Fig 4: Syt11 inhibits endocytosis in neurons. a Statistics showing the decreased transferrin (Tf) uptake of dopaminergic neurons in the SNpc by Syt11 overexpression (OE). Syt11-carrying lentivirus was unilaterally injected into the SNpc in TH-GFP mice and Tf uptake was assessed in the contralateral (Contra) and Syt11-overexpressing (Ipsi, ipsilateral) sides of the SNpc 1 month later (P < 0.001, n represents number of neurons). b TIRF imaging of Clathrin-DsRed showing the inhibitory role of Syt11 in clathrin-mediated endocytosis (CME) in the somata of hippocampal neurons (Hippo soma, data are shown as mean ± s.e.m., P = 0.010, n represents number of imaged neurons). c Representative micrograph and cartoon showing the real-time imaging of synaptophysin-pHluorin during exocytosis and endocytosis. Upper panel, confocal image showing the expression of physin-pHluorin in hippocampal neurons. Lower panel, cartoon showing the fluorescence change of physin-pHluorin during exocytosis and endocytosis. d Sample images showing phsin-pHluorin fluorescence in presynaptic boutons of control (RFP-only) or Syt11 overexpressing (OE) hippocampal neurons before, 2 s after and 40 s after 100 stimuli at 20 Hz. e, f Normalized fluorescence changes and statistics of endocytic tau (t) of synaptophysin-pHluorin in Syt11 overexpressing or control hippocampal synapses (P < 0.001, n represents the number of imaged synaptic boutons). Scale bars, 20 µm for c and 5 µm for d. Unpaired Student’s t-test for b, Mann–Whitney test for a, f, d, box and whisker plots show medians (central line in the box), ranges between 25th and 75th percentiles (box) and minimum–maximum ranges (whiskers). **P < 0.01, ***P < 0.001

Fig 5: Unilateral Syt11 overexpression in the SNpc induces motor abnormalities. a Schematic of unilateral virus injection in the SNpc and time course of experiments. b Representative micrograph of TH staining in an SNpc-containing slice showing that GFP-carrying lentivirus was precisely injected into the SNpc (ipsi) and efficiently infected the dopaminergic neurons. Scale bars, 500 µm. c–f Representative western blots and statistics showing the expression of Syt11 in the bilateral SNpc from mice overexpressing (OE) Syt11 unilaterally and controls 1 month after virus injection (Control, P = 0.691; Syt11-OE, P = 0.022). g, h Methamphetamine (METH) induces progressive asymmetric rotation (rotation latency and rotations in 90 min) of mice with unilateral OE of Syt11 in the SNpc, compared with control virus (latency, P = 0.022, 0.000, 0.000, and 0.000 for 3, 4, 6, and 8 weeks; rotation, P = 0.020, 0.007, 0.003, and 0.000 for 3, 4, 6, and 8 weeks). i–l Footprint data showing the defect in contralateral motor stability of mice with unilateral Syt11-OE in the SNpc. Stride length (i–k, SD, P = 0.003; range, P = 0.004) and front/hind footprint overlap (l, P < 0.001, n = 88 Contra and 84 Ipsi steps from six mice) were quantified, and the dashed red lines in i represent the 5 and 95% percentiles of stride length. m–p Footprint data showing the normal motor stability of mice unilaterally expressing control virus in the SNpc (SD, P = 0.946; range, P = 0.347; foot overlap, P = 0.857, n = 50 Contra and 50 Ipsi steps from six mice). Contra, contralateral; Ipsi, ipsilateral. Data are shown as mean ± s.e.m. for e–h, j, k, n, o. Paired Student’s t-test for e, f, j, k, n, o; unpaired Student’s t-test for g, h; Mann–Whitney test for l, p, box and whisker plots show medians (central line in the box), ranges between 25th and 75th percentiles (box) and minimum–maximum ranges (whiskers). *P < 0.05, **P < 0.01, ***P < 0.001