Slc6a17 Expression in the Mouse Brain.

(A) A schematic diagram illustrating the strategy for the generation of Slc6a17-2A-CreERT2 mice. Crossing of Slc6a17-2A-CreERT2 mice with Ai14 (LSL-tdTomato) mice allowed specific labeling of Slc6a17-expressing neuron after tamoxifen injection. More details in Figure S1A. (B-I) Representative coronal sections of Slc6a17-2A-CreERT2::Ai14 mice. Numbers above images indicate the anteroposterior position of the section from Bregma in millimeters (mm), based on(Paxinos and Franklin, 2019). Scale bars=1 mm. (J) Slc6a17-positive neurons in the basolateral amygdaloid nucleus (BLA) and the central amygdaloid nucleus (CeA). Scale bars=100 μ. (K) Hippocampal expression of Slc6a17. Slc6a17-positive neurons are densely distributed in the granule cell layer of the dentate gyrus (GrDG), and CA3, with little expression in the CA1 and the CA2. Scale bars=200 μm.

Abbreviations: 2Cb, lobule 2 of the cerebellar vermis; 3Cb, lobule 3 of the cerebellar vermis; 4/5Cb, lobule 4 and 5 of the cerebellar vermis; AON, accessory olfactory nucleus; AM, anteromedial thalamic nucleus; AV, anteroventral thalamic nucleus; BLA, basolateral amygdaloid nucleus; Ent, entorhinal cortex; CB, cerebellum; Cg1/2, cingulate ccortex; CIC, central nucleus of the inferior colliculus; Ctx, cortex; Hip, hippocampus; LO, lateral orbital cortex; LS, lateral septal nucleus; M1, primary motor cortex; M2, secondary motor cortex; OB, main olfactory bulb; Pir, piriform cortex; Pn, pontine nuclei; Rt, reticular thalamic nucleus; S1, primary somatosensory cortex; S2, secondary somatosensory cortex; SCN, suprachiasmatic nucleus; SFO, subfornical organ; SuG, superficial gray layer of superior colliculus; Sim, simple lobule; Th, thalamus; V1, primary visual cortex; V2, secondary visual cortex; VPM, ventral posteromedial nucleus.

Slc6a17-KO Mice Exhibited Impaired Memory Formation.

(A) A schematic diagram illustrating the knock-out strategy for generating Slc6a17-KO mice by CRISPR/Cas9. Exon 2 encoding the first 99 amino acids of Slc6a17 was deleted. More details in Figure S2A. (B) A diagram of experiments for contextual and cued fear conditioning (n=16, 23, 18 for Slc6a17+/+, Slc6a17+/- and Slc6a17-/-, respectively). Detailed description in Experimental Procedures. (C) Slc6a17-/- mice exhibited a significant decrease of freezing behavior in both cued and contextual fear conditioning (Day 2 context a, p=0.0062 for Slc6a17+/+ vs. Slc6a17-/-, p<0.0001 for Slc6a17+/- vs. Slc6a17-/-; Day 3 tone, p=0.0093 for Slc6a17+/+ vs. Slc6a17-/-, p=0.0012 for Slc6a17+/- vs. Slc6a17-/-). (D) A diagram of experiments for modified Morris water maze task (n=15, 18, 16 for Slc6a17+/+, Slc6a17+/- and Slc6a17-/-, respectively). Detailed description in Experimental Procedures. (E) Latency to find the hidden platform during the training session. Slc6a17-/- mice differred significantly from Slc6a17+/+ and Slc6a17+/- mice (Two-way ANOVA; main effect of training day: F (4.681, 210.7) = 46.55, p<0.0001; main effect of genotype: F (2, 45) = 21.53, p<0.0001; main effect of training day x genotype: F (12, 270)=3.762, p< 0.0001; Tukey’s multiple comparisons test: day 2, p=0.0004 for Slc6a17+/+ vs. Slc6a17-/-, p=0.0002 for Slc6a17+/- vs. Slc6a17-/-; day 5, p= 0.0002 for Slc6a17+/+ vs. Slc6a17-/-, p= 0.008 for Slc6a17+/- vs. Slc6a17-/-; day 6, p=0.0002 for Slc6a17+/+ vs. Slc6a17-/-, p=0.0027 for Slc6a17+/- vs. Slc6a17-/-; day 7, p<0.0001 for Slc6a17+/+ vs. Slc6a17-/-, p=0.0005 for Slc6a17+/- vs. Slc6a17-/-). (F) Representative swimming traces of each genotype. (G) Percentage of time spent in the target quadrant was significantly different (p=0.0004 for Slc6a17+/+ vs. Slc6a17-/-, p< 0.0001 for Slc6a17+/- vs. Slc6a17-/-). (H) Number of crossings through the platform area was significantly different (p=0.0194 for Slc6a17+/+ vs. Slc6a17-/-, p=0.0054 for Slc6a17+/- vs. Slc6a17-/-). Here and hereafter, data in the Figure are presented as the mean ± SEM, with * indicating p< 0.05, ** indicating p< 0.01, *** indicating p< 0.001, and **** indicating p< 0.0001.

Deficient Memory in Slc6a17P633R Mice.

(A) A diagram illustrating the knock-in strategy for Slc6a17 with a pathogenic point mutation (Slc6a17P633R). It was tagged with 3 repeats of the HA epitope and 3 repeats of the FLAG epitope. More details in Figure S2B. (B) Immunocytochemistry of Slc6a17P633R mouse brains with the anti-HA antibody showed that the subcellular localization of SLC6A17P633R-HA did not co-localized with synaptophysin. It was present in the cytoplasm. Representative view of CA3 was shown. Scale bar=50 μm. (C) Experiments for contextual and cued fear conditioning (n=17, 18, 14 for Slc6a17+/+, Slc6a17P633R/+, Slc6a17P633R/P633R, respectively). Detailed description in Experimental Procedures. (D) Slc6a17P633R homozygous mutants exhibited significant decrease of freezing behavior in both cued and contextual fear conditioning (Day 2 context A, p< 0.0001 for Slc6a17+/+ vs. Slc6a17P633R/P633R, p< 0.0001 for Slc6a17P633R/+ vs. Slc6a17P633R/P633R; Day 3 tone, p= 0.0017 for Slc6a17+/+ vs. Slc6a17P633R/P633R, p= 0.0009 for Slc6a17P633R/+ vs. Slc6a17P633R/P633R). (E) Cartoon figure of experiments for Morris water maze task (n=12, 15, 9 for Slc6a17+/+, Slc6a17P633R/+ and Slc6a17P633R/P633R, respectively). Detailed description in Experimental Procedures. (F) Latency to find the hidden platform during the training session. Slc6a17P633R/P633R mice showed significantly different learning curve from Slc6a17+/+ and Slc6a17P633R/+ mice (Two-way ANOVA; main effect of training day: F (4.381, 144.6) = 20.02, p<0.0001; main effect of genotype: F (2, 33) = 22.65, p<0.0001; main effect of training day x genotype: F (12, 198) = 2.389, p=0.0067; Tukey’s multiple comparisons test: day 2, p= 0.0150 for Slc6a17+/+ vs. Slc6a17P633R/P633R, p= 0.0078 for Slc6a17P633R/+ vs. Slc6a17P633R/P633R; day 3, p= 0.0403 for Slc6a17+/+ vs. Slc6a17P633R/P633R, p= 0.0388 for Slc6a17P633R/+ vs. Slc6a17P633R/P633R; day 5, p= 0.0023 for Slc6a17+/+ vs. Slc6a17P633R/P633R, p= 0.0243 for Slc6a17P633R/+ vs. Slc6a17P633R/P633R; day 6, p= 0.0199 for Slc6a17+/+ vs. Slc6a17P633R/+,p= 0.0010 for Slc6a17+/+ vs. Slc6a17P633R/P633R, p= 0.0077 for Slc6a17P633R/+ vs. Slc6a17P633R/P633R; day 7, p= 0.0074 for Slc6a17+/+ vs. Slc6a17P633R/P633R, p= 0.0129 for Slc6a17P633R/+ vs. Slc6a17P633R/P633R). (G) Representative swimming traces of each genotype. (H) Percentage of time spent in the target quadrant was significantly different (p= 0.0031 for Slc6a17+/+ vs. Slc6a17P633R/P633R, p= 0.0325 for Slc6a17P633R/+ vs. Slc6a17P633R/P633R). (I) Number of crossings through the platform area was significantly different (p= 0.0003 for Slc6a17+/+ vs. Slc6a17P633R/P633R, p= 0.0123 for Slc6a17P633R/+ vs. Slc6a17P633R/P633R). Data in the Figure are presented as the mean ± SEM, with * indicating p< 0.05, ** indicating p< 0.01, *** indicating p< 0.001, and **** indicating p< 0.0001.

Biochemical and Genetically Assisted EM Validation of the Vesicular Localization of SLC6A17.

(A) A schematic diagram illustrating the knock-in strategy for generating Slc6a17-HA-2A-iCre mice. More details in Figure S1D. (B) Higher magnification views of co-immunostaining by anti-Syp and anti-HA antibodies in the hippocampus. Scale bar=10 μ . (C) Immunoisolation of SLC6A17-HA containing vesicles by the anti-HA antibody coated on magnetic beads. SLC6A17-HA fraction was positive for Syp, Syt1, Syb2, v-ATPase, VGluT1, VGluT2, and vGAT, but negative for GluT4 and transferin receptor. (D) Further purification of the LP2 fraction by sucrose gradient showed that SLC6A17-HA was co-immunoisolated with Syp, Syt1 and VGluT1, but not PSD95, ERp72, EEA1, SNAP23, PSMC6 or STX6. (E) A schematic diagram illustrating the APEX2-based labeling strategy with AAV-PHP.eb virus mediated hSLC6A17-APEX2 overexpression in vivo. SLC6A17 was fused in-frame to 3 repeats of the HA tag, a V5 tag, and APEX2. (F) Representative EM image of SVs labeled by hSLC6A17-APEX2 and darkness distribution of DAB positive and DAB negative SVs in sections of Slc6a17-APEX2 mouse brains. Red arrow pointing to APEX2 labeled SVs. White arrow pointing to unlabeled SVs.

Presence of Glutamine in SVs from the Mouse Brain.

(A) A schematic diagram illustrating the procedure to immunoisolate SVs with the anti-Syp antibody for LC-MS analysis of the contents in the SVs. (B) Representative MS results showing Gln signals from SVs immunoisolated by the anti-Syp antibody (anti-Syp) vs the control sample immunoisolated with IgG. (C) Volcano plot of chemical contents in the SVs isolated by anti-Syp vs IgG. The y axis shows p values in log10 and the x axis shows the log2 of the ratio of the level of a molecule immunoisolated by anti-Syp vs IgG. Classical neurotransmitters Glu, GABA and ACh, as well as previously reported substrates of SLC6A17 are listed. (D) Ratios of the level of a chemical immunoisolated by anti-Syp vs IgG (transformed into log2). (E-I) Chemicals were quantified to mole per μg antibody (n=12 for each group): Glu (E, p<0.0001 for anti-Syp vs. IgG); GABA (F, p<0.0001 for anti-Syp vs. IgG); ACh (G, p<0.0001 for anti-Syp vs. IgG); Gln (H, p<0.0001 for anti-Syp vs. IgG); Ser (I, p=0.7553 for anti-Syp vs. IgG).

Gln Enrichment in SVs Containing SLC6A17.

(A) A schematic diagram illustrating the procedure to isolate SLC6A17-containing SVs for chemical analysis of SV contents. (B) Representative result showing MS Gln signals in SVs immunoisolated by the anti-HA beads in SLC6A17HA/+ mice vs those from Slc6a17+/+ mice. (C) Volcano plot of chemical contents in the SVs immunoisolated by anti-HA from Slc6a17HA/+ mice vs Slc6a17+/+ mice. The y axis shows p values in log10 and the x axis shows the log2 of the ratio of the level of a molecule immunoisolated by anti-HA from Slc6a17HA/+ mice vs that from Slc6a17+/+ mice. Classical neurotransmitters such as Glu, GABA and ACh as well as the previously reported substrates of SLC6A17 are listed. (D) Ratios of the level of a chemical immunoisolated by anti-HA from Slc6a17HA/+ mice vs that from Slc6a17+/+ mice (transformed into log2). (E-I) Contents of SLC6A17-containing SVs were quantified to mole per 10 μl anti-HA beads (n=12, for each group): Glu (E, p<0.0001 for Slc6a17HA/+ vs. Slc6a17+/+); GABA (F, p<0.0001 for Slc6a17HA/+ vs. Slc6a17+/+); ACh (G, p<0.0001 for Slc6a17HA/+ vs. Slc6a17+/+); Gln (H, p< 0.0001 for Slc6a17HA/+ vs. Slc6a17+/+); Ser (I, p= 0.7553 for Slc6a17HA/+ vs. Slc6a17+/+).

Increased Gln Transport into SVs Containing SLC6A17 but not into SVs Containing SLC6A17G162R.

(A) A schematic diagram illustrating the strategy for the AAV-PHP.eb virus mediated in vivo overexpression of hSLC6A17-HA and hSLC6A17G162R-HA. (B) Volcano plot of chemical contents in the SVs isolated by anti-HA from the OE-hSLC6A17-HA mice vs WT mice. The y axis shows p values in log10 and the x axis shows the log2 of the ratio of the level of a molecule immunoisolated by anti-HA beads from mice overexpressing hSLC6A17-HA vs that from WT mice. Classical neurotransmitters and previously reported substrates of SLC6A17 are listed.

(C-G) Contents of OE-hSLC6A17-containing SV are quantified to mole per 10 μl HA beads (n=10, for each group): Glu (C, p<0.0001 for OE-hSLC6A17-HA vs. WT); GABA (D, p<0.0001 for OE-hSLC6A17-HA vs. WT); ACh (E, p<0.0001 for OE-hSLC6A17-HA vs. WT); Gln (F, p<0.0001 for OE-hSLC6A17-HA vs. WT); (G, p=0.1655 for OE-hSLC6A17-HA vs. WT). (H) Volcano plot comparing the chemical contents of SVs containing OE-hSLC6A17-HA with those containing OE-hSLC6A17G162R-HA. Classical neurotransmitters and 9 putative substrates of SLC6A17 are listed. Glu is the only classical transmitter significantly increased. Gln is the only substrates significantly increased in hSLC6A17-HA containing SVs vs hSLC6A17G162R-HA containing SVs. (I) Volcano plot comparing the chemical contents of SVs containing OE-hSLC6A17-HA with those containing OE-Syp-HA. Classical neurotransmitters and 9 putative substrates of SLC6A17 are listed. Gln is the only substrates significantly increased in SLC6A17 containing SVs vs Syp-HA containing SVs. (J-N) Contents of SVs containing SCL6A17-HA, SVs containing OE-SLC6A17G162R-HA, SVs containing Syp-HA immunosilated by anti-HA immunoisolation with that from WT mouse brains were quantified to mole per 10 μl HA beads (n=9, 9, 6, 6, for OE-hSLC6A17-HA, OE-hSLC6A17G162R-HA, OE-Syp-HA, and WT, respectively): Glu (J, p<0.0001 for OE-hSLC6A17-HA vs. OE-hSLC6A17G162R-HA; p<0.0001 for OE-hSLC6A17-HA vs. WT; p<0.0001 for OE-hSLC6A17G162R-HA vs. WT; p= 0.0009 for OE-Syp-HA vs. WT; p= 0.1551 for OE-hSLC6A17-HA vs. OE-Syp-HA; p= 0.0618 for OE-hSLC6A17G162R-HA vs. OE-Syp-HA); GABA (K, p= 0.0002 for OE-hSLC6A17-HA vs. WT; p= 0.0002 for OE-hSLC6A17G162R-HA vs. WT; p= 0.0039 for OE-Syp-HA vs. WT; p= 0.9885 for OE-hSLC6A17-HA vs. OE-hSLC6A17G162R-HA; p= 0.9000 for OE-hSLC6A17-HA vs. OE-Syp-HA; p= 0.5840 for OE-hSLC6A17G162R-HA vs. OE-Syp-HA); ACh (L p= 0.0019 for OE-hSLC6A17-HA vs. WT; p= 0.0006 for OE-hSLC6A17G162R-HA vs. WT; p= 0.0467 for OE-Syp-HA vs. WT; p= 0.1054 for OE-hSLC6A17-HA vs. OE-hSLC6A17G162R-HA; p= 0.7563 for OE-hSLC6A17-HA vs. OE-Syp-HA; p= 0.8702 for OE-hSLC6A17G162R-HA vs. OE-Syp-HA); Gln (M, p= 0.001 for OE-hSLC6A17-HA vs. OE-hSLC6A17G162R-HA; p= 0.0018 for OE-hSLC6A17-HA vs. OE-Syp-HA; p= 0.0003 for OE-hSLC6A17-HA vs. WT; P<0.0001 for OE-hSLC6A17G162R-HA vs. WT; p= 0.0189 for OE-Syp-HA vs. WT; p= 0.2749 for OE-hSLC6A17G162R-HA vs. OE-Syp-HA); Ser (N, p= 0.626 for OE-hSLC6A17-HA vs. OE-hSLC6A17G162R-HA; p= 0.8551 for OE-hSLC6A17-HA vs. OE-Syp-HA; p= 0.9874 for OE-hSLC6A17-HA vs. OE-Syp-HA; P<0.0865 for OE-hSLC6A17G162R-HA vs. WT; p= 0.1017 for OE-Syp-HA vs. WT).

Reduced Levels of Gln in the SVs of Slc6a17 KO Mice.

Contents of SVs purified by the anti-Syp antibody from Slc6a17-KO mice were quantified to mole per 10 μg antibody (n=12 for Slc6a17, Slc6a17, Slc6a17): Glu (A, p=0.2749 for Slc6a17+/+ vs. Slc6a17-/-, p=0.2466 for Slc6a17+/- vs. Slc6a17-/-); GABA (B, p=0.665 for Slc6a17+/+ vs. Slc6a17-/-, p=0.2503 for Slc6a17+/- vs. Slc6a17-/-); ACh (C, p=0.9993 for Slc6a17+/+ vs. Slc6a17-/-, p=0.7186 for Slc6a17+/- vs. Slc6a17-/-); Pro (D, p=0.9986 for Slc6a17+/+ vs. Slc6a17-/-, p=0.8035 for Slc6a17+/- vs. Slc6a17-/-); Leu (E, p=0.826 for Slc6a17+/+ vs. Slc6a17-/-, p= 0.61 for Slc6a17+/- vs. Slc6a17-/-); Gly (F, p= 0.3211 for Slc6a17+/+ vs. Slc6a17-/-, p=0.442 for Slc6a17+/- vs. Slc6a17-/-); Ala (G, p=0.9092 for Slc6a17+/+ vs. Slc6a17-/-, p=0.1321 for Slc6a17+/- vs. Slc6a17-/-); Gln (H, p=0.002 for Slc6a17+/+ vs. Slc6a17-/-, p=0.0489 for Slc6a17+/- vs. Slc6a17-/-); Cys (I, p=0.3753 for Slc6a17+/+ vs. Slc6a17-/-, p=0.5718 for Slc6a17+/- vs. Slc6a17-/-); Met (J, p=0.9735 for Slc6a17+/+ vs. Slc6a17-/-, p= 0.13 for +/- vs. Slc6a17-/-); Ser (K, p=0.8678 for Slc6a17+/+ vs. Slc6a17-/-, p=0.7834 for Slc6a17+/- vs. Slc6a17-/-); His (L, p>0.9999 for Slc6a17+/+ vs. Slc6a17-/-, p=0.9922 for Slc6a17+/- vs. Slc6a17-/-).

Physiological Requirement of SLC6A17 for Gln Transport into SVs in vivo.

(A) A schematic diagram illustrating the strategy of Cas9-mediated cleavage of Slc6a17 specifically in Slc6a17 positive neurons, and simultaneous labeling of all SVs in these neurons by Syp-HA. (B) Immunoblot results showing SLC6A17 protein was significantly reduced in targeted neurons, while Syp-HA was efficiently tagged onto the SVs of these neurons. (C) Volcano plot of contents of SVs from AAV-sgRNA/Slc6a17iCre/Cas9+ targeted neurons compared to contents of SVs from control (AAV-sgRNA /Slc6a17iCre/Cas9-) neurons. Glu, GABA, ACh and the 9 previously reported substrates of SLC6A17 are listed. (D) Ratios of the level of a molecule from the SVs of AAV-sgRNA /Slc6a17iCre/Cas9+ neurons vs the level of the same molecule from SVs of AAV-sgRNA /Slc6a17iCre/Cas9- neurons shown as fold change (log2 transformed). GABA level was significantly increased (p=0.0034 for AAV-sgRNA/Slc6a17iCre/Cas9+ vs. AAV-sgRNA /Slc6a17iCre/Cas9-). Gln level was significantly decreased (p< 0.0001 for AAV-sgRNA /Slc6a17iCre/Cas9+ vs. AAV-sgRNA/Slc6a17iCre/Cas9-). (E-H) Contents of SVs from Slc6a17 containing neurons were quantified to mole per 10 μl HA beads (n=18, 27 for Slc6a17 /Cas9 and Slc6a17iCre/Cas9+, respectively): Glu (E, p= 0.0005 for AAV-sgRNA/Slc6a17iCre/Cas9+ vs. AAV-sgRNA /Slc6a17iCre/Cas9-); GABA (F, p=0.0032 for AAV-sgRNA /Slc6a17iCre/Cas9+ vs. AAV-sgRNA /Slc6a17iCre/Cas9-); Gln (G, p<0.0001 for AAV-sgRNA /Slc6a17iCre/Cas9+ vs. AAV-sgRNA /Slc6a17iCre/Cas9-); Ser (H, p=0.0979 for AAV-sgRNA /Slc6a17iCre/Cas9+ vs. AAV-sgRNA/Slc6a17iCre/Cas9-).

Reduced Levels of Gln in the SVs of Slc6a17P633R Mice.

Contents of SVs purified by the anti-Syp antibody from Slc6a17P633R mice were quantified to mole per 10 μ antibody (n=12 for all genotype): Glu (A, p=0.9804 for Slc6a17 vs. Slc6a17P633R/P633R, p=0.9982 for Slc6a17P633R/+ vs. Slc6a17P633R/P633R); GABA (B, p=0.4432 for Slc6a17+/+ vs. Slc6a17P633R/P633R p=0.3898 for Slc6a17P633R/+ vs. Slc6a17P633R/P633R); ACh (C, p=0.8175 for Slc6a17+/+ vs. Slc6a17P633R/P633R, p=0.9804 for Slc6a17P633R/+ vs. Slc6a17P633R/P633R); Pro (D, p=0.9674 for Slc6a17+/+ vs. Slc6a17P633R/P633R, p=0.9989 for Slc6a17P633R/+ vs. Slc6a17P633R/P633R); Leu (E, p=0.7116 for Slc6a17+/+ vs. Slc6a17P633R/P633R, p=0.6696 for Slc6a17P633R/+ vs. Slc6a17P633R/P633R); Gly (F, p=0.7376 for Slc6a17+/+ vs. Slc6a17P633R/P633R, p=0.9971 for Slc6a17P633R/+ vs. Slc6a17P633R/P633R); Ala (G, p=0.9871 for Slc6a17+/+ vs. Slc6a17P633R/P633R, p=0.986 for Slc6a17P633R/+ vs. Slc6a17P633R/P633R); Gln (H, p=0.0029 for Slc6a17+/+ vs. Slc6a17P633R/P633R, p=0.0117 for Slc6a17P633R/+ vs. Slc6a17P633R/P633R); Cys (I, p=0.8143 for Slc6a17+/+ vs. Slc6a17P633R/P633R, p=0.5475 for Slc6a17P633R/+ vs. Slc6a17P633R/P633R); Met (J, p=0.9942 for Slc6a17+/+ vs. Slc6a17P633R/P633R, p=0.995 for Slc6a17P633R/+ vs. Slc6a17P633R/P633R); Ser (K, p= 0.9955 for Slc6a17+/+ vs. Slc6a17P633R/P633R, p=0.8355 for Slc6a17P633R/+ vs. Slc6a17P633R/P633R); His (L, p=0.6177 for Slc6a17+/+ vs. Slc6a17P633R/P633R, p=0.4316 for Slc6a17P633R/+ vs. Slc6a17P633R/P633R).

Generation of Slc6a17 Knock-in Mice.

(A) A schematic diagram illustrating the knock-in strategy for generating Slc6a17-2A-CreERT2 KI mice. This CRISPR/Cas9 mediated strategy allowed CreERT2 to be co-expressed with SLC6A17 protein fused in frame with a T2A sequence. Crossing Slc6a17-2A-CreERT2 mice with Ai14 (LSL-tdTomato) mice specifically labeled Slc6a17-expressing cells after tamoxifen injection. (B-C) Representative sagittal sections of Slc6a17-2A-CreERT2::Ai14 mouse brains at postnatal day 56. Scale bars=1 mm. (D) A schematic diagram illustrating the knock-in strategy for generating Slc6a17-HA-2A-iCre KI mice. Slc6a17 was fused in-frame with 3 repeats of the HA tag, T2A and iCre.

Generation of Slc6a17 Mutant Mice.

(A) A schematic diagram illustrating the strategy for the generation of Slc6a17-KO mice by CRISPR/Cas9. Exon 2 encoding the first 99 amino acids of Slc6a17 was deleted. (B) A schematic diagram illustrating the knock-in strategy of generating Slc6a17P633R point mutation mice by CRISPR/Cas9. The mutated exon 12 was exchanged with endogenous exon 12, while both the HA-and the FLAG-tags were fused in-frame to the C terminus of the mutated Slc6a17. (C) Confocal images of the hippocampal CA3 region (left) and anterior thalamus (right) in both Slc6a17HA/+ and Slc6a17P633R/+ mice after immunocytochemistry with anti-Syp and anti-HA antibodies. Scale bar=10 μm. (D) Sucrose gradient analysis of Slc6a17P633R-HA LP2 fraction. SLC6A17P633R protein is indicated by HA and FLAG tags. HA and FLAG signals were mostly present in the LP2 input but not in layers 2-5 of the sucrose gradient, which were SV rich as shown by Syp and Syb2 positive immunoreactivities. On the other hand, SLC6A17G162R protein is still present in layers 2-5.

Phenotypical Analysis of Slc6a17-KO Mice.

(A) Slc6a17-KO mice were not significantly different from the WT in body weight at the age of 10 week, regardless of sex (male: n=10, 16, 8 for Slc6a17+/+, Slc6a17+/-, Slc6a17-/-, respectively, p=0.8147 for Slc6a17+/+ vs. Slc6a17-/-, p=0.3456 for Slc6a17+/- vs. Slc6a17-/-; female: n=13, 9, 9 for Slc6a17+/+, Slc6a17+/-, Slc6a17-/-, respectively, p> 0.9999 for Slc6a17+/+ vs. Slc6a17-/-, p=0.9896 for Slc6a17+/- vs. Slc6a17-/-). (B) Locomotor activities in the open field test were not significantly different among the three genotypes (n=21, 26, 17 for Slc6a17+/+, Slc6a17+/-, Slc6a17-/-, respectively, p=0.0987 for Slc6a17+/+ vs. Slc6a17-/-, p=0.3233 for Slc6a17+/- vs. Slc6a17-/-). (C) Novel object recognition (NOR) was not significantly different among the three genotypes (n= 22, 22, 20 for Slc6a17+/+, Slc6a17+/-, Slc6a17-/-, respectively, p=0.9318 for Slc6a17+/+ vs. Slc6a17-/-, p=0.7978 for Slc6a17+/- vs. Slc6a17-/-). (D-F) Slc6a17 KO mutants had lower levels of anxiety in elevated plus maze test (n=33, 26, 27 for Slc6a17+/+, Slc6a17+/-, and Slc6a17-/-, respectively): overall locomotion was not significantly different (D, p= 0.3639 for Slc6a17+/+ vs. Slc6a17-/-, p= 0.053 for Slc6a17+/- vs. Slc6a17-/-); percentage of time spend in open arms was significantly increased in Slc6a17-/- mice (E, p<0.0001 for Slc6a17+/+ vs. Slc6a17-/-, p<0.0001 for Slc6a17+/- vs. Slc6a17-/-); the travel distance in open arms was significantly increased in Slc6a17-/- mice (p< 0.0001 for Slc6a17+/+ vs. Slc6a17-/-, p=0.0001 for Slc6a17+/- vs. Slc6a17-/-).

Phenotypical Analysis of Slc6a17P633R Mice.

(A) Slc6a17P633R mice were not significantly different the WT in body weight at the age of 10 week, regardless of sex (male: n=11, 7, 10 for Slc6a17+/+, Slc6a17P633R/+, Slc6a17P633R/P633R, respectively; p=0.7243 for Slc6a17+/+ vs. Slc6a17P633R/P633R, p=0.4078 for Slc6a17P633R/+ vs. Slc6a17P633R/P633R; female: n=11, 19, 12 for Slc6a17+/+, Slc6a17P633R/+, Slc6a17P633R/P633R, respectively; p=0.9698 for Slc6a17+/+ vs. Slc6a17P633R/P633R, p=9982 for Slc6a17P633R/+ vs. Slc6a17P633R/P633R). (B) Locomotor activities of Slc6a17P633R mice were not significantly different from the WT (n=23, 28, 17 for Slc6a17+/+, Slc6a17P633R/+, Slc6a17P633R/P633R, respectively; p>0.9999 for Slc6a17+/+ vs. Slc6a17P633R/P633R, p=0.7803 for Slc6a17P633R/+ vs. Slc6a17P633R/P633R). (C) NOR was not significantly different among Slc6a17P633R mutant and WT mice (n=23, 28, 17 for Slc6a17+/+, Slc6a17P633R/+, Slc6a17P633R/P633R, respectively; p=0.4728 for Slc6a17+/+ vs. Slc6a17P633R/P633R, p=0.5776 for Slc6a17P633R/+ vs. Slc6a17P633R/P633R). (D-F) Slc6a17P633R homozygous mutants had lower level of anxiety in elevated plus maze (n=22, 26, 17 for Slc6a17+/+, Slc6a17P633R/+, Slc6a17P633R/P633R, respectively): overall locomotion was not significantly different (D, p= 0.9974 for Slc6a17+/+ vs. Slc6a17P633R/P633R, P > 0.9999 for Slc6a17P633R/+ vs. Slc6a17P633R/P633R); percentage of time spend in open arms was significantly increased in homozygous Slc6a17P633R mutants (E, p=0.0002 for Slc6a17+/+ vs. Slc6a17P633R/P633R, p=0.0035 for Slc6a17P633R/+ vs. Slc6a17P633R/P633R); travel distance in open arms was significantly increased in homozygous Slc6a17P633R mutants (F, p= 0.0016 for Slc6a17+/+ vs. Slc6a17P633R/P633R, p=0.0044 for Slc6a17P633R/+ vs. Slc6a17P633R/P633R).

Immunoisolation and Immunohistochemistry of Slc6a17-HA Mice.

(A) A schematic diagram illustrating the knock-in strategy for generating Slc6a17-HA-2A-iCre KI mice. (B) Confocal images of the hippocampal region in Slc6a17HA/+ mice after immunocytochemistry with anti-Syp and anti-HA antibodies. Scale bar=50 μm. (C) With fractions of whole brains by differential centrifugation, SLC6A17-HA was co-immunoisolated with synaptic markers including Syp, Syt1, Syb2 and V-ATPase, but not with PSD95. (D) Analysis of SVs isolated by anti-HA beads from Slc6a17-HA-2A-iCre mice and from WT mice. 23 markers for organelles were analyzed to confirm SV specificity, and absence of markers of lysosome, Golgi, mitochondria, ER, endosome, proteasome and postsynaptic cytoplasmic membrane. Scale bar=10 μm.

EM Confirmation of the SV Localization of SLC6A17-APEX2.

(A, C, E, G) Different views of SLC6A17-APEX2 overexpression. (B, D, F, H) Normal distribution of SV electron density in the corresponding images on the left.

MS Analysis of Slc6a17 KO Mice.

TMT labeled quantitative SV proteomics was performed in Slc6a17-KO mice. Briefly, SVs were immunoisolated from LP2 fraction of WT and Slc6a17-KO mice by anti-Syp. SV proteins from different samples were digested and then labeled separately by TMT regents (detailed description in Experimental Procedures). Volcano plot showing SV proteins different between Slc6a17-/- mice and Slc6a17+/+ mice. The y axis shows p values in log10 and the x axis shows the log2 of the ratio of the level of a protein immunoisolated by anti-Syp from Slc6a17-/- mice vs that from Slc6a17+/+ mice. SLC6A17 was the only transporter significantly decreased in Slc6a17-KO mice. Known vesicular transporters are highlighted by blue cycles.

LC-MS Analysis of SVs immunoisolated by the Anti-Syp Antibody.

(A) Analysis of SVs isolated by the anti-Syp antibody. 16 markers for organelles were analyzed to confirm the SV specificity, with no detection of markers for lysosome, Golgi, mitochondria, ER, endosome and postsynaptic cytoplasmic membrane. IgG was used as a control. (B-G) Neurotransmitter contents in SVs isolated by the anti-Syp antibody. Representative LC-MS signals of anti-Syp and IgG group are shown, each graph was normalized: Glu (B), GABA (D) and ACh (F) highly enriched; 5-HT (C), histamine (E) and dopamine (G) moderately enriched.

Virally Mediated Overexpression of SLC6A17-HA and SLC6A17G162R-HA.

(A) Analysis of SVs isolated by anti-HA beads from mice overexpressing OE-Syp-HA, OE-SLC6A17-HA and OE-SLC6A17G162R-HA. High levels of association with SV markers (Syp and Syb2) were detected in both samples, indicating Syp-HA, hSLC6A17-HA and hSLC6A17G162R proteins were all localized on the SVs. LC-MS results normalization was based on immunoblot analysis of SV markers. (B) Volcano plot of the contents from SVs purified by anti-HA immunoisolation from OE-hSLC6A17G162R-HA overexpressing mice compared to that from WT mice. (C) Quantification of His levels (p<0.0001 for OE-hSLC6A17 vs. WT). (D) Quantification of His levels (p=0.2433 for OE-hSLC6A17 vs. OE-hSLC6A17G162R-HA; p=0.0018 for OE-hSLC6A17 vs. Syp-HA; p=0.2735 for OE-hSLC6A17 vs. WT; p= 0.9993 for OE-hSLC6A17G162R-HA vs. WT; p=0.9998 for Syp-HA vs. WT). (E) Sucrose gradient analysis of Slc6a17G162R-HA LP2 fraction. Slc6a17G162R-HA was associated with layers 2-4, which were rich with SV markers such as Syt1, Syp, Syb2 and VGlut1.

CRISPR/Cas9 Mediated Slc6a17 Gene Cleavage in Slc6a17 Expressing Cells of Adult Mice.

(A) A schematic diagram illustrating the strategy for Cas9-mediated cleavage of Slc6a17 specifically in Slc6a17 positive neurons, and simueltaneous labeling of SVs by Syp-HA. A single tRNA-gRNA allowed multiple gRNAs being efficiently produced, which could be precisely excised in vivo by the endogenous RNases, to improve editing efficiency of Cas9 system. (B) SLC6A17 protein in Slc6a17iCre/Cas9+ mice were efficiently removed. The protein level of SLC6A17 was measured in the input supernatant used for immunoisolation.