Figure 7. Increased glutamine (Gln) transport into synaptic vesicles (SVs) containing SLC6A17 but not into SVs containing SLC6A17^G162R.

(A) A schematic diagram illustrating the strategy for the AAV-PHP.eb virus-mediated in vivo overexpression of SLC6A17-HA and SLC6A17^G162R-HA. (B) Volcano plot of chemical contents in the SVs isolated by anti-HA beads from the OE-SLC6A17-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 SLC6A17-HA vs. that from WT mice. Classical neurotransmitters and previously reported substrates of SLC6A17 are listed. (C–G) Contents of OE-SLC6A17-containing SV are quantified to mole per 10 μl HA beads (n = 10, for each group from three different animals with three replicates in two animal and four replicates in one animal): Glu (C, p<0.0001 for OE-SLC6A17-HA vs. WT); GABA (D, p<0.0001 for OE-SLC6A17-HA vs. WT); ACh (E, p<0.0001 for OE-SLC6A17-HA vs. WT); Gln (F, p<0.0001 for OE-SLC6A17-HA vs. WT); (G, p=0.1655 for OE-SLC6A17-HA vs. WT). (H) Volcano plot comparing the chemical contents of SVs containing OE-SLC6A17-HA with those containing OE-SLC6A17^G162R-HA. Classical neurotransmitters and nine putative substrates of SLC6A17 are listed. Glu is the only classical transmitter significantly increased. Gln is the only substrates significantly increased in SLC6A17-HA containing SVs vs. SLC6A17^G162R-HA containing SVs. (I) Volcano plot comparing the chemical contents of SVs containing OE-SLC6A17-HA with those containing OE-Syp-HA. Classical neurotransmitters and nine 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-SLC6A17^G162R-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 and normalized to Syb2 relative abundance in WB (n = 9, 9, 6, and 6 for OE-SLC6A17-HA, OE-SLC6A17^G162R-HA, OE-Syp-HA, and WT, respectively, from different animals with three replicates each): Glu (J, p<0.0001 for OE-SLC6A17-HA vs. OE-SLC6A17^G162R-HA; p<0.0001 for OE-SLC6A17-HA vs. WT; p<0.0001 for OE-SLC6A17^G162R-HA vs. WT; p=0.0009 for OE-Syp-HA vs. WT; p=0.1551 for OE-SLC6A17-HA vs. OE-Syp-HA; p=0.0618 for OE-SLC6A17^G162R-HA vs. OE-Syp-HA); GABA (K, p=0.0002 for OE-SLC6A17-HA vs. WT; p=0.0002 for OE-SLC6A17^G162R-HA vs. WT; p=0.0039 for OE-Syp-HA vs. WT; p=0.9885 for OE-SLC6A17-HA vs. OE-SLC6A17^G162R-HA; p=0.9000 for OE-SLC6A17-HA vs. OE-Syp-HA; p=0.5840 for OE-SLC6A17^G162R-HA vs. OE-Syp-HA); ACh (L, p=0.0019 for OE-SLC6A17-HA vs. WT; p=0.0006 for OE-SLC6A17^G162R-HA vs. WT; p=0.0467 for OE-Syp-HA vs. WT; p=0.1054 for OE-SLC6A17-HA vs. OE-SLC6A17^G162R-HA; p=0.7563 for OE-SLC6A17-HA vs. OE-Syp-HA; p=0.8702 for OE-SLC6A17^G162R-HA vs. OE-Syp-HA); Gln (M, p=0.001 for OE-SLC6A17-HA vs. OE-SLC6A17^G162R-HA; p=0.0018 for OE-SLC6A17-HA vs. OE-Syp-HA; p=0.0003 for OE-SLC6A17-HA vs. WT; p<0.0001 for OE-SLC6A17^G162R-HA vs. WT; p=0.0189 for OE-Syp-HA vs. WT; p=0.2749 for OE-SLC6A17^G162R-HA vs. OE-Syp-HA); Ser (N, p=0.626 for OE-SLC6A17-HA vs. OE-SLC6A17^G162R-HA; p=0.8551 for OE-SLC6A17-HA vs. OE-Syp-HA; p=0.9874 for OE-SLC6A17-HA vs. OE-Syp-HA; p<0.0865 for OE-SLC6A17^G162R-HA vs. WT; p=0.1017 for OE-Syp-HA vs. WT).

Figure 7—figure supplement 1. Virally mediated overexpression of SLC6A17-HA and SLC6A17^G162R-HA.

(A) Analysis of synaptic vesicles (SVs) isolated by anti-HA beads from mice overexpressing OE-Syp-HA, OE-SLC6A17-HA, and OE-SLC6A17^G162R-HA. High levels of association with SV markers (Syp and Syb2) were detected in both samples, indicating Syp-HA, SLC6A17-HA and SLC6A17^G162R proteins were all localized on the SVs. Liquid chromatography coupled to mass spectrometry (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-SLC6A17^G162R-HA overexpressing mice compared to that from WT mice. (C) Quantification of His levels (p<0.0001 for OE-SLC6A17 vs. WT). (D) Quantification of His levels (p=0.2433 for OE-SLC6A17 vs. OE-SLC6A17^G162R-HA; p=0.0018 for OE-SLC6A17 vs. Syp-HA; p=0.2735 for OE-SLC6A17 vs. WT; p=0.9993 for OE-SLC6A17^G162R-HA vs. WT; p=0.9998 for Syp-HA vs. WT). (E) Sucrose gradient analysis of Slc6a17^G162R-HA LP2 fraction. Slc6a17^G162R-HA was associated with layers 2–4, which were rich with SV markers such as Syt1, Syp, Syb2, and VGlut1.