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. 2021 Jul 6;19(7):e3001323.
doi: 10.1371/journal.pbio.3001323. eCollection 2021 Jul.

Synaptotagmin-7-mediated activation of spontaneous NMDAR currents is disrupted in bipolar disorder susceptibility variants

Qiu-Wen Wang  1 , Ying-Han Wang  1 , Bing Wang  1 , Yun Chen  1 , Si-Yao Lu  1 , Jun Yao  1
Affiliations

Synaptotagmin-7-mediated activation of spontaneous NMDAR currents is disrupted in bipolar disorder susceptibility variants

Qiu-Wen Wang et al. PLoS Biol. .

Abstract

Synaptotagmin-7 (Syt7) plays direct or redundant Ca2+ sensor roles in multiple forms of vesicle exocytosis in synapses. Here, we show that Syt7 is a redundant Ca2+ sensor with Syt1/Doc2 to drive spontaneous glutamate release, which functions uniquely to activate the postsynaptic GluN2B-containing NMDARs that significantly contribute to mental illness. In mouse hippocampal neurons lacking Syt1/Doc2, Syt7 inactivation largely diminishes spontaneous release. Using 2 approaches, including measuring Ca2+ dose response and substituting extracellular Ca2+ with Sr2+, we detect that Syt7 directly triggers spontaneous release via its Ca2+ binding motif to activate GluN2B-NMDARs. Furthermore, modifying the localization of Syt7 in the active zone still allows Syt7 to drive spontaneous release, but the GluN2B-NMDAR activity is abolished. Finally, Syt7 SNPs identified in bipolar disorder patients destroy the function of Syt7 in spontaneous release in patient iPSC-derived and mouse hippocampal neurons. Therefore, Syt7 could contribute to neuropsychiatric disorders through driving spontaneous glutamate release.

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Conflict of interest statement

The authors have declared that no competing interests exist.

Figures

Fig 1
Fig 1. Syt7 triggers spontaneous neurotransmitter release.
(A) Design of multiplex CRISPRi system. Upper, schematic illustration of the multiple gene targeting CRISPRi for Syt1/Doc2a/2b and Syt1/Syt7/Doc2a/2b. Lower, qRT-PCR analysis of mRNA expression of hippocampal neurons with multiplex KD of Syt1/Syt7/Doc2a/2b. (B) Sample traces of mEPSCs recorded in cultured hippocampal neurons with multiplex gene KD. (C) Quantification of the frequency (left) and amplitude (right) of mEPSCs. WT, n = 11; tKD, n = 11; qKD, n = 13. (D) Representative traces of mEPSCs recorded from hippocampal CA1 slices. The same neuron was recorded in the presence of extracellular Ca2+ or Sr2+ in the ACSF solution with a 5-min interval. (E) Paired comparison of the frequency of Ca2+- and Sr2+-triggered mEPSCs in hippocampal CA1 slices. WT, n = 13; KO, n = 14. (F) Bar graph of mEPSC amplitude. (G–I) Sample traces (G) and quantification of frequency (H) and amplitude (I) of mEPSCs recorded in the DG slices. WT, n = 15; KO, n = 15. (J) Representative traces of Ca2+- or Sr2+-triggered mEPSCs recorded from cultured WT hippocampal neurons, Syt7 KO neurons, and KO neurons expressing Syt7FL or Syt7CLM. (K) Quantitative analysis of the frequency of mEPSCs. WT, n = 19 for Ca2+, n = 25 for Sr2+; Syt7 KO, n = 18/21; KO + Syt7FL, n = 25/25; KO + Syt7CLM, n = 12/12. (L) Representative traces of mEPSCs recorded from WT and Syt7 KO neurons in a Ca2+ gradient from 0 mM to 10 mM. (M) Quantitative analysis of mEPSC frequency recorded in Ca2+ gradient. Red curves are fitted Hill function. Ca2+ affinity: WT, 1.52 ± 0.53; KO, 1.35 ± 0.52; P = 0.819. Ca2+ cooperativity: WT, 0.46 ± 0.11; KO, 0.77 ± 0.24; P = 0.258. WT, n = 13–19; Syt7 KO, n = 16–20. Student t test; *P < 0.05; **P < 0.001; error bars, SEM. The numerical data underlying this figure are included in S1 Data. CLM, calcium ligand mutant; CRISPRi, CRISPR interference; DG, dentate gyrus; FL, full-length; KD, knockdown; KO, knockout; mEPSC, miniature excitatory postsynaptic current; qKD, quadra KD; qRT-PCR, quantitative reverse transcription PCR; Syt7, synaptotagmin-7; tKD, triple KD; WT, wild-type.
Fig 2
Fig 2. Syt7 triggered spontaneous glutamate release specifically activates GluN2B-NMDARs.
(A) Sample traces of AMPAR/NMDAR-mEPSCs in hippocampal slices of WT and Syt7 KO neurons. (B, C) Bar graphs summarizing the frequency (B) and amplitude (C) of AMPAR/NMDAR-mEPSCs. (D) Average traces of AMPAR/NMDAR-mEPSCs. (E, F) Bar graphs summarizing the decay time (E) and total charge (F). (A–F) WT, n = 14/18/8; KO, n = 14/11/7. Student t test; *P < 0.05; **P < 0.001; error bars, SEM. The numerical data underlying this figure are included in S1 Data. AMPAR, AMPA receptor; KO, knockout; mEPSC, miniature excitatory postsynaptic current; Syt7, synaptotagmin-7; WT, wild-type.
Fig 3
Fig 3. Retargeting Syt7 to the central AZ triggers non-GluN2B spontaneous neurotransmission.
(A, B) qRT-PCR (A) and IB (B) analyses showing the lentiviral expression of HA-conjugated Syt7 in neurons. (C) EM analysis showing that Syt7GAP43-HA (n = 69) was closer to the center of AZ compared to Syt7FL-HA (n = 59). Upper, sample EM images. Scale bar, 200 nm. Lower left, AZ length. Lower right, histogram of Syt7FL-HA/Syt7GAP43-HA distribution. Red and green curves are fitted Gaussian curve. (D) Sample traces of Ca2+/Sr2+-triggered mEPSCs in WT neurons (n = 12/12), Syt7 KO neurons (n = 18/12), and KO neurons expressing Syt7FL (n = 14/12) or Syt7GAP43 (n = 15/12). (E) Analysis of mEPSC frequency (left) and Sr2+/Ca2+ ratio of frequency (right). WT, n = 12/12; KO, n = 18/12; Syt7FL, n = 14/12; Syt7GAP43, n = 15/12. (F) mEPSC amplitude. (G) Average traces showing the kinetics of AMPAR/NMDAR-mEPSCs in Syt7GAP43-expressing neurons following GluN2B blockade. (H, I) Decay time (H) and total charge (I) of mEPSCs. WT, n = 12/9/7; KO, n = 16/13/8; Syt7FL, n = 12/12/8; Syt7GAP43, n = 14/13/11. ANOVA (see S6 Fig); *P < 0.05; **P < 0.001; error bars, SEM. The numerical data underlying this figure are included in S1 Data. AMPAR, AMPA receptor; AZ, active zone; EM, electron microscopy; FL, full-length; IB, immunoblot; KO, knockout; mEPSC, miniature excitatory postsynaptic current; qRT-PCR, quantitative reverse transcription PCR; Syt7, synaptotagmin-7; WT, wild-type.
Fig 4
Fig 4. Human Syt7 variants induce deficits in spontaneous glutamate release and GluN2B activity.
(A) Sample traces of AMPAR/NMDAR-mEPSCs in Syt7 KD neurons expressing Syt7IF4 and Syt7IF4mut. (B, C) Bar graphs summarizing the frequency (B) and amplitude (C) of mEPSCs. Scr, n = 10/10/8; Syt7 KD, n = 8/8/8; Syt7 IF4, n = 16/18/16; Syt7IF4mut, n = 21/20/12. (D) Average traces of AMPAR/NMDAR-mEPSCs. (E, F) Bar graphs summarizing the decay time (E) and total charge (F). (G) Average traces showing changes in the kinetics of AMPAR/NMDAR-mEPSCs in the iPSC-derived DG-like neurons of healthy controls and BD-I patients. (H, I) Bar graphs summarizing the decay time (H) and charge transfer (I) of AMPAR/NMDAR-mEPSCs in the HC neurons, BD-I neurons, and BD-I neurons overexpressing Syt7FL or Syt7IF4mut. For all groups, n = 23–26 neurons of 3 cell lines. Student t test; *P < 0.05; **P < 0.001; error bars, SEM. The numerical data underlying this figure are included in S1 Data. AMPAR, AMPA receptor; BD-I, bipolar disorder I; DG, dentate gyrus; HC, healthy control; iPSC, induced pluripotent stem cell; KD, knockdown; mEPSC, miniature excitatory postsynaptic current; Syt7, synaptotagmin-7.
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This work was supported by National Natural Science Foundation of China (Grant No.31830038, 31771482, 81771466; http://www.nsfc.gov.cn/) and National Key R&D Program of China (Grant No.2016YFA0101900; https://service.most.gov.cn/). The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.