Compromised fidelity of endocytic synaptic vesicle protein sorting in the absence of stonin 2

Abstract

Neurotransmission depends on the exocytic fusion of synaptic vesicles (SVs) and their subsequent reformation either by clathrin-mediated endocytosis or budding from bulk endosomes. How synapses are able to rapidly recycle SVs to maintain SV pool size, yet preserve their compositional identity, is poorly understood. We demonstrate that deletion of the endocytic adaptor stonin 2 (Stn2) in mice compromises the fidelity of SV protein sorting, whereas the apparent speed of SV retrieval is increased. Loss of Stn2 leads to selective missorting of synaptotagmin 1 to the neuronal surface, an elevated SV pool size, and accelerated SV protein endocytosis. The latter phenotype is mimicked by overexpression of endocytosis-defective variants of synaptotagmin 1. Increased speed of SV protein retrieval in the absence of Stn2 correlates with an up-regulation of SV reformation from bulk endosomes. Our results are consistent with a model whereby Stn2 is required to preserve SV protein composition but is dispensable for maintaining the speed of SV recycling.

Fig. 1.

Increased SV pool size and altered short-term plasticity in the absence of Stn2. (A) Electron micrographs of WT and KO MF terminals. (Scale bar, 1 µm.) (B) 3D reconstructions of WT and KO MF terminals: SVs, magenta; cyan, presynaptic membrane; blue, postsynaptic membranes. Cube, 1 × 1 × 1 µm. (C) Mean SV density in WT and Stn2 KO synapses (40 synapses per genotype, **P < 0.01). (Scale bar, 1 µm.) (D–J) MF short-term plasticity is increased in Stn2 KO mice. (D) Representative traces of paired-pulse ratio (PPR) with interstimulus interval of 50 ms for WT and KO. PPR quantification is shown in E (second/first pulse, WT-PPR:2.5 ± 0.08, n = 10 slices, vs. KO-PPR:2.9 ± 0.1, n = 11 slices, *P < 0.01). (F) Frequency facilitation (FF) of MF responses in WT and KO slices. Normalized amplitudes of fEPSPs are depicted in G. (H) Quantification of averaged last five pulses during 1-Hz stimulation (WT-FF: 6.5 ± 0.3, n = 11 slices, vs. KO-FF:7.6 ± 0.3, n = 12 slices, **P < 0.01). (I–J) Train with 400 pulses at 33 Hz. (J) Last 50 pulses of the train, averaged and depicted as bar diagrams (WT:1.7 ± 0.2, n = 10 slices, vs. KO:2.3 ± 0.2, n = 12, *P < 0.05).

Fig. 2.

Repartitioning of Syt1 to the neuronal surface in Stn2 KO brains. (A) AP-2 does not coimmunoprecipitate Syt1 in the absence of Stn2. Brain lysates of WT and KO mice were immunoprecipitated with either AP-2 or Stn2 antibody (control, preimmune serum) and analyzed by immunoblotting for AP-2, Stn2, Syt1, and clathrin light chain (CLC). (B) Syt1 levels in the hippocampus of young and old WT (Bi and Biii) and Stn2 KO (Bii and Biv) mice. Representative epifluorescent images of horizontal brain sections immunostained with Syt1 (green) and synaptobrevin2 (Syb2) (magenta) antibodies. (Scale bar, 40 µm.) (C) Quantification of Syt1 and Syb2 levels in the CA3 stratum lucidum of Stn2 KO mice relative to WT levels (black line), nWT/KO = 7, *P < 0.05. (D) Surface-Syt1 levels in the hippocampus of young WT (Di) and KO (Dii) mice. Epifluorescent images of representative horizontal brain sections immunostained under nonpermeabilizing conditions with Syt1 luminal domain antibody (Syt1 LD). (Scale bar, 100 µm.) (E) Quantification of Syt1 LD levels in CA3 of young and old Stn2 KO mice relative to WT levels (black line) (KO young: 159.28 ± 18.37%, n = 9; KO old: 119.8 ± 15.22%, n = 6, *P < 0.05). All data are given as mean ± SEM. (F) Surface synaptophysin (Syp LD) immunolabeling in the hippocampus of young and old WT (Fi) and Stn2 KO mice (Fii). Representative epifluorescent images of coronal brain sections immunostained under nonpermeabilizing conditions with Syp LD antibodies. (G) Quantification of Syp LD levels in the CA3 stratum lucidum/stratum pyramidale of young and old Stn2 KO mice in relation to WT levels (black line). All data represent mean ± SEM. (Scale bars, 250 µm for all images.)

Fig. 3.

Selective defect in endocytic sorting of Syt1 but not synaptophysin or synaptobrevin 2 in absence of Stn2. (A) Surface-to-total pool ratios of Syt1-, synaptophysin (Syp)-, or synaptobrevin 2 (Syb2)-pHluorin in hippocampal neurons (n = 3 independent experiments, 5–10 coverslips with >50 boutons for each construct and genotype; ***P < 0.0001). (B) Rescue of defective Syt1 sorting by reexpression of Stn2^wt in KO neurons (WT:18.94 ± 2.63%, KO:30.14 ± 2.35%, rescue:14.33 ± 1.59%, *P < 0.05, **P < 0.005).

Fig. 4.

Accumulation of surface Syt1 within the presynaptic boutons in absence of Stn2. (A) Colocalization of Syt1 LD (green) and Bassoon (magenta) in cultured hippocampal neurons from WT (Ai) and Stn2 KO mice (Aii). (Aiii) Distribution pattern of Syt1 LD fluorescence intensity along the Bassoon-positive synaptic bouton in WT (black line) and KO (red line) neurons (two independent experiments, nKO = 28, nWT = 31, synaptic boutons = 50 each, *P < 0.05). (Scale bar, 10 µm.) All data are given as mean ± SEM. (B) Confocal images of MF synapses from young WT (Bi) and Stn2 KO (Bii) mice immunostained with Syt1 LD (green) and Syb2 antibodies (magenta). (Scale bar, 8 µm.) (Biii) Intensity profiles of Syt1 LD (green) and Syb2 (magenta) fluorescence (measured along dotted lines indicated in Bi and Bii) illustrate the accumulation of surface-Syt1 within synaptic boutons of KO MF synapses.

Fig. 5.

Syt1 accumulation at the neuronal surface accelerates SV retrieval. (A) SV retrieval kinetics in WT and Stn2 KO neurons by recording SypHluorin or Syt1-pHluorin fluorescence during and after field stimulation applied at 40 Hz for 5 s. (Ai) Time course of ∆F. (Aii) Decay constants from WT and Stn2 KO neurons obtained by monoexponential fit [f(x) = A₁e^−x/t1 + y₀]. τWT = 39.02 ± 2.44 s, τKO = 29.83 ± 1.94 s, *P < 0.05, mean ± SEM of two independent experiments, >700 boutons per genotype. Facilitated retrieval is rescued by reexpression of Stn2^WT in KO neurons (τKO + Stn2^WT = 38.16 ± 3.41 s, *P < 0.05, mean ± SEM; n = 3 independent experiments, >500 boutons per genotype). (B) Overexpression of Syt1^WT (Bi) or Stn2 binding-defective mutant Syt1^mut (Bii) in WT neurons. Synapses were identified by the AZ marker piccolo (purple), surface Syt1 was visualized with α-FLAG antibodies (green). (Scale bar, 5 μm.) (Biii) Surface-stranded Stn2 binding-defective Syt1^mut displays significantly elevated levels of colocalization with the AZ marker piccolo (Rp = 0.48 ± 0.038) compared with Syt1^WT (Rp = 0.38 ± 0.034, *P < 0.05, mean ± SEM of two independent experiments, >700 boutons per genotype). (C) SypHluorin retrieval in control WT neurons (black line, black bar) and neurons overexpressing Syt1^WT (blue bar) or Stn2 binding-defective Syt1^mut (red line, red bar). Overexpression of Syt1^mut (τWT = 34.69 ± 2.23 s, τWT + Syt1^mut = 28.99 ± 1.70 s, *P < 0.05, mean ± SEM of four independent experiments, >1,000 boutons per condition) but not Syt1^WT (τWT + Syt1^wt = 33.17 ± 3.04 s, mean ± SEM of three independent experiments, >700 boutons per condition) facilitates poststimulation fluorescence decay. (D) Overexpression of Syt1 binding–defective mutant Stn2^δKYE in Stn2 KO neurons (green line, green bar) fails to rescue altered SV retrieval (red bar) (τWT = 43.37 ± 4.44 s, τKO + Stn2^δKYE = 25.10 ± 2.52 s, **P < 0.005, mean ± SEM of four independent experiments, ≥300 boutons per condition).

Fig. 6.

Facilitated SV reformation from endosomes in Stn2 KO mice. (A–D) Accumulation of AP-1 at synaptic boutons of cultured Stn2 KO neurons stimulated with 200 APs at 40 Hz. Representative epifluorescent images of cultured hippocampal neurons from WT (A) and Stn2 KO (B) mice immunostained for the AZ marker piccolo (magenta) and AP-1 (green). (Scale bar, 1 μm.) (C) Elevated levels of AP-1 (i.e., mean gray value) in stimulated neurons from Stn2 KO mice (4.727 ± 0.264) compared with WT littermates (2.423 ± 0.132). ****P < 0.000000005, mean ± SEM, ≥600 boutons per condition. (D) Increased accumulation of AP-1 at piccolo-containing presynaptic AZs in Stn2 KO neurons (Rp = 0.463 ± 0.025) compared with WT neurons (Rp = 0.366 ± 0.033, *P < 0.05, mean ± SEM, ≥600 boutons per condition). (E) Effects of brefeldin A (BFA) on SV exo-endocytosis. Endocytic poststimulation fluorescent decay of Syt1-pHluorin is similar in BFA-treated neurons from Stn2 KO (35.82 ± 4.94) compared with WT mice (37.60 ± 5.61). Time course of ∆F (data point fluorescence-resting fluorescence) was normalized to the peak value (Fpeak). Mean ± SEM of three independent experiments, >1,000 boutons per condition. (F) Electron microscopic images of hippocampal boutons from cultured WT or Stn2 KO neurons stimulated with 200 APs at 40 Hz: sp, spine; az, active zone; black arrows, endosomal structures. (Scale bar, 1 µm.) (G) Mean SV density in cultured Stn2 KO synapses is significantly higher (116 ± 6.93%) compared with WT synapses (100 ± 6.71%). Mean vesicle density in KO synapses normalized to WT (40 synapses per genotype, *P < 0.05). (Scale bar, 1 µm.) (H) The number of endosomes is significantly decreased in Stn2 KO neurons (3.81 ± 0.58) compared with WT neurons (6.14 ± 0.86). Mean ± SEM, 40 synapses per genotype, *P < 0.05. (I) The membrane area of endosomal structures in Stn2 KO neurons is significantly smaller (0.025 ± 0.004 μm²) compared with WT neurons (0.066 ± 0.009 μm²). Mean ± SEM, 40 synapses per genotype, ***P < 0.0005.