Synaptotagmin-1 and -7 Are Redundantly Essential for Maintaining the Capacity of the Readily-Releasable Pool of Synaptic Vesicles

Abstract

In forebrain neurons, Ca(2+) triggers exocytosis of readily releasable vesicles by binding to synaptotagmin-1 and -7, thereby inducing fast and slow vesicle exocytosis, respectively. Loss-of-function of synaptotagmin-1 or -7 selectively impairs the fast and slow phase of release, respectively, but does not change the size of the readily-releasable pool (RRP) of vesicles as measured by stimulation of release with hypertonic sucrose, or alter the rate of vesicle priming into the RRP. Here we show, however, that simultaneous loss-of-function of both synaptotagmin-1 and -7 dramatically decreased the capacity of the RRP, again without altering the rate of vesicle priming into the RRP. Either synaptotagmin-1 or -7 was sufficient to rescue the RRP size in neurons lacking both synaptotagmin-1 and -7. Although maintenance of RRP size was Ca(2+)-independent, mutations in Ca(2+)-binding sequences of synaptotagmin-1 or synaptotagmin-7--which are contained in flexible top-loop sequences of their C2 domains--blocked the ability of these synaptotagmins to maintain the RRP size. Both synaptotagmins bound to SNARE complexes; SNARE complex binding was reduced by the top-loop mutations that impaired RRP maintenance. Thus, synaptotagmin-1 and -7 perform redundant functions in maintaining the capacity of the RRP in addition to nonredundant functions in the Ca(2+) triggering of different phases of release.

Fig 1. Simultaneous ablation of Syt1 and Syt7 decreases the RRP size at inhibitory synapses: Rescue by WT but not mutant Syt1 or Syt7.

A. Ablation of either Syt1 or Syt7 alone does not alter the RRP at inhibitory synapses. Hippocampal neurons were cultured from littermate WT and Syt1 KO mice (for Syt1 analysis) or from WT mice and were then infected at DIV4 with control or Syt7 KD lentiviruses (for Syt7 analysis). At DIV14–16, exocytosis of primed vesicles from the RRP in the neurons was stimulated by application of 0.5 M sucrose for 30 s (gray bars in representative traces on the left), and the RRP size was estimated as the synaptic charge transfer integrated over 30 s (summary graphs on the right). Recordings were performed in the presence of 1 μM tetrodotoxin, 20 μM CNQX, and 50 μM AP5 to isolate inhibitory currents. B. Simultaneous ablation of Syt1 and Syt7 decreases the RRP size of inhibitory synapses in a manner that is rescued by WT Syt7 (Syt1^WT) but not Syt7 with mutations in the top C2 domain sequences containing the Syt7 Ca²⁺ binding sites (Syt7^C2A*^B*). Hippocampal Syt1 KO neurons were infected with control lentiviruses, Syt7 KD lentiviruses without or with expression of either WT or mutant Syt7, or lentiviruses only expressing WT Syt7 (as a control for overexpression effects). RRP was measured as described in A. C. The decreased RRP size in Syt1/7 double-deficient neurons is rescued by WT Syt1 (Syt1^WT) but not Syt1-containing mutations in the top-loop Ca²⁺-binding sequences (Syt1^C2A*^B*). Experiments were performed as described for B. All data are means ± SEM (Standard Error of the Mean); numbers in bars indicate number of neurons/independent cultures analyzed. Statistical significance was assessed by one-way ANOVA (** p < 0.01; *** p < 0.001). The data used to make this figure can be found in S1 Data.

Fig 2. KD of Syt1 in Syt7 KO neurons decreases the RRP size at inhibitory synapses: Rescue by WT but not mutant Syt7.

A & B. Same as 1B, except that Syt7 KO neurons with a Syt1 KD were examined. Hippocampal neurons were cultured from two independent, constitutive Syt7 KO mouse lines (D, Syt7 KO^A from [56]; E, Syt7 KO^N from [55]), and infected with control lentivirus or Syt1 KD lentivirus without or with superinfection with a second lentivirus expressing WT or mutant Syt7. All data are means ± SEM; numbers in bars indicate number of neurons or independent cultures analyzed. Statistical significance was assessed by one-way ANOVA (** p < 0.01; *** p < 0.001). The data used to make this figure can be found in S1 Data.

Fig 3. Simultaneous ablation of Syt1 and Syt7 decreases the RRP size at excitatory synapses.

A & B. Ablation of both Syt1 and Syt7 in hippocampal neurons decreases the RRP size at excitatory synapses in a manner that is rescued by WT Syt7 (Syt7^WT) or Syt1 (Syt1^WT) but not by mutant Syt7 (Syt7^C2A*^B*) or Syt1 (Syt1^C2A*^B*) with altered top-loop sequences containing the Ca²⁺-binding sequences (A, Syt7 rescue; B, Syt1 rescue). Experiments were performed as described for Fig 1, except that picrotoxin (50 μM) was used instead of CNQX. All data are means ± SEM; numbers in bars indicate number of neurons/independent cultures analyzed. Statistical significance was assessed by one-way ANOVA (** p < 0.01, *** p < 0.001). The data used to make this figure can be found in S1 Data.

Fig 4. Syt1/Syt7 double-deficiency does not induce structural changes in synapses as assessed by standard EM.

A. Representative electron micrographs from cultured hippocampal Syt1 KO neurons that were infected with control lentivirus or Syt7 KD lentiviruses without or with expression of WT or mutant Syt7 rescue cDNAs. B. Quantification of the total number of docked vesicles (defined as vesicles touching the membrane), vesicles at the active zone (AZ, defined as vesicles within 100 nm of plasma membrane), total number of vesicles in each bouton, and postsynaptic density (PSD) length and bouton area in EM micrographs. Quantification from two independent cultures was performed manually by an observer blind to the conditions. No significant differences were found for the parameters analyzed. The data used to make this figure can be found in S1 Data.

Fig 5. Differential requirement for Syt1 and Syt7 C2A- and C2B-domain top-loop sequences for RRP maintenance and Ca²⁺ triggering of neurotransmitter release.

A. In Syt1/7 double-deficient neurons, Syt1 mutations in either the C2A-domain (Syt1^C2A*) or C2B domain (Syt1^C2B*) top-loop sequences that include their Ca²⁺ binding sites do not block rescue of the RRP in Syt1/7 double deficient neurons, whereas Syt7 mutations in the C2A-domain (Syt1^C2A*) but not the C2B domain (Syt1^C2B*) Ca²⁺-binding sequence selectively impair rescue of the RRP. Hippocampal neurons were cultured from Syt1 KO mice and infected with control lentivirus, or Syt7 KD lentiviruses co-expressing the indicated Syt1 and Syt7 mutants. Exocytosis of primed vesicles from the RRP was induced by a brief pulse of 0.5 M sucrose, and monitored as IPSCs in the presence of TTX (1 μM), CNQX (20 μM), and AP5 (50 μM). Representative traces are shown on the left, and summary graphs on the right. Note that simultaneous mutation of both Syt1 C2-domains blocks its priming function (Fig 1C). B. Confirmation that mutations of Syt1 in the top-loop sequences containing the Ca²⁺ binding sites of the C2B domain but not the analogous mutations in the C2A domain block rescue of synchronous release in Syt1 KO neurons. Experiments were performed as described for A, except that IPSCs were elicited by 10 Hz, 1 s stimulus trains (left, representative traces; right, summary graphs of the total synaptic charge transfer). Stimulations are indicated by tick marks. All data are means ± SEM; numbers in bars indicate number of neurons/independent cultures analyzed. Statistical significance was assessed by one-way ANOVA (* p < 0.05, ** p < 0.01, *** p < 0.001). The data used to make this figure can be found in S1 Data.

Fig 6. Syt1 and Syt7 both bind to SNARE complexes, and mutations that alter the top-loop Ca²⁺-binding sequences of Syt1 or Syt7 decrease such binding.

A & B. Analysis of the SNARE binding of WT Syt1 and Syt7 (Syt1^WT and Syt7^WT, respectively) and of mutant Syt1 and Syt7-containing top-loop substitutions altering their Ca²⁺-binding sequences (Syt1^C2A*^B* and Syt7^C2A*^B*, respectively; A, Syt1^WT and Syt1^C2A*^B*; B, Syt7^WT and Syt7^C2A*^B*). WT or mutant Syt1 and Syt7 were lentivirally expressed in neurons, and neurons were analyzed at DIV14–16 by immunoprecipitation of the SNARE protein syntaxin-1 followed by immunoblotting for the SNARE protein synaptobrevin-2 (Syb2; to assess the degree of SNARE complex IP) or of Syt1 or Syt7 (to assess the degree of synaptotagmin binding). Data are means ± SEM (n = 3 independent culture experiments). Statistical significance was assessed by one-way ANOVA (*** p < 0.001). The data used to make this figure can be found in S1 Data.

Fig 7. Effect of Syt1 or complexin-1 on SNARE complex assembly when coexpressed in HEK293T cells as a reduced system.

A. Syt1 alone has no effect on SNARE complex assembly. HEK293T cells were cotransfected with plasmids expressing syntaxin-1 (Synt-1), SNAP-25, and synaptobrevin-2 (Syb-2) in a 1:1:1 ratio, together with increasing amounts of Syt1 expression plasmid (0 to 4-fold), and decreasing amounts of emerald expression plasmid (GFP; 4 to 0-fold), to balance the total amount of transfected DNA. Cell lysates were immunoblotted for Syt1, emerald, SNARE complexes (high molecular mass bands), and total SNARE proteins, followed by quantitation (n = 4 independent experiments). B. Complexin increases SNARE complex assembly. Same as (A), except that HEK293T cells were co-transfected with an increasing amount of complexin-1 (Cpx1) expression plasmid (n = 4 independent experiments). All data are means ± SEM; statistical significance was assessed by Student’s t test (* p < 0.05, ** p < 0.01, *** p < 0.001). The data used to make this figure can be found in S1 Data.

Fig 8. Effect of Syt1 and complexin-1 on SNARE complex assembly when coexpressed in HEK293T cells as a reduced system.

Syt1 increases SNARE complex assembly in presence of complexin-1. HEK293T cells were cotransfected with plasmid expressing Synt-1, SNAP-25, Syb-2, and complexin-1 in a 1:1:1:3 ratio, together with an increasing amount of Syt1 expression plasmid (0 to 4-fold), and decreasing amount of emerald expression plasmid (GFP; 4 to 0-fold). Cell lysates were immunoblotted for Syt-1, Cpx1, emerald, SNARE complexes (high molecular mass bands), and total SNARE proteins followed by quantitation (n = 6 independent experiments). All data are means ± SEM; statistical significance was assessed by Student’s t test (* p < 0.05, ** p < 0.01, *** p < 0.001). The data used to make this figure can be found in S1 Data.

Fig 9. Double deficiency of Syt1 and Syt7 does not impair the priming rate of synaptic vesicles during the recovery of the RRP after depletion.

A & B. Simultaneous ablation of Syt1 and Syt7 impairs the RRP size but not the relative rate of RRP recovery after RRP depletion at inhibitory synapses. In A, Syt1 KO neurons without or with induction of the Syt7 KD alone or together with the Syt7 rescue were analyzed; in B, Syt7 KO neurons (from KO strain A) without or with Syt1 KD were tested. The RRP was measured during an initial 10 s application of 0.5 M sucrose which also depleted the RRP, and RRP recovery was monitored after 40 s by a second 10 s application of sucrose (gray bars = sucrose applications). Double deficiency of Syt1 and Syt7 resulted in a reduced RRP size with no effect on the relative rate of RRP recovery (left, representative traces; middle, summary graphs of absolute RRP sizes; right, summary graphs of the ratio of 2nd versus 1st RRP to estimate RRP recovery). Recorded currents were not corrected for the significant changes in access resistance similarly observed for all groups (see S4 Fig). All data are means ± SEM; numbers in bars indicate number of neurons/independent cultures analyzed. Statistical significance was assessed by one-way ANOVA (*** p < 0.001). The data used to make this figure can be found in S1 Data.

Fig 10. Summary of structure–function relations of Syt1 and Syt7 in Ca²⁺ triggering of exocytosis, priming, and clamping of spontaneous mini release.

A. Schematic diagram of the different stages of Ca²⁺-triggered synaptic vesicle exocytosis and of the functions of Syt1 and Syt7 in the maintenance of the RRP size, clamping spontaneous mini release (which is in itself Ca²⁺-dependent via the actions of an as yet unidentified Ca²⁺ sensor [49]), and Ca²⁺ triggering of delayed and fast release. B. The three different functions of Syt1 (top) and Syt7 (bottom) are depicted by arrows; the C2 domain requirement for each function is described on top of the arrows. In addition, schematic diagrams of the Syt1 and Syt7 domain structures are shown, with the top-loop Ca²⁺-binding sequences of a given C2 domain that is required for a particular function indicated in red.