The presence of ATP strengthens binding of Ca 2+ to the main ATP/Ca2+ binding site in the C-domain of SynI
We have previously shown that ATP can bind to SynI also in the absence of Ca2+, questioning the classical view that Ca2+ binding solely serves to regulate ATP binding (Orlando et al., 2014). Here, we first addressed the possibility that Ca2+ and ATP bind independently of each other to the SynI C domain and that Ca2+ binding plays a further functional role in addition to enhancing ATP binding. Figure 1A shows the structure of the SynI C-domain together with the details of the ATP binding site in SynIWT and in the simulated model of the SynIE373K mutant (Fig. 1B; top and bottom panels, respectively). In the SynIWT protein, the binding pocket is covered by the MFL that contacts ATP and prevents water access. The MFL, together with the phosphate binding loop (PBL) and loop 6 (Fig. 1A), also contains residues that are relevant for the formation of SynI oligomers. In the SynIWT structure, the Ca2+ ion is coordinated by all three ATP phosphate groups, according to the second most observed geometry in experimentally determined structures of proteins containing ATP and a divalent cation (Li et al., 2019). Oxygen atoms from glutamic acid residues E373 and E386 also coordinate Ca2+ (Fig. 1B; top panel).
In our model of the SynIE373K mutant, Ca2+ is absent and the ATP molecule interacts with the substituting lysine (Fig. 1B; bottom panel). We next asked whether a Ca2+ ion can bind also in the absence of ATP to the main ATP-Ca2+ binding site of SynIWT C-domain revealed in the crystal structure. Thus, we simulated the SynIWT monomer after removing the ATP molecule and leaving the Ca2+ ion at the site. We observed that, along the entire simulated trajectory (200 ns), the ion remained stably tethered at the same site, tightly coordinated by E373 and E386 and an average number of four water molecules (Fig. 1C; left and middle panels). Indeed, when ATP is not present, although the MFL loop remains closed over the binding pocket as in SynIWT (Orlando et al., 2014), more water molecules can access the site with respect to the WT trajectory, thus providing coordinating oxygen atoms to the Ca2+ ion.
We then investigated how ATP affects the strength of Ca2+ binding to SynI. To this purpose, we ran several simulations in which we pulled the Ca2+ ion away from the main site by applying an external force to linearly increase the distance (we applied a harmonic restraint, or a “spring”, on the distance with a linearly increasing target; see Materials and Methods). Five simulations were carried out with ATP bound, and five without ATP bound, keeping the force constant. We observed that, while in the SynI-noATP simulations Ca2+ moved away from the binding site (Fig. 1C; right panel), in the SynI-ATP simulations the Ca2+-ATP complex remained tightly tethered at the main binding site (Fig. 1C; blue line in right panel). Thus, being in complex with ATP increases the strength of Ca2+ interaction with SynI, indicating that Ca2+ and ATP can reciprocally enhance the binding of the respective partner to the binding site (Orlando et al., 2014). We previously demonstrated that mutations of the ATP binding site in SynI affected its oligomerization dynamics. To examine the possible consequences of E373K mutation on the formation of SynI tetramers, we analyzed the behavior of the tetramer interface residues during the MD simulations of SynIWT and SynIE373K presented in Orlando et al. (2014). We observed that, although ATP remains at the binding site (Fig. 1B; bottom panel) and the MFL is closed over the pocket, the mutation enhances conformational disorder in the PBL (Fig. 1D; left panel) and in the loop 6 (Fig. 1D; right panel). Because these segments contain residues that establish direct contacts between monomers to form the tetramer, their distortion results in the perturbation of the interactions that stabilize the oligomers and are involved in SV clustering.
Expression of SynI E373K in a SynI KO background increases the frequency of miniature postsynaptic currents only in excitatory synapses
Based on the suggestions by MD simulations that Ca2+ binds with high affinity at the ATP binding site and affects the tetramerization surface, we proceeded to physiologically testing the SynIE373K mutant that abolishes the main Ca2+ binding, without affecting the binding of ATP (Orlando et al., 2014). To address the functional role of Ca2+ binding to SynI using lentiviral vectors and low-density primary hippocampal neurons from SynI KO mice, we expressed: (i) a reporter-containing empty vector for the control KO group (SynIKO); (ii) wild type mCherry-SynI (SynIWT); and (iii) mCherry-SynI mutant in the Ca2+ binding site (SynIE373K). Analysis of the fluorescent reporters showed that the vast majority (> 90%) of the neurons were transduced with comparable expression levels (SynIKO: 96.41 ± 0.34; SynIWT: 95.13 ± 0.25; SynIE373K: 94.74 ± 0.27). We first investigated whether the expression of SynIE373K could alter the physiological properties of excitatory (Fig. 2A,B) and inhibitory (Fig. 2C,D) miniature postsynaptic currents (mEPSCs and mIPSCs, respectively) as compared to SynIWT and SynIKO. The expression of the SynIE373K mutant markedly increased the frequency of mEPSCs with respect to SynKO neurons or neurons rescued with SynWT, while the amplitude was not affected (Fig. 2A; lower panels).
On the contrary, no changes in the frequency and amplitude of mIPSCs were observed across the three genotypes (Fig. 2C; lower panels). The analysis of the rise and decay times revealed no significant changes in the kinetics of both mEPSCs (Fig. 2B) and mIPSCs (Fig. 2D) across the three genotypes. The results uncover a strong and selective effect of the SynI Ca2+ mutant on the mEPSC frequency with preservation of the quantal size, thus excluding the presence of secondary postsynaptic effects. The data also confirm that the Syn1deletion per se does not significantly affect the properties of mPSCs, as previously reported (Chiappalone et al., 2009).
SynIE373K does not influence the density of excitatory and inhibitory synapses
An increased frequency of miniature synaptic events is normally attributable to either an increased number of synapses or an increased probability of spontaneous SV fusion. To test the first possibility, we investigated whether the expression of SynI variants could differentially alter the density of excitatory and inhibitory synapses labeled by the presynaptic markers vGLUT1 and vGAT, respectively (Fig. 3A).
The analysis of the Manders’ colocalization coefficient revealed that about 50% of virally expressed Syn isoforms (mCherry-positive puncta) localized at vGLUT1- and vGAT-positive puncta identifying putative inhibitory and excitatory synaptic contacts (Fig. 3B,C; left panels). Next, we evaluated the number of excitatory and inhibitory synapses along proximal dendrites by vGLUT1/vGAT double immunostaining of transduced hippocampal neurons and found no significant differences in the density of both excitatory and inhibitory synaptic contacts (Fig. 3B,C; right panels). This demonstrates that the development of synaptic connectivity is not significantly affected by the E373K mutation and that the increased mEPSC frequency observed in SynIE373K transduced neurons can be tentatively attributed to an increased probability of spontaneous release.
SynIE373K increases the nerve terminal resting Ca2+ concentration
It is known that the spontaneous release of SVs that occurs stochastically in nerve terminals is dependent on the resting intraterminal [Ca2+]i (Kavalali, 2015, 2020). Thus, we addressed the possibility that SynI can actively bind and buffer Ca2+ in the nerve terminal under resting conditions, thus contributing to the resting [Ca2+]i. To ascertain whether deletion of the Ca2+-binding site on SynI alters resting [Ca2+]i, we co-transduced SynI KO hippocampal neurons with the SyGCaMP6f and either SynIWT or SynIE373K tagged with mCherry. SyGCaMP6f is an ultra-sensitive (Kd = 375 nM) genetically encoded fluorescent Ca2+ indicator in which GCaMP6f is fused to the cytoplasmic domain of the integral SV protein synaptophysin, allowing specific measurements of presynaptic Ca2+ concentrations in the nerve terminal cytosol surrounding SVs (Orlando et al., 2019). To effectively investigate the effects of SynIE373K expression on intraterminal resting Ca2+ concentrations, SyGCaMP6f fluorescence was quantitatively analyzed at mCherry-positive boutons (Fig. 4A, left).
Interestingly, under resting conditions, boutons expressing SynIE373K displayed a significantly increased [Ca2+]i with respect to presynaptic sites expressing SynIWT (Fig. 4A, middle), indicating that SynIWT might function as a high affinity Ca2+-buffer within nerve terminals. This possibility was also supported by the observation that no differences in the amplitude of activity dependent Ca2+ transients were observed upon field stimulation in a wide range of stimulation frequencies (Fig. 4A, right). To independently assess the absolute [Ca2+]i at the presynaptic compartment, we loaded both SynIWT and SynIE373K transduced SynI KO neurons with the ratiometric Ca2+ sensor Fura-2 AM and analyzed Fura-2 fluorescence at the level of mCherry-positive puncta. Under resting conditions, SynIE373K-positive boutons showed a significant increase in the intraterminal [Ca2+]i compared to SynIWT-positive boutons (110.1 ± 18.78 nM vs 265.4 ± 30.02 nM; Fig. 4B), confirming the semiquantitative data obtained with SyGCaMP6f.
The effects of SynIE373K on miniature excitatory postsynaptic currents are rescued by intracellular Ca2+ chelators
The higher resting [Ca2+]i in SynIE373K-expressing neurons suggests that the increased frequency of mEPSCs, in the presence of unchanged synaptic density, is attributable to an increased probability of spontaneous release. To obtain independent evidence on the mechanism and its specificity for excitatory synapses, we recorded mPSC events before and after the treatment with BAPTA-AM, a cell-permeable, fast Ca2+-chelator. Both excitatory and inhibitory mPSCs were initially recorded in control extracellular solution for 2 min.
After the addition of BAPTA-AM (2 µM) to the external solution, mPSCs were recorded for further 15 min and analyzed in three consecutive time windows of 5 min. Strikingly, the increased mEPSC frequency that characterized SynI KO neurons transduced with SynIE373K was totally occluded by BAPTA-AM treatment, while mEPSC amplitude resulted similarly unchanged by BAPTA-AM in both genotypes (Fig. 5A). As expected, the BAPTA-AM treatment did not differentially affect SynIWT and SynIE373K expressing inhibitory synapses (Fig. 5B). Taken together, the data indicate that an alteration in the resting presynaptic Ca2+ homeostasis due to lack of Ca2+-binding to SynI could be the mechanistic cause of the specific enhancement of mEPSC frequency.
SynIE373K reduces the amplitude of excitatory, but not inhibitory, evoked postsynaptic currents
Next, we investigated whether the expression of SynIE373K could differentially alter evoked neurotransmitter release by excitatory and inhibitory synapses. Since SynI plays pre- and post- docking roles in synaptic transmission (Cesca et al., 2010; Longhena et al., 2021), we analyzed single-pulse evoked excitatory (eEPSCs) and inhibitory (eIPSCs) synaptic currents in SynIWT- and SynIE373K-expressing SynI KO neurons. eEPSCs were studied in primary autaptic neurons by depolarizing the cell membrane of the excitatory neuron to + 40 mV for 0.5 ms (Fig. 6A; left panel). Excitatory synapses expressing the SynIE373K mutant showed a significant reduction in the amplitude of eEPSCs in response to the first pulse with respect to SynIWT expressing synapses (Fig. 6A; right panel). The different amplitude of eEPSCs brought us to investigate the response to paired-pulse stimulation, in which both excitatory synapses were subjected to two consecutive stimuli at interstimulus intervals (ISI) ranging between 20 ms and 1 s (Fig. 6B; left panel). Excitatory synapses displayed facilitation at short ISIs that vanished at longer stimulation intervals. As previously reported (Rosahl et al., 1995; Chiappalone et al., 2009) SynIKO excitatory synapses displayed increased facilitation at short ISIs that was normalized by the expression of SynIWT. However, no differences in the expression of this short-term plasticity paradigm were found between SynIWT or SynIE373K (Fig. 6B; right panel).
eIPSCs were studied in low-density neuronal cultures, in which the presynaptic neuron was extracellularly stimulated by micromanipulating the stimulating electrode near the putative presynaptic neuron (Fig. 6C; left panel). No changes were observed in the amplitude of eIPSCs evoked in SynIE373K-transduced inhibitory synapses as compared with SynIWT synapses (Fig. 6C; right panel). Inhibitory synapses exhibited a characteristic depression in response to paired-pulse stimulation that was particularly intense at shorter ISIs and attenuated at longer stimulation intervals (Fig. 6D; left panel). This rescue from paired-pulse depression was closely similar in inhibitory synapses from the three genotypes (Fig. 6D right panel).
The decreased amplitude of eEPSC responses indicates that the impaired Ca2+-buffering by mutated SynI under resting conditions does not play a role in the stimulus-dependent Ca2+ entry in the nanodomains of the active zones, suggesting the involvement of an alternative mechanism.
The reduction of eEPSC amplitude by SynI E373K is attributable to a decrease in the size of the RRPsyn
To identify the quantal parameters of release affected by SynIE373K mutation, we performed cumulative ePSC amplitude analysis in excitatory and inhibitory SynI KO neurons transduced with empty vector (SynIKO), SynIWT or SynIE373K. This method analyzes the cumulative amplitude profile during high- frequency trains of stimuli (Schneggenburger et al. 1999; Baldelli et al. 2007), allowing to extract the values of Pr and RRPsyn.
When excitatory neurons were challenged with a 40 Hz train for 2 s, a significant depression of eEPSCs became apparent, irrespective of the amplitude of the first current in the train (Fig. 7A; left panel); the method assumes that Pr during the train approaches unity and that depression during the steady-state phase is limited by a constant recycling of SVs. Accordingly, the cumulative profile showed a rapid rise followed by a slower linear increase, reflecting the equilibrium between depletion and constant replenishment of the RRP (Fig. 7A; right panel). Consistent with previous reports (Chiappalone et al., 2009), the eEPSC amplitude of SynKO excitatory synapses was larger than that of SynWT synapses, due to a pure increase of the RRRsyn size. The decreased eEPSC amplitude of the SynIE373K mutant (see also Fig. 6A) was entirely attributable to a significant drop, approximately of the same extent, in size of the RRPsyn, in the absence of changes in the Pr of evoked release (Fig. 7B) and consistent with the lack of effect on PPR at short time intervals (see Fig. 6B). To verify if the drop in both amplitude and RRPsyn of eEPSCs due to the expression of the SynIE373K mutant was strictly dependent on a presynaptic impairment or the result of a postsynaptic receptor deficit, we performed the same experiments in the presence of CTZ to selectively block AMPA receptor desensitization due to the intense glutamate release (Coombs et al. 2019; Fig. 7C). Interestingly, the desensitization blockade did not change the quantal parameters reported above (Fig. 7D), confirming that the SynIE373K mutant negatively affects excitatory transmission by acting at the presynaptic level. The same cumulative amplitude analysis was performed on inhibitory synapses, using a 40 Hz train for 2.5 s (Fig. 7E). While the decreased eIPSC amplitude of SynIKO synapses was associated with a corresponding decrease in the RRPsyn, as previously reported (Chiappalone et al., 2009), the lack of effect of SynIE373K on the eIPSC amplitude was confirmed by the absence of significant changes in the quantal parameters of synchronous inhibitory transmission (Fig. 7F). Taken together, the data suggest that the SynIE373K mutant affects the activity-dependent refilling of the RRP from the RecP specifically in excitatory synapses.
SynIE373K impairs the recovery from synaptic depression in both excitatory and inhibitory synapses
Central synapses in mice lacking one or more Syn isoforms display a marked decrease in SV density, reflecting a depletion of the RecP (Gitler et al., 2004; Li et al., 1995; Rosahl et al., 1995; Siksou et al., 2007; Takei et al., 1995). Accordingly, most of the available data indicate that Syn deletion enhances synaptic depression during repetitive stimulation (Gitler et al., 2004; Farisello et al., 2013). Excitatory and inhibitory synapses of SynI KO neurons transduced with either SynIWT or SynIE373K were subjected to a prolonged high-frequency stimulation (HFS). The progressive decay of the ePSCs amplitude during the train and the subsequent recovery from depression upon returning the stimulation frequency to 0.1 Hz were exponentially fitted and analyzed for the kinetic parameters and the steady-state current of depression and recovery. During sustained HFS (30 s @ 20 Hz), SynIE373K excitatory synapses presented no significant changes in the depression SSC and in the slow and fast time constants of depression (Fig. 8A,B). However, they showed a significant impairment in the recovery after depression with an almost two-fold difference in the recovery SSC with respect to SynIWT, in the absence of changes in the time constant of recovery (Fig. 8A,C). Under sustained HFS (30 s @ 10 Hz), SynIE373K inhibitory synapses showed a significantly lower depression SSC than SynIWT inhibitory synapses, with no significant differences in the slow and fast time constants of depression (Fig. 8D,E). In addition, inhibitory synapses displayed an impaired recovery after depression similar to that observed at excitatory synapses: the first response after the stimulus train was markedly smaller than in SynIWT synapses and the recovery SSC was two-fold lower, with no change in the time constant of recovery (Fig. 8D,F). These results suggest that the SynIE373K mutant strongly impairs the recovery from depression in both excitatory and inhibitory synapses and the steady state of depression in inhibitory synapses. This suggests that Ca2+ binding to SynI plays a role in the dynamics of SV pools and in SV mobilization during and after sustained HFS in both types of synapses.
SynIE373K impairs SV recycling and slows-down the kinetics of RRP refilling
To further investigate the mechanisms of defective recovery after stimulus in SynIE373K synapses, we performed synaptophysin-pHluorin (sypHy)-based live cell imaging experiments. Primary SynI KO hippocampal neurons were co-transduced at 10 DIV with lentiviral vectors encoding sypHy and either SynIWT or SynIE373K fused to mCherry and analyzed 8–11 days after transduction (Fig. 9A).
We first evaluated the RRP, which in synaptic imaging accounts for both synchronous and asynchronous components of releasable SVs, by stimulating neurons with 20 APs @100 Hz (Ariel et al. 2010). While no genotype-dependent differences were present in the peak fluorescence, a significant increase in the time constant of fluorescence decay was observed in SynIE373K as compared to SynIWT terminals, revealing an impaired SV endocytosis, suggestive of defective recovery after RRP depletion (Fig. 9B). The lack of change in the RRP measured by sypHy imaging suggests that the decreased RRPsyn observed by patch-clamp recordings (see Fig. 7B), is accompanied by an increase of asynchronous release that may be facilitated by the increased resting Ca2+ (see Fig. 4). Next, to measure recovery after sustained stimulation, we performed repetitive RRP stimulations (20 APs @ 100 Hz) before or after a long-lasting stimulation (600 APs @ 20 Hz; Fig. 9C). Consecutive RRP stimuli resulted in a mild and not significant rundown in both SynIWT and SynIE373K synapses. Despite the peak fluorescence evoked by the first round of stimulation was not changed between the two genotypes, the RRP after the long-lasting stimulation was significantly reduced in SynIE373K synapses (Fig. 9C). These data confirmed the defective SV recovery after sustained stimulation in SynIE373K neurons.
Ultrastructural analysis reveals that SynIE373K affects SV trafficking at rest and during activity
To verify if the defects in the excitatory RRPsyn and recovery from synaptic depression in both excitatory and inhibitory synapses were due to alterations in SV trafficking, we evaluated the density and distribution of SVs in presynaptic terminals by electron microscopy of “asymmetric” (i.e., excitatory) and “symmetric” (i.e., inhibitory) synapses expressing either SynIWT or SynIE373K.
Transduced excitatory and inhibitory synapses were analyzed at rest (1 s before the stimulation train) and at following HFS (30 s @ 20 Hz) by quickly fixing the samples at the end of train and during the recovery period (2 min after the train) (Fig. 10A,C). At rest, excitatory SynIE373K synapses showed a significant reduction in total SV density with respect to control SynIWT synapses (Fig. 10B; left panel). Although no genotype-dependent changes were detected in the density of physically docked SVs (Fig. 10B; right panel), the decreased SV content is consistent with an impaired RRP refilling in mutant nerve terminals that could account for the functionally determined decrease in RRPsyn (see Fig. 7B). The total SV density was decreased under the condition of deep synaptic depression reached at the end of the stimulation train, but with no apparent genotype-dependent differences. However, at the end of the recovery period, excitatory synapses failed to recover the basal SV density (Fig. 10B; left panel), recapitulating the defective recovery evaluated electrophysiologically (see Fig. 8A). At rest and the end of the stimulation train, inhibitory SynIE373K synapses were closely similar to control SynIWT synapses in terms of total and docked SV densities (Fig. 10D). However, similarly to excitatory synapses, the total SV density of mutant inhibitory terminals failed to recover the basal SV density, consistent with the electrophysiological data (Fig. 10D; left panel).
Taken together, these results show that the absence of Ca2+ binding to SynI mainly affects excitatory synapses that fail to maintain a correct distribution of SVs under basal conditions. Moreover, in both excitatory and inhibitory synapses, the deletion of the Ca2+ binding site dysregulates the SynI-mediated trafficking of SVs in terminals challenged with sustained HFS.