Abstract
Communication between neurons involves presynaptic neurotransmitter release which can be evoked by action potentials or occur spontaneously as a result of stochastic vesicle fusion. The Ca2+-binding double C2 proteins Doc2a and –b regulate both spontaneous and asynchronous evoked release, but the mechanism remains unclear. Here, we compared wildtype Doc2b with two Ca2+ binding site mutants named DN and 6A, respectively considered gain-and loss-of function mutants and carrying the substitutions D218,220N or D163,218,220,303,357,359A. We found that both mutants bound phospholipids at low free Ca2+ concentrations and were membrane-associated in neurons at rest, mimicking a Ca2+ activated state. Their overexpression in hippocampal primary neurons culture had similar effects on spontaneous and evoked release, inducing higher mEPSC frequencies and increased short-term depression. Together, these data suggest that the DN and 6A mutants both act as gain-of-function mutants at resting conditions but as loss-of-function during neuronal activity.
Introduction
Regulated exocytosis is strictly dependent on Soluble N-ethylmaleimide-sensitive-factor Attachment protein REceptor (SNARE) proteins, Ca2+-sensors and a number of accessory proteins (Südhof, 2013). Neurotransmitter release is either triggered by action potentials (APs) (Südhof, 2004, 2013; Chapman, 2008; Kaeser and Regehr, 2014; Meriney et al., 2014) or occurs spontaneously at resting membrane potential (Fatt and Katz, 1950; Kaeser and Regehr, 2014).
Evoked release consists of synchronous and asynchronous release components (Otsu et al., 2004; Chapman, 2008; Bacaj et al., 2013). Fast, synchronous release triggered by local Ca2+influx (nano & micro-domain) occurs in less than a millisecond (Kaeser and Regehr, 2014; Neher, 2015) and is governed by the fast Ca2+ sensors Syt-1, 2 or 9 (Xu et al., 2007). Another class of high affinity Ca2+ sensors with slow kinetics such as Syt-7 mediates asynchronous release (Saraswati et al., 2007; Xue et al., 2010; Bacaj et al., 2013; Luo and Südhof, 2017). In synapses lacking the fast sensor, diminished synchronous release is accompanied by increased asynchronous release as shown for Syt-1 (Geppert et al., 1994; Maximov and Südhof, 2005) and Syt-2 (Nagy et al., 2006; Pang et al., 2006; Sun et al., 2007).
Unlike evoked release, spontaneous release is AP-independent and occurs as a stochastic process with a probability that appears to be regulated by intracellular Ca2+ (Xu et al., 2009; Groffen et al., 2010; Ermolyuk et al., 2013). Spontaneous release is important for nervous system functioning as it is involved in synapse maturation, maintenance and synaptic plasticity (McKinney et al., 1999; Sutton et al., 2004; Ehlers et al., 2007; Lee et al., 2010). Like asynchronous release, its frequency is suppressed by Syt-1 and Syt2 (Xu et al., 2009; Kochubey and Schneggenburger, 2011; Courtney et al., 2018) and stimulated by double C2(Doc2) proteins (Groffen et al., 2010; Pang et al., 2011; Williams and Smith, 2018).
Doc2a, -b and –c isoforms together constitute the Doc2 protein family. Unlike Doc2a and –b, the third isoform does not have functionally conserved C2 domains. Doc2a is mainly expressed in the adult brain while Doc2b is more widely expressed in the nervous system and various neuroendocrine tissues (Verhage et al., 1997; Korteweg et al., 2000). Both Doc2a and –b contribute to spontaneous release as shown in knockout and knock-down models (Groffen et al., 2010; Pang et al., 2011). A recent study revealed that they regulate spontaneous excitatory and inhibitory activity, respectively (Courtney et al., 2018). In cell-free assays, Doc2b interacts with the SNARE complex to promote fusion of SNARE-liposomes (Groffen et al., 2010; Yao et al., 2011). Doc2b C2 domains bind to phosphatidylserine-containing membranes in a Ca2+-dependent manner (Groffen et al., 2010) but also to PI(4,5)P2, a phospholipid enriched on the cytoplasmic leaflet of the plasma membrane (Michaeli et al., 2017).
The N-terminal domain of Doc2a/b interacts with Munc13 via a Munc13 interacting domain (MID; Figure 1A, B) in HEK293 cells (Duncan et al., 1999), PC12 cells (Orita et al., 1997) chromaffin cells (Friedrich et al., 2010) and neurons (Hori et al., 1999; Xue et al., 2018). This interaction is sufficient for co-translocation of Munc13 together with Doc2 upon phorbol ester (a DAG homologue) stimulation (Duncan et al., 1999; Hori et al., 1999; Groffen et al., 2004). Phorbol esters potentiate exocytosis in a Ca2+-independent way relying on Doc2/Munc-13 interaction (Hori et al., 1999; Duncan et al., 2000; Groffen et al., 2004). Consistently, Doc2 overexpression causes a Ca2+-independent, Munc-13 dependent release increase upon phorbol ester stimulation (Friedrich et al., 2013) and conversely, blockade of the Doc2-Munc13 interaction by synthetic peptides abolishes phorbol ester potentiation (Hori et al., 1999). Munc13-1 is necessary for the Doc2b-induced priming of secretory granules in chromaffin cells (Houy et al., 2017). However, alteration of this interaction by mutations in the MID domain have no effect on Ca2+-induced Doc2b migration to the membrane (Groffen et al., 2004; Gaffaney et al., 2014). The presence of PI(4,5)P2 targets Doc2b to the plasma membrane upon [Ca2+]ielevation (Michaeli et al., 2017). Hence, Doc2b could support exocytosis by both of several mechanisms: i) together with Munc-13 for vesicle priming or superpriming; ii) Ca2+-dependently by enhancing membrane fusion.
Doc2a/b have a high Ca2+ affinity with half-maximal membrane binding at 450 nM and 175 nM respectively in chromaffin cells (Groffen et al., 2006). Ca2+ binding onto their C2 domains requires five acidic aspartate amino acids (Figure 1B) in close proximity to hydrophobic loops which interact with the membrane after Ca2+activation, resulting in reversible translocation (Groffen et al., 2004, 2010). A similar mechanism occurs in Syt-1 (Frazier et al., 2003), except that Syt-1 is anchored to synaptic vesicles by a N-terminal transmembrane domain and has a lower apparent Ca2+ affinity. Doc2b and Syt-1 compete for SNARE protein binding (Groffen et al., 2010; Yao et al., 2011), which suggest a partially shared mechanism in Ca2+-secretion coupling as recently mentioned for spontaneous release (Courtney et al., 2018).
It is still debated whether Doc2b acts as a direct Ca2+-sensor or as a structural element supporting Ca2+-dependent secretion by another process. Neutralization of two critical aspartates D218 and D220 in the C2A domain of Doc2b (Figure 1B-D) induce Ca2+-independent membrane-binding of the domain (Friedrich et al., 2008; Xue et al., 2015). This mutant, designated Doc2bDN, was therefore considered a gain-of-function mutant. When Doc2bDN expression caused a rise of the spontaneous release rate (mEPSCs), this was taken to support a role as Ca2+ sensor (Groffen et al., 2006, 2010). Another Ca2+-ligand mutant designated Doc2b6A, in which six aspartates were substituted by alanines (Figure 1B-D), is unable to bind Ca2+and therefore considered a loss-of-function mutant (Pang et al., 2011). This mutant still rescues spontaneous release in Doc2 double knock-down neurons, suggesting a Ca2+-independent mechanism. Nevertheless, a recent study suggested that this mutant may enhance spontaneous release by mimicking a Ca2+-bound state (Courtney et al., 2018).
As another point of debate, several studies reported reduced asynchronous release in Doc2a knock-out neurons which is rescued by Doc2b expression (Gaffaney et al., 2014; Xue, Gaffaney, & Chapman, 2015; Yao et al., 2011; Figure 1D) and further enhanced by expression of Doc2b mutants, leaving room for debate whether Doc2b acts selectively on spontaneous release, selectively on asynchronous release, or contributes to both processes.
Here we directly compared both mutants and wildtype (WT) Doc2b for their Ca2+-dependent membrane-binding activity, their subcellular localization and their effects on spontaneous and evoked neurotransmission. Under resting conditions both mutants showed a gain-of-function behavior. At high Ca2+ however, both mutants caused significantly higher short-term depression, suggesting a loss of function. These results reconcile previous findings and show that Doc2b can affect both spontaneous release and short-term plasticity.
Results
Aspartate substitutions in Doc2bDNand Doc2b6Acause constitutive membrane association in resting neurons
Activity-dependent plasma membrane binding is an established feature of Doc2b (Groffen et al., 2006). We first tested the Ca2+-dependent membrane binding of the Doc2bDNand Doc2b6Amutants, previously described to be gain-and loss-of function variants respectively (Groffen et al., 2010; Pang et al., 2011) under resting conditions (Figure 2A) and during stimulation (Figure 2B-C). In samples fixed at resting conditions, eGFP-tagged Doc2bWTwas localized homogeneously throughout the cytosol similar to soluble eGFP, as expected for a cytosolic protein (Figure 2A) and in line with previous findings (Groffen et al., 2006). In contrast, eGFP-tagged Doc2bDNand Doc2b6Aboth showed plasma membrane enrichment (Figure 2A), again consistent with previous studies (Friedrich et al., 2008; Houy et al., 2017).
To assess the activity dependence of membrane association, we performed confocal live imaging upon field stimulation at 40Hz for 15 s (Figure 2B). To control for cell-to-cell differences, mCherry-tagged mutants were co-expressed with eGFP-tagged Doc2bWTin the same neurons. Again, both Doc2bDNand Doc2b6Awere membrane-bound while Doc2bWTshowed a homogeneous cytoplasmic localization at rest. During repetitive stimulation, Doc2bWTshowed a clear plasma membrane association, whereas the distribution of both mutants showed only a small change (Figure 2B, C). Doc2bDNslightly enhanced its membrane localization during repetitive stimulation, while Doc2b6Arevealed a slight decrease in plasma membrane binding (Figure 2B, C). Thus, in living neurons, both mutants exhibit increased plasma membrane binding at rest while activity-induced plasma membrane binding is impaired.
Phospholipid-binding properties of Doc2b mutants
The altered subcellular localization of Doc2bDNand Doc2b6Acould result from altered C2-phospholipid interactions. To investigate this, wildtype and mutant C2A and C2AB fragments were expressed as recombinant proteins in bacteria. The mutants were expressed to the expected molecular mass as verified by SDS-PAGE (Figure 3A, I). C2-phospholipid binding was measured in a liposome aggregation assay (Connell, Scott, & Davletov, 2008), using calibrated EGTA-buffered Ca2+solutions and liposomes composed of 25% DOPS and 75% DOPC (Friedrich et al., 2008). To test phospholipid binding by the C2A domain, a glutathione-S-transferase tag was used to induce self-dimerization, so that phospholipid binding causes liposome clustering which can be measured as an absorbance increase at 350 nm. Addition of a GST-C2AWTprotein fragment to a Ca2+-containing solution caused rapid liposome clustering (Figure 3B, E). This activity was strictly Ca2+-and protein-dependent and followed a sigmoid dose dependence giving in EC50 of 435 ± 31 nM in line with previous reports (Friedrich et al., 2008). GST-C2AWTremained membrane-bound at high free Ca2+concentrations in the 1-10 µM range. In contrast, both GST-C2ADNand GST-C2A3A(here called 3A because only 3 of the total 6 aspartates reside in the C2A domain) showed strong phospholipid binding at low free Ca2+concentrations in the range 0 500 nM. At higher [Ca2+]free, both mutants displayed a loss of phospholipid association (Figure3 C-D).
To test phospholipid aggregation by C2AB fragments, the GST tag was removed using thrombin cleavage. Liposome clustering by C2ABWTincreased Ca2+-dependently to reach a half-maximum at 176 ± 38 nM (Figure 3M) consistent with reported data (Groffen et al., 2006; Giladi et al., 2013; Gaffaney et al., 2014). A maximum occurred at approximately 700 nM,followed at higher [Ca2+]free by a strong decrease in the absorbance signal. At the lowest tested [Ca2+]freeof 2.2 nM, the C2ABDNfragment already showed partial activity (Figure 3N). This activity increased with higher [Ca2+]freeThis increase presumably reflects Ca2+-dependent activity from the intact C2B domain. At high Ca2+concentrations above 500 nM, a decrease was observed reminiscent of the data obtained with C2ABWT. The C2AB6Afragment showed near-complete liposome binding at the lowest [Ca2+]freeand no prominent Ca2+dependency in the 0 – 500 nM range of [Ca2+]free(‘rest’, Figure 3O), followed again by a signal decrease above 500 nM. Taken together, in this cell-free liposome clustering assay, both Doc2bDNand Doc2b6Abehave as a gain-of-function mutants at low [Ca2+], but as loss-of-function mutants at high [Ca2].
Doc2bDNand 6Aoverexpression similarly enhances spontaneous release
To measure the effect of Doc2b on synaptic activity, wildtype hippocampal neurons were cultured either in networks (Figure 4A-C) or on glial micro-islands to promote self-innervation (autaptic neurons; Figure 4D-F). In networks, spontaneous release was measured in presence of 1 µM tetrodotoxin (TTX) to block voltage-gated sodium channels. Doc2b was expressed by lentiviral vectors in wildtype neurons. Western-blot confirmed low expression levels of endogenous Doc2b (Korteweg et al., 2000) which were detectable in lysate from brain and cultured cortical neurons (Figure S1C). Viral infection induced Doc2b protein levels much higher than endogenous levels (Figure S1A).
In both networks and autapses, overexpression of wildtype Doc2b did not change the frequency of miniature excitatory postsynaptic currents (mEPSCs), consistent with previous observations (Groffen et al., 2010). In contrast, overexpression of Doc2bDNor Doc2b6Ainduced an approximately 3-fold increase of the mEPSC frequency (Figure 4B,E; see Supplemental Table 1 for statistical tests). In all cases, the mEPSC amplitude, charge and rise time were not changed by Doc2b overexpression.
In autapses, a small but significant effect on mEPSC decay time was observed in both mutants (Figure S2B, effect size (r): 1.260, Supplemental Table 1). This effect on mEPSC decay was not replicated in continental networks and may be attributable to less accurate fitting of exponential decay curves in groups with extremely high mEPSC frequencies. Taken together, in a direct comparison, the aspartate substitutions in Doc2bDNand Doc2b6Ahave similar gain of function effects on the spontaneous release frequency in hippocampal neurons, which confirms previous observations (Groffen et al., 2010; Pang et al., 2011; Courtney et al., 2018). In conclusion, the effect of mutant Doc2b on spontaneous mEPSC frequency parallel the changes in in vitro plasma membrane binding and phospholipid clustering (i.e. a gain of function in resting conditions).
Doc2bDNor Doc2b6Aoverexpression alter evoked release
The above effects on spontaneous release prompt the question whether the Doc2b mutants also affect synaptic release and plasticity during neuronal activity. We next recorded EPSCs induced by a single action potential (AP) or paired APs at varying intervals in wildtype autaptic neurons. Overexpression of Doc2bWTdid not significantly affect the first evoked amplitude and charge but neurons exhibited a slight reduction compared to control cells expressing eGFP alone. In a recent study (Toft-Bertelsen et al., 2016), Doc2bWTwas shown to disperse syntaxin-1 from plasma membrane clusters, thereby inhibiting Ca2+currents through voltage gated Ca2+channels (VGCCs) in chromaffin cells. To test if a similar mechanism occurs in neurons, we compared the single EPSC amplitude and subsequent calcimycin-evoked charge transfer in wildtype autaptic neurons expressing either eGFP control or overexpressing Doc2bWT. The Ca2+ionophore bypasses VGGCs and triggers exocytosis by an artificial Ca2+influx. No significant change was observed in the EPSC amplitude, nor in the charge transfer induced by calcimycin (Figure S3A-D), indicating that Doc2b does not inhibit synaptic strength by modulating Ca2+influx.
When expressing mutant Doc2bDNor Doc2b6Ahowever, the EPSC charge was larger than in cells expressing Doc2bWT(Figure 5A-B). This larger EPSC amplitude was associated with a significantly stronger paired pulse depression at intervals from 20 to 1000 ms, with the strongest difference in the 20 – 200 ms range (Figure 5C). Again, the Doc2bDNand Doc2b6Amutants caused similar changes.
To investigate further how sustained evoked release was affected by Doc2bDNand Doc2b6A, we performed repetitive stimulation with 100 APs at low (5 Hz) and high (40 Hz) frequency (Figure 5D-S). Overexpression of Doc2bDNand Doc2b6Acaused a similar fast depression of the EPSC charge, both at 5 Hz (Figure 5F) and 40 Hz (Figure 5M) yet without altering the total charge transfer (Figure 5H, O). This phenotype was more evident for normalized than for absolute EPSC charges (compare Figure 5F with E and Figure 5M with L respectively), suggesting that the larger initial EPSC charge in mutant expressing neurons contributes to the phenotype. In contrast, overexpression of wildtype Doc2b did not cause any changes in depression compared to GFP-expressing control neurons.
During repetitive stimulation the reduced synchronous release component is accompanied by an asynchronous release component. In view of previous observations implicating Doc2 proteins in asynchronous release (Yao et al., 2011), we tested if Doc2b overexpression affects the proportion of synchronous and asynchronous components to the total EPSC charge (Figure 5J, Q). During repetitive stimulation at 5 Hz, the expression of wildtype or mutant Doc2b did not affect the balance between synchronous and asynchronous release (Figure 5J). Note that the asynchronous component is generally small for low frequency stimulation. Also during 40 Hz stimulation, the balance between synchronous and asynchronous was unaffected by Doc2bWToverexpression. However, a small change in favor of synchronous release was seen in Doc2bDNexpressing neurons (Figure 5Q), but not in Doc2b6A-expressing neurons, coherent with the higher EPSC charge (Figure 5C).
To assess the rate of synaptic recovery (Figure 5R and S) we measured the EPSC from a single AP, triggered 2 seconds after the end of a 5 or 40 Hz train. Interestingly, cells expressing wildtype Doc2b showed a higher recovery rate than GFP control cells. This activity was impaired in both mutants (0.52 ± 0.025, 0.67 ± 0.035, 0.5 ± 0.019, 0.48 ± 0.018 for control, Doc2bWT,DN,6A). Together, these data suggest that both mutants increase the initial release probability and impair prolonged neuronal secretion.
Doc2bDNor Doc2b6Amutants have no effect on synaptogenesis
Doc2b is temporarily and spatially regulated during the embryonic and early postnatal phase (Korteweg et al., 2000) suggesting a role in neuronal development and synaptogenesis. In addition, Syt-7, another high affinity Ca2+-sensor which shares homology with Doc2b, has been implicated in neurite outgrowth (Arantes and Andrews, 2006). One plausible explanation, for the high frequency of spontaneous release in Doc2bDNand Doc2b6Aexpressing cells would be that those mutations affect neurogenesis or development and increase the synaptic density of neurons via altered membrane trafficking. To evaluate this possibility, we performed immunostainings for the synaptic vesicle marker Synaptophysin and the dendritic neuronal marker microtubule associated protein 2 (MAP-2, Figure 6). Quantitative morphometry of autaptic neurons expressing Doc2bWTand mutants did not reveal significant changes in synaptic density, dendritic length, dendritic synapse density, synapse area soma area or synapse distance from the soma (Figure 6B). Thus, the spontaneous release rise induced by Doc2b mutants is not caused by developmental dysregulation.
Doc2bDNor Doc2b6Amutants enhance spontaneous release in Doc2-deficient neurons
To rule out possible effects of endogenous Doc2b in our experiments, we investigated release in Doc2a/b double knock-out (DKO) autaptic neurons (Figure 7). The neurons were infected with Doc2bWT, Doc2bDNor Doc2b6Aand expression was confirmed by immunoblotting (Figure S1B). Expression of Doc2bWTincreased the average mini frequency from 10 ± 1.4 to 13 ± 1.6 Hz in DKO cells. This trend is similar to previous observations (Groffen et al., 2010) but was not significant in this case, likely due to the smaller sample size and overall high mini frequencies in this experiment. Both mutants again caused a strong increase in the mEPSC frequency while the amplitude (Figure 7C), rise and decay (Figure S2C) were unaffected. Quantification of the mEPSC charge showed a small but significant increase for Doc2b6Aexpressing neurons compared to other groups (Figure S2C, effect size (r): 0.838, Supplemental Table 1). Again, the effect size was low. Taken together, the high mEPSC frequency upon Doc2bDNand Doc2b6Aexpression is replicated in DKO neurons, further confirming a gain-of-function effect of these mutations in resting neurons.
Faster synaptic depression in DKO neurons expressing mutant Doc2bDNor 6A
Similar to the results in wildtype cells, expression of Doc2bWTin DKO cells did not affect the 1stevoked EPSC charge (Figure 8A-B). The 1stevoked EPSC charge in Doc2bDNand Doc2b6Amutant expressing cells showed the same tendency to increase (Figure 8B) as in WT neurons, although this increase was not significant. Both mutants caused stronger depression during paired pulse stimulation (Figure 8C), again most notably in the 20-200ms interval range. Faster depression also occurred during repetitive stimulation at 5 and 40 Hz (Figure 8E,H,K,N), again without affecting the overall neurotransmitter release. The balance in synchronous and asynchronous release was slightly shifted by Doc2bWTexpression in favor of the asynchronous component during 5 Hz trains (Figure 8I), suggesting an effect of the wildtype protein on evoked asynchronous release. Again, Doc2bWTexpressing cells showed a more complete recovery of synaptic strength 2 s after repetitive stimulation, an effect that was lacking in both Doc2b mutants (Figure 8P,Q).
Discussion
To shed light on Doc2b protein function in synaptic release, we studied the Ca2+binding site mutants Doc2bDNand Doc2b6A. We found that both mutants i) mimic an activated state at low [Ca2+] resulting in constitutive membrane enrichment and increased spontaneous neurotransmission; ii) have impaired Ca2+-induced membrane enrichment capacity and negatively affect synaptic strength during repetitive activity (paired pulse, train stimulation, recovery after repetitive stimulation). These phenotypes completely mirrored the phospholipid binding behavior in cell-free assays and cultured neurons, suggesting that phospholipid association is a key event in Doc2b’s overall function in secretion.
The gain– or loss–of–function paradox
In previous studies, Doc2bDNhas been interpreted as a gain-of-function mutant based on the increased phospholipid binding at rest (i.e. the mutations were considered to mimick Ca2+-binding). Doc2b6Awas described to be a loss-of-function mutant based on the loss of Ca2+binding capacity. This classification as gain-and loss-of-function mutants had important implications: the enhanced spontaneous release in Doc2bDNoverexpression was taken as evidence that Doc2b functions as a Ca2+sensor (Groffen et al., 2010), while the very similar phenotype in Doc2b6Aoverexpressing neurons was taken to demonstrate a Ca2+-independent function (Pang et al., 2011).
Our data show that Doc2b6Ashows constitutive membrane binding, as supported by other studies (Houy et al., 2017; Courtney et al., 2018). Thus, Doc2b6Ais not a pure loss-of-function mutant. At the same time however, the Doc2bDNmutant is also not a pure gain-of-function mutant, as indicated by several negative effects during repetitive neuronal activity. The dual effect of Ca2+binding site mutations could be attributed to the different surface charge distribution of the aspartates in the Ca2+-and membrane binding site of the C2 domains. In Doc2b wildtype protein, the aspartates residues are neutralized by binding of Ca2+ions. In the mutants, the neutralization of the aspartates could support membrane binding at rest, but this binding may not reach the same affinity as with Ca2+-bound aspartates. Alternatively, the mutations could slightly misplace the membrane-inserting residues in the loops surrounding the aspartates.
The Ca2+-dependent behavior membrane association of both mutants seems slightly more complex as might be expected. At high (>500 nM) [Ca2+]free, isolated C2AB fragments showed a loss of liposome clustering activity using synthetic membranes composed of 75%DOPC and 25%DOPS. Recent investigations revealed a similar effect for the Doc2b6Amutant (termed 6x) as its liposome binding capacity dropped in presence of Ca2+ (Courtney et al., 2018). In our hands a similar reduction in lipid binding occurred at high (>700 nM) [Ca2+]free for Doc2bWTC2AB domain. In live neurons where the physiological membrane composition is different, incomplete detachment was also observed for Doc2b6A(Figure 2C) but not for Doc2bWTor Doc2bDN. Syt-7 was also reported to have a reduced lipid binding activity at high [Ca2+]free, effect that was associated with an inhibitory effect on norepinephrine secretion in PC12 cells (Sugita et al., 2001). Another similar observation is the negative effect of high [Ca2+]freeon Syt-1 in vitro fusion ability (Park et al., 2015), together suggesting that most Ca2+-sensors function in a limited [Ca2+] window.
In a previous study using isothermal calorimetry, the D218,220N mutation did not affect Ca2+ binding activity in a recombinant C2AB fragment named C2ACLMB (for calcium ligand mutant; Gaffaney et al., 2014). However, C2ACLMBCLM carrying the additional mutation D357,359N in the C2B domain abolished Ca2+association to C2domains, suggesting that in the absence of lipids, the C2B domain is solely responsible for the Ca2+binding activity (Gaffaney et al., 2014). The C2ACLMBCLMmutant also greatly increased spontaneous release.
In presence of phospholipids, the negatively charged head groups may stabilize bound Ca2+ions, increasing the apparent Ca2+affinity (Zhang et al., 1998; Radhakrishnan et al., 2009). D220N substitution within the C2A Ca2+-binding-pocket of Doc2b, alone or in combination with other mutations is responsible for the constitutive membrane binding (Xue et al., 2015). A single residue substitution (D303N) completely abolishes Doc2b translocation and this mutant does not rescue spontaneous release (Courtney et al., 2018), confirming the idea that Doc2b acts as a Ca2+-sensor. Considering all these mutants, there is a striking correlation between the Ca2+-dependent phospholipid association of Doc2b and its function in spontaneous neurotransmission at rest.
Ca2+ binding mutants affect synaptic plasticity during repetitive activity
In chromaffin granule secretion, wildtype Doc2b serves both positive and negative roles (Houy et al., 2017), a feature that is shared with other exocytotic proteins such as synaptotagmins, complexins and munc18s. In this system, Doc2bDNand Doc2b6Afavour immediate chromaffin granule fusion but impair sustained release at high [Ca2+]. This phenotype might also be a collateral effect of constant vesicle fusion at rest, exhausting the immediate releasable pool (IRP)
In synapses, we observed a clear modification of evoked release by Ca2+binding mutants. However, overexpression of Doc2bWT(Figure 5 and 8) or the removal of endogenous Doc2b (Groffen et al., 2010) does not importantly affect evoked release, suggesting that this is not a major function of Doc2b in synapses. The effect of Doc2b mutants on evoked release may possibly represent an ectopic function. On the other hand, Doc2bWToverexpression and rescue revealed a consistent effect in post-burst recovery (Figure 5R,S and Figure 8P,Q), absent in DN and 6A expressing neurons. This could occur by increasing the vesicular release probability at high residual [Ca2+] after burst activity. Doc2bDNand 6Aineffectiveness in this potentiation mechanism might be the result of their loss of Ca2+-sensitivity. Additionally, a subtle but significant shift in favour of asynchronous release during repetitive stimulation appeared in DKO neurons rescued with Doc2bWT(Figure 8I) but not with mutants. During intense release, as suggested by the reduced in vitro lipid binding at high [Ca2+], Doc2bWTcould inhibit fast exocytosis acting as a clamp of the fast fusion machinery, sparing the ready releaseable pool (RRP) and preserving a slowly releasable pool (SRP; Houy et al., 2017).
Mutant overexpression caused short-term depression during trains and paired pulse stimulation in both wildtype and DKO neurons. A higher 1stEPSC coupled to short-term depression in mutant expressing neurons suggests an increase in vesicle release probability (Pvr) in the early phase of release. In autapses we found no evidence that Doc2b mutants enhance asynchronous release, as was previously observed for locally stimulated neuronal networks (Gaffaney et al., 2014; Xue et al., 2015). We speculate that an asynchronous release component could possibly build up from delayed synchronous release indirectly connected to the postsynaptic cell.
Conclusion
We conclude that the Doc2bDNand Doc2b6Amutants do not represent divergent gain-and loss-of-function mutants but show similar behavior, characterized by increased activity at rest and impaired activity at high [Ca2+]iduring neuronal activity. When using mutations to investigate the role of Doc2b in asynchronous or spontaneous release, it is important to consider the different performance of the protein at low and high Ca2+. The strict correlation between plasma membrane association and spontaneous release frequency supports a direct role as a Ca2+sensor. In addition, a Ca2+-dependent function in synaptic recovery is also supported by the data. Our collective results provide a unifying explanation for seemingly conflicting data and emphasize the importance of the Ca2+-dependent phospholipid association in Doc2b-mediated secretory regulation.
Material & Methods
Mouse lines
Animals were housed, bred and handled in accordance with Dutch and EU governmental guidelines. Protocols were approved by the VU University Animal Ethics and Welfare Committee (approval number FGA 11-06). Wildtype C57BL/6J mice were obtained from Charles River Laboratories. Doc2 a & b double knockout mice (DKO), maintained on the same C57BL/6J genetic background, were previously described (Groffen et al., 2010). To dissociate brain tissue from DKO mice, hippocampi were isolated at postnatal day 1 (P1). For wildtype mice, E18–stage embryos were used. In this case, pregnant females were sacrificed by cervical dislocation, embryos were obtained by caesarian section, decapitated and used for dissection.
Primary culture of mouse neurons
To isolate mouse neurons, brains were placed in Hanks buffered salt solution (HBSS, Sigma) buffered with 1 mM HEPES (Invitrogen). After meninges removal, hippocampi and cortices were dissociated and separately treated. The tissue was incubated with 0.25% trypsin (Invitrogen) for 20 min at 37°C and washed in DMEM. Cells were dissociated by trituration with a fire-polished Pasteur pipette and counted in a Fuchs-.Rosenthal chamber. Neurons were plated in warmed Neurobasal medium supplemented with 2% B-27, 1.8% 1M HEPES, 0.25% glutamax and 0.1% Pen-strep (all products Invitrogen) as previously established (Wierda et al., 2007).
Electrophysiology experiments were performed in network or autaptic cultures. For network cultures, hippocampal neurons were plated at a density of 25K cells per well in 12-wells plates on etched glass coverslips containing a confluent layer of rat astrocytes (Wierda et al., 2007). For autaptic cultures, 1.5K cells per 12-well or 3K per 6-well were plated on coverslips with astrocyte micro-islands stamps (Wierda et al., 2007). For live imaging, coverslips were coated with rat tail collagen solution (BD Biosciences, Bedford, USA) and cells were plated in low density networks (1K per 12-well) (Wierda et al., 2007). For western blotting, cortical neurons were plated at 300K per well in 6-well plates without coverslips, coated overnight with 0.0005% poly-L-ornithine (Sigma) and 2 µg/ml laminin (Sigma) in PBS and washed with sterile water.
Viral overexpression of Doc2b
For functional assays, Doc2b and EGFP were expressed as separate proteins from a single mRNA using an IRES2 internal ribosome entry site. Wildtype rat Doc2bWT(LIP#1984) was compared to Doc2bDNcarrying the D218, 220N mutation (LIP#1985) (Groffen et al., 2004, 2010) and Doc2b6Acarrying the D163, 218, 220, 303, 357, 359A mutation (LIP#1986) (Pang et al., 2011). Lentiviral infectious particles were packaged in HEK293T human embryonic kidney cells with a passage number lower than 25, maintained in Dulbecco’s Modified Eagle Medium (DMEM) supplemented with 10% fetal calf serum, 50 U/ml penicillin-streptomycin and 1x non-essential amino acids (Gibco). At 2 days in vitro (DIV2) the cells were transfected at 50% confluence with three plasmids: p.MDG2 (encoding the viral envelope protein), pCMVδR8.2 (encoding packaging factors) and a p156RRL-derived plasmid encoding Doc2b. LIP#1984 encoded Doc2bWT, LIP#1985 Doc2bDNand LIP#1986 Doc2b6A. At DIV3 the medium was changed to Optimem + 50 U/ml penicillin-streptomycin without fetal bovine serum. At DIV4, the supernatant containing infectious particles was centrifuged at 1000 x g to remove cell debris. The supernatant was concentrated by ultrafiltration using a 100 kDa cutoff membrane (UFC910024, Millipore, spun at 4000 x g for 20-30 min) to achieve a final volume of 150 µl. The LIPs were diluted to 1 ml with phosphate-buffered saline, filtered through 0.45 µm and stored in aliquots at −80°C until use. Neurons were infected at DIV1 to induce Doc2b expression. To investigate subcellular protein localization, Doc2b was C-terminally tagged with EGFP. Neurons were transduced with Semliki infectious particles 10 to 12 hours before experimentation (SIP#293 encoding Doc2bWT, SIP#244 encoding Doc2bDNand SIP#295 encoding Doc2b6A) as described (Houy et al., 2017).
Electrophysiology in primary hippocampal neuronal networks and autapses
For electrophysiology, both continental and island cells were used between DIV 14 to 21. Doc2b-expressing cells were identified by monitoring EGFP fluorescence. The standard extracellular medium included 140 mM NaCl, 2.4 mM KCl, 4 mM CaCl2, 4 mM MgCl2, 10 mM HEPES, 10 mM glucose, 300 mOsm, pH 7.3. Our standard intracellular (patch pipette) solution was EGTA free to prevent Ca2+buffering; it constituted 125 mM K-gluconate, 10 mM NaCl, 4.6 mM MgCl, 4 mM K2-ATP, 15 mM creatine phosphate and 10 U/ml phosphocreatine kinase, 300 mOsm, pH 7.3. To record spontaneous excitatory events in network cultures, 1 µM tetrodotoxin (TTX, Abcam) and 20 µM gabazine (Sigma Aldrich) were added to the extracellular medium. In autapses, only gabazine was added. Where indicated, the Ca2+ionophore calcimycin (A23187, Sigma) was used at a final concentration of 10 µM and applied by puff for 100 seconds.
The patch pipettes were made of borosilicate and pulled using a multi-step filament pulling (P-1000, Sutter Instruments, Novato, USA) to achieve a pipette resistance of 3 to 5 MOhm. In whole-cell configuration, neurons were voltage clamped at −70 mV with an Axopatch 200B or Multiclamp 700B amplifier (Molecular Devices). Signal was low-pass filtered at 1 kHz and digitized at 10 kHz with a Digidata 1440A or 1550 (Molecular Devices). Neurons with a series resistance (Rs) exceeding 15 MOhm or with an Rs increase beyond 20% of the initial value were excluded. Rs was compensated to 70%. EPSCs were elicited by depolarizing the cell to 0 mV for 1 ms. Standard stimulation paradigms comprised spontaneous activity recording, paired pulse stimuli with intervals from 20 ms to 1 s, two trains of each 100 action potentials at 5 Hz and 40Hz. Each train was followed by a single stimulus at 2s after the last depolarization to test synaptic recovery.
Miniature EPSCs (mEPSCs) were detected using Mini Analysis 6.0 (Synaptosoft Inc.), using thresholds of 7 pA for event amplitude and 15 pC for area. Evoked release events (paired pulse stimulations) were analyzed using an in-house routine in the MATLAB®environment (He et al., 2017 Mathworks) to calculate the paired pulse ratio, the EPSCs charge and amplitude. In AP-induced burst EPSCs, synchronous and asynchronous components were isolated from total response charge as follows. Stimuli artefacts were removed, synchronous charge was determined by a straight line between the EPSC starting point n and the following EPSC starting point n+1. The area below this line was considered as synchronous, the area between the line and before stimulation baseline was considered as asynchronous (see Figure 5K). Both components were calculated using cubic interpolation. Calcimycin evoked responses were quantitated using Clampfit 10.4 (Molecular Devices) by measuring the total charge transfer during the compound application.
Doc2b live microscopy
For Doc2b protein translocation imaging, hippocampal neurons from wildtype mouse at embryonal day E18 were dissociated and platted at 25K per well on glia layer. Cells were double-infected with Semliki virus encoding for Doc2bWT-EGFP and either Doc2bDN-mCherry or Doc2b6A-mCherry between DIV9 and DIV11. The two SIP stocks were first mixed in a 1:1 volume ratio and then added to each coverslip. Live imaging was performed 8–11 h post infection using a Nikon A1R confocal laser microscope controlled by NIS-elements AR software version 4.30 (Laboratory Imaging). Intracellular and extracellular solutions were similar to electrophysiology experiments. Field stimulation of 15 seconds at 40Hz was given after 15 seconds of baseline recording. Trains were triggered from a Master 8 connected to a stimulus isolator (WPI type A385) set to 30 mA output current. ImageJ was used for data analysis. Doc2b translocation was measured by plotting line profiles going through the soma of the neuron.
Solutions for phospholipid-binding assays
Chelated Ca2+/EGTA solutions containing 50 mM HEPES, pH 7.4, 100mM KCl, and varying concentrations of Na2EGTA and CaCl2(0 to 10mM) were made by predicting [Ca2+]free with MaxChelator (http://maxchelator.stanford.edu/CaEGTA-TS.htm). Actual [Ca2+]free was verified in each solution by recording fluorescence excitation spectra of fura-2 (Invitrogen, 0.07 µM) at an emission wavelength of 510 nm on a LS55 fluorescence spectrophotometer (Perkin Elmer). [Ca2+]free was calculated as Kd x [(R-Rmin)/(Rmax-R)] x (Fmax380/Fmin380), where F380is the fluorescence intensity at ʎexcit. = 380 nm and R is the ratio F340/F380. KdEGTA was measured at 34.906 nM. Liposomes were formed by drying chloroform solutions containing 25% 1,2-dioleoyl-sn-glycero-3-phospho-L-serine (DOPS, Avanti Polar Lipids) and 75% 1,2-dioleoyl-sn-glycero-3-phosphocholine (DOPC, Avanti Polar Lipids)under a nitrogen stream. The phospholipids were resuspended in 50 mM HEPES, 100 mM KCl, pH 7.4 to a final concentration of 1 mg/ml, sonicated 5 times for 10 seconds and centrifuged for 90 min at 21,000 x g to clear the liposomes from large aggregates as described previously (Friedrich et al., 2008).
Expression and purification of Doc2b C2A and C2AB fragments
The C2A fragment (aa125-255) and C2AB (115-412) fragment of Doc2bWT, Doc2bDNand Doc2b6Awere expressed as glutathione-S-transferase (GST) fusion proteins in the E. coli strain BL21 and purified as described (Brouwer et al., 2015). GST-C2A was eluted from glutathione-agarose beads by glutathione, leaving the GST tag attached and allowing GST dimerization. C2AB was eluted with thrombin digestion, thus removing the tag. In both cases, Ca2+-dependent C2-membrane interaction causes liposome aggregation which can be measured by an OD 350 nm increase (Connell et al., 2008a; Friedrich et al., 2008). Protein amounts, potential contamination or degradation were verified by SDS gel electrophoresis. To compare lipid-binding activities, recombinant proteins were pooled from the following number of expression cultures: 4 for C2AWT, 5 for C2ADN, 5 for C2A6A, 3 for C2ABWT, 6 for C2ABDN, 6 for C2AB6A.
Phospholipid-binding assays
To measure phospholipid binding, 20 µl of liposomes were mixed with 78, 70, 70 µl for GST-C2AWT, GST-C2ADN, GST-C2A3Arespectively and 73, 70, 60 µl for C2ABWT, C2ABDN, C2AB6Arespectively of buffered Ca2+/EGTA solution in a quartz cuvette to a final concentration of 0.5 mg/ml and the absorption at 350 nm was monitored for 10 minutes at 0.2 s intervals in a Cary 50 UV-Vis spectrophotometer. GST-C2A and C2AB protein used concentration were determined by measurement of their lipids aggregation capacity to reach a maximal OD 350nm around 0.5 and measured afterward by SDS-PAGE. After 60s of baseline recording, GST-C2A or C2AB protein was added to a final concentration of 9 µM, 26 µM, 24 µM for GST-C2AWT, GST-C2ADN, GST-C2A3Arespectively and 0.36 µM, 0.39 µM, 0.9 µM C2ABWT, C2ABDN, C2AB6Arespectively, inducing liposome binding and consequently an increase of A350. The A350 increase was not observed in absence of Ca2+ (Figure 3).The EC50 values were manually calculated using OD 350nm half-maximum X-intercept from raw data.
Immunostaining and confocal imaging for synapse counting
After lentiviral expression of Hippocampal neurons expressing Doc2bWT, Doc2bDNor Doc2b6A, as marked by IRES-eGFP fluorescence, were fixed for 20 minutes at RT in 3.7% paraformaldehyde. After washing with PBS, cells were permeated with 0.5% Triton X-100 for 5 minutes and incubated for 20 minutes with 2% normal goat serum and 0.1% Triton X-100 to prevent aspecific binding. Coverslips were incubated for 2 hours at RT or overnight at 4°C in presence of polyclonal chicken anti-MAP2 (Abcam, ab5392) and polyclonal guinea pig anti-Synaptophysin1 (SySy, 101004) both diluted 1000-fold. After washing, cells were incubated overnight at 4°C with Alexa-546-and 647-conjugated secondary antibodies (1:1000, Invitrogen), washed again and mounted with Mowiol. Images were acquired with a confocal microscope LSM 510 (Carl Zeiss) with 488 nm, 543 nm, 633 nm lasers, using a 40x oil immersion objective and a scan resolution of 1024 x 1024 pixels. Stacks of images with an optical thickness of 0.4 µm were obtained. Neuronal morphology characteristics were analyzed with an automated image analysis MATLAB®(Mathworks) routine (Schmitz et al., 2011).
Western blots
Cortical neuron cultures from WT and Doc2a, b DKO mice were plated to 200 K cells per well, infected at DIV 1 with lentiviral Doc2b constructs (LIP #1984, #1985, #1986 as above) and harvested at DIV 17. Cells were washed 2 times with PBS, lysed in Laemmli sample buffer, loaded with 50% or the totality of each well for WT and DKO cells respectively, separated by SDS-PAGE and blotted on PVDF membrane (Biorad). Membranes were blocked in 2% skim milk powder (Merck) and 0.5% FCS (Gibco) in PBS with 0.01% Tween-20 (Sigma-Aldrich). Doc2B polyclonal antibody 13.2 was used as primary antibody (1:500) for incubation overnight at 4°C. Goat anti rabbit alkaline phosphatase (Jackson lab) was used as secondary and Attophos (Promega) as substrate for 30 min incubation at RT. Reprobing was made for actin immunostaining with monoclonal anti-actin antibody C4 (1:3000 Chemicon) and Goat anti mouse alkaline phosphatase as secondary Ab (1:10000).
Statistical analysis
All statistical analysis was performed using SPSS v.25.00 (IBM Corp., Armonk, NY, USA). All data are reported as mean ±SEM, except when specified. The number of measurements “n”, indicate the number of cells per group and the number of independent observations “N”, represent experimental weeks. Data were checked for normality using Shapiro-Wilk and Kolmogorov-Smirnov tests. Homogeneity of the variance was assessed with Levene’s test and the sphericity assumption was tested with Mauchly’s test. Parametric or non-parametric tests were run depending on homogeneity assumption respect or violation. Moreover, if the sphericity assumption was not met the Greenhouse-Geisser correction was used to adjust the degree of freedom. Considering that groups were independently acquired from one another, independent samples tests were performed i) For two groups a Mann-Whitney U test was performed ii) For more than 2 groups, one-way repeated measure ANOVA or Kruskall-Wallis test were used. For each experiment, the p-value alpha significance threshold was adjusted for multiple testing. The effect size was calculated for the independent samples Mann-Whitney U test as , for one-way repeatedANOVA as and for Kruskall-Wallis test as P-values lower than the accepted α-significance were highlighted in bold and the effect size was reported. Post-hoc tests were all pairwise comparison. In graphs, the empty dots represent single data points, bar plots represent the mean and error bars the SEM. Statistical outliers are depicted by red ‘stars’ symbols.
Competing interests
All authors declare no competing interests.
Funding
This study was financially supported by the EU in the European Neuroscience Campus Network (Cycle 4, project 4) and the Netherlands Organization for Health Research and Development (ZonMW project 91113022).
Author contributions
Quentin Bourgeois-Jaarsma, Formal analysis, Investigation, Writing-review and editing; Matthijs Verhage, Conceptualization, Writing-review and editing; Alexander J. Groffen, Formal analysis, Funding acquisition, Investigation, Writing-review and editing
Acknowledgements
We thank Robbert Zalm, Desiree Schut, Joke Wortel, Joost Hoetjes, Ingrid Saarloos and Eline Kompanje for excellent technical support. We express gratitude to Vincent Huson, Javier Emerador Melero, Rocio Diez Arazola and members of CNCR lab for their critical advice and their scientific support.