Synaptic transmission relies on the release of neurotransmitters by calcium-triggered exocytic fusion of synaptic vesicles (SVs), tiny organelles (~40 nm in diameter) that store and secrete neurotransmitter molecules at specialized active zone (AZ) release sites within presynaptic nerve terminals (Südhof, 2004). Following exocytosis, SVs are locally reformed via compensatory endocytic retrieval of membrane and its protein constituents (i.e., SV proteins) from the cell surface in order to keep the presynaptic surface area constant and to ensure sustained neurotransmission (Dittman and Ryan, 2009; Kononenko and Haucke, 2015). While the core components that mediate the exo- and endocytosis of SVs have been identified and characterized in detail (Rizzoli, 2014; Takamori et al., 2006; Wilhelm et al., 2014), the molecular mechanisms underlying SV endocytosis and reformation have remained controversial (Delvendahl et al., 2016; Kononenko et al., 2014; Soykan et al., 2017; Watanabe et al., 2013; Watanabe et al., 2014; Milosevic, 2018).

Pioneering ultrastructural analyses of stimulated frog neuromuscular junctions using electron microscopy (EM) suggested that SVs are recycled via clathrin-mediated endocytosis (CME) of plasma membrane infoldings or budding from cisternal structures located away from the AZ (Heuser and Reese, 1973). Subsequent studies in neurons and in nonneuronal models showed that CME occurs on a timescale of many seconds and crucially depends on clathrin and its essential adaptor protein complex 2 (AP-2) (Mitsunari et al., 2005), a heterotetramer comprising α, β2, μ2, and σ2 subunits, as well as a plethora of endocytic accessory proteins (Südhof, 2004; Dittman and Ryan, 2009; Kononenko and Haucke, 2015; Rizzoli, 2014; Saheki and De Camilli, 2012; Kaksonen and Roux, 2018). These and other works led to the view that SVs are primarily, if not exclusively, recycled by clathrin/AP-2-dependent CME (Granseth et al., 2006; Granseth et al., 2007).

Recent studies using high-pressure freezing EM paired with optogenetic stimulation have unraveled a clathrin-independent mechanism of SV endocytosis (CIE) in response to single action potential (AP) stimuli that selectively operates at physiological temperature (Watanabe et al., 2013). This ultrafast endocytosis (UFE) pathway is distinct from kiss-and-run or kiss-and-stay exoendocytosis observed in neuroendocrine cells (Alés et al., 1999; Shin et al., 2018), operates on a timescale of hundreds of milliseconds, and results in the generation of endosome-like vacuoles (ELVs) from which SVs can reform via clathrin-mediated budding processes (Kononenko et al., 2014; Watanabe et al., 2014). Temperature-sensitive, clathrin-independent SV endocytosis has also been observed by presynaptic capacitance recordings at cerebellar mossy fiber boutons and by optical imaging at small hippocampal synapses (Delvendahl et al., 2016; Kononenko et al., 2014; Soykan et al., 2017) and is compatible with the accumulation of postendocytic presynaptic vacuoles upon acute or sustained genetic perturbation of clathrin at stimulated fly neuromuscular junctions (Kasprowicz et al., 2014; Kasprowicz et al., 2008; Heerssen et al., 2008) and at mammalian central synapses (Kononenko et al., 2014; Imig et al., 2020). Collectively, these studies suggest that SV endocytosis under physiological conditions is primarily mediated by CIE (e.g., UFE), while the function of clathrin and clathrin adaptors such as AP-2 is limited to the reformation of functional SVs from internal ELVs rather than acting at the plasma membrane proper.

While this model can provide a mechanistic explanation for the observed speed of SV endocytosis, the key question of how SV proteins are sorted to preserve the compositional integrity of SVs (Takamori et al., 2006) remains unresolved. Four points pertaining to this question are to be considered. First, optical imaging-based acid quench experiments in hippocampal neurons indicate that the capacity of UFE is limited to single or few APs, while the majority of SV proteins appear to be internalized on a timescale of several seconds following AP train stimulation (Dittman and Ryan, 2009; Soykan et al., 2017; Kim and Ryan, 2009), that is a timescale compatible with either CME or CIE. Second, as high-pressure freezing EM experiments have not been able to reveal the fate and time course of endocytosis of SV proteins, it is formally possible that UFE proceeds under conditions of clathrin depletion (Watanabe et al., 2014), while SV proteins remain stranded on the neuronal surface. Third, mutational inactivation of the binding motifs for the clathrin adaptor AP-2 in the vesicular transporters for glutamate (VGLUT) and γ-aminobutyric acid (VGAT) severely compromises the speed and efficacy of their endocytic retrieval at room temperature (Voglmaier et al., 2006; Santos et al., 2013; Li et al., 2017; Foss et al., 2013), arguing that at least under these conditions (e.g., low temperature when UFE is blocked) these SV proteins may be retrieved from the cell surface via clathrin/AP-2. Finally, genetic inactivation of clathrin/AP-2-associated endocytic adaptors for the sorting of specific SV proteins, for example Stonin 2, an adaptor for the SV calcium sensor Synaptotagmin, or AP180, an adaptor for Synaptobrevin/VAMP2, causes the accumulation of their respective SV cargos at the neuronal plasma membrane (Kononenko and Haucke, 2015; Cousin, 2017; Kaempf et al., 2015; Koo et al., 2015; Mori and Takamori, 2017). These data could be interpreted to indicate that at least some SV proteins are endocytosed via CME, whereas others may use CIE mechanisms such as UFE. However, such a model bears the problem of how CME and CIE pathways are coordinated and how membrane homeostasis is then maintained.

To solve the question how SV protein sorting is accomplished and how this relates to CME- vs CIE-based mechanisms for SV endocytosis, we have conducted a comprehensive survey of six major SV proteins in primary hippocampal neurons depleted of clathrin or conditionally lacking AP-2. We show that clathrin is dispensable for the endocytosis of all SV proteins at physiological temperature independent of the stimulation paradigm. In contrast, endocytic retrieval of a subset of SV proteins including VGLUT1 and VGAT depends on sorting by AP-2. Our findings highlight a clathrin-independent function of the clathrin adaptor AP-2 in the endocytic retrieval of select SV cargos from the presynaptic plasma membrane and suggest a revised model for SV endocytosis and recycling.

Based on prior works (Dittman and Ryan, 2009; Rizzoli, 2014; Delvendahl et al., 2016; Kononenko et al., 2014; Soykan et al., 2017; Watanabe et al., 2014; Milosevic, 2018; Heuser and Reese, 1973; Saheki and De Camilli, 2012; Takei et al., 1996), three main models for the sorting and endocytic recycling of SV proteins at central mammalian synapses can be envisaged (Figure 1). According to the classical CME-based model of SV endocytosis, SV proteins exocytosed in response to AP trains undergo clathrin/AP-2-mediated sorting and endocytosis from the presynaptic plasma membrane or plasma membrane infoldings (Takei et al., 1996) akin to CME in receptor-mediated endocytosis in nonneuronal cells (Kaksonen and Roux, 2018). This model predicts that loss of either clathrin or its essential adaptor AP-2 delays the endocytic retrieval of all major SV proteins (Figure 1A). A second model supported by elegant high-pressure freezing (Watanabe et al., 2014), electrophysiological (Delvendahl et al., 2016), and optical imaging (Kononenko et al., 2014; Soykan et al., 2017) experiments suggests that exocytosed SV proteins are internalized via clathrin- and AP-2-independent bulk endocytosis. In this model, SV protein sorting occurs from internal ELVs that are formed downstream of the endocytic internalization step. Hence, at physiological temperature the endocytic retrieval of all major SV proteins would proceed unperturbed in the absence of either clathrin or AP-2 (Figure 1B). Finally, it is conceivable that exocytosed SV proteins present on the neuronal surface are sorted by dedicated endocytic adaptors, for example the AP-2 complex, to facilitate their clathrin-independent internalization via CIE. Clathrin, possibly in conjunction with AP-2 and other adaptors then operates downstream of CIE to reform functional SVs from ELVs. In this case, loss of clathrin or AP-2 is predicted to result in distinct phenotypes: While endocytosis of SV proteins is unperturbed upon depletion of clathrin, loss of AP-2 would be expected to selectively affect the rate and efficacy of endocytosis of distinct SV cargos recognized by AP-2 (Figure 1C).

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Endocytic retrieval of SV proteins in hippocampal neurons occurs independent of clathrin at physiological temperature

To distinguish between these models, we optically recorded the stimulation-induced exo-/endocytosis of SV proteins carrying within their luminal domains a pH-sensitive superecliptic green fluorescent protein (SEP, often also referred to as pHluorin) that is dequenched during exocytosis and undergoes requenching as SVs are internalized and reacidified (Miesenböck et al., 1998; Sankaranarayanan et al., 2000). Specifically, we monitored SEP-tagged chimeras of the calcium sensor Synaptotagmin 1 (Syt1), the multispanning glycoprotein SV2A, the SNARE protein Synaptobrevin/VAMP2 (hereafter referred to as Syb2), the tetraspanin Synaptophysin (Syp), the vesicular glutamate transporter 1 (VGLUT1), and the vesicular GABA transporter (VGAT), which have been used extensively to monitor SV recycling in various preparations (Kononenko et al., 2014; Soykan et al., 2017; Granseth et al., 2006; Kim and Ryan, 2009; Voglmaier et al., 2006; Miesenböck et al., 1998; Sankaranarayanan et al., 2000) and constitute the major protein complement of SVs based on their copy numbers (Takamori et al., 2006). We capitalized on the fact that, in hippocampal neurons stimulated with trains of APs, SV endocytosis occurs on a timescale of >10 s at physiological temperature (Soykan et al., 2017), for example a timescale that is much slower than requenching of SEP due to reacidification of newly endocytosed vesicles (Atluri and Ryan, 2006; Egashira et al., 2015). Therefore, under these conditions, the decay of SEP signals can serve as a measure of the time course of SV endocytosis.

We first depleted clathrin heavy chain (CHC) in hippocampal neurons using lentiviral vectors to ~10–25% of the levels found in controls as evidenced by confocal imaging of immunostained samples and by immunoblot analysis (Figure 2—figure supplement 1A–D), in agreement with previous data (Kononenko et al., 2014; Watanabe et al., 2014; Granseth et al., 2006). Lentiviral shRNA-mediated depletion of clathrin potently inhibited uptake of transferrin into cultured neurons, indicating effective blockade of CME (Figure 2A, B). To assess the effects of clathrin loss on the stimulation-induced endocytic retrieval of SV proteins, we stimulated control or clathrin-depleted hippocampal neurons expressing any one of the six major SEP-tagged SV proteins with a high-frequency stimulus train (200 APs applied at 40 Hz) at physiological temperature (35 ± 2°C), and monitored fluorescence rise and decay over time. Strikingly, exo-/endocytosis of all SEP-tagged SV proteins proceeded with unaltered kinetics, that is τ ~ 15–20 s, irrespective of the depletion of clathrin (Figure 2C–N). Similar results were seen if clathrin function was acutely blocked by application of the small molecule inhibitor Pitstop2 (von Kleist et al., 2011; Figure 2O, P), a condition that potently inhibited CME of transferrin (Figure 2—figure supplement 1E,F). When these experiments were repeated under conditions of low-frequency stimulation (200 APs applied at 5 Hz) at room temperature (RT), that is conditions in which the efficacy of CIE is reduced (Watanabe et al., 2014), the endocytic retrieval of Syp-SEP (also often referred to as SypHy) or VGLUT1-SEP was delayed in neurons depleted of clathrin (Figure 2—figure supplement 1G-J), consistent with earlier data using Syt1-SEP as a reporter (Kononenko et al., 2014).

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These results show that in small hippocampal synapses at physiological temperature, endocytosis of all major SV proteins and hence, of SVs as a whole, occurs independent of clathrin via CIE.

CIE of a subset of SV proteins depends on the clathrin adaptor AP-2

We next set out to analyze whether endocytosis of the major SV proteins is also independent of the essential clathrin adaptor complex AP-2. This would be expected, if SV endocytosis was mediated by CIE and the sole function of clathrin/AP-2 was to reform SVs from postendocytic ELVs (Figure 1B). We conditionally ablated AP-2 expression by tamoxifen induction of Cre recombinase in hippocampal neurons from Ap2m1^lox/lox mice crossed with inducible CAG-Cre transgenic mice resulting in a reduction of AP-2 levels to <15% of that detected in WT control neurons (hereafter referred to as AP-2µ KO) (Figure 3—figure supplement 1A,B; Kononenko et al., 2014; Soykan et al., 2017). Further depletion below this level caused neuronal death.

Endocytosis of Syt1-SEP and SV2A-SEP proceeded with similar kinetics in control or AP-2μ KO hippocampal neurons stimulated with 200 APs applied at 40 Hz at physiological temperature, consistent with our earlier findings (Kononenko et al., 2014; Soykan et al., 2017; Figure 3A–D). Surprisingly, however, we found that loss of AP-2 significantly slowed down the endocytic retrieval of other major SV proteins such as Syp, Syb2, and most prominently, of the vesicular neurotransmitter transporters VGLUT1 and VGAT (Figure 3E–L). These phenotypes were specific as plasmid-based reexpression of AP-2μ in AP-2μ KO neurons rescued defective endocytosis of these SEP-tagged SV proteins (Figure 3).

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As elevated pHluorin signals could conceivably arise from defects in endocytosis or vesicle reacidification, we used a quench protocol in which acidic buffer is applied before and after neuronal stimulation with 200 APs to probe the accessibility of VGLUT1- or SV2A-SEP to externally applied acid (Figure 3M–P). These experiments showed that exocytosed VGLUT1-SEP accumulates on the surface of AP-2μ KO neurons (Figure 3M) as quantitatively evidenced by an increased ΔF₂/ΔF₁ ratio (Figure 3N). No difference in the fraction of surface-accumulated SV2A-SEP molecules was observed in AP-2μ KO compared to WT neurons (Figure 3O, P). Defective endocytosis of VGLUT1-SEP in the absence of AP-2 was further confirmed by probing the acid-resistant pool of endocytosed VGLUT1-SEP at 30 s poststimulation with 200 APs in the presence of folimycin, a selective inhibitor of vesicle reacidification by the V-ATPase (Figure 3—figure supplement 1C-F). These experiments demonstrate that AP-2 is required for VGLUT1-SEP endocytosis but is dispensable for vesicle reacidification.

We challenged these data acquired with strong 200 AP stimulation by monitoring SV endocytosis in response to a milder stimulation paradigm that results in the exocytic fusion of the readily releasable pool of SVs (Hua et al., 2011; Murthy and Stevens, 1999), that is 50 APs applied at 20 Hz. While endocytosis of SV2A- and VGLUT1-SEP proceeded unperturbed in hippocampal neurons depleted of clathrin (Figure 4A–D), a substantial delay in the endocytosis of VGLUT1- but not SV2-SEP was observed in neurons lacking AP-2 (Figure 4E–H). No difference was found in the fraction of boutons responding to stimulation with 50 APs between WT and AP-2μ KO neurons (Figure 4—figure supplement 1A,B). At lower stimulation intensities (i.e., 10 or 20 APs), AP-2μ KO neurons displayed significantly attenuated exocytic responses (Figure 4—figure supplement 1B), possibly reflecting a reduced release probability originating from defects in SV reformation, akin to the reported phenotype of clathrin loss in hippocampal neurons (Watanabe et al., 2013; Watanabe et al., 2014).

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Collectively, these data unravel a clathrin-independent role of the clathrin adaptor AP-2 in the endocytic retrieval of select SV cargos including VGLUT1 and VGAT at physiological temperature, while endocytosis of Syt1 or SV2A proceeds with unaltered kinetics in the absence of AP-2.

CIE of endogenous VGAT depends on the clathrin adaptor AP-2

As optical imaging of SEP reporters may lead to artifacts caused by overexpression of exogenous SV proteins (Opazo et al., 2010), we analyzed the internalization kinetics of endogenous VGAT using antibodies directed against its luminal domain coupled to the pH-sensitive fluorophore CypHer5E. The cyanine-based dye CypHer5E is quenched at neutral pH but exhibits bright fluorescence when present in the acidic lumen of SVs (Hua et al., 2011) and, thus can serve as a tracer for the recycling of endogenous SV proteins when it is preloaded into SVs (e.g., by high-frequency stimulation or spontaneous labeling, see Materials and methods) prior to the measurements (Figure 5A). First, we probed the effects of AP-2μ KO on VGAT endocytosis. Loss of AP-2 severely delayed the endocytic retrieval of endogenous VGAT in response to train stimulation with either 200 APs (Figure 5B, C) or 50 APs (Figure 5D, E) at physiological temperature, consistent with our results from exogenously expressed VGAT-SEP (see Figure 3). To determine whether the requirement for AP-2 reflects a function for CME in the retrieval of endogenous VGAT, we examined the effects of genetic or pharmacological blockade of clathrin function. Lentiviral shRNA-mediated depletion of clathrin had no effect on the endocytic retrieval of endogenous VGAT in response to either strong (e.g., train of 200 APs applied at 40 Hz) (Figure 5F, G) or mild stimulation (50 APs at 20 Hz) (Figure 5H, I) at physiological temperature. Similar results were obtained, if clathrin function was perturbed pharmacologically by acute inhibition in the presence of Pitstop2 in either WT or clathrin-depleted neurons (Figure 5B–I). However, block of clathrin function potently inhibited CME of transferrin (Figure 5J) and resulted in a significant reduction of the readily releasable and total recycling vesicle pool sizes probed by consecutive trains of 50 and 900 APs interspersed by a 90-s interstimulus interval (Gerth et al., 2017; Figure 5K). In contrast, the postexocytic retrieval kinetics of endogenous VGAT remained unaltered (Figure 5L).

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We conclude that at physiological temperature the endocytosis of endogenous VGAT from the neuronal surface depends on the clathrin adaptor AP-2, while clathrin function is dispensable. Instead, clathrin may facilitate the reformation of functional SVs from ELVs downstream of CIE to sustain neurotransmission (Kononenko et al., 2014; Watanabe et al., 2014).

AP-2 depletion causes surface stranding of endogenous vesicular neurotransmitter transporters but not of Syt1 and SV2A

As endocytosis of a subset of SV proteins, for example VGLUT1 and VGAT, was impaired in the absence of AP-2, one might expect their partial redistribution to the neuronal surface in AP-2μ KO neurons. To test this, we labeled surface-stranded SV proteins by a membrane-impermeant biotinylating reagent in cultured cerebellar granule neurons derived from AP-2μ KO mice or WT littermate controls. Biotinylated proteins were captured on a streptavidin-conjugated matrix and subsequently analyzed by immunoblotting (Figure 6A). No difference was detected in the plasma membrane levels of Syt1 and SV2A between WT and AP-2μ KO neurons (Figure 6B–E). By contrast, significantly larger amounts of VGLUT1 and VGAT were found at the plasma membrane of AP-2μ KO neurons compared to WT controls (Figure 6F–I), while the total levels of SV proteins assessed either by western blot or immunostaining were unaltered (Figure 6—figure supplement 1A,B).

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To challenge these results by an independent approach, we took advantage of available antibodies that recognize the luminal domains of VGAT, Syt1, and VGLUT1 (Figure 7). Application of these antibodies under nonpermeabilizing conditions to selectively recognize the surface-stranded SV protein pool revealed elevated plasma membrane levels of VGAT (Figure 7F–H) and VGLUT1 (Figure 7—figure supplement 1A-C) in AP-2μ KO hippocampal neurons, while the presynaptic surface pool of Syt1 remained unaltered (Figure 7B–D). Importantly, silencing of neuronal activity in the presence of the sodium channel blocker tetrodotoxin (TTX) rescued surface stranding of VGAT (Figure 7F–H) and VGLUT1 (Figure 7—figure supplement 1A-C), suggesting that the observed plasma membrane accumulation of a subset of SV proteins in AP-2μ KO neurons is a consequence of defective stimulation-induced SV protein retrieval following exocytic SV fusion.

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Collectively, these findings provide strong support for the hypothesis that the clathrin adaptor AP-2 is required for the endocytic retrieval of select SV cargos including VGLUT1 and VGAT under physiological conditions, thereby identifying a clathrin-independent function of AP-2 in the sorting of SV proteins at the presynaptic plasma membrane at central mammalian synapses.

AP-2-binding deficient mutations in vesicular transporters phenocopy loss of AP-2

In a final set of experiments, we set out to determine the molecular basis for the clathrin-independent function of AP-2 in the sorting of SV proteins by focusing on VGLUT1 and VGAT. Previous studies had identified acidic cluster dileucine motifs (Bonifacino and Traub, 2003; Kelly et al., 2008) in the cytoplasmic tails of vesicular neurotransmitter transporters as putative interaction sites for AP-2 and possibly other clathrin adaptors (Figure 8A). As mutational inactivation of these motifs was further reported to delay the kinetics of VGLUT1 and VGAT analyzed at nonphysiological temperature (Voglmaier et al., 2006; Santos et al., 2013; Li et al., 2017), vesicular neurotransmitter transporters were proposed to be internalized via CME mediated by clathrin and AP-2 (Mori and Takamori, 2017). Given our data reported above, we hypothesized that these prior results might reflect the direct recognition of VGLUT1 and VGAT by AP-2 at the neuronal surface to enable their internalization via CIE at physiological temperature (see Figure 1C).

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To probe this hypothesis, we first analyzed the association of the cytoplasmic C-terminal domain of VGLUT1 with the clathrin adaptor complex AP-2 and its close relatives AP-1 and AP-3. Robust binding of the GST-fused cytoplasmic domain of VGLUT1 to AP-2 was observed, whereas no association with AP-3 was detected (Figure 8B, C). Mutational inactivation of the putative AP-2-binding dileucine motif, that is F₅₁₀A/V₅₁₁A (FV/AA) (Voglmaier et al., 2006; Santos et al., 2013; Li et al., 2017), largely abrogated VGLUT1 complex formation with AP-2. We also detected a weak, possibly nonspecific interaction of VGLUT1 with AP-1 that was insensitive to the FV/AA mutation (Figure 8—figure supplement 1A). These results show that VGLUT1 is directly recognized and binds to AP-2 via its acidic cluster dileucine motif.

To probe the functional significance of this interaction we monitored the endocytic retrieval of VGLUT1 and VGAT carrying mutations in their respective AP-2-binding dileucine motifs at physiological temperature. Mutant forms of VGLUT1 or VGAT defective in AP-2 binding displayed significantly slower endocytosis kinetics compared to the respective WT proteins (Figure 8D–G, wild type in black and VGLUT1-FV/AA mutant or VGAT-F₄₄A/AA mutant in purple). These endocytic defects were exacerbated when endocytosis was monitored at RT and under conditions that might favor CME (200 APs, 5 Hz; Kononenko et al., 2014; Figure 8—figure supplement 1B-E), consistent with earlier data (Voglmaier et al., 2006). Importantly, the delayed decay of mutant VGLUT1-FV/AA-SEP signals could not be attributed to defects in reacidification (e.g., caused by internalization into slowly acidifying compartments), because poststimulus application of acid solution effectively quenched its fluorescence (Figure 8—figure supplement 1F,G). To analyze whether the observed kinetic delay in the endocytosis of dileucine mutant VGLUT1 and VGAT variants was caused by loss of their ability to associate with AP-2, we monitored their retrieval in AP-2μ KO neurons. Strikingly, loss of AP-2 not only phenocopied the effect of mutational inactivation of the dileucine motifs in VGLUT1 or VGAT but combined mutational inactivation of the dileucine motifs in VGLUT1 or VGAT and AP-2μ KO did not result in additive phenotypes (Figure 8D–G).

These data show that AP-2 directly recognizes surface-stranded VGLUT1 and VGAT via acidic cluster dileucine motifs contained in their cytoplasmic domains to facilitate their endocytic retrieval from the plasma membrane via CIE.

Our findings based on lentiviral depletion of clathrin and conditional KO of AP-2 in hippocampal neurons reveal a crucial clathrin-independent function of the clathrin adaptor AP-2 in the endocytic sorting of a subset of SV proteins at central synapses. Several lines of evidence support this view: First, comprehensive survey of the endocytic retrieval of six major SV proteins by optical imaging conducted in two independent laboratories provides strong support for the emerging notion (Soykan et al., 2017; Watanabe et al., 2014) that SV endocytosis occurs independent of clathrin, corroborating the prevalence of CIE at physiological temperature. Second and most surprisingly, we find that the endocytic retrieval of a subset of these SV proteins including VGLUT1 and VGAT from the presynaptic plasma membrane depends on sorting by the clathrin adaptor AP-2. This conclusion from SEP- and CypHer5E-based imaging and acid quenching experiments of exogenously expressed or endogenous SV proteins is further corroborated by the observation that a fraction of endogenous VGLUT1 and VGAT molecules remain stranded on the presynaptic plasma membrane of AP-2μ KO neurons, a phenotype that is rescued upon silencing of neuronal activity. Impaired endocytosis of VGLUT1 in the absence of AP-2 may indirectly also impact on the kinetics of Synaptophysin and Synaptobrevin/VAMP2 retrieval (Figure 3I–L), consistent with the proposed function of VGLUT1 as a master orchestrator of the retrieval of a subset of SV proteins (Pan et al., 2015). However, other possibilities such as a direct or indirect association of Synaptophysin and/or Synaptobrevin/VAMP2 with AP-2, cannot be ruled out. Finally, we show that AP-2-mediated efficient sorting of VGLUT1 and VGAT during CIE is achieved by the recognition of acidic cluster dileucine motifs by AP-2, in agreement with earlier biochemical and cell biological experiments (Voglmaier et al., 2006; Santos et al., 2013; Li et al., 2017). Our data thus underscore the importance of AP-2-mediated sorting of select SV cargo during CIE, in the absence of which the compositional integrity of SVs becomes perturbed. This mechanism may also be of pathological relevance in humans. For example, defective endocytosis of VGAT and resulting defects in inhibitory neurotransmission may underlie developmental and epileptic encephalopathy caused by a pathogenic loss-of-function variant of AP-2μ in human patients (Helbig et al., 2019).

Our findings are most consistent with and support a mechanism of SV recycling in which dedicated endocytic adaptors such as AP-2 and others (e.g., AP180, Stonin 2) operate at the presynaptic cell surface where they recognize and recruit SV proteins to sites of CIE. How AP-2 is targeted to presynaptic endocytic sites is unclear, but likely involves coincident detection of phosphatidylinositol 4,5-bisphosphate, a lipid selectively enriched at the plasma membrane, and cargo proteins (e.g., VGLUT1 or VGAT), possibly in conjunction with other endocytic factors. Clathrin assembly only occurs once endocytic vesicles have pinched off from the plasma membrane, that is downstream of CIE, to reform functional SVs by budding from internal ELVs. This process of SV reformation depends on AP-2 and other clathrin-associated endocytic proteins (Figure 8H). Such an integrated model, in which AP-2 acts at two sites, endocytic sites on the presynaptic surface and on internal ELVs together with clathrin, not only explains previous observations pertaining to the speed of SV endocytosis (Delvendahl et al., 2016; Watanabe et al., 2013) and the apparent lack of effect of clathrin loss on SV membrane internalization in various models (Kononenko et al., 2014; Soykan et al., 2017; Kasprowicz et al., 2008; Heerssen et al., 2008), but is also consistent with the slow kinetics of clathrin assembly (McMahon and Boucrot, 2011) and the accumulation of SV proteins on the neuronal surface in the absence of dedicated endocytic adaptors for SV proteins, for example Stonin 2, AP180/CALM (Kononenko and Haucke, 2015; Cousin, 2017; Kaempf et al., 2015; Koo et al., 2015; Mori and Takamori, 2017), and AP-2 (this study). We speculate that the mechanism identified here also operates during UFE. Interestingly, recent quantitative proteomic analysis of rodent brain has revealed AP-2 but not clathrin to be highly enriched on SVs (Taoufiq et al., 2020), suggesting that AP-2 may interact with SV cargos prior to the bona fide endocytic process. This perpetual interaction of AP-2 with SV cargos might thus enable rapid sorting and endocytic internalization, that is during UFE or other forms of CIE. Of note, our findings are also consistent with recent data regarding a calcium-independent form of SV endocytosis that appears to operate independent of clathrin (Orlando et al., 2019).

An important question raised by our work is how AP-2-independent SV cargos such as Synaptotagmin 1 and SV2 are endocytically retrieved from the neuronal surface. While endocytic adaptors for SV2 have not been reported, studies in mouse hippocampal neurons (Kononenko et al., 2013; Lee et al., 2019) and in invertebrate models (Stimson et al., 2001; Phillips et al., 2010; Jung et al., 2007) have identified cargo-selective roles of Stonin 2 and the related SGIP1 protein in the endocytic retrieval of Synaptotagmin 1 from the presynaptic cell surface. Enigmatically however, it was shown that while loss of Stonin 2 causes the partial accumulation of Synaptotagmin 1 at plasma membrane sites near the AZ, the kinetics of SV endocytosis appeared to be even accelerated (Kaempf et al., 2015; Kononenko et al., 2013). One possibility therefore is that Synaptotagmin 1 due to its comparably large presynaptic surface fraction (Pan et al., 2015) does not require active endocytic sorting. Instead, it may reach sites of endocytosis either by lateral diffusion or via confinement near sites of exocytic release (Hua et al., 2011; Kononenko et al., 2013; Willig et al., 2006). SV2 could follow Synaptotagmin 1 via a piggy-back mechanism (Haucke and De Camilli, 1999), consistent with the finding that both proteins can form a stable complex in vivo (Kaempf et al., 2015; Lazzell et al., 2004; Schivell et al., 1996; Yao et al., 2010). A confinement-based mechanism may also support the endocytic retrieval of other SV proteins in the absence of their specific sorting adaptors (e.g., VGLUT1 or VGAT endocytosis in AP-2µ KO neurons). Future studies are needed to address this possibility in more detail.

From a more general perspective our findings dissent from the widely held view that AP-2 obligatorily associates with clathrin to execute its cell physiological functions, at least in central nervous system (CNS) neurons. While the most well-known function of AP-2 is its involvement in CME in mammalian cells and tissues, studies in higher fungi have uncovered a clathrin-independent role of fungal AP-2 in the polar localization of the lipid flippases DnfA and DnfB (Martzoukou et al., 2017). Interestingly, in this system AP-2 is seen to colocalize with endocytic markers and the actin-associated protein AbpA, but not with clathrin (Martzoukou et al., 2017). Because AP-2 also colocalizes with a fungal homolog of Synaptobrevin (Martzoukou et al., 2017), and clathrin-independent SV endocytosis at hippocampal synapses depends on actin polymerization (Soykan et al., 2017), our newly observed function of AP-2 might reflect an unexpectedly widely conserved endocytic mechanism. Conversely, studies in AP-2 KO cells have revealed AP-2-independent forms of CME in mammals that impact on receptor sorting and signaling (Pascolutti et al., 2019).

Taken together our findings together with other studies suggest an unexpected plasticity of endocytic mechanisms in eukaryotes including the mammalian CNS.

All data generated or analyzed during this study are included in the manuscript and supporting files. Raw images and values are available in Source data files.

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Author details

1. Tania López-Hernández

   Leibniz-Forschungsinstitut für Molekulare Pharmakologie (FMP), Berlin, Germany

   Contribution

   Conceptualization, Conducted all experiments involving neurons from AP-2µ KO mice and WT littermates, including SEP-based and cypHer5E-based imaging experiments, transferrin uptakes and the detection of surface SV proteins either by biotinylation assays or immunostainings, Data curation, Formal analysis, Investigation, Methodology, Validation, Writing – original draft, Writing – review and editing

   Contributed equally with

   Koh-ichiro Takenaka

   Competing interests

   No competing interests declared

   "This ORCID iD identifies the author of this article:" 0000-0002-0814-2850

2. Koh-ichiro Takenaka

   Laboratory of Neural Membrane Biology, Graduate School of Brain Science, Doshisha University, Kyoto, Japan

   Contribution

   Conceptualization, Conducted SEP-imaging experiments and analyzed the association of VGLUT1 with adaptor complexes., Data curation, Formal analysis, Investigation, Validation, Writing – original draft, Writing – review and editing

   Contributed equally with

   Tania López-Hernández

   Competing interests

   No competing interests declared

   "This ORCID iD identifies the author of this article:" 0000-0001-5600-6565

3. Yasunori Mori

   Laboratory of Neural Membrane Biology, Graduate School of Brain Science, Doshisha University, Kyoto, Japan

   Contribution

   Methodology, Supervision

   Competing interests

   No competing interests declared

   "This ORCID iD identifies the author of this article:" 0000-0003-0296-3655

4. Pornparn Kongpracha

   Department of Laboratory Medicine, The Jikei University School of Medicine, Tokyo, Japan

   Contribution

   Methodology

   Competing interests

   No competing interests declared

5. Shushi Nagamori

   Department of Laboratory Medicine, The Jikei University School of Medicine, Tokyo, Japan

   Contribution

   Methodology

   Competing interests

   No competing interests declared

   "This ORCID iD identifies the author of this article:" 0000-0003-0203-2754

6. Volker Haucke

   Leibniz-Forschungsinstitut für Molekulare Pharmakologie (FMP), Berlin, Germany

   Contribution

   Conceptualization, Data curation, Funding acquisition, Resources, Supervision, Writing – original draft, Writing – review and editing

   For correspondence

   haucke@fmp-berlin.de

   Competing interests

   No competing interests declared

   "This ORCID iD identifies the author of this article:" 0000-0003-3119-6993

7. Shigeo Takamori

   Laboratory of Neural Membrane Biology, Graduate School of Brain Science, Doshisha University, Kyoto, Japan

   Contribution

   Conceptualization, Data curation, Funding acquisition, Resources, Supervision, Writing – original draft, Writing – review and editing

   For correspondence

   stakamor@mail.doshisha.ac.jp

   Competing interests

   No competing interests declared

   "This ORCID iD identifies the author of this article:" 0000-0003-0215-6124

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