Synaptic Vesicle Retrieval Research Articles

Tissue-type plasminogen activator is a homeostatic regulator of synaptic function in the central nervous system.

Membrane depolarization induces the release of the serine proteinase tissue-type plasminogen activator (tPA) from the presynaptic terminal of cerebral cortical neurons. Once in the synaptic cleft this tPA promotes the exocytosis and subsequent endocytic retrieval of glutamate-containing synaptic vesicles, and regulates the postsynaptic response to the presynaptic release of glutamate. Indeed, tPA has a bidirectional effect on the composition of the postsynaptic density (PSD) that does not require plasmin generation or the presynaptic release of glutamate, but varies according to the baseline level of neuronal activity. Hence, in inactive neurons tPA induces phosphorylation and accumulation in the PSD of the Ca2+/calmodulin-dependent protein kinase IIα (pCaMKIIα), followed by pCaMKIIα-induced phosphorylation and synaptic recruitment of GluR1-containing α-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA) receptors. In contrast, in active neurons with increased levels of pCaMKIIα in the PSD tPA induces pCaMKIIα and pGluR1 dephosphorylation and their subsequent removal from the PSD. These effects require active synaptic N-methyl-D-aspartate (NMDA) receptors and cyclin-dependent kinase 5 (Cdk5)-induced phosphorylation of the protein phosphatase 1 (PP1) at T320. These data indicate that tPA is a homeostatic regulator of the postsynaptic response of cerebral cortical neurons to the presynaptic release of glutamate via bidirectional regulation of the pCaMKIIα /PP1 switch in the PSD.

Tissue-type Plasminogen Activator (tPA) Modulates the Postsynaptic Response of Cerebral Cortical Neurons to the Presynaptic Release of Glutamate.

Tissue-type plasminogen activator (tPA) is a serine proteinase released by the presynaptic terminal of cerebral cortical neurons following membrane depolarization (Echeverry et al., 2010). Recent studies indicate that the release of tPA triggers the synaptic vesicle cycle and promotes the exocytosis (Wu et al., 2015) and endocytic retrieval (Yepes et al., 2016) of glutamate-containing synaptic vesicles. Here we used electron microscopy, proteomics, quantitative phosphoproteomics, biochemical analyses with extracts of the postsynaptic density (PSD), and an animal model of cerebral ischemia with mice overexpressing neuronal tPA to study whether the presynaptic release of tPA also has an effect on the postsynaptic terminal. We found that tPA has a bidirectional effect on the composition of the PSD of cerebral cortical neurons that is independent of the generation of plasmin and the presynaptic release of glutamate, but depends on the baseline level of neuronal activity and the extracellular concentrations of calcium (Ca2+). Accordingly, in neurons that are either inactive or incubated with low Ca2+ concentrations tPA induces phosphorylation and accumulation in the PSD of the Ca2+/calmodulin-dependent protein kinase IIα (pCaMKIIα), followed by pCaMKIIα-mediated phosphorylation and synaptic recruitment of GluR1-containing α-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA) receptors. In contrast, in neurons with previously increased baseline levels of pCaMKIIα in the PSD due to neuronal depolarization in vivo or incubation with high concentrations of either Ca2+ or glutamate in vitro, tPA induces pCaMKIIα and pGluR1 dephosphorylation and their subsequent removal from the PSD. We found that these effects of tPA are mediated by synaptic N-methyl-D-aspartate (NMDA) receptors and cyclin-dependent kinase 5 (Cdk5)-induced phosphorylation of the protein phosphatase 1 (PP1) at T320. Our data indicate that by regulating the pCaMKIIα/PP1 balance in the PSD tPA acts as a homeostatic regulator of the postsynaptic response of cerebral cortical neurons to the presynaptic release of glutamate.

Epsin1 modulates synaptic vesicle retrieval capacity at CNS synapses.

Synaptic vesicle retrieval is an essential process for continuous maintenance of neural information flow after synaptic transmission. Epsin1, originally identified as an EPS15-interacting protein, is a major component of clathrin-mediated endocytosis. However, the role of Epsin1 in synaptic vesicle endocytosis at CNS synapses remains elusive. Here, we showed significantly altered synaptic vesicle endocytosis in neurons transfected with shRNA targeting Epsin1 during/after neural activity. Endocytosis was effectively restored by introducing shRNA-insensitive Epsin1 into Epsin1-depleted neurons. Domain studies performed on neurons in which domain deletion mutants of Epsin1 were introduced after Epsin1 knockdown revealed that ENTH, CLAP, and NPFs are essential for synaptic vesicle endocytosis, whereas UIMs are not. Strikingly, the efficacy of the rate of synaptic vesicle retrieval (the “endocytic capacity”) was significantly decreased in the absence of Epsin1. Thus, Epsin1 is required for proper synaptic vesicle retrieval and modulates the endocytic capacity of synaptic vesicles.

Endosome-mediated endocytic mechanism replenishes the majority of synaptic vesicles at mature CNS synapses in an activity-dependent manner.

Whether synaptic vesicles (SVs) are recovered via endosome-mediated pathways is a matter of debate; however, recent evidence suggests that clathrin-independent bulk endocytosis (CIE) via endosomes is functional and preferentially replenishes SV pools during strong stimulation. Here, using brefeldin-A (BFA) to block CIE, we found that CIE retrieved a minority of SVs at developing CNS synapses during strong stimulation, but its contribution increased up to 61% at mature CNS synapses. Contrary to previous views, BFA not only blocked SV formation from the endosome but also blocked the endosome formation at the plasma membrane. Adaptor protein 1 and 3 (AP-1/3) have key roles in SV reformation from endosomes during CIE, and AP-1 also affects bulk endosome formation from the plasma membrane. Finally, temporary blocking of chronic or acute neuronal activity with tetrodotoxin in mature neurons redirected most SV retrieval to endosome-independent pathways. These results show that during high neuronal activity, CIE becomes the major endocytic pathway at mature CNS synapses. Moreover, mature neurons use clathrin-mediated endocytosis and the CIE pathway to different extents depending on their previous activity; this may result in activity-dependent alterations of the SV composition which ultimately influence transmitter release and contribute to synaptic plasticity.

Synaptic vesicles are "primed" for fast clathrin-mediated endocytosis at the ribbon synapse.

Retrieval of synaptic vesicles can occur 1–10 s after fusion, but the role of clathrin during this process has been unclear because the classical mode of clathrin-mediated endocytosis (CME) is an order of magnitude slower, as during retrieval of surface receptors. Classical CME is thought to be rate-limited by the recruitment of clathrin, which raises the question: how is clathrin recruited during synaptic vesicle recycling? To investigate this question we applied total internal reflection fluorescence microscopy (TIRFM) to the synaptic terminal of retinal bipolar cells expressing fluorescent constructs of clathrin light-chain A. Upon calcium influx we observed a fast accumulation of clathrin within 100 ms at the periphery of the active zone. The subsequent loss of clathrin from these regions reflected endocytosis because the application of a potent clathrin inhibitor Pitstop2 dramatically slowed down this phase by ~3 fold. These results indicate that clathrin-dependent retrieval of synaptic vesicles is unusually fast, most probably because of a “priming” step involving a state of association of clathrin with the docked vesicle and with the endosomes and cisternae surrounding the ribbons. Fluorescence correlation spectroscopy (FCS) and fluorescence recovery after photobleaching (FRAP) showed that the majority of clathrin is moving with the same kinetics as synaptic vesicle proteins. Together, these results indicate that the fast endocytic mechanism operating to retrieve synaptic vesicles differs substantially from the classical mode of CME operating via formation of a coated pit.

Methods for cell-attached capacitance measurements in mouse adrenal chromaffin cell.

Neuronal transmission is an integral part of cellular communication within the brain. Depolarization of the presynaptic membrane leads to vesicle fusion known as exocytosis that mediates synaptic transmission. Subsequent retrieval of synaptic vesicles is necessary to generate new neurotransmitter-filled vesicles in a process identified as endocytosis. During exocytosis, fusing vesicle membranes will result in an increase in surface area and subsequent endocytosis results in a decrease in the surface area. Here, our lab demonstrates a basic introduction to cell-attached capacitance recordings of single endocytic events in the mouse adrenal chromaffin cell. This type of electrical recording is useful for high-resolution recordings of exocytosis and endocytosis at the single vesicle level. While this technique can detect both vesicle exocytosis and endocytosis, the focus of our lab is vesicle endocytosis. Moreover, this technique allows us to analyze the kinetics of single endocytic events. Here the methods for mouse adrenal gland tissue dissection, chromaffin cell culture, basic cell-attached techniques, and subsequent examples of individual traces measuring singular endocytic event are described.

Clathrin/AP-2 Mediate Synaptic Vesicle Reformation from Endosome-like Vacuoles but Are Not Essential for Membrane Retrieval at Central Synapses

Neurotransmission depends on presynaptic membrane retrieval and local reformation of synaptic vesicles (SVs) at nerve terminals. The mechanisms involved in these processes are highly controversial with evidence being presented for SV membranes being retrieved exclusively via clathrin-mediated endocytosis (CME) from the plasma membrane or via ultrafast endocytosis independent of clathrin. Here we show that clathrin and its major adaptor protein 2 (AP-2) in addition to the plasma membrane operate at internal endosome-like vacuoles to regenerate SVs but are not essential for membrane retrieval. Depletion of clathrin or conditional knockout of AP-2 result in defects in SV reformation and an accumulation of endosome-like vacuoles generated by clathrin-independent endocytosis (CIE) via dynamin 1/3 and endophilin. These results together with theoretical modeling provide a conceptual framework for how synapses capitalize on clathrin-independent membrane retrieval and clathrin/AP-2-mediated SV reformation from endosome-like vacuoles to maintain excitability over a broad range of stimulation frequencies.

A Presynaptic Role for the Cytomatrix Protein GIT in Synaptic Vesicle Recycling

Neurotransmission involves the exo-endocytic cycling of synaptic vesicles (SVs) within nerve terminals. Exocytosis is facilitated by a cytomatrix assembled at the active zone (AZ). The precise spatial and functional relationship between exocytic fusion of SVs at AZ membranes and endocytic SV retrieval is unknown. Here, we identify the scaffold G protein coupled receptor kinase 2 interacting (GIT) protein as a component of the AZ-associated cytomatrix and as a regulator of SV endocytosis. GIT1 and its D.melanogaster ortholog, dGIT, are shown to directly associate with the endocytic adaptor stonin 2/stoned B. In Drosophila dgit mutants, stoned B and synaptotagmin levels are reduced and stoned B is partially mislocalized. Moreover, dgit mutants show morphological and functional defects in SV recycling. These data establish a presynaptic role for GIT in SV recycling and suggest a connection between the AZ cytomatrix and the endocytic machinery.

Myosin II Regulates Activity Dependent Compensatory Endocytosis at Central Synapses

Recent evidence suggests that endocytosis, not exocytosis, can be rate limiting for neurotransmitter release at excitatory CNS synapses during sustained activity and therefore may be a principal determinant of synaptic fatigue. At low stimulation frequencies, the probability of synaptic release is linked to the probability of synaptic retrieval such that evoked release results in proportional retrieval even for release of single synaptic vesicles. The exact mechanism by which the retrieval rates are coupled to release rates, known as compensatory endocytosis, remains unknown. Here we show that inactivation of presynaptic myosin II (MII) decreases the probability of synaptic retrieval. To be able to differentiate between the presynaptic and postsynaptic functions of MII, we developed a live cell substrate patterning technique to create defined neural circuits composed of small numbers of embryonic mouse hippocampal neurons and physically isolated from the surrounding culture. Acute application of blebbistatin to inactivate MII in circuits strongly inhibited evoked release but not spontaneous release. In circuits incorporating both control and MIIB knock-out cells, loss of presynaptic MIIB function correlated with a large decrease in the amplitude of evoked release. Using activity-dependent markers FM1-43 and horseradish peroxidase, we found that MII inactivation greatly slowed vesicular replenishment of the recycling pool but did not impede synaptic release. These results indicate that MII-driven tension or actin dynamics regulate the major pathway for synaptic vesicle retrieval. Changes in retrieval rates determine the size of the recycling pool. The resulting effect on release rates, in turn, brings about changes in synaptic strength.

Dynamin phosphorylation controls optimization of endocytosis for brief action potential bursts

Modulation of synaptic vesicle retrieval is considered to be potentially important in steady-state synaptic performance. Here we show that at physiological temperature endocytosis kinetics at hippocampal and cortical nerve terminals show a bi-phasic dependence on electrical activity. Endocytosis accelerates for the first 15-25 APs during bursts of action potential firing, after which it slows with increasing burst length creating an optimum stimulus for this kinetic parameter. We show that activity-dependent acceleration is only prominent at physiological temperature and that the mechanism of this modulation is based on the dephosphorylation of dynamin 1. Nerve terminals in which dynamin 1 and 3 have been replaced with dynamin 1 harboring dephospho- or phospho-mimetic mutations in the proline-rich domain eliminate the acceleration phase by either setting endocytosis at an accelerated state or a decelerated state, respectively. DOI:http://dx.doi.org/10.7554/eLife.00845.001.

Vesicle dynamics: how synaptic proteins regulate different modes of neurotransmission

Central synapses operate neurotransmission in several modes: synchronous/fast neurotransmission (neurotransmitters release is tightly coupled to action potentials and fast), asynchronous neurotransmission (neurotransmitter release is slower and longer lasting), and spontaneous neurotransmission (where small amounts of neurotransmitter are released without being evoked by an action potential). A substantial body of evidence from the past two decades suggests that seemingly identical synaptic vesicles possess distinct propensities to fuse, thus selectively serving different modes of neurotransmission. In efforts to better understand the mechanism(s) underlying the different modes of synaptic transmission, many research groups found that synaptic vesicles used in different modes of neurotransmission differ by a number of synaptic proteins. Synchronous transmission with higher temporal fidelity to stimulation seems to require synaptotagmin 1 and complexin for its Ca²⁺ sensitivity, RIM proteins for closer location of synaptic vesicles (SV) to the voltage operated calcium channels (VGCC), and dynamin for SV retrieval. Asynchronous release does not seem to require functional synaptotagmin 1 as a calcium sensor or complexins, but the activity of dynamin is indispensible for its maintenance. On the other hand, the control of spontaneous neurotransmission remains less clear as deleting a number of essential synaptic proteins does not abolish this type of synaptic vesicle fusion. VGCC distance from the SV seems to have little control on spontaneous transmission, while there is an involvement of functional synaptic proteins including synaptotagmins and complexin. Recently, presynaptic deficits have been proposed to contribute to a number of pathological conditions including cognitive and mental disorders. In this review, we evaluate recent advances in understanding the regulatory mechanisms of synaptic vesicle dynamics and in understanding how different molecular substrates maintain selective modes of neurotransmission. We also highlight the implications of these studies in understanding pathological conditions.

Cooperative Recruitment of Dynamin and BIN/Amphiphysin/Rvs (BAR) Domain-containing Proteins Leads to GTP-dependent Membrane Scission*

Dynamin mediates various membrane fission events, including the scission of clathrin-coated vesicles. Here, we provide direct evidence for cooperative membrane recruitment of dynamin with the BIN/amphiphysin/Rvs (BAR) proteins, endophilin and amphiphysin. Surprisingly, endophilin and amphiphysin recruitment to membranes was also dependent on binding to dynamin due to auto-inhibition of BAR-membrane interactions. Consistent with reciprocal recruitment in vitro, dynamin recruitment to the plasma membrane in cells was strongly reduced by concomitant depletion of endophilin and amphiphysin, and conversely, depletion of dynamin dramatically reduced the recruitment of endophilin. In addition, amphiphysin depletion was observed to severely inhibit clathrin-mediated endocytosis. Furthermore, GTP-dependent membrane scission by dynamin was dramatically elevated by BAR domain proteins. Thus, BAR domain proteins and dynamin act in synergy in membrane recruitment and GTP-dependent vesicle scission.

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

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

Stonin 2 Is a Major Adaptor Protein for Clathrin-Mediated Synaptic Vesicle Retrieval

SummaryAt small synapses in the brain, clathrin-mediated endocytosis (CME) is the dominant mode of synaptic vesicle retrieval following weak stimulation [1–4]. Clathrin cannot bind to membranes or cargo directly and instead uses adaptor proteins to do so [5]. Although the involvement of clathrin and dynamin in synaptic vesicle retrieval is clear, it is unknown which adaptor proteins are used to sort the essential components into the vesicle [1, 4, 6]. In nonneuronal cells, CME of the majority of transmembrane receptors is either directly or indirectly via the heterotetrameric AP-2 complex [5]. In neurons, RNAi of the μ2 subunit of AP-2 resulted in only minor inhibition of synaptic vesicle retrieval [7, 8], a result echoed in C. elegans [9]. These results suggest that alternative adaptors may be employed for vesicle retrieval. Here, we tested which adaptors are required for vesicle retrieval at hippocampal synapses using a targeted RNAi screen coupled with optical measurements. Stonin 2 emerged as a major adaptor, whereas AP-2 played only a minor role in endocytosis at the synapse. Moreover, using chemically induced rerouting of stonin 2 to mitochondria it was possible to switch endocytically competent synapses to an impaired state on a timescale of minutes.

Key Physiological Parameters Dictate Triggering of Activity-Dependent Bulk Endocytosis in Hippocampal Synapses

To maintain neurotransmission in central neurons, several mechanisms are employed to retrieve synaptically exocytosed membrane. The two major modes of synaptic vesicle (SV) retrieval are clathrin-mediated endocytosis and activity-dependent bulk endocytosis (ADBE). ADBE is the dominant SV retrieval mode during intense stimulation, however the precise physiological conditions that trigger this mode are not resolved. To determine these parameters we manipulated rat hippocampal neurons using a wide spectrum of stimuli by varying both the pattern and duration of stimulation. Using live-cell fluorescence imaging and electron microscopy approaches, we established that stimulation frequency, rather than the stimulation load, was critical in the triggering of ADBE. Thus two hundred action potentials, when delivered at high frequency, were sufficient to induce near maximal bulk formation. Furthermore we observed a strong correlation between SV pool size and ability to perform ADBE. We also identified that inhibitory nerve terminals were more likely to utilize ADBE and had a larger SV recycling pool. Thus ADBE in hippocampal synaptic terminals is tightly coupled to stimulation frequency and is more likely to occur in terminals with large SV pools. These results implicate ADBE as a key modulator of both hippocampal neurotransmission and plasticity.

Voltage-Dependent Calcium Channels at the Plasma Membrane, but Not Vesicular Channels, Couple Exocytosis to Endocytosis

Although calcium influx triggers endocytosis at many synapses and non-neuronal secretory cells, the identity of the calcium channel is unclear. The plasma membrane voltage-dependent calcium channel (VDCC) is a candidate, and it was recently proposed that exocytosis transiently inserts vesicular calcium channels at the plasma membrane, thus triggering endocytosis and coupling it to exocytosis, a mechanism suggested to be conserved from sea urchin to human. Here, we report that the vesicular membrane, when inserted into the plasma membrane upon exocytosis, does not generate a calcium current or calcium increase at a mammalian nerve terminal. Instead, VDCCs at the plasma membrane, including the P/Q-type, provide the calcium influx to trigger rapid and slow endocytosis and, thus, couple endocytosis to exocytosis. These findings call for reconsideration of the vesicular calcium channel hypothesis. They are likely to apply to many synapses and non-neuronal cells in which VDCCs control exocytosis, and exocytosis is coupled to endocytosis.

Adaptor Protein Complexes 1 and 3 Are Essential for Generation of Synaptic Vesicles from Activity-Dependent Bulk Endosomes

Activity-dependent bulk endocytosis is the dominant synaptic vesicle retrieval mode during high intensity stimulation in central nerve terminals. A key event in this endocytosis mode is the generation of new vesicles from bulk endosomes, which replenish the reserve vesicle pool. We have identified an essential requirement for both adaptor protein complexes 1 and 3 in this process by employing morphological and optical tracking of bulk endosome-derived synaptic vesicles in rat primary neuronal cultures. We show that brefeldin A inhibits synaptic vesicle generation from bulk endosomes and that both brefeldin A knockdown and shRNA knockdown of either adaptor protein 1 or 3 subunits inhibit reserve pool replenishment from bulk endosomes. Conversely, no plasma membrane function was found for adaptor protein 1 or 3 in either bulk endosome formation or clathrin-mediated endocytosis. Simultaneous knockdown of both adaptor proteins 1 and 3 indicated that they generated the same population of synaptic vesicles. Thus, adaptor protein complexes 1 and 3 play an essential dual role in generation of synaptic vesicles during activity-dependent bulk endocytosis.

Ca²⁺ influx slows single synaptic vesicle endocytosis.

Ca²⁺-dependent synaptic vesicle recycling is critical for maintenance of neurotransmission. However, uncoupling the roles of Ca²⁺ in synaptic vesicle fusion and retrieval has been difficult, as studies probing the role of Ca²⁺ in endocytosis relied on measurements of bulk synaptic vesicle retrieval. Here, to dissect the role of Ca²⁺ in these processes, we used a low signal-to-noise pHluorin-tagged vesicular probe to monitor single synaptic vesicle recycling in rat hippocampal neurons. We show that Ca²⁺ increases synaptic vesicle fusion probability in the classical sense, but surprisingly decreases the rate of synaptic vesicle retrieval. This negative regulation of synaptic vesicle retrieval is blocked by the Ca²⁺ chelator, EGTA, as well as FK506, a specific inhibitor of Ca²⁺-calmodulin-dependent phosphatase calcineurin. The slow time course of aggregate synaptic vesicle retrieval detected during repetitive activity could be explained by a progressive decrease in the rate of synaptic vesicle retrieval during the stimulation train. These results indicate that Ca²⁺ entry during single action potentials slows the pace of subsequent synaptic vesicle recycling.

Synaptophysin Is Required for Synaptobrevin Retrieval during Synaptic Vesicle Endocytosis

The integral synaptic vesicle (SV) protein synaptophysin forms ∼10% of total SV protein content, but has no known function in SV physiology. Synaptobrevin (sybII) is another abundant integral SV protein with an essential role in SV exocytosis. Synaptophysin and sybII form a complex in nerve terminals, suggesting this interaction may have a key role in presynaptic function. To determine how synaptophysin controls sybII traffic in nerve terminals, we used a combination of optical imaging techniques in cultures derived from synaptophysin knock-out mice. We show that synaptophysin is specifically required for the retrieval of the pH-sensitive fluorescent reporter sybII-pHluorin from the plasma membrane during endocytosis. The retrieval of other SV protein cargo reporters still occurred; however, their recapture proceeded with slower kinetics. This slowing of SV retrieval kinetics in the absence of synaptophysin did not impact on global SV turnover. These results identify a specific and selective requirement for synaptophysin in the retrieval of sybII during SV endocytosis and suggest that their interaction may act as an adjustable regulator of SV retrieval efficiency.

Proper synaptic vesicle formation and neuronal network activity critically rely on syndapin I

Synaptic transmission relies on effective and accurate compensatory endocytosis. F-BAR proteins may serve as membrane curvature sensors and/or inducers and thereby support membrane remodelling processes; yet, their in vivo functions urgently await disclosure. We demonstrate that the F-BAR protein syndapin I is crucial for proper brain function. Syndapin I knockout (KO) mice suffer from seizures, a phenotype consistent with excessive hippocampal network activity. Loss of syndapin I causes defects in presynaptic membrane trafficking processes, which are especially evident under high-capacity retrieval conditions, accumulation of endocytic intermediates, loss of synaptic vesicle (SV) size control, impaired activity-dependent SV retrieval and defective synaptic activity. Detailed molecular analyses demonstrate that syndapin I plays an important role in the recruitment of all dynamin isoforms, central players in vesicle fission reactions, to the membrane. Consistently, syndapin I KO mice share phenotypes with dynamin I KO mice, whereas their seizure phenotype is very reminiscent of fitful mice expressing a mutant dynamin. Thus, syndapin I acts as pivotal membrane anchoring factor for dynamins during regeneration of SVs.