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View metadata, citation and similar papers at core.ac.uk brought to you by CORE provided by Elsevier - Publisher Connector Neuron Review Molecular Mechanisms of Presynaptic Membrane Retrieval and Synaptic Vesicle Reformation Natalia L. Kononenko1,2 and Volker Haucke1,2,* 1Leibniz Institut fu¨ r Molekulare Pharmakologie (FMP) and Freie Universita¨ t Berlin, Robert-Roessle-Straße 10, 13125 Berlin, Germany 2Charite Universita¨ tsmedizin, NeuroCure Cluster of Excellence, Virchowweg 6, 10117 Berlin, Germany *Correspondence: [email protected] http://dx.doi.org/10.1016/j.neuron.2014.12.016 The function of the nervous system depends on the exocytotic release of neurotransmitter from synaptic ves- icles (SVs). To sustain neurotransmission, SV membranes need to be retrieved, and SVs have to be reformed locally within presynaptic nerve terminals. In spite of more than 40 years of research, the mechanisms under- lying presynaptic membrane retrieval and SV recycling remain controversial. Here, we review the current state of knowledge in the field, focusing on the molecular mechanism involved in presynaptic membrane retrieval and SV reformation. We discuss the challenges associated with studying these pathways and pre- sent perspectives for future research. Introduction biochemical studies that had identified SVs as store for non- Communication within the nervous system relies on the release peptide neurotransmitters (Whittaker, 1969). Evidence for SV of neurotransmitter by the calcium-triggered fusion of synaptic recycling and their endocytic origin was gathered by morpholog- vesicles (SVs) with the presynaptic plasma membrane at the ical studies based on the uptake of extracellular ‘‘tracers’’ such nerve terminal. The number of vesicles available for fusion and as horseradish peroxidase (HRP) into the SV lumen (Figure 1B) their propensity to undergo exocytosis define the efficacy of (Holtzman et al., 1971) and by electron microscopic analyses neurotransmitter release and fine-tune neuronal function. Given of stimulated nerve-muscle preparations (Heuser and Reese, the distance of the presynaptic terminal from the neuronal cell 1973; Heuser et al., 1979; Miller and Heuser, 1984)(Figures body the replacement of fused SVs by de novo synthesis and 1C–1E). Heuser and Reese (1973) used sustained electrical stim- axonal transport would be far too slow (on the timescale of hours ulation of frog neuromuscular junctions, which resulted in the to days) to sustain neuronal activity. In addition, the continuous depletion of SVs and the appearance of cisternal structures fusion of SVs with the plasma membrane in the absence of labeled with HRP (Figures 1C and 1D) as well as HRP-positive compensatory membrane retrieval would cause a detrimental in- clathrin-coated vesicles and coated endocytic intermediates crease in the area of the presynaptic plasma membrane, loss of in a region located away from the active zone (AZ) (Figure 1E). membrane tension, and the misalignment with postsynaptic These transient structures were apparently converted into structures. Hence, neurons have evolved mechanisms to main- functional SVs if synapses were allowed to rest, suggesting tain presynaptic membrane homeostasis by recycling SV mem- that SV recycling largely occurs by clathrin-mediated endocy- branes locally within their nerve terminals. This specific property tosis (CME), possibly involving endosome-like vacuolar interme- of SVs to undergo a local trafficking cycle has drawn the atten- diates (Heuser and Reese, 1973; Miller and Heuser, 1984). tion of neuroscientists for more than 40 years, but in spite of its Using the same preparation and technique, but a different importance for synaptic transmission, the precise mechanisms stimulation paradigm, Ceccarelli et al. (1972, 1973) observed by which exocytosed SV membranes are recycled and SVs are membrane intermediates devoid of clathrin coats directly at reformed remain enigmatic. the AZ (Figure 1D). As the intermediates corresponded in size to single SVs, these observations gave rise to the notion that Early Studies on SV Recycling SVs could fuse and recycle via a transient fusion pore, while Pioneering work in the 1950s and 1960s established the quantal preserving their identity via a mechanism later on referred to by nature of neurotransmitter release (del Castillo and Katz, 1954; Meldolesi and colleagues as ‘‘kiss and run’’ (Fesce et al., 1994). Katz and Miledi, 1967) and its relationship to the presence of small SVs, hypothesized to be the physical correlate of neuro- Monitoring SV Recycling—Tools, Models, and transmitter quanta (Figure 1A) (De Robertis, 1959; De Robertis Discrepancies and Bennett, 1955). Stunning morphological observations by Since these orginal observations by Heuser and Reese (1973) Heuser and Reese (1973) and Heuser et al. (1979) provided direct and Ceccarelli et al. (1972), we have witnessed a revolution in experimental support for this idea. By relating the amplitude and the techniques used to monitor exo-endocytosis as well as in the quantal content of the endplate potential to the number of ve- the molecular analysis of the components involved in SV recy- sicular openings measured by rapid ‘‘freeze-slam’’ electron mi- cling (Dittman and Ryan, 2009; Jahn and Fasshauer, 2012; Riz- croscopy (EM) they concluded that a single SV indeed contains zoli, 2014; Saheki and De Camilli, 2012). Optical probes for a quantum of neurotransmitter (Heuser and Reese, 1973; Heuser the real-time visualization of membrane recycling at synapses et al., 1979; Miller and Heuser, 1984), a postulate corroborating have been developed (Kavalali and Jorgensen, 2014), which 484 Neuron 85, February 4, 2015 ª2015 Elsevier Inc. Neuron Review Figure 1. Electron Microscopy of Recycling SV Membranes (A) Electron micrograph of a large axonal process (PRSN) of the earthworm, containing numerous synaptic vesicles (SVs). PSN, portion of another axon; ER, endoplasmic reticulum; Y, a specialized synaptic contact. Reproduced with permission from De Robertis and Bennett (1955). (B) SV recycling at the lobster neuromuscular junction, by exposing the synapse to HRP during stimulation for three periods of 10 min each, with 16,000 stimuli in total. HRP is present within many small vesicles in an axon ending, indicated by ar- rows. Scale bar represents 0.5 mm. Reproduced with permission from Holtzman et al. (1971). (C) An example of HRP retained within bulk structures (cisternae, C) at the frog neuromuscular junction stimulated constantly for 15 min and fixed immediately after. Reproduced with permission from Heuser and Reese (1973). (D) Original drawings by Heuser and Reese (1973), illustrating a progressive depletion of synaptic vesicles (open circles) and appearance ofcis- ternae (filled in black) in frog nerve terminal stimulated at 10 Hz for 1 and 15 min. The numbers indicate the area per section of synaptic vesicle membrane + cisternal membrane + surface membrane = total membrane. Reproduced with permission from Heuser and Reese (1973). (E) Clathrin-coated pits and clathrin-coated vesi- cles observed in the vicinity of the plasma mem- brane at a frog nerve terminal stimulated at 10 Hz for 1 min. Reproduced with permission from Heuser and Reese (1973). (F) Uncoated U-shaped intermediates at the active zone of frog nerve terminals stimulated at 2 Hz for 2 hr. Reproduced with permission from Ceccarelli et al. (1972). 1998). In contrast, analysis of SV recycling by fluorescent antibodies directed against the lumenal domain of synaptotagmin 1 (Syt1) or by pHluorin re- have allowed unprecedented insights into the mechanisms porters have challenged these findings and instead suggest that of membrane retrieval and SV reformation. These include exog- following fusion SV proteins intermix with plasma membrane enously applied fluorescent probes such as the styryl dye components (Ferna´ ndez-Alfonso et al., 2006; Wienisch and Klin- FM1-43 and its relatives, which fluoresce upon partitioning into gauf, 2006), where they may form a reservoir of readily retriev- membranes (Betz et al., 1992), the internalization of fluorescent able SV proteins internalized during subsequent exo-endocytic antibodies directed against lumenal epitopes of SV proteins cycles. Such a readily retrievable SV pool (Hua et al., 2011a) (Matteoli et al., 1992), the use of pH-sensitive GFP (pHluorin) may correspond to clusters of surface-stranded SV proteins fused to the lumenal domain of a SV protein (Miesenbo¨ ck visualized by super-resolution microscopy (Hoopmann et al., et al., 1998), and finally, channelrhodopsin-mediated optoge- 2010; Willig et al., 2006). netic stimulation paired with fast-freezing EM (Watanabe et al., Discrepancies also exist regarding the time course of SV 2013a, 2013b)(Table 1). In spite of this impressive arsenal of endocytosis. The earliest estimates of the kinetics of SV endocy- weapons, the precise mechanisms by which SV membranes tosis, originating from ultrastructural analysis of HRP uptake, re- are retrieved and SVs are reformed have remained partially vealed a slow time course of SV endocytosis on the order of tens elusive. For instance, monitoring exo-endocytosis of SVs loaded of seconds (Heuser and Reese, 1973). In contrast, capacitance with FM1-43 has revealed a partial retention of fluorescence measurements in retinal bipolar cells (von Gersdorff and Mat- upon action potential (AP)-triggered exocytosis in at least a frac- thews, 1994) and quantum dot imaging of single SVs in hippo- tion of events. As kiss-and-run is thought to involve only transient

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