Synaptic vesicle recycling

endocytosis at stimulus arrival.

Figure 1.2 SynaptopHluorin: a fluorescent tool for the of study synaptic vesicle recycling.

pHluorin is a mutant version of the fluorescent protein GFP whose brightness varies according to surrounding pH levels. At the low intravesicular pH (∼6.5) created by the proton pump, pHluorin is quenched and therefore undetectable in microscopy imaging. After synaptic vesicle exocytosis, pHluorin faces the neutral pH of the extracellular medium (∼7.4), recovering its maximum fluorescence. The fusion of pHluorin with synaptic vesicle proteins has been called synaptopHluorin, and is used to monitor exo- and endocytosis kinetics.

1.2.3 Synaptic vesicle recycling

After synaptic vesicle exocytosis, compensatory endocytic mechanisms retrieve regions of membrane and their associated proteins from which synaptic vesicles are reformed. This process, called synaptic vesicle recycling, is important for replenishing the pool of vesicles that will undergo exocytosis upon subsequent stimulation rounds, and for keeping the surface area of the synaptic terminal constant (Südhof, 2004). Early evidence for synaptic vesicle recycling came from EM studies: stimulation strength correlates with the amount of HRP labeling taken up into synaptic terminals (Holtzman et al., 1971); the observation of Ω (omega) shapes arising from the plasma membrane and the formation of cisterns in stimulated synapses (Heuser and Reese, 1973); and the finding that synapses exhausted by strong stimulation can resume neurotransmitter release after a recovery period (Ceccarelli et al., 1973). Since then, three main mechanism of synaptic vesicle recycling have been postulated (Figure 1.1).

1.2.3.1 Kiss-and-run

This model suggests that synaptic vesicles do not undergo complete fusion with the plasma

13 membrane. Instead, they form a transient pore with the plasma membrane, through which neurotransmitter can exit. Thereafter, the pore closes and the synaptic vesicle can detach to be ready for a new round of neurotransmitter refilling. A variation of this model proposes that the vesicle could remain attached with the pore open for long periods, while being refilled with neurotransmitter, in what has been called “kiss-and-stay”. The pore could be walled by the fused lipid layers from both membranes, or by a protein complex similar to an ion channel (Ceccarelli et al., 1973, 1979; Fesce et al., 1994; Koenig et al., 1998; Sun et al., 2002; Aravanis et al., 2003; Gandhi and Stevens, 2003). Vesicle fission could be helped by the action of endophilin or dynamin, recruited by synaptic vesicle molecules like synaptophysin (Daly et al., 2000; Llobet et al., 2011). Although this model was postulated several decades ago, undoubtful proof of its existence has been elusive (Rizzoli and Jahn, 2007; Rizzoli, 2014). This model is attractive for the temporal and energetic benefits of not needing to reconstruct a synaptic vesicle from the membrane.

1.2.3.2 Clathrin mediated endocytosis (CME) of synaptic vesicles

A wealth of studies supports the idea that synaptic vesicles completely fuse with the presynaptic membrane. After exocytosis, the vesicular membrane patch could drift away from the active zone for its retrieval by endocytosis (Miller and Heuser, 1984; Roos and Kelly, 1999). Electron micrographs showing an increase in coated pits and coated vesicles following stimulation have suggested that clathrin-mediated endocytosis (CME) is responsible for vesicle reformation (discussed in section 1.1.1.1). Further studies have confirmed that this is the main endocytosis mechanism in conventional synapses (Heuser and Reese, 1973; Zhang et al., 1998; Granseth et al., 2006), being also found in sensory synapses of retinal bipolar cells (Jockusch et al., 2005; Logiudice et al., 2009), photoreceptors (Cooper and McLaughlin, 1983; Fuchs et al., 2014) and auditory cells (Siegel and Brownell, 1986; Lenzi et al., 2002; Duncker et al., 2013; Neef et al., 2014).

The same molecules involved in CME supporting constitutive endocytic pathways have been found to play a role in synaptic vesicle recycling. Hence, synaptic vesicle reformation requires the action of the adaptor protein complex AP2 for coat formation, dynamin for vesicle fission, and amphiphysin for dynamin recruitment (Kosaka and Ikeda, 1983; Takei et al., 1995; Andrews et al., 1996; González-Gaitán and Jäckle, 1997; Shupliakov et al., 1997).

But how is the clathrin machinery specifically recruited to a patch of synaptic vesicle proteins? Synaptotagmin 1 (Syt 1), the Ca²⁺ sensor triggering vesicle exocytosis, seems to be recognized as a cargo molecule by the µ2 and α subunits of the AP2 complex, leading to

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coated pit nucleation. Stonin 2, a protein present in synaptic terminals, seems to facilitate the Syt1-AP2 interaction (Geppert et al., 1994b; Zhang et al., 1994; Haucke et al., 2000; Grass et al., 2004; Diril et al., 2006). In the following step of coat formation, another synapse-specific protein called AP180 has been identified. AP180 interacts with inositides and clathrin, helping in the formation of triskelia. Moreover, AP180 seems to regulate the size of the resultant synaptic vesicles, keeping in this way pool homogeneity (Zhang et al., 1998;

Morgan et al., 1999).

Interestingly, it has been proposed that cycles of phosphoinositide phosphorylation/dephosphorylation may play an important role in synaptic vesicle recycling. This is supported by higher affinity of AP2 and AP180 for phosphorylated forms when anchoring to membranes, and by the presence of the inositol 5-phosphatase synaptojanin in vesicle endocytic intermediates (McPherson et al., 1994, 1996; Cremona and De Camilli, 1997).

1.2.3.3 Bulk endocytosis

Kiss-and-run and CME are the candidate modes of synaptic vesicle recycling happening during physiological stimulation conditions. However, in the early years of synaptic research, scientist often used unphysiological, intense stimulation protocols that revealed a third mode of membrane retrieval: after exhaustion of the RRP and recycling pool, strong stimulation mobilizes the reserve pool to also undergo exocytosis. Such rates of vesicle release outperform the endocytic machinery, creating bulging of the synaptic terminal and inducing deep membrane infoldings, known as bulk membrane retrieval (Ceccarelli et al., 1973; Heuser and Reese, 1973; Fried and Blaustein, 1978; Miller and Heuser, 1984).

Dynamin I dephosphorylation by the Ca²⁺ sensorcalcineurin seems to be important for bulk retrieval activation. After their formation, membrane infoldings detach from the membrane and form intracellular cisterns. During this step, syndapin and dynamin GTPase activity could be involved in membrane curvature and fission, respectively (Evans and Cousin, 2007;

Andersson et al., 2008; Clayton and Cousin, 2009; Clayton et al., 2009; Nguyen et al., 2012).

It is likely that infoldings and cisterns contain mixed amounts of synaptic vesicle and plasma membrane proteins, which are later on selectively segregated by clathrin coat formation and budding (Heuser and Reese, 1973; Richards et al., 2000; Teng and Wilkinson, 2000). In neuromuscular junctions, actin has been implicated in the initiation of bulk membrane retrieval, and together with dynamin in its maturation into cisternae (Nguyen et al., 2012).

15 Although bulk endocytosis is traditionally considered an emergency route to overcome unphysiological high rates of release, new studies have validated it in neuromuscular junctions and the large calyx of Held synapse within physiological stimulation rates (Richards et al., 2000; Wu and Wu, 2007; Clayton et al., 2008). As it will be shown in the results and discussion of this study, bulk endocytosis might have a great importance in synaptic vesicle recycling and normal function of the highly active ribbon-type sensory synapses.

1.2.3.4 Endosomal sorting of recycled vesicles

An additional step of synaptic vesicle processing has been proposed to take place intracellularly: after their retrieval and uncoating, synaptic vesicles from the RRP might rejoin the pool of vesicles waiting for next rounds of release or, alternatively, they might fuse with a sorting endosome. This compartment would fulfill the function of ridding synaptic vesicles of plasma membrane proteins that were fortuitously taken up along with the synaptic vesicle membrane patch. After sorting, regions with only synaptic vesicle proteins could bud, by a still unclear mechanism, in order to produce release-competent synaptic vesicles (Hoopmann et al., 2010).

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