In the central nervous system, proteins of the AZ maintain morphological and physiological in-tegrity of SVs and Ca2+-channels in order to organize fast and synchronous release, thereby reg-ulating synaptic strength (Torres et al., 2017). The molecules of the ribbon AZs and synaptic machinery are partially preserved and partially unique at ribbon synapses. IHC ribbon-type AZs use specialized synaptic proteins, such as RIBEYE (Schmitz et al., 2000; Maxeiner et al., 2016), otoferlin (Yasunaga et al., 1999; Roux et al., 2006) and Cav1.3 L-type Ca2+-channels (Platzer et al., 2000; Brandt et al., 2003) and seem to operate only partially with conventional synaptic proteins (Pangršiˇc et al., 2012, reviewed in Wichmann, 2015). Several synaptic proteins involved in neurotransmission at conventional synapses appear to be absent from HC ribbon synapses or are not functionally required such as synapsins and synaptophysins (Safieddine and Wenthold, 1999), complexins (Strenzke et al., 2009; Uthaiah and Hudspeth, 2010), synaptotagmin 1 and 2 (Safieddine and Wenthold, 1999; Beurg et al., 2010; Reisinger et al., 2011), and neuronal SNARE (soluble NSF attachment protein receptors) proteins (Nouvian et al., 2011). There are also ad-ditional differences between ribbon synapses in different cell types. For instance, the auditory ribbon synapse composition is distinct from the visual system by the absence of Rab3-interacting molecule 1 (RIM1), Munc13s, synaptotagmin 1 and 2, and neuronal SNAREs from IHC AZs (Strenzke et al., 2009; Uthaiah and Hudspeth, 2010; Beurg et al., 2010; Reisinger et al., 2011;
Nouvian et al., 2011; Jung et al., 2015b; Vogl et al., 2015).
RIBEYE
The main protein component of the ribbon is RIBEYE, a protein unique to synaptic ribbons (Schmitz et al., 2000). It consists of two domains, a ribbon specific A domain and a B domain that is identical to the transcriptional co-repressor named C-terminal binding protein 2 (CtBP2) (Schmitz et al., 2000). The A domain is essential for the structural organization of the ribbon due to the assembly of RIBEYE molecules into a large complex, while the B domain is assumed to facilitate tethering of SVs to the ribbon (Schmitz et al., 2000; Schmitz, 2009; Schwarz et al., 2011). Maxeiner and colleagues recently reported that the deletion of RIBEYE leads to a com-plete loss of retinal ribbons from photoreceptors and bipolar cells (Maxeiner et al., 2016). Thus, they could confirm the necessity of RIBEYE to form ribbons in the retina and in addition they demonstrated an impairment of glutamate release by a severe reduction of the fast and sustained components of exocytosis. Maxeiner et al. (2016) concluded that ribbons are required to couple voltage-gated Ca2+-channels to vesicular release sites in order to enable tight control of vesicle fusion (also known as Ca2+-nanodomain coupling), which had already been described by various studies in the ear and eye (Brandt et al., 2005; Bartoletti et al., 2011; Graydon et al., 2011; Wong et al., 2014; Pangršiˇc et al., 2015; Johnson et al., 2017).
Bassoon and piccolo
The two large scaffolding proteins bassoon and piccolo (also termed aczonin) have been described in conventional and ribbon-type synapses (for overview Gundelfinger et al., 2016; Wichmann and Moser, 2015). In conventional synapses, bassoon is structurally related to piccolo (Wang et al., 1999; Fenster et al., 2000). Synaptic ribbons are anchored to the AZ via the presynaptic density at
1.4. Molecular key players of cochlear and utricular HC AZs
the base of the ribbon containing the protein bassoon (Dick et al., 2003; Khimich et al., 2005; tom Dieck et al., 2005; Frank et al., 2010; Jing et al., 2013; Wong et al., 2014). In bassoon-deficient mice, retinal and cochlear ribbon synapses are not properly anchored to the AZ, which results in floating ribbons (Dick et al., 2003; tom Dieck et al., 2005; Khimich et al., 2005; Frank et al., 2010; Jing et al., 2013). Moreover, the organization of Ca2+-channels is impaired and the size of the RRP is decreased, which in turn reduce Ca2+-currents and the fast component of exocy-tosis at IHCs causing a hearing impairment (Khimich et al., 2005; Frank et al., 2010; Jing et al., 2013). Thus, bassoon is essential for the synaptic architecture and neurotransmitter release in ribbon synapses. The lack of bassoon in central synapses reveal no structural changes, but an impaired replenishment of SVs as well as a decline in the RRP size was detected and more silent synapses were observed (Altrock et al., 2003; Hallermann et al., 2010; Mendoza Schulz et al., 2014). The homologous scaffolding protein piccolo is another large AZ protein in conventional synapses (Gundelfinger and Fejtova, 2012; Südhof, 2012). In ribbon-type synapses, the short iso-form of piccolo is expressed, named piccolino, which is a C-terminal truncated piccolo variant (Limbach et al., 2011; Regus-Leidig et al., 2013, 2014). Immunogold electron microscopy using an antibody that recognizes the short and the long isoform revealed that piccolino is localized across the whole ribbon area in photoreceptors (Limbach et al., 2011). In contrast to conventional synapses, piccolino does not colocalize with bassoon (Limbach et al., 2011). In vivo piccol-ino knockdown experiments in photoreceptors resulted in an impairment of the synaptic ribbon morphology (Regus-Leidig et al., 2014) indicating a potential involvement in the structural orga-nization and/or maturation of synaptic ribbons. At conventional synapses, piccolo interacts with several other AZ proteins indicating functions in vesicle trafficking, adhesion, cytoskeletal orga-nization and SV docking and fusion (Fenster et al., 2000; Kim et al., 2003; Gundelfinger et al., 2016, reviewed in Torres et al., 2017). Western blot data associated piccolo as well as bassoon with cytoskeletal structures demonstrating that both proteins organize the AZ (tom Dieck et al., 1998).
In bassoon- and piccolo-deficient conventional synapses, clustering, docking, and density of SVs were reduced, whereas synapses lacking only piccolo showed an increase in short-term synaptic depression and decreased vesicle reloading (Mukherjee et al., 2010; Butola et al., 2017). Piccolo may also be involved in regulating the assembly of presynaptic F-actin, which is known to be important for the SV cycle (Waites et al., 2011; Wagh et al., 2015). Since piccolino in ribbon-type synapses lacks the interaction sites of the long isoform for various AZ proteins like RIM, Munc13, bassoon, CAST/ELKS, and Ca2+-channels (Regus-Leidig et al., 2013), it is highly probable that the long and short isoforms exhibit distinct functions. To date, no data are available about the ex-pression of bassoon and piccolino in vestibular HCs. Therefore, the role of bassoon and piccolino in HCs of the vestibular system remains to be elucidated.
Otoferlin
The protein otoferlin belongs to the ferlin family and is essential for exocytosis in vestibular HCs and mature cochlear IHCs and thus has been proposed to exhibit a multi-functional role, including Ca2+sensing to trigger SV fusion and SV replenishment (Roux et al., 2006; Johnson and Chap-man, 2010; Michalski et al., 2017). Importantly, otoferlin seems not to be functionally required
during the early first postnatal week, where IHC transiently express the neuronal Ca2+ sensors synaptotagmin 1 and 2 (Safieddine and Wenthold, 1999; Uthaiah and Hudspeth, 2010; Beurg et al., 2010; Reisinger et al., 2011). Mutations in this otoferlin gene have been identified to cause a neu-rosensory non-syndromic recessive form of human deafness (DFNB9) (Yasunaga et al., 1999). In otoferlin-deficient cochlear IHCs, Roux et al. (2006) observed a nearly complete abolished exocy-tosis, however, ribbon synapse morphogenesis, numbers of docked SVs, and Ca2+-currents were not affected in the profoundly deaf mice. Further studies could confirm the involvement of otofer-lin in the last steps of IHC exocytosis such as SV tethering and priming (Pangršiˇc et al., 2010;
Vogl et al., 2015) as well as AZ clearance (Pangršiˇc et al., 2010; Jung et al., 2015a). In contrast to cochlear IHCs, vestibular HCs transmit lower frequency head motion stimuli, however, they also operate with high temporal precision in the millisecond range (Huterer and Cullen, 2002). Inter-estingly, fast kinetics of exocytosis with higher Ca2+sensitivity is characteristic for type I utricular HCs, while type II cells show slower kinetics and reduced Ca2+ efficiency (Dulon et al., 2009).
Similar differences in exocytosis have also been reported for cones and rods of the retina (Rabl et al., 2005) as well as in immature vs. mature cochlear IHCs (Johnson et al., 2005). Otoferlin knockout (KO) experiments in utricular HCs suggested that otoferlin is a high affinity Ca2+sensor essential for exocytosis in type I but not type II HCs, which implies an additional unidentified Ca2+sensing mechanism in type II HCs (Dulon et al., 2009).
Ca2+-channels
Hair cell exocytosis also depends on Ca2+-channels that regulate neurotransmitter release and in-teract with otoferlin (Ramakrishnan et al., 2009). In contrast to conventional synapses that mostly express P/Q-, N-, or R-type channels (Catterall, 2011), the predominant Ca2+-channel isoform mediating excitation-secretion coupling at IHC synapses is the L-type Cav1.3 (Platzer et al., 2000;
Brandt et al., 2003). Cav1 (or L-type) Ca2+-channels are characterized by non-inactivating cur-rents with large amplitudes and are mostly found in muscle cells. Using confocal and stimulated emission depletion (STED) microscopy, Cav1.3-channels could be localized at AZs where they form a stripe-like pattern in mature cochlear IHCs (Brandt et al., 2005; Frank et al., 2010; Wong et al., 2014). When Cav1.3 Ca2+-channels are absent from IHCs, exocytosis is completely abol-ished causing deafness (Platzer et al., 2000). On the other hand, exocytosis in vestibular HCs only partially relies on Cav1.3-channels (Bao et al., 2003; Dou et al., 2004). The lack of Cav 1.3-channels leads to deafness, but no obvious vestibular defect has been reported (Dou et al., 2004).
Vestibular HCs, in addition, likely possess the T-type Ca2+-channel Cav3.1 (Nie et al., 2008).
Fundamental understanding of the ultrastructural morphology of AZ proteins and their develop-mental changes at cochlear and vestibular HC ribbon synapses is still insufficient. To decipher the role of ribbons in the presynaptic HC function, it is important to gain more knowledge on the molecular structure and precise topology of presynaptic proteins. This can give new insights into the functions played by particular synaptic proteins and how they work together to reliably transmit sensory input.