Otoferlin

Otoferlin, a member of the ferlin protein family, is a large tail-anchored multi-C2

domain protein encoded by the 48 exons of the OTOF gene (Yasunaga et al., 1999, 2000;

Roux et al., 2006; Lek et al., 2012; PangrŠič, Reisinger and Moser, 2012). Yasunaga et al., 2000 reported the presence of long (~7 kb-long) otoferlin transcripts in human and mouse brain and inner ear tissue. The same research group also detected short (~5 kb-long) otoferlin transcripts in human heart, placenta, liver, pancreas, skeletal muscle, kidney, inner ear, and brain tissue, but not in mouse tissue (Yasunaga et al., 1999, 2000). The long otoferlin isoform (1997 amino acids long) consists of six C2 domains (C2A-C2F) connected via long linker regions, a FerA domain, a FerB domain, and a single C-terminal transmembrane domain. A seventh C2 domain (C2de) was additionally predicted between the C2D and C2E domain of otoferlin (Figure 1.5) (Yasunaga et al., 1999, 2000; Roux et al., 2006; Lek et al., 2012; PangrŠič, Reisinger and Moser, 2012; Harsini et al., 2018). The short isoform (1230 amino acids long), on the contrary, only contains the last three C2 domains and the transmembrane domain (Yasunaga et al., 1999, 2000). Nevertheless, the exact structure of otoferlin, except for the FerA domain and C2A domain (Helfmann et al., 2011;

Harsini et al., 2018), is still unknown (PangrŠič, Reisinger and Moser, 2012; Johnson, 2017).

The expression of otoferlin in auditory HCs first starts at the embryonic stage E16 for IHCs and E18 for OHCs and reaches its peak at approximately postnatal day (P) 6 for both HC types. After P6 its expression starts to change during development and decreases in OHCs until it almost disappears after maturation. In contrast, IHCs continue to express otoferlin throughout their cytosol and plasma membrane even after maturation (Roux et al., 2006; Beurg et al., 2008; Pangrsic et al., 2010; Strenzke et al., 2016).

- 10 -

Pathogenic mutations in the OTOF gene have been linked to autosomal recessive non-syndromic hearing loss (DFNB9) in humans (Yasunaga et al., 1999, 2000; Roux et al., 2006;

Longo-Guess et al., 2007; Rodríguez-Ballesteros et al., 2008; Marlin et al., 2010). Otoferlin knock-out (Otof^-/-) mice lack otoferlin expression and show no characteristic ABR waves when subjected to sound stimuli, but still have a normal OHC function (Roux et al., 2006;

Reisinger et al., 2011). Pachanga (Otof^Pga/Pga) mice, carrying an N-ethyl-N-nitrosourea–

mediated p.Asp1767Gly (D1767G) missense mutation that affects the C2F domain of otoferlin (Schwander et al., 2007; Figure 1.5; Pangrsic et al., 2010), are also profoundly deaf despite having some scarce SGN spiking at high stimulus intensities (>100 dB SPL for less than 10 stimuli/s) and some residual otoferlin expression in their IHCs (Pangrsic et al., 2010). Other otoferlin mutant mouse models like the Otof^C2C/C2C mice, carrying two missense mutations that are predicted to alter the Ca²⁺ binding affinity of the C2C domain (Johnson and Chapman, 2010; Michalski et al., 2017), have normal otoferlin protein levels, but a moderate hearing loss phenotype. Furthermore, the knock-down of otoferlin seems

Figure 1.5 :

Schematic representation of the otoferlin protein and the pathogenic mutations associated with autosomal recessive hearing impairment DFNB9.

The long otoferlin isoform present in auditory IHCs and necessary for hearing consists of six C2 domains:

C2A (aa 1-121), C2B (aa 270-392), C2C (aa 433-556), C2D (aa 976-1109), C2E (aa 1489-1617), and C2F (aa 1729-1890). A possible seventh C2 domain (C2de, aa 1148-1247) has been predicted between the C2D and C2E domain. The protein also possesses a ferlin-specific FerA domain (aa 738-852), a FerB domain (aa 856-933), and a transmembrane domain (TM, aa 1959-1979) at the C-terminus. Different pathogenic otoferlin missense mutations or in-frame deletions associated with OTOF-related hearing loss are presented on the top. Missense mutations linked to temperature sensitive hearing impairment like the p.Ile515Thr (I515T) mutation are displayed in magenta. The p.Asp1767Gly (D1767G) missense mutation is depicted in blue (adapted from PangrŠič, Reisinger and Moser, 2012).

- 11 -

to cause hearing and balance defects in zebrafish (Chatterjee et al., 2015). It was, therefore, suggested that the long otoferlin isoform is essential for normal synaptic transmission in auditory IHCs (Roux et al., 2006; Pangrsic et al., 2010) and plays a role in vestibular HC synaptic transmission as well (Dulon et al., 2009).

Otoferlin is not required for IHC survival and development since Otof^-/- IHCs were morphologically indistinguishable from wild-type IHCs (Roux et al., 2006). This protein additionally seems not to be required for IHC ribbon synapse formation as Otof^-/- mice have normal ribbon synapse numbers at P6 (Roux et al., 2006). However, Otof^-/- (~40%) and Otof^Pga/Pga mice (~19%) have fewer ribbon synapses after the onset of hearing than wild-type animals. These observations indicate that otoferlin is important for IHC ribbon synapse maintenance after the onset of hearing (Roux et al., 2006; Pangrsic et al., 2010).

Research has shown that otoferlin is a major key player in several IHC exocytosis steps (PangrŠič, Reisinger and Moser, 2012; Pangrsic and Vogl, 2018). This protein is crucial for the late steps of RRP exocytosis like SV priming and fusion since Otof^-/- IHCs have almost completely abolished exocytosis despite normal Ca²⁺ currents (Roux et al., 2006; Pangrsic et al., 2010; Reisinger et al., 2011) and SV numbers (Roux et al., 2006; Vogl et al., 2015). It was proposed, in this regard, that otoferlin either takes over the function of the neuronal docking and priming factors CAPS and Munc13, which are not required for IHC exocytosis (Vogl et al., 2015), or interacts with yet unidentified proteins involved in these steps (Johnson, 2017; Pangrsic and Vogl, 2018). This protein additionally seems to plays a role in SV tethering as the length of tethers connecting the SVs to the AZ membrane is altered in Otof^-/- IHCs (Vogl et al., 2015). Otoferlin is not only necessary for the late steps of exocytosis, but also for RRP replenishment (Roux et al., 2006; Pangrsic et al., 2010; Jung et al., 2015; Strenzke et al., 2016; Michalski et al., 2017; Chakrabarti, Michanski and Wichmann, 2018). Otof^Pga/Pga IHCs, unlike Otof^-/- mutants, have normal RRP exocytosis upon short IHC depolarizations up to 10 ms and an unchanged RRP size (Pangrsic et al., 2010).

SV tethering and docking are also unaltered in these mutants (Chakrabarti, Michanski and Wichmann, 2018). The RRP replenishment rates and sustained IHC exocytosis levels upon longer IHC depolarizations are, however, strongly reduced in Otof^Pga/Pga IHCs (Pangrsic et al., 2010). In addition, multiple-tethered and docked SVs seem to accumulate at the AZ

- 12 -

membrane of these mutants, which was attributed to a defect in active zone clearance (Chakrabarti, Michanski and Wichmann, 2018).

Otoferlin is hypothesized to function as a Ca²⁺ sensor that modulates RRP fusion rates and Ca²⁺ dependent RRP replenishment (Roux et al., 2006; Johnson and Chapman, 2010;

Vincent et al., 2014; Meese et al., 2017; Michalski et al., 2017) since both Syt1 and Syt2, which regulate these processes in neurons, are not required for IHC exocytosis (Safieddine and Wenthold, 1999; Beurg et al., 2010; Reisinger et al., 2011). It was further demonstrated that the Ca²⁺ sensor Syt1 and otoferlin cannot substitute each other. Virus-mediated Syt1 expression could not rescue exocytosis in otoferlin deficient IHCs and otoferlin expression could not restore exocytosis in Syt1 knock-out neurons in this regard (Reisinger et al., 2011). In vitro assays further demonstrated that the C2D-C2F domains of otoferlin, but not the C2A domain, can bind Ca²⁺ (Johnson and Chapman, 2010; Meese et al., 2017). The Otof^C2C/C2C otoferlin mutant mouse, carrying two missense mutations that are predicted to change the Ca²⁺ binding affinity of the C2C domain (Johnson and Chapman, 2010; Michalski et al., 2017), had a delayed fast and sustained IHC exocytosis and an increased ribbon-associated SV pool distance to the ribbon (Michalski et al., 2017). Meese et al., 2017 further reported that the C2C domain of otoferlin can only bind Ca²⁺ when phosphorylated in vitro.

In addition, Ca²⁺/calmodulin-dependent serine/threonine kinase II delta (CaMKIIδ) and potentially other kinases seem to regulate the functions of otoferlin by enhancing or decreasing the Ca²⁺ binding affinity of the C2 domains through phosphorylation (Meese et al., 2017). The Cav1.3 Ca²⁺ channels also co-localize with otoferlin in IHCs in vivo (Vincent et al., 2014, 2017). It seems as if this interaction allows IHCs to better synchronize the exocytosis to the transient Ca²⁺ concentration changes by keeping the Ca²⁺ sensor close the Ca²⁺ ion entry sites at the AZ membrane (Johnson, 2017). The ratio of the fast and slow inactivating Cav1.3 splice isoforms is also changed by the presence or absence of otoferlin, indicating that otoferlin can influence Ca²⁺ entry dynamics in IHCs (Vincent et al., 2014).

Nevertheless, it cannot be ruled out that other yet unidentified Ca²⁺-sensing proteins in IHCs might assist otoferlin in its functions (Michalski et al., 2017).

In contrast to many other synapses, IHCs appear to utilize the same Ca²⁺ sensor, otoferlin, in different steps of the SV cycle (Figure 1.4) (PangrŠič, Reisinger and Moser, 2012;

- 13 -

Strenzke et al., 2016; Michalski et al., 2017; Pangrsic and Vogl, 2018). A further role for otoferlin in SV recycling and coupling IHC exocytosis and endocytosis was proposed because of its interaction with proteins involved in these processes. Several in vitro and in vivo studies have reported that otoferlin can interact with the endocytic protein AP-2, the SNAREs, the N-ethylmaleimide sensitive fusion (NSF) protein, the Cav1.3 Ca²⁺ channels, the small GTPase Rab protein 8b (Rab8b), and myosin VI (MyoVI) (Heidrych et al., 2008, 2009;

Ramakrishnan, Drescher and Drescher, 2009; Johnson and Chapman, 2010; Duncker et al., 2013; Ramakrishnan et al., 2014; Vincent et al., 2014; Jung et al., 2015; Hams et al., 2017).

It was even implicated, at this point, that otoferlin might bind to several proteins at the same time with the number and type of interaction partners depending on the amount of Ca²⁺ influx into IHCs (Johnson, 2017). Whether the in vitro interaction of several otoferlin C2 domains with neuronal t-SNARE proteins (Ramakrishnan, Drescher and Drescher, 2009;

Johnson and Chapman, 2010; Hams et al., 2017) is relevant for IHC exocytosis under physiological conditions in vivo is unclear as the presence of neuronal SNARE proteins in IHCs is questionable (see introduction “chapter 1.2”) (Nouvian et al., 2011). The interaction of otoferlin with AP-2 complexes, the phospholipid [PI(4,5)P2], clathrin, and other yet unidentified proteins, in particular, is believed to trigger SVs to pinch off from endosomal vacuoles and plasma membrane invaginations (Kononenko et al., 2014; Pangrsic and Vogl, 2018). Since the AP-2 complex is involved in both AZ clearance and SV reformation it was further suggested that it might function as a sorting factor for otoferlin by inducing the clearance of “used” otoferlin from AZ release sites after SV fusion (Jung et al., 2015).

Another study found that otoferlin immunofluorescence staining correlates with the majority of newly endocytosed organelles in IHCs with the highest correlation found at the basal IHC region and the lowest at the nucleus (Revelo et al., 2014).

It is still unclear whether and to which degree otoferlin can regulate other cellular processes like cargo transport within IHCs, enzymatic activity, and post-translational modifications (Wu et al., 2015; Johnson, 2017). C2 domain proteins are believed to regulate the Ca²⁺-dependent assembly of membrane-trafficking complexes and cellular trafficking events (Lek et al., 2012; Johnson, 2017). In fact, it was shown that ferlins can regulate Ca²⁺ -induced membrane fission and fusion events (Lek et al., 2012; Johnson, 2017). An in vitro

- 14 -

study showed that both the C2C and the C2F domains of otoferlin are able to interact with the phospholipid [PI(4,5)P2] facing the cytoplasmic side of cell membranes (Padmanarayana et al., 2014). This finding led to the assumption that these two domains mediate the targeting of otoferlin towards the plasma membrane and induce fusion events (Johnson and Chapman, 2010; Marty et al., 2014; Padmanarayana et al., 2014). Unlike the synaptotagmins, which only possess two C2 domains (Johnson, 2017), otoferlin can interact with membrane-trafficking proteins like MyoVI (Heidrych et al., 2009; Roux et al., 2009).

Otoferlin’s interaction partners MyoVI and Rab8 are both involved in cargo sorting and endosomal trans-Golgi networking (Heidrych et al., 2008, 2009; Roux et al., 2009; Pangrsic and Vogl, 2018). These interactions further strengthen the theory that otoferlin is involved in trafficking events in IHCs.

A truncated mini-dysferlin protein variant, generated by cleaving some dysferlin splice variants via Ca²⁺-dependent calpain proteolytic cleavage, contributes to the process of membrane repair in straited muscles. Similar to synaptotagmins, the short dysferlin variant only contains the last two C-terminal C2 domains of the protein and its transmembrane domain (Lek et al., 2013). It is unknown whether such a truncated mini-otoferlin variant exists in IHCs and if it mediates similar functions as the truncated mini-dysferlin version.

Several truncated otoferlin forms were supposedly able to rescue hearing and balance in otoferlin knock-down zebrafish (Chatterjee et al., 2015), but were not able to rescue auditory function in otoferlin knock-out mice (Tertrais et al., 2019). These results imply that full-length otoferlin is needed for proper IHC synaptic transmission in mice and probably humans.

In conclusion, otoferlin is a multi-functional protein involved in many cellular processes in auditory IHCs and all six C2 domains of this protein seems to be necessary for proper IHC synaptic transmission. Yet, there are still many open questions regarding the exact role of this protein and how different OTOF mutations interfere with its different functions.

- 15 -