Calcium buffers: Parvalbumin-alpha and Calretinin

Calcium, together with phosphate, are two of the most universal tools in cell functioning and signal transduction, since they can trigger changes in local electrostatic field and therefore in the protein conformation. Thus, calcium is a key ion that can regulate cellular processes that range from the cell shape (by alteration of cytoskeleton structures) to metabolic/enzymatic processes. The intracellular levels of this ion are always kept low (approx. 20000-fold gradient with respect to the extracellular medium). Since low, focal changes in its concentration can trigger a wide plethora of events, cells spend most of its energy in chelating, compartmentalizing or extruding this divalent ion (Clapham, 2007).

In the cochlea, the fast nature of the stimuli that are transduced there and the high metabolic and energetic demands might have obliged the cells to adopt an army of professional calcium binding proteins, including mobile buffers (PV, CR, calbindin(CB)), signaling proteins (calmodulin, Ca²⁺-binding proteins), protein folding (calreticulin, calnexin)(Fettiplace and Nam, 2019) or sensors (otoferlin) (Roux et al., 2006). More concretely, calcium participates in mechanotransduction (vg. maintaining the structure of the tip-links and contributing to the mechanotransduction current), synaptic transmission (vg. glutamate exocytosis from IHC presynaptic terminal requires the influx of calcium through Cav1.3 channels, or regulation of action potential activity through the calcium-activated potassium currents after hearing

75 onset), non-sensory cells physiology processes (vg. intracellular communication through GAP junctions, or ATP-triggered calcium waves) (Ceriani and Mammano, 2012).

In the hair cells and SGNs, the calcium-binding proteins CR, CB-D28k, PV and parvalbumin-beta (also known as oncomodulin) works as mobile cytoplasmic calcium buffers. They spatiotemporally restrict the calcium movement and whose expression is regulated differently across cells, development and species (Fettiplace and Nam, 2019) . Furthermore, all of the members of this family share EF-hand motifs of roughly 30 residues, normally present in adjacent pairs, characterized by the presence of a helix-loop-helix motif that can accommodate calcium or magnesium (Lewit-Bentley and Rety, 2000).

PV has been reported to be in IHCs (Celio, 1990; Pack and Slepecky, 1995; Soto-Prior et al., 1995), in OHCs (only in rats, and mostly in their apical turn (Celio, 1990)), in all subpopulations of type I SGNs (Petitpré et al., 2018; Shrestha et al., 2018) and in the postsynaptic terminals and fibers of type II neurons (Maison et al., 2016). In Figure 19, this pattern can be seen in the cochleae from mouse, rat and gerbil.

The role of PV in the SGN does not seem to be completely understood. It is thought to be involved in many levels of the adaptation to precise and rapid processing of acoustic stimuli.

Taking into consideration other in vitro studies in other cell types, it has been hypothesized that it could work 1) buffering sudden increases of intracellular calcium that occur during sound induced depolarizations, 2) controlling the changes in the cytosolic calcium after depolarization in terms on rise and decay rates and amplitude, regulating the synaptic release of neurotransmitters and intracellular signaling processes and ultimately the auditory transduction (Soto-Prior et al., 1995; Yang et al., 2004).

In IHC, CR, PV and CB decrease the amplitude of the calcium conductance and attenuates its activation, in addition to their role on restricting calcium-dependent glutamate release to the active zones, generating an efficient metabolic control of the calcium-dependent sound encoding (Pangrsic et al., 2015).

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Figure 19. Type I SGN and IHC stained with PV. Left column displays exemplary single slice. They show mid-basal sections where the spiral ganglion, hair cells and auditory nerve can be appreciated. Right column shows the IHC row has been damage and three pieces can be identified outside the IHC row and that in the apical turn of the rat´s cochlea OHCs are also revealed). Background signal allows also to coarsely intuit the cochlear anatomy and occasionally other structures such as vessels (Gerbil´s) or stria vascularis (Rat´s). A. Mouse, B. Gerbil, C. Rat.

Scalebar: 200 µm

The other calcium buffer that we considered was another EF-hand motile calcium buffer: CR.

In IHCs, it has been shown to be homogenously expressed, whereas in SGNs its expression display a gradient among the subtypes of adult type I SGN, being mostly expressed in two subsets, accounting for 35 (Ia, highest levels) and 40% (Ib, mid levels) of the type I neurons (Shrestha et al., 2018). The same study found that CR follows a gradient orthogonal to the tonotopic axis and that its levels seem to correlate with the classical classification of high

77 spontaneous rate (high levels of CR, closer to ST, contacting the pillar side of IHC), mid spontaneous rates and low spontaneous rate (low levels of CR, closer to SV, contacting the modiolar side of IHC). In Figure 20, this staining principle can be seen, the antibody anti-CR labels totally the IHC row, where the ganglion adopts an intermittent pattern, highlighting the fact that is not all the neurons that reside there expresses this calcium buffer.

In a similar fashion to PV, its precise and individual role in each of the cell types has not been fully dissected. However, Iin IHCs, it has been shown its contribution to the Ca²⁺-nanodomain control of vesicle fusion (Pangrsic et al., 2015) . All the SGN expressing calretinin seem to be unitary accommodating neuron (Petitpré et al., 2018) and the ratio calbinidin/CR has been hypothesized to serve as an indicator of the kinetics of the SGN (when the ratio is closer 1, SGNs exhibit longer time constants) (Liu and Davis, 2014). Furthermore, the lower levels of CR in afferent fibers innervating the modiolar side of the IHCs have been related to an increase in their vulnerability to noise trauma (Sharma et al., 2018). All in all, it could be hypothesized that through its calcium buffering capabilities, it can shape the activity-dependent responses and the cells resistance to sound insults.

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Figure 20. IHC and subtype of type I SGN revealed by CR staining. Left column depicts exemplary optical section, where the SGN and IHC can be seen. Since only a subtype of type I SGN is stained, the staining acquires a more intermittent pattern when compared to PV. IHC in the marmoset cochlea seem to be not visible, probably due to an imperfect conservation of organ of Corti after harvesting the sample. Right column displays MIP. A. Mouse (tissue processed in collaboration with Dr. Christian Vogl). B. Gerbil. C. Marmoset. Scalebar: 200 µm.

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