The role of calcium, Akt and ERK signaling in 1 cadmium-induced hair cell death 2

13 14 Exposure to heavy metals has been shown to cause damage to a variety of 15 different tissues and cell types including hair cells, the sensory cells of our inner ears 16 responsible for hearing and balance. Elevated levels of one such metal, cadmium, have 17 been associated with hearing loss and shown to cause hair cell death in multiple 18 experimental models. While the mechanisms of cadmium-induced cell death have been 19 extensively studied in other cell types they remain relatively unknown in hair cells. We 20 have found that calcium signaling, which is known to play a role in cadmium-induced 21 cell death in other cell types through calmodulin and CaMKII activation as well as IP3 22 receptor and mitochondrial calcium uniporter mediated calcium flow, does not appear to 23 play a significant role in cadmium-induced hair cell death. While calmodulin inhibition 24 can partially protect hair cells this may be due to impacts on mechanotransduction 25 activity. Removal of extracellular calcium, and inhibiting CaMKII, the IP3 receptor and 26

the mitochondrial calcium uniporter all failed to protect against cadmium-induced hair 27 cell death. We also found cadmium treatment increased pAkt levels in hair cells and 28 pERK levels in supporting cells. This activation may be protective as inhibiting these 29 pathways enhances cadmium-induced hair cell death rather than protecting cells. Thus 30 cadmium-induced hair cell death appears distinct from cadmium-induced cell death in 31 other cell types where calcium, Akt and ERK signaling all promote cell death. 32

INTRODUCTION
In addition to its association with hearing loss, cadmium has also been linked to a 58 number of other health conditions including cancer, osteoporosis, and dysfunction of the 59 cardiovascular system, kidneys, liver, nervous system and reproductive system. In some cases this increase appears due to the release of calcium from endoplasmic 67 reticulum (ER) stores, as it can be blocked by an inositol 1,4,5-triphosphate (IP3) 68 ROS production (Xu et al., 2011;Yang et al., 2008Yang et al., , 2007Yuan et al., 2013). Cadmium 82 is unable to increase ROS production when directly applied to isolated mitochondria 83 further supporting the role of calcium as a potential intermediary signal in this process 84 (Shih et al., 2005). Mitochondrial calcium also appears important in cadmium-induced 85 cell death as inhibition of the mitochondria calcium uniporter blocks cadmium-induced 86 cytochrome c release, mitochondrial depolarization and cell toxicity (Lee et al., 2005;Li 87 et al., 2003;Shih et al., 2005). 88 Downstream of calcium both calmodulin and calcium/calmodulin-dependent 89 protein kinase II (CaMKII) appear to be playing a role in cadmium-induced cell death. 90 Cadmium is able to directly activate calmodulin as well as increase calcium's activation 91 of calmodulin, and it is believed this may result in a toxic overactivation of this molecule 92 (Chao et al., 1984;Sutoo et al., 1990). Inhibition of both calmodulin and CaMKII can 93 protect cells against cadmium-induced cell death (Liu et al., 2018;Liu and Templeton, 94 2007). 95 Cadmium has also been shown to increase phosphorylated levels of Akt (pAkt), 96 and Extracellular Receptor Kinase (pERK) in vitro in a calcium-dependent manner 97 cell death process as inhibitors of both signaling pathways have been shown to protect 104 hair cells that have been shown to play key roles in the development, maintenance and 127 regeneration of hair cells (reviewed in Monzack and Cunningham, 2013 We have previously shown that cadmium causes reliable hair cell death in the 132 zebrafish lateral line within three hours in a dose-dependent manner, and therefore 133 used that treatment paradigm in this study (Schmid et al., 2020). We first found that 134 removal of extracellular calcium following cadmium treatment caused an increase in hair 135 cell death both in control conditions and following cadmium treatment. We also found 136 that co-treatment of a calmodulin inhibitor with cadmium could significantly protect 137 against cadmium-induced hair cell death. However, when the calmodulin inhibitor was 138 applied after fish were washed out of cadmium, a condition we refer to as 139 posttreatment, protection was no longer seen. Additionally, other calcium signaling 140 pathway inhibitors including a CaMKII inhibitor, inositol IP3 receptor inhibitor, and 141 mitochondria calcium uniporter inhibitor failed to protect against cadmium-induced hair 142 cell death. We also saw an increase in levels of pAkt in hair cells and pERK in 143 cells per neuromast. To normalize data for the zero calcium and ERK inhibitor 197 experiment, the average hair cells/neuromast number for each treatment group was 198 divided by the average hair cells/neuromast number for their respective control groups 199 with no cadmium and then multiplied by 100 to be converted to a percentage. To calculate pAkt and pERK levels fish stained with either the pAkt or pERK 203 antibodies and the otoferlin antibody were imaged on a Zeiss LSM800 confocal 204 microscope using the Zen Blue software. For pAkt, the approximate center of the 205 neuromast was selected and 10 planes separated by 1 µm were imaged for each 206 neuromast. For pERK, there was significant neuronal labeling in the control condition 207 that we did not want to include in our analysis. Therefore, the top and bottom of the 208 neuromast were selected and planes separated by 1µm were imaged throughout the 209 neuromast regardless of number with images ranging from 11-29 planes. Then 10 210 planes with minimal neuronal labeling were selected for subsequent analysis. One 211 neuromast of the anterior lateral line was imaged per fish with the exact neuromast to 212 be imaged selected based on which neuromasts were best positioned to image. Image 213 analysis was conducted in Fiji. Maximum projection images were made for each 10-214 plane stack. The neuromast was outlined using the otoferlin label and the average 215 All experiments were carried out with n=10 animals per group. Statistics were 219 calculated in GraphPad Prism 6 which was also used to generate graphs. For 220 experiments where two or more different groups were tested against a range of calcium 221 or cadmium doses, a two-way ANOVA was used with the either a Tukey's or Šídák's 222 multiple comparisons test depending on which was recommended by Prism. The 223 multiple comparisons test used for each experiment is mentioned in the figure legend. 224  Therefore, to investigate whether calcium signaling was impacting cadmium-induced 249 hair cell death we first wanted to determine if there was continued hair cell death 250 following cadmium removal. If there was, we could potentially separate cadmium entry, 251 which is the phase of cadmium-induced hair cell death most likely to be 252 mechanotransduction dependent, from the subsequent cell death process. To do this 253 we treated fish with 30 µM of cadmium chloride for either 1 or 2 hours, fish were then 254 washed out of cadmium, and either fixed immediately or fixed three hours after the start 255 of the experiment. We found that following both the 1 and 2 hour cadmium chloride 256 treatment continued hair cell death was seen after cadmium removal ( Figure 1 and immediately fixed (squares), treated with cadmium for 1 hour then left in EM for two hours before 263 fixation (triangles), treated with cadmium for 2 hours and then immediately fixed (upside down triangles), 264 or treated with cadmium for 2 hours and then left in EM for 1 hour before fixation (diamonds). Control fish 265 were kept in EM for three hours (circles). There was a significant amount of cell death after cadmium 266 removal for both the 1 hour and 2 hours cadmium treatment paradigms (*** = p<0.001 by ANOVA and

269
As we saw significant continued cadmium-induced hair cell death after cadmium 270 removal, we next wanted to test if this continued cell death could be altered by changing 271 extracellular calcium levels. To do this we used the treatment paradigm where fish were 272 treated with cadmium chloride for one hour, washed out of cadmium, and then fixed two 273 hours later. We first tested a range of calcium concentrations by treating fish with either 274 EM or 30 µM cadmium chloride for 1 hour and then washing them out of that solution 275 and moving them into EM with calcium concentrations ranging from 0-2 mM for 2 hours 276 prior to fixation. We found the only calcium concentration where a significant difference 277 was seen in the amount of cadmium-induced hair cell death as compared to the 278 standard EM calcium concentration of 1mM was 0mM ( Figure 2A). However, rather than 279 protecting hair cells the removal of extracellular calcium appeared to increase cadmium-280 induced hair cell death. We then treated fish with a range of cadmium doses from 0-120 281 µM for 1 hour before washing them out of cadmium and moving them to either standard 282 EM with 1 mM calcium or EM with 0 mM calcium for 2 hours prior to fixation. Again, a 283 significant increase in the amount of cadmium-induced hair cell death was seen in the However, there was also a significant reduction in the number of control hair cells in this 286 condition similar to what has previously been seen when zebrafish are put in calcium-287 free EM . Therefore, to determine whether the incubation in 0 mM 288 calcium EM was sensitizing fish to cadmium-induced hair cell death or simply killing hair 289 cells on its own we normalized these data for both the 1 mM and 0 mM calcium EM 290 groups to the 0 cadmium control for each group. We found in this case while there is still 291 a trend toward a decrease in hair cell number at all cadmium concentrations tested, the 292 only concentration that showed a significant decrease was 120 µM cadmium (Figure 293 However, calmodulin inhibition has also previously been shown to inhibit rapid 329 apical endocytosis in hair cells as measured by rapid FM1-43 uptake, a process 330 dependent on mechanotransduction activity (Seiler and Nicolson, 1999). As mentioned 331 above inhibiting mechanotransduction can protect against cadmium-induced hair cell 332 death (Schmid et al., 2020), therefore, we tested whether inhibiting calmodulin only after 333 cadmium removal would still significantly protect against cadmium-induced hair cell 334 death. To do this fish were treated with varying doses of cadmium chloride from 0-30 DMSO or 30 µM W-13 for two hours before fixation. Using this treatment paradigm we 337 no longer saw protection from calmodulin inhibition ( Figure 3B). This suggests the 338 protection seen with cotreatment with the calmodulin inhibitor may be due to impacts on 339 mechanotransduction-dependent processes, though we can also not rule out a distinct 340 early role for calmodulin in the cell death process. 341 Calmodulin is known to activate CaMKII and inhibiting CaMKII has also been 342 shown to protect against cadmium-induced cell death in multiple cell types (Chen et al.,

Cadmium increases Akt and ERK signaling in neuromasts 396
In many cell types cadmium increases the level of phosphorylated Akt and ERK 397 we treated fish with 30 µM of cadmium chloride for one hour and then fixed and stained with an antibody for either pAkt or pERK. We found that treatment with cadmium 406 significantly increased the levels of pAkt seen in hair cells ( Figure 5A & B). This 407 increase was lost when fish were cotreated with cadmium and 5 µM of the Akt inhibitor 408 MK-2206. We also saw a significant increase in pERK levels following cadmium 409 treatment that was lost when fish were cotreated with cadmium and 3 µM of the MEK 410 inhibitor PD184352 which suppresses ERK phosphorylation ( Figure 5C & D). However, 411 unlike pAkt labeling which appeared localized to hair cells, pERK labeling appears 412

Inhibiting Akt and ERK signaling pathways increases cadmium-induced hair cell 430 death 431
Having seen an increase in pAkt and pERK levels following cadmium treatment, 432 we wanted to determine whether the Akt or ERK signaling pathways impacted To determine if the Akt or ERK pathways were impacting cadmium-induced hair 442 cell death, we cotreated fish with either 5 µM of the Akt inhibitor MK-2206, or 1.5 µM of 443 the MEK inhibitor PD184352, which inhibits ERK activation, and varying doses of 444 cadmium chloride for three hours. A smaller dose of PD184352 was used in this 445 experiment than the pERK levels experiment due to increased hair cell toxicity from 446 PD184352 alone with the longer treatment time. We found both drugs caused a 447 decrease in hair cell number following cadmium treatment particularly, at the 30 and 60 448 µM cadmium chloride doses ( Figure 6A & B). However, as noted above PD184352 449 caused a loss of hair cells independent of cadmium treatment, a similar loss of hair cells 450 from simple ERK inhibition has also previously been seen in cochlear explants induced hair cell death or simply killing hair cells on its own, we normalized the data for 453 both the DMSO and PD184352 groups to the zero cadmium control ( Figure 6C). In 454 doing so we still saw a significant increase in cadmium-induced hair cell death at the 30 455 and 60 µM cadmium chloride doses. Thus, we conclude that unlike in other cell types 456 in hair cells by the fact that calmodulin inhibition also inhibits rapid apical endocytosis in 503 hair cells, a process dependent on mechanotransduction activity (Seiler and Nicolson, 504 1999). As inhibiting mechanotransduction activity is known to inhibit cadmium-induced 505 hair cell death (Schmid et al., 2020) we cannot definitively say that the protection we 506 see from calmodulin inhibition is due to a role for calmodulin in the cell death process. 507 The fact that protection is no longer seen when the calmodulin inhibitor is added after 508 cadmium treatment, rather than as a cotreatment, and the fact that CaMKII inhibition 509 was unable to protect hair cells from cadmium-induced hair cell death further supports the idea that calmodulin inhibition is mainly impacting hair cell mechanotransduction 511 activity and cadmium entry rather than the cell death process. 512 We also failed to see protection against cadmium-induced hair cell death using previously been shown that ERK signaling can be activated in supporting cells even 546 following calcium chelation (Lahne and Gale, 2008). One potential alternative 547 mechanism for the activation of these pathways includes Ras and Raf signaling which 548 has previously been implicated in activating ERK signaling following hair cell damage 549 Cadmium is known to damage support cells (Ding et al., 2020), and thus this 580 ERK activation could be coming from a direct effect of cadmium on the support cells. 581 Alternatively, the damaged hair cells could be sending a signal to the support cells to 582 induce ERK activation. It has been shown that the damage-induced release of ATP and 583 The authors have no competing interests to declare 620