- Open Access
The susceptibility of cochlear outer hair cells to cyclodextrin is not related to their electromotile activity
- Yingjie Zhou†1,
- Satoe Takahashi†2,
- Kazuaki Homma2, 3,
- Chongwen Duan2,
- Jason Zheng2,
- Mary Ann Cheatham1, 3 and
- Jing Zheng1, 2, 3Email authorView ORCID ID profile
© The Author(s). 2018
- Received: 3 August 2018
- Accepted: 13 September 2018
- Published: 24 September 2018
Niemann-Pick Type C1 (NPC1) disease is a fatal neurovisceral disorder caused by dysfunction of NPC1 protein, which plays a role in intracellular cholesterol trafficking. The cholesterol-chelating agent, 2-hydroxypropyl-β-cyclodextrin (HPβCD), is currently undergoing clinical trials for treatment of this disease. Though promising in alleviating neurological symptoms, HPβCD causes irreversible hearing loss in NPC1 patients and outer hair cell (OHC) death in animal models. We recently found that HPβCD-induced OHC death can be significantly alleviated in a mouse model lacking prestin, an OHC-specific motor protein required for the high sensitivity and sharp frequency selectivity of mammalian hearing. Since cholesterol status is known to influence prestin’s electromotility, we examined how prestin contributes to HPβCD-induced OHC death in the disease context using the NPC1 knockout (KO) mouse model (NPC1-KO). We found normal expression and localization of prestin in NPC1-KO OHCs. Whole-cell patch-clamp recordings revealed a significant depolarization of the voltage-operating point of prestin in NPC1-KO mice, suggesting reduced levels of cholesterol in the lateral membrane of OHCs that lack NPC1. OHC loss and elevated thresholds were found for high frequency regions in NPC1-KO mice, whose OHCs retained their sensitivity to HPβCD. To investigate whether prestin’s electromotile function contributes to HPβCD-induced OHC death, the prestin inhibitor salicylate was co-administered with HPβCD to WT and NPC1-KO mice. Neither oral nor intraperitoneal administration of salicylate mitigated HPβCD-induced OHC loss. To further determine the contribution of prestin’s electromotile function, a mouse model expressing a virtually nonelectromotile prestin protein (499-prestin) was subjected to HPβCD treatment. 499-prestin knockin mice showed no resistance to HPβCD-induced OHC loss. As 499-prestin maintains its ability to bind cholesterol, our data imply that HPβCD-induced OHC death is ascribed to the structural role of prestin in maintaining the OHC’s lateral membrane, rather than its motor function.
- Niemann-pick type C1
- Outer hair cells
Cyclodextrins (CDs) comprise a family of amphipathic cyclic oligosaccharides with a hydrophobic core and a hydrophilic outer surface. Because CDs can complex with hydrophobic molecules to enhance their solubility, they have been widely used in the pharmaceutical industry to facilitate drug delivery . More recently, CDs themselves have become attractive therapeutic candidates that can extract lipids and cholesterol from cell membranes to treat cardiovascular and neurodegenerative diseases . One such example is 2-hydroxypropyl-β-cyclodextrin (HPβCD), which is currently undergoing a clinical trial for the treatment of a rare genetic disease called Niemann-Pick Type C1 (NPC1) [34, 35]. In NPC1 disease, lack or dysfunction of NPC1 protein that resides in endosomal and lysosomal membranes  disturbs intracellular cholesterol trafficking, resulting in progressive neural degeneration and early death in affected individuals. HPβCD administration drastically improves the neurological symptoms of both NPC1 animal models and human patients; however, hearing loss is reported to be a common outcome associated with HPβCD treatment [12, 13, 17, 22, 55].
Since hearing loss is an unavoidable adverse effect of HPβCD therapy in NPC1 patients, understanding the mechanisms underlying its ototoxicity is of high interest. In animal models, HPβCD-induced hearing loss is associated with loss of outer hair cells (OHCs) . OHCs function as cochlear amplifiers by undergoing somatic length changes, called electromotility , which are required for sensitivity and frequency selectivity in mammalian hearing . OHC electromotility is mediated by a distinctive motor protein, prestin , which belongs to a family of solute carrier protein 26 (SLC26) anion transporters. Prestin (SLC26A5) is unusual among the SLC26 family as it exhibits robust voltage-dependent conformational changes that confer electromotility. Recently, we discovered that HPβCD-induced OHC death is exacerbated by the presence of prestin, as OHCs lacking prestin were less vulnerable to HPβCD . We further demonstrated that prestin can directly interact with cholesterol, suggesting that the extraction of cholesterol by HPβCD may disrupt the prestin-rich membrane, resulting in rapid OHC death . Although prestin contributes to the OHC’s sensitivity to cholesterol depletion, it is not clear how prestin influences HPβCD-evoked OHC death or whether prestin’s motile function contributes to its vulnerability to HPβCD treatment.
The OHC’s lateral membrane is packed with ~ 11 nm particles composed of prestin tetramers [25, 54, 58]. In fact, removing prestin results in a 40% decrease in OHC length , indicating that cholesterol concentration and the lipid environment are probably wild-type (WT)-like, as cell surface area decreases proportionally in OHCs lacking prestin. Although cholesterol molecules bound to WT prestin could be the reason that OHCs are more sensitive to HPβCD treatment, we cannot rule out the possibility that prestin’s motile function may also contribute to susceptibility, as cholesterol is known to influence both motility and oligomerization [21, 37]. Prestin’s motile function is based on its ability to change its conformation when membrane voltage is changed. As β-cyclodextrin is capable of changing protein conformation , whether prestin’s motile function contributes to its vulnerability to HPβCD treatment requires investigation.
In this study, we sought to understand the molecular mechanisms underlying HPβCD-induced OHC death in the disease context. NPC1 patients present with variable degrees of hearing impairment even before receiving their first HPβCD treatment . The NPC1-knockout (KO) mouse model (also known as NPCnih) that lacks NPC1 expression exhibits hearing impairment well before the onset of overt neurological symptoms . These observations suggest that NPC1 plays important roles directly or indirectly in hearing, as decreased OHC performance was detected by measuring distortion product otoacoustic emissions (DPOAE). Since cholesterol has an enormous influence on prestin’s function and structure [21, 37], we first tested whether the expression, localization, and/or function of prestin is influenced in the NPC1 disease context using the NPC1-KO mouse model. Our data show that lack of NPC1 protein does not affect the normal distribution pattern of prestin protein. Using an electrophysiological method, we assessed function of prestin by measuring nonlinear capacitance (NLC), a proxy for electromotility. OHCs isolated from NPC1-KOs exhibited robust NLC indicating that prestin-based somatic electromotility is present, although its sensitivity and voltage dependence are altered relative to WT prestin. Consistent with normal prestin expression, OHCs from NPC1-KOs are as sensitive to HPβCD as WT. In order to determine whether the motile function of prestin contributes to the HPβCD-induced ototoxicity, we utilized the prestin inhibitor salicylate, a commonly used painkiller and anti-inflammatory drug known as aspirin. Salicylate competes with prestin’s substrates such as chloride and bicarbonate, thereby reversibly inhibiting function . Co-administration of salicylate and HPβCD did not mitigate HPβCD-induced OHC death, indicating that inhibition of prestin’s electromotility did not affect the sensitivity of OHCs to HPβCD. We further tested the contribution of prestin’s motile function using a prestin knockin (KI) mouse model that expresses virtually nonfunctional 499-prestin protein (499-prestin-KI) . 499-prestin KI mice were as sensitive to HPβCD-induced OHC loss as WT, suggesting that prestin’s motor action is not the key factor underlying the OHC’s sensitivity to HPβCD. Since 499-prestin targets the lateral membrane and interacts with cholesterol as in WT prestin, OHC loss appears to be determined by the presence of cholesterol-interacting prestin proteins that confer normal OHC stiffness and length, rather than to its electromotile function.
All experimental procedures were conducted in accordance with the Guide for the Care and Use of Laboratory Animals, and were approved by Northwestern University’s Animal Care and Use Committee and the National Institutes of Health. NPC1-KO mice (BALB/cNctr-Npc1m1N, also known as NPCnih) were obtained from The Jackson Laboratory (Stock No: 003092). Wild-type (WT) and NPC1-KO mice were obtained by heterozygous breeding. Genotyping was outsourced to Transnetyx (Cordova, TN). Mice younger than 2.5 months (age) were used to avoid complications from neurological dysfunction due to loss of NPC1. 499-prestin-KI mice that carry the V499G/Y501H mutation in the prestin gene were maintained on the original 129S6/C57Bl6J background . 499-KI mice younger than 1 month of age were used to minimize OHC loss . In all experiments, both males and females were tested.
HPβCD and HPβCD/salicylate treatments
WT and 499-prestin-KI mice were injected with saline or HPβCD (Sigma, H107) dissolved in saline (0.9% NaCl) subcutaneously as described previously . For low-dose (4000 mg/kg) treatment, mice were repeatedly injected four times once per week for four weeks. For high-dose (8000 mg/kg) treatment, mice were injected once. For oral administration of salicylate, adult mice (P32–43) were supplied with drinking water containing 3 mg/ml sodium salicylate for 7 days prior to high-dose HPβCD subcutaneous injection. Salicylate water bottles were changed twice per week. Animals were returned to regular water 24 h after HPβCD injection. For intraperitoneal administration of salicylate, mice (P32–45) were first injected with sodium salicylate (245 mg/kg) a day before HPβCD treatment. Approximately, 16 h later, a second salicylate injection was administered as described before . Within 1 h, mice were screened using DPOAEs and auditory brainstem responses (ABR), and then injected with 8000 mg/kg HPβCD subcutaneously, i.e., ~ 1 h after the second salicylate injection. Since salicylate is known to be eliminated from the blood after 8 h in mice , these animals were also supplied with water containing 3 mg/ml sodium salicylate, but returned to regular water 24 h after HPβCD injection. Control mice were supplied with regular water and injected with saline. Auditory function was measured before and after HPβCD injection.
Cochlear in vivo physiology
WT and NPC1-KO mice were anaesthetized with intraperitoneal injections of ketamine (100 mg kg− 1) and xylazine (10 mg kg− 1). Additional doses were given throughout the experiment to maintain a surgical level of anesthesia. During data collection, body temperature was maintained using a heating blanket. DPOAE were measured by presenting the two stimulating primaries (f2/f1 = 1.2) at 70 dB SPL or when the level of f1 was 50 dB SPL and the level of f2 as 35 dB SPL. Growth or input-output functions were also acquired for f2 = 12 kHz and for f2 = 27 kHz. For these measurements, the level of f1 was always 10 dB higher than that for f2. In order to obtain a measure of sensitivity, DPOAE thresholds were determined as the level of f1 that generated 2f1-f2 of 0 dB. ABRs to tone-burst stimuli were then collected to document the output of the cochlea, as well as the brainstem. For these recordings, subcutaneous electrodes were inserted at the vertex and the mastoid with each response measured relative to the indifferent electrode inserted into the opposite shoulder/neck region. Calibration was performed quasi-free field as reported previously . Thresholds were determined by noting the signal level where the ABR waveform disappeared into the noise. Because the number of averages increased as signal level decreased (5 dB step size), the noise level was ~ 0.2 microvolts. Further details on how these recordings are made appear in a previous publication . All DPOAE and ABR measurements were completed on the same day.
Nonlinear capacitance (NLC) measurements
where α is the slope factor of the voltage-dependence of charge transfer, Qmax is the maximum charge transfer, Vm is the membrane potential, Vpkcm is the voltage at which the maximum charge movement is attained, and Clin is the linear capacitance .
Immunostaining and anatomical measurements
Mice were cardiac perfused with 4% paraformaldehyde and cochleae extracted. After post-fixation and decalcification, cochleae were dissected following the Eaton-Peabody Laboratory cochlear dissection protocol . In order to detect prestin, N-terminal prestin rabbit antisera  was used at 1:1000 with goat anti-rabbit Alexa Fluor 488 (Thermo) as the secondary antibody at 1:500. Alexa 546-conjugated phalloidin and Hoechst 33342 (Thermo) were also used to stain actin and nuclei, respectively, as described before . Stained cochlear sections were mounted onto slides using Dako fluorescent mounting medium (Agilent). Images were captured on a Nikon A1R confocal microscope with Plan Fluor 10X and Plan Apo 20X objectives (Nikon) controlled by NIS Element software. Basilar membrane length was measured using ImageJ, and the numbers of remaining OHCs determined. A mouse cochlear place-frequency map  was used to determine the corresponding frequencies.
Plasmids, cell line and cell culture
To generate pIZ-499-prestin-ceGFP, pIZ-gPres-ceGFP that contained full-length gerbil prestin with the C-terminal V5 and GFP tag in the pIZ-V5/His vector (Thermo Fisher)  was mutagenized using QuickChange Site-Directed Mutagenesis Kit (Agilent) following the manufacturer’s instructions. To introduce 499 mutations (V499G/Y501H) , the following primers were used: gPres V499A/Y501H A (5′- CATTGCTCTGCTGACTGGGATCCACAGAACCCAGAGTCC -3′) and gPres V499A/Y501H B (5′- GGACTCTGGGTTCTGTGGATCCCAGTCAGCAGAGCAATG -3′). Sf9 cells (Thermo Fisher) were maintained in Sf-900 III SFM supplemented with 5% fetal bovine serum (Gibco) and 1X antibiotic antimycotic solution (Sigma). To generate stable Sf9 cells expressing 499-prestin protein, Sf9 cells were transfected with pIZ-499-prestin-V5_ceGFP using Effectene (Qiagen), and selected with 1 μg/μl zeocin (Thermo Fisher). A single clone was chosen to establish the stable cell line. Generation of the stable sf9-prestin-ceGFP cell line, which expresses WT prestin, was previously reported .
Cholesterol binding assay
Pre-washed CarboxyLink coupling gel was processed with or without cholesteryl hemisuccinate to prepare cholesterol-beads and unconjugated control-beads for non-specific binding as described before . Cell lysates containing membrane fractions isolated from stable Sf9 cells expressing WT-prestin and 499-prestin were mixed with cholesterol-beads or unconjugated control beads and incubated for 1 h at room temperature. The reaction mix was then centrifuged and washed 5 times with 50 mM Tris-Cl (pH 7.5) buffer containing 50 mM NaCl and 20 mM DDM (n-dodecyl β-D-maltoside), and eluted with 2X Laemmli Sample Buffer (BioRad). Eluates were analyzed by Western blotting as described .
Preservation of prestin expression and motor function in OHCs of NPC1-KO mice
To evaluate prestin’s electromotility, we measured and compared nonlinear capacitance (NLC) of OHCs isolated from WT and NPC1-KO mice. Figures 1d-g summarize the NLC measurements, which provide a signature of prestin’s motor activity [2, 40]. There was no statistically significant difference in the charge density (Fig. 1e) between WT and NPC1-KO. This parameter, charge density (CD, defined as Qmax/ Clin), normalizes the amount of prestin activity to cell size, such that Qmax correlates with the amount of functional prestin expressed in the cell membrane, and Clin is an indication of OHC size. These data are consistent with the staining results, indicating that similar amounts of prestin protein were expressed in adult WT and NPC1-KO OHCs. We did, however, observe significant changes in alpha (Fig. 1f), which indicates voltage sensitivity, as well as a depolarizing shift in Vpkcm (Fig. 1g). These observations are consistent with previous reports showing that cholesterol affects the sensitivity of prestin, and decreasing cholesterol in the membrane shifts Vpkcm in the depolarizing direction [21, 37, 45]. Hence, the fact that OHCs from NPC1-KO mice have a more depolarized Vpkcm suggests that the cholesterol content of the membrane in NPC1-KO mice could be lower than that of OHCs from WT mice . Taken together, lack of NPC1 does not affect prestin expression and membrane targeting. However, prestin’s sensitivity and Vpkcm are altered, which could relate to a reduction in the cholesterol content of the OHC’s plasma membrane in NPC1-KO mice.
Reduced sensitivity and OHC loss in NPC1-KO mice
OHCs from NPC1-KO mice are still sensitive to HPβCD
Since some NPC1-KO mice were resistant to HPβCD-induced OHC loss and reductions in DPOAEs, we increased the HPβCD dosage to a single administration of 8000 mg/kg, which is known to cause OHC death within hours . As shown in Fig. 4a, a single injection of 8000 mg/kg HPβCD (HD for high dose) caused a statistically significant threshold shift in both WT and NPC-KO mice compared to their corresponding untreated groups (Ctrl) (Fig. 4a, p < 0.001). Although this particular NPC1-KO mouse retained slightly more OHCs with 8000 mg/kg HPβCD (Fig. 4b, green) than WT littermates (Fig. 4b, blue), this animal still suffered a vast reduction in the overall numbers of surviving OHCs. Taken together, NPC1-KO mice remain susceptible to HPβCD-induced OHC loss but exhibit large variations in their sensitivity to the low dosage of HPβCD when compared to WT.
Co-administration of salicylate with HPβCD did not mitigate HPβCD-induced loss of sensitivity
HP βCD-induced ototoxicity does not depend on OHC electromotility
NPC1 disease affects the homeostasis of cellular cholesterol, which can have profound effects on cellular functions. Our evaluation of prestin protein expression and function in OHC of NPC1-KO mice was unaffected, except for the slight yet significant depolarization of the voltage operating point (Vpkcm, Fig. 1g). This shift of Vpkcm in the depolarizing direction indicates a decrease in the amount of cholesterol in the OHC’s plasma membrane in NPC1-KO mice, which is plausible considering the altered cellular trafficking of cholesterol  and the high frequency threshold shifts in NPC1-KOs evident at weaning (Fig. 2). Although abnormal accumulation of cholesterol was observed in other cell types including spiral ganglion neurons and cells in the stria vascularis of NPC1-KO cochleae , OHC loss may be the predominant cause of threshold shifts in the basal high-frequency region in NPC1-KO mice (Fig. 3). High variability observed in this region of the cochlea in NPC1-KO mice may underlie individual variations in the progression of disease, as often noted in NPC1 patients .
It is generally understood that HPβCD helps reduce cholesterol accumulation in NPC1 disease by releasing lysosomal cholesterol into the cytoplasm [30, 39, 49]. However, in the cochlea, rapid OHC loss has been observed in animal models in a dose-dependent manner regardless of the mode of HPβCD administration [12, 13]. Since prestin is a lateral membrane protein HPβCD likely acts directly on plasma membrane cholesterol to confer its cytotoxic effect on OHCs. Curiously, some of the NPC1-KO mice receiving low-dose HPβCD (4000 mg/kg × 4) were resistant to the cytotoxic effect (Fig. 4a-b). This resilience may simply be a result of individual variations; however, it may also relate to the reduced plasma membrane cholesterol level in OHCs of NPC1-KO mice, revealed by NLC measurement (Fig. 1d, g). We do not fully understand what factors contribute to the variations observed in our data or the data in other publications. For example, multiple injections of 4000 mg/kg HPβCD did not cause permanent ototoxicity in FVB/NJ mice , while BALB/c mice lost a significant number of OHCs after 4 injections of 4000 mg/kg HPβCD. This information suggests that OHC sensitivity to HPβCD is influenced by strain background, which may also underlie the variations observed in human patients.
As one of the contributors to HPβCD-induced ototoxicity, prestin provides a potential molecular target for ameliorating unwanted side effect of HPβCD. Because salicylate is a small-molecule inhibitor of prestin’s motile function that was shown to have no adverse effect on NPC1-KO mice , we tested both oral and systemic administration protocols. Oral administration of salicylate failed to confer an inhibitory effect on prestin function (Fig. 5a). In contrast, direct systemic injection of salicylate increased ABR and DPOAE thresholds, indicative of prestin inhibition (Fig. 5b). However, it did not mitigate the ototoxic effects of HPβCD in either WT or in NPC1-KO mice. Thus, although potentially attractive, inhibition of prestin’s function by salicylate did not mitigate HPβCD-induced threshold shifts at the doses used in this report.
This conclusion was also corroborated by using 499-prestin-KI mice that express mutated prestin protein with virtually no motile function in vivo. Unlike prestin-KO mice, 499-prestin-KIs were as sensitive to HPβCD as WT (Fig. 6a-c), indicating that somatic electromotility per se is not directly linked to HPβCD susceptibility. The lateral wall of the OHC is highly specialized and consists of a trilaminate structure that includes an actin/spectrin-based cortical lattice and subsurface cisternae, linked to the prestin-embedded plasma membrane to form a distinct functional domain . As an integral component of this trilaminate structure, prestin plays an important structural role . Since prestin can directly interact with cholesterol , it is likely that the prestin-cholesterol interaction contributes to the stability of this specialized membrane domain. As 499-prestin retains its ability to bind cholesterol (Fig. 6d), it is similarly affected by HPβCD treatment as in WT.
Our study provides a detailed characterization of prestin expression and function in OHCs in the context of NPC1 disease. Although potentially promising, our study indicates that specifically targeting prestin did not provide protection of OHCs in response to HPβCD. Future efforts for the treatment of NPC1 disease should include effective drug delivery to avoid cochlear exposure, development of alternative small molecules that are more specific or non-toxic [4, 47], and gene therapy .
OHCs in NPC1-KO mice have normal prestin expression and motor function.
HPβCD-induced ototoxicity is not dependent on prestin’s motile function.
Salicylate, at the doses used in this report, does not mitigate HPβCD-induced ototoxicity in NPC1-KO mice.
Imaging was performed at the Northwestern University’s Center for Advanced Microscopy generously supported by an NCI CCSG P30 CA06553 award to the Robert H Lurie Comprehensive Cancer Center. This work was supported by the Ara Parseghian Medical Research Fund to J.Z. and a Hugh Knowles Leadership Fund Award to J.Z. by the Knowles Hearing Center.
YZ performed drug admission, ABR and DPOAE measurement, and collected OHCs for NLC measurement. ST performed immunostaining and anatomical measurements of WT and NPC-KO mice, analyzed data, and prepared figures. KH performed NLC measurement. CD performed cholesterol binding assay. JZ performed immunostaining and anatomical measurements of 499-KI mice. MAC involved in experimental design. JZ established Sf9 stable cell lines and designed the study. ST, MAC, and JZ wrote the manuscript with input from all other authors. All authors read and approved the final manuscript.
The authors declare that they have no competing interests.
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