It is not often that a series of publications from a single laboratory illustrates the path from intriguing cellular responses in vitro to functional auditory preservation against widely used, clinically relevant agents in vivo. Yet data published in recent years by Lisa Cunningham, PhD, and colleagues used preclinical models of ototoxicity to do just that.
Bactericidal drugs like aminoglycosides (e.g., kanamycin) and anticancer drugs like cisplatin induce acquired, permanent hearing loss, primarily by killing the sensory hair cells within the cochlea. Preventing the loss of cochlear and vestibular hair cells is critical to maintaining auditory function and quality of life in patients treated with these lifesaving drugs.
While with the Medical University of South Carolina in 2006, Dr. Cunningham reported that the exposure of inner-ear (utricular) explants to heat (43°C for 30 minutes) induced the expression of heat shock proteins (HSPs; J Assoc Res Otolaryngol 2006;7:299-307 http://link.springer.com/article/10.1007%2Fs10162-006-0043-x/fulltext.html), a family of highly conserved molecular chaperones that are ubiquitously upregulated following cellular stress.
Heat shock proteins assist in protein folding for proper function, prevent unwanted protein aggregation during shock, aid in protein transport across membranes, and can mark proteins for degradation. Preconditioning with heat to induce expression of HSPs decreased the susceptibility of explanted utricles to aminoglycosides and cisplatin ( J Assoc Res Otolaryngol 2006;7:299-307 http://link.springer.com/article/10.1007%2Fs10162-006-0043-x/fulltext.html).
A subsequent in vitro study published in 2008 revealed that heat-induced expression of Hsp70 protected utricular hair cells against aminoglycoside-induced cytotoxicity ( J Assoc Res Otolaryngol 2008;9:277-289 http://link.springer.com/article/10.1007%2Fs10162-008-0122-2/fulltext.html).
Heat shock only protected hair cells in utricular explants that came from wild-type mice, not from mutant mice lacking Hsp70 expression. This finding was corroborated when utricular explants from mice constitutively overexpressing Hsp70 were protected against aminoglycoside exposure in the absence of heat shock, unlike wild-type explants ( J Assoc Res Otolaryngol 2008;9:277-289 http://link.springer.com/article/10.1007%2Fs10162-008-0122-2/fulltext.html).
These in vitro studies were then verified in vivo by the finding that mice constitutively overexpressing Hsp70 were also protected against kanamycin-induced cochlear hair cell death (cochleotoxicity) and hearing loss ( Cell Stress Chaperones 2009;14:427-437 http://link.springer.com/article/10.1007%2Fs12192-008-0097-2). A well-established murine ototoxicity protocol was used ( Hear Res 2001;158[1-2]:165-178 http://www.sciencedirect.com/science/article/pii/S0378595501003033).
The studies provided substantial evidence for thermal preconditioning to induce a cellular response that protects inner-ear hair cells against clinically relevant ototoxic drugs.
STRATEGIES FOR PRECONDITIONING
Thermal preconditioning is one strategy to induce otoprotection, but this approach, in vivo, suffers from poor targeting or is poorly tolerated.
Therefore, to investigate as proof of principle whether an otoprotective cellular response could be initiated pharmacologically, celastrol, a known antioxidant and anti-inflammatory compound that induces transcription of HSPs, was tested ( Cell Death Dis 2011;2:e195 http://www.nature.com/cddis/journal/v2/n8/full/cddis201176a.html).
Celastrol induced mRNA and protein expression of Hsp70 and Hsp32 in explanted utricles, and it produced partial protection against kanamycin-induced hearing loss and cochlear hair cell loss in mice ( Cell Death Dis 2011;2:e195 http://www.nature.com/cddis/journal/v2/n8/full/cddis201176a.html).
While celastrol has a long history in traditional Chinese medicine and is well tolerated, the safety and efficacy of the compound for exploitation by pharmaceutical companies remain unknown, substantially delaying any potential translation into clinical practice for otoprotection.
Otoprotective preconditioning strategies have been proposed previously. These approaches primarily have used sound to protect against noise trauma ( Hear Res 2000;148[1-2]:213-219 http://www.sciencedirect.com/science/article/pii/S0378595500001611; Hear Res 1995;84[1-2]:112-124 http://www.sciencedirect.com/science/article/pii/0378595595000205) or age-related hearing loss ( Laryngoscope 2009;119:1374-1379 http://onlinelibrary.wiley.com/doi/10.1002/lary.20244/abstract?systemMessage=Wiley+Online+Library+will+be+disrupted+Saturday%2C+15+March+from+10%3A00-12%3A00+GMT+%2806%3A00-08%3A00+EDT%29+for+essential+maintenance), or, remarkably, kanamycin to protect against noise-induced hearing loss ( J Assoc Res Otolaryngol 2010;11:235-244 http://link.springer.com/article/10.1007/s10162-009-0204-9/fulltext.html).
Sound Preconditioning Therapy Inhibits Ototoxic Hearing Loss in Mice
Roy S, Ryals MM, Van den Bruele AB, Fitzgerald TS, Cunningham LL
J Clin Invest http://www.jci.org/articles/view/71353
In the first paper selected for this month's Journal Club, Dr. Cunningham's group, now at the National Institute on Deafness and Other Communication Disorders in Rockville, MD, used preconditioning sound therapy to replicate their earlier data and provide a translational proof of principle.
Preconditioning sound therapy (8- to 16-kHz octave band at 90 dB SPL for two hours) induced temporary threshold shifts of 9 dB to 22 dB that returned to preexposure auditory thresholds within one week.
Coincident with these temporary threshold shifts, the sound therapy also upregulated the mRNA expression of HSPs, particularly Hsp32 and Hsp70, in the cochlea only.
Having verified the localized effect of this preconditioning protocol to induce HSP expression only in the cochlea, the researchers then successfully developed a novel protocol to evoke cisplatin-induced ototoxicity in a reproducible way.
Previously published models of cisplatin-induced ototoxicity have been limited to rapid experimental endpoints. The protocol reported in this paper extends through three rounds of daily cisplatin dosing for four days followed by recovery periods, similar to clinical chemotherapy paradigms.
Sound preconditioning occurred prior to and once during the dosing periods, as well as during the recovery periods.
Two weeks after the final dose of cisplatin, auditory brainstem response threshold shifts were significantly and substantially reduced in the cisplatin-plus-sound therapy group compared with the cisplatin-alone group. In addition, sound preconditioning reduced hair cell loss in the middle and upper basal turns of the murine cochlea.
The preconditioning strategy was modified for aminoglycoside-induced cochleotoxicity, and reductions in drug-induced threshold shifts and hair cell loss were also observed. Translation of these preclinical findings into clinical trials is now eagerly awaited.
Although an understanding of the biological pathways at play in a clinically viable strategy is not necessarily a prerequisite for a clinical trial, Dr. Cunningham's group has partially uncovered some of the mechanisms involved in HSP protection of hair cells during ototoxic drug treatment.
Inner-Ear Supporting Cells Protect Hair Cells by Secreting Hsp70
May LA, Kramarenko II, Brandon CS, et al
J Clin Invest http://www.jci.org/articles/view/68480
The second paper highlighted in this month's Journal Club demonstrates that the supporting cells within the organ of Corti release Hsp70, protecting the adjacent hair cells against aminoglycoside-induced hair cell death.
Heat shock induced Hsp70 expression in supporting cells but not in hair cells. In addition, adenoviral infection of supporting cells with Hsp70 vectors induced Hsp70 expression that protected hair cells from aminoglycoside-induced death without heat shock.
An elegant series of experiments (see figure) then demonstrated that coculture of heat-shocked utricular explants expressing Hsp70 protects hair cells in non-heat-shocked utricular explants from aminoglycoside-induced hair cell death.
This finding suggested that supporting cells’ secretion of Hsp70 was responsible for the protective effect in the hair cells of non-heat-shocked utricles. The interpretation was verified when heat-shocked wild-type utricles also protected hair cells in utricular explants from Hsp70-knockout mice.
In the reverse experiment, heat-shocked utricular explants from Hsp70 knockout mice were unable to protect hair cells in cocultured utricular explants from wild-type mice that had not been heat shocked.
Additional experiments also revealed that extracellular Hsp70 is crucial for hair cell survival during aminoglycoside exposure in non-heat-shocked utricular explants.
These data demonstrate the importance of supporting cells for hair cell survival during cochlear stress. As the authors point out, researchers have only recently begun to explore how integral supporting cells are to maintaining hair cell populations during aminoglycoside challenge.
Unravelling these mechanisms promises to reveal more intricate means of cooperation and codependency between cochlear hair cells and supporting cells to ensure optimal auditory perception and exquisite cochlear function.