Here we study links between aminoglycoside-induced mistranslation, protein misfolding and neuropathy. suggesting a high selective pressure during evolution to minimize errors in protein synthesis.1 In bacteria, erroneous protein synthesis induces protein misfolding.2 In higher eukaryotes, protein misfolding results in endoplasmatic reticulum (ER) stress and initiates the unfolded protein response (UPR), a cascade of integrated pathways regulating gene expression. The UPRER is usually mediated by three ubiquitously expressed transmembrane protein in the ER: inositol-requiring enzyme 1 (IRE1), PKR-like ER kinase (PERK) and activating transcription factor 6 (ATF6).3, 4, 5, 6, 7 Under normal conditions, the luminal domains of IRE1, PERK and ATF6 are bound by the ER chaperone-binding immunoglobulin protein (BiP), which inhibits self-dimerization and activation of the cytosolic domain name.8, 9 Under ER stress, BiP is released resulting in dimerization of IRE1 and ATF6 and oligomerization of PERK, 849217-68-1 manufacture initiating the UPR signaling cascades.8, 9 The initial UPR response is protective, increasing the expression of chaperone proteins promoting refolding and, if unsuccessful, the degradation of misfolded proteins.10, 11, 12, 13 Prolonged or severe stress triggers additional pathways that eventually lead to cellular apoptosis.14, 15, 16 Aminoglycoside antibiotics are well known to affect translational fidelity in bacteria and lower eukaryotes17, 18, 19, 20 but only few reports suggest that aminoglycoside antibiotics may also induce misreading in higher eukaryotes.21, 22, 23 Aminoglycoside-mediated readthrough activity has been exploited for therapy of human genetic diseases associated with premature stop codons.24, 25, 26, 27 In addition, aminoglycosides have been shown to induce apoptosis in human cell cultures, accompanied by ER stress and mitochondrial cytochrome c release.28, 29 It was suggested that the observed ER stress could be the result of protein misfolding, reflecting aminoglycoside-induced mistranslation.28 Despite this potential for misreading induced by aminoglycosides in eukaryotes, aminoglycoside treatment in experimental animals and in patients is well tolerated. Side effects are highly organ specific, limited to the kidney and the inner ear,30 while toxicity to the nervous system is usually not evident even in 849217-68-1 manufacture long-term aminoglycoside administration.31 In the case of ototoxicity, the primary drug target are the sensory hair cells, as convincingly demonstrated in various animal models, regardless of whether the drug is given systemically32 or directly introduced into the cochlea.33 Degeneration of spiral ganglion cells (SGCs) observed after ototoxic 849217-68-1 manufacture dosages of aminoglycosides are thought to occur only as a sequel to the loss of sensory hair cells in the vast majority of cases. Surprisingly, however, a few analyses of human temporal bones have suggested that spiral ganglia can be affected by aminoglycosides without overt insult to the hair cells.34, 35 This rare pathology, unexplained by the treatment modus, suggests individual variability possibly based on genetic factors. Prompted by the anecdotal reports of aminoglycoside-induced selective spiral ganglion damage and the potential of aminoglycosides to induce mistranslation, the objective of this study was to assess the contribution of ER stress to ototoxicity. We first investigated aminoglycoside-induced misreading and UPR responses in HEK293 cells mouse model36 with a compromised ER stress response because of X-box binding protein-1 (XBP1) haploinsufficiency37 in order to probe potential links between aminoglycoside neurotoxicity, translation fidelity and protein misfolding. Results Aminoglycosides alter translation fidelity Drug-induced inhibition of translation was used to assess aminoglycoside Rabbit polyclonal to HPX activity on the eukaryotic ribosome. IC50 values were 0.3?-untreated cells revealed a broad transcriptional response totaling 705 genes (selected for a fold change >1.2, BenjaminiCHochberg corrected were examined from base-to-apex 3 849217-68-1 manufacture weeks after drug injection. OHCs were present in all parts of the cochlea in both wild-type and XBP1+/? mice except for some scattered loss at the very end of the basal turn (Supplementary Physique S5c). Quantitation of hair cell loss along the entire cochlea confirmed only minor damage at the extreme, the basal turn with no difference between wild-type and XBP1+/? mice. In the absence of any discernible defects on hair cell honesty and prompted by 849217-68-1 manufacture the results, we then analyzed spiral ganglion density and synaptic connections. Three weeks after gentamicin injection, the SGCs were counted on mid-modiolar cryosections stained.