Pivotal role of
            <i>Harakiri</i>
            in the induction and prevention of gentamicin-induced hearing loss

Kalinec, Gilda M.; Fernández-Zapico, Martín E.; Urrutia, Raúl; Esteban-Cruciani, Nora; Chen, Shanping; Kalinec, Federico

doi:10.1073/pnas.0508053102

Cited by 64 publications

(55 citation statements)

References 27 publications

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“…To ensure the specificity, multiplicity controls were employed (17). Two Dp5 small hairpin RNAs (shRNAs), shdp5a and shdp5b, targeting the 19-nucleotide AGAGAAAC-GGGATGTCATT (938 -956) or GACGGAGCGTGATTT-CTAA (1386 -1404) in the 3Ј-untranslated region of dp5 mRNA (NCBI accession number NM_057130) were designed (18). The targeted regions showed no significant homology with any other genes by BLAST searches.…”

Section: Methodsmentioning

confidence: 99%

dp5/HRK Is a c-Jun Target Gene and Required for Apoptosis Induced by Potassium Deprivation in Cerebellar Granule Neurons

Ying

Zhang

et al. 2007

Journal of Biological Chemistry

105

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In cerebellar granule neurons, a BH3-only Bcl-2 family member, death protein 5/harakiri, is up-regulated in a JNK-dependent manner during apoptosis induced by potassium deprivation. However, it is not clear whether c-Jun is directly involved in the induction of dp5. Here, we showed that the up-regulation of dp5, but not fas ligand and bim, after potassium deprivation was suppressed by the expression of a dominant negative form of c-Jun. Deletion analysis of the 5-flanking sequence of the dp5 gene revealed that a major responsive element responsible for the induction by potassium deprivation is an ATF binding site located at ؊116 to ؊109 relative to the transcriptional start site. Mutation of this site completely abolished promoter activation. Furthermore, a gel shift assay showed that a specific complex containing c-Jun and ATF2 recognized this site and increased in potassium-deprived cerebellar granule neurons. Chromatin immunoprecipitation demonstrated that c-Jun was able to bind to this site in vivo. Finally, we demonstrated that knockdown of Dp5 by small interfering RNA rescued neurons from potassium deprivation-induced apoptosis. Taken together, these results suggest that dp5 is a target gene of c-Jun and plays a critical role in potassium deprivation-induced apoptosis in cerebellar granule neurons.The Bcl-2 family proteins can be divided into three major subgroups (1). Antiapoptotic proteins, such as Bcl-2, Bcl-X L , and Mcl-1, typically share four conserved motifs termed Bcl-2 homology (BH) 3 domains and inhibit mitochondrial cytochrome c release and apoptosis. Multidomain proapoptotic proteins, the second subgroup, such as Bax, Bak, and Bok, typically have three BH domains but promote cytochrome c release and apoptosis. The third, and the most structurally diverse subgroup, is the BH3-only proteins, including Dp5/HRK (death protein 5/harakiri), Bim (Bcl2-interacting mediator of cell death), Bid, Bad, Puma, and Noxa, which share the BH3 domain. The BH3-only proteins are critical initiators of apoptosis. Upon challenge, BH3-only proteins translocate to mitochondria and promote the chromec release by neutralizing the antiapoptotic action of Bcl-2 family members. BH3-only proteins are stringently regulated at the transcriptional and post-translational levels during apoptosis, such as Dp5, Bim, and Puma, depending on the cell type and apoptotic stimulus (2-6). Among the BH3-only proteins, Dp5 is of particular interest to studies of apoptosis in the nervous system. In rodents, the expression of Dp5 is largely restricted to and is developmentally regulated in the nervous system (2, 7). Dp5 is the first found BH3-only protein to be induced by NGF deprivation in sympathetic neurons (2). dp5 is highly homologous to the human gene harakiri (HRK) cloned by a two-hybrid screen with Bcl-2 and Bcl-X L (3). As well as being induced in NGF-deprived sympathetic neurons, the induction of dp5 is also observed in cerebellar granule neurons (CGNs) deprived of potassium, cortical neurons exposed to toxic concentrations of amyl...

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Section: Methodsmentioning

confidence: 99%

dp5/HRK Is a c-Jun Target Gene and Required for Apoptosis Induced by Potassium Deprivation in Cerebellar Granule Neurons

Ying

Zhang

et al. 2007

Journal of Biological Chemistry

105

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“…2C. a M. smegmatis wild-type rRNA loss, which is initiated by mitoribosomal misreading, subsequently via activation of downstream signaling pathways, such as MAPK and JNK (44,45), misreading results in hair cell death through apoptosis.…”

Section: Table 2 Aminoglycoside-induced Inhibition Of Aug(uuu)12-drimentioning

confidence: 99%

Genetic analysis of interactions with eukaryotic rRNA identify the mitoribosome as target in aminoglycoside ototoxicity

Hobbie

Subramanian

Kalapala

et al. 2008

Proc. Natl. Acad. Sci. U.S.A.

164

180

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Aminoglycoside ototoxicity has been related to a surprisingly large number of cellular structures and metabolic pathways. The finding that patients with mutations in mitochondrial rRNA are hypersusceptible to aminoglycoside-induced hearing loss has indicated a possible role for mitochondrial protein synthesis. To study the molecular interaction of aminoglycosides with eukaryotic ribosomes, we made use of the observation that the drug binding site is a distinct domain defined by the small subunit rRNA, and investigated drug susceptibility of bacterial hybrid ribosomes carrying various alleles of the eukaryotic decoding site. Compared to hybrid ribosomes with the A site of human cytosolic ribosomes, susceptibility of mitochondrial hybrid ribosomes to various aminoglycosides correlated with the relative cochleotoxicity of these drugs. Sequence alterations that correspond to the mitochondrial deafness mutations A1555G and C1494T increased drug-binding and rendered the ribosomal decoding site hypersusceptible to aminoglycoside-induced mistranslation and inhibition of protein synthesis. Our results provide experimental support for aminoglycoside-induced dysfunction of the mitochondrial ribosome. We propose a pathogenic mechanism in which interference of aminoglycosides with mitochondrial protein synthesis exacerbates the drugs' cochlear toxicity, playing a key role in sporadic dosedependent and genetically inherited, aminoglycoside-induced deafness.decoding ͉ mitochondria ͉ ribosomes ͉ toxicity ͉ translation

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“…The mechanisms involved in aminoglycoside ototoxicity have been a matter of debate (17,(20)(21)(22)(23)(24), but compelling evidence now suggests a causal involvement of reactive oxygen species (ROS) (25,26). The origin of ROS generation remains unresolved, however, and both nonenzymatic and enzymatic mechanisms have been suggested (27).…”

mentioning

confidence: 99%

Dissociation of antibacterial activity and aminoglycoside ototoxicity in the 4-monosubstituted 2-deoxystreptamine apramycin

Matt

Lang

et al. 2012

Proc. Natl. Acad. Sci. U.S.A.

182

259

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Aminoglycosides are potent antibacterials, but therapy is compromised by substantial toxicity causing, in particular, irreversible hearing loss. Aminoglycoside ototoxicity occurs both in a sporadic dose-dependent and in a genetically predisposed fashion. We recently have developed a mechanistic concept that postulates a key role for the mitochondrial ribosome (mitoribosome) in aminoglycoside ototoxicity. We now report on the surprising finding that apramycin, a structurally unique aminoglycoside licensed for veterinary use, shows little activity toward eukaryotic ribosomes, including hybrid ribosomes which were genetically engineered to carry the mitoribosomal aminoglycoside-susceptibility A1555G allele. In ex vivo cultures of cochlear explants and in the in vivo guinea pig model of chronic ototoxicity, apramycin causes only little hair cell damage and hearing loss but it is a potent antibacterial with good activity against a range of clinical pathogens, including multidrug-resistant Mycobacterium tuberculosis. These data provide proof of concept that antibacterial activity can be dissected from aminoglycoside ototoxicity. Together with 3D structures of apramycin-ribosome complexes at 3.5-Å resolution, our results provide a conceptual framework for further development of less toxic aminoglycosides by hypothesis-driven chemical synthesis.antibiotic | translation | misreading | deafness | mycobacteria

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Pivotal role of Harakiri in the induction and prevention of gentamicin-induced hearing loss

Cited by 64 publications

References 27 publications

dp5/HRK Is a c-Jun Target Gene and Required for Apoptosis Induced by Potassium Deprivation in Cerebellar Granule Neurons

dp5/HRK Is a c-Jun Target Gene and Required for Apoptosis Induced by Potassium Deprivation in Cerebellar Granule Neurons

Genetic analysis of interactions with eukaryotic rRNA identify the mitoribosome as target in aminoglycoside ototoxicity

Dissociation of antibacterial activity and aminoglycoside ototoxicity in the 4-monosubstituted 2-deoxystreptamine apramycin

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