student research day - Case Western Reserve University School of ...

Aaron Lindsay
Inner Ear Protein Networks and Biomarkers in a mouse model for deafness in Usher Syndrome 1F and DFNB23.
Aaron Lindsay, Mark Chance, Shuqing Liu, Daniel H.-C. Chen, Qing Y. Zheng, Kumar Alagramam
Center for Proteomics and Mass Spectrometry, Department of Otolaryngology
Introduction: This study identifies candidate biomarkers of inner ear pathogenesis in the Ames waltzer (av) mouse, a model
for deafness in Usher syndrome 1F (USH1F). Additionally, candidate protein networks active in the degeneration process
are identified with the aid of mapping software. Methods: Cochleae from av and normal mice at postnatal day 30, a time
point were cochlear pathology is well established in the av model, were dissected and homogenized in ice-cold lysis buffer.
The protein was extracted, labeled by Cydye, prefractionated by 2-D DIGE, and analyzed by mass spectrometry. DeCyder
software 6.5 was used to quantify protein spots and Metacore software was used to predict protein interactions and
establish statistically significant candidate networks. To validate the proteomics analysis, two protein products (p53 and
caspase-3) were further analyzed by qPCR and enzyme activity assays, respectively. Results: 43 proteins were found to be
up-regulated and 26 down-regulated in the cochleae of av mice compared to controls. Cochlin was identified in 20 peptide
spots of both increased and decreased expression. 7 statistically significant candidate protein networks that are predicted
to be active in USH1F were identified. Some key proteins predicted to be up-regulated in the networks include c-JUN, NFkB,
p53, and caspase-3. Enzyme activity assays for caspase-3 and qPCR analysis for p53 confirmed increased activity and
transcript levels, respectively, in av cochleae compared to controls. Conclusions: This study identifies candidate biomarkers
and protein networks of inner ear pathogenesis in an USH1F model. Cochlin, which has been associated with other forms of
hearing loss, was found to be both up-regulated and down-regulated in the USH1F model. Factors known to be associated
with apoptosis and degeneration, including c-JUN, p53, and caspase-3, are predicted to be active in the degeneration of hair
cells and spiral ganglion cells in the USH1F model.
This work was supported by a grant from Rainbow Babies and Children’s Hospital, University Hospitals Case
Medical Center, Cleveland, Ohio 44106.
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