Graduate Thesis Or Dissertation

 

Characterizing the Functional Properties of Otoferlin, Essential for Neurotransmission in Inner Hair Cells of the Cochlea Public Deposited

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  • Hearing loss is one of the most common defect, affecting 360 million people worldwide due to several factors including congenital, present at or soon after birth or acquired with age. Congenital hearing loss affects 32 million children in the world. The economic impact of hearing loss is estimated to cost society an average of 300,000 dollars over the lifetime of a person. It also has serious impacts on quality of life including the literacy rate in children. Although, hearing loss is clearly a major health problem its genetic basis for the pathology is poorly understood. To date, over 60 pathogenic mutations in otoferlin have been found to be associated with inherited, non-syndromic congenital hearing loss and temperature sensitive auditory neuropathy (TSAN). The sense of hearing depends on reliable and temporally precise neurotransmitter release at the synapses of inner hair cells (IHCs) of the cochlea. Inner hair cells of the cochlea derive their name from hair bundle (stereocilia) protruding at the apical tip of the cell, that are arranged in rows of graded height. The nanometer displacement of the hair bundle opens mechanically gated ion channels that depolarize the cell. This change in membrane potential triggers calcium dependent fusion of synaptic vesicles with the plasma membrane and the release of neurotransmitter. In IHCs, Calcium-regulated exocytosis and neurotransmitter release exhibit fast kinetics in achieving exquisite temporal fidelity. To aid in fidelity and for allowing high rates of sustained synaptic neurotransmission, IHCs contain specialized structures called synaptic ribbons for tethering synaptic vesicles at release sites. The calcium triggered synaptic vesicle fusion with the plasma membrane is believed to be driven by the assembly of SNARE proteins. However, SNARE proteins are insensitive to calcium. In conventional neurotransmission in neurons, synaptotagmin 1 confers calcium sensitivity to SNARE-mediated fast synchronous neurotransmission. However, Yasunaga et al. 1999 and Beurg et al. 2010 have reported that synaptotagmin 1 was not detected in mature IHCs and it has been suggested that IHCs have evolved a unique calcium sensor, otoferlin for calcium-regulated synaptic neurotransmission. The evidence for calcium sensor hypothesis of otoferlin comes from Roux et al., 2006 who reported that mice lacking otoferlin were profoundly deaf and lack synaptic vesicle exocytosis in IHCs. Otoferlin has also been shown to be required for calcium dependent synaptic exocytosis at immature outer hair cells (OHCs) and vestibular hair cells. Otoferlin belongs to ferlin family of proteins and consists of six C2 domains (C2A- C2F) linked in tandem followed by a single-pass C-terminal transmembrane region. C2 domains are known to bind to calcium and lipids. The lipid and calcium binding properties of synaptic proteins are critical characteristics that define and shape the release properties of a synapse, and thus, without a quantitative characterization of these activities, an understanding of otoferlin’s function in hair cells will remain elusive. This dissertation addresses the intrinsic calcium binding properties of each C2 domain of otoferlin and also the lipid binding specificity and the effects of lipids on calcium binding were also assessed using liposome sedimentation assays and laurdan fluorescence measurements. On the basis of our results, we proposed that the C2C and C2F domains of otoferlin preferentially bind PI(4,5)P2 and that PI(4,5)P2 may serve to target otoferlin to the presynapse in a calcium-independent manner. This positioning would facilitate fast calcium-dependent exocytosis at the hair cell synapse. Many C2 domains involved in exocytosis also tilt, orient and partially insert into membranes to aid facilitating membrane fusion. To determine the orientation and structural characterization of the C2F domain at the membrane interface, we used label free sum frequency generation vibrational spectroscopy. We found that the C2F domain of the otoferlin interact with membranes DPPC:DPPS and orients 32° normal to the membrane. Our results represent the first structural view of any C2 domain of otoferlin docked at the membrane interface. In my dissertation, I also characterized the temperature sensitive mutants of otoferlin. Deletion of glutamic acid at position 1804 in otoferlin results in a temperature-sensitive mutant in the C2F domain (C2F-TS) that causes temporary deafness in febrile patients. our results using CD-spectroscopy, tryptophan fluorescence and urea denaturation methods, we found that C2F-TS may have minor structural changes localized in the active site.
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  • 2017-08-09 to 2019-02-15

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