Bachelor of Arts, Northwestern University, Genetics and Molecular Biology
Doctor of Philosophy, Universitat Hamburg (2014)
Sound detection happens in the inner ear via the mechanical deflection of the hair bundle of cochlear hair cells. The hair bundle is an apical specialization consisting of actin-filled membrane protrusions (called stereocilia) connected by tip links (TLs) that transfer the deflection force to gate the mechanotransduction channels. Here, we identified the hearing loss-associated Loxhd1/DFNB77 gene as being required for the mechanotransduction process. LOXHD1 consists of 15 polycystin lipoxygenase alpha-toxin (PLAT) repeats, which in other proteins can bind lipids and proteins. LOXHD1 was distributed along the length of the stereocilia. Two LOXHD1 mouse models with mutations in the 10th PLAT repeat exhibited mechanotransduction defects (in both sexes). While mechanotransduction currents in mutant inner hair cells (IHCs) were similar to wild-type (WT) levels in the first postnatal week, they were severely affected by postnatal day 11. The onset of the MET phenotype was consistent with the temporal progression of postnatal LOXHD1 expression/localization in the hair bundle. The mechanotransduction defect observed in Loxhd1-mutant IHCs was not accompanied by a morphological defect of the hair bundle or a reduction in TL number. Using immunolocalization, we found that two proteins of the upper and lower TL protein complexes (Harmonin and LHFPL5) were maintained in the mutants, suggesting that the mechanotransduction machinery was present but not activatable. This work identified a novel LOXHD1-dependent step in hair bundle development that is critical for mechanotransduction in mature hair cells as well as for normal hearing function in mice and humans.SIGNIFICANCE STATEMENT:Hair cells detect sound-induced forces via the hair bundle, which consists of membrane protrusions connected by tip links. The mechanotransduction machinery forms protein complexes at the tip-link ends. The current study showed that LOXHD1, a multi-repeat protein responsible for hearing loss in humans and mice when mutated, was required for hair-cell mechanotransduction, but only after the first postnatal week. Using immunochemistry, we demonstrated that this defect was not caused by the mislocalization of the tip-link complex proteins Harmonin or LHFPL5, suggesting that the mechanotransduction protein complexes were maintained. This work identified a new step in hair bundle development, which is critical for both hair-cell mechanotransduction and hearing.
View details for DOI 10.1523/JNEUROSCI.0975-20.2021
View details for PubMedID 33707295
The RNA-binding protein TDP-43 is heavily implicated in neurodegenerative disease. Numerous patient mutations in TARDBP, the gene encoding TDP-43, combined with data from animal and cell-based models, imply that altered RNA regulation by TDP-43 causes Amyotrophic Lateral Sclerosis and Frontotemporal Dementia. However, underlying mechanisms remain unresolved. Increased cytoplasmic TDP-43 levels in diseased neurons suggest a possible role in this cellular compartment. Here, we examined the impact on translation of overexpressing human TDP-43 and the TDP-43A315T patient mutant protein in motor neuron-like cells and primary cultures of cortical neurons. In motor-neuron like cells, TDP-43 associates with ribosomes without significantly affecting global translation. However, ribosome profiling and additional assays revealed enhanced translation and direct binding of Camta1, Mig12, and Dennd4a mRNAs. Overexpressing either wild-type TDP-43 or TDP-43A315T stimulated translation of Camta1 and Mig12 mRNAs via their 5'UTRs and increased CAMTA1 and MIG12 protein levels. In contrast, translational enhancement of Dennd4a mRNA required a specific 3'UTR region and was specifically observed with the TDP-43A315T patient mutant allele. Our data reveal that TDP-43 can function as an mRNA-specific translational enhancer. Moreover, since CAMTA1 and DENND4A are linked to neurodegeneration, they suggest that this function could contribute to disease.
View details for PubMedID 30357366
Cardiac allograft vasculopathy (CAV) affects approximately 30% of cardiac transplant patients at 5 years post-transplantation. To date, there are few CAV treatment or prevention options, none of which are highly effective. The aim of the study was to investigate the effect of thalidomide on the development of CAV. The effect of thalidomide treatment on chronic rejection was assessed in rat orthotopic aortic transplants in allogeneic F344 or syngeneic Lew rats (n = 6 per group). Animals were left untreated or received thalidomide for 30 days post-transplant, and evidence of graft CAV was determined by histology (trichrome and immunohistochemistry) and intragraft cytokine measurements. Animals that received thalidomide treatment post-transplant showed markedly reduced luminal obliteration, with concomitant rescue of smooth muscle cells (SMCs) in the aortic media of grafts. Thalidomide counteracted neointimal hyperplasia by preventing dedifferentiation of vascular SMCs. Measurement of intragraft cytokine levels after thalidomide treatment revealed downregulation of matrix metalloproteinase 8 and monocyte chemotactic protein 1, cytokines involved in tissue remodelling and inflammation, respectively. Importantly, no negative side effects of thalidomide were observed. Thalidomide treatment prevents CAV development in a rodent model and is therefore potentially useful in clinical applications to prevent post-transplant heart rejection.
View details for PubMedID 28672061
During cap-dependent eukaryotic translation initiation, ribosomes scan messenger RNA from the 5' end to the first AUG start codon with favourable sequence context. For many mRNAs this AUG belongs to a short upstream open reading frame (uORF), and translation of the main downstream ORF requires re-initiation, an incompletely understood process. Re-initiation is thought to involve the same factors as standard initiation. It is unknown whether any factors specifically affect translation re-initiation without affecting standard cap-dependent translation. Here we uncover the non-canonical initiation factors density regulated protein (DENR) and multiple copies in T-cell lymphoma-1 (MCT-1; also called MCTS1 in humans) as the first selective regulators of eukaryotic re-initiation. mRNAs containing upstream ORFs with strong Kozak sequences selectively require DENR-MCT-1 for their proper translation, yielding a novel class of mRNAs that can be co-regulated and that is enriched for regulatory proteins such as oncogenic kinases. Collectively, our data reveal that cells have a previously unappreciated translational control system with a key role in supporting proliferation and tissue growth.
View details for DOI 10.1038/nature13401
View details for PubMedID 25043021
The central importance of translational control by post-translational modification has spurred major interest in regulatory pathways that control translation. One such pathway uniquely adds hypusine to eukaryotic initiation factor 5A (eIF5A), and thereby affects protein synthesis and, subsequently, cellular proliferation through an unknown mechanism. Using a novel conditional knockout mouse model and a Caenorhabditis elegans knockout model, we found an evolutionarily conserved role for the DOHH-mediated second step of hypusine synthesis in early embryonic development. At the cellular level, we observed reduced proliferation and induction of senescence in 3T3 Dohh-/- cells as well as reduced capability for malignant transformation. Furthermore, mass spectrometry showed that deletion of DOHH results in an unexpected complete loss of hypusine modification. Our results provide new biological insight into the physiological roles of the second step of the hypusination of eIF5A. Moreover, the conditional mouse model presented here provides a powerful tool for manipulating hypusine modification in a temporal and spatial manner, to analyse both how this unique modification normally functions in vivo as well as how it contributes to different pathological conditions.
View details for DOI 10.1242/dmm.014449
View details for PubMedID 24832488
Dramatic structural changes in microtubules (MT) and the assembly of complicated intercellular connections are seen during the development of the cellular matrix of the sense organ for hearing, the organ of Corti. This report examines the expression of marshalin, a minus-end binding protein, during this process of cochlear development. We discovered that marshalin is abundantly expressed in both sensory hair cells and supporting cells. In the adult, prominent marshalin expression is observed in the cuticular plates of hair cells and in the noncentrosomal MT organization centers (MTOC) of Deiters' and pillar cells. Based upon differences in marshalin expression patterns seen in the organ of Corti, we identified eight isoforms ranging from 863 to 1280 amino acids. mRNAs/proteins associated with marshalin's isoforms are detected at different times during development. These isoforms carry various protein-protein interacting domains, including coiled-coil (CC), calponin homology (CH), proline-rich (PR), and MT-binding domains, referred to as CKK. We, therefore, examined membranous organelles and structural changes in the cytoskeleton induced by expressing two of these marshalin isoforms in vitro. Long forms containing CC and PR domains induce thick, spindle-shaped bundles, whereas short isoforms lacking CC and PR induce more slender variants that develop into densely woven networks. Together, these data suggest that marshalin is closely associated with noncentrosomal MTOCs, and may be involved in MT bundle formation in supporting cells. As a scaffolding protein with multiple isoforms, marshalin is capable of modifying cytoskeletal networks, and consequently organelle positioning, through interactions with various protein partners present in different cells.
View details for PubMedID 24244856
View details for PubMedCentralID PMC3828766
We report on a secreted protein found in mammalian cochlear outer hair cells (OHC) that is a member of the carcinoembryonic antigen-related cell adhesion molecule (CEACAM) family of adhesion proteins. Ceacam16 mRNA is expressed in OHC, and its protein product localizes to the tips of the tallest stereocilia and the tectorial membrane (TM). This specific localization suggests a role in maintaining the integrity of the TM as well as in the connection between the OHC stereocilia and TM, a linkage essential for mechanical amplification. In agreement with this role, CEACAM16 colocalizes and coimmunoprecipitates with the TM protein ?-tectorin. In addition, we show that mutation of CEACAM16 leads to autosomal dominant nonsyndromic deafness (ADNSHL) at the autosomal dominant hearing loss (DFNA4) locus. In aggregate, these data identify CEACAM16 as an ?-tectorin-interacting protein that concentrates at the point of attachment of the TM to the stereocilia and, when mutated, results in ADNSHL at the DFNA4 locus.
View details for DOI 10.1073/pnas.1005842108
View details for Web of Science ID 000288120400074
View details for PubMedID 21368133
Prestin is the motor protein responsible for cochlear outer hair cell (OHC) somatic electromotility. Eliminating this abundant basolateral membrane protein not only causes loss of frequency selectivity and hearing sensitivity, but also leads to OHC death. A membrane-based yeast two-hybrid approach was used to screen an OHC-enriched cDNA (complementary Deoxyribonucleic Acid) library in order to identify prestin-associated proteins. Several proteins were recognized as potential prestin partners, including vesicle-associated membrane protein associated protein A (VAPA or VAP-33). VAPA is an integral membrane protein that plays an important role in membrane trafficking, endoplasmic reticulum homeostasis, and the stress-signaling system. The connection between VAPA and prestin was confirmed through co-immunoprecipitation experiments. This new finding prompted the investigation of the interaction between VAPA and prestin in outer hair cells. By comparing VAPA expression between wild-type OHCs and OHCs derived from prestin-knockout mice, we found that VAPA is expressed in OHCs and the quantity of VAPA expressed is related to the presence of prestin. In other words, less VAPA protein is found in OHCs lacking prestin. Thus, prestin appears to modify the expression of VAPA protein in OHCs. Intriguingly, more prestin protein appears at the plasma membrane when VAPA is co-expressed with prestin. These data suggest that VAPA could be involved in prestin's transportation inside OHCs and may facilitate the targeting of this abundant OHC protein to the plasma membrane.
View details for DOI 10.1016/j.bbamcr.2010.03.017
View details for Web of Science ID 000279093300003
View details for PubMedID 20359505
Cystic fibrosis transmembrane conductance regulator (CFTR) is a cAMP-activated chloride channel that is present in a variety of epithelial cell types, and usually expressed in the luminal membrane. In contrast, prestin (SLC26A5) is a voltage-dependent motor protein, which is present in the basolateral membrane of cochlear outer hair cells (OHCs), and plays an important role in the frequency selectivity and sensitivity of mammalian hearing. By using in situ hybridization and immunofluorescence, we found that both mRNA and protein of CFTR are present in OHCs, and that CFTR localizes in both the apical and the lateral membranes. CFTR was not detected in the lateral membrane of inner hair cells (IHCs) or in that of OHCs derived from prestin-knockout mice, i.e., in instances where prestin is not expressed. These results suggest that prestin may interact physically with CFTR in the lateral membrane of OHCs. Immunoprecipitation experiments confirmed a prestin-CFTR interaction. Because chloride is important for prestin function and for the efferent-mediated inhibition of cochlear output, the prestin-directed localization of CFTR to the lateral membrane of OHCs has a potential physiological significance. Aside from its role as a chloride channel, CFTR is known as a regulator of multiple protein functions, including those of the solute carrier family 26 (SLC26). Because prestin is in the SLC26 family, several members of which interact with CFTR, we explored the potential modulatory relationship associated with a direct, physical interaction between prestin and CFTR. Electrophysiological experiments demonstrated that cAMP-activated CFTR is capable of enhancing voltage-dependent charge displacement, a signature of OHC motility, whereas prestin does not affect the chloride conductance of CFTR.
View details for DOI 10.1016/j.bbamem.2010.02.001
View details for Web of Science ID 000278321200002
View details for PubMedID 20138822
Cadherin 23 (CDH23), a transmembrane protein localized near the tips of hair cell stereocilia in the mammalian inner ear, is important for delivering mechanical signals to the mechano-electric transducer channels. To identify CDH23-interacting proteins, a membrane-based yeast two-hybrid screen of an outer hair cell (OHC) cDNA library was performed. EHD4, a member of the C-terminal EH domain containing a protein family involved in endocytic recycling, was identified as a potential interactor. To confirm the interaction, we first demonstrated the EHD4 mRNA expression in hair cells using in situ hybridization. Next, we showed that EHD4 co-localizes and co-immunoprecipitates with CDH23 in mammalian cells. Interestingly, the co-immunoprecipitation was found to be calcium-sensitive. To investigate the role of EHD4 in hearing, compound action potentials were measured in EHD4 knock-out (KO) mice. Although EHD4 KO mice have normal hearing sensitivity, analysis of mouse cochlear lysates revealed a 2-fold increase in EHD1, but no increase in EHD2 or EHD3, in EHD4 KO cochleae compared with wild type, suggesting that a compensatory increase in EHD1 levels may account for the absence of a hearing defect in EHD4 KO mice. Taken together, these data indicate that EHD4 is a novel CDH23-interacting protein that could regulate CDH23 trafficking/localization in a calcium-sensitive manner.
View details for DOI 10.1074/jbc.M109.025668
View details for Web of Science ID 000268097400039
View details for PubMedID 19487694
Although outer hair cells (OHCs) play a key role in cochlear amplification, it is not fully understood how they amplify sound signals by more than 100 fold. Two competing or possibly complementary mechanisms, stereocilia-based and somatic electromotility-based amplification, have been considered. Lacking knowledge about the exceptionally rich protein networks in the OHC plasma membrane, as well as related protein-protein interactions, limits our understanding of cochlear function. Therefore, we focused on finding protein partners for two important membrane proteins: Cadherin 23 (cdh23) and prestin. Cdh23 is one of the tip-link proteins involved in transducer function, a key component of mechanoelectrical transduction and stereocilia-based amplification. Prestin is a basolateral membrane protein responsible for OHC somatic electromotility.Using the membrane-based yeast two-hybrid system to screen a newly built cDNA library made predominantly from OHCs, we identified two completely different groups of potential protein partners using prestin and cdh23 as bait. These include both membrane bound and cytoplasmic proteins with 12 being de novo gene products with unknown function(s). In addition, some of these genes are closely associated with deafness loci, implying a potentially important role in hearing. The most abundant prey for prestin (38%) is composed of a group of proteins involved in electron transport, which may play a role in OHC survival. The most abundant group of cdh23 prey (55%) contains calcium-binding domains. Since calcium performs an important role in hair cell mechanoelectrical transduction and amplification, understanding the interactions between cdh23 and calcium-binding proteins should increase our knowledge of hair cell function at the molecular level.The results of this study shed light on some protein networks in cochlear hair cells. Not only was a group of de novo genes closely associated with known deafness loci identified, but the data also indicate that the hair cell tip link interacts directly with calcium binding proteins. The OHC motor protein, prestin, also appears to be associated with electron transport proteins. These unanticipated results open potentially fruitful lines of investigation into the molecular basis of cochlear amplification.
View details for DOI 10.1186/1471-2164-10-127
View details for Web of Science ID 000265792600001
View details for PubMedID 19320974