Open Access Journals at IU Indianapolis
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Hurricane St. Agatha through the Eyes of a Coastal Florida Timeshare Purchased through a Costco Membership circa 2017
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Heart to Heart: A Journey in Cardiovascular Research: Interview with Rohan Dharmakumar, PhD
No full textFeature: Visual Art, Kathleen Ho
Get PDFKathleen Ho, MS3 showcases her artwork. Works include:
Upper Cataract Falls, Bloomington (Oil paint, 8x10")
Venice, Italy (Oil paint, 8x10")
Downtown Indianapolis (Oil paint, 18x24”)
Flower Market (Oil paint, 18x24”)
Fort Harrison State Park, Indianapolis (Oil paint, 10x12”
Reduced Endocochlear Potential in vivo Prevents Hair Cell Degeneration in Tmprss3- deficient Mice
Get PDFBackground: Transmembrane serine protease 3 (TMPRSS3) is a transmembrane serine protease with proteolytic activity essential for normal auditory function in mice and humans. While Tmprss3 mutations are the most common gene variant adain cochlear implant recipients, details behind its cellular mechanism remain elusive. Tmprss3-mutant mice exhibit normal hair cell (HC) development until postnatal day 12 (P12), followed by rapid HC degeneration within 48 hours, resulting in deafness. The HC degeneration temporally correlates with the rapid rise in endocochlear potential (EP) that is required for hearing. This phenotype mirrors other mouse models with defects in genes expressing tight junctions (TJs). Thus, we hypothesize that TMPRSS3 regulates tight junctions and cell death is mediated through high EP.
Methods: Our laboratory has previously demonstrated that performing cochlear explants at P7 followed by in vitro cultures for an additional 7 days leads to complete preservation of HCs in Tmprss3-mutant mice. It is unknown if the observed HC survival is due directly from removing EP or from other extracellular factors. Here we investigated the role of EP in Tmprss3-deficient mice using in vivo experiments. We crossed the Tmprss3-mutant mice with Pou3f4-mutant mice, which fail to generate EP. Cochlear whole mounts were dissected, fixed, and stained for four groups of mice: wild-type, Tmprss3-mutant, Pou3f4-mutant, and double-mutant mice. Inner and outer hair cells were quantified within a span of 125 μm and compared between groups.
Results: We found significant preservation of HCs (p<0.001) in double mutant mice with reduced EP compared to Tmprss3-mutant mice. Thus, HC degeneration in Tmprss3-deficient mice is due to endocochlear potential driven K+ toxicity. Tmprss3-deficient mice likely have faulty apical TJs that result in leakage of K+ ions from the endolymph to the basolateral side of HCs, leading to HC degeneration.
Conclusion: Future research should work to elucidate TMPRSS3’s proteolytic target and its mechanism of TJ-related regulation
Affinity Purification of Anti-Parkin Antibodies from Rabbit Serum for their Application in Immunohistochemistry
Get PDFBackground:Parkin is a mitochondrial autophagy protein that is associated with Parkinson’s Disease (PD), as well as auditory function. Parkin dysfunction in the context of PD allows accumulation of protein aggregates, leading to dopaminergic neurotoxicity. Previous work has demonstrated that Parkin plays an essential role in normal auditory function, as Prkn-/- mice exhibit significantly reduced hearing sensitivity. However, Prkn-/- mice also experience a protective effect from cochlear hair cell death and hearing loss caused by aminoglycosides, which are commonly used antibiotics that can cause permanent hearing loss. Multiple antibodies marketed as suitable for immunohistochemistry (IHC) have been validated using Prkn-/- tissues; however, they exhibit non-specific activity. This project aims to generate a highly specific antibody for accurate and reliable detection of Parkin expression.
Project Methods:Rabbits were immunized using two Parkin peptides, notated P1 and P2, and the resulting serum was collected. A two-step purification process was utilized in this project, first isolating the IgGs from serum via protein A/G columns, and secondly using affinity purification to obtain specific antibodies against antigens P1 and P2. Additionally, two different protocols for affinity purification were tested and compared. Purity of serum, IgG, and specific antibodies was assessed by Western blot and immunofluorescence.
Results:Utilizing the first purification protocol, IHC antibodies were applied in Western blot, and all demonstrated a prominent ~50kD band specific to Parkin, with some degree of nonspecific binding. Immunostaining confirmed functionality of the IHC antibodies and revealed those against P2 exhibited higher binding specificity. The second purification protocol generated IHC antibodies of similar, if not slightly superior specific interaction.
Conclusion/Implications:Further investigation of Parkin expression would facilitate a better understanding of how it may be associated with the development of PD, as well as auditory function, with the potential of utilizing Parkin as a therapeutic target in PD treatment and preventing aminoglycoside ototoxicity