Summary Mutations in the gene encoding the type II transmembrane protease 3 ( TMPRSS3 ) cause human hearing loss, although the underlying mechanisms that result in TMPRSS3 -related hearing loss are still unclear. We combined the use of stem cell-derived inner ear organoids with single-cell RNA sequencing to investigate the role of TMPRSS3. Defective Tmprss3 leads to hair cell apoptosis without altering the development of hair cells and the formation of the mechanotransduction apparatus. Prior to degeneration, Tmprss3 -KO hair cells demonstrate reduced numbers of BK channels and lower expressions of genes encoding calcium ion-binding proteins, suggesting a disruption in intracellular homeostasis. A proteolytically active TMPRSS3 was detected on cell membranes in addition to ER of cells in inner ear organoids. Our in vitro model recapitulated salient features of genetically associated inner ear abnormalities and will serve as a powerful tool for studying inner ear disorders.

The brain and muscle aryl hydrocarbon receptor nuclear translocator-like protein (BMAL)-1 constitutes a major transcriptional regulator of the circadian clock. Here, we explored the impact of conditional deletion of Bmal1 in endothelium and hematopoietic cells in murine models of microvascular and macrovascular injury. We used two models of Bmal1;Tek-Cre mice, a retinal ischemia/reperfusion model and a neointimal hyperplasia model of the femoral artery. Eyes were enumerated for acellular capillaries and were stained for oxidative damage markers using nitrotyrosine immunohistochemistry. LSK (lineage-negative, stem cell antigen-1-positive, c-Kit-positive) cells were quantified and proliferation assessed. Hematopoiesis is influenced by innervation to the bone marrow, which we assessed using IHC analysis. The number of acellular capillaries increased threefold, and nitrotyrosine staining increased 1.5-fold, in the retinas of Bmal1;Tek-Cre mice. The number of LSK cells from the Bmal1;Tek-Cre mice decreased by 1.5-fold and was accompanied by a profound decrease in proliferative potential. Bmal1;Tek-Cre mice also exhibited evidence of bone marrow denervation, demonstrating a loss of neurofilament-200 staining. Injured femoral arteries showed a 20% increase in neointimal hyperplasia compared with similarly injured wild-type controls. Our study highlights the importance of the circadian clock in maintaining vascular homeostasis and demonstrates that specific deletion of BMAL1 in endothelial and hematopoietic cells results in phenotypic features similar to those of diabetes.

PurposeDiabetes leads to the downregulation of the retinal Kir4.1 channels and Müller cell dysfunction. The insulin receptor substrate-1 (IRS-1) is a critical regulator of insulin signaling in Müller cells. Circadian rhythms play an integral role in normal physiology; however, diabetes leads to a circadian dysrhythmia. We hypothesize that diabetes will result in a circadian dysrhythmia of IRS-1 and Kir4.1 and disturbed clock gene function will have a critical role in regulating Kir4.1 channels.MethodsWe assessed a diurnal rhythm of retinal IRS-1 and Kir4.1 in db/db mice. The Kir4.1 function was evaluated using a whole-cell recording of Müller cells. The rat Müller cells (rMC-1) were used to undertake in vitro studies using a siRNA.ResultsThe IRS-1 exhibited a diurnal rhythm in control mice; however, with diabetes, this natural rhythm was lost. The Kir4.1 levels peaked and troughed at times similar to the IRS-1 rhythm. The IRS-1 silencing in the rMC-1 led to a decrease in Kir4.1 and BMAL1. The insulin treatment of retinal explants upregulated Kir4.1 possibly via upregulation of BMAL1 and phosphorylation of IRS-1 and Akt-1.ConclusionsOur studies highlight that IRS-1, by regulating BMAL1, is an important regulator of Kir4.1 in Müller cells and the dysfunctional signaling mediated by IRS-1 may be detrimental to Kir4.1.

PURPOSE. Diabetic retinopathy (DR) is a leading cause of visual impairment. Müller cells in DR are dysfunctional due to downregulation of the inwardly rectifying potassium channel Kir4.1. Metformin, a commonly used oral antidiabetic drug, is known to elicit its action through 5 adenosine monophosphate-activated protein kinase (AMPK), a cellular metabolic regulator; however, its effect on Kir4.1 channels is unknown. For this study, we hypothesized that metformin treatment would correct circadian rhythm disruption and Kir4.1 channel dysfunction in db/db mice. METHODS. Metformin was given orally to db/db mice. Wheel-running activity, retinal levels of Kir4.1, and AMPK phosphorylation were determined at study termination. In parallel, rat retinal Müller cell line (rMC-1) cells were treated using metformin and 5aminoimidazole-4-carboxamide ribonucleotide (AICAR) to assess the effect of AMPK activation on the Kir4.1 channel. RESULTS. The wheel-running activity of the db/db mice was improved following the metformin treatment. The Kir4.1 level in Müller cells was corrected after metformin treatment. Metformin treatment led to an upregulation of clock regulatory genes such as melanopsin (Opn4) and aralkylamine N-acetyltransferase (Aanat). In rMC-1 cells, AMPK activation via AICAR and metformin resulted in increased Kir4.1 and intermediate core clock component Bmal-1 protein expression. The silencing of Prkaa1 (gene for AMPKα1) led to decreased Kir4.1 and Bmal-1 protein expression. CONCLUSIONS. Our findings demonstrate that metformin corrects abnormal circadian rhythm and Kir4.1 channels in db/db mouse a model of type 2 diabetes. Metformin could represent a critical pharmacological agent for preventing Müller cell dysfunction observed in human DR.

Introduction: Diabetes is the leading cause of microvascular disorders such as diabetic retinopathy (DR). There are no treatments for DR and finding new drug targets is of considerable interest. SGLT2 inhibitors, a newer class of antidiabetics are promising in the management of diabetes, however, the potential role of SGLT2 in diabetic retinal microvasculature remains unknown. We hypothesized that diabetes will lead to an increase in SGLT2 in the retina and that its inhibition will be beneficial in protecting retinal vasculature from the insult of diabetes milieu. Methods: The retinal sections of diabetic (db/db; an animal model of type 2 diabetes) and control (db/m) mice were analyzed for SGLT2 expression using confocal microscopy. In parallel, the mRNA levels of SGLT2 were determined using qRT-PCR. Human retinal endothelial cells (HRECs) were treated with the SGLT2 inhibitor dapagliflozin (0.1, 1, 10 nM) to perform a glucose uptake assay and to determine its effects on mRNA levels of SGLT2. Results: The mRNA levels of SGLT2 were significantly higher (p<0.05) in the retina of db/db mice, 1.1 ± 0.46, n=5, when compared to db/m retinas, 0.06 ± 0.02, n=4. The confocal microscopy revealed SGLT2 expression throughout the retina and around the retinal blood vessels, brighter fluorescence was observed in the db/db retina. Treatment of HRECs with 10 nM dapagliflozin led to a significant decrease in SGLT2 mRNA (p<0.05); dapagliflozin: 7.95 ± 4.7, n=4; vehicle: 26 ± 3.6, n=8. The dapagliflozin inhibition demonstrated a profound decrease in glucose uptake in HRECs at all concentrations. Untreated: 2012 ± 388, n=3; 0.1 nM: 545.7 ± 296, 1 nM: 358.2 ± 241.5, 10 nM: 70.67 ± 138.9; p<0.01 as compared to untreated; n=6. Conclusion: Our studies suggest that SGLT2 is significantly upregulated in the retina during diabetes and that SGLT2 potentially plays a critical role in retinal glucose transport. In future, SGLT2 inhibition could be a useful treatment option to slow down or prevent the onset of diabetic retinopathy. Disclosure S.P. Leley: None. Q. Luo: None. A.L. Alex: None. A.D. Bhatwadekar: None. Funding National Eye Institute; Indiana University Center for Diabetes and Metabolic Diseases