A redox-mediated conformational change in NQO1 controls binding to microtubules and α-tubulin acetylation.

The localization of NQO1 near acetylated microtubules has led to the hypothesis that NQO1 may work in concert with the NAD+-dependent deacetylase SIRT2 to regulate acetyl α-tubulin (K40) levels on microtubules. NQO1 catalyzes the oxidation of NADH to NAD+ and may supplement levels of NAD+ near microtubules to aid SIRT2 deacetylase activity. While HDAC6 has been shown to regulate the majority of microtubule acetylation at K40, SIRT2 is also known to modulate microtubule acetylation (K40) in the perinuclear region. In this study we examined the potential roles NQO1 may play in modulating acetyl α-tubulin levels. Knock-out or knock-down of NQO1 or SIRT2 did not change the levels of acetyl α-tubulin in 16HBE human bronchial epithelial cells and 3T3-L1 fibroblasts; however, treatment with a mechanism-based inhibitor of NQO1 (MI2321) led to a short-lived temporal increase in acetyl α-tubulin levels in both cell lines without impacting the intracellular pools of NADH or NAD+. Inactivation of NQO1 by MI2321 resulted in lower levels of NQO1 immunostaining on microtubules, consistent with redox-dependent changes in NQO1 conformation as evidenced by the use of redox-specific, anti-NQO1 antibodies in immunoprecipitation studies. Given the highly dynamic nature of acetylation-deacetylation reactions at α-tubulin K40 and the crowded protein environment surrounding this site, disruption in the binding of NQO1 to microtubules may temporally disturb the physical interactions of enzymes responsible for maintaining the microtubule acetylome.

Transcriptional profiling of murine retinas undergoing semi-synchronous cone photoreceptor differentiation.

Uncovering the gene regulatory networks that control cone photoreceptor formation has been hindered because cones only make up a few percent of the retina and form asynchronously during development. To overcome these limitations, we used a γ-secretase inhibitor, DAPT, to disrupt Notch signaling and force proliferating retinal progenitor cells to rapidly adopt neuronal identity. We treated mouse retinal explants at the peak of cone genesis with DAPT and examined tissues at several time-points by histology and bulk RNA-sequencing. We found that this treatment caused supernumerary cone formation in an overwhelmingly synchronized fashion. This analysis revealed several categorical patterns of gene expression changes over time relative to DMSO treated control explants. These were placed in the temporal context of the activation of Otx2, a transcription factor that is expressed at the onset of photoreceptor development and that is required for both rod and cone formation. One group of interest had genes, such as Mybl1, Ascl1, Neurog2, and Olig2, that became upregulated by DAPT treatment before Otx2. Two other groups showed upregulated gene expression shortly after Otx2, either transiently or permanently. This included genes such as Mybl1, Meis2, and Podxl. Our data provide a developmental timeline of the gene expression events that underlie the initial steps of cone genesis and maturation. Applying this strategy to human retinal organoid cultures was also sufficient to induce a massive increase in cone genesis. Taken together, our results provide a temporal framework that can be used to elucidate the gene regulatory logic controlling cone photoreceptor development.

Combinatorial regulation of a Blimp1 (Prdm1) enhancer in the mouse retina.

The mouse retina comprises seven major cell types that exist in differing proportions. They are generated from multipotent progenitors in a stochastic manner, such that the relative frequency of any given type generated changes over time. The mechanisms determining the proportions of each cell type are only partially understood. Photoreceptors and bipolar interneurons are derived from cells that express Otx2. Within this population, Blimp1 (Prdm1) helps set the balance between photoreceptors and bipolar cells by suppressing bipolar identity in most of the cells. How only a subset of these Otx2+ cells decides to upregulate Blimp1 and adopt photoreceptor fate is unknown. To understand this, we investigated how Blimp1 transcription is regulated. We identified several potential Blimp1 retinal enhancer elements using DNase hypersensitivity sequencing. Only one of the elements recapitulated Blimp1 spatial and temporal expression in cultured explant assays and within the retinas of transgenic mice. Mutagenesis of this retinal Blimp1 enhancer element revealed four discrete sequences that were each required for its activity. These included highly conserved Otx2 and ROR (retinoic acid receptor related orphan receptor) binding sites. The other required sequences do not appear to be controlled by Otx2 or ROR factors, increasing the complexity of the Blimp1 gene regulatory network. Our results show that the intersection of three or more transcription factors is required to correctly regulate the spatial and temporal features of Blimp1 enhancer expression. This explains how Blimp1 expression can diverge from Otx2 and set the balance between photoreceptor and bipolar fates.

In vitro assessment of induced phrenic nerve cryothermal injury.

BACKGROUND: Phrenic nerve injury, both left and right, is considered a significant complication of cryoballoon ablation for treatment of drug-refractory atrial fibrillation, and functional recovery of the phrenic nerve can take anywhere from hours to months. OBJECTIVE: The purpose of this study was to focus on short periods of cooling to determine the minimal amount of cooling that may terminate nerve function related to cryo ablation. METHODS: Left and/or right phrenic nerves were dissected from the pericardium and connective tissue of swine (n = 35 preparations). Nerves were placed in a recording chamber modified with a thermocouple array. This apparatus was placed in a digital water bath to maintain an internal chamber temperature of 37°C. Nerves were stimulated proximally with a 1-V, 0.1-ms square wave. Bipolar compound action potentials were recorded proximal and distal to the site of ablation both before and after ablation, then analyzed to determine changes in latency, amplitude, and duration. Temperatures were recorded at a rate of 5 Hz, and maximum cooling rates were calculated. RESULTS: Phrenic nerves were found to elicit compound action potentials upon stimulation for periods up to 4 hours minimum. Average conduction velocity was 56.7 ± 14.7 m/s preablation and 49.8 ± 16.6 m/s postablation (P = .17). Cooling to mild subzero temperatures ceased production of action potentials for >1 hour. CONCLUSION: Taking into account the data presented here, previous publications, and a conservative stance, during cryotherapy applications, cooling of the nerve to below 4°C should be avoided whenever possible.