Faculty Bibliography

Rationale: Loss-of-function of the cardiac sodium channel Na_V1.5 causes conduction slowing and arrhythmias. Na_V1.5 is differentially distributed within subcellular domains of cardiomyocytes, with sodium current (I_Na) being enriched at the intercalated discs (ID). Various pathophysiological conditions associated with lethal arrhythmias display ID-specific I_Na reduction, but the mechanisms underlying microdomain-specific targeting of Na_V1.5 remain largely unknown. Objective: To investigate the role of the microtubule (MT) plus-end tracking proteins end binding protein 1 (EB1) and CLIP-associated protein 2 (CLASP2) in mediating Na_V1.5 trafficking and subcellular distribution in cardiomyocytes.Methods and Results: EB1 overexpression in human induced pluripotent stem cell-derived cardiomyocytes (hiPSC-CMs) resulted in enhanced whole-cell I_Na, increased action potential (AP) upstroke velocity (V_max), and enhanced Na_V1.5 localization at the plasma membrane as detected by multi-color stochastic optical reconstruction microscopy (STORM). Fluorescence recovery after photobleaching (FRAP) experiments in HEK293A cells demonstrated that EB1 overexpression promoted Na_V1.5 forward trafficking. Knockout of MAPRE1 in hiPSC-CMs led to reduced whole-cell I_Na, decreased V_max and AP duration (APD) prolongation. Similarly, acute knockout of the MAPRE1 homolog in zebrafish (mapre1b) resulted in decreased ventricular conduction velocity and V_max as well as increased APD. STORM imaging and macropatch I_Na measurements showed that subacute treatment (2-3 hours) with SB216763 (SB2), a GSK3β inhibitor known to modulate CLASP2-EB1 interaction, reduced GSK3β localization and increased Na_V1.5 and I_Na preferentially at the ID region of wild type murine ventricular cardiomyocytes. By contrast, SB2 did not affect whole cell I_Na or Na_V1.5 localization in cardiomyocytes from Clasp2-deficient mice, uncovering the crucial role of CLASP2 in SB2-mediated modulation of NaV1.5 at the ID. Conclusions: Our findings demonstrate the modulatory effect of the MT plus-end tracking protein EB1 on Na_V1.5 trafficking and function, and identify the EB1-CLASP2 complex as a target for preferential modulation of I_Na within the ID region of cardiomyocytes.

KdpFABC is an oligomeric K⁺ transport complex in prokaryotes that maintains ionic homeostasis under stress conditions. The complex comprises a channel-like subunit (KdpA) from the superfamily of K⁺ transporters and a pump-like subunit (KdpB) from the superfamily of P-type ATPases. Recent structural work has defined the architecture and generated contradictory hypotheses for the transport mechanism. Here, we use substrate analogs to stabilize four key intermediates in the reaction cycle and determine the corresponding structures by cryogenic electron microscopy. We find that KdpB undergoes conformational changes consistent with other representatives from the P-type superfamily, whereas KdpA, KdpC, and KdpF remain static. We observe a series of spherical densities that we assign as K⁺ or water and which define a pathway for K⁺ transport. This pathway runs through an intramembrane tunnel in KdpA and delivers ions to sites in the membrane domain of KdpB. Our structures suggest a mechanism where ATP hydrolysis is coupled to K⁺ transfer between alternative sites in KdpB, ultimately reaching a low-affinity site where a water-filled pathway allows release of K⁺ to the cytoplasm.

Many military veterans who experienced blast-related traumatic brain injuries (TBI) in the conflicts in Iraq and Afghanistan currently suffer from chronic cognitive and mental health problems including post-traumatic stress disorder (PTSD). Besides static symptoms, new symptoms may emerge or existing symptoms may worsen. TBI is also a risk factor for the later development of neurodegenerative diseases. Rats exposed to repetitive low-level blast overpressure (BOP) develop robust and enduring cognitive and PTSD-related behavioral traits that are present for at least one year after blast exposure. Here we determined the time-course of these traits appearance by testing rats in the immediate post-blast period. Three cohorts of rats examined within the first eight weeks exhibited no behavioral phenotype, or in one cohort features of anxiety. None showed the altered cued fear responses, or impaired novel object recognition characteristic of the fully developed phenotype. Two cohorts retested 36 to 42 weeks after blast exposure exhibited the expanded behavioral phenotype including anxiety as well as altered cued fear learning and impaired novel object recognition. Combined with previous work the chronic behavioral phenotype has been observed in six cohorts of blast-exposed rats studied at 3-4 months or longer after blast injury and the three cohorts studied here document the progressive nature of the cognitive/behavioral phenotype. These studies suggest the existence of a latent, delayed emerging, and progressive blast-induced cognitive and behavioral phenotype. The delayed onset has implications for the evolution of post-blast neurobehavioral syndromes in military veterans and its modeling in experimental animals.

Hepatic uptake and biosynthesis of fatty acids (FA), as well as the partitioning of FA into oxidative, storage, and secretory pathways are tightly regulated processes. Dysregulation of one or more of these processes can promote excess hepatic lipid accumulation, ultimately leading to systemic metabolic dysfunction. Angiopoietin-like-4 (ANGPTL4) is a secretory protein that inhibits lipoprotein lipase (LPL) and modulates triacylglycerol (TAG) homeostasis. To understand the role of ANGPTL4 in liver lipid metabolism under normal and high-fat fed conditions, we generated hepatocyte specific Angptl4 mutant mice (Hmut). Using metabolic turnover studies, we demonstrate that hepatic Angptl4 deficiency facilitates catabolism of TAG-rich lipoprotein (TRL) remnants in the liver via increased hepatic lipase (HL) activity, which results in a significant reduction in circulating TAG and cholesterol levels. Consequently, depletion of hepatocyte Angptl4 protects against diet-induce obesity, glucose intolerance, liver steatosis, and atherogenesis. Mechanistically, we demonstrate that loss of Angptl4 in hepatocytes promotes FA uptake which results in increased FA oxidation, ROS production, and AMPK activation. Finally, we demonstrate the utility of a targeted pharmacologic therapy that specifically inhibits Angptl4 gene expression in the liver and protects against diet-induced obesity, dyslipidemia, glucose intolerance, and liver damage, which likely occurs via increased HL activity. Notably, this novel inhibition strategy does not cause any of the deleterious effects previously observed with neutralizing antibodies.

BACKGROUND:Appropriate passive-active immunoprophylaxis effectively reduces mother-to-child transmission (MTCT) of hepatitis B virus (HBV), but the immunoprophylaxis failure was still more than 5% under the current strategy. The study objective was to investigate the effects of high dose of HB vaccine on MTCT and immune response for infants born to hepatitis B surface antigen (HBsAg)-positive mothers. METHODS:This was a prospective, multicenter, large-sample cohort study in four sites of China, and 955 pairs of HBsAg-positive mothers and their infants were enrolled in our investigation. The infants were given 10 μg or 20 μg HB vaccine (at age 0, 1, and 6 months) plus HB immunoglobulin (at age 0 and 1 month). Serum HBsAg, antibody to HBsAg (anti-HBs), and/or HBV DNA levels in the infants were determined at age 12 months. The safety of 20 μg HB vaccine was evaluated by adverse events and observing the growth indexes of infants. RESULTS:IU/mL, 20 μg HB vaccine did not present these above response advantages. The 20 μg HB vaccine showed good safety for infants. CONCLUSIONS:IU/mL. TRIAL REGISTRATION/BACKGROUND:Chinese Clinical Trial Registry, ChiCTR-PRC-09000459.

The commonly mutated human KRAS oncogene encodes two distinct KRAS4A and KRAS4B proteins generated by differential splicing. We demonstrate here that coordinated regulation of both isoforms through control of splicing is essential for development of Kras mutant tumors. The minor KRAS4A isoform is enriched in cancer stem-like cells, where it responds to hypoxia, while the major KRAS4B is induced by ER stress. KRAS4A splicing is controlled by the DCAF15/RBM39 pathway, and deletion of KRAS4A or pharmacological inhibition of RBM39 using Indisulam leads to inhibition of cancer stem cells. Our data identify existing clinical drugs that target KRAS4A splicing, and suggest that levels of the minor KRAS4A isoform in human tumors can be a biomarker of sensitivity to some existing cancer therapeutics.

Osteoarthritis (OA), the most prevalent joint disease, is a major cause of disability worldwide. Growth hormone (GH) has been suggested to play significant roles in maintaining articular chondrocyte function and ultimately articular cartilage (AC) homeostasis. In humans, the age-associated decline in GH levels was hypothesized to play a role in the etiology of OA. We studied the impact of adult-onset isolated GH deficiency (AOiGHD) on the life span and skeletal integrity including the AC, in 23- to 30-month-old male and female mice on C57/BL6 genetic background. Reductions in GH during adulthood were associated with extended life span and reductions in body temperature in female mice only. However, end-of-life pathology revealed high levels of lymphomas in both sexes, independent of GH status. Skeletal characterization revealed increases in OA severity in AOiGHD mice, evidenced by AC degradation in both femur and tibia, and significantly increased osteophyte formation in AOiGHD females. AOiGHD males showed significant increases in the thickness of the synovial lining cell layer that was associated with increased markers of inflammation (IL-6, iNOS). Furthermore, male AOiGHD showed significant increases in matrix metalloproteinase-13 (MMP-13), p16, and β-galactosidase immunoreactivity in the AC as compared to controls, indicating increased cell senescence. In conclusion, while the life span of AOiGHD females increased, their health span was compromised by high-grade lymphomas and the development of severe OA. In contrast, AOiGHD males, which did not show extended life span, showed an overall low grade of lymphomas but exhibited significantly decreased health span, evidenced by increased OA severity.

The organization of an integrated coronary vasculature requires the specification of immature endothelial cells (ECs) into arterial and venous fates based on their localization within the heart. It remains unclear how spatial information controls EC identity and behavior. Here we use single-cell RNA sequencing at key developmental timepoints to interrogate cellular contributions to coronary vessel patterning and maturation. We perform transcriptional profiling to define a heterogenous population of epicardium-derived cells (EPDCs) that express unique chemokine signatures. We identify a population of Slit2+ EPDCs that emerge following epithelial-to-mesenchymal transition (EMT), which we term vascular guidepost cells. We show that the expression of guidepost-derived chemokines such as Slit2 are induced in epicardial cells undergoing EMT, while mesothelium-derived chemokines are silenced. We demonstrate that epicardium-specific deletion of myocardin-related transcription factors in mouse embryos disrupts the expression of key guidance cues and alters EPDC-EC signaling, leading to the persistence of an immature angiogenic EC identity and inappropriate accumulation of ECs on the epicardial surface. Our study suggests that EC pathfinding and fate specification is controlled by a common mechanism and guided by paracrine signaling from EPDCs linking epicardial EMT to EC localization and fate specification in the developing heart.

Congenital myasthenia (CM) is a devastating neuromuscular disease, and mutations in DOK7, an adaptor protein that is crucial for forming and maintaining neuromuscular synapses, are a major cause of CM^1,2. The most common disease-causing mutation (DOK7^{1124_1127 dup}) truncates DOK7 and leads to the loss of two tyrosine residues that are phosphorylated and recruit CRK proteins, which are important for anchoring acetylcholine receptors at synapses. Here we describe a mouse model of this common form of CM (Dok7^CM mice) and a mouse with point mutations in the two tyrosine residues (Dok7^2YF). We show that Dok7^CM mice had severe deficits in neuromuscular synapse formation that caused neonatal lethality. Unexpectedly, these deficits were due to a severe deficiency in phosphorylation and activation of muscle-specific kinase (MUSK) rather than a deficiency in DOK7 tyrosine phosphorylation. We developed agonist antibodies against MUSK and show that these antibodies restored neuromuscular synapse formation and prevented neonatal lethality and late-onset disease in Dok7^CM mice. These findings identify an unexpected cause for disease and a potential therapy for both DOK7 CM and other forms of CM caused by mutations in AGRIN, LRP4 or MUSK, and illustrate the potential of targeted therapy to rescue congenital lethality.

Study question: Do human sperm contain novel LINE-1 insertions and are they affected by paternal age? Summary answer: Human sperm contain novel LINE-1 insertions. Their location or number are not affected by paternal age. What is known already: LINE-1 comprises 17% of the human genome and some LINE-1s are the only autonomous retrotransposons in humans. Retrotransposons influence genomic instability and/or regulation if new retrotransposition events disrupt coding or regulatory regions in the host genome. Demethylation during germ cell development de-represses retrotransposons. Advanced paternal age is associated with genomic instability. Previously we showed that sperm LINE-1 copy number decreases with paternal age. We hypothesize that human sperm exhibit De novo retrotransposition and that sperm from older men contain increased novel LINE-1 insertions. Study design, size, duration: Cross-sectional case-control study with semen samples collected between February to July 2020. Participants/materials, setting, methods: Normospermic sperm samples (n=10; 5 <35 years old and 5 >=45 years old) obtained from consenting men undergoing IVF at NYU Fertility Center were submitted to a novel method, single cell Transposon Insertion Profiling by Sequencing (scTIPseq) to identify and map LINE-1 insertions in human sperm. TIPseqHunter, a custom bioinformatics pipeline, compared the architecture of sperm LINE-1 to known LINE-1 insertions from the European database of human specific LINE-1 (L1Hs) retrotransposon insertions in humans (euL1db). Main results and the role of chance: TIPseq identified 17 novel insertions in sperm, 8 from older (>= 45 years) and 9 in younger men (<35 years). New insertions were mainly intergenic or intronic, including AC007402 (2/10), TMEM163 (2/7), CTTNBP2NL (3/5), AC107023 (3/3), TMC2 (2/19), MacroD2 (2/6), RAB3C (3/4), LINC02664 (1/1), AC079052 (2/3) and AC017091 (4/4). One novel insertion (<35 years old) hits a known regulatory element. Only one sample (>= 45 years old) did not exhibit any new insertion. The location or number of novel insertions did not differ by paternal age. Limitations, reasons for caution: The small sample-size and use of normospermic specimens limit interpretation of paternal age effect on LINE-1. Besides, the novel insertions could be polymorphic sites that have low allele frequency and thus have not yet been described. Wider implications of the findings: This study for the first time reports novel LINE-1 insertions in human sperm, demonstrating that scTIPseq method is a feasible technique, and identifying new contributions to genetic diversity in the human germ line. Further studies are needed to evaluate the impact of these insertions on sperm function