The signaling mechanisms that choreograph the assembly of the highly asymmetric pre-and postsynaptic structures are still poorly defined. Using synaptosome fractionation, immunostaining, and coimmunoprecipitation, we found that Celsr3 and Vangl2, core components of the planar cell polarity (PCP) pathway, are localized at developing glutamatergic synapses and interact with key synaptic proteins. Pyramidal neurons from the hippocampus of Celsr3 knockout mice exhibit loss of ∼50% of glutamatergic synapses, but not inhibitory synapses, in culture. Wnts are known regulators of synapse formation, and our data reveal that Wnt5a inhibits glutamatergic synapses formed via Celsr3. To avoid affecting earlier developmental processes, such as axon guidance, we conditionally knocked out Celsr3 in the hippocampus 1 week after birth. The CA1 neurons that lost Celsr3 also showed a loss of ∼50% of glutamatergic synapses in vivo without affecting the inhibitory synapses assessed by miniature excitatory postsynaptic current (mEPSC) and electron microscopy. These animals displayed deficits in hippocampus-dependent behaviors in adulthood, including spatial learning and memory and fear conditioning. In contrast to Celsr3 conditional knockouts, we found that the conditional knockout of Vangl2 in the hippocampus 1 week after birth led to a large increase in synaptic density, as evaluated by mEPSC frequency and spine density. PCP signaling is mediated by multiple core components with antagonizing functions. Our results document the opposing roles of Celsr3 and Vangl2 in glutamatergic synapse formation.Celsr3 | Vangl2 | glutamatergic | synapse formation G lutamatergic synapses, the predominant excitatory synapses in the brain, are asymmetric cell-cell junctions formed from distinct pre-and postsynaptic components involving highly organized complexes of hundreds of proteins across the 20-nm synaptic cleft (1, 2). The signaling pathway that directly assembles these asymmetric protein complexes has not been well understood. Understanding mechanisms of glutamatergic synapse formation will provide important insights into the function and plasticity as well as dysfunction of glutamatergic synapses, which underlie numerous nervous system disorders.Many epithelial tissues show planar cell polarity, the global asymmetry of cellular and tissue morphology and/or structure along the tissue plane (3, 4). The conserved core planar cell polarity (PCP) components, Frizzled, Dishevelled, Diego, Prickle, Vang(l), and Flamingo (Fmi)/Celsr, form asymmetric complexes at the cadherin-mediated adherens junctions that connect neighboring epithelial cells (3, 4). Recent studies suggest that mutations of some components of the PCP signaling pathway, Celsr3/Fmi and Vangl2, affect GABAergic circuit development in zebrafish retina, GABAergic motoneuron synapse development in Caenorhabditis elegans, and hippocampal/cortical glutamatergic and GABAergic synapse formation (5-11). PCP components are critical regulators of neuronal migration and axon guidance, which take p...

Rationale: Bioavailable and free 25-hydroxyvitamin D (25(OH)D) are emerging measurements of vitamin D. Whether serum bioavailable or free 25(OH)D level is associated with mortality in patients with coronary artery disease (CAD) is unknown. Objective: Our aim is to determine the potential association between serum total, bioavailable, and free 25(OH)D levels and the risk of mortality among patients with CAD. Methods and Results: We measured serum 25(OH) levels in 1387 patients with angiographically confirmed CAD from the Guangdong Coronary Artery Disease Cohort. Serum DBP (vitamin D-binding protein) levels were measured using a polyclonal immunoassay, and serum-free 25(OH)D levels were measured using a 2-step immunoassay. Bioavailable 25(OH)D levels were calculated using a previously validated formula. By the median follow-up time of 6.7 years, 205 patients had died, including 134 deaths from cardiovascular diseases. In multivariate analyses, low serum bioavailable 25(OH)D level was significantly associated with increased risks of mortality, independent of established cardiovascular risk factors, features and treatments of CAD, factors associated with vitamin D and mineral metabolism, and CRP (C-reactive protein). The multivariable-adjusted hazard ratios across quartiles of bioavailable 25(OH)D were 1.79, 1.35, 1.36, and 1.00 for all-cause mortality ( P for trend=0.01) and 2.58, 1.85, 1.73, and 1.00 for cardiovascular mortality ( P for trend=0.001), respectively. Serum-free 25(OH)D level was inversely associated with the risk of mortality, with the extreme-quartile hazard ratios of 1.64 for all-cause mortality ( P for trend=0.024) and 1.97 for cardiovascular mortality ( P for trend=0.013). In contrast, serum total 25(OH)D level was not significantly associated with all-cause mortality or cardiovascular mortality. Conclusions: Lower serum bioavailable and free 25(OH)D levels rather than total 25(OH)D level are independently associated with an increased risk of all-cause mortality and cardiovascular mortality in a population-based CAD cohort.

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The prevalence of hypertension is about twofold higher in diabetic than in nondiabetic subjects. Hypertension aggravates the progression of diabetic complications, especially diabetic nephropathy. However, the mechanisms for the development of hypertension in diabetes have not been elucidated. We hypothesized that enhanced constrictive responsiveness of renal afferent arterioles (Af-Art) to angiotensin II (ANG II) mediated by ANG II type 1 (AT1) receptors contributes to the development of hypertension in diabetes. In response to an acute bolus intravenous injection of ANG II, alloxan-induced diabetic mice exhibited a higher mean arterial pressure (MAP) (119.1 ± 3.8 vs. 106.2 ± 3.5 mmHg) and a lower renal blood flow (0.25 ± 0.07 vs. 0.52 ± 0.14 ml/min) compared with nondiabetic mice. In response to chronic ANG II infusion, the MAP measured with telemetry increased by 55.8 ± 6.5 mmHg in diabetic mice, but only by 32.3 ± 3.8 mmHg in nondiabetic mice. The mRNA level of AT1 receptor increased by ~10-fold in isolated Af-Art of diabetic mice compared with nondiabetic mice, whereas ANG II type 2 (AT2) receptor expression did not change. The ANG II dose-response curve of the Af-Art was significantly enhanced in diabetic mice. Moreover, the AT1 receptor antagonist, losartan, blocked the ANG II-induced vasoconstriction in both diabetic mice and nondiabetic mice. In conclusion, we found enhanced expression of the AT1 receptor and exaggerated response to ANG II of the Af-Art in diabetes, which may contribute to the increased prevalence of hypertension in diabetes.

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