Yu-Te Yeh

Proper formation of the complex neurovascular unit (NVU) along with the blood-brain barrier is critical for building and sustaining a healthy, functioning central nervous system. The RNA binding protein argonaute2 (Ago2) mediates microRNA (miRNA)–mediated gene silencing, which is critical for many facets of brain development, including NVU development. Here, we found that Ago2 in glutamatergic neurons was critical for NVU formation in the developing cortices of mice. Glutamatergic neuron–specific loss of Ago2 diminished synaptic formation, neuronal-to-endothelial cell contacts, and morphogenesis of the brain vasculature, ultimately compromising the integrity of the blood-brain barrier. Ago2 facilitated miRNA targeting of phosphatase and tensin homolog ( Pten ) mRNA, which encodes a phosphatase that modulates reelin-dependent phosphatidylinositol 3-kinase (PI3K)–Akt signaling within the glutamatergic subpopulation. Conditionally deleting Pten in Ago2 -deficient neurons restored Akt2 phosphorylation as well as postnatal development and survival. Several mutations in AGO2 impair small RNA silencing and are associated with Lessel-Kreienkamp syndrome, a neurodevelopmental disorder. When expressed in a neuronal cell line, these human AGO2 loss-of-function variants failed to suppress PTEN, resulting in attenuated PI3K-Akt signaling, further indicating that dysregulation of Ago2 function may contribute to both impaired development and neurological disorders. Together, these results identify Ago2 as central to the engagement of neurons with blood vessels in the developing brain.

Background and Aims: Alcohol-associated liver disease (ALD) and NAFLD often coexist in Western societies that consume energy-rich and cholesterol-containing Western diets. Increased rates of ALD mortality in young people in these societies are likely attributable to binge drinking. It is largely unknown how alcohol binge causes liver damage in the setting of Western diets. Approach and Results: In this study, we showed that a single ethanol binge (5 g/kg body weight) induced severe liver injury as shown by marked increases in serum activities of the 2 aminotransferases AST and ALT in C57BL/6J mice that have been fed a Western diet for 3 weeks. The Western diet plus binge ethanol-fed mice also displayed severe lipid droplet deposition and high contents of triglycerides and cholesterol in the liver, which were associated with increased lipogenic and reduced fatty acid oxidative gene expression. These animals had the highest Cxcl1 mRNA expression and myeloperoxidase (MPO)-positive neutrophils in the liver. Their hepatic ROS and lipid peroxidation were the highest, but their hepatic levels of mitochondrial oxidative phosphorylation proteins remained largely unaltered. Hepatic levels of several ER stress markers, including mRNAs for CHOP, ERO1A, ERO1B, BIM, and BIP, as well as Xbp1 splicing and proteins for BIP/GRP78 and IRE-α were also the highest in these animals. Interestingly, Western diet feeding for 3 weeks or ethanol binge dramatically increased hepatic caspase 3 cleavage, and the combination of the 2 did not further increase it. Thus, we successfully established a murine model of acute liver injury by mimicking human diets and binge drinking. Conclusions: This simple Western diet plus single ethanol binge model recapitulates major hepatic phenotypes of ALD, including steatosis and steatohepatitis characterized by neutrophil infiltration, oxidative stress, and ER stress.

Chinese olive (Canarium album L.) has been highlighted for its remarkable health benefits. We previously showed that the ethyl acetate fraction of Chinese olive (COE) is an effective anti-inflammatory agent. In this study, we used a luciferase-based RAW 264.7 cell platform to detect the transcriptional activity of NF-κB, a key mediator of inflammation, and the promoter activity of its downstream target, COX-2. Through functional-oriented screening using these platforms, we further divided COE into several subfractions. Subsequently, we used silica gel column chromatography for purification, and the active compounds were separated and isolated by thin-layer chromatography (TLC) and high-performance liquid chromatography (HPLC). The structure of the resulting compound with high anti-inflammatory activity was then identified as scoparone. Our results showed that scoparone not only inhibited lipopolysaccharide (LPS)-induced secretion of nitric oxide and suppressed M1 macrophage markers (iNOS, Il-6, Ccl2, and Tnf-α) but also markedly decreased the production of pro-inflammatory cytokines (IL-6, CCL2, and TNF-α). Treatment with scoparone significantly reduced the protein level of TNF-α in LPS-treated bone-marrow-derived macrophages (BMDMs). In addition, scoparone promoted macrophages toward an M2 anti-inflammatory phenotype, as determined by the significantly increased gene expression of M2 macrophage markers (Arg1, Ym1, Mrc1, Il-10, and Cd206) and the protein level of Arg1. This study indicates that COE fruit has high therapeutic potential for various inflammatory diseases as a result of switching the macrophage phenotype from pro-inflammatory M1 to anti-inflammatory M2.

BACKGROUND: Pathophysiology of type 1 diabetes (T1D) is illustrated by pancreatic islet infiltration of inflammatory lymphocytes, including CD8 ⁺ T cells; however, the molecular factors mediating their recruitment remain unknown. We hypothesized that single-cell RNA-sequencing (scRNA-Seq) analysis of immune cell populations isolated from islets of NOD mice captured gene expression dynamics providing critical insight into autoimmune diabetes pathogenesis. METHODS: Pancreatic sections from human donors were investigated, including individuals with T1D, autoantibody-positive (aAb ⁺ ) individuals, and individuals without diabetes who served as controls. IHC was performed to assess islet hormones and both novel and canonical immune cell markers that were identified from unbiased, state-of-the-art workflows after reanalyzing murine scRNA-Seq data sets. RESULTS: Computational workflows identified cell adhesion molecule 1–mediated ( Cadm1 -mediated) homotypic binding among the most important intercellular interactions among all cell clusters, as well as Cadm1 enrichment in macrophages and DCs from pancreata of NOD mice. Immunostaining of human pancreata revealed an increased number of CADM1 ⁺ glucagon ⁺ cells adjacent to CD8 ⁺ T cells in sections from T1D and aAb ⁺ donors compared with individuals without diabetes. Numbers of CADM1 ⁺ CD68 ⁺ peri-islet myeloid cells adjacent to CD8 ⁺ T cells were also increased in pancreatic sections from both T1D and aAb ⁺ donors compared with individuals without diabetes. CONCLUSION: Increased detection of CADM1 ⁺ cells adjacent to CD8 ⁺ T cells in pancreatic sections of individuals with T1D and those who were aAb ⁺ validated workflows and indicated CADM1-mediated intercellular contact may facilitate islet infiltration of cytotoxic T lymphocytes and serve as a potential therapeutic target for preventing T1D pathogenesis. FUNDING: The Johns Hopkins All Children’s Foundation Institutional Research Grant Program, the National Natural Science Foundation of China (grant 82071326), and the Deutsche Forschungsgemeinschaft (grants 431549029–SFB1451, EXC2030–390661388, and 411422114-GRK2550).

The heart primarily uses fatty acids as energy substrates. Adipose lipolysis is a major source of fatty acids, particularly under stress conditions. In this study, we showed that mice with selective inactivation of the lipolytic coactivator comparative gene identification-58 (CGI-58) in adipose tissue (FAT-KO mice), relative to their littermate controls, had lower circulating FA levels in the fed and fasted states due to impaired adipose lipolysis. They preferentially utilized carbohydrates as energy fuels and were more insulin sensitive and glucose tolerant. Under cold stress, FAT-KO versus control mice had >10-fold increases in glucose uptake in the hearts but no increases in other tissues examined. Plasma concentrations of atrial natriuretic peptide and cardiac mRNAs for atrial and brain-type natriuretic peptides, two sensitive markers of cardiac remodeling, were also elevated. After one week of cold exposure, FAT-KO mice showed reduced cardiac expression of several mitochondrial oxidative phosphorylation proteins. After one month of cold exposure, hearts of these animals showed depressed functions, reduced SERCA2 protein, and increased proteins for MHC-β, collagen I proteins, Glut1, Glut4 and phospho-AMPK. Thus, CGI-58-dependent adipose lipolysis critically regulates cardiac metabolism and function, especially during cold adaptation. The adipose-heart axis may be targeted for the management of cardiac dysfunction.