Rosmarinic acid liposomes suppress ferroptosis in ischemic brain via inhibition of TfR1 in BMECs.

BACKGROUND: Iron deposition and ferroptosis are involved in ischemic stroke injury, but the choice of drugs for treatment is limited. PURPOSE: To investigate the potential neuroprotective effects of Rosmarinic acid (RosA) encapsulated within nanoliposomes (RosA-LIP) on ischemic stroke. METHODS: Wild-type (WT) and TfR1EC cKO (specific knockout of the TfR1 gene in BMECs) mice used to establish a dMCAO model, with simultaneous administration of RosA-LIP (20 mg/kg/d, i.p.) or RosA (20 mg/kg/d, i.p.). RESULTS: The successful synthesis of RosA-LIP resulted in enhanced stability and precise delivery in both the serum and brain. The administration of RosA-LIP effectively mitigated ischemia-induced behavioral abnormalities and pathological damage. RosA-LIP inhibited ferroptosis by ameliorating mitochondrial abnormalities, increasing GPX4 levels, and decreasing ACSL4/LPCAT3/Lox-dependent lipid peroxidation. RosA-LIP effectively improved blood‒brain barrier (BBB) permeability, increased tight junctions (TJs) protein expression and reduced iron levels in ischemic tissue and brain microvascular endothelial cells (BMECs) by modulating FPN1 and TfR1 levels. Furthermore, RosA-LIP suppressed TfR1 to attenuate ACSL4/LPCAT3/Lox-mediated ferroptosis in TfR1EC cKO mice subjected to dMCAO. CONCLUSION: RosA-LIP effectively increased the brain level of RosA and protected against ferroptosis through the regulation of TfR1 in BMECs.

Rosmarinic Acid Liposomes Downregulate Hepcidin Expression via BMP6-SMAD1/5/8 Pathway in Mice with Iron Overload.

The objective of this study is to examine the potential protective effect of rosmarinic acid (RosA) encapsulated within nanoliposomes (RosA-LIP) on hepatic damage induced by iron overload. The characteristics, stability, and release of RosA-LIP in vitro were identified. The mice were randomly assigned to five groups: Control, Model, Model+DFO (DFO), Model+RosA (RosA), and Model+RosA-LIP (RosA-LIP). The iron overload model was induced by administering iron dextran (i.p.). The DFO, RosA, and RosA-LIP groups received iron dextran and were subsequently treated with DFO, RosA, and RosA-LIP for 14 days. We developed a novel formulation of RosA-LIP that exhibited stability and controlled release properties. Firstly, RosA-LIP improved liver function and ameliorated pathological changes in a mouse model of iron overload. Secondly, RosA-LIP demonstrated the ability to enhance the activities of T-SOD, GSH-Px, and CAT, while reducing the levels of MDA and 4-HNE, thereby effectively mitigating oxidative stress damage induced by iron overload. Thirdly, RosA-LIP reduced hepatic iron levels by downregulating FTL, FTH, and TfR1 levels. Additionally, RosA-LIP exerted a suppressive effect on hepcidin expression through the BMP6-SMAD1/5/8 signaling pathway. Furthermore, RosA-LIP upregulated FPN1 expression in both the liver and duodenum, thereby alleviating iron accumulation in these organs in mice with iron overload. Notably, RosA exhibited a comparable iron chelation effect, and RosA-LIP demonstrated superior efficacy in mitigating liver damage induced by excessive iron overload. RosA-LIP exhibited favorable sustained release properties, targeted delivery, and efficient protection against iron overload-induced liver damage. A schematic representation of the proposed protective mechanism of rosmarinic acid liposome during iron overload. Once RosA-LIP is transported into cells, RosA is released. On the one hand, RosA attenuates the BMP6-SMAD1/5/8-SMAD4 signaling pathway activation, leading to inhibiting hepcidin transcription. Then, the declined hepcidin contacted the inhibitory effect of FPN1 in hepatocytes and duodenum, increasing iron mobilization. On the other hand, RosA inhibits TfR1 and ferritin expression, which decreases excessive iron and oxidative damage.

New species of the tribe Meconematini (Orthoptera: Tettigoniidae: Meconematinae: Meconematini) from Sichuan, China.

Two new species of the subfamily Meconematinae are described, i.e. Neocyrtopsis (Neocyrtopsis) shimianensis Wang Shi, sp. nov. and Doicholobosa acuta Wang Shi, sp. nov. from Sichuan, China. All examined specimens are deposited in the Museum of Hebei University.

Myeloid Acat1/Soat1 KO attenuates pro-inflammatory responses in macrophages and protects against atherosclerosis in a model of advanced lesions.

Cholesterol esters are a key ingredient of foamy cells in atherosclerotic lesions; their formation is catalyzed by two enzymes: acyl-CoA:cholesterol acyltransferases (ACATs; also called sterol O-acyltransferases, or SOATs) ACAT1 and ACAT2. ACAT1 is present in all body cells and is the major isoenzyme in macrophages. Whether blocking ACAT1 benefits atherosclerosis has been under debate for more than a decade. Previously, our laboratory developed a myeloid-specific Acat1 knockout (KO) mouse (Acat1-M/-M), devoid of ACAT1 only in macrophages, microglia, and neutrophils. In previous work using the ApoE KO (ApoE-/-) mouse model for early lesions, Acat1-M/-M significantly reduced lesion macrophage content and suppressed atherosclerosis progression. In advanced lesions, cholesterol crystals become a prominent feature. Here we evaluated the effects of Acat1-M/-M in the ApoE KO mouse model for more advanced lesions and found that mice lacking myeloid Acat1 had significantly reduced lesion cholesterol crystal contents. Acat1-M/-M also significantly reduced lesion size and macrophage content without increasing apoptotic cell death. Cell culture studies showed that inhibiting ACAT1 in macrophages caused cells to produce less proinflammatory responses upon cholesterol loading by acetyl low-density lipoprotein. In advanced lesions, Acat1-M/-M reduced but did not eliminate foamy cells. In advanced plaques isolated from ApoE-/- mice, immunostainings showed that both ACAT1 and ACAT2 are present. In cell culture, both enzymes are present in macrophages and smooth muscle cells and contribute to cholesterol ester biosynthesis. Overall, our results support the notion that targeting ACAT1 or targeting both ACAT1 and ACAT2 in macrophages is a novel strategy to treat advanced lesions.

Gpnmb secreted from liver promotes lipogenesis in white adipose tissue and aggravates obesity and insulin resistance.

Metabolism in mammals is regulated by complex interplay among different organs. Fatty acid synthesis is increased in white adipose tissue (WAT) when it is inhibited in the liver. Here we identify glycoprotein non-metastatic melanoma protein B (Gpnmb) as one liver-WAT cross-talk factor involved in lipogenesis. Inhibition of the hepatic sterol regulatory element-binding protein pathway leads to increased transcription of Gpnmb and promotes processing of the membrane protein to a secreted form. Gpnmb stimulates lipogenesis in WAT and exacerbates diet-induced obesity and insulin resistance. In humans, Gpnmb is tightly associated with body mass index and is a strong risk factor for obesity. Gpnmb inhibition by a neutralizing antibody or liver-specific knockdown improves metabolic parameters, including weight gain reduction and increased insulin sensitivity, probably by promoting the beiging of WAT. These results suggest that Gpnmb is a liver-secreted factor regulating lipogenesis in WAT, and that Gpnmb inhibition may provide a therapeutic strategy in obesity and diabetes.

A LIMA1 variant promotes low plasma LDL cholesterol and decreases intestinal cholesterol absorption.

A high concentration of low-density lipoprotein cholesterol (LDL-C) is a major risk factor for cardiovascular disease. Although LDL-C levels vary among humans and are heritable, the genetic factors affecting LDL-C are not fully characterized. We identified a rare frameshift variant in the LIMA1 (also known as EPLIN or SREBP3) gene from a Chinese family of Kazakh ethnicity with inherited low LDL-C and reduced cholesterol absorption. In a mouse model, LIMA1 was mainly expressed in the small intestine and localized on the brush border membrane. LIMA1 bridged NPC1L1, an essential protein for cholesterol absorption, to a transportation complex containing myosin Vb and facilitated cholesterol uptake. Similar to the human phenotype, Lima1-deficient mice displayed reduced cholesterol absorption and were resistant to diet-induced hypercholesterolemia. Through our study of both mice and humans, we identify LIMA1 as a key protein regulating intestinal cholesterol absorption.

Corrigendum: Cholesterol and fatty acids regulate cysteine ubiquitylation of ACAT2 through competitive oxidation.

This corrects the article DOI: 10.1038/ncb3551.

Cholesterol and fatty acids regulate cysteine ubiquitylation of ACAT2 through competitive oxidation.

Ubiquitin linkage to cysteine is an unconventional modification targeting protein for degradation. However, the physiological regulation of cysteine ubiquitylation is still mysterious. Here we found that ACAT2, a cellular enzyme converting cholesterol and fatty acid to cholesteryl esters, was ubiquitylated on Cys277 for degradation when the lipid level was low. gp78-Insigs catalysed Lys48-linked polyubiquitylation on this Cys277. A high concentration of cholesterol and fatty acid, however, induced cellular reactive oxygen species (ROS) that oxidized Cys277, resulting in ACAT2 stabilization and subsequently elevated cholesteryl esters. Furthermore, ACAT2 knockout mice were more susceptible to high-fat diet-associated insulin resistance. By contrast, expression of a constitutively stable form of ACAT2 (C277A) resulted in higher insulin sensitivity. Together, these data indicate that lipid-induced stabilization of ACAT2 ameliorates lipotoxicity from excessive cholesterol and fatty acid. This unconventional cysteine ubiquitylation of ACAT2 constitutes an important mechanism for sensing lipid-overload-induced ROS and fine-tuning lipid homeostasis.

The GARP Complex Is Involved in Intracellular Cholesterol Transport via Targeting NPC2 to Lysosomes.

Proper intracellular cholesterol trafficking is critical for cellular function. Two lysosome-resident proteins, NPC1 and NPC2, mediate the egress of low-density lipoprotein-derived cholesterol from lysosomes. However, other proteins involved in this process remain largely unknown. Through amphotericin B-based selection, we isolated two cholesterol transport-defective cell lines. Subsequent whole-transcriptome-sequencing analysis revealed two cell lines bearing the same mutation in the vacuolar protein sorting 53 (Vps53) gene. Depletion of VPS53 or other subunits of the Golgi-associated retrograde protein (GARP) complex impaired NPC2 sorting to lysosomes and caused cholesterol accumulation. GARP deficiency blocked the retrieval of the cation-independent mannose 6-phosphate receptor (CI-MPR) to the trans-Golgi network. Further, Vps54 mutant mice displayed reduced cellular NPC2 protein levels and increased cholesterol accumulation, underscoring the physiological role of the GARP complex in cholesterol transport. We conclude that the GARP complex contributes to intracellular cholesterol transport by targeting NPC2 to lysosomes in a CI-MPR-dependent manner.

Cholesterol Modification of Smoothened Is Required for Hedgehog Signaling.

Hedgehog (Hh) has been known as the only cholesterol-modified morphogen playing pivotal roles in development and tumorigenesis. A major unsolved question is how Hh signaling regulates the activity of Smoothened (SMO). Here, we performed an unbiased biochemical screen and identified that SMO was covalently modified by cholesterol on the Asp95 (D95) residue through an ester bond. This modification was inhibited by Patched-1 (Ptch1) but enhanced by Hh. The SMO(D95N) mutation, which could not be cholesterol modified, was refractory to Hh-stimulated ciliary localization and failed to activate downstream signaling. Furthermore, homozygous SmoD99N/D99N (the equivalent residue in mouse) knockin mice were embryonic lethal with severe cardiac defects, phenocopying the Smo-/- mice. Together, the results of our study suggest that Hh signaling transduces to SMO through modulating its cholesterylation and provides a therapeutic opportunity to treat Hh-pathway-related cancers by targeting SMO cholesterylation.

AAV9-NPC1 significantly ameliorates Purkinje cell death and behavioral abnormalities in mouse NPC disease.

Niemann-Pick type C (NPC) disease is a fatal inherited neurodegenerative disorder caused by loss-of-function mutations in the NPC1 or NPC2 gene. There is no effective way to treat NPC disease. In this study, we used adeno-associated virus (AAV) serotype 9 (AAV9) to deliver a functional NPC1 gene systemically into NPC1-/- mice at postnatal day 4. One single AAV9-NPC1 injection resulted in robust NPC1 expression in various tissues, including brain, heart, and lung. Strikingly, AAV9-mediated NPC1 delivery significantly promoted Purkinje cell survival, restored locomotor activity and coordination, and increased the lifespan of NPC1-/- mice. Our work suggests that AAV-based gene therapy is a promising means to treat NPC disease.

Inhibition of the sterol regulatory element-binding protein pathway suppresses hepatocellular carcinoma by repressing inflammation in mice.

Obesity is a critical risk factor for hepatocellular carcinoma (HCC). However, it remains unknown whether inhibition of de novo lipid biosynthesis can suppress HCC. In this study, we blocked the sterol regulatory element-binding protein (SREBP) pathway, one of the key determinants of lipid homeostasis, by ablating 78-kDa cell-surface glycoprotein or SREBP cleavage-activating protein in hepatocytes, as well as by administering a chemical compound called betulin. We found that either genetically or pharmacologically inhibiting the SREBP pathway dramatically reduced diethylnitrosamine-induced HCC progression by down-regulating tumor-promoting cytokines, including interleukin (IL)-6, tumor necrosis factor alpha, and IL-1ß. CONCLUSION: Inhibition of de novo lipid biosynthesis by suppressing the SREBP pathway prevents HCC. This study identifies a previously underappreciated role of the SREBP pathway in HCC and suggests a novel metabolic strategy to control liver cancer. (Hepatology 2017;65:1936-1947).

Identification and characterization of NPC1L1 variants in Uygur and Kazakh with extreme low-density lipoprotein cholesterol.

BACKGROUND: Plasma levels of low-density lipoprotein cholesterol (LDL-C) are a major risk factor for cardiovascular disease and are influenced by both heredity and dietary habits. The Niemann-Pick C1 like 1 (NPC1L1) protein mediates efficient dietary cholesterol absorption and contributes to variations in human LDL-C levels. METHODS: In the present study, using high throughput sequencing we identified three non-synonymous (NS) variations and 64 synonymous variations in the NPC1L1 gene from subsets of Chinese Han, Uygur and Kazakh populations with high or low LDL-C. Subsequently, three NS variations encoding R174H, V177I and V1284L substitutions were observed only in Uygur and Kazakh individuals with limited maximal plasma LDL-C levels. RESULTS: In further experiments, we investigated cholesterol-regulated recycling and glycosylation and stability of these NS NPC1L1 variants. However, no significant differences between WT and variant NPC1L1 proteins were observed using in vivo assays in mouse livers with adenovirus-mediated expression, demonstrating that none of the three NPC1L1 NS variants caused decreased uptake of biliary cholesterol. CONCLUSIONS: Simultaneously, these data indicate that R174H, V177I and V1284L NPC1L1 variations in high or low LDL-C individuals may not directly influence cholesterol absorption by NPC1L1.

Genome editing with CRISPR/Cas9 in postnatal mice corrects PRKAG2 cardiac syndrome.

PRKAG2 cardiac syndrome is an autosomal dominant inherited disease resulted from mutations in the PRKAG2 gene that encodes γ2 regulatory subunit of AMP-activated protein kinase. Affected patients usually develop ventricular tachyarrhythmia and experience progressive heart failure that is refractory to medical treatment and requires cardiac transplantation. In this study, we identify a H530R mutation in PRKAG2 from patients with familial Wolff-Parkinson-White syndrome. By generating H530R PRKAG2 transgenic and knock-in mice, we show that both models recapitulate human symptoms including cardiac hypertrophy and glycogen storage, confirming that the H530R mutation is causally related to PRKAG2 cardiac syndrome. We further combine adeno-associated virus-9 (AAV9) and the CRISPR/Cas9 gene-editing system to disrupt the mutant PRKAG2 allele encoding H530R while leaving the wild-type allele intact. A single systemic injection of AAV9-Cas9/sgRNA at postnatal day 4 or day 42 substantially restores the morphology and function of the heart in H530R PRKAG2 transgenic and knock-in mice. Together, our work suggests that in vivo CRISPR/Cas9 genome editing is an effective tool in the treatment of PRKAG2 cardiac syndrome and other dominant inherited cardiac diseases by selectively disrupting disease-causing mutations.

Numb directs the subcellular localization of EAAT3 through binding the YxNxxF motif.

Excitatory amino acid transporter type 3 (EAAT3, also known as SLC1A1) is a high-affinity, Na(+)-dependent glutamate carrier that localizes primarily within the cell and at the apical plasma membrane. Although previous studies have reported proteins and sequence regions involved in EAAT3 trafficking, the detailed molecular mechanism by which EAAT3 is distributed to the correct location still remains elusive. Here, we identify that the YVNGGF sequence in the C-terminus of EAAT3 is responsible for its intracellular localization and apical sorting in rat hepatoma cells CRL1601 and Madin-Darby canine kidney (MDCK) cells, respectively. We further demonstrate that Numb, a clathrin adaptor protein, directly binds the YVNGGF motif and regulates the localization of EAAT3. Mutation of Y503, N505 and F508 within the YVNGGF motif to alanine residues or silencing Numb by use of small interfering RNA (siRNA) results in the aberrant localization of EAAT3. Moreover, both Numb and the YVNGGF motif mediate EAAT3 endocytosis in CRL1601 cells. In summary, our study suggests that Numb is a pivotal adaptor protein that mediates the subcellular localization of EAAT3 through binding the YxNxxF (where x stands for any amino acid) motif.

Potentiating the antitumour response of CD8(+) T cells by modulating cholesterol metabolism.

CD8(+) T cells have a central role in antitumour immunity, but their activity is suppressed in the tumour microenvironment. Reactivating the cytotoxicity of CD8(+) T cells is of great clinical interest in cancer immunotherapy. Here we report a new mechanism by which the antitumour response of mouse CD8(+) T cells can be potentiated by modulating cholesterol metabolism. Inhibiting cholesterol esterification in T cells by genetic ablation or pharmacological inhibition of ACAT1, a key cholesterol esterification enzyme, led to potentiated effector function and enhanced proliferation of CD8(+) but not CD4(+) T cells. This is due to the increase in the plasma membrane cholesterol level of CD8(+) T cells, which causes enhanced T-cell receptor clustering and signalling as well as more efficient formation of the immunological synapse. ACAT1-deficient CD8(+) T cells were better than wild-type CD8(+) T cells at controlling melanoma growth and metastasis in mice. We used the ACAT inhibitor avasimibe, which was previously tested in clinical trials for treating atherosclerosis and showed a good human safety profile, to treat melanoma in mice and observed a good antitumour effect. A combined therapy of avasimibe plus an anti-PD-1 antibody showed better efficacy than monotherapies in controlling tumour progression. ACAT1, an established target for atherosclerosis, is therefore also a potential target for cancer immunotherapy.

Design and synthesis of simple, yet potent and selective non-ring-A pyripyropene A-based inhibitors of acyl-coenzyme A: cholesterol acyltransferase 2 (ACAT2).

A series of pyripyropene A-based compounds were designed and synthesized by opening the upper section of the A-ring, which significantly simplifies the structure and synthesis from commercially available starting materials. Representative compound (-)-3 exhibited potent activity against ACAT2 and greater selectivity for ACAT2 than for ACAT1.