RAB21 controls autophagy and cellular energy homeostasis by regulating retromer-mediated recycling of SLC2A1/GLUT1.

The endosomal system maintains cellular homeostasis by coordinating multiple vesicular trafficking events, and the retromer complex plays a critical role in endosomal cargo recognition and sorting. Here, we demonstrate an essential role for the small GTPase RAB21 in regulating retromer-mediated recycling of the glucose transporter SLC2A1/GLUT1 and macroautophagy/autophagy. RAB21 depletion mis-sorts SLC2A1 to lysosomes and affects glucose uptake, thereby activating the AMPK-ULK1 pathway to increase autophagic flux. RAB21 depletion also increases lysosome function. Notably, RAB21 depletion does not overtly affect retrograde transport of IGF2R/CI-M6PR or WLS from endosomes to the trans-Golgi network. We speculate that RAB21 regulates fission of retromer-decorated endosomal tubules, as RAB21 depletion causes accumulation of the SNX27-containing retromer complex on enlarged endosomes at the perinuclear region. Functionally, RAB21 depletion sensitizes cancer cells to energy stress and inhibits tumor growth in vivo, suggesting an oncogenic role for RAB21. Overall, our study illuminates the role of RAB21 in regulating endosomal dynamics and maintaining cellular energy homeostasis and suggests RAB21 as a potential metabolic target for cancer therapy.

Vitamin D-VDR (vitamin D receptor) regulates defective autophagy in renal tubular epithelial cell in streptozotocin-induced diabetic mice via the AMPK pathway.

Diabetic nephropathy (DN) has become a major cause of end-stage renal disease, and autophagy disorder is implicated in the pathogenesis of DN. Our previous studies found that vitamin D (VD) and VDR (vitamin D receptor) played a renoprotective role by inhibiting inflammation and fibrosis. However, whether VD-VDR regulates autophagy disorders in DN remains unclear. In this study, we established a streptozotocin (STZ)-induced diabetic model in vdr knockout (vdr-KO) mice and VDR specifically overexpressed in renal proximal tubular epithelial cells (Vdr-OE) mice. Our results showed that paricalcitol (an activated vitamin D analog) or Vdr-OE could alleviate STZ-induced ALB (albumin) excretion, renal tubule injury and inflammation, while these were worsened in vdr-KO mice. Defective autophagy was observed in the kidneys of STZ mice, which was more pronounced in vdr-KO mice and could be partially restored by paricalcitol or Vdr-OE. In high glucose-induced HK-2 cells, defective autophagy and decreased PRKAA1/AMPK phosphorylation was observed, which could be partially restored by paricalcitol in a VDR-dependent manner. AMPK inhibitor abolished paricalcitol-induced autophagy activation, and AMPK activator restored the defective autophagy in high glucose-induced HK-2 cells. Furthermore, paricalcitol-mediated AMPK activation was abrogated by CAMKK2/CaMKKß inhibition, but not by STK11/LKB1 knockout. Meanwhile, paricalcitol rescued the decreased Ca2+ concentration induced by high glucose. In conclusion, VD-VDR can restore defective autophagy in the kidney of STZ-induced diabetic mice, which could be attributed to the activation of the Ca2+-CAMKK2-AMPK pathway in renal tubular epithelial cells.Abbreviations: ACTB/ß-actin: actin beta;AGE: advanced glycation end-products;AMPK: AMP-activated protein kinase;CAMKK2/CaMKKß: calcium-calmodulin dependent protein kinase kinase 2;CQ: chloroquine;DN: diabetic nephropathy;HG: high levels of glucose;KO: knockout;LG: low levels of glucose;MAP1LC3/LC3: microtubule associated protein 1 light chain 3;NOD2: nucleotide binding oligomerization domain containing 2;OE: overexpression;PAS: periodic acid Schiff; Pari: paricalcitol;PTECs: proximal renal tubule epithelial cells;RT: room temperature;SQSTM1/p62: sequestosome 1;STK11/LKB1: serine/threonine kinase 11;STZ: streptozotocin;TEM: transmission electron microscopy;VD: vitamin D;VDR: vitamin D receptor;WT: wild-type.

ATP13A2 facilitates HDAC6 recruitment to lysosome to promote autophagosome-lysosome fusion.

Mutations in ATP13A2 cause Kufor-Rakeb syndrome, an autosomal recessive form of juvenile-onset atypical Parkinson's disease (PD). Recent work tied ATP13A2 to autophagy and other cellular features of neurodegeneration, but how ATP13A2 governs numerous cellular functions in PD pathogenesis is not understood. In this study, the ATP13A2-deficient mouse developed into aging-dependent phenotypes resembling those of autophagy impairment. ATP13A2 deficiency impaired autophagosome-lysosome fusion in cultured cells and in in vitro reconstitution assays. In ATP13A2-deficient cells or Drosophila melanogaster or mouse tissues, lysosomal localization and activity of HDAC6 were reduced, with increased acetylation of tubulin and cortactin. Wild-type HDAC6, but not a deacetylase-inactive mutant, restored autophagosome-lysosome fusion, antagonized cortactin hyperacetylation, and promoted lysosomal localization of cortactin in ATP13A2-deficient cells. Mechanistically, ATP13A2 facilitated recruitment of HDAC6 and cortactin to lysosomes. Cortactin overexpression in cultured cells reversed ATP13A2 deficiency-associated impairment of autophagosome-lysosome fusion. PD-causing ATP13A2 mutants failed to rescue autophagosome-lysosome fusion or to promote degradation of protein aggregates and damaged mitochondria. These results suggest that ATP13A2 recruits HDAC6 to lysosomes to deacetylate cortactin and promotes autophagosome-lysosome fusion and autophagy. This study identifies ATP13A2 as an essential molecular component for normal autophagy flux in vivo and implies potential treatments targeting HDAC6-mediated autophagy for PD.

Cardiopulmonary effects of overnight indoor air filtration in healthy non-smoking adults: A double-blind randomized crossover study.

BACKGROUND: More than 90% of the world's population lives in areas where outdoor air pollution levels exceed health-based limits. In these areas, individuals may use indoor air filtration, often on a sporadic basis, in their residences to reduce exposure to respirable particles (PM2.5). Whether this intervention can lead to improvements in health outcomes has not been evaluated. METHODS: Seventy non-smoking healthy adults, aged 19 to 26 years, received both true and sham indoor air filtration in a double-blinded randomized crossover study. Each filtration session was approximately 13 h long. True and sham filtration sessions were separated by a two-week washout interval. The study was carried out in a suburb of Shanghai. RESULTS: During the study period, outdoor PM2.5 concentrations ranged from 18.6 to 106.9 µg/m3, which overlapped with levels measured in Western Europe and North America. Compared to sham filtration, true filtration on average decreased indoor PM2.5 concentration by 72.4% to 10.0 µg/m3 and particle number concentration by 59.2% to 2316/cm3. For lung function measured immediately after the end of filtration, true filtration significantly lowered airway impedance at 5 Hz (Z5) by 7.1% [95% CI: 2.4%, 11.9%], airway resistance at 5 Hz (R5) by 7.4% [95% CI: 2.4%, 12.5%], and small airway resistance (R5-R20) by 20.3% [95% CI: 0.1%, 40.5%], reflecting improved airway mechanics especially for the small airways. However, no significant improvements for spirometry indicators (FEV1, FVC) were observed. True filtration also significantly lowered von Willebrand factor (VWF) by 26.9% [95% CI: 7.3%, 46.4%] 24 h after the end of filtration, indicating reduced risk for thrombosis. Stratified analysis in male and female participants showed that true filtration significantly decreased pulse pressure by 3.3% [95% CI: 0.8%, 7.4%] in females, and significantly reduced VWF by 42.4% [95% CI: 17.4%, 67.4%] and interleukin-6 by 22.6% [95% CI: 0.4%, 44.9%] in males. Effect modification analyses indicated that filtration effects in male and female participants were not significantly different. CONCLUSION: A single overnight residential air filtration, capable of reducing indoor particle concentrations substantially, can lead to improved airway mechanics and reduced thrombosis risk.

PINK1-PRKN/PARK2 pathway of mitophagy is activated to protect against renal ischemia-reperfusion injury.

Damaged or dysfunctional mitochondria are toxic to the cell by producing reactive oxygen species and releasing cell death factors. Therefore, timely removal of these organelles is critical to cellular homeostasis and viability. Mitophagy is the mechanism of selective degradation of mitochondria via autophagy. The significance of mitophagy in kidney diseases, including ischemic acute kidney injury (AKI), has yet to be established, and the involved pathway of mitophagy remains poorly understood. Here, we show that mitophagy is induced in renal proximal tubular cells in both in vitro and in vivo models of ischemic AKI. Mitophagy under these conditions is abrogated by Pink1 and Park2 deficiency, supporting a critical role of the PINK1-PARK2 pathway in tubular cell mitophagy. Moreover, ischemic AKI is aggravated in pink1 andpark2 single- as well as double-knockout mice. Mechanistically, Pink1 and Park2 deficiency enhances mitochondrial damage, reactive oxygen species production, and inflammatory response. Taken together, these results indicate that PINK1-PARK2-mediated mitophagy plays an important role in mitochondrial quality control, tubular cell survival, and renal function during AKI.

Taxifolin enhances osteogenic differentiation of human bone marrow mesenchymal stem cells partially via NF-κB pathway.

Taxifolin, a flavonoid compound, has been reported to stimulate osteogenic differentiation in osteoblasts. The present study investigated whether taxifolin affects the osteogenic differentiation of human bone marrow mesenchymal stem cells (hBMSCs) and the molecular mechanisms involved. The proliferation and osteogenic differentiation of hBMSCs in the presence of taxifolin were examined by CCK-8 assay, alkaline phosphatase (ALP) activity, ALP staining and Alizarin red staining. The expression of osteogenic differentiation markers were detected by real-time quantitative PCR (RT-PCR) analysis and western blot assay. The activation of potential related pathways was examined by luciferase reporter assay, immunofluorescence and western blot analysis. Taxifolin treatment increased osteogenic differentiation of hBMSCs without cytotoxicity. Luciferase reporter assay showed that taxifolin could not activate estrogen receptor pathway, but inhibit TNF-α-induced NF-κB signaling pathway activation in osteogenic induction condition. Moreover, the nucleus translocation of NF-κB under TNF-α treatment was inhibited by taxifolin treatment. The taxifolin-induced osteogenic differentiation effects of hBMSCs were abolished by TNF-α treatment. In conclusion, our results suggested that taxifolin could promote osteogenesis of hBMSCs, partially through antagonism of NF-κB signaling pathway.

BNIP3 Protein Suppresses PINK1 Kinase Proteolytic Cleavage to Promote Mitophagy.

Mutations in PINK1 (PTEN-induced putative kinase 1) cause early onset familial Parkinson's disease (PD). PINK1 accumulates on the outer membrane of damaged mitochondria followed by recruiting parkin to promote mitophagy. Here, we demonstrate that BCL2/adenovirus E1B 19-kDa interacting protein 3 (BNIP3), a mitochondrial BH3-only protein, interacts with PINK1 to promote the accumulation of full-length PINK1 on the outer membrane of mitochondria, which facilitates parkin recruitment and PINK1/parkin-mediated mitophagy. Inactivation of BNIP3 in mammalian cells promotes PINK1 proteolytic processing and suppresses PINK1/parkin-mediated mitophagy. Hypoxia-induced BNIP3 expression results in increased expression of full-length PINK1 and mitophagy. Consistently, expression of BNIP3 in Drosophila suppresses muscle degeneration and the mitochondrial abnormality caused by PINK1 inactivation. Together, the results suggest that BNIP3 plays a vital role in regulating PINK1 mitochondrial outer membrane localization, the proteolytic process of PINK1 and PINK1/parkin-mediated mitophagy under physiological conditions. Functional up-regulation of BNIP3 may represent a novel therapeutic strategy to suppress the progression of PD.

PINK1/Parkin-mediated mitophagy alleviates chlorpyrifos-induced apoptosis in SH-SY5Y cells.

Chlorpyrifos (CPF) is one of the most widely used organophosphorous insecticides. There are links between CPF exposure and neurological disorders. Mitochondrial damage has been implicated to play a key role in CPF-induced neurotoxicity. Mitophagy, the selective autophagic elimination of mitochondria, is an important mitochondrial quality control mechanism. However, the role of mitophagy in CPF-induced neurotoxicity remains unclear. In this study, CPF-caused mitochondrial damage, role and mechanism of mitophagy on CPF-induced neuroapoptosis were extensively studied by using SH-SY5Y cells. We showed that CPF treatment caused mitochondrial fragmentation, excessive ROS generation and mitochondrial depolarization, thus led to cell apoptosis. Moreover, CPF treatment also resulted in increased colocalizaton of mitochondria with LC3, decreased levels of mitochondrial proteins, PINK1 stabilization and mitochondrial accumulation of Parkin. These data suggested that CPF treatment induced PINK1/Parkin-mediated mitophagy in SH-SY5Y cells. Furthermore, knockdown of Parkin dramatically increased CPF-induced neuroapoptosis. On the other hand, overexpression of Parkin markedly alleviated CPF-induced SH-SY5Y cell apoptosis. Together, these findings implicate a protective role of PINK1/Parkin-mediated mitophagy against neuroapoptosis and that enhancing mitophagy provides a potential therapeutic strategy for CPF-induced neurological disorders.

Baicalein induces human osteosarcoma cell line MG-63 apoptosis via ROS-induced BNIP3 expression.

Baicalein, a flavonoid compound, is one of the active constituents of the root of Scutellariae Radix. Its antitumor effects have attracted widespread attention worldwide. One of its major functions is to induce the apoptosis of tumor cells, but the antitumor mechanism is currently unclear. In the present study, we found that baicalein increased MG-63 cell mortality in a dose-dependent manner. Meanwhile, baicalein activated apoptosis through induced intracellular reactive oxygen species (ROS) generation, and that ROS scavenger N-acetyl-cysteine (NAC), glutathione (GSH), and superoxide dismutase (SOD) apparently inhibited intracellular ROS production, consequently attenuating the baicalein-induced apoptosis. Baicalein also induce the mitochondrial fragmentation which precedes the cell apoptosis. This morphological alteration is accompanied by an increase in the expression of the protein BNIP3 as well as Mul1 and Drp1. Furthermore, we show that the inhibition of BNIP3 expression can inhibit cell apoptosis by baicalein treatment. Taken together, our results bring the evidence of a mechanism that links apoptosis and ROS-induced BNIP3 expression in MG-63 cells with bacalein treatment and suggest that baicalein has a good potential as an anti-osteosarcoma drug.