In 2008 we published the first set of guidelines for standardizing research in autophagy. Since then, research on this topic has continued to accelerate, and many new scientists have entered the field. Our knowledge base and relevant new technologies have also been expanding. Accordingly, it is important to update these guidelines for monitoring autophagy in different organisms. Various reviews have described the range of assays that have been used for this purpose. Nevertheless, there continues to be confusion regarding acceptable methods to measure autophagy, especially in multicellular eukaryotes. A key point that needs to be emphasized is that there is a difference between measurements that monitor the numbers or volume of autophagic elements (e.g., autophagosomes or autolysosomes) at any stage of the autophagic process vs. those that measure flux through the autophagy pathway (i.e., the complete process); thus, a block in macroautophagy that results in autophagosome accumulation needs to be differentiated from stimuli that result in increased autophagic activity, defined as increased autophagy induction coupled with increased delivery to, and degradation within, lysosomes (in most higher eukaryotes and some protists such as Dictyostelium) or the vacuole (in plants and fungi). In other words, it is especially important that investigators new to the field understand that the appearance of more autophagosomes does not necessarily equate with more autophagy. In fact, in many cases, autophagosomes accumulate because of a block in trafficking to lysosomes without a concomitant change in autophagosome biogenesis, whereas an increase in autolysosomes may reflect a reduction in degradative activity. Here, we present a set of guidelines for the selection and interpretation of methods for use by investigators who aim to examine macroautophagy and related processes, as well as for reviewers who need to provide realistic and reasonable critiques of papers that are focused on these processes. These guidelines are not meant to be a formulaic set of rules, because the appropriate assays depend in part on the question being asked and the system being used. In addition, we emphasize that no individual assay is guaranteed to be the most appropriate one in every situation, and we strongly recommend the use of multiple assays to monitor autophagy. In these guidelines, we consider these various methods of assessing autophagy and what information can, or cannot, be obtained from them. Finally, by discussing the merits and limits of particular autophagy assays, we hope to encourage technical innovation in the field.

Guidelines for the use and interpretatoin of assays for monitoring autophagy

Ferroptosis is recently identified, an iron- and reactive oxygen species- (ROS-) dependent form of regulated cell death. This study was designed to determine the existence of ferroptosis in the pathogenesis of type 2 diabetic osteoporosis and confirm that melatonin can inhibit the ferroptosis of osteoblasts through activating Nrf2/HO-1 signaling pathway to improve bone microstructure in vivo and in vitro. We treated MC3T3-E1 cells with different concentrations of melatonin (1, 10, or 100 μM) and exposed them to high glucose (25.5 mM) for 48 h in vitro. Our data showed that high glucose can induce osteoblast cytotoxicity and the accumulation of lipid peroxide, the mitochondria of osteoblast show the same morphology changes as the erastin treatment group, and the expression of ferroptosis-related proteins glutathione peroxidase 4 (GPX4) and cystine-glutamate antiporter (SLC7A11) is downregulated, but these effects were reversed by ferroptosis inhibitor ferrastatin-1 and iron chelator deferoxamine (DFO). Furthermore, western blot and real-time polymerase chain reaction were used to detect the expression levels of nuclear factor erythroid 2-related factor 2 (Nrf2) and heme oxygenase-1 (HO-1); osteogenic capacity was evaluated by alizarin red S staining and the expression of osteoprotegerin, osteocalcin, and alkaline phosphatase; the results showed that the expression levels of these proteins in osteoblasts with 1, 10, or 100 μM melatonins were significantly higher than the high glucose group, but after using Nrf2-SiRNA interference, the therapeutic effect of melatonin was significantly inhibited. We also performed in vivo experiments in a diabetic rat model treated with two concentrations of melatonin (10, 50 mg/kg). Dynamic bone histomorphometry and micro-CT were used to observe the rat bone microstructure, and the expression of GPX4 and Nrf2 was determined by immunohistochemistry. Here, we first report that high glucose induces ferroptosis via increased ROS/lipid peroxidation/glutathione depletion in type 2 diabetic osteoporosis. More importantly, melatonin significantly reduced the level of ferroptosis and improved the osteogenic capacity of MC3T3-E1 through activating the Nrf2/HO-1 pathway in vivo and in vitro.

/pdf/melatonin-suppresses-ferroptosis-induced-by-high-glucose-via-z3r0mhd23e.pdf

Melatonin Suppresses Ferroptosis Induced by High Glucose via Activation of the Nrf2/HO-1 Signaling Pathway in Type 2 Diabetic Osteoporosis

Bone metabolic disorders include osteolysis, osteoporosis, osteoarthritis and rheumatoid arthritis. Osteoblasts and osteoclasts are two major types of cells in bone constituting homeostasis. The imbalance between bone formation by osteoblasts and bone resorption by osteoclasts has been shown to have a direct contribution to the onset of these diseases. Recent evidence indicates that autophagy and mitophagy, the selective autophagy of mitochondria, may play a vital role in regulating the proliferation, differentiation and function of osteoblasts and osteoclasts. Several signaling pathways, including PINK1/Parkin, SIRT1, MAPK8/FOXO3, Beclin-1/BECN1, p62/SQSTM1, and mTOR pathways, have been implied in the regulation of autophagy and mitophagy in these cells. Here we review the current progress about the regulation of autophagy and mitophagy in osteoblasts and osteoclasts in these bone metabolic disorders, as well as the molecular signaling activated or deactivated during this process. Together, we hope to draw attention to the role of autophagy and mitophagy in bone metabolic disorders, and their potential as a new target for the treatment of bone metabolic diseases and the requirements of further mechanism studies.

/pdf/the-role-of-autophagy-and-mitophagy-in-bone-metabolic-3jx4qzu9om.pdf

The Role of Autophagy and Mitophagy in Bone Metabolic Disorders.

Kaempferol has been shown to protect cells against cerebral ischemia/reperfusion injury through inhibition of apoptosis. In the present study, we sought to investigate whether ferroptosis is involved in the oxygen-glucose deprivation/reperfusion (OGD/R)-induced neuronal injury and the effects of kaempferol on ferroptosis in OGD/R-treated neurons. Western blot, immunofluorescence, and transmission electron microscopy were used to analyze ferroptosis, whereas cell death was detected using lactate dehydrogenase (LDH) release. We found that OGD/R attenuated SLC7A11 and glutathione peroxidase 4 (GPX4) levels as well as decreased endogenous antioxidants including nicotinamide adenine dinucleotide phosphate (NADPH), glutathione (GSH), and superoxide dismutase (SOD) in neurons. Notably, OGD/R enhanced the accumulation of lipid peroxidation, leading to the induction of ferroptosis in neurons. However, kaempferol activated nuclear factor-E2-related factor 2 (Nrf2)/SLC7A11/GPX4 signaling, augmented antioxidant capacity, and suppressed the accumulation of lipid peroxidation in OGD/R-treated neurons. Furthermore, kaempferol significantly reversed OGD/R-induced ferroptosis. Nevertheless, inhibition of Nrf2 by ML385 blocked the protective effects of kaempferol on antioxidant capacity, lipid peroxidation, and ferroptosis in OGD/R-treated neurons. These results suggest that ferroptosis may be a significant cause of cell death associated with OGD/R. Kaempferol provides protection from OGD/R-induced ferroptosis partly by activating Nrf2/SLC7A11/GPX4 signaling pathway.

https://www.mdpi.com/2218-273X/11/7/923/pdf

Kaempferol Ameliorates Oxygen-Glucose Deprivation/Reoxygenation-Induced Neuronal Ferroptosis by Activating Nrf2/SLC7A11/GPX4 Axis

Cetuximab is approved for the treatment of metastatic colorectal cancer (mCRC) with RAS wild-type. Nevertheless, the prognosis remains poor and the effectiveness of cetuximab is limited in KRAS mutant mCRC. Recently, emerging evidence has shown that ferroptosis, a newly discovered form of nonapoptotic cell death, is closely related to KRAS mutant cells. Here, we further investigated whether cetuximab-mediated regulation of p38/Nrf2/HO-1 promotes RSL3-induced ferroptosis and plays a pivotal role in overcoming drug resistance in KRAS mutant colorectal cancer (CRC). In our research, we used two KRAS mutant CRC cell lines, HCT116 and DLD-1, as models of intrinsic resistance to cetuximab. The viability of cells treated with the combination of RSL3 and cetuximab was assessed by the CCK-8 and colony formation assays. The effective of cetuximab to promote RSL3-induced ferroptosis was investigated by evaluating lipid reactive oxygen species accumulation and the expression of the malondialdehyde and the intracellular iron assay. Cetuximab therapy contributed to regulating the p38/Nrf2/HO-1 axis, as determined by western blotting and transfection with small interfering RNAs. Cetuximab promoted RSL3-induced ferroptosis by inhibiting the Nrf2/HO-1 in KRAS mutant CRC cells, and this was further demonstrated in a xenograft nude mouse model. Our work reveals that cetuximab enhances the cytotoxic effect of RSL3 on KRAS mutant CRC cells and that cetuximab enhances RSL3-induced ferroptosis by inhibiting the Nrf2/HO-1 axis through the activation of p38 MAPK.

Cetuximab promotes RSL3-induced ferroptosis by suppressing the Nrf2/HO-1 signalling pathway in KRAS mutant colorectal cancer.

High glucose downregulates the effects of autophagy on osteoclastogenesis via the AMPK/mTOR/ULK1 pathway.

The highly selective magnesium transporter non-imprinted in Prader-Willi/Angelman syndrome region protein 2 (NIPA2) has recently been associated with the development and progression of type 2 diabetes osteoporosis, but the mechanisms involved are still poorly understood. Because mitophagy is involved in the pathology of type 2 diabetes osteoporosis, the present study aimed to explore the relationship among NIPA2, mitophagy and osteoblast osteogenic capacity. NIPA2 expression was reduced in C57BKS background db/db mice and in vitro models of type 2 diabetes osteoporosis, and the activation of mitophagy in primary culture osteoblast-derived from db/db mice and in high glucose-treated human fetal osteoblastic cells (hFOB1.19) was observed. Knockdown, overexpression of NIPA2 and pharmacological inhibition of peroxisome proliferator-activated receptor γ coactivator 1-α (PGC-1α) showed that NIPA2 increased osteoblast function, which was likely regulated by PTEN induced kinase 1 (PINK1)/E3 ubiquitin ligase PARK2 (Parkin)-mediated mitophagy via the PGC-1α/forkhead box O3a(FoxO3a)/mitochondrial membrane potential (MMP) pathway. Furthermore, the negative effect of mitophagy on osteoblast function was confirmed by pharmacological regulation of mitophagy and knockdown of Parkin. Taken together, these results suggest that NIPA2 positively regulates the osteogenic capacity of osteoblasts via the mitophagy pathway in type 2 diabetes.

/pdf/nipa2-regulates-osteoblast-function-by-modulating-mitophagy-4ow3rxvskf.pdf

NIPA2 regulates osteoblast function by modulating mitophagy in type 2 diabetes osteoporosis

Alzheimer's disease (AD) and osteoporosis (OP) are 2 common progressive age‑associated diseases, primarily affecting the elderly worldwide. Accumulating evidence has demonstrated that patients with AD are more likely to suffer from bone mass loss and even OP, but whether it is a pathological feature of AD or secondary to motor dysfunction remains poorly understood. The present study aimed to investigate whether amyloid‑β1‑42 (Aβ1‑42), the typical pathological product of AD, exhibited a negative effect on the proliferation of bone marrow mesenchymal stem cells (BMSCs) and the role of autophagy. The proliferation of BMSCs was measured using a Cell Counting Kit‑8 assay, cell cycle analysis and 5‑ethynyl‑2'‑deoxyuridine (EdU) staining. The autophagy‑associated proteins microtubule‑associated proteins 1A/1B light chain 3B and sequestosome 1 (p62) were evaluated by western blot analysis and autophagosomes were detected by transmission electron microscopy and immunofluorescence. The activity of the protein kinase B (AKT)/mammalian target of rapamycin (mTOR) signaling pathway was measured using western blot analysis, and the autophagy inducer rapamycin (RAPA), inhibitor 3‑methyladenine (3‑MA) and the AKT activator SC79 were also used to investigate the role of AKT/mTOR signaling pathway and autophagy in the proliferation of BMSCs. The results suggested that the proliferation of BMSCs treated with Aβ1‑42 was inhibited, with the autophagy level increasing following treatment with Aβ1‑42 in a dose‑dependent manner, while the AKT/mTOR signaling pathway participated in the regulation of the autophagy level. Activation of autophagy using RAPA inhibited the decrease in proliferation of BMSCs, while suppression of autophagy by 3‑MA and activation of the AKT/mTOR signaling pathway increased the decrease in proliferation of BMSCs caused by Aβ1‑42. It was concluded that Aβ1‑42, as an external stimulus, suppressed the proliferation of BMSCs directly and that the AKT/mTOR signaling pathway participated in the regulation of the level of autophagy. Concomitantly, autophagy may serve as a resistance mechanism in inhibiting the decreased proliferation of BMSCs treated with Aβ1‑42.

/pdf/autophagy-alleviates-the-decrease-in-proliferation-of-zv3ynswm9p.pdf

Wei Zhao

Papers

Melatonin Suppresses Ferroptosis Induced by High Glucose via Activation of the Nrf2/HO-1 Signaling Pathway in Type 2 Diabetic Osteoporosis

High glucose downregulates the effects of autophagy on osteoclastogenesis via the AMPK/mTOR/ULK1 pathway.

NIPA2 regulates osteoblast function by modulating mitophagy in type 2 diabetes osteoporosis

Autophagy alleviates the decrease in proliferation of amyloid β1‑42‑treated bone marrow mesenchymal stem cells via the AKT/mTOR signaling pathway.

NIPA2 regulates osteoblast function via its effect on apoptosis pathways in type 2 diabetes osteoporosis.