LC3 Protein Research Articles

Tripartite motif 25 inhibits protein aggregate degradation during PRRSV infection by suppressing p62-mediated autophagy.

Viral infection causes endoplasmic reticulum stress and protein metabolism disorder, influencing protein aggregates formation or degradation that originate from misfolded proteins. The mechanism by which host proteins are involved in the above process remains largely unknown. The present study found that porcine reproductive and respiratory syndrome virus (PRRSV) infection promoted the degradation of intracellular ubiquitinated protein aggregates via activating autophagy. The host cell E3 ligase tripartite motif-containing (TRIM)25 promoted the recruitment and aggregation of polyubiquitinated proteins and impeded their degradation caused by PRRSV. TRIM25 interacted with ubiquitinated aggregates and was part of the aggregates complex. Next, the present study investigated the mechanisms by which TRIM25 inhibited the degradation of protein aggregates, and it was found that TRIM25 interacted with both Kelch-like ECH-associated protein 1 (KEAP1) and nuclear factor E2-related factor 2 (Nrf2), facilitated the nuclear translocation of Nrf2 by targeting KEAP1 for K48-linked ubiquitination and proteasome degradation, and activated Nrf2-mediated p62 expression. Further studies indicated that TRIM25 interacted with p62 and promoted its K63-linked ubiquitination via its E3 ligase activity and thus caused impairment of its oligomerization, aggregation, and recruitment for the autophagic protein LC3, leading to the suppression of autophagy activation. Besides, TRIM25 also suppressed the p62-mediated recruitment of ubiquitinated aggregates. Activation of autophagy decreased the accumulation of protein aggregates caused by TRIM25 overexpression, and inhibition of autophagy decreased the degradation of protein aggregates caused by TRIM25 knockdown. The current results also showed that TRIM25 inhibited PRRSV replication by inhibiting the KEAP1-Nrf2-p62 axis-mediated autophagy. Taken together, the present findings showed that the PRRSV replication restriction factor TRIM25 inhibited the degradation of ubiquitinated protein aggregates during viral infection by suppressing p62-mediated autophagy.IMPORTANCESequestration of protein aggregates and their subsequent degradation prevents proteostasis imbalance and cytotoxicity. The mechanisms controlling the turnover of protein aggregates during viral infection are mostly unknown. The present study found that porcine reproductive and respiratory syndrome virus (PRRSV) infection promoted the autophagic degradation of ubiquitinated protein aggregates, whereas tripartite motif-containing (TRIM)25 reversed this process. It was also found that TRIM25 promoted the expression of p62 by activating the Kelch-like ECH-associated protein 1 (KEAP1) and nuclear factor E2-related factor 2 (Nrf2) pathway and simultaneously prevented the oligomerization of p62 by promoting its K63-linked ubiquitination, thus suppressing its recruitment of the autophagic adaptor protein LC3 and ubiquitinated aggregates, leading to the inhibition of PRRSV-induced autophagy activation and the autophagic degradation of protein aggregates. The present study identified a new mechanism of protein aggregate turnover during viral infection and provided new insights for understanding the pathogenic mechanism of PRRSV.

Morin Improves the Bone Histomorphology and Biochemical Markers in an Animal Model of Ovariectomy‐Induced Osteoporosis by Suppressing Autophagy and Apoptosis

ABSTRACTOsteoporosis (OP) is the most prevalent metabolic bone disease and an important postmenopausal consequence. This study aimed to investigate the effects of morin, a flavonoid with beneficial properties, on ovariectomy‐induced OP. Animals were ovariectomized (OVX) and treated with different doses of morin (15, 30, and 45 mg/kg/day) or estradiol (10 μg/kg/day) for 10 weeks by gavage. Then bone histo‐stereology, bone‐related biochemical indicators, and gene and protein levels of autophagy and apoptosis‐related markers were analyzed. In comparison to controls, OVX significantly decreased the number of osteoblasts (5.78 × 106 vs. 1.66 × 106) and osteocytes (32.55 × 106 vs. 11.92 × 106), whereas increasing the number of osteoclasts (83.38 × 103 vs. 392.1 × 103). Moreover, OVX caused a remarkable decrease in bone structures and Ca, P, and estradiol levels while increasing ALP and OC (p < 0.001). The administration of 45 mg/kg/day morin restored the effects of OP on bone histomorphology and biochemical markers (p < 0.05). Further studies revealed that morin caused a 7.1% and 36.6% decrease in the bone level of LC3 and BECN1 proteins, respectively, compared to the OVX group. Also, morin caused a significant decrease of 47.4% in the CASP3 level and a significant increase of 23.6% in the BCL‐2 level compared to OVX animals (p < 0.001). The present findings showed that morin is potentially able to improve the bone‐related histomorphological and biochemical changes caused by osteoporosis, which is probably attributed to the suppression of apoptosis‐ and autophagy‐caused cell death.

Apache is a neuronal player in autophagy required for retrograde axonal transport of autophagosomes

Neurons are dependent on efficient quality control mechanisms to maintain cellular homeostasis and function due to their polarization and long-life span. Autophagy is a lysosomal degradative pathway that provides nutrients during starvation and recycles damaged and/or aged proteins and organelles. In neurons, autophagosomes constitutively form in distal axons and at synapses and are trafficked retrogradely to the cell soma to fuse with lysosomes for cargo degradation. How the neuronal autophagy pathway is organized and controlled remains poorly understood. Several presynaptic endocytic proteins have been shown to regulate both synaptic vesicle recycling and autophagy. Here, by combining electron, fluorescence, and live imaging microscopy with biochemical analysis, we show that the neuron-specific protein APache, a presynaptic AP-2 interactor, functions in neurons as an important player in the autophagy process, regulating the retrograde transport of autophagosomes. We found that APache colocalizes and co-traffics with autophagosomes in primary cortical neurons and that induction of autophagy by mTOR inhibition increases LC3 and APache protein levels at synaptic boutons. APache silencing causes a blockade of autophagic flux preventing the clearance of p62/SQSTM1, leading to a severe accumulation of autophagosomes and amphisomes at synaptic terminals and along neurites due to defective retrograde transport of TrkB-containing signaling amphisomes along the axons. Together, our data identify APache as a regulator of the autophagic cycle, potentially in cooperation with AP-2, and hypothesize that its dysfunctions contribute to the early synaptic impairments in neurodegenerative conditions associated with impaired autophagy.

Small Molecules Inducing Autophagic Degradation of Expanded Polyglutamine Protein through Interaction with Both Mutant ATXN3 and LC3.

Polyglutamine (polyQ)-mediated spinocerebellar ataxia (SCA), including SCA1, 2, 3, 6, 7, and 17, are caused by mutant genes with expanded CAG repeats, leading to the intracellular accumulation of aggregated proteins, the production of reactive oxygen species, and cell death. Among SCA, SCA3 is caused by a mutation in the ATXN3 (ataxin-3) gene. In a circumstance of polyQ aggregation, the autophagic pathway is induced to degrade the aggregated proteins, thereby suppressing downstream deleterious effects and promoting neuronal survival. In this study, we tested the effects of synthetic indole (NC009-1, -2, -3, -6) and coumarin (LM-022, -031) derivatives as chemical chaperones to assist mutant ATXN3-Q75 folding, as well as autophagy inducers to clear aggregated protein. Among the tested compounds, NC009-1, -2, and -6 and LM-031 interfered with Escherichia coli-derived ATXN3-Q75 aggregation in thioflavin T binding and filter trap assays. In SH-SY5Y cells expressing GFP-fused ATXN3-Q75, these compounds displayed aggregation-inhibitory and neurite growth-promoting potentials compared to untreated cells. Furthermore, these compounds activated autophagy by increasing the phosphatidylethanolamine-conjugated LC3 (microtubule associated protein 1 light chain 3)-II:cytosolic LC3-I ratio in these cells. A biochemical co-immunoprecipitation assay by using a mixture of HEK 293T cell lysates containing recombinant ATXN3-Q75-Venus-C-terminus (VC) or Venus-N-terminus (VN)-LC3 protein indicated that NC009-1 and -2 and LM-031 served as an autophagosome-tethering compound (ATTEC) to interact with ATXN3-Q75 and LC3, and the interaction was further confirmed by bimolecular fluorescence complementation analysis in cells co-expressing both ATXN3-Q75-VC and VN-LC3 proteins. The study results suggest the potential of NC009-1 and -2 and LM-031 as an ATTEC in treating SCA3 and, probably, other polyQ diseases.

Experimental and proteomics evidence revealed the protective mechanisms of ShemaZhichuan Liquid in attenuating neutrophilic asthma

BackgroundNeutrophilic asthma (NA) is one of the most important phenotypes of non-Th2 asthma and is often insensitive to glucocorticoid therapy, making current treatment difficult. Shemazhichuan Liquid (SMZCL), a Chinese medicine compound preparation, has unique advantages in the treatment of asthma. However, the underlying mechanisms of SMZCL in treating NA are not fully understood. PurposeThe efficacy and underlying mechanisms of SMZCL on NA were investigated by TMT-labeled quantitative proteomics analysis and in vivo and in vitro experiments. MethodsNA mouse model was constructed by OVA/CFA sensitization followed by a 10-day challenge with 5% OVA. Lung histopathology, leukocyte counts and cell sorting counts, inflammatory cytokines levels, as well as expression of autophagy markers were then assessed. The specific pathways and proteins of SMZCL for treating NA were further illustrated through TMT-based quantitative proteomics. In addition, RAW264.7 cells were induced by LPS to further explore the mechanism of the main active ingredient of SMZCL on autophagy pathway. ResultsIn vivo, SMZCL contributed to attenuating airway inflammation and collagen disposition, markedly reduced the number of leukocytes, especially neutrophils in bronchoalveolar lavage fluid (BALF), as well as decreased IgE and inflammatory cytokine levels (TNF-α, IL-1β, IL-6 and IL-8) in BALF and serum. Besides, SMZCL elevated the levels of LC3 and ATG5 while inhibiting the expression of p62 and mTOR. Mzb1 and Rab3ip were identified as the critical overlapping DEPs whose expression was inhibited by SMZCL and rapamycin. KEGG enrichment analysis showed that necroptosis process was a key pathway for SMZCL to treat NA airway inflammation. IHC and WB results confirmed that SMZCL and rapamycin inhibited the phosphorylation of RIPK1, PIPK3 and MLKL. In vitro, ATG5 and LC3 proteins were obviously increased while p-mTOR expression was inhibited after amygdalin treatment. ConclusionSMZCL attenuated airway inflammation in NA mainly through inhibition of the mTOR pathway, along with inhibition of the necroptosis pathway regulated by the RIPK1/RIPK3/MLKL axis and inhibition of Mzb1 and Rab3ip expression.

Heme deficiency in skeletal muscle exacerbates sarcopenia and impairs autophagy by reducing AMPK signaling

Heme serves as a prosthetic group in hemoproteins, including subunits of the mammalian mitochondrial electron transfer chain. The first enzyme in vertebrate heme biosynthesis, 5-aminolevulinic acid synthase 1 (ALAS1), is ubiquitously expressed and essential for producing 5-aminolevulinic acid (ALA). We previously showed that Alas1 heterozygous mice at 20–35 weeks (aged-A1+/−s) manifested impaired glucose metabolism, mitochondrial malformation in skeletal muscle, and reduced exercise tolerance, potentially linked to autophagy dysfunction. In this study, we investigated autophagy in A1+/−s and a sarcopenic phenotype in A1+/−s at 75–95 weeks (senile-A1+/−s). Senile-A1+/−s exhibited significantly reduced body and gastrocnemius muscle weight, and muscle strength, indicating an accelerated sarcopenic phenotype. Decreases in total LC3 and LC3-II protein and Map1lc3a mRNA levels were observed in aged-A1+/−s under fasting conditions and in Alas1 knockdown myocyte-differentiated C2C12 cells (A1KD-C2C12s) cultured in high- or low-glucose medium. ALA treatment largely reversed these declines. Reduced AMP-activated protein kinase (AMPK) signaling was associated with decreased autophagy in aged-A1+/−s and A1KD-C2C12s. AMPK modulation using AICAR (activator) and dorsomorphin (inhibitor) affected LC3 protein levels in an AMPK-dependent manner. Our findings suggest that heme deficiency contributes to accelerated sarcopenia-like defects and reduced autophagy in skeletal muscle, primarily due to decreased AMPK signaling.

Association of GLOD4 with Alzheimer's Disease in Humans and Mice.

Glyoxalase domain containing protein 4 (GLOD4), a protein of an unknown function, is associated with Alzheimer's disease (AD). Three GLOD4 isoforms are known. The mechanism underlying GLOD4's association with AD was unknown. To assess GLOD4's role in the central nervous system by studying GLOD4 isoforms expression in human frontal cerebral cortical tissues from AD patients and in brains of Blmh-/-5xFAD mouse AD model of AD. GLOD4 protein and mRNA were quantified in human and mouse brains by western blotting and RT-qPCR, respectively. Mouse brain amyloid-β (Aβ) was quantified by western blotting. Behavioral assessments of mice were performed by cognitive/neuromotor testing. Glod4 gene in mouse neuroblastoma N2a-APPswe cells was silenced by RNA interference and Glod4, Aβ precursor protein (Aβpp), Atg5, p62, and Lc3 proteins and mRNAs were quantified. GLOD4 mRNA and protein isoforms were downregulated in cortical tissues from AD patients compared to non-AD controls. Glod4 mRNA was downregulated in brains of Blmh-/-5xFAD mice compared to Blmh+/+5xFAD sibling controls, but not in Blmh-/- mice without the 5xFAD transgene compared to Blmh+/+ sibling controls. The 5xFAD transgene downregulated Glod4 mRNA in Blmh-/- mice of both sexes and in Blmh+/+ males but not females. Attenuated Glod4 was associated with elevated Aβ and worsened memory/sensorimotor performance in Blmh-/-5xFAD mice. Glod4 depletion in N2a-APPswe cells upregulated AβPP, and downregulated autophagy-related Atg5, p62, and Lc3 genes. These findings suggest that GLOD4 interacts with AβPP and the autophagy pathway, and that disruption of these interactions leads to Aβ accumulation and cognitive/neurosensory deficits.

TDRD3 functions as a selective autophagy receptor with dual roles in autophagy and modulation of stress granule stability.

Tudor Domain Containing 3 (TDRD3) is a methylarginine-reader protein that functions as a scaffold in the nucleus facilitating transcription, however TDRD3 is also recruited to stress granules (SGs) during the Integrated Stress Response (ISR) although its function therein remains largely unknown. We previously showed that TDRD3 is a novel antiviral restriction factor that is cleaved by virus 2A protease, and plays complex modulatory roles in both interferon and inflammatory signaling during stress and enterovirus infections. Here we have found that TDRD3 contains structural motifs similar to known selective autophagy receptors such as p62/SQSTM1, sharing ubiquitin associated domains (UBA) and LC3 interacting regions (LIR) that anchor cargo destined for autophagosomes to activated LC3 protein coating autophagosome membranes. This is of interest since enteroviruses hijack autophagy machinery to facilitate formation of viral replication factories, virus assembly and egress from the infected cell. Here we explored possible roles of TDRD3 in autophagy, hypothesizing that TDRD3 may function as a specialized selective autophagy receptor. We found that KO of TDRD3 in HeLa cells significantly reduces starvation induced autophagy, while its reintroduction restores it in a dose-dependent manner. Autophagy receptors are degraded during autophagy and expression levels decrease during this time. We found that TDRD3 levels decrease to the same extent as the autophagy receptor p62/SQSTM1 during autophagy, indicating autophagy-targeted turnover in that role. Knockout of TDRD3 or G3BP1 did not make significant changes in overall cell localization of LC3B or p62/SQSTM1, but did result in greater concentration of Lamp2 phagosome marker for phagosomes and phagolysosomes. To test the potential roles of TDRD3 in autophagic processes, we created a series of deletion mutants of TDRD3 lacking either UBA domain or the various LIR motifs that are predicted to interact with LC3B. Microscopic examination of starved cells expressing these variants of TDRD3 showed ΔLIR-TDRD3 had defects in colocalization with LC3B or Lamp2. Further, super resolution microscopy revealed ring structures with TDRD3 interfacing with p62/SQSTM1. In examination of arsenite induced stress granules we found recruitment of TDRD3 variants disrupted normally tight SG condensation, altered the decay rate of SGs upon release from stress and the kinetics of SG formation. We found evidence that the LIR3 motif on TDRD3 is involved in TDRD3 interaction with LC3B in coIP experiments, colocalization studies, and that this motif plays a key role in TDRD3 recruitment to SGs and SG resolution. Overall, these data support a functional role of TDRD3 in selective autophagy in a mode similar to p62/SQSTM1, with specific roles in SG stability and turnover. Enterovirus cleavage of TDRD3 likely affects both antiviral and autophagic responses that the virus controls for replication.

Coxsackievirus B3 Activates Macrophages Independently of CAR-Mediated Viral Entry.

Enteroviruses are a genus of small RNA viruses that are responsible for approximately one billion global infections annually. These infections range in severity from the common cold and flu-like symptoms to more severe diseases, such as viral myocarditis, pancreatitis, and neurological disorders, that continue to pose a global health challenge with limited therapeutic strategies currently available. In the current study, we sought to understand the interaction between coxsackievirus B3 (CVB3), which is a model enterovirus, and macrophage cells, as there is limited understanding of how this virus interacts with macrophage innate immune cells. Our study demonstrated that CVB3 can robustly activate macrophages without apparent viral replication in these cells. We also showed that myeloid cells lacked the viral entry receptor coxsackievirus and adenovirus receptor (CAR). However, the expression of exogenous CAR in RAW264.7 macrophages was unable to overcome the viral replication deficit. Interestingly, the CAR expression was associated with altered inflammatory responses during prolonged infection. Additionally, we identified the autophagy protein LC3 as a novel stimulus for macrophage activation. These findings provide new insights into the mechanisms of CVB3-induced macrophage activation and its implications for viral pathogenesis.

Extracorporeal Shock Wave and Melatonin Alleviate Joint Capsule Fibrosis after Knee Trauma in Rats by Regulating Autophagy.

Joint contracture is a common clinical problem affecting joint function. Capsule fibrosis plays a pivotal role in the progression of Joint contracture. Previous studies have reported that autophagy plays a regulatory role in visceral fibrosis. This study aimed to investigate whether extracorporeal shock wave therapy (ESWT) and melatonin alleviate joint capsule fibrosis in rats with extended knee joint contracture by regulating autophagy. A rat knee joint extension contracture model was made. Then, the rats were treated with ESWT, melatonin, ESWT + melatonin, or ESWT + melatonin + mTOR agonist for 4 weeks. The range of motion (ROM) of the knee joints was measured. Joint capsules were collected and observed for pathological changes by H&E and Masson staining. LC3B protein expression was evaluated by immunofluorescence staining. TGF-β1, MMP-1, Col-Ⅰ, Col-Ⅲ, LC3, ATG7, Beclin1, p-AMPK, p-mTOR and p-ULK1 protein expressions were measured by Western blot assay. The intervention groups had significantly improved ROM of knee joint (P < 0.05), significantly improved pathological changes on HE and Masson staining, significantly decreased protein expressions of TGF-β1, MMP-1, Col-Ⅰ, Col-Ⅲ and pmTOR (P < 0.05), and significantly increased protein expressions of LC3B, LC3II/LC3I ratio, ATG7, Beclin1, p-AMPK, and p-ULK1 (P < 0.05). Among these groups, the effects demonstrated by the ESWT + melatonin group were the best. With the mTOR agonist supplement, the therapeutic effects of extracorporeal shock waves and melatonin were significantly reduced. ESWT plus melatonin alleviated knee joint capsule fibrosis in rats by regulating autophagy.

Thiouracil and triazole conjugate induces autophagy through the downregulation of Wnt/β-catenin signaling pathway in human breast cancer cells.

Autophagy is vital for maintaining cellular homeostasis by breaking down unnecessary organelles and proteins within cells. Its activity varies abnormally in several diseases, including cancer, making it a potential target for therapeutic strategies. The Wnt/β-catenin signaling pathway significantly impacts cancer by stabilizing β-catenin protein and promoting the transcription of its target genes. Therefore, we aimed to identify candidate substances targeting this signaling pathway. We designed and tested a thiouracil conjugate, discovering that TTP-8 had anti-tumor effects on human breast cancer cell lines MCF-7 and MDA-MB231. Our findings showed that TTP-8 upregulated the expression of LC3 protein, a marker of autophagy in breast cancer cells, suggesting that TTP-8 might induce autophagy. Further analysis confirmed an increase in autophagy-related proteins, with consistent results obtained from flow cytometry and confocal microscopy. Interestingly, the induction of LC3 expression by TTP-8 was even more pronounced in MCF-7 and MDA-MB231 cells transfected with β-catenin siRNA. Thus, our research supports the idea that the Wnt/β-catenin signaling pathway influences the regulation of autophagy-related proteins, thereby inducing autophagy. This suggests that TTP-8 could serve as a novel agent for treating breast cancer.

The role of rapamycin in the PINK1/Parkin signaling pathway in mitophagy in podocytes.

This study aimed to clarify the role of rapamycin in the PINK1/Parkin signaling pathway in mitophagy in podocytes and the role of voltage-dependent anion channel 1 (VDAC1) in the PINK1/Parkin signaling pathway in mouse glomerular podocytes. For this purpose, podocytes were cultured with rapamycin and observed using microscopy. The apoptosis rate of podocytes was detected by flow cytometry. Changes in the mitochondrial membrane potential were measured. The autophagy-related proteins VDAC1, PINK1, Parkin, and LC3 were detected, and mitochondrial autophagosomes were observed via transmission electron microscopy. In the present study, we demonstrated that the number of podocytes treated with rapamycin was significantly reduced. Compared with those in the control group, the apoptosis rate of podocytes and the degree of mitochondrial membrane potential depolarization were significantly higher. We also found the expression levels of VDAC1, PINK1, Parkin, and LC3 were significantly increased. In the rapamycin-treated group, the numbers of swollen mitochondria and mitochondrial autophagosomes were significantly higher. Finally, we showed that rapamycin can upregulate the expression of VDAC1, PINK1, Parkin, and LC3 in glomerular podocytes, which is correlated with mitophagy. VDAC1 is involved in mitophagy and is related to the PINK1/Parkin signaling pathway, serving as an indicator of mitophagy in podocytes.

Tangningtongluo Tablet ameliorates pancreatic damage in diabetic mice by inducing autophagy and inhibiting the PI3K/Akt/mTOR signaling pathway

BackgroundDiabetes is a metabolic disease characterized by hyperglycaemia. Tangningtongluo Tablet (TNTL) is an inpatient formula extensively utilized to treat diabetes mellitus (DM), but the protective mechanism is not clear. This study aimed to investigate the relevant mechanisms by which TNTL affects pancreatic damage in diabetic mice and autophagy. MethodsThe impact of TNTL on pancreatic damage in diabetic mice in vitro and in vivo was investigated via glucose and lipid metabolism analyses, HE staining, CCK-8, TUNEL staining, Annexin V/PI, and Western blotting. Molecular docking and Western blotting were used to verify the results of network pharmacological analysis, which was carried out to explore the mechanism by which TNTL affects DM. The autophagosome levels were visualized via RFP-GFP-LC3 and transmission electron microscopy, and lysosomal function was evaluated via Lysotracker red staining. Western blot, immunohistochemistry and immunofluorescence staining were used to detect the expression of the autophagy proteins LC3, p62 and LAMP2. ResultsCompared with the model group, TNTL protected pancreas from oxidative stress, decreased the level of MDA, increased the levels of SOD and GSH-px, induced the occurrence of autophagy and decreased the levels of apoptotic factors. Moreover, TNTL inhibited the protein expression of p-PI3K, p-Akt and p-mTOR, increased the levels of LC3 and LAMP2 and decreased the level of p62, and the autophagy inhibitor CQ blocked the protective effect of TNTL on pancreatic damage in diabetic mice. ConclusionThese results demonstrated that TNTL ameliorated pancreatic damage in diabetic mice by inhibiting the PI3K/Akt/mTOR signaling and regulating autophagy.

Scutellarin ameliorates mitochondrial dysfunction and apoptosis in OGD/R-insulted HT22 cells through mitophagy induction

Scutellarin (Scu), a flavonoid from herbal Erigeron breviscapus (Vaniot) Hand-Mazz, exerts neuroprotective effects against cerebral ischemia. However, whether the effects of Scu are related to mitochondrial protection needs further investigation. In this study, we aimed to clarify the mechanisms of Scu against HT22 cells injury caused by oxygen-glucose deprivation and reperfusion (OGD/R). Our results proved that Scu significantly reduced the overload of intracellular reactive oxygen species (cellar ROS) and mitochondria reactive oxygen species (mito-ROS), ameliorating oxidative stress damage. TUNEL positive rate, Caspase-3 activity, and Cytochrome c (Cyto-c) expression remarkably decreased following Scu treatment. Meanwhile, Scu could maintain mitochondrial morphology and reverse ultrastructure changes. And mitochondrial membrane potential (MMP), oxygen consumption rate (OCR), adenosine triphosphate (ATP) production and Na+/K+-ATPase activity were obviously promoted. Additionally, Scu was found to stimulate mitophagy level by increasing the expression of LC3, Beclin1, PINK1 and Parkin proteins, as well as promoting the degradation of p62. More importantly, the regulatory effects of Scu on mito-ROS, MMP, ATP, Na+/K+-ATPase, cell viability and lactate dehydrogenase (LDH) were markedly limited by Mdivi-1 (a mitophagy inhibitor). Of note, the inhibitor also reversed Scu-mediated apoptosis suppression, evidenced by the diminished apoptosis rate, the down-regulated expression activities of Cyto-c, Bax and cleaved Caspase-3, as well as the elevated level of Bcl-2 protein. Collectively, Scu could improve mitochondrial dysfunction and inhibit apoptosis by stimulating mitophagy, thereby attenuating OGD/R-induced HT22 cells injury.

Expression of Autophagy-Related Proteins in Microlissencephaly Associated with a Novel Variant in the WDR81 Gene

Introduction: Microlissencephaly is a subtype of congenital microcephaly characterized by extreme microcephaly with simplified gyral pattern. Other brain malformations may accompany it. WDR81 encodes a multi-domain transmembrane protein that is predominantly expressed in the brain and is thought to play a role in endolysosomal trafficking and autophagy. Methods: We reported two siblings with microlissencephaly born to consanguineous Turkish parents and reviewed all previously reported patients with WDR81 variants presenting with severe microcephaly accompanied by congenital brain malformations. Whole-exome sequencing was performed on both siblings. Sanger DNA sequencing was performed on the patients’ parents. We also examined LC3, p62, and Beclin 1 mRNA and protein expression levels from blood samples of both siblings using real-time PCR and Western blotting, respectively. Results: Whole-exome sequencing revealed a novel biallelic c.4157+5G>A splice variant in the WDR81 gene in both siblings. In our study, LC3, p62, and Beclin 1 mRNA expression levels were high, LC3 protein expression level was also high and p62 and Beclin 1 protein expression levels were low. Conclusion: High LC3 and low p62 protein expression levels supported studies concluding that WDR81 inhibits autophagy through PI3KC3 inhibition, while low Beclin 1 protein expression level supported studies concluding that autophagy is suppressed in case of loss of function variants in the WDR81 gene. We suggest that due to its role in endolysosomal trafficking, WDR81-related diseases should be included in vesicular trafficking disorders.

Protective effect of irbesartan against hepatic ischemia-reperfusion injury in rats: role of ERK, STAT3, and PPAR-γ inflammatory pathways in rats.

This study aimed to elucidate the possible hepatocellular protective role of irbesartan during hepatic ischemia-reperfusion injury (HIRI) and the probable underlying mechanisms. Wistar rats were allocated into four groups: sham; HIRI (control); irbesartan (50 mg/kg) + HIRI; irbesartan (100 mg/kg) + HIRI; irbesartan + GW9662 (1 mg/kg, i.p.) + HIRI. Rats pretreated orally with irbesartan or vehicle for 14 days underwent 45-min hepatic ischemia followed by 60-min reperfusion. Irbesartan preconditioning diminished alanine transaminase (ALT) and aspartate transaminase (AST) serum levels, and reduced extracellular signal-regulated kinase (ERK) and signal transducer and activator of transcription 3 (STAT3). Irbesartan decreased proapoptotic BAX (bcl-2-like protein 4), increased anti-apoptotic B-cell lymphoma 2 (BCL2) hepatic content, and thereby reduced BAX/BCL2 ratio. Moreover, irbesartan preconditioning reduced autophagy-related proteins Beclin1 and LC3 II, and elevated p62 (protein responsible for autophagosome degradation). It elevated proliferator-activated receptor γ (PPAR-γ), and reduced tumor necrosis factor-alpha (TNF-α) and interleukin-6 (IL-6) hepatic gene expression. Also, hepatic protein expressions of nuclear factor kappa-B p65 (NF-κB p65) and caspase-3 were lessoned by irbesartan pretreatment in HIRI rats. However, GW9662 abrogated irbesartan's effect on HIRI. The protective effect of irbesartan on HIRI may be mediated by alleviation of ERK, STAT3, and PPAR-γ inflammatory pathways, exerting anti-apoptotic and anti-autophagic effects in HIRI in rats.

Conjugation of ATG8s to single membranes at a glance.

Autophagy refers to a set of degradative mechanisms whereby cytoplasmic contents are targeted to the lysosome. This is best described for macroautophagy, where a double-membrane compartment (autophagosome) is generated to engulf cytoplasmic contents. Autophagosomes are decorated with ubiquitin-like ATG8 molecules (ATG8s), which are recruited through covalent lipidation, catalysed by the E3-ligase-like ATG16L1 complex. LC3 proteins are ATG8 family members that are often used as a marker for autophagosomes. In contrast to canonical macroautophagy, conjugation of ATG8s to single membranes (CASM) describes a group of non-canonical autophagy processes in which ATG8s are targeted to pre-existing single-membrane compartments. CASM occurs in response to disrupted intracellular pH gradients, when the V-ATPase proton pump recruits ATG16L1 in a process called V-ATPase-ATG16L1-induced LC3 lipidation (VAIL). Recent work has demonstrated a parallel, alternative axis for CASM induction, triggered when the membrane recruitment factor TECPR1 recognises sphingomyelin exposed on the cytosolic face of a membrane and forms an alternative E3-ligase-like complex. This sphingomyelin-TECPR1-induced LC3 lipidation (STIL) is independent of the V-ATPase and ATG16L1. In light of these discoveries, this Cell Science at a Glance article summarises these two mechanisms of CASM to highlight how they differ from canonical macroautophagy, and from each other.

Transplanting mitochondria from gastric epithelial cells reduces the malignancy of gastric cancer

Mitochondria are essential for energy supplementation and metabolic homeostasis of cancer cells. Using mitochondria transplantation to reduce the malignancy of gastric cancer (GC) cells is herein proposed. In our study normal human gastric mucous epithelium cell line (GES-1) showed a lower mitochondrial membrane potential (MMP) compared to immortalized human vascular endothelial cell line (EAhy 926) and human gastric adenocarcinoma cell line (AGS). The transplantation of GES-1 mitochondria to AGS were confirmed both by confocal microscopy and flow cytometry. After transplanting GES-1 mitochondria, the AGS showed a reduced cell migration, and invasion without affecting cell viability and apoptosis. Investigating the expression of proteins involved in epithelial-mesenchymal-transition (EMT), transplanted GES-1 mitochondria reduced the expression of mesenchymal markers α-SMA, MMP-9, snail, vimentin and N-cadherin, whereas the epithelial markers E-cadherin and clauding-1 were not changed. The proteins implicated in the cell cycle such as cyclin B1 and D1 were decreased. In mice, inoculation with AGS carrying the transplanted GES-1 mitochondria resulted in smaller sized tumors. Further investigating the mitochondrial balance, the transplanted GES-1 mitochondria were more stably preserved compared to endogenous AGS mitochondria. The MMP, ATP production and mitochondrial mass decreased in GES-1 mitochondria and the mitophagic proteins LC3 II and PINK1 were up-regulated. In conclusion the decreased malignancy of AGS was a result of exogenous GES-1 mitochondria transplantation. This suggests for a therapy with low efficiency mitochondria transplantation in the treatment of cancer cells.

Baicalin suppresses type 2 dengue virus-induced autophagy of human umbilical vein endothelial cells by inhibiting the PI3K/AKT pathway

To investigate the effect of type 2 dengue virus (DENV-2) infection on autophagy in human umbilical vein endothelial cells (HUVECs) and the mechanism mediating the inhibitory effect of baicalin against DENV-2 infection. Cultured HUVECs with DENV-2 infection were treated with different concentrations of baicalin, and the changes in autophagy of the cells were detected using transmission electron microscopy. Lyso Tracker Red staining was used to examine pH changes in the lysosomes of the cells, and the expressions of ATG5, beclin-1, LC3, P62, STX17, SNAP29, VAMP8, and PI3K/AKT signaling pathway-related proteins were detected by Western blotting. DENV-2 replication in the cells were evaluated using RT-qPCR. The differentially expressed proteins in DENV-2-infected HUVECs were identified by proteomics screening. Treatment with baicalin did not significantly affect the viability of cultured HUVECs. Proteomic studies suggested that the PI3K-AKT pathway played an important role in mediating cell injury induced by DENV-2 infection. The results of RT-qPCR demonstrated that baicalin dose-dependently inhibited DENV-2 replication in HUVECs and produced the strongest inhibitory effect at the concentration of 50 μg/mL. Transmission electron microscopy, Lyso Tracker Red staining, RT-qPCR, and Western blotting all showed significant inhibitory effect of baicalin on DENV-2-induced autophagy in HUVECs. DENV-2 infection of HUVECs caused increased cellular expressions of LC3 and P62 proteins, which were significantly lowered by treatment with LY294002 (a PI3K inhibitor). Baicalin inhibits DENV-2 replication in HUVECs and suppresses DENV-2-induced cell autophagy by inhibiting the PI3K/AKT signaling pathway.

Protective effects of CY-09 and astaxanthin on NaIO3-induced photoreceptor inflammation via the NLRP3/autophagy pathway.

To study the effect of the NLRP3/autophagy pathway on the photoreceptor inflammatory response and the protective mechanism of CY-09 and astaxanthin (AST). ICR mice were intraperitoneally injected NaIO3, CY-09, AST successively and divided into 5 groups, including the control, NaIO3, NaIO3+CY-09, NaIO3+AST, and NaIO3+CY-09+AST groups. Spectral domain optical coherence tomography and flash electroretinogram were examined and the retina tissues were harvested for immunohistochemistry, enzyme linked immunosorbent assay (ELISA), and Western blotting. Retinal pigment epithelium cell line (ARPE-19 cells) and mouse photoreceptor cells line (661W cells) were also treated with NaIO3, CY-09, and AST successively. Cell proliferation was assessed by cell counting kit-8 (CCK-8) assay. Apoptosis was analyzed by flow cytometry. Changes in autophagosome morphology were observed by transmission electron microscopy. Quantitative polymerase chain reaction (qPCR) was used to detect NLRP3 and caspase-1. NLRP3, caspase-1, cleaved caspase-1, p62, Beclin-1, and LC3 protein levels were measured by Western blotting. IL-1β and IL-18 were measured by ELISA. Compared with the control group, the activity of NaIO3-treated 661W cells decreased within 24 and 48h, apoptosis increased, NLRP3, caspase-1, IL-1β and IL-18 levels increased, and autophagy-related protein levels increased (P<0.05). Compared with NaIO3 group, CY-09 and AST inhibited apoptosis (P<0.05), reduced NLRP3, caspase-1, IL-1β and IL-18 expression (P<0.05), and inhibited autophagy. Compared with the other groups, CY-09 combined with AST significantly decreased NLRP3 expression and inhibited the expression of the autophagy-related proteins p62, Beclin-1, and LC3 in vitro and in vivo (P<0.05). CY-09 and AST inhibit NaIO3-induced inflammatory damage through the NLRP3/autophagy pathway in vitro and in vivo. CY-09 and AST may protect retina from inflammatory injury.