Autophagic Markers Research Articles

Dantrolene alleviates mitochondrial dysfunction and neuroinflammation in traumatic brain injury by modulating the NF-ĸβ/Akt pathway

Traumatic brain injury (TBI) triggers a bevy of changes including mitochondrial dysfunction, apoptosis, oxidative stress, neurobehavioural impairment, and neuroinflammation, among others. Dantrolene (DNT), a muscle relaxant which inhibits intracellular Ca2+ signaling from the ER, has been repurposed as a potential neuroprotective agent in various neurological diseases. However, there have been limited studies on whether it can mitigate TBI-induced deficits and restore impaired mitochondrial dynamics. This study sought to evaluate whether Dantrolene can potentially provide neuroprotection in an in vivo model of TBI. Male wistar rats subjected to TBI were treated with DNT (10 mg/kg) 1 h and 12 h post surgery. Animals were assessed 24 h post-TBI to evaluate neurobehavioural deficits and cerebral edema. We evaluated the protein expressions of apoptotic, autophagic, and neuroinflammatory markers by immunoblotting, as well as Mitochondrial Membrane Potential (MMP) and Reactive Oxygen Species (ROS) via Flow Cytometry to ascertain the effects of DNT on TBI. We further analysed immunofluorescence staining with Glial Fibrillary Acidic Protein (GFAP) and immunohistochemistry with NF-κβ to investigate neuroinflammation. H&E staining was also performed post-TBI. Our findings revealed DNT administration inhibits mitochondria-mediated apoptotis and reduces heightened oxidative stress. DNT treatment was also found to reverse neurobehavioural impairments and offer neuroprotection by preserving neuronal architechture. We also demonstrated that DNT inhibits neuronal autophagy and alleviates neuroinflammation following TBI by modulating the NF-κβ/Akt signaling pathway. Thus, our results suggest a novel application of DNT in ameliorating the multitude of deficits induced by TBI, thereby conferring neuroprotection.

Abemaciclib and Vacuolin-1 decrease aggregate-prone TDP-43 accumulation by accelerating autophagic flux

(Macro)autophagy is a cellular degradation system for unnecessary materials, such as aggregate-prone TDP-43, a central molecule in neurodegenerative diseases including amyotrophic lateral sclerosis and frontotemporal lobar degeneration. Abemaciclib (Abe) and vacuolin-1 (Vac) treatments are known to induce vacuoles characterized by an autophagosome and a lysosome component, suggesting that they facilitate autophagosome-lysosome fusion. However, it remains unknown whether Abe and Vac suppress the accumulation of aggregate-prone TDP-43 by accelerating autophagic flux. In the present study, the Abe and Vac treatment dose-dependently reduced the GFP/RFP ratio in SH-SY5Y neuroblastoma cells stably expressing the autophagic flux marker GFP-LC3-RFP-LC3ΔG. Abe and Vac also increased the omegasome marker GFP-ATG13 signal and the autophagosome marker mCherry-LC3 localized to the lysosome marker LAMP1-GFP. The Abe and Vac treatment decreased the intracellular level of the lysosome marker LAMP1-GFP in SH-SY5Y cells stably expressing LAMP1-GFP, but did not increase the levels of LAMP1-GFP, the autophagosome marker LC3-II, or the multivesicular body marker TSG101 in the extracellular vesicle-enriched fraction. Moreover, Abe and Vac treatment autophagy-dependently inhibited GFP-tagged aggregate-prone TDP-43 accumulation. The results of a PI(3)P reporter assay using the fluorescent protein tagged-2 × FYVE and LAMP1-GFP indicated that Abe and Vac increased the intensity of the PI(3)P signal on lysosomes. A treatment with the VPS34 inhibitor wortmannin (WM) suppressed Abe-/Vac-facilitated autophagic flux and the degradation of GFP-tagged aggregate-prone TDP-43. Collectively, these results suggest that Abe and Vac degrade aggregate-prone TDP-43 by accelerating autophagosome formation and autophagosome-lysosome fusion through the formation of PI(3)P.

Immunohistochemical expression of Beclin-1 and LC3B in benign and borderline ovarian tumors

Background Epithelial ovarian tumors account for ⁓90% of ovarian cancers. Recently conducted studies have demonstrated autophagy role in tumor development and progression. Autophagic markers include Beclin-1, essential for autophagosome formation and LC3B, required for the elongation step during autophagy. Aim To investigate Beclin-1 and LC3B expression in benign and borderline epithelial ovarian tumors along with its relation to clinicopathologic parameters. Materials and methods Thirty-six cases of epithelial ovarian neoplasms were subjected to hematoxylin and eosin staining and immunohistochemical staining with Beclin-1 and LC3B. Results Beclin-1 was expressed in the nucleus and/or the cytoplasm of tumor cells. The cytoplasmic expression of Beclin-1 was significantly more frequent in borderline cases compared with benign tumors. High Beclin-1 cytoplasmic expression was significantly associated with tumor size, gross appearance, and tumor histological type. Benign Brenner tumors were the only benign tumors expressing cytoplasmic Beclin-1 localization. Regarding LC3B expression, nuclear and cytoplasmic subcellular localizations were also detected. Cytoplasmic LC3B localization was significantly more frequent in the borderline groups. A significant relation was observed between high cytoplasmic LC3B expression and tumor size, tumor gross appearance, and histological type. The majority of benign Brenner tumors showed a high cytoplasmic LC3B expression. Conclusions The expression of Beclin-1 and LC3B varies in benign versus borderline epithelial ovarian tumors. Cytoplasmic expression is predominant in borderline tumors. Beclin-1 and LC3B cytoplasmic expression is significantly high in small-sized and solid benign tumors. Benign Brenner tumors are the only benign tumors showing cytoplasmic Beclin-1 and LC3B localization.

Differential effects of cholecalciferol and calcitriol on muscle proteolysis and oxidative stress in angiotensin II-induced C2C12 myotube atrophy.

Renin-angiotensin system activation contributes to skeletal muscle atrophy in aging individuals with chronic diseases. We aimed to explore the effects of cholecalciferol (VD3) and calcitriol (1,25VD3) on signaling of muscle proteolysis and oxidative stress in myotubes challenged with angiotensin II (AII). The mouse C2C12 myotubes were assigned to vehicle, AII, AII + VD3, AII + 1,25VD3, and AII + losartan groups. The expression levels of muscle-specific E3 ubiquitin ligase proteins, autophagy-related proteins, and oxidative stress markers were investigated. We demonstrated the diverse effects of VD3 and 1,25VD3 on AII-induced myotube atrophy. The myotube diameter was preserved by treatment with 100 nM VD3 and losartan, while 1 and 10 nM 1,25VD3 increased levels of FoxO3a, MuRF1, and atrogin-1 protein expression in myotubes exposed to AII. Treatment with AII + 10 nM 1,25VD3 resulted in the upregulation of LC3B-II, LC3B-II/LC3B-I, and mature cathepsin L, which are autophagic marker proteins. The p62/SQSTM1 protein was downregulated and vitamin D receptor was upregulated after treatment with AII + 10 nM 1,25VD3. A cellular redox imbalance was observed as AII + 10 nM 1,25VD3-induced reactive oxygen species and NADPH oxidase-2 overproduction, and these changes were associated with an inadequate response of antioxidant superoxide dismutase-1 and catalase proteins. Collectively, these findings provide a translational perspective on the role of vitamin D3 in alleviating muscle atrophy related to high levels of AII.

Antioxidant and anti-ageing effects of oleuropein aglycone in canine skeletal muscle cells

Reactive oxygen species (ROS) are normally produced in skeletal muscle. However, an imbalance in their regulatory systems can lead to their accumulation and ultimately to oxidative stress, which is one of the causes of the ageing process. Companion dogs share the same environment and lifestyle as humans, making them an excellent comparative model for the study of ageing, as well as they constitute a growing market for bioactive molecules that improve the quality of life of pets. The anti-ageing properties of oleuropein aglycone (OLE), a bioactive compound from olive leaves known for its antioxidant properties, were investigated in Myok9 canine muscle cell model. After incubation with OLE, senescence was induced in the canine cellular model by hydrogen peroxide (H2O2). Analyses were performed on cells after seven days of differentiation. The oxidative stress induced by H2O2 treatment on differentiated canine muscle cells led to a significant increase in ROS formation, which was reduced by OLE pretreatment alone or in combination with H2O2 by about 34% and 32%, respectively. Cells treated with H2O2 showed a 48% increase the area of senescent cells stained by SA-β-gal, while OLE significantly reduced the coloured area by 52%. OLE, alone or in combination with H2O2, showed a significant antioxidant activity, possibly through autophagy activation, as indicated by the expression of autophagic markers.

Abstract 1483: Sugar analogs inhibit KSHV replication by blocking N-glycosylation and inducing the unfolded protein response

Abstract Background: Kaposi´s Sarcoma (KS) herpesvirus (KSHV) is one of the seven known human oncogenic viruses. KSHV infection precedes the development of KS, an AIDS-associated malignancy. While the incidence of AIDS-KS globally has seen a decline, this endothelial cancer is refractory to treatment. The standard of care for AIDS-KS has gone virtually unchanged for over 20 years and effective treatments against the oncovirus KSHV have yet to be developed. 2-DG (2-deoxy-D-glucose), 2-FDG (2-fluoro-deoxy-D-glucose), and2-DFM (2-deoxy-2-fluoro-D-mannose) are sugar analogs. 2-DG and 2-FDG are D-glucose analogs and strong glycolysis inhibitors. 2-DG is also a D-mannose analog so that can inhibit N-glycosylation. In this work we aimed to further characterize and compare the inhibition of the three drug analogs on KSHV replication and pathogenesis. Design/Methods: We determined the ability of each drug to inhibit KSHV reactivation and infection by flow cytometry using the KSHV producer cell line iSLK.219 Virus titers were measured by de novo infection of naïve AdHEK293 cells and quantifying the number of AdHEK293 infected cells. We further characterized sugar analog inhibition assessing viral glycoprotein expression by western blot and quantifying virion production by qPCR. To show that N-glycosylation impairment is the main contributor of virus replication inhibition, we carried out D-mannose rescue experiments and analyzed activation of the unfolded protein response (UPR). When UPR cannot be resolved, it leads to either apoptosis or autophagy. For this reason, we analyzed the cellular fate of reactivated sugar analog-treated cells by western blot using apoptotic and autophagic markers. Additionally, we calculated the cell death index using the IncuCyte® machine. Results: All three sugar analogs negatively affected KSHV replication and virion infectivity, but 2-DFM displayed the strongest inhibition. Viral glycoproteins K8.1 and gB were down-regulated and virion production was diminished especially with 2-FDG and 2-DFM sugar analogs. D-mannose could rescue viral glycoprotein expression and virion infectivity, but could not reverse the number of reactivated cells, indicating that N-glycosylation is the main target of the drugs. In line with this, all analogs triggered UPR and even overcame KSHV-induced suppression of the PERK pathway. Finally, 2-DG, 2-FDG, and 2-DFM played a protective role in reactivated cells, displaying the autophagic marker instead of the apoptotic signature observed in the untreated cells. Conclusion: Collectively, this work identifies the non-toxic inhibition of N-glycosylation by sugar analogs as an important viral target and reinforces the potential of 2-DG as an antiviral for KSHV and other enveloped viruses. We also found that 2-DFM is a more potent and target-oriented compound and a promising therapeutic antiviral agent against an oncogenic virus. Citation Format: Mariana Schlesinger, Christian McDonald, Anuj Ahuja, Carolina Alvez-Canete, Zelmira Nunez Del Prado, Julian Naipauer, Theodore Lampidis, Enrique Mesri, Noula Shembade. Sugar analogs inhibit KSHV replication by blocking N-glycosylation and inducing the unfolded protein response [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 1483.

Autophagy Induced by Low Shear Stress Leads to Endothelial Glycocalyx Disruption

Introduction: Previous studies have confirmed that low shear stress (LSS) induces glycocalyx disruption, leading to endothelial dysfunction. However, the role of autophagy in LSS-induced glycocalyx disruption and relevant mechanism are not clear. In this study, we hypothesized that LSS may promote autophagy, disrupting the endothelium glycocalyx. Methods: Human umbilical vein endothelial cells were subjected to physiological shear stress and LSS treatments, followed by the application of autophagy inducers and inhibitors. Additionally, cells were treated with specific matrix metalloproteinase-2 (MMP-2) and matrix metalloproteinase-9 (MMP-9) inhibitor. The expression of autophagic markers, glycocalyx, MMP-2, and MMP-9 was measured. Results: LSS impacted the expression of endothelium autophagy markers, increasing the expression of LC3II.LC3I⁻¹ and Beclin⁻¹, and decreasing the levels of p62, accompanied by glycocalyx disturbance. Moreover, LSS upregulated the expression of MMP-2 and MMP-9 and downregulated the levels of syndecan-1 and heparan sulfate (HS). Additionally, expression of MMP-2 and MMP-9 was increased by an autophagy promoter but was decreased by autophagy inhibitor treatment under LSS. Autophagy and MMP-2 and MMP-9 further caused glycocalyx disruption. Conclusion: LSS promotes autophagy, leading to glycocalyx disruption. Autophagy increases the expression of MMP-2 and MMP-9, which are correlated with the glycocalyx destruction induced by LSS.

Carbon-Coated Iron Oxide Nanoparticles Promote Reductive Stress-Mediated Cytotoxic Autophagy in Drug-Induced Senescent Breast Cancer Cells.

The surface modification of magnetite nanoparticles (Fe3O4 NPs) is a promising approach to obtaining biocompatible and multifunctional nanoplatforms with numerous applications in biomedicine, for example, to fight cancer. However, little is known about the effects of Fe3O4 NP-associated reductive stress against cancer cells, especially against chemotherapy-induced drug-resistant senescent cancer cells. In the present study, Fe3O4 NPs in situ coated by dextran (Fe3O4@Dex) and glucosamine-based amorphous carbon coating (Fe3O4@aC) with potent reductive activity were characterized and tested against drug-induced senescent breast cancer cells (Hs 578T, BT-20, MDA-MB-468, and MDA-MB-175-VII cells). Fe3O4@aC caused a decrease in reactive oxygen species (ROS) production and an increase in the levels of antioxidant proteins FOXO3a, SOD1, and GPX4 that was accompanied by elevated levels of cell cycle inhibitors (p21, p27, and p57), proinflammatory (NFκB, IL-6, and IL-8) and autophagic (BECN1, LC3B) markers, nucleolar stress, and subsequent apoptotic cell death in etoposide-stimulated senescent breast cancer cells. Fe3O4@aC also promoted reductive stress-mediated cytotoxicity in nonsenescent breast cancer cells. We postulate that Fe3O4 NPs, in addition to their well-established hyperthermia and oxidative stress-mediated anticancer effects, can also be considered, if modified using amorphous carbon coating with reductive activity, as stimulators of reductive stress and cytotoxic effects in both senescent and nonsenescent breast cancer cells with different gene mutation statuses.

Sulforaphane's role in Redefining autophagic Responses in depression associated with polycystic ovarian syndrome: Unveiling the SIRT1/AMPK/LKB1 pathway connection

Polycystic ovarian syndrome (PCOS) has been associated with depression and suicidal ideations in females. Studies have highlighted the role of autophagic deficiency in depression pathogenesis. Sulforaphane (SFN) is a natural product that improved autophagic deficiency and showed antidepressant activity in depressed patients. Herein, the study aimed to evaluate the impact of using SFN on depression-associated with PCOS via hippocampal energy sensors and cellular bioenergetics. PCOS was induced by administering letrozole (1 mg/kg, p. o.) for 21 days, followed by SFN treatment (0.5 mg/kg, i. p.) for one week. Two days before euthanasia, PCOS rats showed anhedonic behavior in the sucrose preference test and increased immobility time in the forced swimming test. Depressed rats showed a reduction in nuclear SIRT1 and an elevated cytoplasmic one. This was associated with a reduction in phosphorylation of energy sensors, liver kinase B1 (LKB1), and adenosine monophosphate kinase (AMPK), along with an imbalance of autophagic markers such as Beclin-1, microtubule-associated protein I/II light chain 3, autophagy enzyme 7 and selective autophagy receptor P62. Additionally, Nrf2 and KEAP1 levels were decreased. These abnormalities were alleviated by SFN treatment, as evidenced by the nuclear translocation of SIRT1 and the repression of downstream proteins, including FOXO1, NF-κB, and TNF-α production. These changes were reflected in improved behavioral performance in the sucrose preference test (SPT) and forced swimming test (FST). The antidepressant effects of SFN were counteracted by an autophagic inhibitor, 3-methyladenine. Eventually, SFN, as a nutraceutical, has a promising antidepressant effect via restoring autophagic-related depression in the PCOS rat model.

Microarray data analysis of antileukemic action of Cinnamoylated benzaldehyde LQB-461 in Jurkat cell line.

Leukemias stand out for being the main type of childhood cancer in the world. Current treatments have strong side effects for patients, and there is still a high rate of development of resistance to multidrug therapy. Previously, our research group developed a structure-activity study with novel synthetic molecules analogous to LQB-278, described as an essential molecule with in vitro antileukemic action. Among these analogs, LQB-461 stood out, presenting more significant antileukemic action compared to its derivative LQB-278, with cytostatic and cytotoxicity effect by apoptosis, inducing caspase-3, and increased sub-G1 phase on cell cycle analysis. Deepening the study of the mechanism of action of LQB-461 in Jurkat cells in vitro, a microarray assay was carried out, which confirmed the importance of the apoptosis pathway in the LQB-461 activity. Through real-time PCR, we validated an increased expression of CDKN1A and BAX genes, essential mediators of the apoptosis intrinsic pathway. Through the extrinsic apoptosis pathway, we found an increased expression of the Fas receptor by flow cytometry, showing the presence of a more sensitive population and another more resistant to death. Considering the importance of autophagy in cellular resistance, it was demonstrated by western blotting that LQB-461 decreased LC-3 protein expression, an autophagic marker. These results suggest that this synthetic molecule LQB-461 induces cell death by apoptosis in Jurkat cells through intrinsic and extrinsic pathways and inhibits autophagy, overcoming some mechanisms of cell resistance related to this process, which differentiates LQB-461 of other drugs used for the leukemia treatment.

Methods to Assess Autophagic Activity in Dictyostelium.

Autophagy is an intracellular clearance and recycling pathway that delivers different types of cargos to lysosomes for degradation. In recent years, autophagy has attracted considerable medical interest, and many different techniques are being developed to study this process in experimental models such as Dictyostelium. Here we describe the use of different autophagic markers in confocal microscopy, in vivo and also in fixed cells. In particular, we describe the use of the GFP-Atg8-RFP-Atg8ΔG marker and the optimization of the GFP-PgkA cleavage assay to detect small differences in autophagy flux.

Cytotoxic Impact of Naringenin-Loaded Solid Lipid Nanoparticles on RIN5F Pancreatic β Cells via Autophagy Blockage.

Autophagy plays a crucial role in modulating the proliferation of cancer diseases. However, the application of Naringenin (Nar), a compound with potential benefits against these diseases, has been limited due to its poor solubility and bioavailability. This study aimed to develop solid lipid nanoparticles (Nar-SLNs) loaded with Nar to enhance their therapeutic impact. In vitro experiments using Rin-5F cells exposed to Nar and Nar-SLNs were carried out to investigate the protective effects of Nar and its nanoformulation against the pancreatic cancer cell line of Rin-5F. Treatment with Nar and Nar-SLN led to an increase in autophagic markers (Akt, LC3, Beclin1, and ATG genes) and a decrease in the level of miR-21. Both Nar and Nar-SLN treatments inhibited cell proliferation and reduced the expression of autophagic markers. Notably, Nar-SLNs exhibited greater efficacy compared to free Nar. These findings suggest that SLNs effectively enhance the cytotoxic impact of Nar, making Nar-SLNs a promising candidate for suppressing or preventing Rin-5F cell growth.

Fucoidan alleviated autoimmune diabetes in NOD mice by regulating pancreatic autophagy through the AMPK/mTOR1/TFEB pathway.

The present study investigated the effect and its underlying mechanisms of fucoidan on Type 1 diabetes mellitus (T1DM) in non-obese diabetic (NOD) mice. Twenty 7-week-old NOD mice were used in this study, and randomly divided into two groups (10 mice in each group): the control group and the fucoidan treatment group (600 mg/kg. body weight). The weight gain, glucose tolerance, and fasting blood glucose level in NOD mice were detected to assess the development of diabetes. The intervention lasted for 5 weeks. The proportions of Th1/Th2 cells from spleen tissues were tested to determine the anti-inflammatory effect of fucoidan. Western blot was performed to investigate the expression levels of apoptotic markers and autophagic markers. Apoptotic cell staining was visualized through TdT-mediated dUTP nick-end labeling (TUNEL). The results suggested that fucoidan ameliorated T1DM, as evidenced by increased body weight and improved glycemic control of NOD mice. Fucoidan down-regulated the Th1/Th2 cells ratio and decreased Th1 type pro-inflammatory cytokines' level. Fucoidan enhanced the mitochondrial autophagy level of pancreatic cells and increased the expressions of Beclin-1 and LC3B II/LC3B I. The expression of p-AMPK was up-regulated and p-mTOR1 was inhibited, which promoted the nucleation of transcription factor EB (TFEB), leading to autophagy. Moreover, fucoidan induced apoptosis of pancreatic tissue cells. The levels of cleaved caspase-9, cleaved caspase-3, and Bax were up-regulated after fucoidan treatment. Fucoidan could maintain pancreatic homeostasis and restore immune disorder through enhancing autophagy via the AMPK/mTOR1/TFEB pathway in pancreatic cells.

PGC‐1a inhibition promotes autophagy in induced pluripotent stem cell‐derived retinal pigment epithelial cells generated from age‐related macular degeneration patients

Aims/Purpose: Dysfunctional protein clearance, oxidative stress, mitochondrial damage, and chronic inflammation cause retinal pigment epithelial (RPE) cell damage and the development of age‐related macular degeneration (AMD). PGC‐1α can alleviate oxidative stress, is a key player in mitochondrial quality control and could be considered as a treatment target in AMD. Here, we assessed the effect of PGC‐1α inhibition on induced pluripotent stem cell (iPSC)‐derived RPE cells exposed to hydroquinone‐induced oxidative stress.Methods: iPSC‐derived RPE cells from two neovascular AMD patients or one healthy age‐matched control were cultured for 8 weeks on transwell cell culture inserts until mature. Cells were exposed to the PGC‐1α inhibitor SR‐18292 for 1 h before an exposure to hydroquinone for an additional 4 h. Lactate dehydrogenase (LDH) was measured from collected medium samples to assess cellular viability. Cells were lysed in 3x Laemmli sample buffer for analysis by western blotting or subjected to RNA extraction, followed by RT‐PCR analysis.Results: Hydroquinone caused a robust increase in LDH release to the medium, which was not modulated by PGC‐1α inhibition. In neovascular AMD‐derived iPSC‐RPE cells, hydroquinone caused an accumulation of p62 and LC3‐II, an effect that was reversed by SR‐18292 pre‐treatment. In control iPSC‐RPE cells, neither hydroquinone nor SR‐18292 affected autophagic markers p62 or LC3. RT‐PCR analysis showed an increase in p62 mRNA levels following hydroquinone exposure in both control‐ and AMD‐derived iPSC‐RPE cells while no change was observed in the RNA levels of LC3B or mTOR.Conclusions: Increased oxidative stress caused autophagy dysregulation in AMD‐derived iPSC‐RPE cells but not in healthy age‐matched control cells. Interestingly, PGC‐1α inhibition promoted autophagy and the clearance of p62, but further studies are necessary to explore the underlying pathways and the disease‐specific differences observed between healthy and disease‐derived RPE cells.

Low dose ribosomal DNA P-loop mutation affects development and enforces autophagy in Arabidopsis

ABSTRACT Arabidopsis contains hundreds of ribosomal DNA copies organized within the nucleolar organizing regions (NORs) in chromosomes 2 and 4. There are four major types of variants of rDNA, VAR1–4, based on the polymorphisms of 3’ external transcribed sequences. The variants are known to be differentially expressed during plant development. We created a mutant by the CRISPR-Cas9-mediated excision of ~ 25 nt from predominantly NOR4 ribosomal DNA copies, obtaining mosaic mutational events on ~ 5% of all rDNA copies. The excised region consists of P-loop and Helix-82 segments of 25S rRNA. The mutation led to allelic, dosage-dependent defects marked by lateral root inhibition, reduced size, and pointy leaves, all previously observed for defective ribosomal function. The mutation in NOR4 led to dosage compensation from the NOR2 copies by elevated expression of VAR1 in mutants and further associated single-nucleotide variants, thus, resulting in altered rRNA sub-population. Furthermore, the mutants exhibited rRNA maturation defects specifically in the minor pathway typified by 32S pre-rRNA accumulation. Density-gradient fractionation and subsequent RT-PCR of rRNA analyses revealed that mutated copies were not incorporated into the translating ribosomes. The mutants in addition displayed an elevated autophagic flux as shown by the autophagic marker GFP-ATG8e, likely related to ribophagy.

Apoptotic and Autophagic Cell Death Effects of the Hexane Extract of Tropical Marine Algae Halymenia durvillei against Human Glioblastoma Cells: In vitro and in silico Studies

Glioblastoma (GBM) considered as aggressive brain cancer with high mortality rate in patients even after surgical resection. Resistant to chemotherapy is the major problem in GBM therapy. Discovery of novel bioactive compounds from algae is being investigated as alternative sources for potential treatment as well as prevention in glioblastoma. This study revealed the effects of marine red algae extract from hexane solvent fraction of Halymenia durvillei (HDHE) on proliferation and cell death in A172 human GBM cells. HDHE decreased proliferation and promoted cell cycle arrest at G2/M phase. HDHE induced apoptotic cell death in A172 cells through mitochondrial membrane dysfunction, the decrease of anti-apoptotic Bcl-2 protein expression, and activation of caspase 3/7. Moreover, HDHE increased intracellular reactive oxygen species (ROS) production and accumulation of LC3-II, an autophagic marker. The docked conformation of palmitic acid, a major component of HDHE, showed a high affinity binding to TP53 and Beclin-1 as cell death-related target molecules. This research conclusively demonstrated that HDHE might serve as a potent anticancer agent against glioblastoma by promoting apoptotic and autophagic cell death in A172 human GBM cells. HIGHLIGHTS The palmitic acid-enriched extract of red alga Halymenia durvillei (HDHE) could inhibit proliferation of A172 human glioblastoma cells by arresting cell cycle at G2/M phase HDHE could induce both caspase-dependent apoptotic and autophagic death of A172 cells Palmitic acid, a major component of HDHE, showed a high affinity binding to TP53 and Beclin-1 The extract could be beneficial to develop as chemopreventive agents or food supplements against glioblastoma GRAPHICAL ABSTRACT

Muscle diffusion MRI reveals autophagic buildup in a mouse model for Pompe disease

Quantitative muscle MRI is increasingly important in the non-invasive evaluation of neuromuscular disorders and their progression. Underlying histopathotological alterations, leading to changes in qMRI parameters are incompletely unraveled. Early microstructural differences of unknown origin reflected by Diffusion MRI in non-fat infiltrated muscles were detected in Pompe patients. This study employed a longitudinal approach with a Pompe disease mouse model to investigate the histopathological basis of these changes. Monthly scans of Pompe (Gaa6neo/6neo) and wildtype mice (age 1–8 months) were conducted using diffusion MRI, T2-mapping, and Dixon-based water-fat imaging on a 7 T scanner. Immunofluorescence studies on quadriceps muscles were analyzed for lysosomal accumulations and autophagic buildup and correlated with MRI outcome measures. Fat fraction and water-T2 did not differ between groups and remained stable over time. In Pompe mice, fractional anisotropy increased, while mean diffusivity (MD) and radial diffusivity (RD) decreased in all observed muscles. Autophagic marker and muscle fibre diameter revealed significant negative correlations with reduced RD and MD, while lysosomal marker did not show any change or correlation. Using qMRI, we showed diffusion changes in muscles of presymptomatic Pompe mice without fat-infiltrated muscles and correlated them to autophagic markers and fibre diameter, indicating diffusion MRI reveals autophagic buildup.

Nanogels with covalently bound and releasable trehalose for autophagy stimulation in atherosclerosis

Atherosclerosis, cholesterol-driven plaque formation in arteries, is a complex multicellular disease which is a leading cause of vascular diseases. During the progression of atherosclerosis, the autophagic function is impaired, resulting in lipid accumulation-mediated foam cell formation. The stimulation of autophagy is crucial for the recovery of cellular recycling process. One of the potential autophagy inducers is trehalose, a naturally occurring non-reducing disaccharide. However, trehalose has poor bioavailability due to its hydrophilic nature which results in poor penetration through cell membranes. To enhance its bioavailability, we developed trehalose-releasing nanogels (TNG) for the treatment of atherosclerosis. The nanogels were fabricated through copolymerization of 6-O-acryloyl-trehalose with the selected acrylamide-type monomers affording a high trehalose conjugation (~ 58%, w/w). TNG showed a relatively small hydrodynamic diameter (dH, 67 nm) and a uniform spherical shape and were characterized by negative ζ potential (-18 mV). Thanks to the trehalose-rich content, TNG demonstrated excellent colloidal stability in biological media containing serum and were non-hemolytic to red blood cells. In vitro study confirmed that TNG could stimulate autophagy in foam cells and enhance lipid efflux and in vivo study in ApoE−/− mice indicated a significant reduction in atherosclerotic plaques, while increasing autophagic markers. In conclusion, TNG hold great promise as a trehalose delivery system to restore impaired autophagy-mediated lipid efflux in atherosclerosis and subsequently reduce atherosclerotic plaques.Graphical abstract

Early-Life Exposure to the Mycotoxin Fumonisin B1 and Developmental Programming of the Ovary of the Offspring: The Possible Role of Autophagy in Fertility Recovery.

Mycotoxins are produced by more than one hundred fungi and produce secondary metabolites that contaminate various agricultural commodities, especially rice and corn. Their presence in the food chain is considered a serious problem worldwide. In recent years, a link between exposure to mycotoxins and impaired fertility has been suggested. Consequently, it has become vital to investigate the interactive effects of these mycotoxins on ovarian function. In this study, we investigated the intergenerational effects of the mycotoxin fumonisin B1 (FB1) on ovarian structure and function. Virgin Wistar albino female rats were separated into control and FB1 treatment groups and examined from day 6 of pregnancy until delivery (20 and 50 mg/kg b.w./day). The obtained female rats of the first (F1) and second generations (F2) were euthanized at 4 weeks of age, and ovary samples were collected. We found that the ovary weight index increased with the high dose of the treatment (50 mg/kg b.w./day) among both F1 and F2, in a manner similar to that observed in polycystic ovary syndrome. As expected, FB1 at a high dose (50 mg/kg b.w.) reduced the number of primordial follicles in F1 and F2, leading to an accelerated age-related decline in reproductive capacity. Moreover, it reduced the fertility rate among the F1 female rats by affecting follicle growth and development, as the number of secondary and tertiary follicles decreased. Histopathological changes were evidenced by the altered structures of most of the growing follicle oocytes, as revealed by a thinning irregular zona pellucida and pyknosis in granulosa cells. These findings are concomitant with steroidogenesis- and folliculogenesis-related gene expression, as evidenced by the decrease in CYP19 activity and estrogen receptor beta (ESR2) gene expression. Additionally, GDF-9 mRNA levels were significantly decreased, and IGF-1 mRNA levels were significantly increased. However, the results from the ovaries of the F2 treatment groups were different and unexpected. While there was no significant variation in CYP19 activity compared to the control, the ESR2 significantly increased, leading to stereological and histopathological changes similar to those of the control, except for some altered follicles. The hallmark histological feature was the appearance of vacuolar structures within the oocyte and between granulosa cell layers. Interestingly, the autophagic marker LC3 was significantly increased in the F2 offspring, whereas this protein was significantly decreased in the F1 offspring. Therefore, we suggest that the promotion of autophagy in the ovaries of the F2 offspring may be considered a recovery mechanism from the effect of prenatal FB1 exposure. Thus, autophagy corrected the effect of FB1 during the early life of the F1 female rats, leading to F2 offspring with ovarian structure and function similar to those of the control. However, the offspring, treated female rats may experience early ovarian aging because their ovarian pool was affected.

Carbon black nanoparticles and cadmium co-exposure aggravates bronchial epithelial cells inflammation via autophagy-lysosome pathway

Carbon black nanoparticles (CBNPs) and cadmium (Cd) are major components of various air pollutants and cigarette smoke. Autophagy and inflammation both play critical roles in understanding the toxicity of particles and their components, as well as maintaining body homeostasis. However, the effects and mechanisms of CBNPs and Cd (CBNPs-Cd) co-exposure on the human respiratory system remain unclear. In this study, a CBNPs-Cd exposure model was constructed to explore the respiratory toxicity and combined mechanism of these chemicals on the autophagy-lysosome pathway in the context of respiratory inflammation. Co-exposure of CBNPs and Cd significantly increased the number of autophagosomes and lysosomes in human bronchial epithelial cells (16HBE) and mouse lung tissues compared to the control group, as well as the groups exposed to CBNPs and Cd alone. Autophagic markers, LC3II and P62 proteins, were up-regulated in 16HBE cells and mouse lung tissues after CBNPs-Cd co-exposure. However, treatment with Cq inhibitor (an indicator of lysosomal acid environment) resulted in a substantial decreased co-localization fluorescence of LC3 and lysosomes in the CBNPs-Cd combination group compared with the CBNPs-Cd single and control groups. No difference in LAMP1 protein expression was observed among the exposed groups. Adding 3 MA alleviated inflammatory responses, while applying the Baf-A1 inhibitor aggravated inflammation both in vitro and in vivo following CBNPs-Cd co-exposure. Factorial analysis showed no interaction between CBNPs and Cd in their effects on 16HBE cells. We demonstrated that co-exposure to CBNPs-Cd increases the synthesis of autophagosomes and regulates the acidic environment of lysosomes, thereby inhibiting autophagy-lysosome fusion and enhancing the inflammatory response in both 16HBE cells and mouse lung. These findings provide evidence for a comprehensive understanding of the interaction between CBNPs and Cd in mixed pollutants, as well as for the prevention and control of occupational exposure to these two chemicals.