Autophagic Markers Research Articles

Intermittent Fasting Ameliorates Age-Induced Morphological Changes in Aged Albino Rat Kidney via Autophagy Activation and Reduction of Apoptosis and Inflammation

Abstract Aging is a biological process with gradual decrease of cell function. Kidneys are one of the organs with higher susceptibility to the development of age-dependent tissue damage. Intermittent fasting has several beneficial effects on age-related degenerative changes. The aim of this study was to investigate the possible beneficial effect of intermittent fasting in delaying age-related renal changes and the possible mechanisms of this effect. Thirty male albino rats were classified into three groups: control, adult rats aged 3 months; aged group, 15-month-old rats and maintained until the age of 18 months; and intermittent fasting-aged groups, 15-month-old rats maintained on intermittent fasting for 3 months. Kidneys were processed for histological and immunohistochemical study. Aging resulted in a significant reduction in renal function and significant several degenerative changes in renal corpuscles and tubules which showed abnormal histological structure with increased collagen deposition. Aging caused significant reduction in the expression of autophagic marker light chain 3 with increased expression of active caspase-3 and inducible nitric oxide synthase. Intermittent fasting significantly improved these age-related renal changes. Intermittent fasting effectively prevents age-related renal changes through the reduction of age-related oxidative stress, inflammation, apoptosis, and activation of autophagy.

9317 Diabetogenic Stress-Mediated Beta Cell Extracellular Vesicle:Autophagy Pathway Crosstalk

Abstract Disclosure: A. Roy: None. G. Ariyaratne: None. A. Matveyenko: None. N. Javeed: None. Diabetogenic stressors including elevated free fatty acids (lipotoxicity), low-grade systemic inflammation, and proteotoxicity are central to the development of Type 2 Diabetes (T2D). Due to their high protein synthetic burden, β-cells employ autophagy and endo/lysosomal pathways to reduce protein aggregates, and protect β-cell mass and function. Recently, extracellular vesicles (EVs; 30-150 nm sized nanoparticles) have been shown to work in tandem with autophagy to facilitate cellular adaptation and intercellular communication. As diabetogenic stressors have been shown to perturb autophagy via lysosomal defects, our group has noted enhanced EV secretion. Thus, we hypothesize that diabetogenic factors enhance EV secretion through activation of an EV-based secretory autophagy pathway and that re-balancing of both pathways could improve β-cell function in T2D. To test this, MIN6 cells were exposed to free fatty acid palmitate (PAL; 24h), or pro-inflammatory cytokines IL-1β, TNFα, and IFNγ (CYTO; 48h). Expression of autophagic markers, LC3 and p62 were increased in CYTO and PAL conditions suggesting defects in auto/lysosomal function. Upon PAL/CYTO treatment in MIN6 cells, conditioned media EVs were isolated using differential ultracentrifugation. We noted enhanced protein expression of LC3-II and p62 in PAL- and cytoEVs, suggesting a diversion of immature autophagosome components into EVs due to lysosomal inhibition. To restore balance between autophagy:EV flux, we used EV biogenesis inhibitor, GW4869 (GW; 24h) or autophagy inducer, rapamycin (Rapa; 24h) on MIN6 cells exposed to PAL or CYTO. Isolated EVs were subjected to Nanoparticle Tracking Analysis (NTA) which showed a significant reduction in EV concentration with GW or Rapa addition (p<.05). Isolated mouse islet exposed to CYTO+GW/Rapa or PAL+GW/Rapa showed significant improvements in glucose stimulated insulin secretion (vs. CYTO or PAL; p<.05). Taken together, these data implicate activation of EV-based secretory autophagy in response to diabetogenic stressors and thus, potential targets to improve functional β-cell mass. Presentation: 6/2/2024

Exendin-4 exhibits cardioprotective effects against high glucose-induced mitochondrial abnormalities: Potential role of GLP-1 receptor and mTOR signaling

Mitochondrial dysfunction is associated with hyperglycemic conditions and insulin resistance leading to cellular damage and apoptosis of cardiomyocytes in diabetic cardiomyopathy. The dysregulation of glucagon-like peptide-1 (GLP-1) receptor and mammalian target of rapamycin (mTOR) is linked to cardiomyopathies and myocardial dysfunctions mediated by hyperglycemia. However, the involvements of mTOR for GLP-1 receptor-mediated cardioprotection against high glucose (HG)-induced mitochondrial disturbances are not clearly identified. The present study demonstrated that HG-induced cellular stress and mitochondrial damage resulted in impaired ATP production and oxidative defense markers such as catalase and SOD2, along with a reduction in survival markers such as Bcl-2 and p-Akt, while an increased expression of pro-apoptotic marker Bax was observed in H9c2 cardiomyoblasts. In addition, the autophagic marker LC3-II was considerably reduced, together with the disruption of autophagy regulators (p-mTOR and p-AMPKα) under the hyperglycemic state. Furthermore, there was a dysregulated expression of several indicators related to mitochondrial homeostasis, including MFN2, p-DRP1, FIS1, MCU, UCP3, and Parkin. Remarkably, treatment with either exendin-4 (GLP-1 receptor agonist) or rapamycin (mTOR inhibitor) significantly inhibited HG-induced mitochondrial damage while co-treatment of exendin-4 and rapamycin completely reversed all mitochondrial abnormalities. Antagonism of GLP-1 receptors using exendin-(9–39) abolished these cardioprotective effects of exendin-4 and rapamycin under HG conditions. In addition, exendin-4 attenuated HG-induced phosphorylation of mTOR, and this inhibitory effect was antagonized by exendin-(9–39), indicating the regulation of mTOR by GLP-1 receptor. Therefore, improvement of mitochondrial dysfunction by stimulating the GLP-1 receptor/AMPK/Akt pathway and inhibiting mTOR signaling could ameliorate cardiac abnormalities caused by hyperglycemic conditions.

MTOR is an essential gate in adapting the functional response of ovine trophoblast cells under stress-inducing environments

IntroductionDuring the early stage of pregnancy trophoblast cells adapt to adverse uterine environments characterized by oxygen and nutrient deprivation. Autophagy is an intracellular degradation process that aims to promote cell survival in response to stressful conditions. Autophagy activation passes through the mechanistic target of rapamycin (mTOR), also known as a placental nutrient sensor. Here, we tested the hypothesis that ovine trophoblast cells may adapt to a suboptimal environment through an mTOR dependent regulation of cell survival with relevant implications for key placental functionality. MethodsPrimary ovine trophoblast cells subjected to mTOR inhibitor and low-nutrient conditions were used to explore how autophagy affects cellular functionality and expression of solute carriers’ genes (SLCs). ResultsAutophagy activation was confirmed both in rapamycin-treated and low-nutrient conditions, through the detection of specific autophagic markers. However, p-mTOR activation seems to be severely modified only following rapamycin treatment whereas 24h of starvation allowed p-mTOR reactivation. Starvation promoted migration compared to normal culture conditions whereas all trophoblast functional activities were decreased in rapamycin treatment. Interestingly in both conditions, the autophagy-activated environment did not affect the progesterone release. mRNA expression of amino acid transporters remains largely undisturbed except for SLC43A2 and SLC38A4 which are downregulated in starved and rapamycin-treated cells, respectively. DiscussionThe study demonstrates that sheep trophoblast cells can adapt to adverse conditions in the early stage of placentation by balancing, in an mTOR dependent manner, nutrient recycling and transport with relevant effects for in vitro functional properties, which could potentially impact conceptus development and survival.

A Decrease in Autophagy Increases the Level of Collagen Type I Expression in Scleral Fibroblasts.

Autophagy dysregulation triggers extracellular matrix remodeling via changes in cellular collagen levels and protease secretion. However, the effect of autophagy on scleral extracellular matrix remodeling in the context of myopia is not fully understood. In this study, we measured the level of autophagy in sclera of form deprivation myopic guinea pigs; we also sought a correlation between the level of autophagy in human scleral fibroblasts and the extent of COL1A1 synthesis. We measured the level of COL1A1 expression and the levels of autophagic protein markers in scleral tissues in vivo using a form deprivation myopic guinea pig model. Rapamycin and chloroquine were respectively used to activate and inhibit autophagy in cultured human scleral fibroblasts. COL1A1 gene and protein expression levels were analyzed via quantitative real-time polymerase chain reaction, Western blotting, and immunofluorescence. Levels of autophagy-related proteins were assessed via Western blotting. The sclera of form deprivation myopic guinea pig eyes exhibited decreased expression of COL1A1 and increased expression level of autophagy. After chloroquine exposure, human scleral fibroblasts exhibited decreased autophagy and increased COL1A1 expression. Inhibition of scleral fibroblast autophagy increased COL1A1 expression at the gene and protein levels, thus explaining the effect of autophagy on collagen synthesis by scleral fibroblasts.

The Antitumor Potential of Sicilian Grape Pomace Extract: A Balance between ROS-Mediated Autophagy and Apoptosis.

From the perspective of circular economy, it is extremely useful to recycle waste products for human health applications. Among the health-beneficial properties of bioactive phyto-compounds, grape pomace represents a precious source of bioactive molecules with potential antitumor properties. Here, we describe the effects of a Sicilian grape pomace hydroalcoholic extract (HE) in colon and breast cancer cells. The characterization of HE composition revealed the predominance of anthoxanthins and phenolic acids. HE treatment was more effective in reducing the viability of colon cancer cells, while breast cancer cells appeared more resistant. Indeed, while colon cancer cells underwent apoptosis, as shown by DNA fragmentation, caspase-3 activation, and PARP1 degradation, breast cancer cells seemed to not undergo apoptosis. To elucidate the underlying mechanisms, reactive oxygen species (ROS) were evaluated. Interestingly, ROS increased in both cell lines but, while in colon cancer, cells' ROS rapidly increased and progressively diminished over time, in breast cancer, cells' ROS increase was persistent up to 24 h. This effect was correlated with the induction of pro-survival autophagy, demonstrated by autophagosomes formation, autophagic markers increase, and protection by the antioxidant NAC. The autophagy inhibitor bafilomycin A1 significantly increased the HE effects in breast cancer cells but not in colon cancer cells. Overall, our data provide evidence that HE efficacy in tumor cells depends on a balance between ROS-mediated autophagy and apoptosis. Therefore, inhibiting pro-survival autophagy may be a tool to target those cells that appear more resistant to the effect of HE.

Copper-induced pro-apoptotic response and its alleviation by Quercetin through autophagic modulation in HEPG2 cells

BackgroundRecent studies indicated that the liver is susceptible to Cu-induced stress as it stores and distributes the metal to other cellular organelles. To counteract the hepatocytic damage, a known polyphenol, quercetin, was employed for its remarkable antioxidant properties. Thus, the study aimed to assess quercetin’s potency against Cu-induced toxicity in HEPG2 cells. MethodsThe cellular viability of HEPG2 cells was carried out by MTT assay. All the cellular experiments were divided into control, Cu 100 µM, Cu 100 µM (with Q μM), Cu 300 µM, Cu 300 µM (with Q 50 nM), and only quercetin (50 nM). Following this, reactive oxygen species (ROS) levels and mitochondrial membrane potential (MMP) were evaluated in co-exposure studies. Moreover, rhodamine-123, Hoechst stain, monodansylcadaverine (MDC), and acridine orange (AO) stain were used to visualize the morphological changes under bright field and fluorescent microscopy. Subsequently, western blotting was adopted to determine the expression level of apoptotic and autophagic marker proteins. ResultsCopper increased intracellular ROS, resulted in morphological abnormalities, nuclear condensation, and disrupted MMP. Moreover, Cu caused apoptotic cell deaths characterized by overexpressed pro-apoptotic proteins such as poly (ADP-ribose) polymerase (PARP), cysteine-dependent aspartate-specific proteases 3 (Caspase 3), and Bcl-2-associated X protein (Bax) and downregulated anti-apoptotic proteins such as B-cell lymphoma 2 (Bcl-2), respectively. However, quercetin restored overexpressed pro-apoptotic proteins and induced autophagosome-bound microtubule-associated protein light chain-3 (LC3II) conversion from LC3I. Furthermore, Cu-modulated autophagy marker proteins, including sequestosome-1 (p62), heat shock cognate proteins (Hsc 70, Hsc 90), lysosome-associated membrane protein (LAMP-2A), and AMP-associated protein kinase (AMPK). ConclusionThis study promotes the nutraceutical ability of quercetin to combat Cu-induced hepatotoxicity by understanding the intricate biological signaling pathways within cells.

Functional OmpA of Salmonella Typhimurium provides protection from lysosomal degradation and inhibits autophagic processes in macrophages.

Our previous study showed that OmpA-deficient Salmonella Typhimurium (STM) failed to retain LAMP-1, quit Salmonella-containing vacuole (SCV) and escaped to the host cytosol. Here we show that the cytosolic population of STM ΔompA sequestered autophagic markers, syntaxin17 and LC3B in a sseL-dependent manner and initiated lysosomal fusion. Moreover, inhibition of autophagy using bafilomycinA1 restored its intracellular proliferation. Ectopic overexpression of OmpA in STM ΔsifA restored its vacuolar niche and increased interaction of LAMP-1, suggesting a sifA-independent role of OmpA in maintaining an intact SCV. The OmpA extracellular loops impaired the LAMP-1 recruitment to SCV and caused bacterial release into the cytosol of macrophages, but unlike STM ΔompA, they retained their outer membrane stability and didn't activate the lysosomal degradation pathway aiding in their intra-macrophage survival. Finally, OmpA extracellular loop mutations protected the cytosolic STM ΔsifA from the lysosomal surveillance, revealing a unique OmpA-dependent strategy of STM for its intracellular survival.

Baicalin induces cell death of non-small cell lung cancer cells via MCOLN3-mediated lysosomal dysfunction and autophagy blockage

BackgroundNon-small cell lung cancer (NSCLC) accounts for 85 % of lung cancer, becoming the most mortality of all cancers globally. Blockage of autophagy in NSCLC represents a promising therapeutic strategy that inhibits angiogenesis and overcomes drug resistance. Natural ingredients in anti-tumor adjuvants are increasingly reported to promote cell death with less side effects and the potential to increase chemotherapeutic drugs sensitivity. Baicalin, a Scutellaria baicalensis-extracted flavonoid glycoside, is reported to induce death of NSCLC cells, however, its effects on autophagy in NSCLC cells remain unclear. PurposeThis study aimed to investigate the effect of baicalin on autophagic flux in NSCLC cells, unraveling the underlying mechanism including potential target and its role in cell death of NSCLC cells. MethodsIn vitro anti-cancer effects of baicalin were verified by evaluating proliferation, clone formation, cell cycle, and cell migration in three NSCLC cell lines (A549, H1299, and PC-9). In vivo anti-tumor efficacies of baicalin were evaluated in subcutaneous xenograft tumor model in nude mice. Autophagy characterization in NSCLC cells included autophagic marker detection by western blot and immunofluorescence staining, subcellular structure observation by TEM, lysosomal function by RNA-seq and fluorescence staining (LysoTracker®, LysoSensor®, and acridine orange). Based on RNA-seq and molecular biological verification using apoptotic, autophagic, and lysosomal inhibitors, potential target molecule of baicalin was verified via Ca2+ flux assay, MCOLN3 knockdown by shRNA, and virtual molecular docking. ResultsBaicalin inhibited NSCLC cell proliferation and migration, and suppressed tumor growth in vivo. Baicalin blocked the autophagic flux via activating the membranal cation channel MCOLN3 of lysosome, which disrupted its Ca2+ balance and induced lysosome dysfunction, leading to failure of autolysosome degradation. The cytoplasmic Ca2+ imbalance further resulted in depolarization of mitochondrial membrane potentials and ROS accumulation in NSCLC cells, mediating autophagy-related apoptosis. ConclusionThis study demonstrated that baicalin inhibited autolysosome degradation by activating MCOLN3, leading to dysfunction in lysosomal pH elevation, thereby inhibiting autophagy in NSCLC, leading to apoptotic death of NSCLC cells. These findings enriched the existing theories of cancer therapy based on autophagy inhibition and underlying mechanisms of flavonoids as antitumor agents, paving the way for their clinical application in future.

Enhanced kidney damage induced by increasing nonylphenol doses: impact on autophagy-related proteins and proinflammatory cytokines in rats

Nonylphenol (NP) is an organic pollutant and endocrine disruptor chemical that has harmful effects on the environment and living organisms. This study looked at whether kidney tissues subjected to increasing doses of nonylphenol generated alterations in histopathologic, pro-inflammatory, and autophagic markers. Fifty rats were divided into five groups of ten each: group I: healthy group, II: control (corn oil), group III: 25 μl/kg NP, group IV: 50 μl/kg NP, group V: 75 μl/kg NP. The kidney tissue samples were obtained for histopathological, immunohistochemical, and biochemical analyses. The histological deteriorations observed in all NP groups included tubular epithelial cell degeneration, inflammation areas, and hemorrhage. The immunohistochemical investigations showed that NP significantly elevated the autophagy markers (Beclin-1, LC3A/B, p62), pro-inflammatory cytokines (TNF-α, IL-6), HIF-1α, and eNOS in group III, IV and V compared with group I and II. The biochemical analysis also revealed that pro-inflammatory cytokines (TNF-α, IL-1β, and IL-6) increased in correlation with the NP doses, but only IL-1β reached statistical significance in NP treated rats kidney tissue. The biochemical findings have been confirmed by the histological studies. The damage to renal tissue caused by NP exposure may worsen it by increasing inflammatory and autophagic markers.

Chrysin-loaded PEGylated liposomes protect against alloxan-induced diabetic neuropathy in rats: the interplay between endoplasmic reticulum stress and autophagy

BackgroundDiabetic neuropathy (DN) is recognized as a significant complication arising from diabetes mellitus (DM). Pathogenesis of DN is accelerated by endoplasmic reticulum (ER) stress, which inhibits autophagy and contributes to disease progression. Autophagy is a highly conserved mechanism crucial in mitigating cell death induced by ER stress. Chrysin, a naturally occurring flavonoid, can be found abundantly in honey, propolis, and various plant extracts. Despite possessing advantageous attributes such as being an antioxidant, anti-allergic, anti-inflammatory, anti-fibrotic, and anticancer agent, chrysin exhibits limited bioavailability. The current study aimed to produce a more bioavailable form of chrysin and discover how administering chrysin could alter the neuropathy induced by Alloxan in male rats.MethodsChrysin was formulated using PEGylated liposomes to boost its bioavailability and formulation. Chrysin PEGylated liposomes (Chr-PLs) were characterized for particle size diameter, zeta potential, polydispersity index, transmission electron microscopy, and in vitro drug release. Rats were divided into four groups: control, Alloxan, metformin, and Chr-PLs. In order to determine Chr- PLs’ antidiabetic activity and, by extension, its capacity to ameliorate DN, several experiments were carried out. These included measuring acetylcholinesterase, fasting blood glucose, insulin, genes dependent on autophagy or stress in the endoplasmic reticulum, and histopathological analysis.ResultsAccording to the results, the prepared Chr-PLs exhibited an average particle size of approximately 134 nm. They displayed even distribution of particle sizes. The maximum entrapment efficiency of 90.48 ± 7.75% was achieved. Chr-PLs effectively decreased blood glucose levels by 67.7% and elevated serum acetylcholinesterase levels by 40% compared to diabetic rats. Additionally, Chr-PLs suppressed the expression of ER stress-related genes (ATF-6, CHOP, XBP-1, BiP, JNK, PI3K, Akt, and mTOR by 33%, 39.5%, 32.2%, 44.4%, 40.4%, 39.2%, 39%, and 35.9%, respectively). They also upregulated the miR-301a-5p expression levels by 513% and downregulated miR-301a-5p expression levels by 65%. They also boosted the expression of autophagic markers (AMPK, ULK1, Beclin 1, and LC3-II by 90.3%, 181%, 109%, and 78%, respectively) in the sciatic nerve. The histopathological analysis also showed that Chr-PLs inhibited sciatic nerve degeneration.ConclusionThe findings suggest that Chr-PLs may be helpful in the protection against DN via regulation of ER stress and autophagy.Graphical

The Autophagic and Apoptotic Death of Forebrain Neurons of Rats with Global Brain Ischemia Is Diminished by the Intranasal Administration of Insulin: Possible Mechanism of Its Action.

Insulin is a promising neuroprotector. To better understand the mechanism of insulin action, it was important to show its ability to diminish autophagic neuronal death in animals with brain ischemic and reperfusion injury. In forebrain ischemia and reperfusion, the number of live neurons in the hippocampal CA1 region and frontal cortex of rats decreased to a large extent. Intracerebroventricular administration of the autophagy and apoptosis inhibitors to ischemic rats significantly increased the number of live neurons and showed that the main part of neurons died from autophagy and apoptosis. Intranasal administration of 0.5 IU of insulin per rat (before ischemia and daily during reperfusion) increased the number of live neurons in the hippocampal CA1 region and frontal brain cortex. In addition, insulin significantly diminished the level of autophagic marker LC3B-II in these forebrain regions, which markedly increased during ischemia and reperfusion. Our studies demonstrated for the first time the ability of insulin to decrease autophagic neuronal death, caused by brain ischemia and reperfusion. Insulin administered intranasally activated the Akt-kinase (activating the mTORC1 complex, which inhibits autophagy) and inhibited the AMP-activated protein kinase (which activates autophagy) in the hippocampus and frontal cortex of rats with brain ischemia and reperfusion.

Autophagy is involved in granulosa cell death and follicular atresia in ewe ovaries

In mammalian ovaries, most follicles do not ovulate and are eliminated by atresia, which primarily depends on granulosa cell (GC) apoptosis. Autophagy is an alternative mechanism involved in follicle depletion in mammals through independent or tandem action with apoptosis. However, follicular autophagy has not yet been investigated in sheep; therefore, the present study aimed to investigate the involvement of autophagy in atresia among a pool of growing antral follicles in ewe ovaries. The abundance of the autophagic marker LC3B-II was determined using western blotting in GCs collected from ewe antral follicles. The antral follicles were classified as healthy or atretic based on morphological criteria and steroid measurements in follicular fluid (FF). Immunofluorescence and confocal microscopy analyses were performed on GCs to evaluate the presence of autophagic proteins and their subcellular localisation. Caspase-3 and DNA fragmentation were assessed using western blotting and TUNEL assays, respectively, in the same GC population to investigate the simultaneous apoptosis. The novel results of this study demonstrated enhanced LC3B-II protein expression in GCs of atretic follicles compared to that of healthy ones (1.3-fold increase; P = 0.0001, ANOVA), indicating a correlation between autophagy enhancement in GCs and antral follicular atresia. Autophagy, either functioning independently or in tandem with apoptosis, may be involved in the atresia of growing antral follicles in ewe ovaries because atretic GCs also showed high levels of apoptotic markers. The findings of this study might have important implication on scientific understanding of ovarian follicle dynamics.

Immunohistochemistry analysis of autophagy-related proteins Beclin-1, p62/SQSTM1, and LC3B in breast carcinoma progression to bone metastasis

Autophagy is a catabolic pathway involved both in tissue homeostasis and in cellular response to stress. The precise role of autophagy in cancer is still undefined and seems to depend on the tumor stage, appearing tumor-suppressive in physiological conditions and helpful to tumor progression in the established tumor. Here we analyzed by immunohistochemistry Beclin-1, p62, and LC3B, autophagic markers, in human specimens of normal breast, bone metastasis together with pair-matched invasive breast carcinoma of no special type (IBC-NST) as well as non-metastatic breast carcinoma, to disclose the possibility that they could be early prognostic indicators of the evolution of the disease toward the worst outcome. Different regions of metastatic carcinomas, i.e., areas adjacent to the tumor without signs of neoplastic growth, dysplastic lesions, and areas with invasive growth were considered. The pattern of autophagic parameters showed differences among the stages of breast carcinoma progression with a trend that indicated the activation of autophagic process in normal breast (Beclin-1 more elevated than p62), a pattern that was maintained in non-metastatic carcinoma. As the neoplasia proceeds with malignancy, the modification of the pattern of expression of autophagic markers (low ratio between Beclin-1 and p62) in areas of invasive growth of carcinomas suggested inhibition of the process. Of note, the parameters showed a different pattern in bone metastasis with respect to bone metastatic (bm)-IBC-NST, suggesting the reactivation of the autophagic process in the new growth site, helpful to the colonization. The course of autophagy markers during tumor progression could have a prognostic value towards bone metastasis and reveal different roles of the process in different phases of neoplastic growth. The understanding of the role of autophagy in bone metastasis could disclose new therapeutic targets to improve the conditions of patients.

Deoxynivalenol Induces Drp-1-Mediated Mitochondrial Dysfunction via Elevating Oxidative Stress.

Mitochondrial dysfunction is often linked to neurotoxicity and neurological diseases and stems from oxidative stress, yet effective therapies are lacking. Deoxynivalenol (DON or vomitoxin) is one of the most common and hazardous type-B trichothecene mycotoxins, which contaminates crops used for food and animal feed. Despite the abundance of preliminary reports, comprehensive investigations are scarce to explore the relationship between these fungal metabolites and neurodegenerative disorders. The present study aimed to elucidate the precise role of DON in mitochondrial dynamics and cell death in neuronal cells. Excessive mitochondrial fission is associated with the pathology of several neurodegenerative diseases. Human SH-SY5Y cells were treated with different concentrations of DON (250-1000 ng/mL). Post 24 and 48 h DON treatment, the indexes were measured as follows: generation of reactive oxygen species (ROS), ATP levels, mitochondrial membrane potential, calcium levels, and cytotoxicity in SH-SY5Y cells. The results showed that cytotoxicity, intracellular calcium levels, and ROS in the DON-treated group increased, while the ATP levels and mitochondrial membrane potential decreased in a dose-dependent manner. With increasing DON concentrations, the expression levels of P-Drp-1, mitochondrial fission proteins Mff, and Fis-1 were elevated with reduced activities of MFN1, MFN2, and OPA1, further resulting in an increased expression of autophagic marker LC3 and beclin-1. The reciprocal relationship between mitochondrial damage and ROS generation is evident as ROS can instigate structural and functional deficiencies within the mitochondria. Consequently, the impaired mitochondria facilitate the release of ROS, thereby intensifying the cycle of damage and exacerbating the overall process. Using specific hydroxyl, superoxide inhibitors, and calcium chelators, our study confirmed that ROS and Ca2+-mediated signaling pathways played essential roles in DON-induced Drp1 phosphorylation. Therefore, ROS and mitochondrial fission inhibitors could provide critical research tools for drug development in mycotoxin-induced neurodegenerative diseases.

Altered autophagic flux in GNE mutant cells of Indian origin: Potential drug target for GNE myopathy

Autophagy phenomenon in the cell maintains proteostasis balance by eliminating damaged organelles and protein aggregates. Imbalance in autophagic flux may cause accumulation of protein aggregates in various neurodegenerative disorders. Regulation of autophagy by either calcium or chaperone play a key role in the removal of protein aggregates from the cell. The neuromuscular rare genetic disorder, GNE Myopathy, is characterized by accumulation of rimmed vacuoles having protein aggregates of β-amyloid and tau that may result from altered autophagic flux. In the present study, the autophagic flux was deciphered in HEK cell-based model for GNE Myopathy harbouring GNE mutations of Indian origin. The refolding activity of HSP70 chaperone was found to be reduced in GNE mutant cells compared to wild type controls. The autophagic markers LC3II/I ratio was altered with increased number of autophagosome formation in GNE mutant cells compared to wild type cells. The cytosolic calcium levels were also increased in GNE mutant cells of Indian origin. Interestingly, treatment of GNE mutant cells with HSP70 activator, BGP-15, restored the expression and refolding activity of HSP70 along with autophagosome formation. Treatment with calcium chelator, BAPTA-AM restored the cytoplasmic calcium levels and autophagosome formation but not LC3II/I ratio significantly. Our study provides insights towards GNE mutation specific response for autophagy regulation and opens up a therapeutic advancement area in calcium signalling and HSP70 function for GNE related Myopathy.

The Autophagy-Related Protein ATG8 Orchestrates Asexual Development and AFB1 Biosynthesis in Aspergillus flavus.

Autophagy, a conserved cellular recycling process, plays a crucial role in maintaining homeostasis under stress conditions. It also regulates the development and virulence of numerous filamentous fungi. In this study, we investigated the specific function of ATG8, a reliable autophagic marker, in the opportunistic pathogen Aspergillus flavus. To investigate the role of atg8 in A. flavus, the deletion and complemented mutants of atg8 were generated according to the homologous recombination principle. Deletion of atg8 showed a significant decrease in conidiation, spore germination, and sclerotia formation compared to the WT and atg8C strains. Additionally, aflatoxin production was found severely impaired in the ∆atg8 mutant. The stress assays demonstrated that ATG8 was important for A. flavus response to oxidative stress. The fluorescence microscopy showed increased levels of reactive oxygen species in the ∆atg8 mutant cells, and the transcriptional result also indicated that genes related to the antioxidant system were significantly reduced in the ∆atg8 mutant. We further found that ATG8 participated in regulating the pathogenicity of A. flavus on crop seeds. These results revealed the biological role of ATG8 in A. flavus, which might provide a potential target for the control of A. flavus and AFB1 biosynthesis.

Deciphering the potential of a novel cytostatic autophagy modulator for the treatment of brain cancer along with reduction in brain inflammation

Background and Purpose: Glioblastoma multiforme (GBM) is the most aggressive form of brain tumor, with significant failure of current treatment options, with the five-year survival rate of GBM being less than 7%. Temozolomide (TMZ), the standard chemotherapy, leads to severe toxicity, whereas radiation therapy leads to necrosis. Moreover, GBM leads to severe neuroinflammation, leading to severe fatalities and, thus, poor quality of life in patients. Therefore, owing to the pressing need for safer treatment alternatives, we repurposed a novel FDA-approved acetamide, BRZ, which not only exhibited a significant curbing of GBM progression but also reduced neuroinflammation. Thus, our study provides a treatment option that can also lead to better quality of life in GBM patients. Methods: Sulfarhodamine B cytotoxicity assay was performed on human (SF268, SF295, SF188) and murine (CT2A-LUC) GBM cell lines for both BRZ and TMZ. In the quest for figuring out the mode of cell-growth suppression, we performed annexin and acridine orange flow cytometry assays along with b-galactosidase cellular senescence staining assay. Cellular staining was also confirmed using confocal microscopy. Oncogenic modulation due to BRZ was studied by performing western blotting. In order to study the effect of BRZ on neuroinflammation, we examined the modulation of different neuroinflammatory genes on LPS-stimulated human and murine microglia using RT-qPCR and western blotting. We corroborated our in vitro findings by investigating the tumor progression in intracranially-injected GBM tumors in C57/BL6 mice. Mice weight and organ weights were recorded for both vehicle and BRZ-treated mice to assess toxicity profiles. Results: BRZ exhibited ~300 times more cytotoxicity in GBM cells as compared to TMZ, making it a more potent treatment option. BRZ lead to an increase in acridine orange in a concentration-dependent manner, hinting towards autophagic cell death. b-galactosidase staining assay and confocal microscopy exhibited a dose-dependent increase in b-galactosidase, showcasing a probable cytostatic effect. Expression of autophagic markers such as Atg5, Atg101, LC3A/B, and senescence markers b-galactosidase, p16, and p21 were increased due to BRZ confirming a cytostatic autophagic modulation in GBM cells. Immunoblotting revealed oncogenic modulation due to BRZ wherein Notch-1, Gli-1, Akt, and p-STAT3 were significantly downregulated, as observed in western blotting. Neuroinflammatory genes such as CXCL10, IL6, and ISG15 were downregulated in LPS-stimulated microglia using BRZ in a concentration-dependent manner, hinting towards the potential of BRZ in brain inflammation. BRZ significantly reduced the progression of orthotopic GBM tumors in C57/BL6 mice. There was no significant difference observed in body and organ weights in the vehicle and BRZ-treated mice, hinting toward the safety profile of BRZ. Conclusions and future direction: Our study provides a novel and safer treatment option for GBM in the form of a cytostatic autophagy modulator, BRZ, which also reduces neuroinflammation with the potential to increase patient’s quality of life. In the future, we aim to perform ex vivo analyses of mice brain tumors and perform the neuroinflammation study in GBM mice. This is the full abstract presented at the American Physiology Summit 2024 meeting and is only available in HTML format. There are no additional versions or additional content available for this abstract. Physiology was not involved in the peer review process.

Short-term treatment of Tat-Beclin1 and endurance exercise enhances autophagy and increases grip strength in old male mice but not in female

Sarcopenia is a geriatric disease causing loss of muscle mass and function, ultimately declining quality of life and causing death. Despite substantial efforts to prevent sarcopenia, there are no approved drugs for treating this muscle disease. Autophagy, a critical process for maintaining muscle health, offers potential avenues for intervention. However, an integrative therapy comprised of an autophagic-specific compound and endurance exercise in old animals has yet to be tested. We hypothesize that combining Tat-Beclin1 (a potent autophagy-specific inducer) and endurance exercise improves muscle force and autophagy in both male and female old mice. Female and male mice at 23 months of age were divided into four groups: Control, Tat-Beclin1 (TB), endurance exercise (Exe), and TB+Exe. Short-term combined treatment underwent for one month. Exercise training at moderate intensity (70% of running peak test) was performed on Mondays, Wednesdays, and Fridays. Tat-Beclin1 administration (15 mg/kg/day) was conducted on Tuesdays and Thursdays. We assessed muscle function through forelimb grip strength and muscle autophagic markers by western blotting. There was no difference in grip strength in female groups. In contrast, there was an increase in grip strength in treated male mice (p<0.05; TB, Exe, and TB+Exe vs. Control group). Notably, the combination treatment in the TB+Exe group outperformed the individual treatments (TB and Exe groups) in enhancing grip strength (p=0.05). Beclin1 and GLIPR2 (a novel autophagic suppressor) were unchanged in both male and female mice. Sequestosome/p62 was decreased in males only, indicating autophagy cargo degradation. Our findings demonstrate that a short-term treatment of Tat-Beclin1 combined with endurance exercise improves grip strength (muscle force) and autophagy in old male mice. Notably, female mice did not exhibit a similar response to the short-term treatment, suggesting that a more extended therapy duration may be necessary to explore potential benefits for female mice. This synergy of an autophagic-specific agonist with endurance exercise may represent a novel strategy to tackle sarcopenia. Camden Health Research Initiative - Startup fundings. This is the full abstract presented at the American Physiology Summit 2024 meeting and is only available in HTML format. There are no additional versions or additional content available for this abstract. Physiology was not involved in the peer review process.

The role of labile iron on brain proteostasis; could it be an early event of neurodegenerative disease?

Iron deposits in the brain are a natural consequence of aging. Iron accumulation, especially in the form of labile iron, can trigger a cascade of adverse effects, eventually leading to neurodegeneration and cognitive decline. Aging also increases the dysfunction of cellular proteostasis. The question of whether iron alters proteostasis is now being pondered. Herein, we investigated the effect of ferric citrate, considered as labile iron, on various aspects of proteostasis of neuronal cell lines, and also established an animal model having a labile iron diet in order to evaluate proteostasis alteration in the brain along with behavioral effects. According to an in vitro study, labile iron was found to activate lysosome formation but inhibits lysosomal clearance function. Furthermore, the presence of labile iron can alter autophagic flux and can also induce the accumulation of protein aggregates. RNA-sequencing analysis further reveals the upregulation of various terms related to proteostasis along with neurodegenerative disease-related terms. According to an in vivo study, a labile iron-rich diet does not induce iron overload conditions and was not detrimental to the behavior of male Wistar rats. However, an iron-rich diet can promote iron accumulation in a region-dependent manner. By staining for autophagic markers and misfolding proteins in the cerebral cortex and hippocampus, an iron-rich diet was actually found to alter autophagy and induce an accumulation of misfolding proteins. These findings emphasize the importance of labile iron on brain cell proteostasis, which could be implicated in developing of neurological diseases.