The mechanistic target of rapamycin complex 2 (mTORC2), a key regulator of cellular metabolism, growth, and survival, remains poorly characterized in the context of dopaminergic neurotoxicity. In this study, we investigated the role of mTORC2 signaling in the survival of SH-SY5Y neuroblastoma cells exposed to the Parkinsonian neurotoxins 1-methyl-4-phenylpyridinium (MPP⁺) and 6-hydroxydopamine (6-OHDA), and examined its interplay with oxidative stress and major stress-responsive signaling pathways. Both neurotoxins induced oxidative stress and mitochondrial damage, accompanied by PINK1 accumulation and culminating in caspase-3 activation, PARP1 cleavage, and apoptotic cell death. These effects were associated with reactive oxygen species (ROS)-dependent phosphorylation of the mTORC2 components Rictor and SIN1, as well as the downstream mTORC2 target Akt (Ser473), indicating activation of mTORC2 signaling in response to neurotoxic insult. RNA interference-mediated depletion of the mTORC2 subunits Rictor, SIN1, or mLST8 reduced Akt phosphorylation and potentiated 6-OHDA-induced cytotoxicity by exacerbating oxidative stress, mitochondrial damage, PINK1 accumulation, and the apoptotic cleavage of caspase-3 and PARP1. In contrast, only Rictor depletion, but not SIN1 or mLST8 knockdown, increased the susceptibility of SH-SY5Y cells to MPP⁺-induced toxicity. Genetic inactivation of mTORC2 reduced basal phosphorylation of the cellular energy sensor AMP-activated protein kinase (AMPK), but did not alter neurotoxin-induced phosphorylation of AMPK or the mitogen-activated protein kinases ERK and JNK. Together, these findings demonstrate a protective role of mTORC2 components against 6-OHDA-induced, and to a lesser extent, MPP+-induced, mitochondrial damage and apoptotic cell death.

Introduction: Autophagy is a process that cells use to eliminate old, unused, and damaged cytoplasmic components. The interaction between autophagy and cytokines could be one of the mechanisms that coordinate the activity of the innate and adaptive immune systems. Depending on the cell type and activation pathway, autophagy and pro-inflammatory cytokines have different mutual effects. Understanding the balance between these two processes is necessary to realize the therapeutic potential of autophagy regulation in various infectious, inflammatory, and autoimmune diseases. Aim: The aim was to investigate the role of pharmacological modulation of autophagy on the transcription of mRNA for the proinflammatory cytokines TNF, IL-1, and IL-6 in the monocytic cell line THP-1. Material and methods: The pharmacological modulation of autophagy by bafilomycin and trehalose was determined by measuring the autophagic flux, the conversion of LC3-II after blocking its degradation, by the immunoblot method. LC3 represents a marker of autophagy and LC3-II levels are thought to correlate with the number of autophagosomes. Using the RTqPCR, it was determined if bafilomycin and trehalose, through the modulation of autophagy, affect the transcription of genes for proinflammatory cytokines TNF, IL-1, and IL-6 by measuring mRNA concentrations of these cytokines. For the statistical analysis, a One-Sample t-test was used. Results: Immunoblot analysis confirmed that bafilomycin blocks autophagic flux by increasing intracellular levels of LC3-II. Trehalose increased the level of LC3-II, both in the presence and absence of bafilomycin, inducing LC3-II conversion in THP-1 cells. RT-qPCR analysis of THP-1 cells treated with trehalose showed a significant increase in transcription of the genes TNF, IL-1, and IL-6, whereas cells treated with bafilomycin exhibited a significant decrease in the transcription of these genes. Conclusion: Based on the results obtained in the research, it can be concluded that autophagy activates the expression of pro-inflammatory cytokines by increasing the transcription of their genes

Introduction: Coronavirus disease 2019 (COVID-19) is a contagious respiratory infection caused by the novel severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). A significant contributor to the pathogenesis of COVID-19 is the SARS-CoV-2 regulatory protein open reading frame 3a (ORF3a), which drives an excessive inflammatory response associated with adverse clinical outcomes and mortality. Notably, c-Jun N-terminal kinase (JNK), a part of the mitogen-activated protein kinase (MAPK) family, is essential in orchestrating the cellular innate and adaptive immune response initiated by diverse viral infections. Aim: This study aims to elucidate the involvement of JNK in the proinflammatory response induced by SARS-CoV-2 ORF3a in the H460 non-small cell lung cancer cell line, specifically focusing on the expression of proinflammatory cytokines IL-1β and IL-8. Material and methods: The H460 cell line was transfected with a DNA plasmid encoding the SARS-CoV-2 ORF3a. Pharmacological inhibition of JNK in H460 cells was achieved using JNK inhibitor SP600215. The expression of p-JNK and JNK was confirmed by immunoblotting, while RT-qPCR was used to quantify IL-1β and IL-8 mRNA levels. Student’s t-test was used for statistical analysis. Results: H460 cells expressing SARS-CoV-2 ORF3a displayed a significant increase in JNK phosphorylation and IL-1β and IL-8 mRNA levels, as verified by immunoblot analysis and RT-qPCR respectively (p < 0.05). Subsequently, JNK inhibition in SARS-CoV-2 ORF3a -expressing cells resulted in a significant reduction of IL-1β and IL-8 mRNA expression (p < 0.05), thus confirming its involvement in the proinflammatory response elicited by SARSCoV-2 ORF3a. Conclusion:The results of the study demonstrated that JNK plays a crucial role in the expression of proinflammatory cytokines IL-1β and IL-8 triggered by SARS-CoV-2 ORF3a. Therefore, targeted JNK inhibition holds promise for mitigating SARS-CoV-2 ORF3a-driven inflammation and providing innovative treatment alternatives for severe COVID-19 cases.

AMP-activated protein kinase (AMPK) is a key sensor and regulator of intracellular energy balance. During energy stress, AMPK helps restore cellular ATP levels by preventing anabolic and promoting catabolic processes, such as autophagy. AMPK activates autophagy both posttranslationally and transcriptionally, by suppressing the mechanistic target of rapamycin complex 1 activity and stimulating the activation of unc-51 like autophagy activating kinase (ULK), autophagosome-lysosome fusion, and expression of autophagy-related genes. Recent research, however, suggests an unexpected role of AMPK in energy stress, where AMPK inhibits ULK and suppresses ATP-consuming autophagic response, possibly to save energy and maintain the autophagic machinery for subsequent activation once the stress subsides. The present review elucidates this dual nature of AMPK in autophagy regulation while highlighting its molecular mechanisms and importance for therapeutic approaches involving AMPK modulation.

The protein kinase known as the mammalian/mechanistic target of rapamycin (mTOR) is present in numerous cells and plays a vital role in regulating cellular growth, metabolism, and survival through two complexes, mTORC1 and mTORC2. Even though the role of mTORC1 in development and metabolism has been extensively studied, the regulatory signals of mTORC2 and diversity of its function in the cell still need to be fully understood. The body of research shows that mTORC2 plays an important role in regulating cellular metabolism, and its dysregulation is associated to many diseases such as cancer, diabetes, and neurodegenerative disorders. Studies have reported altered mTORC2 signaling in several neurodegenerative and neurodevelopmental disorders. Furthermore, genetic studies have revealed the crucial role of mTORC2 in maintaining physiological structure and function in neurons, as well as oligodendrocytes myelination. This protein complex is responsible for various processes related to the structural organization and movement of neurons, interneuronal communication, and their adaptive ability. These processes include organizing the actin cytoskeleton, control of ion channels' function and neurotransmitter receptors, and regulating signal transduction. This review aims to provide an overview of the current understanding of mTORC2, including its components and known functions, in regulating various cellular processes, with special accent on its role in the nervous system.

Autophagy is a lysosome-mediated self-degradation process of central importance for cellular quality control. It also provides macromolecule building blocks and substrates for energy metabolism during nutrient or energy deficiency, which are the main stimuli for autophagy induction. However, like most biological processes, autophagy itself requires ATP, and there is an energy threshold for its initiation and execution. We here present the first comprehensive review of this often-overlooked aspect of autophagy research. The studies in which ATP deficiency suppressed autophagy in vitro and in vivo were classified according to the energy pathway involved (oxidative phosphorylation or glycolysis). A mechanistic insight was provided by pinpointing the critical ATP-consuming autophagic events, including transcription/translation/interaction of autophagy-related molecules, autophagosome formation/elongation, autophagosome fusion with the lysosome, and lysosome acidification. The significance of energy-dependent fine-tuning of autophagic response for preserving the cell homeostasis, and potential implications for the therapy of cancer, autoimmunity, metabolic disorders, and neurodegeneration are discussed.

We examined the role of endoplasmic reticulum (ER) stress and the ensuing unfolded protein response (UPR) in the development of the central nervous system (CNS)-directed immune response in the rat model of experimental autoimmune encephalomyelitis (EAE). The induction of EAE with syngeneic spinal cord homogenate in complete Freund's adjuvant (CFA) caused a time-dependent increase in the expression of ER stress/UPR markers glucose-regulated protein 78 (GRP78), X-box-binding protein 1 (XBP1), C/EBP homologous protein (CHOP), and phosphorylated eukaryotic initiation factor 2α (eIF2α) in the draining lymph nodes of both EAE-susceptible Dark Agouti (DA) and EAE-resistant Albino Oxford (AO) rats. However, the increase in ER stress markers was more pronounced in AO rats. CFA alone also induced ER stress, but the effect was weaker and less sustained compared to full immunization. The ultrastructural analysis of DA lymph node tissue by electron microscopy revealed ER dilatation in lymphocytes, macrophages, and plasma cells, while immunoblot analysis of CD3-sorted lymph node cells demonstrated the increase in ER stress/UPR markers in both CD3+ (T cell) and CD3− (non-T) cell compartments. A positive correlation was observed between the levels of ER stress/UPR markers in the CNS-infiltrated mononuclear cells and the clinical activity of the disease. Finally, the reduction of EAE clinical signs by ER stress inhibitor ursodeoxycholic acid was associated with the decrease in the expression of mRNA encoding pro-inflammatory cytokines TNF and IL-1β, and encephalitogenic T cell cytokines IFN-γ and IL-17. Collectively, our data indicate that ER stress response in immune cells might be an important pathogenetic factor and a valid therapeutic target in the inflammatory damage of the CNS.

The effects of trehalose, an autophagy-inducing disaccharide with neuroprotective properties, on the neurotoxicity of parkinsonian mimetics 6-hydroxydopamine (6-OHDA) and 1-methyl-4-phenylpiridinium (MPP+) are poorly understood. In our study, trehalose suppressed 6-OHDA-induced caspase-3/PARP1 cleavage (detected by immunoblotting), apoptotic DNA fragmentation/phosphatidylserine externalization, oxidative stress, mitochondrial depolarization (flow cytometry), and mitochondrial damage (electron microscopy) in SH-SY5Y neuroblastoma cells. The protection was not mediated by autophagy, autophagic receptor p62, or antioxidant enzymes superoxide dismutase and catalase. Trehalose suppressed 6-OHDA-induced activation of c-Jun N-terminal kinase (JNK), p38 mitogen-activated protein kinase (MAPK), and AMP-activated protein kinase (AMPK), as revealed by immunoblotting. Pharmacological/genetic inhibition of JNK, p38 MAPK, or AMPK mimicked the trehalose-mediated cytoprotection. Trehalose did not affect the extracellular signal-regulated kinase (ERK) and mechanistic target of rapamycin complex 1 (mTORC1)/4EBP1 pathways, while it reduced the prosurvival mTORC2/AKT signaling. Finally, trehalose enhanced oxidative stress, mitochondrial damage, and apoptosis without decreasing JNK, p38 MAPK, AMPK, or AKT activation in SH-SY5Y cells exposed to MPP+. In conclusion, trehalose protects SH-SY5Y cells from 6-OHDA-induced oxidative stress, mitochondrial damage, and apoptosis through autophagy/p62-independent inhibition of JNK, p38 MAPK, and AMPK. The opposite effects of trehalose on the neurotoxicity of 6-OHDA and MPP+ suggest caution in its potential development as a neuroprotective agent.

Trehalose is a natural, non-reducing disaccharide synthesized in some bacteria, fungi, plants, and insects. Due to its advantageous physical and chemical properties, trehalose can stabilize proteins and membranes, and protect cells from desiccation, heating, and freezing. Vertebrates do not synthesize trehalose, but the beneficial effects of trehalose have been demonstrated in numerous diseases as it eliminates aggregates, misfolded proteins, and damaged organelles, and reduces hyperinflammation and oxidative stress. Trehalose induces autophagy through nuclear translocation and activation of transcription factor EB (TFEB) in an mTOR-independent manner, but increases the expression of SQSTM1/p62 and has antioxidant properties in an autophagy-independent manner. Furthermore, trehalose induces apoptosis in tumor cells by increasing membrane fluidity through the activation of caspase 3, 6 and the JNK (c-Jun N-terminal kinase) pathway. Overall, in this review, previous knowledge on the therapeutic potential of trehalose in various diseases such as dry eye syndrome, neurodegenerative diseases, and tumors, was summarized, focusing on the underlying molecular mechanisms.

We previously reported that macrolide antibiotics, such as clarithromycin (CAM), blocked autophagy flux, and simultaneous proteasome and autophagy inhibition by bortezomib (BTZ) plus CAM resulted in enhanced apoptosis induction in multiple myeloma (MM) cells via increased endoplasmic reticulum (ER) stress loading. However, in actual therapeutic settings, cell adhesion-mediated drug resistance between bone marrow stromal cells (BMSC) and MM cells has been known to be a barrier to treatment. To investigate whether CAM could enhance BTZ-induced cytotoxicity in MM cells under direct cell adhesion with BMSC, we established a co-culture system of EGFP-labeled MM cells with BMSC. The cytotoxic effect of BTZ on MM cells was diminished by its interaction with BMSC; however, the attenuated cytotoxicity was recovered by the co-administration of CAM, which upregulates ER stress loading and NOXA expression. Knockout of NOXA in MM cells canceled the enhanced cell death by CAM, indicating that NOXA is a key molecule for cell death induction by the co-administration of CAM. Since NOXA is degraded by autophagy as well as proteasomes, blocking autophagy with CAM resulted in the sustained upregulation of NOXA in MM cells co-cultured with BMSC in the presence of BTZ. Our data suggest that BMSC-associated BTZ resistance is mediated by the attenuation of ER stress loading. However, the addition of CAM overcomes BMSC-associated resistance via upregulation of NOXA by concomitantly blocking autophagy-mediated NOXA degradation and transcriptional activation of NOXA by ER stress loading.

As autophagy can promote or inhibit inflammation, we examined autophagy-inflammation interplay in COVID-19. Autophagy markers in the blood of 19 control subjects and 26 COVID-19 patients at hospital admission and one week later were measured by ELISA, while cytokine levels were examined by flow cytometric bead immunoassay. The antiviral IFN-α and proinflammatory TNF, IL-6, IL-8, IL-17, IL-33, and IFN-γ were elevated in COVID-19 patients at both time points, while IL-10 and IL-1β were increased at admission and one week later, respectively. Autophagy markers LC3 and ATG5 were unaltered in COVID-19. In contrast, the concentration of autophagic cargo receptor p62 was significantly lower and positively correlated with TNF, IL-10, IL-17, and IL-33 at hospital admission, returning to normal levels after one week. The expression of SARS-CoV-2 proteins NSP5 or ORF3a in THP-1 monocytes caused an autophagy-independent decrease or autophagy-inhibition-dependent increase, respectively, of intracellular/secreted p62, as confirmed by immunoblot/ELISA. This was associated with an NSP5-mediated decrease in TNF/IL-10 mRNA and an ORF3a-mediated increase in TNF/IL-1β/IL-6/IL-10/IL-33 mRNA levels. A genetic knockdown of p62 mimicked the immunosuppressive effect of NSP5, and a p62 increase in autophagy-deficient cells mirrored the immunostimulatory action of ORF3a. In conclusion, the proinflammatory autophagy receptor p62 is reduced inacute COVID-19, and the balance between autophagy-independent decrease and autophagy blockade-dependent increase of p62 levels could affect SARS-CoV-induced inflammation.

Type 2 diabetes is a major health burden to the society. Macrophages and liver inflammation emerged as important factors in its development. We investigated ultrastructural changes in the liver, with a special emphasis on macrophages in high fat diet (HFD) fed C57BL/6 J mice treated with metformin or simvastatin, two drugs that are used frequently in diabetes. Both metformin and simvastatin reduced the liver damage in HFD fed animals, manifested as the prevention of nonalcoholic steatohepatitis development and reduced activation and number of macrophages in the liver, as well as the percentage of these cells with lipid droplets in the cytoplasm compared to untreated HFD animals. In contrast with untreated HFD-fed animals, lipid droplets were not observed in lysosomes of macrophages in HFD animals treated with metformin and simvastatin. These findings provide new insight into the effects of metformin and simvastatin on the liver in this experimental model of type 2 diabetes and provide further rationale for implementation of statins in the therapeutic regimens in this disease.

Mott cells are plasma cells that have multiple spherical Russell bodies packed in their cytoplasm. Russell bodies are dilated endoplasmic reticulum cisternae filled with aggregates of immunoglobulins that are neither secreted nor degraded. Mott cells were observed in our study by light and electron microscope in the lymph nodes of rats with experimental autoimmune encephalomyelitis (EAE), an animal model of multiple sclerosis. Mott cells were detected on hematoxylin and eosin (HE)-stained lymph node sections as vacuolated cells with eccentrically positioned nuclei and large number of faint blue spherical inclusions in the cytoplasm. Electron microscopic investigation revealed the presence of Russell bodies of the "medusa" form inside Mott cells in lymph node ultra-thin sections of EAE animals. Mott cells expressed the plasma cell marker CD138 and either kappa or lambda immunoglobulin light chains, indicating their origin from polyclonally activated B cells. Finally, Mott cells were associated with active EAE, as they were not found in the lymph nodes of EAE-resistant Albino Oxford rats. The presence of Russell bodies implies an excessive production of immunoglobulins in EAE, thus further emphasizing the role of B cells, and among them Mott cells, in the pathogenesis of this animal model of multiple sclerosis.

OBJECTIVE:We have recently shown that priming of synovial fibroblasts (SFs) drives arthritis flares. Pathogenic priming of SFs is essentially mediated by epigenetic reprogramming. Bromodomain and extraterminal motif (BET) proteins translate epigenetic changes into transcription. Here, we used a BET inhibitor (I-BET151) to target inflammatory tissue priming and to reduce flare severity in a murine experimental arthritis model.METHODS:BALB/c mice were treated by intraperitoneal injection or by local injection in the paw with I-BET151, which blocks the interaction of BET proteins with acetylated histones. We assessed the effects of I-BET151 on acute arthritis and/or inflammatory tissue priming in a model of repeated injections of monosodium urate crystals or zymosan into the mouse paw. I-BET151 was given before arthritis induction, at peak inflammation, or after healing of the first arthritis bout. We performed transcriptomic (RNA-Seq), epigenomic (ATAC-Seq), and functional (invasion, cytokine production, migration, senescence, metabolic flux) analyses of murine and human SFs treated with I-BET151 in vitro or in vivo.RESULTS:Systemic I-BET151 administration did not affect acute inflammation but abolished inflammatory tissue priming and diminished flare severity in both preventive and therapeutic treatment settings. I-BET151 was also effective when applied locally in the joint. BET inhibition also inhibited osteoclast differentiation, while macrophage activation in the joint was not affected. Flare reduction after BET inhibition was mediated, at least in part, by rolling back the primed transcriptional, metabolic, and pathogenic phenotype of SFs.CONCLUSION:Inflammatory tissue priming is dependent on transcriptional regulation by BET proteins, making them promising therapeutic targets for prevention of arthritis flares in previously affected joints.

AMP-activated protein kinase (AMPK) is an intracellular energy sensor that regulates metabolic and immune functions mainly through the inhibition of the mechanistic target of rapamycin (mTOR)-dependent anabolic pathways and the activation of catabolic processes such as autophagy. The AMPK/mTOR signaling pathway and autophagy markers were analyzed by immunoblotting in blood mononuclear cells of 20 healthy control subjects and 23 patients with an acute demyelinating form of Guillain–Barré syndrome (GBS). The activation of the liver kinase B1 (LKB1)/AMPK/Raptor signaling axis was significantly reduced in GBS compared to control subjects. In contrast, the phosphorylated forms of mTOR activator AKT and mTOR substrate 4EBP1, as well as the levels of autophagy markers LC3-II, beclin-1, ATG5, p62/sequestosome 1, and NBR1 were similar between the two groups. The downregulation of LKB1/AMPK signaling, but not the activation status of the AKT/mTOR/4EBP1 pathway or the levels of autophagy markers, correlated with higher clinical activity and worse outcomes of GBS. A retrospective study in a diabetic cohort of GBS patients demonstrated that treatment with AMPK activator metformin was associated with milder GBS compared to insulin/sulphonylurea therapy. In conclusion, the impairment of the LKB1/AMPK pathway might contribute to the development/progression of GBS, thus representing a potential therapeutic target in this immune-mediated peripheral polyneuropathy.

We investigated the ability of the ascorbic acid (AA) and menadione (MD) combination, the well-known reactive oxidative species- (ROS-) generating system, to induce autophagy in human U251 glioblastoma cells. A combination of AA and MD (AA+MD), in contrast to single treatments, induced necrosis-like cell death mediated by mitochondrial membrane depolarization and extremely high oxidative stress. AA+MD, and to a lesser extent MD alone, prompted the appearance of autophagy markers such as autophagic vacuoles, autophagosome-associated LC3-II protein, degradation of p62, and increased expression of beclin-1. While both MD and AA+MD increased phosphorylation of AMP-activated protein kinase (AMPK), the well-known autophagy promotor, only the combined treatment affected its downstream targets, mechanistic target of rapamycin complex 1 (mTORC1), Unc 51-like kinase 1 (ULK1), and increased the expression of several autophagy-related genes. Antioxidant N-acetyl cysteine reduced both MD- and AA+MD-induced autophagy, as well as changes in AMPK/mTORC1/ULK1 activity and cell death triggered by the drug combination. Pharmacological and genetic autophagy silencing abolished the toxicity of AA+MD, while autophagy upregulation enhanced the toxicity of both AA+MD and MD. Therefore, by upregulating oxidative stress, inhibiting mTORC1, and activating ULK1, AA converts MD-induced AMPK-dependent autophagy from nontoxic to cytotoxic. These results suggest that AA+MD or MD treatment in combination with autophagy inducers could be further investigated as a novel approach for glioblastoma therapy.

We investigated the mechanisms and the role of autophagy in the differentiation of HL-60 human acute myeloid leukemia cells induced by protein kinase C (PKC) activator phorbol myristate acetate (PMA). PMA-triggered differentiation of HL-60 cells into macrophage-like cells was confirmed by cell-cycle arrest accompanied by elevated expression of macrophage markers CD11b, CD13, CD14, CD45, EGR1, CSF1R, and IL-8. The induction of autophagy was demonstrated by the increase in intracellular acidification, accumulation/punctuation of autophagosome marker LC3-II, and the increase in autophagic flux. PMA also increased nuclear translocation of autophagy transcription factors TFEB, FOXO1, and FOXO3, as well as the expression of several autophagy-related (ATG) genes in HL-60 cells. PMA failed to activate autophagy inducer AMP-activated protein kinase (AMPK) and inhibit autophagy suppressor mechanistic target of rapamycin complex 1 (mTORC1). On the other hand, it readily stimulated the phosphorylation of mitogen-activated protein (MAP) kinases extracellular signal-regulated kinase (ERK) and c-Jun N-terminal kinase (JNK) via a protein kinase C-dependent mechanism. Pharmacological or genetic inhibition of ERK or JNK suppressed PMA-triggered nuclear translocation of TFEB and FOXO1/3, ATG expression, dissociation of pro-autophagic beclin-1 from its inhibitor BCL2, autophagy induction, and differentiation of HL-60 cells into macrophage-like cells. Pharmacological or genetic inhibition of autophagy also blocked PMA-induced macrophage differentiation of HL-60 cells. Therefore, MAP kinases ERK and JNK control PMA-induced macrophage differentiation of HL-60 leukemia cells through AMPK/mTORC1-independent, TFEB/FOXO-mediated transcriptional and beclin-1-dependent post-translational activation of autophagy.