The Dysregulated MAD in Mad: A Neuro-theranostic Approach Through the Induction of Autophagic Biomarkers LC3B-II and ATG.

The word mad has historically been associated with the psyche, emotions, and abnormal behavior. Dementia is a common symptom among psychiatric disorders or mad (schizophrenia, depression, bipolar disorder) patients. Autophagy/mitophagy is a protective mechanism used by cells to get rid of dysfunctional cellular organelles or mitochondria. Autophagosome/mitophagosome abundance in autophagy depends on microtubule-associated protein light chain 3B (LC3B-II) and autophagy-triggering gene (ATG) which functions as an autophagic biomarker for phagophore production and quick mRNA disintegration. Defects in either LC3B-II or the ATG lead to dysregulated mitophagy-and-autophagy-linked dementia (MAD). The impaired MAD is closely associated with schizophrenia, depression, and bipolar disorder. The pathomechanism of psychosis is not entirely known, which is the severe limitation of today's antipsychotic drugs. However, the reviewed circuit identifies new insights that may be especially helpful in targeting biomarkers of dementia. Neuro-theranostics can also be achieved by manufacturing either bioengineered bacterial and mammalian cells or nanocarriers (liposomes, polymers, and nanogels) loaded with both imaging and therapeutic materials. The nanocarriers must cross the BBB and should release both diagnostic agents and therapeutic agents in a controlled manner to prove their effectiveness against psychiatric disorders. In this review, we highlighted the potential of microRNAs (miRs) as neuro-theranostics in the treatment of dementia by targeting autophagic biomarkers LC3B-II and ATG. Focus was also placed on the potential for neuro-theranostic nanocells/nanocarriers to traverse the BBB and induce action against psychiatric disorders. The neuro-theranostic approach can provide targeted treatment for mental disorders by creating theranostic nanocarriers.

Extracellular vesicles produced by primary human keratinocytes in response to TLR agonists induce stimulus-specific responses in antigen-presenting cells.

Cells can communicate through the extracellular vesicles (EVs) they secrete. Pathogen associated molecular patterns (PAMPs), alter the biophysical and communicative properties of EVs released from cells, but the functional consequences of these changes are unknown. Characterization of keratinocyte-derived EVs after poly(I:C) treatment (poly(I:C)-EVs) showed slight differences in levels of EV markers TSG101 and Alix, a loss of CD63 and were positive for autophagosome marker LC3b-II and the cytokine IL36γ compared to EVs from unstimulated keratinocytes (control-EVs). Flagellin treatment (flagellin-EVs) led to an EV marker profile like control-EVs but lacked LC3b-II. Flagellin-EVs also lacked IL-36γ despite nearly identical intracellular levels. While poly(I:C) treatment led to the clear emergence of a > 200 nm diameter EV sub-population, these were not found in flagellin-EVs. EV associated IL-36γ colocalized with LC3b-II in density gradient analysis, equilibrating to 1.10 g/mL, indicating a common EV species. Poly(I:C), but not flagellin, induced intracellular vesicles positive for IL-36γ, LC3b-II, Alix and TSG101, consistent with fusion of autophagosomes and multivesicular bodies. Simultaneous rapamycin and flagellin treatment induced similar intracellular vesicles but was insufficient for the release of IL-36γ+/LC3b-II+ EVs. Finally, a qRT-PCR array screen showed eight cytokine/chemokine transcripts were altered (p < 0.05) in monocyte-derived Langerhans cells (LCs) when stimulated with poly(I:C)-EVs while three were altered when LCs were stimulated with flagellin-EVs compared to control-EVs. After independent confirmation, poly(I:C)-EVs upregulated BMP6 (p = 0.035) and flagellin-EVs upregulated CXCL8 (p = 0.005), VEGFA (p = 0.018) and PTGS2 (p = 0.020) compared to control-EVs. We conclude that exogenous signals derived from pathogens can alter keratinocyte-mediated modulation of the local immune responses by inducing changes in the types of EVs secreted and responses in antigen presenting cells.

Mir-139 Regulates Autophagy in Prostate Cancer Cells Through Beclin-1 and mTOR Signaling Proteins.

BACKGROUND/AIM: We previously identified a panel of five miRNAs (including miR-139) associated with biochemical recurrence and metastasis in prostate cancer patients. MATERIALS AND METHODS: We examined miR-139 transfected PC3, DU145 and LNCaP cells by morphology as well as by cell-based assays, confocal microscopy and immunoblotting. RESULTS: We found that treatment of prostate cancer cells with miR-139 resulted in phenotypic changes characteristic of autophagic cells. MiR-139 increased the autophagy-related conversion of the microtubule-associated protein light chain 3 (LC3-I to LC3-II) that was specifically inhibited by the miR-139 antagomir. The upregulation of LC3 II was further confirmed by confocal microscopy. miR-139 regulated activation of both mTOR and Beclin1 the two important autophagy-related molecules. We found that upon miR-139 treatment, the cargo adaptor protein p62 which is degraded during autophagy, accumulates. CONCLUSION: These results suggest that miR-139 is inducing autophagic flux blockade leading to apoptosis in prostate cancer cells through the mTOR and Beclin-1 proteins.

An Observation of the Role of Autophagy in Patients with Endometriosis of Different Stages during Secretory Phase and Proliferative Phase.

BACKGROUND: Autophagy exists widely in various physiological and pathological conditions. Lots of investigations have verified that the autophagic activity is always related to the occurrence and the development of cancer. Endometriosis (EMs) is a disease that endometrium-like tissues abnormally grow outside the uterus and also considered to possess the characters of tumor because of its malignant biological behavior. INTRODUCTION: Recently, several studies have already revealed that autophagy may play a potential role in proliferative-phase EMs. However, the function of autophagic activity in secretory-phase EMs is still unclear. METHODS: In our work, we explored autophagic activity between normal endometrium and EMs lesion endometrium during different menstrual phases and EMs stages. The clinical endometrium samples from 73 women were selected in this study, including 30 healthy individuals and 43 patients with EMs (endometrium samples include eutopic and its matched ectopic endometrium). All the participants were divided into two groups according to the menstrual cycle, namely proliferative-phase and secretive- phase group. Among the patients with EMs, 22 individuals in proliferative phase and the other 21 individuals in secretory phase were further classified into the groups of Stage I-II and Stage III-IV according to revised-American Fertility Society (r-AFS). Two autophagy-related proteins microtubuleassociated protein 1 light chain 3 beta-II (LC3B-II) and sequestosome protein (P62), which are believed to be the indicators of autophagy activity, were chosen in the study. Immunohistochemical (IHC) staining, Western blot assay and Real-Time quantitative Polymerase Chain Reaction (RTqPCR) were used to examine the expression of LC3B-II and P62 in protein and mRNA level accordingly. RESULT: It showed that the expression of LC3B-II both in protein and mRNA level decreased and that of P62 increased in secretory phase of the healthy group (P<0.05), but showed no significant difference in ectopic and its eutopic endometrium group during proliferative and secretory phase (P>0.05). In addition, the expression of LC3B-II in ectopic endometrium group was significantly lower than that of its eutopic endometrium group (P<0.05), and the expression of P62 was significantly higher accordingly (P<0.05). At the same time, both LC3B-II and P62 levels remained same between eutopic endometrium group and control group (P>0.05). Furthermore, compared to Stage I-II EMs group, the expression of LC3B-II was significantly lower (P<0.05) and P62 was significantly higher (P<0.05) in Stage III-IV EMs during secretory phase. CONCLUSION: Taken together, the periodicity-losing in EMs and the decreased autophagic activity in ectopic endometrium may exert a potential role in the pathogenesis of EMs. Down-regulated autophagy of ectopic endometrium in secretory phase may be related to the progression of EMs.

Pharmacological Inhibition of Lysosomal Activity as a Method For Monitoring Thyroid Hormone-induced Autophagic Flux in Mammalian Cells In Vitro.

Autophagy is an evolutionarily conserved intracellular catabolic process that is essential for cellular housekeeping and nutrient homeostasis. Recently, we provided evidence that thyroid hormone (TH) is a major inducer of autophagy in mammalian cells. Here, we describe a method for detecting TH-induced autophagic flux in hepatic, muscle, and brown adipocyte cells using lysosomal inhibitor bafilomycin A1 (BafA1) and conventional Western blot techniques.

Silver nanoparticles induce lysosomal-autophagic defects and decreased expression of transcription factor EB in A549 human lung adenocarcinoma cells.

Although silver nanoparticles (AgNPs) are widely used in consumer and medical products, the mechanism by which AgNPs cause pulmonary damage is unclear. AgNPs are incorporated into cells and processed via the autophagy pathway. We examined the effects of AgNP exposure on autophagic flux and expression of transcription factor EB (TFEB) in A549 lung adenocarcinoma cells. In cells exposed to citrate-coated 60-nm AgNPs, confocal laser microscopic examination showed a decrease in the LysoTracker fluorescence signal and an increase in that of Cyto-ID, indicating lysosomal pH alkalization and autophagosome formation, respectively. The proteins p62 and microtubule-associated protein light chain 3B-II (LC3B-II) are both degraded by autophagy, and their levels increased depending on AgNP dose. Furthermore, AgNP-induced increase in LC3B-II was not enhanced by treatment with the autophagic inhibitor bafilomycin A1. TFEB mRNA levels, and protein levels in cytosolic and nuclear fractions, were suppressed by exposure to AgNPs, suggesting transcriptional inhibition of TFEB expression. Overexpression of TFEB did not suppress AgNP-induced LC3B-II accumulation and cellular damage, indicating that impairment of autophagic flux and cellular damage by AgNPs might not be primarily caused by reduced TFEB expression. The present study suggests that AgNP-induced lysosomal dysfunction plays a principal role in the autophagic flux defect.

P2X7 Receptor Activation Modulates Autophagy in SOD1-G93A Mouse Microglia.

Autophagy and inflammation play determinant roles in the pathogenesis of Amyotrophic Lateral Sclerosis (ALS), an adult-onset neurodegenerative disease characterized by deterioration and final loss of upper and lower motor neurons (MN) priming microglia to sustain neuroinflammation and a vicious cycle of neurodegeneration. Given that extracellular ATP through P2X7 receptor constitutes a neuron-to-microglia alarm signal implicated in ALS, and that P2X7 affects autophagy in immune cells, we have investigated if autophagy can be directly triggered by P2X7 activation in primary microglia from superoxide dismutase 1 (SOD1)-G93A mice. We report that P2X7 enhances the expression of the autophagic marker microtubule-associated protein 1 light chain 3 (LC3)-II, via mTOR pathway and concomitantly with modulation of anti-inflammatory M2 microglia markers. We also demonstrate that the autophagic target SQSTM1/p62 is decreased in SOD1-G93A microglia after a short stimulation of P2X7, but increased after a sustained challenge. These effects are prevented by the P2X7 antagonist A-804598, and the autophagy/phosphoinositide-3-kinase inhibitor wortmannin (WM). Finally, a chronic in vivo treatment with A-804598 in SOD1-G93A mice decreases the expression of SQSTM1/p62 in lumbar spinal cord at end stage of disease. These data identify the modulation of the autophagic flux as a novel mechanism by which P2X7 activates ALS-microglia, to be considered for further investigations in ALS.

Identification of Novel Vacuolin-1 Analogues as Autophagy Inhibitors by Virtual Drug Screening and Chemical Synthesis.

Autophagy is a fundamental cellular degradation process which is essential for cell homeostasis, and dysfunctional autophagy has been associated with a variety of human diseases, such as cancer. Several autophagy chemical modulators have been applied in a number of preclinical or clinical trials against these autophagy related diseases, especially cancer. Small molecule vacuolin-1 potently and reversibly inhibits both endosomal-lysosomal trafficking and autophagosome-lysosome fusion, yet the molecular mechanisms underlying vacuolin-1 mediated autophagy inhibition remain unknown. Here, we first performed the virtual drug screening and identified 14 vacuolin-1 analogues as autophagy inhibitors. Based on these virtual screening results, we further designed and synthesized 17 vacuolin-1 analogues, and found that 13 of them are autophagy inhibitors and a couple of them are as potent as vacuolin-1. In summary, these studies expanded the pool of useful autophagy inhibitors and reveal the structural-activity relationship of vacuolin-1 analogues, which is useful for future development of vacuolin-1 analogues with high potency and for identification of the molecular targets of vacuolin-1.

4-Acetylantroquinonol B suppresses autophagic flux and improves cisplatin sensitivity in highly aggressive epithelial cancer through the PI3K/Akt/mTOR/p70S6K signaling pathway.

Targeting residual self-renewing, chemoresistant cancerous cells may represent the key to overcoming therapy resistance. The entry of these quiescent cells into an activated state is associated with high metabolic demand and autophagic flux. Therefore, modulating the autophagy pathway in aggressive carcinomas may be beneficial as a therapeutic modality. In this study, we evaluated the anti-tumor activities of 4-acetylantroquinonol B (4-AAQB) in chemoresistant ovarian cancer cells, particularly its ability to modulate autophagy through autophagy-related genes (Atg). Atg-5 was overexpressed in invasive ovarian cancer cell lines and tissue (OR: 5.133; P=0.027) and depleting Atg-5 in ES-2 cell lines significantly induced apoptosis. 4-AAQB effectively suppressed viability of various subtypes of ovarian cancer. Cells with higher cisplatin-resistance were more responsive to 4-AAQB. For the first time, we demonstrate that 4-AAQB significantly suppress Atg-5 and Atg-7 expression with decreased autophagic flux in ovarian cancer cells via inhibition of the PI3K/Akt/mTOR/p70S6K signaling pathway. Similar to Atg-5 silencing, 4-AAQB-induced autophagy inhibition significantly enhanced cell death in vitro. These results are comparable to those of hydroxychloroquine (HCQ). In addition, 4-AAQB/cisplatin synergistically induced apoptosis in ovarian cancer cells. In vivo, 4-AAQB/cisplatin also significantly induced apoptosis and autophagy in an ES-2 mouse xenografts model. This is the first report demonstrating the efficacy of 4-AAQB alone or in combination with cisplatin on the suppression of ovarian cancer via Atg-5-dependent autophagy. We believe these findings will be beneficial in the development of a novel anti-ovarian cancer therapeutic strategy.

Downregulation of KDM4A Suppresses the Survival of Glioma Cells by Promoting Autophagy.

Glioma is the most common type of primary intracranial tumor and has a poor prognosis. It has been reported that lysine-specific demethylase 4A (KDM4A) can promote tumor progression; however, its role in human glioma remains unclear. Western blot and qRT-PCR analyses showed that KDM4A was highly expressed in U87MG and T98G cells. 48 h after transfection with siKDM4A, the protein level of KDM4A was significantly downregulated. The silenced expression of KDM4A in T98G or U87MG cells inhibited cell viability and invasion, and aggravated cell apoptosis. We found that the siKDM4A led to a significant increase in acidic vesicular organelles (AVOs) and upregulated the expression of autophagy-related proteins, including LC3B-phosphatidylethanolamine conjugate, a cytosolic form of LC3B (LC3B-II/LC3B-I) and Beclin 1 in T98G and U87MG cells. Further studies demonstrated that after pretreatment with 3-MA (3 mmol/L) for 48 h, siKDM4A-transfected cells showed a prominent decrease in LC3B-II/LC3B-I and Beclin 1, accompanied by increased viability and invasion and decreased apoptosis. Our results suggest that the inhibition of KDM4A expression might efficiently suppress glioma cell survival by promoting autophagy, providing a promising agent for treating malignant gliomas.

AUTEN-67, an autophagy-enhancing drug candidate with potent antiaging and neuroprotective effects.

Autophagy is a major molecular mechanism that eliminates cellular damage in eukaryotic organisms. Basal levels of autophagy are required for maintaining cellular homeostasis and functioning. Defects in the autophagic process are implicated in the development of various age-dependent pathologies including cancer and neurodegenerative diseases, as well as in accelerated aging. Genetic activation of autophagy has been shown to retard the accumulation of damaged cytoplasmic constituents, delay the incidence of age-dependent diseases, and extend life span in genetic models. This implies that autophagy serves as a therapeutic target in treating such pathologies. Although several autophagy-inducing chemical agents have been identified, the majority of them operate upstream of the core autophagic process, thereby exerting undesired side effects. Here, we screened a small-molecule library for specific inhibitors of MTMR14, a myotubularin-related phosphatase antagonizing the formation of autophagic membrane structures, and isolated AUTEN-67 (autophagy enhancer-67) that significantly increases autophagic flux in cell lines and in vivo models. AUTEN-67 promotes longevity and protects neurons from undergoing stress-induced cell death. It also restores nesting behavior in a murine model of Alzheimer disease, without apparent side effects. Thus, AUTEN-67 is a potent drug candidate for treating autophagy-related diseases.

Disruption of sphingolipid metabolism augments ceramide-induced autophagy in preeclampsia.

Bioactive sphingolipids including ceramides are involved in a variety of pathophysiological processes by regulating cell death and survival. The objective of the current study was to examine ceramide metabolism in preeclampsia, a serious disorder of pregnancy characterized by oxidative stress, and increased trophoblast cell death and autophagy. Maternal circulating and placental ceramide levels quantified by tandem mass spectrometry were elevated in pregnancies complicated by preeclampsia. Placental ceramides were elevated due to greater de novo synthesis via high serine palmitoyltransferase activity and reduced lysosomal breakdown via diminished ASAH1 expression caused by TGFB3-induced E2F4 transcriptional repression. SMPD1 activity was reduced; hence, sphingomyelin degradation by SMPD1 did not contribute to elevated ceramide levels in preeclampsia. Oxidative stress triggered similar changes in ceramide levels and acid hydrolase expression in villous explants and trophoblast cells. MALDI-imaging mass spectrometry localized the ceramide increases to the trophophoblast layers and syncytial knots of placentae from pregnancies complicated by preeclampsia. ASAH1 inhibition or ceramide treatment induced autophagy in human trophoblast cells via a shift of the BOK-MCL1 rheostat toward prodeath BOK. Pharmacological inhibition of ASAH1 activity in pregnant mice resulted in increased placental ceramide content, abnormal placentation, reduced fetal growth, and increased autophagy via a similar shift in the BOK-MCL1 system. Our results reveal that oxidative stress-induced reduction of lysosomal hydrolase activities in combination with elevated de novo synthesis leads to ceramide overload, resulting in increased trophoblast cell autophagy, and typifies preeclampsia as a sphingolipid storage disorder.