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Microgravity-induced muscle atrophy experienced by astronauts shares similar physiological changes to muscle wasting experienced by older adults, known as sarcopenia. These shared attributes provide a rationale for investigating microgravity-induced molecular changes in human bioengineered muscle cells that may also mimic the progressive underlying pathophysiology of sarcopenia. Here, we report the results of an experiment that incorporated three-dimensional myobundles derived from muscle biopsies from young and older adults, that were integrated into an autonomous CubeLab™, and flown to the International Space Station (ISS) aboard SpaceX CRS-21 in December 2020 as part of the NIH/NASA funded Tissue Chips in Space program. Global transcriptomic RNA-Seq analysis comparing the myobundles in space and on the ground revealed downregulation of shared transcripts related to myoblast proliferation and muscle differentiation for those in space. The analysis also revealed differentially expressed gene pathways related to muscle metabolism unique to myobundles derived from the older cohort exposed to the space environment compared to ground controls. Gene classes related to inflammatory pathways were uniquely modulated in flight samples cultured from the younger cohort compared to ground controls. Our muscle tissue chip platform provides a novel approach to studying the cell autonomous effects of microgravity on muscle cell biology that may not be appreciated on the whole organ or organism level and sets the stage for continued data collection from muscle tissue chip experimentation in microgravity. Thus, we also report on the challenges and opportunities for conducting autonomous tissue-on-chip CubeLabTM payloads on the ISS.

Dopaminergic degeneration is a central feature of Parkinson’s disease (PD), but glial dysfunction may accelerate or trigger neuronal death. In fact, astrocytes play a key role in maintenance of the blood-brain barrier and detoxification. 6-hydroxydopamine (6OHDA) is used to induce PD in rodent models due to its specific toxicity to dopaminergic neurons, but its effect on astrocytes has been poorly investigated. Here, we show that 6OHDA dose-dependently impairs autophagy in human U373 and primary murine astrocytes in the absence of cell death. LC3II downregulation was observed 6 to 48 hours after treatment. Interestingly, 6OHDA enhanced NRH:quinone oxidoreductase 2 (NQO2) expression and activity in U373 cells, even if 6OHDA is not its substrate. The autophagic flux was restored by inhibition of NQO2 with S29434, which correlated with a partial reduction of oxidative stress in response to 6OHDA in human and murine astrocytes. NQO2 inhibition also increased neuroprotective capability of U373 cells, since S29434 protected dopaminergic SHSY5Y cells from 6OHDA-induced cell death when co-cultured with astrocytes. Silencing of NQO2 attenuated toxic effects of 6OHDA on autophagy. Finally, the analysis of Gene Expression Ominibus datasets showed elevated NQO2 gene expression in the blood cells of early-stage PD patients. These data support a toxifying function of NQO2 in dopaminergic degeneration via negative regulation of autophagy and neuroprotection in astrocytes, suggesting a novel pharmacological target in PD.

Microphysiological systems (MPS), also referred to as tissue chips, incorporating 3D skeletal myobundles are a novel approach for physiological and pharmacological studies to uncover new medical treatments for sarcopenia. We characterize a MPS in which engineered skeletal muscle myobundles derived from donor-specific satellite cells that model aged phenotypes are encapsulated in a perfused tissue chip platform containing platinum electrodes. Our myobundles were derived from CD56

International audience; Dietary flavonoids stimulate autophagy and prevent liver dysfunction, but the upstream signaling pathways triggered by these compounds are not well understood. Certain polyphenols bind directly to NRH-quinone oxidoreductase 2 (NQO2) and inhibit its activity. NQO2 is highly expressed in the liver, where it participates in quinone metabolism, but recent evidence indicates that it may also play a role in the regulation of oxidative stress and autophagy. Here, we addressed a potential role of NQO2 in autophagy induction by flavonoids. The pro-autophagic activity of seven flavonoid aglycons correlated perfectly with their ability to inhibit NQO2 activity, and flavones such as apigenin and luteolin showed the strongest activity in all assays. The silencing of NQO2 strongly reduced flavone-induced autophagic flux, although it increased basal LC3-II levels in HepG2 cells. Both flavones induced AMP kinase (AMPK) activation, while its reduction by AMPK beta (PRKAB1) silencing inhibited flavone-induced autophagy. Interestingly, the depletion of NQO2 levels by siRNA increased the basal AMPK phosphorylation but abrogated its further increase by apigenin. Thus, NQO2 contributes to the negative regulation of AMPK activity and autophagy, while its targeting by flavones releases pro-autophagic signals. These findings imply that NQO2 works as a flavone receptor mediating autophagy and may contribute to other hepatic effects of flavonoids.

Nonalcoholic fatty liver disease (NAFLD) is the most common cause of chronic liver disease worldwide (Younossi et al., 2019). As more individuals develop obesity, diabetes, and metabolic syndrome, the global prevalence of NAFLD is likewise increasing, affecting as high as a billion people. NAFLD is a spectrum disease that includes a progression from benign hepatic steatosis to nonalcoholic steatohepatitis (NASH) in its most severe form. Approximately 17% of the population advances to liver fibrosis and cancer making NAFLD/NASH the second-most common reason for liver transplantation (Mikolasevic et al., 2018). With the shortage of organ donors, limited supply of healthy liver tissue, and failure of animal models to accurately predict human toxicity, there is a growing interest in hepatocyte-based in vitro systems for research to generate results that better predict human pathophysiology. In this chapter, we will discuss the inherent challenges involved in recapitulating the complex liver tissue in vitro. We will highlight the recent advances in human-induced pluripotent stem cell (hiPSC) technology including three-dimensional (3D) self-organized cell culture models such as spheroids and organoids and the emergence of microphysiological systems that have facilitated the use of iPSC-derived hepatocytes. These hepatocytelike cells isolated from healthy individuals and patients with liver disease or who harbor single nucleotide polymorphisms that make them high risk of developing fatty liver disease have become promising platforms to model in vitro NAFLD for dissecting basic liver biology and for applications in therapeutic screening and clinical evaluation.

Non-alcoholic fatty liver disease (NAFLD) has a large impact on global health. At the onset of disease, NAFLD is characterized by hepatic steatosis defined by the accumulation of triglycerides stored as lipid droplets. Developing therapeutics against NAFLD and progression to non-alcoholic steatohepatitis (NASH) remains a high priority in the medical and scientific community. Drug discovery programs to identify potential therapeutic compounds have supported high throughput/high-content screening of in vitro human-relevant models of NAFLD to accelerate development of efficacious anti-steatotic medicines. Human induced pluripotent stem cell (hiPSC) technology is a powerful platform for disease modeling and therapeutic assessment for cell-based therapy and personalized medicine. In this study, we applied AstraZeneca&rsquo, s chemogenomic library, hiPSC technology and multiplexed high content screening to identify compounds that significantly reduced intracellular neutral lipid content. Among 13,000 compounds screened, we identified hits that protect against hiPSC-derived hepatic endoplasmic reticulum stress-induced steatosis by a mechanism of action including inhibition of the cyclin D3-cyclin-dependent kinase 2-4 (CDK2-4)/CCAAT-enhancer-binding proteins (C/EBP&alpha, )/diacylglycerol acyltransferase 2 (DGAT2) pathway, followed by alteration of the expression of downstream genes related to NAFLD. These findings demonstrate that our phenotypic platform provides a reliable approach in drug discovery, to identify novel drugs for treatment of fatty liver disease as well as to elucidate their underlying mechanisms.

Mannose-binding lectin (MBL)-Associated Serine Proteases (MASP)-1 and 3, key enzymes in the lectin complement pathway of innate immune response, are also expressed in glioma cell lines. We investigated MASP-1 and MASP-3 expression during dibutyryl cyclic AMP (dbcAMP)- or Interleukin-6 (rIL-6)-induced astrocytic differentiation of C6 glioma cells. Our results demonstrate that C6 cells express basal levels of MASP-1 and MASP-3 and following exposure to dbcAMP or IL-6, a consistent MASP-1 and MASP-3 mRNA up-regulation was found, with a behavior similar to that showed by the fibrillary acidic protein (GFAP). Furthermore, in cell conditioned media, rIL-6 stimulated MASP-3 secretion which reached levels similar to those obtained by dbcAMP treatment. Moreover, the detection of a 46-kDa MASP-3 suggested its processing to the mature form in the extracellular cell medium. Interestingly, the H89 PKA inhibitor, mostly affected dbcAMP-induced MASP-1 and MASP-3 mRNA levels, compared to that of rIL-6, suggesting that cAMP/PKA pathway contributes to MASP-1 and MASP-3 up-regulation. MASP-1 and MASP-3 expression increase was concomitant with dbcAMP- or rIL-6-induced phosphorylation of STAT3. Our findings suggest that the increase in intracellular cAMP concentration or rIL-6 stimulation can play a role in innate immunity enhancing MASP-1 and MASP-3 expression level in C6 glioma cells.

Bergamot flavonoids counteract dyslipidemia and hyperglycemia but fail to induce a significant weight loss. Here, we evaluated the efficacy of bergamot polyphenol extract complex (BPE-C), a novel bergamot juice-derived formulation enriched with flavonoids and pectins, on several metabolic syndrome parameters. Obese patients with atherogenic index of plasma (AIP) over 0.34 and mild hyperglycemia were recruited to a double-blind randomized trial comparing two doses of BPE-C (650 and 1300 mg daily) with placebo. Fifty-two subjects met the inclusion criteria and were assigned to three experimental groups. Fifteen subjects per group completed 90 days-trial. BPE-C reduced significantly fasting glucose by 18.1%, triglycerides by 32% and cholesterol parameters by up to 41.4%, leading to a powerful reduction of AIP (below 0.2) in the high dose group. The homeostasis model assessment of insulin resistance (HOMA-IR) and insulin levels were also reduced. Moreover, BPE-C decreased body weight by 14.8% and body mass index by 15.9% in BPE-C high group. This correlated with a significant reduction of circulating hormones balancing caloric intake, including leptin, ghrelin and upregulation of adiponectin. All effects showed a dose-dependent tendency. This study suggests that food supplements, containing full spectrum of bergamot juice components, such as BPE-C efficiently induce a combination of weight loss and insulin sensitivity effects together with a robust reduction of atherosclerosis risk.

Hepatic steatosis, a reversible state of metabolic dysregulation, can promote the onset of nonalcoholic steatohepatitis (NASH), and its transition is thought to be critical in disease evolution. The association between endoplasmic reticulum (ER) stress response and hepatocyte metabolism disorders prompted us to characterize ER stress-induced hepatic metabolic dysfunction in human induced pluripotent stem cell-derived hepatocytes (hiPSC-Hep), to explore regulatory pathways and validate a phenotypic in vitro model for progression of liver steatosis. We treated hiPSC-Hep with a ratio of unsaturated and saturated fatty acids in the presence of an inducer of ER stress to synergistically promote triglyceride accumulation and dysregulate lipid metabolism. We monitored lipid accumulation by high-content imaging and measured gene regulation by RNA sequencing and reverse transcription quantitative PCR analyses. Our results show that ER stress potentiated intracellular lipid accumulation by 5-fold in hiPSC-Hep in the absence of apoptosis. Transcriptome pathway analysis identified ER stress pathways as the most significantly dysregulated of all pathways affected. Obeticholic acid dose dependently inhibited lipid accumulation and modulated gene expression downstream of the farnesoid X receptor. We were able to identify modulation of hepatic markers and gene pathways known to be involved in steatosis and nonalcoholic fatty liver disease (NAFLD), in support of a hiPSC-Hep disease model that is relevant to clinical data for human NASH. Our results show that the model can serve as a translational discovery platform for the understanding of molecular pathways involved in NAFLD, and can facilitate the identification of novel therapeutic molecules based on high-throughput screening strategies., Summary: Our study demonstrates expanded use of human induced pluripotent stem cell-derived hepatocytes for molecular studies and drug screening, to evaluate new therapeutics with an antisteatotic mechanism of action for nonalcoholic fatty liver disease.

Nonalcoholic fatty liver disease (NAFLD) is the most common liver disease in industrialized countries. Defective autophagy of lipid droplets (LDs) in hepatocytes, also known as lipophagy, has recently been identified as a possible pathophysiological mechanism of NAFLD. Experimental and epidemiological evidence suggests that dietary polyphenols may prevent NAFLD. To address this hypothesis and analyze the underlying mechanisms, we supplemented bergamot polyphenol fraction (BPF) to cafeteria (CAF) diet-fed rats, a good model for pediatric metabolic syndrome and NAFLD. BPF treatment (50 mg/kg/day supplemented with drinking water, 3 months) potently counteracted the pathogenic increase of serum triglycerides and had moderate effects on blood glucose and obesity in this animal model. Importantly, BPF strongly reduced hepatic steatosis as documented by a significant decrease in total lipid content (-41.3% ± 12% S.E.M.), ultrasound examination and histological analysis of liver sections. The morphometric analysis of oil-red stained sections confirmed a dramatic reduction in LDs parameters such as total LD area (48.5% ± 15% S.E.M.) in hepatocytes from CAF+BPF rats. BPF-treated livers showed increased levels of LC3 and Beclin 1 and reduction of SQSTM1/p62, suggesting autophagy stimulation. Consistent with BPF stimulation of lipophagy, higher levels of LC3II were found in the LD subcellular fractions of BPF-expose livers. This study demonstrates that the liver and its lipid metabolism are the main targets of bergamot flavonoids, supporting the concept that supplementation of BPF is an effective strategy to prevent NAFLD.

Autophagy dysfunction has been implicated in the pathogenesis of nonalcoholic fatty liver disease (NAFLD). Natural compounds present in bergamot polyphenol fraction (BPF) prevent NAFLD and induce autophagy in rat livers. Here, we employed HepG2 cells expressing DsRed-LC3-GFP, a highly sensitive model system to screen for proautophagic compounds present in BPF. BPF induced autophagy in a time- and dose-dependent fashion and the effect was amplified in cells loaded with palmitic acid. Autophagy was mediated by the hydrophobic fraction of acid-hydrolyzed BPF (A-BPF), containing six flavanone and flavone aglycones as identified by liquid chromatography-high-resolution mass spectrometry. Among them, naringenin, hesperitin, eriodictyol and diosmetin were weak inducers of autophagy. Apigenin showed the strongest and dose-dependent proautophagic activity at early time points (6 h). Luteolin induced a biphasic autophagic response, strong at low doses and inhibitory at higher doses. Both flavones were toxic in HepG2 cells and in differentiated human liver progenitors HepaRG upon longer treatments (24 h). In contrast, BPF and A-BPF did not show any toxicity, but induced a persistent increase in autophagic flux. A mixture of six synthetic aglycones mimicking A-BPF was sufficient to induce a similar autophagic response, but it was mildly cytotoxic. Thus, while six main BPF flavonoids fully account for its proautophagic activity, their combined effect is not sufficient to abrogate cytotoxicity of individual compounds. This suggests that a natural polyphenol phytocomplex, such as BPF, is a safer and more effective strategy for the treatment of NAFLD than the use of pure flavonoids.

BACKGROUND AND PURPOSE The mechanisms of paraquat (PQ)-induced toxicity are poorly understood and PQ poisoning is often fatal due to a lack of effective antidotes. In this study we report the effects of N-[2-(2-methoxy-6H-dipyrido{2,3-a:3,2-e}pyrrolizin-11-yl)ethyl]-2-furamide (NMDPEF), a melatonin-related inhibitor of quinone oxidoreductase2 (QR2) on the toxicity of PQ in vitro & in vivo. EXPERIMENTAL APPROACH Prevention of PQ-induced toxicity was tested in different cells, including primary pneumocytes and astroglial U373 cells. Cell death and reactive oxygen species (ROS) were analysed by flow cytometry and fluorescent probes. QR2 silencing was achieved by lentiviral shRNAs. PQ (30 mg·kg(-1)) and NMDPEF were administered i.p. to Wistar rats and animals were monitored for 28 days. PQ toxicity in the substantia nigra (SN) was tested by a localized microinfusion and electrocorticography. QR2 activity was measured by fluorimetry of N-benzyldihydronicotinamide oxidation. KEY RESULTS NMDPEF potently antagonized non-apoptotic PQ-induced cell death, ROS generation and inhibited cellular QR2 activity. In contrast, the cytoprotective effect of melatonin and apocynin was limited and transient compared with NMDPEF. Silencing of QR2 attenuated PQ-induced cell death and reduced the efficacy of NMDPEF. Significantly, NMDPEF (4.5 mg·kg(-1)) potently antagonized PQ-induced systemic toxicity and animal mortality. Microinfusion of NMDPEF into SN prevented severe behavioural and electrocortical effects of PQ which correlated with inhibition of malondialdehyde accumulation in cells and tissues. CONCLUSIONS AND IMPLICATIONS NMDPEF protected against PQ-induced toxicity in vitro and in vivo, suggesting a key role for QR2 in the regulation of oxidative stress.

Wrong alimentary behaviors and so-called &ldquo, junk food&rdquo, are a driving force for the rising incidence of non-alcoholic fatty liver disease (NAFLD) among children and adults. The &ldquo, toxicity can be studied in &ldquo, cafeteria&rdquo, (CAF) diet animal model. Young rats exposed to CAF diet become obese and rapidly develop NAFLD. We have previously showed that bergamot (Citrus bergamia Risso et Poiteau) flavonoids, in the form of bergamot polyphenol fraction (BPF), effectively prevent CAF diet-induced NAFLD in rats. Here, we addressed if BPF can accelerate therapeutic effects of weight loss induced by a normocaloric standard chow (SC) diet. 21 rats fed with CAF diet for 16 weeks to induce NAFLD with inflammatory features (NASH) were divided into three groups. Two groups were switched to SC diet supplemented or not with BPF (CAF/SC&plusmn, BPF), while one group continued with CAF diet (CAF/CAF) for 10 weeks. BPF had no effect on SC diet-induced weight loss, but it accelerated hepatic lipid droplets clearance and reduced blood triglycerides. Accordingly, BPF improved insulin sensitivity, but had little effect on leptin levels. Interestingly, the inflammatory parameters were still elevated in CAF/SC livers compared to CAF/CAF group after 10 weeks of dietary intervention, despite over 90% hepatic fat reduction. In contrast, BPF supplementation decreased hepatic inflammation by reducing interleukin 6 (Il6) mRNA expression and increasing anti-inflammatory Il10, which correlated with fewer Kupffer cells and lower inflammatory foci score in CAF/SC+BPF livers compared to CAF/SC group. These data indicate that BPF mediates a specific anti-inflammatory activity in livers recovering from NASH, while it boosts lipid-lowering and anti-diabetic effects of the dietary intervention.

Protease Nexin-1, a 43-kDa glycoprotein, is a major physiological thrombin inhibitor involved in the modulation of nerve cell plasticity. Recombinant rat Protease Nexin-1 (rPN-1) was efficiently produced in Escherichia coli using a T7 RNA polymerase based expression system and purified by heparin-sepharose affinity chromatography yielding 3 mg of protein per liter of cell culture. The purity and chemical identity of rPN-1 were assessed by SDS-PAGE, Reverse Phase- High Performance Liquid Chromatography, mass spectrometry and two-dimensional-gel electrophoresis. Conformational analysis by circular dichroism and fluorescence spectroscopy revealed the presence of mixed α/β secondary structure and the prevailing localization of Trp-residues in rather polar environments. Fluorescence titration of rPN-1 with heparin indicated that rPN-1 binds heparin with high affinity. Furthermore, the formation of a SDS-stable 1:1 thrombin–rPN-1 complex, monitored by SDS-PAGE, confirmed the native-like structure of rPN-1. Finally, the cellular effects of rPN-1, such as its ability to promote neurite outgrowth in neuroblastoma cells, were found to be very similar to those elicited by natural PN-1. Altogether, our results demonstrate that glycosylation does not alter neither structure nor function of PN-1 and that E. coli is a suitable expression system for obtaining milligram quantities of pure and fully active rPN-1 for structural and functional studies.

Oxidative stress (OS) stimulates autophagy in different cellular systems, but it remains controversial if this rule can be generalized. We have analyzed the effect of chronic OS induced by the parkinsonian toxin paraquat (PQ) on autophagy in astrocytoma cells and primary astrocytes, which represent the first cellular target of neurotoxins in the brain. PQ decreased the basal levels of LC3-II and LC3-positive vesicles, and its colocalization with lysosomal markers, both in the absence and presence of chloroquine. This was paralleled by increased number and size of SQSTM1/p62 aggregates. Downregulation of autophagy was also observed in cells chronically exposed to hydrogen peroxide or nonlethal concentrations of PQ, and it was associated with a reduced astrocyte capability to protect dopaminergic cells from OS in co-cultures. Surprisingly, PQ treatment led to inhibition of MTOR, activation of MAPK8/JNK1 and MAPK1/ERK2-MAPK3/ERK1 and upregulation of BECN1/Beclin 1 expression, all signals typically correlating with induction of autophagy. Reduction of OS by NMDPEF, a specific NQO2 inhibitor, but not by N-acetylcysteine, abrogated the inhibitory effect of PQ and restored autophagic flux. Activation of NQO2 by PQ or menadione and genetic manipulation of its expression confirmed the role of this enzyme in the inhibitory action of PQ on autophagy. PQ did not induce NFE2L2/NRF2, but when it was co-administered with NMDPEF NFE2L2 activity was enhanced in a SQSTM1-independent fashion. Thus, a prolonged OS in astrocytes inhibits LC3 lipidation and impairs autophagosome formation and autophagic flux, in spite of concomitant activation of several pro-autophagic signals. These findings outline an unanticipated neuroprotective role of astrocyte autophagy and identify in NQO2 a novel pharmacological target for its positive modulation.