Autophagic Impairment Research Articles

Abstract P138: Diet Influences the Expression of Renal Sodium-Glucose Cotransporters in Juvenile Male Rats

Poor nutrition and metabolic dysfunction are becoming more prevalent among children, as evident by increasing childhood obesity in recent years. The kidney sodium-glucose cotransporter 2 (SGLT2) is a contributor to overall metabolic homeostasis, with inhibition of SGLT2 significantly reducing circulating glucose and lipids. The study objective was to determine if diet influences the expression of renal glucose transporters in young rats. Weanling male Wistar Kyoto rats received 1) standard laboratory chow, 2) AIN-93G diet, or 3) a high-fat diet (60% kcal, HFD). Each dietary group was then further subdivided to receive either filtered water or 5% (w/v) sucrose solution ad libitum . Four weeks later, animals were euthanized and kidneys were collected and processed. mRNA expression of Sglt1 , Sglt2 , Sqstm1 , and Nlrp3 was determined using gene specific primers and RT-PCR. Renal SGLT2 protein expression was visualized using immunohistochemistry and quantified using Imagej software. Diet significantly influenced the renal expression of both Sglt1 ( P =0.005) and Sglt2 (P=0.046), while beverage did not have a significant effect on either ( P =0.7 and P =0.4, respectively). Rats fed the HFD had more than 30% reduction in Sglt1 mRNA expression as compared to rats fed chow, and ~20% reduction in Sglt2 as compared to rats fed AIN. Preliminary data suggest that diet and beverage significantly altered renal protein expression of SGLT2, with rats fed chow having the lowest renal expression that was slightly increased with sugar consumption (1.5% versus 2.7%, respectively). Rats fed AIN had the greatest expression, but was drastically reduced in rats consuming sugar (4% versus 1.2%, respectively). To determine if autophagy and inflammasome activation is involved, the expression of Sqstm1 and Nlrp3 was measured by RT-PCR. Similarly, rats fed HFD had the lowest expression of both Sqstm1 ( P<0.001) and Nlrp3 ( P=0.03). Sqstm1 and Nlrp3 mRNA expression was decreased with sugar consumption in rats fed chow and AIN, whereas Sqstm1 ( P =0.01) and Nlrp3 ( P <0.001) mRNA expression was increased in rats fed HFD with sugar beverage. Our data suggest that diet significantly influences renal glucose transporter expression in juvenile rats in a way that may be mediated by autophagic impairment. The modulatory effect of sugar consumption on SGLT2 protein, Sqstm1, and Nlrp3 was diet-dependent, highlighting the importance of nutrition to renal physiology within weeks after weaning.

Traumatic brain injury heterogeneity affects cell death and autophagy.

Traumatic brain injury (TBI) mechanism and severity are heterogenous clinically, resulting in a multitude of physical, cognitive, and behavioral deficits. Impact variability influences the origin, spread, and classification of molecular dysfunction which limits strategies for comprehensive clinical intervention. Indeed, there are currently no clinically approved therapeutics for treating the secondary consequences associated with TBI. Thus, examining pathophysiological changes from heterogeneous impacts is imperative for improving clinical translation and evaluating the efficacy of potential therapeutic strategies. Here we utilized TBI models that varied in both injury mechanism and severity including severe traditional controlled cortical impact (CCI), modified mild CCI (MTBI), and multiple severities of closed-head diffuse TBI (DTBI), and assessed pathophysiological changes. Severe CCI induced cortical lesions and necrosis, while both MTBI and DTBI lacked lesions or significant necrotic damage. Autophagy was activated in the ipsilateral cortex following CCI, but acutely impaired in the ipsilateral hippocampus. Additionally, autophagy was activated in the cortex following DTBI, and autophagic impairment was observed in either the cortex or hippocampus following impact from each DTBI severity. Thus, we provide evidence that autophagy is a therapeutic target for both mild and severe TBI. However, dramatic increases in necrosis following CCI may negatively impact the clinical translatability of therapeutics designed to treat acute dysfunction in TBI. Overall, these results provide evidence that injury sequalae affiliated with TBI heterogeneity is linked through autophagy activation and/or impaired autophagic flux. Thus, therapeutic strategies designed to intervene in autophagy may alleviate pathophysiological consequences, in addition to the cognitive and behavioral deficits observed in TBI.

The role and mechanism of macrophage autophagy in the experimental model of chronic obstructive pulmonary disease.

Macrophages play an important role in chronic obstructive pulmonary disease (COPD). Cigarette smoke (CS) impairs autophagy in alveolar macrophages from COPD patients, and autophagic impairment leads to reduced clearance of protein aggregates, dysfunctional mitochondria, and defective bacterial delivery to lysosomes. However, the exact function of lung macrophage autophagy in the pathogenesis of CS-induced COPD remains largely unknown. Western blot detected the expression of autophagy-related proteins induced by CSE. The model of COPD mice was established by CS exposure combined with CSE intraperitoneal injection. Double immunofluorescence was used to measure the CD206+LC3B+ cells. The morphological changes and effects on lung function were observed. Masson staining detected the changes in collagen fibers in lung tissue. The expression levels of E-cadherinb and N-cadherinb were detected by immunohistochemistry. Western blot detected the expression of ATP6V1E1 in lung tissue. At 24 hours of exposure to CSE, the expression levels of LC3B (microtubule-associated protein 1A/1B-light chain 3B) and P62 (nucleoporin 62) were highest at 1% CSE and AGT5 (nucleoporin 62) at 2.5% CSE; at 48 hours, the expression levels of LC3B, P62 and AGT5 were highest at 2.5% CSE, and as the intervention time increased.CD206+LC3B+ cells were significantly higher in the COPD group. Enhanced macrophage autophagy may promote emphysema formation and aggravate lung function damage. The expression of E-cadherinb in lung tissue of the COPD group was decreased, and N-cadherinb expression was increased; the expression of E-cadherinb was increased, and N-cadherinb expression was decreased in ATG5myeΔ COPD mice. The expression of ATP6V1E1 in the lung tissue was increased in the COPD group; ATP6V1E1 expression was decreased in the lung tissues of ATG5myeΔ COPD mice. CSE enhanced macrophage autophagy, leads to increased lung function impairment and collagenous fiber in lung tissue, as well as promotes epithelial-mesenchymal transition, and eventually leads to small airway remodeling, which may be achieved through the ATG5/ATP6V1E1 pathway.

RNF213 variant and autophagic impairment: A pivotal link to endothelial dysfunction in moyamoya disease

Moyamoya disease (MMD) is closely associated with the Ring Finger Protein 213 (RNF213), a susceptibility gene for MMD. However, its biological function remains unclear. We aimed to elucidate the role of RNF213 in the damage incurred by human endothelial cells under oxygen-glucose deprivation (OGD). We analyzed autophagy in peripheral blood mononuclear cells (PBMCs) derived from patients carrying either RNF213 wildtype (WT) or variant (p.R4810K). Subsequently, human umbilical vein endothelial cells (HUVECs) were transfected with RNF213 WT (HUVECWT) or p.R4810K (HUVECR4810K) and exposed to OGD for 2 h. Immunoblotting was used to analyze autophagy marker proteins, and endothelial function was analyzed by tube formation assay. Autophagic vesicles were observed using transmission electron microscopy. Post-OGD exposure, we administered rapamycin and cilostazol as potential autophagy inducers. The RNF213 variant group during post-OGD exposure (vs. pre-OGD) showed autophagy inhibition, increased protein expression of SQSTM1/p62 (p < 0.0001) and LC3-II (p = 0.0039), and impaired endothelial function (p = 0.0252). HUVECR4810K during post-OGD exposure (versus pre-OGD) showed a remarkable increase in autophagic vesicles. Administration of rapamycin and cilostazol notably restored the function of HUVECR4810K and autophagy. Our findings support the pivotal role of autophagy impaired by the RNF213 variant in MMD-induced endothelial cell dysfunction.

A partial Drp1 knockout improves autophagy flux independent of mitochondrial function

BackgroundDynamin-related protein 1 (Drp1) plays a critical role in mitochondrial dynamics. Partial inhibition of this protein is protective in experimental models of neurological disorders such as Parkinson’s disease and Alzheimer’s disease. The protective mechanism has been attributed primarily to improved mitochondrial function. However, the observations that Drp1 inhibition reduces protein aggregation in such neurological disorders suggest the involvement of autophagy. To investigate this potential novel protective mechanism of Drp1 inhibition, a model with impaired autophagy without mitochondrial involvement is needed.MethodsWe characterized the effects of manganese (Mn), which causes parkinsonian-like symptoms in humans, on autophagy and mitochondria by performing dose-response studies in two cell culture models (stable autophagy HeLa reporter cells and N27 rat immortalized dopamine neuronal cells). Mitochondrial function was assessed using the Seahorse Flux Analyzer. Autophagy flux was monitored by quantifying the number of autophagosomes and autolysosomes, as well as the levels of other autophagy proteins. To strengthen the in vitro data, multiple mouse models (autophagy reporter mice and mutant Drp1+/− mice and their wild-type littermates) were orally treated with a low chronic Mn regimen that was previously reported to increase α-synuclein aggregation and transmission via exosomes. RNAseq, laser captured microdissection, immunofluorescence, immunoblotting, stereological cell counting, and behavioural studies were used.Results in vitrodata demonstrate that at low non-toxic concentrations, Mn impaired autophagy flux but not mitochondrial function and morphology. In the mouse midbrain, RNAseq data further confirmed autophagy pathways were dysregulated but not mitochondrial related genes. Additionally, Mn selectively impaired autophagy in the nigral dopamine neurons but not the nearby nigral GABA neurons. In cells with a partial Drp1-knockdown and Drp1+/− mice, Mn induced autophagic impairment was significantly prevented. Consistent with these observations, Mn increased the levels of proteinase-K resistant α-synuclein and Drp1-knockdown protected against this pathology.ConclusionsThis study demonstrates that improved autophagy flux is a separate mechanism conferred by Drp1 inhibition independent of its role in mitochondrial fission. Given that impaired autophagy and mitochondrial dysfunction are two prominent features of neurodegenerative diseases, the combined protective mechanisms targeting these two pathways conferred by Drp1 inhibition make this protein an attractive therapeutic target.Graphical

Control of NAD+ homeostasis by autophagic flux modulates mitochondrial and cardiac function

Impaired autophagy is known to cause mitochondrial dysfunction and heart failure, in part due to altered mitophagy and protein quality control. However, whether additional mechanisms are involved in the development of mitochondrial dysfunction and heart failure in the setting of deficient autophagic flux remains poorly explored. Here, we show that impaired autophagic flux reduces nicotinamide adenine dinucleotide (NAD+) availability in cardiomyocytes. NAD+ deficiency upon autophagic impairment is attributable to the induction of nicotinamide N-methyltransferase (NNMT), which methylates the NAD+ precursor nicotinamide (NAM) to generate N-methyl-nicotinamide (MeNAM). The administration of nicotinamide mononucleotide (NMN) or inhibition of NNMT activity in autophagy-deficient hearts and cardiomyocytes restores NAD+ levels and ameliorates cardiac and mitochondrial dysfunction. Mechanistically, autophagic inhibition causes the accumulation of SQSTM1, which activates NF-κB signaling and promotes NNMT transcription. In summary, we describe a novel mechanism illustrating how autophagic flux maintains mitochondrial and cardiac function by mediating SQSTM1-NF-κB-NNMT signaling and controlling the cellular levels of NAD+.

Chlorpromazine affects autophagy in association with altered Rag GTPase-mTORC1-TFEB signaling.

Autophagy is a critical protein and organelle quality control system, which regulates cellular homeostasis and survival. Growing pieces of evidence suggest that autophagic dysfunction is strongly associated with many human diseases, including neurological diseases and cancer. Among various autophagic regulators, microphthalmia (MiT)/TFE transcription factors, including transcription factor EB (TFEB), have been shown to act as the master regulators of autophagosome and lysosome biogenesis in both physiological and pathological conditions. According to the previous studies, chlorpromazine (CPZ), an FDA-approved antipsychotic drug, affects autophagy in diverse cell lines, but the underlying mechanism remains elusive. In our present study, we find that CPZ treatment induces TFEB nuclear translocation through Rag GTPases, the upstream regulators of mechanistic target of rapamycin complex 1 (mTORC1) signaling. Meanwhile, CPZ treatment also blocks autophagosome-lysosome fusion. Notably, we find a significant accumulation of immature autophagosome vesicles in CPZ-treated cells, which may impede cellular homeostasis due to the dysfunction of the autophagy-lysosome pathway. Interestingly and importantly, our data suggest that the expression of the active form of Rag GTPase heterodimers helps in reducing the accumulation of autophagosomes in CPZ-treated cells, further suggesting a major contribution of the Rag GTPase-mTORC1-TFEB signaling axis in CPZ-induced autophagic impairment.

MALAT1 Mediates α-Synuclein Expression through miR-23b-3p to Induce Autophagic Impairment and the Inflammatory Response in Microglia to Promote Apoptosis in Dopaminergic Neuronal Cells.

Parkinson's disease (PD) is a very common neurodegenerative disease that adversely affects the physical and mental health of many patients, but there is currently no effective treatment. To this end, this study focused on investigating the potential mechanisms leading to dopaminergic neuronal apoptosis in PD. Rotenone induces damage in dopaminergic neuronal MN9D cells. Apoptosis was detected by flow cytometry, and the expression of apoptosis-related proteins was detected by western blot. RT-qPCR was used to detect the expression of MALAT1 and miR-23b-3p. The expression of α-synuclein was detected by ELISA. A dual luciferase gene reporter assay was used to determine the targeted regulatory relationship between MALAT1 and miR-23b-3p and miR-23b-3p and α-synuclein. MN9D supernatant was cocultured with BV-2 cells, or BV-2 cells were treated with exogenous α-synuclein and then treated with an autophagy inhibitor (3-MA) and autophagy activator (RAPA). The expression of α-synuclein in BV-2 cells was detected by immunofluorescence. The expression of MIP-1α, a marker of microglial activation, was detected by ELISA. The nuclear translocation of NF-κB p65 was detected by immunofluorescence. The expression of proinflammatory cytokines was detected by ELISA. Western blotting was used to detect the expression of autophagy-related proteins. Apoptosis of MN9D cells was detected after coculture of BV-2 supernatant with MN9D. The expression of MALAT1 and α-synuclein was upregulated, while the expression of miR-23b-3p was downregulated in damaged MN9D cells, resulting in cell apoptosis. MALAT1 can negatively regulate the expression of miR-23b-3p, while miR-23b-3p negatively regulates the expression of α-synuclein. α-synuclein can enter BV-2 cells through cell phagocytosis. Coculture of BV-2 cells with α-synuclein or with MN9D supernatant overexpressing MALAT1 resulted in a decrease in the autophagy level of BV-2 cells and an inflammatory reaction. However, miR-23b-3p mimics and knockdown of α-synuclein reversed the effect of MALAT1 on autophagy and the inflammatory response of BV-2 cells. In addition, after coculture of BV-2 cells with α-synuclein, the level of autophagy further decreased when 3-MA was added, while the opposite result occurred when RAPA was added. After coculture of α-synuclein-treated BV-2 cell supernatant with MN9D cells, autophagy-impaired BV-2 promoted the apoptosis of MN9D cells, and 3-MA aggravated the autophagy disorder of BV-2 and further promoted the apoptosis of MN9D cells, while RAPA reversed the autophagy disorder of BV-2 and alleviated the apoptosis of MN9D cells. MALAT1 can promote α-synuclein expression by regulating miR-23b-3p, thereby inducing microglial autophagy disorder and an inflammatory response leading to apoptosis of dopaminergic neurons. This newly discovered molecular mechanism may provide a potential target for the treatment of PD.

Impaired Autophagy in Krabbe Disease: The Role of BCL2 and Beclin-1 Phosphorylation

Autophagic impairment was identified in many lysosomal storage diseases and adult neurodegenerative diseases. It seems that this defect could be directly related to the appearance of a neurodegenerative phenotype and could contribute to worsen metabolite accumulation and lysosomal distress. Thus, autophagy is becoming a promising target for supportive therapies. Autophagy alterations were recently identified also in Krabbe disease. Krabbe disease is characterized by extensive demyelination and dysmyelination and it is due to the genetic loss of function of the lysosomal enzyme galactocerebrosidase (GALC). This enzyme leads to the accumulation of galactosylceramide, psychosine, and secondary substrates such as lactosylceramide. In this paper, we induced autophagy through starvation and examined the cellular response occurring in fibroblasts isolated from patients. We demonstrated that the inhibitory AKT-mediated phosphorylation of beclin-1 and the BCL2-beclin-1 complex concur to reduce autophagosomes formation in response to starvation. These events were not dependent on the accumulation of psychosine, which was previously identified as a possible player in autophagic impairment in Krabbe disease. We believe that these data could better elucidate the capability of response to autophagic stimuli in Krabbe disease, in order to identify possible molecules able to stimulate the process.

Mesenchymal stem cell-derived extracellular vesicles carrying miR-99b-3p restrain microglial activation and neuropathic pain by stimulating autophagy.

Neuropathic pain is a complex condition that seriously affects human quality of life. This study aimed to investigate the therapeutic mechanism of mesenchymal stem cell-derived extracellular vesicles (MSC-EVs) and try to discover new targets for alleviating neuropathic pain. Extracellular vesicles were isolated and identified via ultracentrifugation. BV-2 microglial cells were stimulated with lipopolysaccharide (LPS) in the presence or absence of MSC-EVs. Further, microglial activation and neuroinflammation were evaluated by flow cytometry, RT-qPCR, and ELISA. High-throughput sequencing analysis was performed to reveal the differentially expressed (DE) miRNAs in BV-2 microglia. Autophagy-related regulators were assessed by Western blotting and Immunofluorescence staining. Chronic constriction injury (CCI) model was used to induce neuropathic pain in rats, and the mechanical withdrawal threshold (MWT) was measured. High-throughput sequencing analysis identified 17 DE miRNAs, which were mainly enriched in PI3K-AKT and mTOR signaling pathways. MSC-EVs inhibited the activation of PI3K/AKT/mTOR signaling pathway in LPS-stimulated microglia. Moreover, MSC-EVs treatment enhanced the autophagy level in activated microglia, whereas autophagy inhibitor 3-MA reversed the suppressing effects of MSC-EVs on microglial activation and neuroinflammation. The MSC-EV-mediated transfer of miR-99b-3p was verified to promote microglial autophagy, and miR-99b-3p overexpression suppressed the expression of pro-inflammatory factors in activated microglia. During in vivo studies, intrathecal injection of MSC-EVs significantly up-regulated the expression of miR-99b-3p, and alleviated mechanical allodynia caused by activated microglia in the spinal cord dorsal horn of CCI rats. Moreover, MSC-EVs treatment repaired CCI-induced autophagic impairment by stimulating autophagy in the spinal cord. Collectively, our findings demonstrated that MSC-EVs had an analgesic effect on neuropathic pain via promoting autophagy, and these antinociceptive effects were at least in part caused by MSC-EV-mediated transfer of miR-99b-3p, thereby inhibiting microglial activation and pro-inflammatory cytokines expression.

NLRP3 Inflammasome: From Pathophysiology to Therapeutic Target in Major Depressive Disorder

Major depressive disorder (MDD) is a highly prevalent psychiatric disorder, whose pathophysiology has been linked to the neuroinflammatory process. The increased activity of the Nod-like receptor pyrin containing protein 3 (NLRP3) inflammasome, an intracellular multiprotein complex, is intrinsically implicated in neuroinflammation by promoting the maturation and release of proinflammatory cytokines such as interleukin (IL)-1β and IL-18. Interestingly, individuals suffering from MDD have higher expression of NLRP3 inflammasome components and proinflammatory cytokines when compared to healthy individuals. In part, intense activation of the inflammasome may be related to autophagic impairment. Noteworthy, some conventional antidepressants induce autophagy, resulting in less activation of the NLRP3 inflammasome. In addition, the fast-acting antidepressant ketamine, some bioactive compounds and physical exercise have also been shown to have anti-inflammatory properties via inflammasome inhibition. Therefore, it is suggested that modulation of inflammasome-driven pathways may have an antidepressant effect. Here, we review the role of the NLRP3 inflammasome in the pathogenesis of MDD, highlighting that pathways related to its priming and activation are potential therapeutic targets for the treatment of MDD.

Preclinical reserpine models recapitulating motor and non-motor features of Parkinson's disease: Roles of epigenetic upregulation of alpha-synuclein and autophagy impairment.

Reserpine is an effective drug for the clinical treatment of hypertension. It also induces Parkinson’s disease (PD)-like symptoms in humans and animals possible through the inhibition of monoamine vesicular transporters, thus decreasing the levels of monoamine neurotransmitters in the brain. However, the precise mechanisms remain unclear. Herein, we aimed to develop a preclinical reserpine model recapitulating the non-motor and motor symptoms of PD and investigate the underlying potential cellular mechanisms. Incubation of reserpine induced apoptosis, led to the accumulation of intracellular reactive oxygen species (ROS), lowered DNA methylation of alpha-synuclein gene, resulted in alpha-synuclein protein deposition, and elevated the ratio of LC3-II/LC3-Ⅰ and p62 in cultured SH-SY5Y cells. Feeding reserpine dose-dependently shortened the lifespan and caused impairment of motor functions in male and female Drosophila. Moreover, long-term oral administration of reserpine led to multiple motor and non-motor symptoms, including constipation, pain hypersensitivity, olfactory impairment, and depression-like behaviors in mice. The mechanistic studies showed that chronic reserpine exposure caused hypomethylation of the alpha-synuclein gene and up-regulated its expression and elevated the ratio of LC3-II/LC3-Ⅰ and expression of p62 in the substantia nigra of mice. Thus, we established preclinical animal models using reserpine to recapitulate the motor and non-motor symptoms of PD. Chronic reserpine exposure epigenetically elevated the levels of alpha-synuclein expression possible by lowering the DNA methylation status and inducing autophagic impairment in vitro and in vivo.

Abstract P3114: Intermittent Fasting Potentiates Anthracycline Cardiotoxicity

Background: Anthracyclines such as doxorubicin (Dox) cause cardiotoxicity and associated autophagic impairment, though controversy exists increasing autophagy ameliorates or exacerbates Dox cardiotoxicity. Because intermittent fasting (IF), a potent stimulus of autophagy, is presently under clinical investigation in multiple human clinical trials of cancer patients receiving chemotherapy, we tested whether IF would ameliorate or exacerbate Dox cardiotoxicity in multiple murine models. Methods: Chow-fed C57BL/6 mice (n=150) were randomized to ad libitum feeding ( adlib ) or IF (every other day) and treated with vehicle or Dox (5 mg/kg IP x 4 doses). Alternatively, to stimulate autophagy and lysosomal biogenesis transcriptionally, we employed adeno-associated virus 9 (AAV9)-driven overexpression of transcription factor EB (TFEB) vs null, followed by vehicle or Dox (n=40). We further analyzed TFEB nuclear content in myocardial tissue from patients with anthracycline cardiomyopathy vs non-failing donors and patients with other non-ischemic cardiomyopathies (n=17). Results: In Dox-treated mice (but not in vehicle controls), IF significantly reduced survival (18% absolute reduction or AR) ( figure A ), left ventricular ejection fraction (LVEF) (8% AR) ( figure B ) and heart weight index (12% relative reduction) ( figure C ) compared to adlib group. Mechanistically, in Dox-treated mice, IF significantly decreased phosphorylation of mammalian target of rapamycin, increased active, nuclear TFEB content, and increased levels of the atrophy factor muscle RING-finger protein-1. In myocardial tissue from patients with anthracycline cardiomyopathy, we observed a 3.5-fold increase (p&lt;0.01) in nuclear TFEB content compared to both tissues from donors without heart failure and tissues from patients with other non-ischemic cardiomyopathies. Overexpression of TFEB exacerbated Dox-induced cardiotoxicity, including significant decreases in LVEF, left ventricular mass index, and survival. Conclusions: IF exacerbates Dox-related mortality and cardiomyopathy, likely by stimulating the autophagy-lysosomal transcriptional regulator TFEB. Our data raise concerns about the safety of IF and augmentation of TFEB activity in the context of anthracycline chemotherapy.

Integrated regulation of stress responses, autophagy and survival by altered intracellular iron stores

Iron is a mineral essential for blood production and a variety of critical cellular functions. Altered iron metabolism has been increasingly observed in many diseases and disorders, but a comprehensive and mechanistic understanding of the cellular impact of impaired iron metabolism is still lacking. We examined the effects of iron overload or iron deficiency on cellular stress responses and autophagy which collectively regulate cell homeostasis and survival. Acute iron loading led to increased mitochondrial ROS (mtROS) production and damage, lipid peroxidation, impaired autophagic flux, and ferroptosis. Iron-induced mtROS overproduction is the mechanism of increased lipid peroxidation, impaired autophagy, and the induction of ferroptosis. Iron excess-induced ferroptosis was cell-type dependent and regulated by activating transcription factor 4 (ATF4). Upregulation of ATF4 mitigated iron-induced autophagic dysfunction and ferroptosis, whereas silencing of ATF4 expression impaired autophagy and resulted in increased mtROS production and ferroptosis. Employing autophagy-deficient hepatocytes and different autophagy inhibitors, we further showed that autophagic impairment sensitized cells to iron-induced ferroptosis. In contrast, iron deficiency activated the endoplasmic reticulum (ER) stress response, decreased autophagy, and induced apoptosis. Decreased autophagy associated with iron deficiency was due to ER stress, as reduction of ER stress by 4-phenylbutyric acid (4-PBA) improved autophagic flux. The mechanism of decreased autophagy in iron deficiency is a disruption in lysosomal biogenesis due to impaired posttranslational maturation of lysosomal membrane proteins. In conclusion, iron excess and iron deficiency cause different forms of cell stress and death in part through the common mechanism of impaired autophagic function.

α-synuclein suppresses microglial autophagy and promotes neurodegeneration in a mouse model of Parkinson's disease.

The cell‐to‐cell transfer of α‐synuclein (α‐Syn) greatly contributes to Parkinson's disease (PD) pathogenesis and underlies the spread of α‐Syn pathology. During this process, extracellular α‐Syn can activate microglia and neuroinflammation, which plays an important role in PD. However, the effect of extracellular α‐Syn on microglia autophagy is poorly understood. In the present study, we reported that extracellular α‐Syn inhibited the autophagy initiation, as indicated by LC3‐II reduction and p62 protein elevation in BV2 and cultured primary microglia. The in vitro findings were verified in microglia‐enriched population isolated from α‐Syn‐overexpressing mice induced by adeno‐associated virus (AAV2/9)‐encoded wildtype human α‐Syn injection into the substantia nigra (SN). Mechanistically, α‐Syn led to microglial autophagic impairment through activating toll‐like receptor 4 (Tlr4) and its downstream p38 and Akt‐mTOR signaling because Tlr4 knockout and inhibition of p38, Akt as well as mTOR prevented α‐Syn‐induced autophagy inhibition. Moreover, inhibition of Akt reversed the mTOR activation but failed to affect p38 phosphorylation triggered by α‐Syn. Functionally, the in vivo evidence showed that lysozyme 2 Cre (Lyz2 cre)‐mediated depletion of autophagy‐related gene 5 (Atg5) in microglia aggravated the neuroinflammation and dopaminergic neuron losses in the SN and exacerbated the locomotor deficit in α‐Syn‐overexpressing mice. Taken together, the results suggest that extracellular α‐Syn, via Tlr4‐dependent p38 and Akt‐mTOR signaling cascades, disrupts microglial autophagy activity which synergistically contributes to neuroinflammation and PD development.

Abstract 11407: E-Cigarettes Induce Dysregulation of Autophagy Leading to Endothelial Dysfunction in an Ipsc Model of Pulmonary Arterial Hypertension

Introduction: Given the increasing popularity of electronic-cigarettes (e-cigs), it is imperative to evaluate the potential health risks of e-cigs especially in users with pre-existing health concerns. Although we have recently demonstrated that acute exposure to e-cig exacerbates endothelial dysfunction, a prominent feature of pulmonary arterial hypertension (PAH), it is currently unknown whether the use of e-cig represents an even higher risk factor in PAH patients. The aim of the present study is to assess if differential susceptibility exists between healthy and PAH patients following e-cig exposure and mechanisms contributing to it. Methods and Results: Using patient-specific induced pluripotent stem cells (iPSCs) derived from both PAH patients and healthy controls, we showed that PAH iPSC-derived endothelial cells (iPSC-ECs) exhibited increased susceptibility against e-cig exposure compared to healthy iPSC-ECs leading to significantly decreased cell viability, increased reactive oxygen species generation and apoptosis, and reduced tube formation and migration. Transcriptomic analyses revealed that differential expression of AKT3 may be responsible for triggering enhanced autophagic flux impairment in PAH iPSC-ECs compared to healthy iPSC-ECs which underlies increased susceptibility upon e-cig exposure. Consistent with sequencing data, western blot confirmed that AKT3 level was decreased in PAH iPSC-ECs following e-cig exposure and AKT3 proteasomal degradation was mediated by K48-linked ubiquitination. Moreover, knockdown of AKT3 in healthy iPSC-ECs significantly induced autophagic flux impairment and increased endothelial dysfunction and these vascular toxicities were further increased when exposed to e-cig, thus mimicking the PAH cell phenotype after e-cig exposure. Finally, rapamycin treatment reversed e-cig-induced decreased AKT3 level, endothelial dysfunction, and autophagy impairment in PAH iPSC-ECs. Conclusions: Taken together, these data suggest a potential link between autophagy and Akt3-mediated increased susceptibility to e-cig in PAH.

Lipopolysaccharide exacerbates chronic restraint stress-induced neurobehavioral deficits: Mechanisms by redox imbalance, ASK1-related apoptosis, autophagic dysregulation

Major depressive disorder (MDD) is the foremost leading psychiatric illness prevailing around the globe. It usually exists along with anxiety and other clinical conditions (cardiovascular, cancer, neurodegenerative diseases, and infectious diseases). Chronic restraint stress (RS) and LPS-induce neurobehavioral alterations in rodent models however their interaction studies in association with the pathogenesis of MDD are still unclear. Therefore, the current study was aimed to investigate the LPS influence on chronic RS mediated redox imbalance, apoptosis, and autophagic dysregulation in the hippocampus (HIP) and frontal cortex (FC) of mice brain. Male Balb/c mice were exposed to 28 days consecutive stress (6h/day) with a single-dose LPS challenge (0.83 mg/kg, i.p.) on the last day (Day 28). In addition, we also carried out separate study to understand physiological relevance, where we used the DSS (dextran sulfate sodium), a water soluble polysaccharide (negatively charged) and studied its influence on RS induced neurobehavioral and certain neurochemical anomalies. The obtained results in RS and RS + LPS animal groups showed significant immune dysfunction, depleted monoamines, lowered ATP & NAD level, elevated serum CORT level, serum and brain tissues IL-1β/TNF-α/IL-6, SOD activity but reduced CAT activity. Furthermore, the redox perturbation was found where significantly upregulated P-NFκB p65, Keap-1, Prx-SO3 and downregulated Nrf2, Srx1, Prx2 protein expression was seen in RS + LPS mice. The apoptosis signaling (P-ASK1, P-p38 MAPK, P-SAPK/JNK, cleaved PARP, cleaved Caspase-3, Cyto-C), autophagic impairment (p62, LC3II/I) were noticed in HIP and FC of RS and RS + LPS grouped animals. Our new findings provide a complex interplay of chemical (LPS) and physical (RS) stressors where both single dose LPS challenge and 3% DSS in drinking water (for 7 days) exaggerated chronic RS-induced inflammation, lowered redox status, increased apoptosis and dysregulated autophagy leading drastic neurobehavioral alterations in the mice.

Inhibition of mitochondrial pyruvate carrier 1 by lapatinib ditosylate mitigates Alzheimer's-like disease in D-galactose/ovariectomized rats

Mitochondrial, autophagic impairment, excitotoxicity, and also neuroinflammation are implicated in Alzheimer's disease (AD) pathophysiology. We postulated that inhibiting the mitochondrial pyruvate carrier-1 (MPC-1), which inhibits the activation of the mammalian target of rapamycin (mTOR), may ameliorate the neurodegeneration of hippocampal neurons in the rat AD model. To assess this, we used lapatinib ditosylate (LAP), an anti-cancer drug that inhibits MPC-1 through suppression of estrogen-related receptor-alpha (ERR-α), in D-galactose/ovariectomized rats. AD characteristics were developed in ovariectomized (OVX) rats following an 8-week injection of D-galactose (D-gal) (150 mg/kg, i.p.). The human epidermal growth factor receptor-2 (HER-2) inhibitor, LAP (100 mg/kg, p.o.) was daily administered for 3 weeks. LAP protected against D-gal/OVX-induced changes in cortical and hippocampal neurons along with improvement in learning and memory, as affirmed using Morris water maze (MWM) and novel object recognition (NOR) tests. Furthermore, LAP suppressed the hippocampal expression of Aβ1-42, p-tau, HER-2, p-mTOR, GluR-II, TNF-α, P38-MAPK, NOX-1, ERR-α, and MPC-1. Also, LAP treatment leads to activation of the pro-survival PI3K/Akt pathway. As an epilogue, targeting MPC-1 in the D-gal-induced AD in OVX rats resulted in the enhancement of autophagy, and suppression of neuroinflammation and excitotoxicity. Our work proves that alterations in metabolic signaling as a result of inhibiting MPC-1 were anti-inflammatory and neuroprotective in the AD model, revealing that HER-2, MPC-1, and ERR-α may be promising therapeutic targets for AD.

Metformin-Enhanced Cardiac AMP-Activated Protein Kinase/Atrogin-1 Pathways Inhibit Charged Multivesicular Body Protein 2B Accumulation in Ischemia-Reperfusion Injury.

Background: Cardiac autophagic flux is impaired during myocardial ischemia/reperfusion (MI/R). Impaired autophagic flux may exacerbate MI/R injury. Charged multivesicular body protein 2B (CHMP2B) is a subunit of the endosomal sorting complex required for transport (ESCRT-III) complex that is required for autophagy. However, the reverse role of CHMP2B accumulation in autophagy and MI/R injury has not been established. The objective of this article is to elucidate the roles of AMP-activated protein kinase (AMPK)/atrogin-1 pathways in inhibiting CHMP2B accumulation in ischemia–reperfusion injury.Methods: Male C57BL/6 mice (3–4 months) and H9c2 cardiomyocytes were used to evaluate MI/R and hypoxia/reoxygenation (H/R) injury in vivo and in vitro, respectively. MI/R was built by a left lateral thoracotomy and occluded the left anterior descending artery. H9c2 cells were firstly treated in 95% N2 and 5% CO2 for 15 h and reoxygenation for 1 h. Metformin (100 mg/kg/d) and CHMP2B (Ad-CHMP2B) transfected adenoviruses were administered to the mice. The H9c2 cells were treated with metformin (2.5 mM), MG-132 (10 μM), bafilomycin A1 (10 nM), and compound C (20 μM).Results: Autophagic flux was found to be inhibited in H/R-treated cardiomyocytes and MI/R mice, with elevated cardiac CHMP2B accumulation. Upregulated CHMP2B levels in the in vivo and in vitro experiments were shown to inhibit autophagic flux leading to the deterioration of H/R-cardiomyocytes and MI/R injury. This finding implies that CHMP2B accumulation increases the risk of myocardial ischemia. Metformin suppressed CHMP2B accumulation and ameliorated H/R-induced autophagic dysfunction by activating AMPK. Activated AMPK upregulated the messenger RNA expression and protein levels of atrogin-1, a muscle-specific ubiquitin ligase, in the myocardium. Atrogin-1 significantly enhanced the interaction between atrogin-1 and CHMP2B, therefore, promoting CHMP2B degradation in the MI/R myocardium. Finally, this study revealed that metformin-inhibited CHMP2B accumulation induced autophagic impairment and ischemic susceptibility in vivo through the AMPK-regulated CHMP2B degradation by atrogin-1.Conclusion: Impaired CHMP2B clearance in vitro and in vivo inhibits autophagic flux and weakens the myocardial ischemic tolerance. Metformin treatment degrades CHMP2B through the AMPK-atrogin-1-dependent pathway to maintain the homeostasis of autophagic flux. This is a novel mechanism that enriches the understanding of cardioprotection.

Berberine Protects Against NLRP3 Inflammasome via Ameliorating Autophagic Impairment in MPTP-Induced Parkinson's Disease Model.

The NLR family pyrin domain containing 3 (NLRP3) inflammasome was reported to be regulated by autophagy and activated during inflammatory procession of Parkinson’s disease (PD). Berberine (BBR) is well-studied to play an important role in promoting anti-inflammatory response to mediate the autophagy activity. However, the effect of Berberine on NLRP3 inflammasome in PD and its potential mechanisms remain unclear. Hence, in this study, we investigated the effects of BBR on 1-Methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP)-induced PD mice, by evaluating their behavioral changes, dopaminergic (DA) neurons loss, neuroinflammation, NLRP3 inflammasome and autophagic activity. BBR was also applied in BV2 cells treated with 1-methyl-4-pehnyl-pyridine (MPP+). The autophagy inhibitor 3-Methyladenine (3-MA) was administrated to block autophagy activity both in vivo and in vitro. In our in vivo studies, compared to MPTP group, mice in MPTP + BBR group showed significant amelioration of behavioral disorders, mitigation of neurotoxicity and NLRP3-associated neuroinflammation, enhancement of the autophagic process in substantia nigra (SN). In vitro, compared to MPP+ group, BBR significantly decreased the level of NLRP3 inflammasome including the expressions of NLRP3, PYD and CARD domain containing (PYCARD), cleaved caspase 1 (CASP1), and mature interleukin 1 beta (IL1B), via enhancing autophagic activity. Furthermore, BBR treatment increased the formation of autophagosomes in MPP+-treated BV2 cells. Taken together, our data indicated that BBR prevents NLRP3 inflammasome activation and restores autophagic activity to protect DA neurons against degeneration in vivo and in vitro, suggesting that BBR may be a potential therapeutic to treat PD.