Impaired Autophagy Research Articles

Tannic Acid Mitigates Rotenone-Induced Dopaminergic Neurodegeneration by Inhibiting Inflammation, Oxidative Stress, Apoptosis, and Glutamate Toxicity in Rats.

Parkinson's disease (PD), a movement disorder, is a neurodegenerative disease characterized by the degeneration of dopaminergic neurons in the substantia nigra pars compacta (SNpc) region of the brain. The etiopathogenesis of PD involves increased oxidative stress, augmented inflammation, impaired autophagy, accumulation of α-synuclein, and α-Glutamate neurotoxicity. The treatment of PD is limited and there is a lack of agents to prevent the disease/delay its progression and inhibit the onset of pathogenic events. Many agents of natural and synthetic origin have been investigated employing experimental models of PD, mimicking human PD. In the present study, we assessed the effect of tannic acid (TA) in a rodent model of PD induced by rotenone (ROT), a pesticide and an environmental toxin of natural origin reported to cause PD in agricultural workers and farmers. Rotenone (2.5 mg/kg/day, i.p.) was administered for 28 days, and TA (50 mg/kg, orally) was administered 30 min before ROT injections. The study results showed an increase in oxidative stress, as evidenced by the depletion of endogenous antioxidants and enhanced formation of lipid peroxidation products, along with the onset of inflammation following a rise in inflammatory mediators and proinflammatory cytokines. ROT injections have also augmented apoptosis, impaired autophagy, promoted synaptic loss, and perturbed α-Glutamate hyperpolarization in rats. ROT injections also induced the loss of dopaminergic neurons subsequent to the activation of microglia and astrocytes. However, TA treatment was observed to reduce lipid peroxidation, prevent loss of endogenous antioxidants, and inhibit the release and synthesis of proinflammatory cytokines, in addition to the favorable modulation of apoptosis and autophagic pathways. Treatment with TA also attenuated the activation of microglia and astrocytes along with preservation of dopaminergic neurons following reduced loss of dopaminergic neurodegeneration and inhibition of synaptic loss and α-Glutamate cytotoxicity. The effects of TA in ROT-induced PD were attributed to the antioxidant, anti-inflammatory, antiapoptotic, and neurogenesis properties. Based on the present study findings, it can be concluded that TA may be a promising novel therapeutic candidate for pharmaceutical as well as nutraceutical development owing to its neuroprotective properties in PD. Further regulatory toxicology and translational studies are suggested for future clinical usage in PD.

Rev1 deficiency induces a metabolic shift in MEFs that can be manipulated by the NAD+ precursor nicotinamide riboside

Replication stress, caused by Rev1 deficiency, is associated with mitochondrial dysfunction, and metabolic stress. However, the overall metabolic alterations and possible interventions to rescue the deficits due to Rev1 loss remain unclear. Here, we report that loss of Rev1 leads to intense changes in metabolites and that this can be manipulated by NAD + supplementation. Autophagy decreases in Rev1−/− mouse embryonic fibroblasts (MEFs) and can be restored by supplementing the NAD+ precursor nicotinamide riboside (NR). The abnormal mitochondrial morphology in Rev1−/− MEFs can be partially reversed by NR supplementation, which also protects the mitochondrial cristae from rotenone-induced degeneration. In nematodes rev-1 deficiency causes sensitivity to oxidative stress but this cannot be rescued by NR supplementation. In conclusion, Rev1 deficiency leads to metabolic dysregulation of especially lipid and nucleotide metabolism, impaired autophagy, and mitochondrial anomalies, and all of these phenotypes can be improved by NR replenishment in MEFs.

Corynoxine B targets at HMGB1/2 to enhance autophagy for α-synuclein clearance in fly and rodent models of Parkinson's disease.

Parkinson's disease (PD) is the most common neurodegenerative movement disease. It is featured by abnormal alpha-synuclein (α-syn) aggregation in dopaminergic neurons in the substantia nigra. Macroautophagy (autophagy) is an evolutionarily conserved cellular process for degradation of cellular contents, including protein aggregates, to maintain cellular homeostasis. Corynoxine B (Cory B), a natural alkaloid isolated from Uncaria rhynchophylla (Miq.) Jacks., has been reported to promote the clearance of α-syn in cell models by inducing autophagy. However, the molecular mechanism by which Cory B induces autophagy is not known, and the α-syn-lowering activity of Cory B has not been verified in animal models. Here, we report that Cory B enhanced the activity of Beclin 1/VPS34 complex and increased autophagy by promoting the interaction between Beclin 1 and HMGB1/2. Depletion of HMGB1/2 impaired Cory B-induced autophagy. We showed for the first time that, similar to HMGB1, HMGB2 is also required for autophagy and depletion of HMGB2 decreased autophagy levels and phosphatidylinositol 3-kinase III activity both under basal and stimulated conditions. By applying cellular thermal shift assay, surface plasmon resonance, and molecular docking, we confirmed that Cory B directly binds to HMGB1/2 near the C106 site. Furthermore, invivo studies with a wild-type α-syn transgenic drosophila model of PD and an A53T α-syn transgenic mouse model of PD, Cory B enhanced autophagy, promoted α-syn clearance and improved behavioral abnormalities. Taken together, the results of this study reveal that Cory B enhances phosphatidylinositol 3-kinase III activity/autophagy by binding to HMGB1/2 and that this enhancement is neuroprotective against PD.

Stevioside protects primary articular chondrocytes against IL-1β-induced inflammation and catabolism by targeting integrin.

Osteoarthritis (OA) is a common, progressive, and chronic disorder of the joints that is characterized by the inflammation and degradation of articular cartilage and is known to significantly impair quality of daily life. Stevioside (SVS) is a natural diterpenoid glycoside that has anti-inflammatory benefits. Hence, in the current research, it was hypothesized that SVS might exert anti-inflammatory effects on articular chondrocytes and alleviate cartilage degradation in mice with OA. The expression of inflammatory cytokines, like inducible nitric oxide synthase (iNOS), NOD-, LRR- and pyrin domain-containing protein 3 (NLRP3), and cyclooxygenase-2 (COX-2) in chondrocytes after interleukin-1β (IL-1β) exposure, was inhibited by the pretreatment of SVS. As well, SVS inhibited the reduction of collagen II and sry-box transcription factor 9 (SOX9) in chondrocytes stimulated by IL-1β and suppressed the expression of MMP3 and MMP13. Further, after treatment with SVS, cell cytometry, autophagy flux, and related protein expression showed diminished cell apoptosis and reduced autophagy impairment. Moreover, SVS blocked the activation of phosphoinositide-3-kinase/Akt/nuclear factor-kappa beta (PI3K/Akt/NF-κB) and mitogen-activated protein kinase (MAPK) signaling pathways stimulated by IL-1β. This resulted in decreased cellular inflammation. In vivo experiments with intra-articular injections of SVS in mice with the DMM mouse model demonstrated a decrease in cartilage degradation and an improvement in subchondral bone remodeling. After the integrin αVβ3-related knockdown using siRNA, a reversed effect was observed on the anti-inflammatory, anabolic promoting, catabolic blocking, and NF-κB and MAPK signaling pathway inhibition of SVS on chondrocytes treated with IL-1β. The above findings highlighted that SVS blocked IL-1β, triggered an inflammatory response in mice chondrocytes, and prevented cartilage degradation in vivo through integrin αVβ3. This suggested that SVS might serve as a novel therapeutic option for OA.

Oleic acid improves hepatic lipotoxicity injury by alleviating autophagy dysfunction

Lipotoxicity caused by excess free fatty acids, particularly saturated fatty acids (SFAs) such as palmitic acid (PA), is one of the most important pathogenesis of nonalcoholic fatty liver disease (NAFLD). However, unsaturated fatty acids (UFAs), such as oleic acid (OA), are nontoxic and can combat SFA-induced toxicity through alleviation of cell apoptosis, endoplasmic reticulum stress (ER stress) and lipids metabolism disorder. However, whether OA is able to regulate autophagy is largely unknown. So, this study aims to investigate the mechanism underlying OA mediated modulation of autophagy in hepatocytes and mice with NAFLD. In vitro, human hepatoma cell line HepG2 cells, human normal liver cells L-02 and mouse normal liver cells AML12 were treated with palmitic acid (PA)/tunicamycin (TM) or/and OA for 48 h. In vivo, C57/BL6 mice were fed with high fat diet (HFD) to induce NAFLD. And the HFD was partial replaced by olive oil to observe the protective effects of olive oil. We demonstrated that PA/TM impaired cell viability and induced cellular apoptosis in HepG2 cells and L-02 cells. Moreover, PA/TM induced autophagy impairment by reducing the nuclear translocation of transcription factor EB (TFEB) and inhibiting the activity of CTSB. However, OA substantially alleviated PA/TM induced cellular apoptosis and autophagy dysfunction in hepatocytes. Additionally, restoring autophagy function is able to reduce ER stress. Similarly, HFD for 20 weeks successfully established NAFLD model in C57/BL6 mice, and significant autophagy impairment were observed in liver tissues. Noteworthily, 30% replacement of HFD with olive oil had profoundly reversed NAFLD. It significantly impoved steatosis, and reduced autophagy dysfunction, ER stress and apoptosis in liver tissue. Conclusively, these data demonstrated that OA is able to effectively impove autophagy dysfunction under the context of both PA and ER stress inducer induced lipotoxicity, and OA mediated regulation of lysosome dysfunction through TFEB plays an important role, suggesting that the regulation of ER stress-autophagy axis is a critical mechanism in OA driven protection in NAFLD.

Uncovering Novel Therapeutic Targets for Parkinson's Disease.

Parkinson's disease (PD) is the second most prevailing progressive disorder leading to neurodegeneration, typically in people above 65 years of age. Motor clinical manifestations of PD appear in a much later stage and include rigidity, tremors, akinesia, and gait dysfunction. There are also nonmotor symptoms like GI and olfactory dysfunction. However, they cannot be considered for diagnosis of the disease, as they are unspecific. PD pathogenesis is mainly characterized by deposits of inclusion bodies on dopaminergic (DA) neurons in substantia nigra pars compacta region (SNpc) of the brain. The major component of these inclusion bodies, are α-synuclein aggregates. α-Synuclein undergoes misfolding and oligomerization to form aggregates and fibrils. These aggregates gradually propagate PD pathology. Other prominent features of this pathological development include mitochondrial dysfunction, neuroinflammation, oxidative stress, and impaired autophagy. These all contribute to neuronal degeneration. Besides this, there are many underlying factors which influence these processes. These factors comprise molecular proteins and signaling cascades. In this review, we have listed out underexplored molecular targets that may aid in development of neoteric and advanced therapeutics.

Enhanced expression of the autophagosomal marker LC3-II in detergent-resistant protein lysates from a CLN3 patient's post-mortem brain

• Neuronal Ceroido Lipofuscinoses (NCL) are inherited, neurodegenerative disorders associated with lysosomal storage.• Impaired autophagy plays a pathogenetic role in several NCL forms, including CLN3 disease, but study on human brains are lacking.• In post-mortem brain samples of a CLN3 patient the LC3-I to LC3-II shift was consistent with activated autophagy. However, the autophagic process seemed to be ineffective due to the presence of lysosomal storage markers.• After fractionation with buffers of increasing detergent-denaturing strength, a peculiar solubility pattern of LC3-II was observed in CLN3 patient's samples, suggesting a different lipid composition of the membranes where LC3-II is stacked.

Senescence-related impairment of autophagy induces toxic intraneuronal amyloid-β accumulation in a mouse model of amyloid pathology

Aging is the main risk factor for Alzheimer’s disease (AD) and other neurodegenerative pathologies, but the molecular and cellular changes underlying pathological aging of the nervous system are poorly understood. AD pathology seems to correlate with the appearance of cells that become senescent due to the progressive accumulation of cellular insults causing DNA damage. Senescence has also been shown to reduce the autophagic flux, a mechanism involved in clearing damaged proteins from the cell, and such impairment has been linked to AD pathogenesis. In this study, we investigated the role of cellular senescence on AD pathology by crossing a mouse model of AD-like amyloid-β (Aβ) pathology (5xFAD) with a mouse model of senescence that is genetically deficient for the RNA component of the telomerase (Terc−/−). We studied changes in amyloid pathology, neurodegeneration, and the autophagy process in brain tissue samples and primary cultures derived from these mice by complementary biochemical and immunostaining approaches. Postmortem human brain samples were also processed to evaluate autophagy defects in AD patients. Our results show that accelerated senescence produces an early accumulation of intraneuronal Aβ in the subiculum and cortical layer V of 5xFAD mice. This correlates with a reduction in amyloid plaques and Aβ levels in connecting brain regions at a later disease stage. Neuronal loss was specifically observed in brain regions presenting intraneuronal Aβ and was linked to telomere attrition. Our results indicate that senescence affects intraneuronal Aβ accumulation by impairing autophagy function and that early autophagy defects can be found in the brains of AD patients. Together, these findings demonstrate the instrumental role of senescence in intraneuronal Aβ accumulation, which represents a key event in AD pathophysiology, and emphasize the correlation between the initial stages of amyloid pathology and defects in the autophagy flux.

Gα13-Mediated Signaling Cascade Is Related to the Tau Pathology Caused by Anesthesia and Surgery in 5XFAD Transgenic Mice.

Our previous studies indicated that anesthesia and surgery could aggravate cognitive impairment of 5XFAD transgenic (Tg) mice, and this aggravation was associated with tau hyperphosphorylation. We previously identified that GNA13 (the gene encoding Gα13) was a hub gene with tau hyperphosphorylation. This study aims to further investigate the mechanism that whether the Gα13-mediated signaling pathway acts as an instigator to regulate cofilin activation and autophagy impairment in this process. 5XFAD Tg mice and their littermate (LM) mice were randomly allocated into four groups: LM Control group, LM Anesthesia/Surgery group, AD Control group, and AD Anesthesia/Surgery group. For mice in the Anesthesia/Surgery groups, abdominal surgery was performed under 1.4% isoflurane anesthesia followed by sustaining anesthetic inhalation for up to 2 h. Compared with the AD Control group, protein levels of Gα13, ROCK2, LPAR5, and p-tau/tau46 ratio were increased, while p-cofilin/cofilin protein expression ratio was decreased in the AD Anesthesia/Surgery group. However, the differences in these protein levels were not significant among LM groups. This study demonstrated that anesthesia and surgery might exacerbate p-tau accumulation in 5XFAD Tg mice but not in LM mice. And this might be closely related to cofilin activation via Gα13-mediated signaling cascade.

"Dirty Dancing" of Calcium and Autophagy in Alzheimer's Disease.

Alzheimer's disease (AD) is the most common cause of dementia. There is a growing body of evidence that dysregulation in neuronal calcium (Ca2+) signaling plays a major role in the initiation of AD pathogenesis. In particular, it is well established that Ryanodine receptor (RyanR) expression levels are increased in AD neurons and Ca2+ release via RyanRs is augmented in AD neurons. Autophagy is important for removing unnecessary or dysfunctional components and long-lived protein aggregates, and autophagy impairment in AD neurons has been extensively reported. In this review we discuss recent results that suggest a causal link between intracellular Ca2+ signaling and lysosomal/autophagic dysregulation. These new results offer novel mechanistic insight into AD pathogenesis and may potentially lead to identification of novel therapeutic targets for treating AD and possibly other neurodegenerative disorders.

Inhibition of NADPH Oxidase (NOX) 2 Mitigates Colitis in Mice with Impaired Macrophage AMPK Function.

Macrophage adenosine monophosphate-activated protein kinase (AMPK) limits the development of experimental colitis. AMPK activation inhibits NADPH oxidase (NOX) 2 expression, reactive oxygen species (ROS) generation, and pro-inflammatory cytokine secretion in macrophages during inflammation, while increased NOX2 expression is reported in experimental models of colitis and inflammatory bowel disease (IBD) patients. Although there are reductions in AMPK activity in IBD, it remains unclear whether targeted inhibition of NOX2 in the presence of defective AMPK can reduce the severity of colitis. Here, we investigate whether the inhibition of NOX2 ameliorates colitis in mice independent of AMPK activation. Our study identified that VAS2870 (a pan-Nox inhibitor) alleviated dextran sodium sulfate (DSS)-induced colitis in macrophage-specific AMPKβ1-deficient (AMPKβ1LysM) mice. Additionally, VAS2870 blocked LPS-induced TLR-4 and NOX2 expression, ROS production, nuclear translocation of NF-κB, and pro-inflammatory cytokine secretion in bone marrow-derived macrophages (BMDMs) from AMPKβ1LysM mice, whereas sodium salicylate (SS; AMPK β1 activator) did not. Both VAS2870 and SS inhibited LPS-induced NOX2 expression, ROS production, and pro-inflammatory cytokine secretions in bone marrow-derived macrophages (BMDMs) from wildtype (AMPKβ1fl/fl) mice but only VAS2870 inhibited these effects of LPSs in AMPKβ1LysM BMDMs. Furthermore, in macrophage cells (RAW 264.7), both SS and VAS2870 inhibited ROS production and the secretion of pro-inflammatory cytokines and reversed the impaired autophagy induced by LPSs. These data suggest that inhibiting NOX2 can reduce inflammation independent of AMPK in colitis.

Inosine attenuates rotenone-induced Parkinson's disease in rats by alleviating the imbalance between autophagy and apoptosis.

Growing evidence points to impaired autophagy as one of the major factors implicated in the pathophysiology of Parkinson's disease (PD). Autophagy is a downstream target of adenosine monophosphate-activated protein kinase (AMPK). Inosine has already demonstrated a neuroprotective effect against neuronal loss in neurodegenerative diseases, mainly due its anti-inflammatory and antioxidant properties. We, herein, aimed at investigating the neuroprotective effects of inosine against rotenone-induced PD in rats and to focus on the activation of AMPK-mediated autophagy. Inosine successfully increased p-AMPK/AMPK ratio in PD rats and improved their motor performance and muscular co-ordination (assessed by rotarod, open field, and grip strength tests, as well as by manual gait analysis). Furthermore, inosine was able to mitigate the rotenone-induced histopathological alterations and to restore the tyrosine hydroxylase immunoreactivity in PD rats' substantia nigra. Inosine-induced AMPK activation resulted in an autophagy enhancement, as demonstrated by the increased striatal Unc-S1-like kinase1 and beclin-1 expression, and also by the increment light chain 3II to light chain 3I ratio, along with the decline in striatal mammalian target of rapamycin and p62 protein expressions. The inosine-induced stimulation of AMPK also attenuated neuronal apoptosis and promoted antioxidant activity. Unsurprisingly, these neuroprotective effects were antagonized by a preadministration of dorsomorphin (an AMPK inhibitor). In conclusion, inosine exerted neuroprotective effects against the rotenone-induced neuronal loss via an AMPK activation and through the restoration of the imbalance between autophagy and apoptosis. These findings support potential application of inosine in PD treatment.

Cardiovascular Protective Effects of NP-6A4, a Drug with the FDA Designation for Pediatric Cardiomyopathy, in Female Rats with Obesity and Pre-Diabetes.

Obese and pre-diabetic women have a higher risk for cardiovascular death than age-matched men with the same symptoms, and there are no effective treatments. We reported that obese and pre-diabetic female Zucker Diabetic Fatty (ZDF-F) rats recapitulate metabolic and cardiac pathology of young obese and pre-diabetic women and exhibit suppression of cardio-reparative AT2R. Here, we investigated whether NP-6A4, a new AT2R agonist with the FDA designation for pediatric cardiomyopathy, mitigate heart disease in ZDF-F rats by restoring AT2R expression. ZDF-F rats on a high-fat diet (to induce hyperglycemia) were treated with saline, NP-6A4 (10 mg/kg/day), or NP-6A4 + PD123319 (AT2R-specific antagonist, 5 mg/kg/day) for 4 weeks (n = 21). Cardiac functions, structure, and signaling were assessed by echocardiography, histology, immunohistochemistry, immunoblotting, and cardiac proteome analysis. NP-6A4 treatment attenuated cardiac dysfunction, microvascular damage (-625%) and cardiomyocyte hypertrophy (-263%), and increased capillary density (200%) and AT2R expression (240%) (p < 0.05). NP-6A4 activated a new 8-protein autophagy network and increased autophagy marker LC3-II but suppressed autophagy receptor p62 and autophagy inhibitor Rubicon. Co-treatment with AT2R antagonist PD123319 suppressed NP-6A4's protective effects, confirming that NP-6A4 acts through AT2R. NP-6A4-AT2R-induced cardioprotection was independent of changes in body weight, hyperglycemia, hyperinsulinemia, or blood pressure. Cardiac autophagy impairment underlies heart disease induced by obesity and pre-diabetes, and there are no drugs to re-activate autophagy. We propose that NP-6A4 can be an effective drug to reactivate cardiac autophagy and treat obesity- and pre-diabetes-induced heart disease, particularly for young and obese women.

Autophagy and mitophagy: physiological implications in kidney inflammation and diseases.

Autophagy is a ubiquitous intracellular cytoprotective quality control program that maintains cellular homeostasis by recycling superfluous cytoplasmic components (lipid droplets, protein, or glycogen aggregates) and invading pathogens. Mitophagy is a selective form of autophagy that by recycling damaged mitochondrial material, which can extracellularly act as damage-associated molecular patterns, prevents their release. Autophagy and mitophagy are indispensable for the maintenance of kidney homeostasis and exert crucial functions during both physiological and disease conditions. Impaired autophagy and mitophagy can negatively impact the pathophysiological state and promote its progression. Autophagy helps in maintaining structural integrity of the kidney. Mitophagy-mediated mitochondrial quality control is explicitly critical for regulating cellular homeostasis in the kidney. Both autophagy and mitophagy attenuate inflammatory responses in the kidney. An accumulating body of evidence highlights that persistent kidney injury-induced oxidative stress can contribute to dysregulated autophagic and mitophagic responses and cell death. Autophagy and mitophagy also communicate with programmed cell death pathways (apoptosis and necroptosis) and play important roles in cell survival by preventing nutrient deprivation and regulating oxidative stress. Autophagy and mitophagy are activated in the kidney after acute injury. However, their aberrant hyperactivation can be deleterious and cause tissue damage. The findings on the functions of autophagy and mitophagy in various models of chronic kidney disease are heterogeneous and cell type- and context-specific dependent. In this review, we discuss the roles of autophagy and mitophagy in the kidney in regulating inflammatory responses and during various pathological manifestations.

Arctigenin improves neuropathy via ameliorating apoptosis and modulating autophagy in streptozotocin-induced diabetic mice.

Oxidative stress mediates the pathophysiology of diabetic neuropathy (DN) with activation of apoptotic pathway and reduction of autophagy. Arctigenin (ARC) is a natural lignan isolated from some plants of the Asteraceae family that shows antioxidant property. The present study aimed to explore the mechanistic neuroprotective effect of ARC on animal model for DN. DN was induced using streptozotocin (STZ) at a dose of 45 mg/kg, i.p, for five consecutive days and ARC was administered orally (25 or 50 mg) for 3 weeks. The mechanical sensitivity and thermal latency were determined using von Frey and hotplate, respectively. Beclin, p62, and LC3 were detected as markers for autophagy by western blot. Levels of reduced glutathione, lipid peroxides, and activities of catalase and superoxide dismutase were detected as readout for oxidative stress. Apoptotic parameters and histopathological changes were revealed in all experimental groups. The present study showed deterioration of the function and structure of neurons as a result of hyperglycemia. Oxidative stress and impaired autophagy were observed in diabetic neurons as well as the activation of apoptotic pathway. ARC improved the behavioral and histopathological changes of diabetic mice. ARC combated oxidative stress through diminishing lipid peroxidation and improving the activity of antioxidant enzymes. This was concomitant by reducing the biomarkers of apoptosis. ARC augmented the expression of Beclin and LC3 while it lessened the expression of p62 indicating the activation of autophagy. These findings suggest that ARC can ameliorate DN by combating apoptosis and oxidative stress and improving autophagy.

Neuroprotection of Andrographolide against Neurotoxin MPP+-Induced Apoptosis in SH-SY5Y Cells via Activating Mitophagy, Autophagy, and Antioxidant Activities.

Parkinson's disease (PD) is associated with dopaminergic neuron loss and alpha-synuclein aggregation caused by ROS overproduction, leading to mitochondrial dysfunction and autophagy impairment. Recently, andrographolide (Andro) has been extensively studied for various pharmacological properties, such as anti-diabetic, anti-cancer, anti-inflammatory, and anti-atherosclerosis. However, its potential neuroprotective effects on neurotoxin MPP+-induced SH-SY5Y cells, a cellular PD model, remain uninvestigated. In this study, we hypothesized that Andro has neuroprotective effects against MPP+-induced apoptosis, which may be mediated through the clearance of dysfunctional mitochondria by mitophagy and ROS by antioxidant activities. Herein, Andro pretreatment could attenuate MPP+-induced neuronal cell death that was reflected by reducing mitochondrial membrane potential (MMP) depolarization, alpha-synuclein, and pro-apoptotic proteins expressions. Concomitantly, Andro attenuated MPP+-induced oxidative stress through mitophagy, as indicated by increasing colocalization of MitoTracker Red with LC3, upregulations of the PINK1-Parkin pathway, and autophagy-related proteins. On the contrary, Andro-activated autophagy was compromised when pretreated with 3-MA. Furthermore, Andro activated the Nrf2/KEAP1 pathway, leading to increasing genes encoding antioxidant enzymes and activities. This study elucidated that Andro exhibited significant neuroprotective effects against MPP+-induced SH-SY5Y cell death in vitro by enhancing mitophagy and clearance of alpha-synuclein through autophagy, as well as increasing antioxidant capacity. Our results provide evidence that Andro could be considered a potential supplement for PD prevention.

Loss of function of FIP200 in human pluripotent stem cell-derived neurons leads to axonal pathology and hyperactivity

FIP200 plays important roles in homeostatic processes such as autophagy and signaling pathways such as focal adhesion kinase (FAK) signaling. Furthermore, genetic studies suggest an association of FIP200 mutations with psychiatric disorders. However, its potential connections to psychiatric disorders and specific roles in human neurons are not clear. We set out to establish a human-specific model to study the functional consequences of neuronal FIP200 deficiency. To this end, we generated two independent sets of isogenic human pluripotent stem cell lines with homozygous FIP200KO alleles, which were then used for the derivation of glutamatergic neurons via forced expression of NGN2. FIP200KO neurons exhibited pathological axonal swellings, showed autophagy deficiency, and subsequently elevated p62 protein levels. Moreover, monitoring the electrophysiological activity of neuronal cultures on multi-electrode arrays revealed that FIP200KO resulted in a hyperactive network. This hyperactivity could be abolished by glutamatergic receptor antagonist CNQX, suggesting a strengthened glutamatergic synaptic activation in FIP200KO neurons. Furthermore, cell surface proteomic analysis revealed metabolic dysregulation and abnormal cell adhesion-related processes in FIP200KO neurons. Interestingly, an ULK1/2-specific autophagy inhibitor could recapitulate axonal swellings and hyperactivity in wild-type neurons, whereas inhibition of FAK signaling was able to normalize the hyperactivity of FIP200KO neurons. These results suggest that impaired autophagy and presumably also disinhibition of FAK can contribute to the hyperactivity of FIP200KO neuronal networks, whereas pathological axonal swellings are primarily due to autophagy deficiency. Taken together, our study reveals the consequences of FIP200 deficiency in induced human glutamatergic neurons, which might, in the end, help to understand cellular pathomechanisms contributing to neuropsychiatric conditions.

Abstract 360: Role Of Mitochondrial Dynamics In Vascular Smooth Muscle Cell Death

Background: Vascular smooth muscle cell (VSMC) death has been linked to the pathogenesis of various vascular diseases. Excessive mitochondrial fission in response to pathological stimuli contributes to cell death, thus, inhibition of mitochondrial fission can be a promising therapeutic target in vascular diseases. Mitochondrial division inhibitor 1 (mdivi-1) inhibits Drp1-dependent mitochondrial fission by attenuation of its GTPase activity. As association between fission inhibition &amp; VSMCs cell death has been implicated in vascular diseases, the underlying mechanism warrants further investigation. We assessed the effects of mdivi-1 (an inhibitor of Drp1-mediated mitochondrial fission) on staurosporine (STS)-induced mitochondrial dysfunction, impaired autophagy, &amp; cell death in aortic VSMC. Methods &amp; Results: The flow cytometry analysis demonstrated reduced cell death in VSMCs-treated with mdivi-1. The mitochondrial fission was evaluated using Mitotracker Red staining. mdivi-1-treated VSMCs showed significantly higher mitochondrial networks, mean branch length, &amp; mitochondrial footprint compared to STS-treated cells indicating reduced mitochondrial fragmentation. Inhibitory effects of mdivi-1 on fission were exerted by reduced Drp1 phosphorylation at 616. Mitochondrial function via evaluation of mitochondrial permeability transition pore opening, mitochondrial membrane potential &amp; bioenergetic profile showed restored mitochondrial function &amp; metabolic potential in the mdivi-1-treated cells compared to the STS group. The mdivi-1-treated VSMCs showed significantly reduced ROS generation indicating suppressed oxidative stress. The improved mitochondrial structural integrity due to inhibition of fission was associated with reduced mitophagy &amp; cell death-related proteins. Conclusion: In response to apoptotic stimuli, excessive Drp1-dependent mitochondrial fission leads to mitochondrial damage, playing a key role in VSMC death. Inhibition of Drp1-dependent mitochondrial fission by mdivi-1 attenuated mitochondrial fission &amp; prevented cell death in VSMCs. Pharmacological inhibition of mitochondrial fission provides a potential therapeutic target in vascular diseases associated with loss of VSMC.

Acute inhibition of autophagy impairs maximal diaphragm force generation in old mice

Transdiaphragmatic pressure (Pdi) measurements are used to determine diaphragm force generation across various motor behaviors. Previous studies in the diaphragm muscle found selective age-related changes at fast-twitch fatigable motor units including increased susceptibility to neuromuscular transmission failure and denervation. Accordingly, age-related changes at diaphragm motor units predominantly impair the ability to accomplish higher force behaviors, and these aging effects are evident prior to the onset of muscle atrophy. Recent findings suggest that autophagy plays a critical role in the development of age-related neuromuscular dysfunction; however, the possible contribution of autophagy impairment to the maintenance of diaphragm force generation in old age is unknown. We hypothesized that inhibition of autophagy (using chloroquine) decreases diaphragm force-generating capacity (Pdi) in mice susceptible to age-related neuromuscular dysfunction. Adult male and female C57BL/6 mice at 16-28 months of age were randomized to receive a single intraperitoneal injection of 50 mg/kg of chloroquine or vehicle (n= 9 per treatment group) four hours before Pdi measurements, with the experimenter blinded. Pdi was measured during eupnea (room air breathing), exposure to hypoxia/hypercapnia (10% O 2 /5% CO 2 ), tracheal occlusion, spontaneous deep breaths (“sighs”) and during maximal activation elicited by bilateral phrenic nerve stimulation. Consistent with our hypothesis, there was an ~25% reduction in maximal Pdi in the chloroquine group (46 ± 12 cm H 2 O) compared to the vehicle control (61 ± 16 cm H 2 O; p &lt; 0.05). There was no effect of chloroquine on Pdi during eupnea, hypoxia/hypercapnia, tracheal occlusion, or sigh behaviors. Acute inhibition of autophagy results in reduced maximal Pdi, consistent with that evident in old mice. Thus, we propose that autophagy impairment may be an important contributor to the reduced diaphragm force generation associated with aging. These findings help us further understand the mechanisms regulating age-related neuromuscular dysfunction and may serve as a foundation to develop novel treatments to mitigate aging effects. Support: NIH R01 AG057052. This is the full abstract presented at the American Physiology Summit 2023 meeting and is only available in HTML format. There are no additional versions or additional content available for this abstract. Physiology was not involved in the peer review process.

Aging Associated Changes in Autophagy Markers in Mouse Lumbar Motor Neurons

Autophagy is a process through which damaged cytoplasmic structures are recycled and degraded within the cell. Aging can affect autophagy regulation in different steps and lead to the accumulation of altered proteins, leading to cell damage and death. Motor neuron (MN) loss is an important contributor to sarcopenia age-dependent motor dysfunction. Understanding the regulatory mechanisms of autophagy can help elucidate the motor dysfunction that occurs with aging and provide a target for developing future therapeutic agents. Previous immunofluorescence imaging studies in phrenic MNs showed an age-dependent increase in key autophagy proteins LC3 and p62, reflecting autophagosome accumulation and autophagy impairment. Accordingly, we hypothesized that aging in lumbar MNs leads to autophagy impairment reflected in regulatory protein expression. The current study assessed autophagy in choline acetyltransferase-positive MNs from spinal levels L1-L6, focused on crural muscles (extensor digitorum longus and soleus), using confocal immunofluorescence imaging. Expression of LC3 and p62 proteins in MNs were determined in male and female C57BL/6 mice at 6-, 18- and 24- months of age. Increased expression of LC3 in lumbar MNs was observed with aging across all three age groups and p62 expression in lumbar MNs was significantly increased by 24- and 18-months compared to 6-months (p&lt;0.001). There was no sex effect observed in LC3, but p62 was significantly higher in lumbar MNs of female mice (p=0.007). Expression of both LC3 and p62 increased diffusely in ventral lumbar gray matter regions with age (p&lt;0.001), an effect that was predominant in male and not female mice. Taken together, the results of this study provide evidence for that age-dependent changes in autophagy markers expression occurs in regions of the spinal cord (motor neurons and ventral horn gray matter). The neuronal mechanisms of aging effects include dysregulation of autophagy. Supported by NIH R01 AG057052. This is the full abstract presented at the American Physiology Summit 2023 meeting and is only available in HTML format. There are no additional versions or additional content available for this abstract. Physiology was not involved in the peer review process.