Regulation of FSP1 myristoylation by NADPH: A novel mechanism for ferroptosis inhibition.

Excitotoxicity is a prevalent pathological event in neurodegenerative diseases. The involvement of ferroptosis in the pathogenesis of excitotoxicity remains elusive. Transcriptome analysis has revealed that cytoplasmic reduced nicotinamide adenine dinucleotide phosphate (NADPH) levels are associated with susceptibility to ferroptosis-inducing compounds. Here we show that exogenous NADPH, besides being reductant, interacts with N-myristoyltransferase 2 (NMT2) and upregulates the N-myristoylated ferroptosis suppressor protein 1 (FSP1). NADPH increases membrane-localized FSP1 and strengthens resistance to ferroptosis. Arg-291 of NMT2 is critical for the NADPH-NMT2-FSP1 axis-mediated suppression of ferroptosis. This study suggests that NMT2 plays a pivotal role by bridging NADPH levels and neuronal susceptibility to ferroptosis. We propose a mechanism by which the NADPH regulates N-myristoylation, which has important implications for ferroptosis and disease treatment.

PHLDA1 contributes to hypoxic ischemic brain injury in neonatal rats via inhibiting FUNDC1-mediated mitophagy.

Hypoxia-ischemia (HI) is one of the main causes of neonatal brain injury. Mitophagy has been implicated in the degradation of damaged mitochondria and cell survival following neonatal brain HI injury. Pleckstrin homology-like domain family A member 1 (PHLDA1) plays vital roles in the progression of various disorders including the regulation of oxidative stress, the immune responses and apoptosis. In the present study we investigated the role of PHLDA1 in HI-induced neuronal injury and further explored the mechanisms underlying PHLDA1-regulated mitophagy in vivo and in vitro. HI model was established in newborn rats by ligation of the left common carotid artery plus exposure to an oxygen-deficient chamber with 8% O2 and 92% N2. In vitro studies were conducted in primary hippocampal neurons subjected to oxygen and glucose deprivation/-reoxygenation (OGD/R). We showed that the expression of PHLDA1 was significantly upregulated in the hippocampus of HI newborn rats and in OGD/R-treated primary neurons. Knockdown of PHLDA1 in neonatal rats via lentiviral vector not only significantly ameliorated HI-induced hippocampal neuronal injury but also markedly improved long-term cognitive function outcomes, whereas overexpression of PHLDA1 in neonatal rats via lentiviral vector aggravated these outcomes. PHLDA1 knockdown in primary neurons significantly reversed the reduction of cell viability and increase in intracellular reactive oxygen species (ROS) levels, and attenuated OGD-induced mitochondrial dysfunction, whereas overexpression of PHLDA1 decreased these parameters. In OGD/R-treated primary hippocampal neurons, we revealed that PHLDA1 knockdown enhanced mitophagy by activating FUNDC1, which was abolished by FUNDC1 knockdown or pretreatment with mitophagy inhibitor Mdivi-1 (25 µM). Notably, pretreatment with Mdivi-1 or the knockdown of FUNDC1 not only increased brain infarct volume, but also abolished the neuroprotective effect of PHLDA1 knockdown in HI newborn rats. Together, these results demonstrate that PHLDA1 contributes to neonatal HI-induced brain injury via inhibition of FUNDC1-mediated neuronal mitophagy.

Nicotinamide riboside attenuates myocardial ischemia-reperfusion injury via regulating SIRT3/SOD2 signaling pathway.

Ischemic heart disease invariably leads to devastating damage to human health. Nicotinamide ribose (NR), as one of the precursors of NAD+ synthesis, has been discovered to exert a protective role in various neurological and cardiovascular disorders. Our findings demonstrated that pretreatment with 200 mg/kg NR for 3 h significantly reduced myocardial infarct area, decreased levels of CK-MB and LDH in serum, and improved cardiac function in the rats during myocardial ischemia-reperfusion (I/R) injury. Meanwhile, 0.5 mM NR also effectively increased the viability and decreased the LDH release of H9c2 cells during OGD/R. We had provided evidence that NR pretreatment could decrease mitochondrial reactive oxygen species (mtROS) production and MDA content, and enhance SOD activity, thereby mitigating mitochondrial damage and inhibiting apoptosis during myocardial I/R injury. Further investigations revealed that NR increased NAD+ content and upregulated SIRT3 protein expression in myocardium. Through using of SIRT3 small interfering RNA and the SIRT3 deacetylase activity inhibitor 3-TYP, we had confirmed that the cardioprotective effect of NR on cardiomyocytes was largely dependent on the inhibition of mitochondrial oxidative stress via SIRT3-SOD2 axis. Overall, our study suggested that exogenous supplementation with NR mitigated mitochondrial damage and inhibited apoptosis during myocardial I/R injury by reducing mitochondrial oxidative stress via SIRT3-SOD2-mtROS pathway.

TIGAR exacerbates obesity by triggering LRRK2-mediated defects in macroautophagy and chaperone-mediated autophagy in adipocytes.

Obesity is one of the most common metabolic diseases around the world, which is distinguished by the abnormal buildup of triglycerides within adipose cells. Recent research has revealed that autophagy regulates lipid mobilization to maintain energy balance. TIGAR (Trp53 induced glycolysis regulatory phosphatase) has been identified as a glycolysis inhibitor, whether it plays a role in the metabolism of lipids is unknown. Here, we found that TIGAR transgenic (TIGAR+/+) mice exhibited increased fat mass and trended to obesity phenotype. Non-target metabolomics showed that TIGAR caused the dysregulation of the metabolism profile. The quantitative transcriptome sequencing identified an increased levels of LRRK2 and RAB7B in the adipose tissue of TIGAR+/+ mice. It was confirmed in vitro that TIGAR overexpression increased the levels of LRRK2 by inhibiting polyubiquitination degradation, thereby suppressing macroautophagy and chaperone-mediated autophagy (CMA) while increasing lipid accumulation which were reversed by the LRRK2 inhibitor DNL201. Furthermore, TIGAR drove LRRK2 to interact with RAB7B for suppressing lysosomal degradation of lipid droplets, while the increased lipid droplets in adipocytes were blocked by the RAB7B inhibitor ML282. Additionally, fat-specific TIGAR knockdown of TIGAR+/+ mice alleviated the symptoms of obesity, and adipose tissues-targeting superiority DNL201 nano-emulsion counteracted the obesity phenotype in TIGAR+/+ mice. In summary, the current results indicated that TIGAR performed a vital function in the lipid metabolism through LRRK2-mediated negative regulation of macroautophagy and CMA in adipocyte. The findings suggest that TIGAR has the potential to serve as a viable therapeutic target for treating obesity and its associated metabolic dysfunction.

The Multiple Roles of Autophagy in Neural Function and Diseases.

Autophagy involves the sequestration and delivery of cytoplasmic materials to lysosomes, where proteins, lipids, and organelles are degraded and recycled. According to the way the cytoplasmic components are engulfed, autophagy can be divided into macroautophagy, microautophagy, and chaperone-mediated autophagy. Recently, many studies have found that autophagy plays an important role in neurological diseases, including Alzheimer's disease, Parkinson's disease, Huntington's disease, neuronal excitotoxicity, and cerebral ischemia. Autophagy maintains cell homeostasis in the nervous system via degradation of misfolded proteins, elimination of damaged organelles, and regulation of apoptosis and inflammation. AMPK-mTOR, Beclin 1, TP53, endoplasmic reticulum stress, and other signal pathways are involved in the regulation of autophagy and can be used as potential therapeutic targets for neurological diseases. Here, we discuss the role, functions, and signal pathways of autophagy in neurological diseases, which will shed light on the pathogenic mechanisms of neurological diseases and suggest novel targets for therapies.

Exogenous NADPH exerts a positive inotropic effect and enhances energy metabolism via SIRT3 in pathological cardiac hypertrophy and heart failure.

BACKGROUND: Therapies are urgently required to ameliorate pathological cardiac hypertrophy and enhance cardiac function in heart failure. Our preliminary experiments have demonstrated that exogenous NADPH exhibits a positive inotropic effect on isolated heart. This study aims to investigate the positive inotropic effects of NADPH in pathological cardiac hypertrophy and heart failure, as well as the underlying mechanisms involved. METHODS: Endogenous plasma NADPH contents were determined in patients with chronic heart failure and control adults. The positive inotropic effects of NADPH were investigated in isolated toad heart or rat heart. The effects of NADPH were investigated in isoproterenol (ISO)-induced cardiac hypertrophy or transverse aortic constriction (TAC)-induced heart failure. The underlying mechanisms of NADPH were studied using SIRT3 knockout mice, echocardiography, Western blotting, transmission electron microscopy, and immunoprecipitation. FINDINGS: The endogenous NADPH content in the blood of patients and animals with pathological cardiac hypertrophy or heart failure was significantly reduced compared with age-sex matched control subjects. Exogenous NADPH showed positive inotropic effects on the isolated normal and failing hearts, while antagonism of ATP receptor partially abolished the positive inotropic effect of NADPH. Exogenous NADPH administration significantly reduced heart weight indices, and improved cardiac function in the mice with pathological cardiac hypertrophy or heart failure. NADPH increased SIRT3 expression and activity, deacetylated target proteins, improved mitochondrial function and facilitated ATP production in the hypertrophic myocardium. Importantly, inhibition of SIRT3 abolished the positive inotropic effect of NADPH, and the anti-heart failure effect of NADPH was significantly reduced in the SIRT3 Knockout mice. INTERPRETATION: Exogenous NADPH shows positive inotropic effect and improves energy metabolism via SIRT3 in pathological cardiac hypertrophy and heart failure. NADPH thus may be one of the potential candidates for the treatment of pathological cardiac hypertrophy or heart failure. FUNDING: This work was supported by grants from the National Natural Science Foundation of China (No. 81973315, 82173811, 81730092), Natural Science Foundation of Jiangsu Higher Education (20KJA310008), Jiangsu Key Laboratory of Neuropsychiatric Diseases (BM2013003) and the Priority Academic Program Development of the Jiangsu Higher Education Institutes (PAPD).

Cucurbitacin B Exerts Significant Antidepressant-Like Effects in a Chronic Unpredictable Mild Stress Model of Depression: Involvement of the Hippocampal BDNF-TrkB System.

BACKGROUND: Although depression has been a serious neuropsychiatric disorder worldwide, current antidepressants used in clinical practice have various weaknesses, including delayed onset and low rates of efficacy. Recently, the development of new antidepressants from natural herbal medicine has become one of the important research hotspots. Cucurbitacin B is a natural compound widely distributed in the Cucurbitaceae and Cruciferae families and has many pharmacological activities. The present study aimed to investigate whether cucurbitacin B possess antidepressant-like effects in mice. METHODS: The antidepressant-like effects of cucurbitacin B on mice behaviors were explored using the forced swim test, tail suspension test, open field test, sucrose preference test, and a chronic unpredictable mild stress model of depression together. Then, western blotting and immunofluorescence were used to examine the effects of cucurbitacin B on the brain-derived neurotrophic factor (BDNF)-tyrosine kinase B (TrkB) signaling cascade and neurogenesis in the hippocampus of mice. Furthermore, BDNF-short hairpin RNA, K252a, and p-chlorophenylalanine methyl ester were adopted together to determine the antidepressant mechanism of cucurbitacin B. RESULTS: It was found that administration of cucurbitacin B indeed produced notable antidepressant-like effects in mice, which were accompanied with significant promotion in both the hippocampal BDNF-TrkB pathway and neurogenesis. The antidepressant mechanism of cucurbitacin B involves the hippocampal BDNF-TrkB system but not the serotonin system. CONCLUSIONS: Cucurbitacin B has the potential to be a novel antidepressant candidate.

Alleviation of ischemic brain injury by exercise preconditioning is associated with modulation of autophagy and mitochondrial dynamics in cerebral cortex of female aged mice.

Evidence from clinical studies and preclinical studies supports that exercise preconditioning can not only reduce the risk of stroke but also improve brain tissue and functional outcome after stroke. It has been demonstrated that autophagy and mitochondrial dynamics are involved in ischemic stroke. However, it is still unclear whether exercise preconditioning-induced neuroprotection against stroke is associated with modulation of autophagy and mitochondrial dynamics. Although age and sex interactively affect ischemic stroke risk, incidence, and outcome, studies based on young male animals are most often used to explore the role of exercise preconditioning in the prevention of ischemic stroke. In the current study, we examined whether exercise preconditioning could modulate autophagy and mitochondrial dynamics in a brain ischemia and reperfusion (I/R) model of female aged mice. The results showed that exercise preconditioning reduced infarct volume and improved neurological deficits. Additionally, increased levels of autophagy-related proteins LC3-II/LC3-I, LC3-II, p62, Atg7, and mitophagy-related proteins Bnip3L and Parkin, as well as increased levels of mitochondrial fusion modulator Mfn2 and mitochondrial fission modulator Drp1 in the ischemic cortex of female aged mice at 12 h after I/R were present. Our results could contribute to a better understanding of exercise preconditioning-induced neuroprotection against ischemic stroke for the elderly.

Neuroprotection of NAD+ and NBP against ischemia/reperfusion brain injury is associated with restoration of sirtuin-regulated metabolic homeostasis.

Background: Ischemic stroke seriously threatens human health because of high rates of morbidity, mortality and disability. This study compared the effects of nicotinamide adenine dinucleotide (NAD+) and butylphthalide (NBP) on in vitro and in vivo ischemic stroke models. Methods: Transient middle cerebral artery occlusion/reperfusion (t-MCAO/R) model was established in mice, and the cultured primary cortical neurons were subjected to oxygen-glucose deprivation/reoxygenation (OGD/R). Cerebral infarct volume, neurobehavioral indices, antioxidant activity, ATP level and lactic acid content were determined. The neuroprotective effects of NAD+ or NBP were compared using sirtuin inhibitor niacinamide (NAM). Results: Intraperitoneal injection of NBP within 4 h or intravenous injection of NAD+ within 1 h after t-MCAO/R significantly reduced the volume of infarcts, cerebral edema, and neurological deficits. Administration of NAD+ and NBP immediately after t-MCAO/R in mice showed similar neuroprotection against acute and long-term ischemic injury. Both NAD+ and NBP significantly inhibited the accumulation of MDA and H2O2 and reduced oxidative stress. NAD+ was superior to NBP in inhibiting lipid oxidation and DNA damage. Furthermore, although both NAD+ and NBP improved the morphology of mitochondrial damage induced by ischemia/reperfusion, NAD+ more effectively reversed the decrease of ATP and increase of lactic acid after ischemia/reperfusion compared with NBP. NAD+ but not NBP treatment significantly upregulated SIRT3 in the brain, but the sirtuin inhibitor NAM could abolish the protective effect of NAD+ and NBP by inhibiting SIRT1 or SIRT3. Conclusions: These results confirmed the protective effects of NAD+ and NBP on cerebral ischemic injury. NBP and NAD+ showed similar antioxidant effects, while NAD+ had better ability in restoring energy metabolism, possibly through upregulating the activity of SIRT1 and SIRT3. The protection provided by NBP against cerebral ischemia/reperfusion may be achieved through SIRT1.

RRx-001 Exerts Neuroprotection Against LPS-Induced Microglia Activation and Neuroinflammation Through Disturbing the TLR4 Pathway.

Neuroinflammation plays an important role in the pathogenesis of many central nervous system diseases. Here, we investigated the effect of an anti-cancer compound RRx-001 on neuroinflammation and its possible new applications. BV2 cells and primary microglia cells were used to evaluate the role of RRx-001 in LPS-induced microglial activation and inflammatory response in vitro. And, we found that the increase in the synthesis and release of cytokines and the up-regulation of pro-inflammatory factors in LPS-treated microglial cells were significantly reduced by RRx-001 pretreatment. As the most classical inflammatory pathways, NF-κB and MAPK signaling pathways were activated by LPS, but were inhibited by RRx-001. Transcription of NLRP3 was also reduced by RRx-001. In addition, LPS induced oxidative stress by increasing the expression of Nox mediated by transcription factors NF-κB and AP-1, while RRx-001 pretreatment ameliorated Nox-mediated oxidative stress. LPS-induced activation of TAK1, an upstream regulator of NF-κB and MAPK pathways, was significantly inhibited by RRx-001 pretreatment, whereas recruitment of MyD88 to TLR4 was not affected by RRx-001. LPS-primed BV2 condition medium induced injury of primary neurons, and this effect was inhibited by RRx-001. Furthermore, we established a neuroinflammatory mouse model by stereotactic injection of LPS into the substantia nigra pars compacta (SNpc), and RRx-001 dose-dependently reduced LPS-induced microglial activation and loss of TH + neurons in the midbrain. In conclusion, the current study found that RRx-001 suppressed microglia activation and neuroinflammation through targeting TAK1, and may be a candidate for the treatment of neuroinflammation-related brain diseases.

Mitochondrial-derived damage-associated molecular patterns amplify neuroinflammation in neurodegenerative diseases.

Both mitochondrial dysfunction and neuroinflammation are implicated in neurodegeneration and neurodegenerative diseases. Accumulating evidence shows multiple links between mitochondrial dysfunction and neuroinflammation. Mitochondrial-derived damage-associated molecular patterns (DAMPs) are recognized by immune receptors of microglia and aggravate neuroinflammation. On the other hand, inflammatory factors released by activated glial cells trigger an intracellular cascade, which regulates mitochondrial metabolism and function. The crosstalk between mitochondrial dysfunction and neuroinflammatory activation is a complex and dynamic process. There is strong evidence that mitochondrial dysfunction precedes neuroinflammation during the progression of diseases. Thus, an in-depth understanding of the specific molecular mechanisms associated with mitochondrial dysfunction and the progression of neuroinflammation in neurodegenerative diseases may contribute to the identification of new targets for the treatment of diseases. In this review, we describe in detail the DAMPs that induce or aggravate neuroinflammation in neurodegenerative diseases including mtDNA, mitochondrial unfolded protein response (mtUPR), mitochondrial reactive oxygen species (mtROS), adenosine triphosphate (ATP), transcription factor A mitochondria (TFAM), cardiolipin, cytochrome c, mitochondrial Ca2+ and iron.

Reduced nicotinamide adenine dinucleotide phosphate in redox balance and diseases: a friend or foe?

The nicotinamide adenine dinucleotide (NAD+/NADH) and nicotinamide adenine dinucleotide phosphate (NADP+/NADPH) redox couples function as cofactors or/and substrates for numerous enzymes to retain cellular redox balance and energy metabolism. Thus, maintaining cellular NADH and NADPH balance is critical for sustaining cellular homeostasis. The sources of NADPH generation might determine its biological effects. Newly-recognized biosynthetic enzymes and genetically encoded biosensors help us better understand how cells maintain biosynthesis and distribution of compartmentalized NAD(H) and NADP(H) pools. It is essential but challenging to distinguish how cells sustain redox couple pools to perform their integral functions and escape redox stress. However, it is still obscure whether NADPH is detrimental or beneficial as either deficiency or excess in cellular NADPH levels disturbs cellular redox state and metabolic homeostasis leading to redox stress, energy stress, and eventually, to the disease state. Additional study of the pathways and regulatory mechanisms of NADPH generation in different compartments, and the means by which NADPH plays a role in various diseases, will provide innovative insights into its roles in human health and may find a value of NADPH for the treatment of certain diseases including aging, Alzheimer's disease, Parkinson's disease, cardiovascular diseases, ischemic stroke, diabetes, obesity, cancer, etc.

Long-term running exercise alleviates cognitive dysfunction in APP/PSEN1 transgenic mice via enhancing brain lysosomal function.

Amyloid-ß peptide (Aß) aggregation is the hallmark of Alzheimer's disease (AD). The imbalance between the production and clearance of Aß results in the accumulation and aggregation of Aß in the brain. Thus far, few drugs are available for AD treatment, but exercise has been recognized for its cognition-enhancing properties in AD patients. The underlying mechanisms remain unclear. Our recent study showed that long-term running exercise could activate the lysosomal function in the brains of mice. In this study, we investigated whether exercise could reduce Aß accumulation by activating lysosomal function in APP/PSEN1 transgenic mice. Started at the age of 5 months, the mice were trained with a running wheel at the speed of 18 r/min, 40 min/d, 6 d/week for 5 months, and were killed at the end of the 10th month, then brain tissue was collected for biochemical analyses. The cognitive ability was assessed in the 9th month. We showed that long-term exercise significantly mitigated cognitive dysfunction in AD mice, accompanied by the enhanced lysosomal function and the clearance of Aß in the brain. Exercise significantly promoted the nuclear translocation of transcription factor EB (TFEB), and increased the interaction between nuclear TFEB with AMPK-mediated acetyl-CoA synthetase 2, thus enhancing transcription of the genes associated with the biogenesis of lysosomes. Exercise also raised the levels of mature cathepsin D and cathepsin L, suggesting that more Aß peptides could be degraded in the activated lysosomes. This study demonstrates that exercise may improve the cognitive dysfunction of AD by enhancing lysosomal function.

NADPH is superior to NADH or edaravone in ameliorating metabolic disturbance and brain injury in ischemic stroke.

Our previous studies confirm that exogenous reduced nicotinamide adenine dinucleotide phosphate (NADPH) exerts a neuroprotective effect in animal models of ischemic stroke, and its primary mechanism is related to anti-oxidative stress and improved energy metabolism. However, it is unknown whether nicotinamide adenine dinucleotide (NADH) also plays a neuroprotective role and whether NADPH is superior to NADH against ischemic stroke? In this study we compared the efficacy of NADH, NADPH, and edaravone in ameliorating brain injury and metabolic stress in ischemic stroke. Transient middle cerebral artery occlusion/reperfusion (t-MCAO/R) mouse model and in vitro oxygen glucose deprivation/reoxygenation (OGD/R) model were established. The mice were intravenously administered the optimal dose of NADPH (7.5 mg/kg), NADH (22.5 mg/kg), or edaravone (3 mg/kg) immediately after reperfusion. We showed that the overall efficacy of NADPH in ameliorating ischemic injury was superior to NADH and edaravone. NADPH had a longer therapeutic time window (within 5 h) after reperfusion than NADH and edaravone (within 2 h) for ischemic stroke. In addition, NADPH and edaravone were better in alleviating the brain atrophy, while NADH and NADPH were better in increasing the long-term survival rate. NADPH showed stronger antioxidant effects than NADH and edaravone; but NADH was the best in terms of maintaining energy metabolism. Taken together, this study demonstrates that NADPH exerts better neuroprotective effects against ischemic stroke than NADH and edaravone.

TIGAR plays neuroprotective roles in KA-induced excitotoxicity through reducing neuroinflammation and improving mitochondrial function.

Excitotoxicity refers to the ability of excessive extracellular excitatory amino acids to damage neurons via receptor activation. It is a crucial pathogenetic process in neurodegenerative diseases. TP53 is confirmed to be involved in excitotoxicity. It is demonstrated that TP53 induced glycolysis and apoptotic regulator (TIGAR)-regulated metabolic pathway can protect against neuronal injury. However, the role of TIGAR in excitotoxicity and specific mechanisms is still unknown. In this study, an in vivo excitotoxicity model was constructed via stereotypical kainic acid (KA) injection into the striatum of mice. KA reduced TIGAR expression levels, neuroinflammatory responses and mitochondrial dysfunction. TIGAR overexpression could reverse KA-induced neuronal injury by reducing neuroinflammation and improving mitochondrial function, thereby exerting neuroprotective effects. Therefore, this study could provide a potential therapeutic target for neurodegenerative diseases.

Pharmacology and Potential Implications of Nicotinamide Adenine Dinucleotide Precursors.

Coenzyme I (nicotinamide adenine dinucleotide, NAD+/NADH) and coenzyme II (nicotinamide adenine dinucleotide phosphate, NADP+/NADPH) are involved in various biological processes in mammalian cells. NAD+ is synthesised through the de novo and salvage pathways, whereas coenzyme II cannot be synthesised de novo. NAD+ is a precursor of coenzyme II. Although NAD+ is synthesised in sufficient amounts under normal conditions, shortage in its supply due to over consumption and its decreased synthesis has been observed with increasing age and under certain disease conditions. Several studies have proved that in a wide range of tissues, such as liver, skin, muscle, pancreas, and fat, the level of NAD+ decreases with age. However, in the brain tissue, the level of NADH gradually increases and that of NAD+ decreases in aged people. The ratio of NAD+/NADH indicates the cellular redox state. A decrease in this ratio affects the cellular anaerobic glycolysis and oxidative phosphorylation functions, which reduces the ability of cells to produce ATP. Therefore, increasing the exogenous NAD+ supply under certain disease conditions or in elderly people may be beneficial. Precursors of NAD+ have been extensively explored and have been reported to effectively increase NAD+ levels and possess a broad range of functions. In this review article, we discuss the pharmacokinetics and pharmacodynamics of NAD+ precursors.

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 (Lyz2cre )-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.

Biomarkers of Autophagy.

Biomarkers (short for biological markers) are biological measures of a biological state. Autophagy biomarkers play an important role as an indicator of autophagy during normal physiological processes, pathogenic processes or pharmacological responses to drugs. In this chapter, some biomarkers of different types of autophagy, including macroautophagy, selective autophagy, chaperone-mediated autophagy, and microautophagy, as well as the lysosomal biomarkers are introduced. The described biomarkers may be used to detect the level of autophagy in cells or tissues in a dynamic, real-time, and quantitative manner. However, each biomarker has its specific significance and limitation. Therefore, the analysis of the autophagy level in cells or tissues through the detection of autophagy biomarkers should be carried out carefully.

NADPH Oxidases in the Central Nervous System: Regional and Cellular Localization and the Possible Link to Brain Diseases.

Significance: The significant role of reduced nicotinamide adenine dinucleotide phosphate (NADPH) oxidase (Nox) in signal transduction is mediated by the production of reactive oxygen species (ROS), especially in the central nervous system (CNS). The pathogenesis of some neurologic and psychiatric diseases is regulated by ROS, acting as a second messenger or pathogen. Recent Advances: In the CNS, the involvement of Nox-derived ROS has been implicated in the regulation of multiple signals, including cell survival/apoptosis, neuroinflammation, migration, differentiation, proliferation, and synaptic plasticity, as well as the integrity of the blood/brain barrier. In these processes, the intracellular signals mediated by the members of the Nox family vary among different tissues. The present review illuminates the regions and cellular, subcellular localization of Nox isoforms in the brain, the signal transduction, and the role of NOX enzymes in pathophysiology, respectively. Critical Issues: Different signal transduction cascades are coupled to ROS derived from various Nox homologues with varying degrees. Therefore, a critical issue worth noting is the varied role of the homologues of NOX enzymes in different signaling pathways and also they mediate different phenotypes in the diverse pathophysiological condition. This substantiates the effectiveness of selective Nox inhibitors in the CNS. Future Directions: Further investigation to elucidate the role of various homologues of NOX enzymes in acute and chronic brain diseases and signaling mechanisms, and the development of more specific NOX inhibitors for the treatment of CNS disease are urgently needed. Antioxid. Redox Signal. 35, 951-973.