The p53 target DRAM1 modulates calcium homeostasis and ER stress by promoting contact between lysosomes and the ER through STIM1.

It is well established that DNA Damage Regulated Autophagy Modulator 1 (DRAM1), a lysosomal protein and a target of p53, participates in autophagy. The cellular functions of DRAM1 beyond autophagy remain elusive. Here, we show p53-dependent upregulation of DRAM1 in mitochondrial damage-induced Parkinson's disease (PD) models and exacerbation of disease phenotypes by DRAM1. We find that the lysosomal location of DRAM1 relies on its intact structure including the cytosol-facing C-terminal domain. Excess DRAM1 disrupts endoplasmic reticulum (ER) structure, triggers ER stress, and induces protective ER-phagy. Mechanistically, DRAM1 interacts with stromal interacting molecule 1 (STIM1) to tether lysosomes to the ER and perturb STIM1 function in maintaining intracellular calcium homeostasis. STIM1 overexpression promotes cellular health by restoring calcium homeostasis, ER stress response, ER-phagy, and AMP-activated protein kinase (AMPK)-Unc-51 like autophagy activating kinase 1 (ULK1) signaling in cells with excess DRAM1. Thus, by promoting organelle contact between lysosomes and the ER, DRAM1 modulates ER structure and function and cell survival under stress. Our results suggest that DRAM1 as a lysosomal protein performs diverse roles in cellular homeostasis and stress response. These findings may have significant implications for our understanding of the role of the p53/DRAM1 axis in human diseases, from cancer to neurodegenerative diseases.

KA-mediated excitotoxicity induces neuronal ferroptosis through activation of ferritinophagy.

OBJECTIVES: This study aims to elucidate the role of Fe2+ overload in kainic acid (KA)-induced excitotoxicity, investigate the involvement of ferritinophagy selective cargo receptor NCOA4 in the pathogenesis of excitotoxicity. METHODS: Western blotting was used to detect the expression of FTH1, NCOA4, Lamp2, TfR, FPN, and DMT1 after KA stereotaxic injection into the unilateral striatum of mice. Colocalization of Fe2+ with lysosomes in KA-treated primary cortical neurons was observed by using confocal microscopy. Desferrioxamine (DFO) was added to chelate free iron, a CCK8 kit was used to measure cell viability, and the Fe2+ levels were detected by FerroOrange. BODIPY C11 was used to determine intracellular lipid reactive oxygen species (ROS) levels, and the mRNA levels of PTGS2, a biomarker of ferroptosis, were measured by fluorescent quantitative PCR. 3-Methyladenine (3-MA) was employed to inhibit KA-induced activation of autophagy, and changes in ferritinophagy-related protein expression and the indicated biomarkers of ferroptosis were detected. Endogenous NCOA4 was knocked down by lentivirus transfection, and cell viability and intracellular Fe2+ levels were observed after KA treatment. RESULTS: Western blot results showed that the expression of NCOA4, DMT1, and Lamp2 was significantly upregulated, while FTH1 was downregulated, but there were no significant changes in TfR and FPN. The fluorescence results indicated that KA enhanced the colocalization of free Fe2+ with lysosomes in neurons. DFO intervention could effectively rescue cell damage, reduce intracellular lipid peroxidation, and decrease the increased transcript levels of PTGS2 caused by KA. Pretreatment with 3-MA effectively reversed KA-induced ferritinophagy and ferroptosis. Endogenous interference with NCOA4 significantly improved cell viability and reduced intracellular free Fe2+ levels in KA-treated cells. CONCLUSION: KA-induced excitotoxicity activates ferritinophagy, and targeting ferritinophagy effectively inhibits downstream ferroptosis. Interference with NCOA4 effectively attenuates KA-induced neuronal damage. This study provides a potential therapeutic target for excitotoxicity related disease conditions.

The P2X7 receptor mediates NADPH transport across the plasma membrane.

Nicotinamide Adenine Dinucleotide Phosphate (NADPH) plays a vital role in regulating redox homeostasis and reductive biosynthesis. However, if exogenous NADPH can be transported across the plasma membrane has remained elusive. In this study, we present evidence supporting that NADPH can traverse the plasma membranes of cells through a mechanism mediated by the P2X7 receptor (P2X7R). Notably, we observed an augmentation of intracellular NADPH levels in cultured microglia upon exogenous NADPH supplementation in the presence of ATP. The P2X7R-mediated transmembrane transportation of NADPH was validated with P2X7R antagonists, including OX-ATP, BBG, and A-438079, or through P2X7 knockdown, which impeded NADPH transportation into cells. Conversely, overexpression of P2X7 resulted in an enhanced capacity for NADPH transport. Furthermore, transfection of hP2X7 demonstrated the ability to complement NADPH uptake in native HEK293 cells. Our findings provide evidence for the first time that NADPH is transported across the plasma membrane via a P2X7R-mediated pathway. Additionally, we propose an innovative avenue for modulating intracellular NADPH levels. This discovery holds promise for advancing our understanding of the role of NADPH in redox homeostasis and neuroinflammation.

BNIP3-mediated mitophagy attenuates hypoxic-ischemic brain damage in neonatal rats by inhibiting ferroptosis through P62-KEAP1-NRF2 pathway activation to maintain iron and redox homeostasis.

As a major contributor to neonatal death and neurological sequelae, hypoxic-ischemic encephalopathy (HIE) lacks a viable medication for treatment. Oxidative stress induced by hypoxic-ischemic brain damage (HIBD) predisposes neurons to ferroptosis due to the fact that neonates accumulate high levels of polyunsaturated fatty acids for their brain developmental needs but their antioxidant capacity is immature. Ferroptosis is a form of cell death caused by excessive accumulation of iron-dependent lipid peroxidation and is closely associated with mitochondria. Mitophagy is a type of mitochondrial quality control mechanism that degrades damaged mitochondria and maintains cellular homeostasis. In this study we employed mitophagy agonists and inhibitors to explore the mechanisms by which mitophagy exerted ferroptosis resistance in a neonatal rat HIE model. Seven-days-old neonatal rats were subjected to ligation of the right common carotid artery, followed by exposure to hypoxia for 2 h. The neonatal rats were treated with a mitophagy activator Tat-SPK2 peptide (0.5, 1 mg/kg, i.p.) 1 h before hypoxia, or in combination with mitochondrial division inhibitor-1 (Mdivi-1, 20 mg/kg, i.p.), and ferroptosis inhibitor Ferrostatin-1 (Fer-1) (2 mg/kg, i.p.) at the end of the hypoxia period. The regulation of ferroptosis by mitophagy was also investigated in primary cortical neurons or PC12 cells in vitro subjected to 4 or 6 h of OGD followed by 24 h of reperfusion. We showed that HIBD induced mitochondrial damage, ROS overproduction, intracellular iron accumulation, lipid peroxidation and ferroptosis, which were significantly reduced by the pretreatment with Tat-SPK2 peptide, and aggravated by the treatment with Mdivi-1 or BNIP3 knockdown. Ferroptosis inhibitors Fer-1 and deferoxamine B (DFO) reversed the accumulation of iron and lipid peroxides caused by Mdivi-1, hence reducing ferroptosis triggered by HI. We demonstrated that Tat-SPK2 peptide-activated BNIP3-mediated mitophagy did not alleviate neuronal ferroptosis through the GPX4-GSH pathway. BNIP3-mediated mitophagy drove the P62-KEAP1-NRF2 pathway, which conferred ferroptosis resistance by maintaining iron and redox homeostasis via the regulation of FTH1, HO-1, and DHODH/FSP1-CoQ10-NADH. This study may provide a new perspective and a therapeutic drug for the treatment of neonatal HIE.

GPR50 regulates neuronal development as a mitophagy receptor.

Neurons rely heavily on high mitochondrial metabolism to provide sufficient energy for proper development. However, it remains unclear how neurons maintain high oxidative phosphorylation (OXPHOS) during development. Mitophagy plays a pivotal role in maintaining mitochondrial quality and quantity. We herein describe that G protein-coupled receptor 50 (GPR50) is a novel mitophagy receptor, which harbors the LC3-interacting region (LIR) and is required in mitophagy under stress conditions. Although it does not localize in mitochondria under normal culturing conditions, GPR50 is recruited to the depolarized mitochondrial membrane upon mitophagy stress, which marks the mitochondrial portion and recruits the assembling autophagosomes, eventually facilitating the mitochondrial fragments to be engulfed by the autophagosomes. Mutations Δ502-505 and T532A attenuate GPR50-mediated mitophagy by disrupting the binding of GPR50 to LC3 and the mitochondrial recruitment of GPR50. Deficiency of GPR50 causes the accumulation of damaged mitochondria and disrupts OXPHOS, resulting in insufficient ATP production and excessive ROS generation, eventually impairing neuronal development. GPR50-deficient mice exhibit impaired social recognition, which is rescued by prenatal treatment with mitoQ, a mitochondrially antioxidant. The present study identifies GPR50 as a novel mitophagy receptor that is required to maintain mitochondrial OXPHOS in developing neurons.

NADPH mimics the antidepressant effects of exercise in a chronic unpredictable stress rat model.

Exercise is known to be an effective intervention for depression. NADPH has been demonstrated to have neuroprotective effects in our previous studies. This study aimed to investigate if NADPH has antidepressant effects and can mimic the effects of exercise in a chronic unpredictable stress (CUS) rat model. CUS rats underwent an 8-week swimming exercise (30 min/d, 5d/w) or were intraperitoneally administered 4 mg/kg or 8 mg/kg NADPH. The open field test (OFT), sucrose preference test (SPT), novelty-suppressed feeding test (NSFT), and forced swimming test (FST) were used to examine the antidepressant-like behaviors of the rats. Exercise, 4 mg/kg, and 8 mg/kg NADPH similarly reduced anxiety, as demonstrated by the number of fecal pellets. Meanwhile, exercise and 8 mg/kg NADPH significantly increased locomotion activity in the OFT. Exercise, 4 mg/kg, and 8 mg/kg NADPH effectively reversed CUS-induced anhedonia in rats in the SPT. Exercise, 4 mg/kg, and 8 mg/kg NADPH had no impact on appetite of depressed rats; however, 8 mg/kg NADPH increased the rats' exploratory activity in the NSFT. Exercise, 4 mg/kg, and 8 mg/kg NADPH significantly reduced the immobility time of CUS model rats, while exercise and 8 mg/kg NADPH postponed the early CUS-induced "immobility" in the FST. These results demonstrated that NADPH has similar antidepressant-like effects to exercise in CUS-induced depression model rats and is a potential exercise-mimicking antidepressant.

Nicotinamide riboside restores nicotinamide adenine dinucleotide levels and alleviates brain injury by inhibiting oxidative stress and neuroinflammation in a mouse model of intracerebral hemorrhage.

Hemorrhagic stroke is a global health problem owing to its high morbidity and mortality rates. Nicotinamide riboside is an important precursor of nicotinamide adenine dinucleotide characterized by a high bioavailability, safety profile, and robust effects on many cellular signaling processes. This study aimed to investigate the protective effects of nicotinamide riboside against collagenase-induced hemorrhagic stroke and its underlying mechanisms of action. An intracerebral hemorrhage model was constructed by stereotactically injecting collagenase into the right striatum of adult male Institute for Cancer Research mice. After 30 minutes, nicotinamide riboside was administered via the tail vein. The mice were sacrificed at different time points for assessments. Nicotinamide riboside reduced collagenase-induced hemorrhagic area, significantly reduced cerebral water content and histopathological damage, promoted neurological function recovery, and suppressed reactive oxygen species production and neuroinflammation. Nicotinamide riboside exerts neuroprotective effects against collagenase-induced intracerebral hemorrhage by inhibiting neuroinflammation and oxidative stress.

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.

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.

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.

TIGAR reduces neuronal ferroptosis by inhibiting succinate dehydrogenase activity in cerebral ischemia.

Ischemia Stroke (IS) is an acute neurological condition with high morbidity, disability, and mortality due to a severe reduction in local cerebral blood flow to the brain and blockage of oxygen and glucose supply. Oxidative stress induced by IS predisposes neurons to ferroptosis. TP53-induced glycolysis and apoptosis regulator (TIGAR) inhibits the intracellular glycolytic pathway to increase pentose phosphate pathway (PPP) flux, promotes NADPH production and thus generates reduced glutathione (GSH) to scavenge reactive oxygen species (ROS), and thus shows strong antioxidant effects to ameliorate cerebral ischemia/reperfusion injury. However, in the current study, prolonged ischemia impaired the PPP, and TIGAR was unable to produce NADPH but was still able to reduce neuronal ferroptosis and attenuate ischemic brain injury. Ferroptosis is a form of cell death caused by free radical-driven lipid peroxidation, and the vast majority of ROS leading to oxidative stress are generated by mitochondrial succinate dehydrogenase (SDH) driving reverse electron transfer (RET) via the mitochondrial electron transport chain. Overexpression of TIGAR significantly inhibited hypoxia-induced enhancement of SDH activity, and TIGAR deficiency further enhanced SDH activity. We also found that the inhibitory effect of TIGAR on SDH activity was related to its mitochondrial translocation under hypoxic conditions. TIGAR may inhibit SDH activity by mediating post-translational modifications (acetylation and succinylation) of SDH A through interaction with SDH A. SDH activity inhibition reduces neuronal ferroptosis by decreasing ROS production, eliminating MitoROS levels and attenuating lipid peroxide accumulation. Notably, TIGAR-mediated inhibition of SDH activity and ferroptosis was not dependent on the PPP-NADPH-GPX4 pathways. In conclusion, mitochondrial translocation of TIGAR in prolonged ischemia is an important pathway to reduce neuronal ferroptosis and provide sustainable antioxidant defense for the brain under prolonged ischemia, further complementing the mechanism of TIGAR resistance to oxidative stress induced by IS.

GOLGA7 is essential for NRAS trafficking from the Golgi to the plasma membrane but not for its palmitoylation.

NRAS mutations are most frequently observed in hematological malignancies and are also common in some solid tumors such as melanoma and colon cancer. Despite its pivotal role in oncogenesis, no effective therapies targeting NRAS has been developed. Targeting NRAS localization to the plasma membrane (PM) is a promising strategy for cancer therapy, as its signaling requires PM localization. However, the process governing NRAS translocation from the Golgi apparatus to the PM after lipid modification remains elusive. This study identifies GOLGA7 as a crucial factor controlling NRAS' PM translocation, demonstrating that its depletion blocks NRAS, but not HRAS, KRAS4A and KRAS4B, translocating to PM. GOLGA7 is known to stabilize the palmitoyltransferase ZDHHC9 for NRAS and HRAS palmitoylation, but we found that GOLGA7 depletion does not affect NRAS' palmitoylation level. Further studies show that loss of GOLGA7 disrupts NRAS anterograde trafficking, leading to its cis-Golgi accumulation. Remarkably, depleting GOLGA7 effectively inhibits cell proliferation in multiple NRAS-mutant cancer cell lines and attenuates NRASG12D-induced oncogenic transformation in vivo. These findings elucidate a specific intracellular trafficking route for NRAS under GOLGA7 regulation, highlighting GOLGA7 as a promising therapeutic target for NRAS-driven cancers.

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.

RP58 knockdown contributes to hypoxia-ischemia-induced pineal dysfunction and circadian rhythm disruption in neonatal rats.

Hypoxia-ischemia (HI) of the brain not only impairs neurodevelopment but also causes pineal gland dysfunction, which leads to circadian rhythm disruption. However, the underlying mechanism of circadian rhythm disruption associated with HI-induced pineal dysfunction remains unknown. The zinc finger protein repressor protein with a predicted molecular mass of 58 kDa (RP58) is involved in the development and differentiation of nerve cells. In this study, we established an HI model in neonatal rats to investigate the expression of RP58 and its role in pineal dysfunction and circadian rhythm disruption induced by HI. We demonstrated that RP58 was highly expressed in the pineal gland under normal conditions and significantly downregulated in the pineal gland and primary pinealocytes following HI. Knockdown of RP58 decreased the expression of enzymes in the melatonin (Mel) synthesis pathway (tryptophan hydroxylase 1 [TPH1], acetylserotonin O-methyltransferase [ASMT], and arylalkylamine N-acetyltransferase [AANAT]) and clock genes (circadian locomotor output cycles kaput [CLOCK] and brain and muscle ARNT-like 1 [BMAL1]), and it also reduced the production of Mel, caused pineal cell injury, and disrupted circadian rhythms in vivo and in vitro. Similarly, HI reduced the expression of Mel synthesis enzymes (TPH1, ASMT, and AANAT) and clock genes (CLOCK and BMAL1), and caused pineal injury and circadian rhythm disruption, which were exacerbated by RP58 knockdown. The detrimental effect of RP58 knockdown on pineal dysfunction and circadian rhythm disruption was reversed by the addition of exogenous Mel. Furthermore, exogenous Mel reversed HI-induced pineal dysfunction and circadian rhythm disruption, as reflected by improvements in Mel production, voluntary activity periods, and activity frequency, as well as a diminished decrease in the expression of Mel synthesis enzymes and clock genes. The present study suggests that RP58 is an endogenous source of protection against pineal dysfunction and circadian rhythm disruption after neonatal HI.

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.