Astragaloside IV-mediated inhibition of oxidative stress by upregulation of ghrelin in type 2 diabetes-induced cognitive impairment.

This study is to observe the upregulation effect of astragaloside IV on ghrelin in diabetic cognitive impairment (DCI) rats and to investigate the pathway in prevention and treatment by reducing oxidative stress. The DCI model was induced with streptozotocin (STZ) in conjunction with a high-fat and high-sugar diet and divided into three groups: model, low-dose (40 mg/kg), and high-dose (80 mg/kg) astragaloside IV. After 30 days of gavage, the learning and memory abilities of rats, as well as their body weight and blood glucose levels, were tested using the Morris water maze and then detection of insulin resistance, SOD activity, and serum MDA levels. The whole brain of rats was sampled for hematoxylin-eosin and Nissl staining to observe pathological changes in the hippocampal CA1 region. Immunohistochemistry was used to detect ghrelin expression in the hippocampal CA1 region. A Western blot was used to determine changes in GHS-R1α/AMPK/PGC-1α/UCP2. RT-qPCR was used to determine the levels of ghrelin mRNA. Astragaloside IV reduced nerve damage, increased superoxide dismutase (SOD) activity, decreased MDA levels, and improved insulin resistance. Ghrelin levels and expression increased in serum and hippocampal tissues, and ghrelin mRNA levels increased in rat stomach tissues. According to Western blot, it increased the expression of the ghrelin receptor GHS-R1α and upregulated the mitochondrial function associated-protein AMPK-PGC-1α-UCP2. Astragaloside IV increases ghrelin expression in the brain to reduce oxidative stress and delay diabetes-induced cognitive impairment. It may be related to the promotion of ghrelin mRNA levels.

Facile synthesis of novel helical imprinted fibers based on zucchini-derived microcoils for efficient recognition of target protein in biological sample.

Highly selective recognition and purification of target proteins from complex biological matrices remains a challenging subject in natural and life sciences. Compared with natural recognition receptors, artificial imprinted polymers are an ideal alternative candidate. In this study, we report a novel method to prepare helical protein imprinted fibers (HPIFs) with zucchini-derived microcoils as a carrier, firstly. Inspired by the self-polymerization of adhesive proteins in mussels, dopamine and 3,4-dihydroxyphenylacetic acid were chosen as bifunctional monomers for the first time to form a biocompatible imprinted layer. The chemical/physical properties and recognition performance of HPIFs were studied in a series of experiments. Additionally, the practicability of HPIFs was verified by specifically recognizing target protein in complex egg white sample. The one-step synthesis process and excellent binding performance of HPIFs make them a promising material for protein recognition and purification, and endow HPIFs with potential application value in the food, chemical and pharmaceutical fields.

Dopamine D1 receptors mediate methamphetamine-induced dopaminergic damage: involvement of autophagy regulation via the AMPK/FOXO3A pathway.

RATIONALE: Clinical studies have revealed that methamphetamine abuse increases risk for developing Parkinson's diseases. It is thus important to elucidate the mechanisms by which methamphetamine damages dopaminergic neurons. OBJECTIVES: The present study was designed to elucidate the role of the dopamine D1 receptor in methamphetamine-mediated dopaminergic neuronal damage and its underlying mechanisms. METHODS: Mice were treated for 4 days with vehicle, methamphetamine, or the D1 agonist SKF38393 and then assessed for locomotion and performance in the pole and rotarod tests. Cellular indices of autophagy, LC3, P62, and Beclin-1, tyrosine hydroxylase, and the AMPK/FOXO3A pathway were analyzed in striatal tissue from treated mice, in PC12 cells, and in D1 receptor mutant mice. RESULTS: Repeated treatment with a relatively high dose of methamphetamine for 4 days induced both loss of dopaminergic neurons and activation of autophagy in the striatum as evidenced by increased expression of LC3 and P62. However, such treatment did not induce either loss of dopaminergic neurons or activation of autophagy in D1 receptor knockout mice. D1 receptor-mediated activation of autophagy was also confirmed in vitro using dopaminergic neuronal PC12 cells. Further studies demonstrated that the AMPK/FOXO3A signaling pathway is responsible for D1 receptor-mediated activation of autophagy. CONCLUSIONS: The present data indicate a novel mechanism for methamphetamine-induced dopaminergic neuronal damage and reveal an important role for D1 receptors in the neurotoxicity of this drug.

Dysregulation of iron homeostasis and methamphetamine reward behaviors in Clk1-deficient mice.

Chronic administration of methamphetamine (METH) leads to physical and psychological dependence. It is generally accepted that METH exerts rewarding effects via competitive inhibition of the dopamine transporter (DAT), but the molecular mechanism of METH addiction remains largely unknown. Accumulating evidence shows that mitochondrial function is important in regulation of drug addiction. In this study,  we investigated the role of Clk1, an essential mitochondrial hydroxylase for ubiquinone (UQ), in METH reward effects. We showed that Clk1+/- mutation significantly suppressed METH-induced conditioned place preference (CPP), accompanied by increased expression of DAT in plasma membrane of striatum and hippocampus due to Clk1 deficiency-induced inhibition of DAT degradation without influencing de novo synthesis of DAT. Notably, significantly decreased iron content in striatum and hippocampus was evident in both Clk1+/- mutant mice and PC12 cells with Clk1 knockdown. The decreased iron content was attributed to increased expression of iron exporter ferroportin 1 (FPN1) that was associated with elevated expression of hypoxia-inducible factor-1α (HIF-1α) in response to Clk1 deficiency both in vivo and in vitro. Furthermore, we showed that iron played a critical role in mediating Clk1 deficiency-induced alteration in DAT expression, presumably via upstream HIF-1α. Taken together, these data demonstrated that HIF-1α-mediated changes in iron homostasis are involved in the Clk1 deficiency-altered METH reward behaviors.

Stimulation of astrocytic sigma-1 receptor is sufficient to ameliorate inflammation- induced depression.

Astrocytes play important roles in the development of depression. As a promising target for antidepressant development, sigma-1 receptor (Sig-1R) is reported to promote activation of astrocyte in chronic stress-induced depression in our previous study. However, astrocytes are hyper-activated in inflammation-induced depression, raising concerns of whether stimulation of astrocytic Sig-1R would exert antidepressant-like effect in inflammation-induced depression. Here we reported that specific stimulation of astrocytic Sig-1R using adeno-associated virus (AAV) significantly attenuated lipopolysaccharide (LPS)- induced depressive-like behavior in the forced swim test (FST), tail suspension test (TST), sucrose preference test, and improved the memory function in novel object recognition test. Besides, specific stimulation of astrocytic Sig-1R decreased the activation of astrocyte and microglia, as well as increased brain-derived neurotrophic factor (BDNF) in LPS-induced depression. In primary cultured astrocytes, overexpression of Sig-1R also reduced the expression of IL-1ß, TNF-α, iNOS during inflammation-treated astrocyte. Taken together, the results suggest that specific stimulation of astrocytic Sig-1R ameliorates inflammation-induced depressive-like behavior, providing the evidence that astrocytic Sig-1R could represent a reliable therapeutic target for depression.

Sigma-1 receptor regulates mitophagy in dopaminergic neurons and contributes to dopaminergic protection.

Mitochondria are essential for neuronal survival and function, and mitochondrial dysfunction plays a critical role in the pathological development of Parkinson's disease (PD). Mitochondrial quality control is known to contribute to the survival of dopaminergic (DA) neurons, with mitophagy being a key regulator of the quality control system. In this study, we show that mitophagy is impaired in the substantia nigra pars compacta (SNc) of the 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP)-induced mouse model of PD. Treatment with the sigma-1 receptor (Sig 1R) agonist 2-morpholin-4-ylethyl 1-phenylcyclohexane-1-carboxylate (PRE-084) reduced loss of DA neurons, restored motor ability and MPTP-induced damage to mitophagy activity in the SNc of PD-like mice. Additionally, knockdown of Sig 1R in SH-SY5Y DA cells inhibited mitophagy and enhanced 1-methyl-4-phenylpyridinium ion (MPP+) neurotoxicity, whereas application of the Sig 1R selective agonist SKF10047 promoted clearance of damaged mitochondria. Moreover, knockdown of Sig 1R in SH-SY5Y cells resulted in decreased levels of p-ULK1 (Unc-51 Like Autophagy Activating Kinase 1) (Ser555), p-TBK1 (TANK Binding Kinase 1) (Ser172), p-ubiquitin (Ub) (Ser65), Parkin recruitment, and stabilization of PTEN-induced putative kinase 1 (PINK1) in mitochondria. The present data provide the first evidence for potential roles of PINK1/Parkin in Sig 1R-modulated mitophagy in DA neurons.

PHLDA1 promotes microglia-mediated neuroinflammation via regulating K63-linked ubiquitination of TRAF6.

Microglia-mediated neuroinflammation plays an important role in the progression of neurodegenerative diseases including Parkinson's disease (PD). Pleckstrin homology-like domain family A member 1 (PHLDA1) plays an important role in immunological regulation, particularly in the Toll-like receptor-mediated immune response. Here, we explored the potential roles of PHLDA1 in microglia-mediated inflammation and neuronal protection. We found that PHLDA1 expression was rapidly increased in response to inflammatory stimuli in microglia cells in vivo or in vitro. Knockdown of PHLDA1 using adeno-associated virus serotype (AAV) ameliorated MPTP-induced motor deficits and inhibited neuroinflammation in mice. In support of this observation in vivo, we found that LPS-induced proinflammatory gene expression, including TNF-α, IL-1ß, iNOS, and COX-2, was decreased in PHLDA1-deficient microglial cells. Mechanistic studies demonstrated that increased expression of PHLDA1, upon LPS stimulation in microglia, led to direct interaction with TRAF6 and enhanced its K63-linked ubiquitination-mediated NF-κB signaling activation. PHLDA1 deficiency interfered with TRAF6 K63-linked ubiquitination and inhibited microglial inflammatory responses. These findings reveal the first evidence that PHLDA1 is an important modulator of microglial function that is associated with microglia-mediated dopaminergic neurotoxicity. The data therefore provided the first evidence that PHLDA1 may be a potent modulator for neuroinflammation, and PHLDA1 may be a novel drug target for treatment of neuroinflammation-related diseases such as PD.

The oncometabolite 2-hydroxyglutarate inhibits microglial activation via the AMPK/mTOR/NF-κB pathway.

Microglia, the brain-resident macrophage, is known as the innate immune cell type in the central nervous system. Microglia is also the major cellular component of tumor mass of gliomas that plays a key role in glioma development. Mutations of isocitrate dehydrogenases 1 and 2 (IDH1/2) frequently occur in gliomas, which leads to accumulation of oncometabolic product 2-hydroxyglutarate (2HG). Moreover, IDH1/2 mutations were found to correlate with better prognosis in glioma patients. In the present study, we investigated the effects of the 2HG on microglial inflammatory activation. We showed that the conditioned media (CM) from GL261 glioma cells stimulated the activation of BV-2 microglia cells, evidenced by markedly increased expression of interleukin-6 (IL-6), IL-1ß, tumor necrosis factor-α (TNF-α), CCL2 (C-C motif chemokine ligand 2) and CXCL10 (C-X-C motif chemokine 10). CM-induced expression of proinflammatory genes was significantly suppressed by pretreatment with a synthetic cell-permeable 2HG (1 mM) or a nuclear factor-κB (NF-κB) inhibitor BAY11-7082 (10 µM). In lipopolysaccharide (LPS)- or TNF-α-stimulated BV-2 microglia cells and primary microglia, pretreatment with 2HG (0.25-1 mM) dose-dependently suppressed the expression of proinflammatory genes. We further demonstrated that 2HG significantly suppressed LPS-induced phosphorylation of IκB kinase α/ß (IKKα/ß), IκBα and p65, IκB degradation, and nuclear translocation of p65 subunit of NF-κB, as well as NF-κB transcriptional activity. Similarly, ectopic expression of mutant isocitrate dehydrogenase 1 (IDH1) (R132H) significantly decreased TNF-α-induced activation of NF-κB signaling pathway. Finally, we revealed that activation of adenosine 5'-monophosphate-activated protein kinase (AMPK) and subsequent inhibition of mammalian target of rapamycin (mTOR) signaling contributed to the inhibitory effect of 2HG on NF-κB signaling pathway in BV-2 cells. Taken together, these results, for the first time, show that oncometabolite 2HG inhibits microglial activation through affecting AMPK/mTOR/NF-κB signaling pathway and provide evidence that oncometabolite 2HG may regulate glioma development via modulating microglial activation in tumor microenvironment.

Activation of AMPK/mTORC1-Mediated Autophagy by Metformin Reverses Clk1 Deficiency-Sensitized Dopaminergic Neuronal Death.

The autophagy-lysosome pathway (ALP) plays a critical role in the pathology of Parkinson's disease (PD). Clk1 (coq7) is a mitochondrial hydroxylase that is essential for coenzyme Q (ubiquinone) biosynthesis. We have reported previously that Clk1 regulates microglia activation via modulating microglia metabolic reprogramming, which contributes to dopaminergic neuronal survival. This study explores the direct effect of Clk1 on dopaminergic neuronal survival. We demonstrate that Clk1 deficiency inhibited dopaminergic neuronal autophagy in cultured MN9D dopaminergic neurons and in the substantia nigra pars compacta of Clk+/- mutant mice and consequently sensitized dopaminergic neuron damage and behavioral defects. These mechanistic studies indicate that Clk1 regulates the AMP-activated protein kinase (AMPK)/rapamycin complex 1 pathway, which in turn impairs the ALP and TFEB nuclear translocation. As a result, Clk1 deficiency promotes dopaminergic neuronal damage in vivo and in vitro, which ultimately contributes to sensitizing 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP)-induced dopaminergic neuronal death and behavioral impairments in Clk1-deficient mice. Moreover, we found that activation of autophagy by the AMPK activator metformin increases dopaminergic neuronal survival in vitro and in the MPTP-induced PD model in Clk1 mutant mice. These results reveal that Clk1 plays a direct role in dopaminergic neuronal survival via regulating ALPs that may contribute to the pathologic development of PD. Modulation of Clk1 activity may represent a potential therapeutic target for PD.

Clk1 deficiency promotes neuroinflammation and subsequent dopaminergic cell death through regulation of microglial metabolic reprogramming.

Clock (Clk)1/COQ7 is a mitochondrial hydroxylase that is necessary for the biosynthesis of ubiquinone (coenzyme Q or UQ). Here, we investigate the role of Clk1 in neuroinflammation and consequentially dopaminergic (DA) neuron survival. Reduced expression of Clk1 in microglia enhanced the LPS-induced proinflammatory response and promoted aerobic glycolysis. Inhibition of glycolysis abolished Clk1 deficiency-induced hypersensitivity to the inflammatory stimulation. Mechanistic studies demonstrated that mTOR/HIF-1α and ROS/HIF-1α signaling pathways were involved in Clk1 deficiency-induced aerobic glycolysis. The increase in neuronal cell death was observed following treatment with conditioned media from Clk1 deficient microglia. Increased DA neuron loss and microgliosis were observed in Clk1+/- mice after treatment with MPTP, a rodent model of Parkinson's disease (PD). This increase in DA neuron loss was due to an exacerbated microglial inflammatory response, rather than direct susceptibility of Clk1+/- DA cells to MPP+, the active species of MPTP. Exaggerated expressions of proinflammatory genes and loss of DA neurons were also observed in Clk1+/- mice after stereotaxic injection of LPS. Our results suggest that Clk1 regulates microglial metabolic reprogramming that is, in turn, involved in the neuroinflammatory processes and PD.

Accessible Method for the Development of Novel Sterol Analogues with Dipeptide-like Side Chains That Act as Neuroinflammation Inhibitors.

A number of novel sterol derivatives with dipeptide-like side chains were synthesized using an Ugi four-component condensation method and assayed to test their anti-inflammatory effects in activated microglial cells. Compound 18b ((3S,10R,13S)-N-((R)-1-(tert-butylamino)-1-oxo-3-phenylpropan-2-yl)-3-hydroxy-N,10,13-trimethyl-2,3,4,7,8,9,10,11,12,13,14,15-dodecahydro-1H-cyclopenta[a]phenanthrene-17-carboxamide) was identified as the most potent anti-inflammatory agent in the series of compounds analyzed. Compound 18b markedly inhibited the expression of proinflammatory factors, including inducible nitric oxide synthase, interleukin (IL)-6, IL-1ß, tumor necrosis factor-α, and cyclooxygenase-2 in lipopolysaccharide-stimulated microglial cells. Further studies showed that compound 18b significantly suppressed the transcriptional activity of AP-1 and NF-κB in activated microglial cells, which was likely mediated by the inhibition of the p38 MAPK and JNK signal transduction pathways. In addition, compound 18b displayed neuroprotective effects in a microglial-conditioned medium/neuron coculture and an experimental focal ischemic mouse model.