Cuiqing Zhu

Acteoside protects human neuroblastoma SH-SY5Y cells against β-amyloid-induced cell injury

Amyloid beta-peptide (Abeta) has been implicated in the pathogenesis of AD. It can cause cell death in AD by evoking a cascade of oxidative damage to neurons. So antioxidant compounds may throw a light on the treatment of AD. In the present study, we investigated the protective effect of acteoside (AS), an antioxidative phenylethanoid glycoside, on Abeta(25-35)-induced SH-SY5Y cell injury. Exposure of cells to 25 muM Abeta(25-35) for 24 h caused viability loss, apoptotic increase and reactive oxygen species (ROS) increase, pre-treatment with acteoside for 1.5 h significantly reduced the viability loss, apoptotic rate and attenuated Abeta-mediated ROS production. In addition, AS strikingly inhibited Abeta(25-35)-induced mitochondrial dysfunctions, including lowered membrane potential, increased Bax/Bcl-2 ratio, cytochrome c release and the cleavage of caspase-3. Taken together, these results indicated that acteoside could protect SH-SY5Y cells against beta-amyloid-induced cell injury by the attenuating ROS production and the modulating apoptotic signal pathway through Bcl-2 family, cytochrome c, and caspase-3.

Astaxanthin upregulates heme oxygenase-1 expression through ERK1/2 pathway and its protective effect against beta-amyloid-induced cytotoxicity in SH-SY5Y cells.

Astaxanthin (ATX), the most abundant flavonoids in propolis, has been proven to exert neuroprotective property against glutamate-induced neurotoxicity and ischemia-reperfusion-induced apoptosis. Previous study have revealed that ATX can rescue PC12 cells from Aβ(25-35)-induced apoptotic death. However, the mechanisms by which ATX mediates its therapeutic effects in vitro are unclear. In the present study, we explored the underlying mechanisms involved in the protective effects of ATX on the Aβ(25-35)-induced cytotoxicity in SH-SY5Y cells. Pre-treatment with ATX for 4h significantly reduced the Aβ(25-35)-induced viability loss, apoptotic rate and attenuated Aβ-mediated ROS production. In addition, ATX inhibited Aβ(25-35)-induced lowered membrane potential, decreased Bcl-2/Bax ratio. We also demonstrated that ATX could prevent the activation of p38MAPK kinase pathways induced by Aβ. Moreover, we for the first time have revealed the ATX increased antioxidant enzyme heme oxygenase-1 (HO-1) expression in concentration-dependent and time-dependent manners, which were correlated with its protective effect against Aβ(25-35)-induced injury. Because the inhibitor of HO-1 activity, ZnPP reversed the protective effect of ATX against Aβ(25-35)-induced cell death. We also demonstrated that the specific ERK inhibitor, PD98059, concentration-dependently blocked on ATX-induced HO-1 expression, and meanwhile PD98059 reversed the protective effect of ATX against Aβ25-35-induced cell death. Taken together, these findings suggest that astaxanthin can induce HO-1 expression through activation of ERK signal pathways, thereby protecting the SH-SY5Y cells from Aβ(25-35)-induced oxidative cell death.

Neuroprotective role of δ-opioid receptors against mitochondrial respiratory chain injury

It is recognized in recent years that activation of delta-opioid receptor (DOR) elicits neuroprotection against hypoxia and ischemia. However, the underlying mechanisms are not well understood yet. Mitochondrial dysfunction plays a key role in hypoxic neuronal injury, but the effect of DOR activation on neurons with a mitochondrial respiratory chain deficiency is poorly elucidated. In this study we tested the effects of DOR activation and inhibition on cultured cortical neurons after inhibiting mitochondrial respiratory chain with sodium azide (NaN(3)) in days 8 cultures. Neuronal injury was assessed by lactate dehydrogenase release. Changes in DOR proteins were investigated using an antibody against the N-terminus of the DOR, which recognizes the 60, 48, and 32 kDa proteins. Our main findings are that 1) delta- but not mu-opioid receptor activation reduces NaN(3)-induced neuronal damage, and this neuroprotective effect is abolished by DOR antagonist (naltrindole, NTI); 2) prolonged DOR inhibition with NTI further increases NaN(3)-induced neuronal damage; 3) NaN(3) treatment down-regulates DOR protein levels in neurons, and the 60 and 32 kDa proteins are particularly sensitive; 4) DADLE, besides activating DOR directly, also reverses the decrease of neuronal DOR protein levels induced by NaN(3), which may contribute greatly to its neuroprotective effect; 5) NTI reverses NaN(3)-induced down-regulation of DOR proteins as well, the effect of NTI amplifying NaN(3)-induced neuronal damage therefore is probably due to its inhibition on DOR activity only. In conclusion, these data suggest that DOR activation plays an important role in neuroprotection against mitochondrial respiratory chain injury.

Chronic restraint stress alters the expression and distribution of phosphorylated tau and MAP2 in cortex and hippocampus of rat brain

Microtubule-associated proteins (MAPs) play a critical role in maintaining normal cytoskeletal architecture and functions. In the present study, we aim to explore the effects of the emotional stressor, chronic restraint stress, on the expression levels and localization of tau and MAP2. We found that after chronic restraint stress, soluble hyperphosphorylated tau was greatly increased, whereas MAP2 was decreased. Moreover, immunohistochemistry analysis demonstrated that phosphorylated tau and MAP2 displayed the similar subcellular distribution pattern after chronic restraint stress. Robust hyperphosphorylated tau immunolabeling was observed both in cortex and hippocampus of stressed animals and mainly located to perikaryal/dendritic elements. After stress, the MAP2 was mainly distributed in the perikaryal compartments, discontinuous dendrites and neuropil. Moreover, the distribution pattern of MAP2 in hippocampus significantly changed. Immunofluorescence double labeling indicated that hyperphosphorylated tau increased in the regions where there displayed an decrease of MAP2. These results suggest that the involvement of repeated restraint stress may not only induce phosphorylation state of tau and distribution of phosphorylated tau, but also alter the content and neuronal distribution of MAP2. Tau and MAP2 are most important MAPs for neuronal cells, the subcellular distribution change of them might be link to functional change of neurons after emotional stress.

NRSF/REST neuronal deficient mice are more vulnerable to the neurotoxin MPTP

Parkinsons disease (PD) is characterized by progressing loss of dopaminergic neurons in the midbrain. Abnormal gene expression plays a critical role in its pathogenesis. Neuron-restrictive silencer factor (NRSF)/neuronal repressor element-1 silencing transcription factor (REST), a member of the zinc finger transcription factors, inhibits the expression of neuron-specific genes in nonneuronal cells, and regulates neurogenesis. Our previous work showed that 1-methyl-4-phenyl-pyridinium ion triggers dynamic changes of messenger RNA and protein expression of NRSF in human dopaminergic SH-SY5Y cells, and alteration of NRSF expression exacerbates 1-methyl-4-phenyl-pyridinium ion-induced cell death. The purpose of this study was to explore the in vivo role of NRSF in the progress of PD by using NRSF/REST neuron-specific conditional knockout mice (cKO). 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP) was adopted to generate PD models in the cKO mice and wild type littermates. At 1, 3, 7, 14, 21, and 28 days after MPTP injection, behavioral tests were performed, and cKO mice displayed some impairments in locomotor activities. Also, the reduction of tyrosine hydroxylase protein in the striatum and the loss of dopaminergic neurons in the substantia nigra were more severe in the cKO mice. Meanwhile, the cKO mice exhibited a more dramatic depletion of striatal dopamine, accompanied by an increase in glial fibrillary acidic protein (GFAP) expression and sustained interleukin-1β transcription. These results suggested that NRSF/REST neuronal cKO mice are more vulnerable to the dopaminergic neurotoxin MPTP. Disturbance of the homeostasis of NRSF and its target genes, gliogenesis, and inflammation may contribute to the higher MPTP sensitivity in NRSF/REST neuronal cKO mice.

Overexpression of F0F1-ATP synthase α suppresses mutant huntingtin aggregation and toxicity in vitro

Huntingtons disease (HD) and other polyglutamine (polyQ) neurodegenerative diseases are characterized by neuronal accumulation of the disease protein, suggesting that the cellular ability to handle abnormal proteins is compromised. As a multi-subunit protein localized in the mitochondria of eukaryotic cells, the F(0)F(1)-ATP synthase alpha belongs to the family of stress proteins HSP60. Currently, mounting evidences indicate F(0)F(1)-ATP synthase alpha may play a role in neurodegenerative diseases, including Alzheimers disease (AD) and Parkinsons disease (PD). Recently, ATP synthase alpha was reported to have protective and therapeutic roles in primary cardiacmyocytes of iron-overloaded rats by lowering ROS production. However, little is understood about the role of ATP synthase alpha in cell death and neurodegeneration. Here, we demonstrate that overexpression of ATP synthase alpha suppresses huntingtin (htt) polyQ aggregation and toxicity in transfected SH-SY5Y cell lines. Overexpression of ATP synthase alpha is able to protect cell death caused by polyglutamine-expanded htt. Transient overexpression of ATP synthase alpha suppresses the aggregate formation by estimation of polyQ aggregation, Western blot analysis, and filter trap assay (FTA) in transfected SH-SY5Y cells. These results indicated that ATP synthase alpha has a strong inhibitory effect on polyglutamine aggregate formation and toxicity in vitro, and suggest a novel neuroprotective role of ATP synthase alpha.

Effect of ginsenoside Rg3 on tyrosine hydroxylase and related mechanisms in the forced swimming-induced fatigue rats.

ETHNOPHARMACOLOGICAL RELEVANCE Ginsenoside Rg3 has shown multiple pharmacological activities and been considered as one of the most promising approaches for fatigue treatment. However, little is known about the cellular and molecular mechanisms of Rg3 on anti-fatigue and the effect of Rg3 on dopaminergic system has not been reported yet. The major aim of this study is to investigate the effect of Rg3 on TH expression and the related biochemical parameters, such as PKAα, ERK1/2, Akt and α-synuclein in brain of fatigue rats. MATERIALS AND METHODS Weight-loaded forced swimming was performed to establish an animal model of fatigue. Rg3 (10mg/kg, 50mg/kg and 100mg/kg) was intragastrically administrated before swimming. The effect of Rg3 on the expression and phosphorylation of TH and TH-related proteins in fatigue rats or in SH-SY5Y cells was assessed with western blotting. HPLC was used to examine the level of DA and DOPAC in the fatigue rats tissues. RESULTS TH and phosphorylated TH were decreased in different brain regions of which ventral midbrain were less affected in weight-loaded forced swimming rats. Pretreatment with Rg3 significantly suppressed fatigue-induced decrease expression of TH and TH phosphorylation. Also treatment with Rg3 reversed the decrease expression of PKAα as well as the phosphorylation of ERK1/2 and Akt which were induced by weight-loaded forced swimming. Moreover, weight-loaded swimming could induce the increase expression of α-synuclein in hippocampus and midbrain, while suppressed α-synuclein expression in striatum and prefrontal cortex. Furthermore, Rg3 could induce the increase of TH expression and phosphorylation which was accompanied with elevated expression and phosphorylation of related kinase proteins in vitro, while the inhibitors of kinase proteins could suppress these effects of Rg3. In addition, HPLC results showed that Rg3 could reverse the weight-loaded swimming-induced increase of DOPAC/DA ratio. CONCLUSION Our data suggest that fatigue can induce the decrease of DA which might partially result from the change of TH expression and phosphorylation, and Rg3 can reverse these fatigue-induced changes. The underling mechanisms may include the activity changes of PKAα, ERK1/2, Akt and α-synuclein.

Antibody binding to cell surface amyloid precursor protein induces neuronal injury by deregulating the phosphorylation of focal adhesion signaling related proteins

The biological function of full-length amyloid-beta protein precursor (APP), the precursor of Abeta, is not fully understood. Mounting studies reported that antibody binding to cell surface APP causes neuronal injury. However, the mechanism of cell surface APP mediating neuronal injury remains to be determined. Colocalization of APP with integrin on cell surface leads us to suppose that focal adhesion (FA) related mechanism is involved in surface APP-mediated neuronal injury. In the present study, results demonstrated that primary cultured neurons treated with antibody against APP-N-terminal not only caused neuronal injury and aberrant morphologic changes of neurite, but also induced reaction of FA proteins appearing an acute increase then decrease pattern. Moreover, the elevation of tyrosine phosphorylation of FA proteins including paxillin and focal adhesion kinase (FAK), and down-regulated expression of protein tyrosine phosphatase (PTP1B) induced by APP antibody were prevented by inhibitor of Src protein kinases 4-amino-5-(4-chlorophenyl)-7(t-butyl) pyrazol (3,4-D) pyramide (PP2) and G protein inhibitor pertussis toxin (PTX), implying that Src family kinase and G protein play roles in APP-induced FA signals. In addition, pretreatment with PTX and PP2 was able to suppress APP-antibody induced neuronal injury. Taken together, the results suggest a novel mechanism for APP mediating neuronal injury through deregulating FA signals.

Beta amyloid-induced upregulation of death receptor 6 accelerates the toxic effect of N-terminal fragment of amyloid precursor protein

Amyloid precursor protein (APP) plays essential roles in the development of the Alzheimers disease. Although full-length APP has been thoroughly studied, the role of the cleavage fragments especially the N-terminal fragments (N-APPs) in Alzheimers disease pathogenesis was still elusive. In this study, we demonstrated that application of recombinant APP₁₈₋₂₈₆ could enhance beta amyloid (Aβ)-induced neuronal injuries which were related to the activation of apoptosis proteins. Aβ treatment could induce a slight increase of N-APPs release. In addition, expression of death receptor 6 (DR6) was increased in Aβ-treated neurons and APP transgenic mice. Moreover, the effect of APP₁₈₋₂₈₆ on Aβ-induced injuries could be suppressed by the application of recombinant DR6₄₁₋₃₄₁ and DR6 antibody. Furthermore, pull-down assay revealed that APP₁₈₋₂₈₆ could bind both exogenous and endogenous DR6. Aβ promoted APP₁₈₋₂₈₆ targeting to neuron which was accompanied with the increase of DR6 expression, whereas downregulation of DR6 by interference RNA could alleviate the binding of N-APPs to neuron and also suppressed Aβ-dependent toxic effect with N-APPs. These results suggested that APP N-terminal fragments might play neurotoxic roles in Aβ-induced neuronal injuries through cell surface DR6.

Effects of puerarin on cholinergic enzymes in the brain of ovariectomized guinea pigs

Estrogen has beneficial effects on neurodegenerative disorders and cognitive function of postmenopausal women. Puerarin, isolated from Pueraria lobota, has been classified as a phytoestrogen, which can be highly effective against cerebrovascular diseases. In this study, the effects of puerarin on neural cholinergic system in the brain of ovariectomized guinea pigs were studied. The puerarin at the doses used (15 mg/kg body weight (bw)/day and 30 mg/kg bw/day) for 10 days had the estrogenic activity indicated by the attenuation of the reduction of uterine weight induced by ovariectomy. In brain, puerarin treatment increased choline acetyltransferase (ChAT) activity and expression in hippocampus, and increased ChAT immnuopositive signals in septal diagonal region. Puerarin treatment could suppress the increase of acetylcholinesterase expression and activity to the levels of the intact group, although they were not significantly different from those of the ovariectomized animals. Moreover, puerarin decreased the β-amyloid immunopositive staining in hippocampus. In brief, the present study suggests that puerarin prevents the dysfunction of the neuronal cholinergic system and ameliorates the increase of β-amyloid caused by estrogen deficiency.