Inactivation of glycogen synthase kinase-3beta protects against kainic acid-induced neurotoxicity in vivo

Many neurodegenerative diseases involve oxidative stress and excitotoxic cell death. In an attempt to further elucidate the signal transduction pathways involved in the cell death/cell survival associated with excitotoxicity, we have used an in vivo model of excitotoxicity employing kainic acid (KA)-induced neurotoxicity. Here, we show that extracellular signal-related kinase (ERK) 2, but not ERK 1, is phosphorylated and thereby activated in the hippocampus and cerebellum of kainic acid-treated mice. Phosphorylation and hence inactivation of glycogen synthase kinase 3β (GSK-3β), a general survival factor, is often a downstream consequence of mitogen-activated protein kinase pathway activation. Indeed, GSK-3β phosphorylation occurred in response to kainic acid exclusively in the affected hippocampus, but not as a consequence of ERK activation. This may represent a compensatory attempt at self-protection by the cells in this particular brain region. A role for GSK-3β inhibition in cell survival was further supported by the fact that pharmacological inhibition of GSK-3β using lithium chloride was protective against kainic acid-induced excitotoxicity in hippocampal slice cultures. This work supports a role for GSK-3β in cell death in response to excitotoxins in vivo and further confirms that GSK-3β plays a role in cell death/cell survival pathways.     

Activation of Akt signaling in rat brain by intracerebroventricular injection of ouabain: A rat model for mania

Intracerebroventricular (ICV) injection of ouabain, a specific Na–K ATPase inhibitor, induces behavioral changes in rats resembling the manic phenotypes of bipolar disorder. The binding of ouabain to the Na–K ATPase affects signal events in vitro including Akt, a possible molecular target of mood disorders. However, the effects of ouabain on Akt in the brain need further clarification. In this study, we investigated changes in the phosphorylation state of Akt in the rat brain after ICV injection of ouabain. Consistent with our previous report, the locomotor activity of rats within 30 min after ouabain ICV injection changed according to the dose with higher doses of ouabain, 0.5 and 1 mM, inducing significant hyperactivity. In addition, ouabain administration induced a dose-dependent increase in the immunoreactivity of p-Akt (Ser473) in the frontal cortex, striatum, and hippocampus after 30 min, and reached statistical significance with 1 mM of ouabain. Phosphorylation of GSK-3β (Ser9), FOXO1 (Ser256), and eNOS (Ser1177), which are downstream molecules of Akt, was also increased in a dose-dependent manner within the same brain regions. Moreover, hyperactivity was seen for 8 h after a single 1 mM injection of ouabain and increased phosphorylation of Akt (Ser473), GSK-3β (Ser9), FOXO1 (Ser256), and eNOS (Ser1177) was also observed in the cortex, striatum, and hippocampus. Thus, intrabrain injection of ouabain induces activation of Akt signaling accompanied by hyperactivity, suggesting the possible role of Akt in ouabain rat model of mania.     

α-Lipoic Acid Interaction with Dopamine D2 Receptor-Dependent Activation of the Akt/GSK-3β Signaling Pathway Induced by Antipsychotics: Potential Relevance for the Treatment of Schizophrenia

Chronic administration of antipsychotics has been associated with dopamine D2 receptor (D2R) upregulation and tardive dyskinesia. We have previously shown that haloperidol, a first-generation antipsychotic (FGA), exerted an increase in D2R expression and oxidative stress and that (±)-α-lipoic acid reversed its effect. Previous studies have implicated the Akt/glycogen synthase kinase-3β (GSK-3β) signaling pathway in antipsychotic action. These findings led us to examine whether the Akt/GSK-3β pathway was involved in D2R upregulation and oxidative stress elicited by antipsychotics and, in (±)-α-lipoic acid-induced reversal of these phenomena, in SH-SY5Y cells. Antipsychotics increased phosphorylation of Akt and GSK-3β, and additive effects were observed with (±)-α-lipoic acid. GSK-3β inhibitors reversed haloperidol-induced overexpression of D2R mRNA levels but did not affect haloperidol-induced oxidative stress. Sustained antipsychotic treatment increased β-arrestin-2 and D2R receptor interaction. Regarding Akt/GSK-3β downstream targets, antipsychotics increased β-catenin levels, whereas (±)-α-lipoic acid induced an elevation of mTOR activation. These results suggest (1) that the effect of antipsychotics on the Akt/GSK-3β pathway in SH-SY5Y cells is reminiscent of their in vivo action, (2) that (±)-α-lipoic acid partially synergizes with antipsychotic drugs (APDs) on the same pathway, and (3) that the Akt/GSK-3β signaling cascade is not involved in the preventive effect of (±)-α-lipoic acid on antipsychotics-induced D2R upregulation.     

Selectively silencing GSK-3 isoforms reduces plaques and tangles in mouse models of Alzheimer's disease

Glycogen synthase kinase-3 (GSK-3) is linked to the pathogenesis of Alzheimer's disease (AD), senile plaques (SPs), and neurofibrillary tangles (NFTs), but the specific contributions of each of the GSK-3 α and β isoforms to mechanisms of AD have not been clarified. In this study, we sought to elucidate the role of each GSK-3α and GSK-3β using novel viral and genetic approaches. First, we developed recombinant adeno-associated virus 2/1 short hairpin RNA constructs which specifically reduced expression and activity of GSK-3α or GSK-3β. These constructs were injected intraventricularly in newborn AD transgenic (tg) mouse models of SPs (PDAPP?/?), both SPs and NFTs (PDAPP?/?;PS19?/?), or wild-type controls. We found that knockdown (KD) of GSK-3α, but not GSK-3β, reduced SP formation in PDAPP?/? and PS19?/?;PDAPP?/? tg mice. Moreover, both GSK-3α and GSK-3β KD reduced tau phosphorylation and tau misfolding in PS19?/?;PDAPP?/? mice. Next, we generated triple tg mice using the CaMKIIα-Cre (α-calcium/calmodulin-dependent protein kinase II-Cre) system to KD GSK-3α in PDAPP?/? mice for further study of the effects of GSK-3α reduction on SP formation. GSK-3α KD showed a significant effect on reducing SPs and ameliorating memory deficits in PDAPP?/? mice. Together, the data from both approaches suggest that GSK-3α contributes to both SP and NFT pathogenesis while GSK-3β only modulates NFT formation, suggesting common but also different targets for both isoforms. These findings highlight the potential importance of GSK-3α as a possible therapeutic target for ameliorating behavioral impairments linked to AD SPs and NFTs.     

Oestrogen receptors and signalling pathways: implications for neuroprotective effects of sex steroids in Parkinson's disease

Parkinson's disease (PD) is an age-related neurodegenerative disorder with a higher incidence in the male population. In the 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP) mouse model of PD, 17β-oestradiol but not androgens were shown to protect dopamine (DA) neurones. We report that oestrogen receptors (ER)α and β distinctly contribute to neuroprotection against MPTP toxicity, as revealed by examining the membrane DA transporter (DAT), the vesicular monoamine transporter 2 (VMAT2) and tyrosine hyroxylase in ER wild-type (WT) and knockout (ERKO) C57Bl/6 male mice. Intact ERKOβ mice had lower levels of striatal DAT and VMAT2, whereas ERKOα mice were the most sensitive to MPTP toxicity compared to WT and ERKOβ mice and had the highest levels of plasma androgens. In both ERKO mice groups, treatment with 17β-oestradiol did not provide neuroprotection against MPTP, despite elevated plasma 17β-oestradiol levels. Next, the recently described membrane G protein-coupled oestrogen receptor (GPER1) was examined in female Macaca fascicularis monkeys and mice. GPER1 levels were increased in the caudate nucleus and the putamen of MPTP-monkeys and in the male mouse striatum lesioned with methamphetamine or MPTP. Moreover, neuroprotective mechanisms in response to oestrogens transmit via Akt/glycogen synthase kinase-3 (GSK3) signalling. The intact and lesioned striata of 17β-oestradiol treated monkeys, similar to that of mice, had increased levels of pAkt (Ser 473)/βIII-tubulin, pGSK3 (Ser 9)/βIII-tubulin and Akt/βIII-tubulin. Hence, ERα, ERβ and GPER1 activation by oestrogens is imperative in the modulation of ER signalling and serves as a basis for evaluating nigrostriatal neuroprotection.     

Parkinson's Disease-Associated Dj-1 Mutations Increase Abnormal Phosphorylation of Tau Protein through Akt/Gsk-3β Pathways

Hyperphosphorylated tau protein is the main component of neurofibrillary tangles found in Alzheimer's disease and Parkinson's disease (PD). Mutations in DJ-1 have been identified as the causative gene for Parkinson's disease 7 (PARK7)-linked PD. DJ-1_L166P and DJ-1_D149A, two types of DJ-1 mutations, are most commonly studied as the loss-of-function mutations responsible for early-onset familial PD. Whether mutations in DJ-1 result in tauopathy is as yet unknown. In this study, we found that the L166P and D149A mutant isoforms of DJ-1 associated with familial PD cause tau phosphorylation at Ser202, Ser262, and PHF1 (396/404) sites in neuroblastoma 2a cells. Glycogen synthase kinase (GSK)-3β phosphorylation at serine 9 (Ser9) decreases around 50 % in DJ-1_L166P- or DJ-1_D149A-transfected cells, while there is no change in total levels of GSK-3β. Our results also indicate that overexpression of DJ-1_L166P or DJ-1_D149A leads to a significant decrease in the level of phosphorylation of Akt at Thr308, which plays a critical role in phosphorylating GSK-3β at Ser9 and inhibiting its kinase activity. Importantly, insulin, the activator for Akt, effectively attenuates the reduced phosphorylation level of GSK-3β at Ser9 induced by DJ-1_L166P. Neither the expression of cyclin-dependent kinase 5 nor the level of PP2A activity was found to have changed, suggesting that the familial PD-associated DJ-1_L166P and DJ-1_D149A mutations increase tau phosphorylation by increasing the activity of GSK-3β. Finally, we found that administration of lithium chloride, a well-known GSK-3β inhibitor, resulted in decreased levels of phosphorylated tau in DJ-1_L166P-transfected cells.     

Ketogenic diet protects the hippocampus from kainic acid toxicity by inhibiting the dissociation of bad from 14-3-3

The ketogenic diet (KD) is often effective for intractable epilepsy, but its antiepileptic mechanisms remain largely unknown. Within the cell death/survival pathway, Akt and its downstream protein Bad play an important role in kainic acid (KA)-induced cell death. Therefore, we investigated the effects of a KD on KA-induced changes in the Akt/Bad/14-3-3 signaling pathway by evaluating Akt, Bad, 14-3-3, and cleaved caspase-3 expression levels as well as their relative interactions. Our results showed that a KD did not affect the expression levels of Akt, Bad, Bcl-xL, Bax, and 14-3-3 but increased phospho-Akt [serine 473; p-Akt (Ser473)] and phospho-Bad [serine 136; p-Bad (Ser136)] expression levels as well as decreased cleaved caspase-3 levels following a KA-induced seizure in the hippocampus. Furthermore, we found that a KD increased the protein-protein interaction between 14-3-3 and p-Bad (Ser136), which might be phosphorylated by p-Akt (Ser473), and decreased interaction of Bad and Bcl-xL. These results suggest that a KD might protect, at least partially, the hippocampus from KA-induced cell death via inhibiting the dissociation of Bad from 14-3-3.     

Evidence that AKT and GSK-3β pathway are involved in acute hyperhomocysteinemia

Homocysteine is a neurotoxic amino acid that accumulates in several disorders including homocystinuria, neurodegenerative and neuroinflammatory diseases. In the present study we evaluated the effect of acute and chronic hyperhomocysteinemia on Akt, NF-κB/p65, GSK-3β, as well as Tau protein in hippocampus of rats. For acute treatment, rats received a single injection of homocysteine (0.6 μmol/g body weight) or saline (control). For chronic treatment, rats received daily subcutaneous injections of homocysteine (0.3-0.6 μmol/g body weight) or saline (control) from the 6th to the 28th days-of-age. One or 12h after the last injection, rats were euthanized, the hippocampus was removed and samples were submitted to electrophoresis followed by Western blotting. Results showed that acute hyperhomocysteinemia increases Akt phosphorylation, cytosolic and nuclear immunocontent of NF-κB/p65 subunit and Tau protein phosphorylation, but reduces GSK-3β phosphorylation at 1h after homocysteine injection. However, 12h after acute hyperhomocysteinemia there is no effect on Akt and GSK-3β phosphorylation. Furthermore, chronic hyperhomocysteinemia did not alter Akt and GSK-3β phosphorylation at 1h and 12h after the last administration of this amino acid. Our data showed that Akt, NF-κB/p65, GSK-3β and Tau protein are activated in hippocampus of rats subjected to acute hyperhomocysteinemia, suggesting that these signaling pathways may be, at least in part, important contributors to the neuroinflammation and/or brain dysfunction observed in some hyperhomocystinuric patients.     

Mice transgenic for the human Huntington's disease mutation have reduced sensitivity to kainic acid toxicity

The mechanism underlying the pathology of Huntington's disease (HD) is unknown, although there is substantial evidence supporting a role for excitotoxicity. The discovery of abnormal aggregations of protein in the brains of patients with HD, as well as in the brains of transgenic mice modeling this disease, has led to the suggestion that these "inclusions" have a pathogenic role. However, the relationship between inclusion formation and the progressive neurodegeneration in HD remains unclear. Here, we used mice transgenic for the first exon of the HD gene and an expanded CAG repeat (R6/2 line) to examine the role of neuronal intranuclear inclusions in kainic acid (KA) excitotoxicity. Unexpectedly, we found that the toxicity of KA was markedly attenuated in R6/2 mice compared with wild-type mice. In particular, the number and severity of KA-induced seizures in R6/2 mice was significantly reduced. When seizures occurred in 3-week-old R6/2 mice, we found lesions in the CA3 region of the hippocampus. However, neuronal intranuclear inclusions were not induced by KA in 3-week-old mice. Further, in older mice (9 weeks), the pre-existence of inclusions in CA3 neurons did not increase the vulnerability of neurons to KA, since no lesions were seen in 9-week R6/2 mouse brain. Our results suggest that an increased susceptibility to excitotoxic stimuli does not underlie the early phase of the neurological phenotype in R6/2 mice, although a role in later stages is not excluded by our findings. The significance of these findings is discussed in the context of the R6/2 mouse as a model for HD.     

Activation of the Akt/GSK3beta signaling pathway mediates survival of vulnerable hippocampal neurons after transient global cerebral ischemia in rats.

Recent studies have revealed that the phosphatidylinositol 3-kinase (PI3-K) pathway is involved in apoptotic cell death after experimental cerebral ischemia. The serine-threonine kinase, Akt, functions in the PI3-K pathway and prevents apoptosis by phosphorylation at Ser473 after a variety of cell death stimuli. After phosphorylation, activated Akt inactivates other apoptogenic factors, including glycogen synthase kinase-3beta (GSK3beta), thereby inhibiting cell death. However, the role of Akt/GSK3beta signaling in the delayed death of hippocampal neurons in the CA1 subregion after transient global cerebral ischemia (tGCI) has not been clarified. Transient global cerebral ischemia for 5 mins was induced by bilateral common carotid artery occlusion combined with hypotension. Western blot analysis showed a significant increase in phospho-Akt (Ser473) and phospho-GSK3beta (Ser9) in the hippocampal CA1 subregion after tGCI. Immunohistochemistry showed that expression of phospho-Akt (Ser473) and phospho-GSK3beta (Ser9) was markedly increased in the vulnerable CA1 subregion, but not in the ischemic-tolerant CA3 subregion. Double staining with phospho-GSK3beta (Ser9) and terminal deoxynucleotidyl transferase-mediated uridine 5'-triphosphate-biotin nick end labeling showed different cellular distributions in the CA1 subregion 3 days after tGCI. Phosphorylation of Akt and GSK3beta was prevented by LY294002, a PI3-K inhibitor, which facilitated subsequent DNA fragmentation 3 days after tGCI. Moreover, transgenic rats that overexpress copper/zinc-superoxide dismutase, which is known to be neuroprotective against delayed hippocampal CA1 injury after tGCI, had enhanced and persistent phosphorylation of both Akt and GSK3beta after tGCI. These findings suggest that activation of the Akt/GSK3beta signaling pathway may mediate survival of vulnerable hippocampal CA1 neurons after tGCI.     

PTEN, Akt, and GSK3beta signalling in rat primary cortical neuronal cultures following tumor necrosis factor-alpha and trans-4-hydroxy-2-nonenal treatments

PTEN is a dual phosphatase that negatively regulates the phosphatidylinositol 3-kinase (PI3K)/Akt signalling pathway important for cell survival. We determined effects of the inflammation and oxidative stresses of tumor necrosis factor-alpha (TNFalpha) and trans-4-hydroxy-2-nonenal (HNE), respectively, on PTEN, Akt, and GSK3beta signalling in rat primary cortical neurons. The inhibitors bisperoxovanadium [bpV(Pic)] and LY294002 were also used to determine PTEN and PI3K involvement in TNFalpha and HNE modulation of neuronal cell death. PTEN inhibition with bpV(Pic) alone did not affect Ser(473)Akt or Ser(9)GSK3beta phosphorylation. Instead, effects of this inhibitor were manifest when it was used together with TNFalpha and to a lesser extent with HNE. TNFalpha together with PTEN inhibition increased phosphorylation of Ser(473)Akt and Ser(9)GSK3beta. TNFalpha and HNE both gave decreased numbers of viable and increased numbers of early apoptotic neurons. PTEN inhibition partially reversed the toxic effect of TNFalpha as shown by an increased number of viable and a decreased number of early apoptotic neurons. All effects were reversed by PI3K inhibition. HNE together with inhibition of PTEN gave increased Ser(473)Akt but not Ser(9)GSK3beta phosphorylation and no effects on the number of viable or early apoptotic cells. In conclusion, PTEN inhibition gives a mild reversal of TNFalpha- but not HNE-induced cell death via the PI3K pathway.     

Striatal Akt/GSK3 signaling pathway in the development of L-Dopa-induced dyskinesias in MPTP monkeys

L-Dopa treatment, the gold standard therapy for Parkinson's disease, is hampered by motor complications such as dyskinesias. Recently, impairment of striatal Akt/GSK3 signaling was proposed to play a role in the mechanisms implicated in development of L-Dopa-induced dyskinesias in a rodent model of Parkinson's disease. The present experiment investigated in 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP) monkeys, the effects on Akt/GSK3 of chronic L-Dopa treatment inducing dyskinesias compared to L-Dopa with CI-1041 (NMDA receptor antagonist) or a low dose of cabergoline (dopamine D2 receptor agonist) preventing dyskinesias. The extensive dopamine denervation induced by MPTP was associated with a decrease by about half of phosphorylated Akt(Ser473) levels in posterior caudate nucleus, anterior and posterior putamen; smaller changes were observed for phosphorylated Akt(Thr308) levels that did not reach statistical significance. Dopamine depletion reduced phosphorylated GSK3β(Ser9) levels, mainly in posterior putamen whereas pGSK3β(Tyr216) and pGSK3α(Ser21) were unchanged. In posterior caudate nucleus, anterior and posterior putamen of dyskinetic L-Dopa-treated MPTP monkeys, pAkt(Ser473) and pGSK3β(Ser9) were elevated whereas L-Dopa+cabergoline treated MPTP monkeys without dyskinesias had lower values in posterior striatum as vehicle-treated MPTP monkeys. In non-dyskinetic MPTP monkeys treated with L-Dopa+CI-1041, putamen pAkt(Ser473) and pGSK3β(Ser9) levels remained elevated as in dyskinetic monkeys while in posterior caudate nucleus, these levels were low as vehicle-treated and lower than L-Dopa treated MPTP monkeys. Extent of phosphorylation of Akt and GSK3β in putamen correlated positively with dyskinesias scores of MPTP monkeys; these correlations were higher with dopaminergic drugs (L-Dopa, cabergoline) suggesting implication of additional mechanisms and/or signaling molecules in the NMDA antagonist antidyskinetic effect. In conclusion, our results showed that in MPTP monkeys, loss of striatal dopamine decreased Akt/GSK3 signaling and that increased phosphorylation of Akt and GSK3β was associated with L-Dopa-induced dyskinesias.     

Activation of glycogen synthase kinase-3 induces Alzheimer-like tau hyperphosphorylation in rat hippocampus slices in culture

Summary. Formation of neurofibrillary tangle from hyperphosphorylated tau is one of the hallmark lesions seen in Alzheimer’s disease (AD) brain, and neuronal deregulation of glycogen synthase kinase-3 (GSK-3) activity plays key role in tau hyperphosphorylation. In the present study, the role of GSK-3 on tau phosphorylation in hippocampus slice culture was examined by incubating the slice with wortmannin (WT), an inhibitor of phosphatidylinositol 3-kinase (PI3K) and GF-109203X (GFX), an inhibitor of protein kinase C (PKC). It was found that treatment of the slices with GFX or WT separately induced tau hyperphosphorylation both at Ser396/Ser404 (PHF-1) and Ser199/Ser202 (Tau-1) sites. The phosphorylation rate of tau at PHF-1 and Tau-1 epitopes was further increased when GFX and WT were used in combination, and at this condition, AD-like tau accumulation was observed. GSK-3 activity was significantly increased with a concurrently decreased level of inactivated form of GSK-3. Lithium chloride (LiCl), a GSK-3 inhibitor, prevented tau from WT- and GFX-induced hyperphosphorylation. It suggests that GSK-3 is regulated through PI3K and PKC pathway, and activation of GSK-3 not only induces hyperphosphorylation of tau but also leads to accumulation of tau in cultured rat brain slice. Authors contributed equally to the paper     

Proteins of the Akt1/GSK-3β signaling pathway in peripheral blood mononuclear cells of patients with affective disorders

The Akt1/GSK-3β signaling pathway is involved in the regulation of biological processes in nerve cells. Here we studied the intracellular proteins Akt1 and GSK-3β and their phosphoforms in patients with affective disorders. Sixty patients with diagnoses of depressive episodes, major depressive disorder, and bipolar affective disorder (BAD) and 34 mentally and somatically healthy subjects were examined. Proteins of the Akt1/GSK-3β signaling pathway (total glycogen synthase kinase 3β (GSK-3β), phospho-serine-9-GSK-3β, total protein kinase Akt1, phospho-serine-473-Akt1) were measured in peripheral blood mononuclear cells using the immunoblotting technique. High levels of total GSK-3β were found in groups of patients with affective disorders. Patients with recurrent depressive disorder had an increased level of total GSK-3β as compared with the level in depressive episode. The level of total Akt1 in patients decreased as compared with the control group. Patients with depressive disorder had decreased levels of phospho-serine-473-Akt1. Our results confirm the hypothesis of the involvement of glycogen synthase kinase-3β in the pathogenesis of affective disorders.     

Leptin receptor deficiency confers resistance to behavioral effects of fluoxetine and desipramine via separable substrates

Depression is a complex, heterogeneous mental disorder. Currently available antidepressants are only effective in about one-third to one-half of all patients. The mechanisms underlying antidepressant response and treatment resistance are poorly understood. Recent clinical evidence implicates the involvement of leptin in treatment response to antidepressants. In this study, we determined the functional role of the leptin receptor (LepRb) in behavioral responses to the selective serotonergic antidepressant fluoxetine and the noradrenergic antidepressant desipramine. While acute and chronic treatment with fluoxetine or desipramine in wild-type mice elicited antidepressant-like effects in the forced swim test, mice null for LepRb (db/db) displayed resistance to treatment with either fluoxetine or desipramine. Fluoxetine stimulated phosphorylation of Akt(Thr308) and GSK-3β(Ser9) in the hippocampus and prefrontal cortex (PFC) of wild-type mice but not in db/db mice. Desipramine failed to induce measurable changes in Akt, GSK-3β or ERK1/2 phosphorylation in the hippocampus and PFC, as well as hypothalamus of either genotype of mice. Deletion of LepRb specifically from hippocampal and cortical neurons resulted in fluoxetine insensitivity in the forced swim test and tail suspension test while leaving the response to desipramine intact. These results suggest that functional LepRb is critically involved in regulating the antidepressant-like behavioral effects of both fluoxetine and desipramine. The antidepressant effects of fluoxetine but not desipramine are dependent on the presence of functional LepRb in the hippocampus and cortex.     

PI3 K/Akt信号在阿尔茨海默病样细胞中的作用及依达拉奉对其影响

目的探讨PI3K/Akt信号在β样淀粉蛋白(Aβ1-40)引起的PC12细胞凋亡中的作用及依达拉奉(MCI-186)对其影响。方法采用流式细胞学检测细胞凋亡,Western blot法检测磷酸化Akt及总Akt水平,观察MCI-186对其保护作用。结果模型组中各时间点Akt Ser473的磷酸化水平与对照组比较均降低而保护组各时间点Akt Ser473的磷酸化水平均有明显的升高(P<0.05),保护组中细胞凋亡率较模型组显著降低(P<0.01)。结论Aβ1-40主要通过抑制磷酸化Akt水平,进而诱导PC12细胞凋亡。MC-186通过激活PI3K/Akt信号传导途径,发挥拮抗细胞凋亡的作用,最终达到保护神经细胞的目的。     

Inhibition of glycogen synthase kinase-3β by Angelica sinensis extract decreases β-amyloid-induced neurotoxicity and tau phosphorylation in cultured cortical neurons

Increasing evidence has shown that β-amyloid (Aβ) induces hyperphosphorylation of tau and contributes to Aβ toxicity. Recently, tau hyperphosphorylation by glycogen synthase kinase-3β (GSK-3β) activation has been emphasized as one of the pathogenic mechanisms of Alzheimer's disease (AD). The phosphoinositide 3 kinase (PI3K)/Akt pathway is known as an upstream element of GSK-3β. The inhibitory control of GSK-3β, via the PI3K/Akt pathway, is an important mechanism of cell survival. In the present study, we investigated the neuroprotective effects of Angelica sinensis (AS), a traditional Chinese herbal medicine, against Aβ(1-42) toxicity in cultured cortical neurons and also the potential involvement of PI3K/Akt/GSK-3β signal pathway. We revealed that AS extract significantly attenuated Aβ(1-42) -induced neurotoxicity and tau hyperphosphorylation at multiple AD-related sites in a dose-dependent manner. Simultaneously, it increased the levels of phospho-Ser(473) -Akt and down-regulated GSK-3β activity by PI3K activation. The neuroprotective effects of AS extract against Aβ(1-42) -induced neurotoxicity and tau hyperphosphorylation were blocked by LY294002 (10 μM), a PI3K inhibitor. In addition, AS extract reversed the Aβ(1-42) -induced decrease in phosphorylation cyclic AMP response element binding protein (CREB), which could be blocked by the PI3K inhibitor. These results suggest that AS-mediated neuroprotection against Aβ toxicity is likely mediated by the PI3K/Akt/GSK-3β signal pathway.     

Implication of the ERK/MAPK Pathway in Antipsychotics-induced Dopamine D2 Receptor Upregulation and in the Preventive Effects of (±)-α-lipoic acid in SH-SY5Y Neuroblastoma Cells

Chronic administration of antipsychotics (APs) has been associated with dopamine D2 receptor (D2R) upregulation and tardive dyskinesia. We previously showed that haloperidol, a first-generation AP, exerted a more robust increase in D2R expression than amisulpride, a second-generation AP and that (±)-α-lipoic acid pre-treatment reversed the AP-induced D2R upregulation. We also demonstrated that the Akt/GSK-3β/β-catenin pathway is involved in the control of D2R expression levels, but is unlikely implicated in the preventive effects of (±)-α-lipoic acid since co-treatment with haloperidol and (±)-α-lipoic acid exerts synergistic effects on Akt/GSK-3β activation. These findings led us to examine whether the ERK/MAPK signaling pathway may be involved in D2R upregulation elicited by APs, and in its reversal by (±)-α-lipoic acid, in SH-SY5Y human neuroblastoma cells. Our results revealed that haloperidol, in parallel with an elevation in D2R mRNA levels, induced a larger increase of ERK (p42/p44) phosphorylation than amisulpride. Pre-treatment with the selective ERK inhibitor U0126 attenuated haloperidol-induced increase in D2R upregulation. Furthermore, (±)-α-lipoic acid prevented AP-induced ERK activation. These results show that (1) the ERK/MAPK pathway is involved in haloperidol-induced D2R upregulation; (2) the preventive effect of (±)-α-lipoic acid on haloperidol-induced D2R upregulation is in part mediated by an ERK/MAPK-dependent signaling cascade. Taken together, our data suggest that (±)-α-lipoic acid exerts synergistic effects with haloperidol on the Akt/GSK-3β pathway, potentially involved in the therapeutic effects of APs, and antagonism of ERK activation and D2R upregulation, potentially involved in tardive dyskinesia and treatment resistance.     

Selective regulation of axonal growth from developing hippocampal neurons by tumor necrosis factor superfamily member APRIL

APRIL (A Proliferation-Inducing Ligand, TNFSF13) is a member of the tumor necrosis factor superfamily that regulates lymphocyte survival and activation and has been implicated in tumorigenesis and autoimmune diseases. Here we report the expression and first known activity of APRIL in the nervous system. APRIL and one of its receptors, BCMA (B-Cell Maturation Antigen, TNFRSF17), are expressed by hippocampal pyramidal cells of fetal and postnatal mice. In culture, these neurons secreted APRIL, and function-blocking antibodies to either APRIL or BCMA reduced axonal elongation. Recombinant APRIL enhanced axonal elongation, but did not influence dendrite elongation. The effect of APRIL on axon elongation was inhibited by anti-BCMA and the expression of a signaling-defective BCMA mutant in these neurons, suggesting that the axon growth-promoting effect of APRIL is mediated by BCMA. APRIL promoted phosphorylation and activation of ERK1, ERK2 and Akt and serine phosphorylation and inactivation of GSK-3β in cultured hippocampal pyramidal cells. Inhibition of MEK1/MEK2 (activators of ERK1/ERK2), PI3-kinase (activator of Akt) or Akt inhibited the axon growth-promoting action of APRIL, as did pharmacological activation of GSK-3β and the expression of a constitutively active form of GSK-3β. These findings suggest that APRIL promotes axon elongation by a mechanism that depends both on ERK signaling and PI3-kinase/Akt/GSK-3β signaling.     

Roles of adenosine receptors in the regulation of kainic acid-induced neurotoxic responses in mice

Kainic acid (KA) is a well-known excitatory and neurotoxic substance. In ICR mice, morphological damage of hippocampus induced by KA administered intracerebroventricularly (i.c.v.) was markedly concentrated on the hippocampal CA3 pyramidal neurons. In the present study, the possible role of adenosine receptors in hippocampal cell death induced by KA (0.1 microg) administered i.c.v. was examined. It has been shown that 3,7-dimethyl-1-propargylxanthine (DMPX; A2 adenosine receptors antagonist, 20 microg) reduced KA-induced CA3 pyramidal cell death. KA dramatically increased the phosphorylated extracellular signal-regulated kinase (p-ERK) immunoreactivities (IR) in dentate gyrus (DG) and mossy fibers. In addition, c-Jun, c-Fos, Fos-related antigen 1 (Fra-1) and Fos-related antigen 2 (Fra-2) protein levels were increased in hippocampal area in KA-injected mice. DMPX attenuated KA-induced p-ERK, c-Jun, Fra-1 and Fra-2 IR. However, 1,3-dipropyl-8-(2-amino-4-chlorophenyl)-xanthine (PACPX; A1 adenosine receptor antagonist, 20 microg) did not affect KA-induced p-ERK, c-Jun, Fra-1 and Fra-2 IR. KA also increased the complement receptor type 3 (OX-42) IR in CA3 region of hippocampus. DMPX, but not PACPX, blocked KA-induced OX-42 IR. Our results suggest that p-ERK and c-Jun may function as important regulators responsible for the hippocampal cell death induced by KA administered i.c.v. in mice. Activated microglia, which was detected by OX-42 IR, may be related to phagocytosis of degenerated neuronal elements by KA excitotoxicity. Furthermore, it is implicated that A2, but not A1, adenosine receptors appear to be involved in hippocampal CA3 pyramidal cell death induced by KA administered i.c.v. in mice.