Curcumin enhances neuronal survival in N-methyl-d-aspartic acid toxicity by inducing RANTES expression in astrocytes via PI-3K and MAPK signaling pathways

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Abstract

Object

Neuroinflammation, which is characterized by the overproduction of cytokines and chemokines, plays an important role in neurodegenerative diseases, especially in Alzheimer's disease (AD). In the brain, chemokines are predominantly released by astrocytes and microglias. Expression of RANTES, as well as other cytokines, is involved in the inflammatory cascade that contributes to neurodegeneration in AD. Expression of RANTES may also have a neuroprotective effect. We sought to investigate whether curcumin exhibited neuroprotective and antioxidant activity via enhanced RANTES expression by astrocytes in cortical neuron cultures. We evaluated the neuroprotective and anti-neurodegenerative effects of curcumin in NMDA toxicity and in long-term cultures.

Methods

Pregnant female Sprague–Dawley (SD) rats were used for primary culture of cortical neurons, and neonatal 0- to 2-day-old SD rats were used for primary culture of astrocytes. Cultured astrocytes were conditioned with curcumin to prepare astrocyte-conditioned medium (ACM). Real-time polymerase chain reaction was performed to assess RANTES and iNOS mRNA expression in astrocytes following curcumin treatment. ELISA was used to detect astrocyte-secreted RANTES protein in ACM with curcumin treatment. JAK/STAT, PI-3K, PKC and MAPK inhibitors were used to ascertain whether the effects of curcumin involved these signaling pathways. To evaluate the effects of curcumin-enhanced astrocytes on neuronal survival, cultured cortical neurons treated or untreated with NMDA were incubated in ACM with or without curcumin treatment. Long-term culture (15 days in vitro, DIV) was performed to investigate the effects of curcumin-treated astrocytes on the survival of cultured cortical neurons. Neuronal survival rate was assessed by using 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) reduction activity assay (for cell viability), and the lactate dehydrogenase (LDH) release assay (for cell death).

Results

We demonstrated that curcumin enhanced RANTES expression in primary cultured astrocytes, and that this effect was related to activation of PI-3K and MAPK signaling pathways. We found that curcumin inhibited iNOS expression in primary cultured astrocytes in non-stressed condition. We also found that neurons exposed to NMDA and cultured with curcumin treated ACM, which characteristically exhibited elevated RANTES expression showed higher level of cell viability and lower level of cell death. Using a small interfering RNA (siRNA) knockdown model, we found evidence that the basal level of RANTES expression in non-stimulated astrocytes provided neuroprotection.

Conclusion

We postulate that the enhanced neuronal survival by curcumin treatment in NMDA toxicity and long-term cultures was in part attributable to elevated astrocyte-derived RANTES expression via activation of PI3K/MAPK signaling pathways.

Research Highlights

► Curcumin enhances RANTES expression in primary astrocytes. ► Activation of PI-3K and MAPK augments RANTES expression in astrocytes. ► Curcumin inhibits iNOS expression in primary astrocytes. ► Curcumin-treated ACM protects neurons from NMDA toxicity.

Introduction

Neuroinflammation plays an important role in the pathogenesis of several neurodegenerative disorders, such as Alzheimer's disease (AD), multiple sclerosis, stroke, and Parkinson's disease (Cartier et al., 2005, Galimberti et al., 2006, Xia and Hyman, 1999). Astrocytes, the major non-neuronal cell type in the central nervous system (CNS), rapidly release chemokines and cytokines in response to inflammatory insults. Chemokines are a family of proinflammatory cytokines that are involved in the regulation of inflammation and the immune system (Mennicken et al., 1999). Chemokines are subdivided into four groups (CXC, CC, CX3C, and XC) based on the number and position of cysteine residues in their structures. The CXC subfamily includes interleukin (IL)-8, stromal cell-derived factor (SDF)-1alpha, and interferon-induced protein (IP)-10. The CC subfamily contains the largest number of chemokines, including regulated on activation normal T expressed and secreted (RANTES), and macrophage inflammatory protein (MIP)-1α. Chemokines interact with guanine-protein-coupled receptors (GPCRs) named CXCR, CCR, or CX3R.

There is increasing evidence that interactions between astrocytes and neurons play greatly important roles in brain function. Many pathological conditions of the CNS are accompanied by astrogliosis. The mechanisms that lead to astrogliosis remain unclear (Röhl et al., 2007). It has been postulated that activated microglia play a role in astrogliosis. It has been found that activated microglia increase the number of astrocytes but do not have a hypertrophic effect on astrocytes (Röhl et al., 2007).The interaction between astrocytes and neurons appears to be important for regulation of brain energy metabolism (Escartin et al., 2006). There is evidence from in vitro and in vivo studies in rodents that glutamate and Na+ uptake in astrocytes provides an important signal for glucose regulation in the brain (Escartin et al., 2006). The disturbance of the interactions between astrocytes and neurons is related to numerous neurologic disorders, including cerebral ischemia, neurodegeneration, cerebral edema, and hepatic encephalopathy (Lin et al., 2009). The role of astrocytes in protecting neurons has been an area of significant research interest and has emerged as a potential therapeutic target in the treatment of neurodegenerative diseases.

Numerous studies have found that the expression of RANTES and MIP-1α, among other chemokines and their GPCRs, are involved in neurodegenerative diseases, especially AD (Heneka and O'Banion, 2007, Huang et al., 2000, McGeer et al., 2006, Mennicken et al., 1999). However, the effects of such chemokines are diverse, and both neurotoxic and neuroprotective effects have been described. An increase in MIP-1α expression from Parkinson's disease-producing neurotoxin MPTP (1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine), has been reported in vivo (Pattarini et al., 2007). There is also a correlation between MIP-1α level and cognitive dysfunction in patients with Down's syndrome and Alzheimer-like dementia (Carta et al., 2002). Both RANTES and MIP-1α have also been shown to be produced by astrocytes in vivo after challenge with amyloid-beta peptide, which mimics the neuroinflammation and cell death observed in AD (Smits et al., 2002). In contrast, a study using cDNA microarrays found that a large number of RANTES-responsive genes in cultured neurons appeared to be involved in neuronal survival and differentiation (Tripathy et al., 2010). More recently, Tripathy et al. (2010) demonstrated that treatment with RANTES in primary cultures of cortical neurons enhanced neuronal survival, and that pre-treatment with RANTES resulted in neuroprotection against toxicity of thrombin and sodium nitroprusside. Accordingly, any novel treatment that modulates the multifunctional mediator, RANTES, in augmenting neuroprotection and diminishing neuroinflammation would be a valuable therapeutic advancement in neurodegenerative disorder therapies.

Originally identified as a T-cell specific gene (Schall et al., 1988), RANTES has been shown to be involved in the ontogenetic development of the brain. RANTES is expressed by 5-week old human brains, and induces proliferation of astrocyte cultures (Bakhiet et al., 2001). RANTES has also been shown to protect mixed cortical cultures from human immunodeficiency virus (HIV)-tat or N-methyl-d-aspartic acid (NMDA)-induced apoptosis (Eugenin et al., 2003). Pretreatment with RANTES in the murine hippocampal cell line HT22 enhances HT22 cell viability in the presence of amyloid-beta peptide, via its GPCR, GPR75, which mediates the activation of the antiapoptotic mitogen activated protein kinase (MAPK) via the phospholipase-C (PLC)/phosphatidylinositol 3-kinase (PI-3K)/Akt signaling pathway (Ignatov et al., 2006). This enhanced survival of HT22 cells is explained by the activation of Akt and MAPK, which is known to promote cell proliferation, differentiation, growth, and survival (Li et al., 2003).

In AD, a complex array of mediators in the inflammatory cascade contributes to neurodegeneration and accumulation of beta-amyloid peptide. These include RANTES, MIP-1α, IL-8, reactive oxygen species (ROS), and inducible nitric-oxide synthase (iNOS)-mediated production of nitric oxide species (NOS) (Akiyama et al., 2000). Excitotoxic stimulation of the NMDA receptor by glutamate is implicated in neurodegenerative diseases including AD (Reisberg et al., 2003).

The curry spice, curcumin, from a plant (Curcuma longa) from the Ginger family, has been proposed as a potential therapeutic for neurodegenerative diseases such as AD, for its anti-inflammatory, antioxidant, and immunomodulatory activities (Menon and Sudheer, 2007). The effects of curcumin have been demonstrated to result from its modulation of important molecular targets that include transcription factors, enzymes, cell cycle proteins, receptors, and cell surface adhesion molecules (Shishodia et al., 2005). Curcumin inhibits aggregation of beta-amyloid and promotes its disaggregation (Garcia-Alloza et al., 2007), and antagonizes several mediators of the inflammatory cascade, including activation of nuclear factor-kB, and iNOS (Weber et al., 2006). Curcumin is also a superior scavenger of nitric oxide (NO) compared to vitamin E (Chan et al., 1998). Many of curcumin's protective effects are attainable at a dose of 1 μM or less (Cole et al., 2007). It has been recently demonstrated that curcumin exhibited neuroprotective effects via activation of brain-derived neutrophic factor/TrkB-dependent MAPK and PI-3K cascades in rodent cortical neurons (Wang et al., 2010). In the present study, we sought to investigate whether curcumin can induce RANTES expression and secretion in primary cultured astrocytes without stress, and if so, whether the PI-3K and MAPK signal pathways are involved, and whether RANTES has a protective effect with regard to NMDA-treated and aged neurons. We hypothesized that curcumin would upregulate RANTES expression via PI-3K and MAPK signaling pathways, and that this would contribute to enhanced neuronal survival in NMDA toxicity, representing neurodegenerative diseases, and long-term cultures, representing aging.

Section snippets

Chemicals and reagents

NMDA, curcumin, U0126 and MTT were purchased from Sigma-Aldrich (St. Louis, MO). AG490 and RO-318220 were obtained from Calbiochem (San Diego, CA). LY294002 was purchased from Calbiochem (Cambridge, MA). All common chemicals were from Sigma (St Louis, MO, USA) unless otherwise indicated.

Animals

Pregnant female Sprague–Dawley (SD) rats and neonatal 1- to 2-day-old SD rats, obtained from the National Institute of Experimental Animal Research, Taipei, Taiwan, were used in this study for the primary

Curcumin enhances RANTES mRNA expression and protein secretion in non-stimulated cultured astrocytes

To evaluate the effects of curcumin on RANTES expression by astrocytes, we treated primary cultured astrocytes with a low dose of 1 μΜ curcumin. We did not use a high-dose of curcumin because it was likely that a high concentration would have been excitotoxic to astrocytes. Real-time PCR analysis showed that treatment with curcumin in primary cultured astrocytes resulted in a 14-fold increase in RANTES mRNA expression (Fig. 1) when compared to control (P < 0.001). ACM from cultured astrocytes

Discussion

We found that in non-stressed conditions, curcumin enhanced RANTES mRNA expression in non-stimulated cultured astrocytes, and that this effect was blocked by PI-3K and MAPK inhibitors. RANTES mRNA expression by non-stimulated astrocytes was significantly elevated at doses greater than 1 μΜ, but decreased after treatment with 5 μΜ curcumin, presumably because of excitotoxic activity of curcumin at high doses. We next investigated whether curcumin would enhance neuronal survival in NMDA toxicity

Disclosure

  • Muh-Shi Lin: Conception and design; acquisition of data; analysis and interpretation of data; and drafting the article

  • Kuo-Sheng Hung: Conception and design; critically revising the article; approve it for submission; and study supervision

  • Wen-Ta Chiu: Conception and design; critically revising the article; reviewed final version of manuscript and approved it for submission; and study supervision

  • Yu-Yo Sun: Acquisition of data and statistical analysis

  • Shin-Han Tsai:

Acknowledgments

This work serves as the doctoral thesis of MS Lin. The authors acknowledge support for this work by Taipei Medical University-Wan Fang Hospital (grant no. 98-wf-phd-02 and 99-wf-phd-02) and DOH99-TD-B-111-003, Center of Excellence for Clinical Trial and Research in Neuroscience, NHRI-EX99-9940NI from National Health Research Institute, Aim for the Top University Plan 99ACB11-2 from Ministry of Education, NSC 99-2323-B-038 -003 and NSC 99-2323-B-038 -003 from National Science Council, Taiwan.

References (41)

  • H.A. Smits et al.

    Amyloid-beta-induced chemokine production in primary human macrophages and astrocytes

    J Neuroimmunol

    (2002)
  • D. Tripathy et al.

    RANTES upregulation in the Alzheimer's disease brain: a possible neuroprotective role

    Neurobiol Aging

    (2010)
  • R. Wang et al.

    Curcumin produces neuroprotective effects via activating brain-derived neurotrophic factor/TrkB-dependent MAPK and PI-3K cascades in rodent cortical neurons

    Prog Neuropsychopharmacol Biol Psychiatry

    (2010)
  • W.M. Weber et al.

    TPA-induced up-regulation of activator protein-1 can be inhibited or enhanced by analogs of the natural product curcumin

    Biochem Pharmacol

    (2006)
  • Z. Yavin et al.

    Survival and maturation of cerebral neurons on poly(l-lysine) surfaces in the absence of serum

    Dev Biol

    (1980)
  • Y. Zhang et al.

    RANTES-mediated chemokine transcription in astrocytes involves activation and translocation of p90 ribosomal S6 protein kinase (RSK)

    J Biol Chem

    (2002)
  • M. Bakhiet et al.

    RANTES promotes growth and survival of human first-trimester forebrain astrocytes

    Nat Cell Biol

    (2001)
  • N.B. Butchi et al.

    Analysis of the neuroinflammatory response to TLR7 stimulation in the brain: comparison of multiple TLR7 and/or TLR8 agonists

    J Immunol

    (2008)
  • M.G. Carta et al.

    Chemokines and pro-inflammatory cytokines in Down's syndrome: an early marker for Alzheimer-type dementia?

    Psychother Psychosom

    (2002)
  • S.Y. Chou et al.

    Expanded-polyglutamine huntingtin protein suppresses the secretion and production of a chemokine (CCL5/RANTES) by astrocytes

    J Neurosci

    (2008)
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