The endogenous α7 nicotinic acetylcholine receptor antagonist kynurenic acid modulates amyloid-β-induced inflammation in BV-2 microglial cells

https://doi.org/10.1016/j.jns.2014.06.032Get rights and content

Highlights

  • Kynurenic acid and amyloid-β do not change α7nAchR quantity on BV-2 microglial cells

  • Kynurenic acid reduces amyloid-β induced pro-inflammatory cytokines TNF-α and IL-6.

  • Kynurenic acid decreases amyloid-β phagocytosis in BV-2 microglial cells

Abstract

Amyloid-β has been shown to interact with the α7 nicotinic acetylcholine receptor on neuronal cells. Not much is known on the effect on microglial cells and whether this effect can be modulated by the endogenous α7 nicotinic acetylcholine receptor antagonist kynurenic acid. Our aim was to investigate the effect of kynurenic acid on amyloid-β-treated BV-2 microglial cells with respect to α7 nicotinic acetylcholine receptor expression, cell viability, cytokine production and phagocytotic abilities. Therefore BV-2 cells were treated with oligomeric or fibrillar forms of amyloid-β1–40 and co-treated with kynurenic acid. α7 nicotinic acetylcholine receptor quantity was investigated using Western blotting. Cell viability was assessed by staining cells with fluorescein diacetate and propidium iodide. Pro-inflammatory cytokines were measured in cell culture supernatants of treated cells with ELISAs; NO with Griess reagents and amyloid-β uptake were investigated with fluorescence-activated cell sorting and verified by Western blotting. Amyloid-β nor kynurenic acid did have an effect on the protein level of the α7 nicotinic acetylcholine receptor. Amyloid-Beta induced cell mortality was unchanged after addition of kynurenic acid. However, kynurenic acid co-treatment reduced the pro-inflammatory cytokines tumour necrosis factor-α and IL-6 and amyloid-β phagocytosis.

We provide evidence for an immunomodulating effect of the endogenous α7 nicotinic acetylcholine receptor antagonist kynurenic acid. Our findings indicate a role for kynurenic acid in amyloid-β associated neuroinflammation in Alzheimer disease.

Introduction

Alzheimer disease (AD) is the most common neurodegenerative disease affecting more than 30 million people in the world. The main pathological hallmarks are the extracellular formation of amyloid-β (Aβ) plaques and the intracellular formation of neurofibrillary tangles, composed of hyperphosphorylated tau-protein [5]. Post-mortem analysis of AD brains also shows the involvement of activated microglial cells [23]. It has already been shown that soluble oligomeric forms of Aβ are able to induce microglial activation [34] and some therapeutic approaches, such as non-steroidal anti-inflammatory drugs, are able to prevent microglial actication in vitro. Cholinergic neurons in the nucleus basalis Meynert seem to be extremely vulnerable to Aβ toxicity and cognitive dysfunctions are especially connected to the loss of these cholinergic neurons [3]. There is evidence that Aβ oligomers interact with the α7 nicotinic acetylcholine receptor (α7nAChR) on neuronal cells in vitro [36]. The interaction of Aβ and the α7nAchR results in elevated intracellular calcium levels in neurons [26] pointing to the role of Aβ as an α7nAChR agonist. Even subtle changes in intracellular calcium levels induce a plethora of signalling events resulting in augmentation of Aβ formation as well as hyperphosphorylation of tau protein [19]. To date the role of the interaction of Aβ and α7nAChR on microglial cells is not clear. Expression of the α7nAChR on primary mouse microglia and BV-2 microglial cells has been reported as well as the involvement of this receptor in the modulation of microglial activation [22], [29]. Considerable empirical evidence suggests a neuroprotective role of nicotine in an endogenous anti-inflammatory pathway, in which activation of monocytes is regulated by α7nAChR [35]. However, it is not clear whether this effect is due to activation or inhibition of the α7nAChR. It is assumed that the anti-inflammatory effect of nicotine is not due to its agonistic activity on the α7nAchR, because very high concentrations of nicotine that even desensitize the receptor are needed to obtain an anti-inflammatory effect [6]. Astrocytes are believed, among others, to control microglial activation and are able to synthesize ACh as well as the endogenous potent α7nAChR antagonist kynurenic acid (KYNA); thus, microglial activation might be regulated via cholinergic signalling [17], [37]. KYNA and quinolinic acid (QA) are by-products of tryptophan degradation. QA exerts toxic effects on neurons [33] and KYNA is able to reduce this toxic effects, most likely due to its antagonistic activity on α7nAchR [12]. In states of inflammation enhanced concentration of indoleamin 2,3-dioxygenase can be detected leading to an increase in QA [27]. In addition, it has been shown that Aβ-activated human microglial cells synthesize larger amounts of quinolinic acid [11], pointing to a possible regulatory role of KYNA in Aβ-induced neuroinflammation.

Interestingly in human serum and cerebrospinal fluid of AD patients lower concentrations of KYNA compared to healthy controls could be detected, whereas the level of KYNA is enhanced in the putamen and caudate nucleus of AD patients [2], [12], [13]. In conclusion it has to be stated that the role of KNYA in AD is unclear so far. Thus, it is of considerable interest to investigate the role of the endogenous α7nAchR antagonist KYNA on Aβ-induced microglial neuroinflammation.

Section snippets

Material and methods

All chemicals were obtained from Sigma-Aldrich, St. Louis, MO, USA, unless indicated otherwise.

Aβ and KYNA do not influence the quantity of α7nAchR in BV-2 cell lysates

To investigate the quantity of α7nAchR on BV-2 cells, BV-2 cell lysates were subjected to Western blotting and α7nAchR was detected with a monoclonal antibody (sc-5544). In addition, cells were treated with 5 μM Aβ oligomers and/or 1 μM, 10 μM and 100 μM KYNA for 24 h to investigate the influence of those on the α7nAchR expression, because an influence on α7nAchR expression on neuronal cells has been previously shown in AD brains [8]. However no difference in α7nAchR expression was observed in BV-2

Discussion

There is considerable evidence that microglial cells exert an influence on the development and progression of AD. Aβ is able to induce neuroinflammation [15] and also reduces the viability of neurons [1]. KYNA has been assigned a neuroprotective role in neurodegeneration as well as in ischemia [17], [38] and it has been shown that KYNA binds to the α7nAchR and promotes an antagonistic effect [17]. As a member of the cholinergic anti-inflammatory pathway the α7nAchR plays a pivotal role in the

Conflict of interest

The authors declare that there are no conflicts of interest.

Acknowledgements

We would like to thank Christine Forbach for excellent technical assistance.

References (38)

  • K. Takata et al.

    Galantamine-induced amyloid-{beta} clearance mediated via stimulation of microglial nicotinic acetylcholine receptors

    J Biol Chem

    (Dec 17 2010)
  • S. Neumann et al.

    The non-neuronal cholinergic system in peripheral blood cells: effects of nicotinic and muscarinic receptor antagonists on phagocytosis, respiratory burst and migration

    Life Sci

    (May 30 2007)
  • H. Braak et al.

    Alzheimer's disease: striatal amyloid deposits and neurofibrillary changes

    J Neuropathol Exp Neurol

    (May 1990)
  • K. Morimoto et al.

    Expression profiles of cytokines in the brains of Alzheimer's disease (AD) patients compared to the brains of non-demented patients with and without increasing AD pathology

    J Alzheimers Dis

    (2011)
  • S. Walter et al.

    Role of the toll-like receptor 4 in neuroinflammation in Alzheimer's disease

    Cell Physiol Biochem

    (2007)
  • R.T. Bartus et al.

    The cholinergic hypothesis of geriatric memory dysfunction

    Science

    (Jul 30 1982)
  • H.Y. Wang et al.

    Amyloid peptide Abeta(1-42) binds selectively and with picomolar affinity to alpha7 nicotinic acetylcholine receptors

    J Neurochem

    (Sep 2000)
  • S. Oddo et al.

    Chronic nicotine administration exacerbates tau pathology in a transgenic model of Alzheimer's disease

    Proc Natl Acad Sci U S A

    (Feb 22 2005)
  • F.M. LaFerla

    Calcium dyshomeostasis and intracellular signalling in Alzheimer's disease

    Nat Rev Neurosci

    (Nov 2002)
  • Cited by (21)

    • N-acetylcholine receptors regulate cytokines expression and neutrophils recruitment via MAPK/ERK signaling in zebrafish

      2022, Developmental and Comparative Immunology
      Citation Excerpt :

      Recent studies have shown that α7 nAChR was an essential regulator and pharmacological target of central inflammation responses (de Jonge and Ulloa, 2007; Wang et al., 2003). The α7 nAChR was expressed in hippocampal astrocytes and activation of this receptor inhibited amyloid-induced inflammation in astrocytes (Wang et al., 2012); the endogenous α7 nAChR antagonist, kynurenic acid, modulated β-amyloid induced immune responses in BV-2 microglial cells (Steiner et al., 2014). Although these few studies have revealed the functions of nAChRs in immune responses in the central neural system, the roles of nAChRs in the dynamic behaviors of immune cells such as neutrophils in the peripheral system were barely reported and the potential regulatory mechanism has not yet been elucidated.

    • Stimulation of toll-like receptor 4 downregulates the expression of α7 nicotinic acetylcholine receptors via histone deacetylase in rodent microglia

      2020, Neurochemistry International
      Citation Excerpt :

      Until now, there are no antibodies which are clearly validated specificity against α7 nAChR (Garg and Loring, 2017). As an alternate indirect indicator of changes in α7 nAChR expression, changes in α7 nAChR function following treatment with either nicotine or specific agonists have been reported (Ke et al., 2017; Steiner et al., 2014; Zhang et al., 2017b). Until direct assessment methods are available, alternate indicators of change of α7 nAChR expression, such as changes in α7 nAChR function, are needed to elaborate the role of microglial α7 nAChR in the inflammatory state.

    View all citing articles on Scopus
    1

    Equal contributors.

    View full text