Lithium-induced neuroprotective activity in neuronal and microglial cells: A purinergic perspective
Introduction
Lithium is the mainstay of the pharmacotherapy available for treating bipolar disorder (BD). It has well-established clinical applications, and has been used for over 60 years, mainly due its ability to maintain euthymia, even in those at a high risk for suicide (Geddes and Miklowitz 2013). Some studies have described an action on the stabilization and control of the electrolyte balance in monoaminergic neurons and on GSK3β activity (Bielecka and Obuchowicz 2008, Wang, Zhang et al. 2013); additionally, neurotrophic, anti-inflammatory and antioxidant actions of this molecule are well accepted (Jinhua, Sawmiller et al. 2019). However, despite its wide use in the clinic, its mechanisms of action are still not well defined.
Importantly, consistent findings show a neuroprotective action of lithium (Rowe and Chuang 2004). In vitro studies have demonstrated neuroprotection in several cell lines, including cerebellar granular cells, cerebral cortical cells, hippocampal neurons (Nonaka, Katsube et al. 1998) and neuronal PC12 cells (Bournat, Brown et al. 2000). Additionally, pre-treatment with therapeutic levels of lithium protect neurons from glutamatergic excitotoxicity, mediated by the N-methyl-D-aspartate receptor (NMDA) (Nonaka, Katsube et al. 1998). On the other hand, a post-mortem study showed that BD patients who were not using lithium presented reductions in glial cell counts in amygdala, when compared to those under treatment (Bowley, Drevets et al. 2002). Thus, lithium appears to act on both neuronal and glial cells.
Microglia are immunocompetent cells of the Central Nervous System (CNS) (Pessac, Godin et al. 2001), producing either a neuroprotective or an inflammatory response (Stertz, Magalhães et al. 2013), adopting distinctive phenotypes including the classically activated - M1 state and the alternatively - M2 state in response to stimulation. The M1-like phenotype is characterized by the production of pro-inflammatory cytokines including IL-1β, TNF-α and MCP-1 as well as inducible nitric oxide synthase (iNOS), while the M2 phenotype, which is considered the anti-inflammatory profile is characterized by the increase of IL-10 (Zhou, Huang et al. 2017). Multiple lines of evidence suggest that BD is a multi-systemic inflammatory disease and progressive impairment of cognitive functions and brain atrophy have been consistently described in BD indicating that the disease is progressive with important components of excitotoxicity and neuroinflammation (Rao, Harry et al. 2010, Lewandowski, Cohen et al. 2011).
Previous studies have demonstrated, in hippocampus slices – which have complex cellular composition and cellular interactions - that lithium also has neuroprotective effects against cellular death induced by the extracellular purinergic molecule, adenosine 5’-triphosphate (ATP), which is considered to be a danger signal, or a damage-associated molecular pattern (DAMP) (Wilot, Bernardi et al. 2007). This effect was mediated by the P2 × 7 receptor (P2 × 7R), an ATP–binding ligand-gated ion channel that is activated by high concentrations of extracellular ATP (North 2002). Furthermore, recent evidence has been shown a role for this receptor in the pathophysiology of bipolar disorder in both clinic and pre-clinic stages (Gubert, Rodrigo Fries et al. 2013, Gubert, Fries et al. 2016, Gubert, Andrejew et al. 2019, Gubert et al., 2019 ). Additionally, P2 × 7R has been reported as a crucial factor in microglial activation, inducing the production and release of inflammatory cytokines and leading to an inflammatory response (Suzuki, Hide et al. 2004, Bours, Swennen et al. 2006, Di Virgilio 2007). Speciffically, activation of P2 × 7R by extracellular ATP, promotes the processing and release of the cytokine interleukin-1β (IL-1β), inducing activation of microglia and macrophages associated with cell death (Brough, Le Feuvre et al. 2002).
The concentrations of extracellular nucleotides (e.g. ATP, UTP) and nucleosides (adenosine) are controlled by ectonucleotidases such as members of the ectonucleoside triphosphate diphosphohydrolase (E-NTPDase) family and ecto- 5’-nucleotidase/CD73 respectively (Robson, Sévigny et al. 2006). NTPDase1/CD39 hydrolyzes ATP and ADP with comparable rates producing the rapid formation of AMP as a final product and are expressed by microglia (Braun, Sévigny et al. 2000). NTPDase3 reveal an intermediary rate preference for ATP over ADP and are expressed by neurons (Vorhoff, Zimmermann et al. 2005). AMP is further hydrolyzed to adenosine by CD73 and are expressed by the majority of cells (Burnstock, Krügel et al. 2011). Interestingly, lithium was able to increase ATP and AMP hydrolysis in hippocampal synaptosomes of chronically-treated rats (Wilot, Bernardi et al. 2007), indicating a promising action of this drug on neuronal ectonucleotidases modulation. Therefore, we hypothesise that lithium plays a purinergic role for its neuroprotective effects, specifically by preventing activation via P2 × 7R in microglial cells and by modulating ectonucleotidases in neuronal cells.
Elucidating lithium's mechanism of action may not only shed light on the pathophysiology of BD but may also uncover new targets for the treatment of this devastating disorder. As such, the main goal of this study was to focus on purinergic signaling as a promising new pathway. Since previous studies suggest that the purinergic system is involved in lithium's neuroprotective action, the specific aim of this study is to better understand the neuroprotective action of lithium against ATP-induced cellular death, in both neuronal and microglial cellular lineages, from a purinergic perspective.
Section snippets
Materials
RPMI 1640, Dulbecco Modified Eagle Medium (DMEM), penicillin/streptomycin, trypsin/EDTA solution and fungizone were purchased from Gibco (Gibco®/Invitrogen, São Paulo, Brazil)). Fetal bovine serum (FBS) was purchased from Cultilab (Cultilab, Campinas, Brazil) and horse serum was from LGC standards (United Kingdom, UK). Lithium chloride (LiCl), adenosine 5′-triphosphate (ATP), LPS from Escherichia coli O111:B4 and IL4 were purchased from Sigma Aldrich (Merck Group, St Louis, MO, USA). Nitrite
LiCl can prevent cellular death induced by ATP in neuronal PC12 cells
Fig. 1 depicts the effect of LiCl on PC12 cells exposed to 3mM ATP. LiCl treatment had a protective effect on cell viability (F(3,12)=34.27, p<0.0001); the post hoc test showed that LiCl treatment per se increased the number of viable cells, in comparison with control (p<0.01), while, as expected, 3mM ATP significantly decreased cellular viability when compared to the control and LiCl groups (p<0.001 and p<0.0001 respectively) (Fig. 1a). Interestingly, pre-treatment with LiCl was able to
Discussion
Our study shows that the treatment of PC12 neuronal cells with ATP induces cellular death through apoptosis, and that lithium is able to prevent this cellular toxicity. However, ATP activates the N9 microglial cell line via the P2 × 7R, and lithium treatment cannot prevent this microglial phenotype switch.
At high concentrations of ATP, such as those used in this study, it is well established that ATP causes cytotoxic effects (Lemmens, Vanduffel et al. 1996), especially in cells of the central
Conclusion
Taken together, our results indicate that cells of different neural origins respond to ATP by inducing neuronal excitotoxicity and microglial switching to the M1 activated phenotype. Lithium, in turn, modulates the response to ATP in PC12 cells, preventing ATP-induced cell death. On the other hand, in N9 microglial cells, lithium was unable to prevent ATP-induced activation via modulation of P2 × 7R. Further studies are needed to better characterize how the purinergic system mediates lithium's
Funding and disclosure
This study was financed in part by the Coordenação de Aperfeiçoamento de Pessoal de Nível Superior - Brasil (CAPES) - Finance Code 001. This work was also supported by grants from the National Science and Technology Institute for Translational Medicine (INCT-TM) (Project 573671/2008-7), INCT for excitotoxicity and neuroprotection (INCT-EN) (Project 465671/2014-4), Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq) (Project No 303264/2013-6). The funding agencies did not have
Author Contribution Statement
CG was involved in experimental design, collection of data, data analysis and manuscript writing. RA, FF and LB were involved in data collection, analysis and editing of the manuscript. FK, PVSM and AMO were involved in experimental design, project management and funding, data analysis and drafting of the manuscript.
Conflict of Interest
The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest
Acknowledgments
CG and RA were recipients of scholarships from Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq). PVSM is supported by a CNPq productivity fellowship. Currently CG is recipient of a Post-Doctoral Fellowship (PDE – Pos Doutorado no Exterior) from CNPq – Conselho Nacional de Desenvolvimento Cientifico e Tecnologico, of the Ministry of Science, Technology, Innovation, Communication of Brazil and a University of Melbourne Early Career Researcher Award.
References (45)
- et al.
"Adenosine 5′-triphosphate and adenosine as endogenous signaling molecules in immunity and inflammation
Pharmacol Ther
(2006) - et al.
"Low glial numbers in the amygdala in major depressive disorder
Biol Psychiatry
(2002) "A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of protein-dye binding
Anal Biochem
(1976)- et al.
"Purinergic (P2X7) receptor activation of microglia induces cell death via an interleukin-1-independent mechanism
Mol Cell Neurosci
(2002) - et al.
"Purinergic signalling: from normal behaviour to pathological brain function
Prog Neurobiol
(2011) - et al.
"A direct colorimetric assay for Ca2+ -stimulated ATPase activity
Anal Biochem
(1986) "Liaisons dangereuses: P2X(7) and the inflammasome
Trends Pharmacol Sci
(2007)- et al.
"Treatment of bipolar disorder
Lancet
(2013) - et al.
Bipolar disorder and 1513A>C P2RX7 polymorphism frequency
Neurosci Lett
(2019) - et al.
"N9 microglial cells polarized by LPS and IL4 show differential responses to secondary environmental stimuli
Cell Immunol
(2012)
"Lipopolysaccharide-induced microglial activation in culture: temporal profiles of morphological change and release of cytokines and nitric oxide
Neurosci Res
"The pro- and anti-inflammatory properties of the cytokine interleukin-6
Biochim Biophys Acta
"Production and release of neuroprotective tumor necrosis factor by P2X7 receptor-activated microglia
J Neurosci
"Inhibition of glycogen synthase kinase-3β by lithium chloride suppresses 6-hydroxydopamine-induced inflammatory response in primary cultured astrocytes
Neurochem Int
"Mood stabilizers have differential effects on endogenous ADP ribosylation in C6 glioma cells
Eur J Pharmacol
"Switching of the Microglial Activation Phenotype Is a Possible Treatment for Depression Disorder
Front Cell Neurosci
"Antiapoptotic action of lithium and valproate
Pharmacol Rep
"Wnt-1 dependent activation of the survival factor NF-kappaB in PC12 cells
J Neurosci Res
"Assignment of ecto-nucleoside triphosphate diphosphohydrolase-1/cd39 expression to microglia and vasculature of the brain
Eur J Neurosci
"P2X7 Purinergic Receptor Is Involved in the Pathophysiology of Mania: a Preclinical Study
Mol Neurobiol
"Role of P2X7 Receptor in an Animal Model of Mania Induced by D-Amphetamine
Mol Neurobiol
"The P2X7R purinergic receptor as a molecular target in bipolar disorder
Neuropsychiatry & Neuropsychology
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- 1
Florey Institute of Neuroscience and Mental Health, University of Melbourne, Melbourne VIC 3010, Australia