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L-Carnitine Inhibits Lipopolysaccharide-Induced Nitric Oxide Production of SIM-A9 Microglia Cells Emily L Gill, Shreya Raman, Richard A Yost, Timothy J. Garrett, and Vinata Vedam-Mai ACS Chem. Neurosci., Just Accepted Manuscript • DOI: 10.1021/acschemneuro.7b00468 • Publication Date (Web): 25 Jan 2018 Downloaded from http://pubs.acs.org on January 26, 2018
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ACS Chemical Neuroscience
L-Carnitine Inhibits Lipopolysaccharide-Induced Nitric Oxide Production of SIM-A9 Microglia Cells Emily L. Gill, ¶ Shreya Raman, ¥ Richard A. Yost, ¶, § Timothy J. Garrett, § and Vinata Vedam-Mai ¥, * ¶
Department of Chemistry, University of Florida, Gainesville, Florida, 32611 USA Department of Neurosurgery, University of Florida, Gainesville, Florida, 32610 USA § Department of Pathology, Immunology and Laboratory Medicine, University of Florida, Gainesville, Florida, 32610 USA ¥
ABSTRACT: Microglia are the resident immune effector cells of the central nervous system. They account for approximately 1015% of all cells found in the brain and spinal cord, acting as macrophages, sensing and engaging in phagocytosis to eliminate toxic proteins. Microglia are dynamic and can change their morphology in response to cues from their milieu. Parkinson’s disease is a neurodegenerative disease, associated with reactive gliosis, neuroinflammation, and oxidative stress. It is thought that Parkinson’s disease is caused by the accumulation of abnormally folded alpha-synuclein protein, accompanied by persistent neuroinflammation, oxidative stress, and subsequent neuronal injury/death. There is evidence in the literature for mitochondrial dysfunction in Parkinson’s disease as well as fatty acid beta-oxidation, involving L-carnitine. Here we investigate L-carnitine in the context of microglial activation, suggesting a potential new strategy of supplementation for PD patients. Preliminary results from our studies suggest that the treatment of activated microglia with the endogenous antioxidant L-carnitine can reverse the effects of detrimental neuroinflammation in vitro. Key Words: Parkinson’s disease, Microglia, Lipopolysaccharide, L-carnitine, Gliosis, Neuroinflammation Parkinson’s disease (PD) is neurodegenerative disorder characterized by the degradation of dopaminergic neurons in the substantia nigra portion of the basal ganglia.1 While the exact reason for the degradation is still unknown, it is theorized that oxidative stress plays a crucial role, resulting in an imbalance in neuronal redox potential and the accumulation of abnormally folded alpha-synuclein (α-syn), ultimately causing their demise.2 One of the hallmarks of PD pathophysiology is chronic neuroinflammation, wherein the release of proinflammatory cytokines via activated microglia and astrocytes leads to neuronal degeneration in the substantia nigra pars compacta (SNPc).3 Once almost ignored, microglia have recently emerged into the spotlight, for the roles they play in central nervous system (CNS) health and disease.4 They are the resident macrophages of the CNS and survey their parenchymal milieu in the quiescent, or ramified form. Whilst surveying, they are able to detect cellular signals and can respond to these signals, thereby maintaining brain homeostasis. Microglia respond to disease by exhibiting a morphological change from the normally ramified form to an amoeboid form (Figure 1), engulfing or degrading toxic proteins (such as aberrant α-syn) and releasing inflammatory cytokines. The role of α-syn in PD pathology has been well documented, although the normal functionality of the proteins remains unclear.5 In PD, pathology has revealed that patients present with chronic neuroinflammation, with the presence of microglia in the vicinity of the degenerating dopaminergic neurons.6 However, the specifics of microglial interactions and their role in dopaminergic cell death are yet to be clarified. It is now known that microglia have both neuroprotective and neurotoxic effects.4 As PD progresses,
inflammation is thought to outweigh the neuroprotective effects of the microglia, providing a counterproductive immune response.4 Therefore, regulating microglial activation may assist in treating PD.7 Mitochondrial dysfunction and disturbances in the mitochondrial dynamics of dopaminergic neurons have been well studied and are thought to cause neuronal degeneration in PD.8 However, there is no clear line of evidence to suggest that mitochondrial impairment in microglia plays a direct role in neurodegeneration, which is a consequence of neuroinflammation. A recent study by Sarkar et al. demonstrates the existence of a complex, drawn out interaction between microglia mitochondrial impairment, inflammasome signaling and the consequent persistent neuroinflammation observed in PD.9 Markers of microglia activation include the release of cytotoxic cytokines, reactive oxygen species (ROS) and nitric oxide (NO).10 ROS can damage cell components including proteins, DNA and lipids and this can result in untimely demise of the cells. Microglia induced dopaminergic cell injury results in the release of NO. Studies have demonstrated the use of L-ergothioneine,11 vitamin E12 and Ltaurine,10 for quenching NO release, in a variety of cell lines, using different cytotoxic stimuli. Furthermore, Koc et al. demonstrated the inhibition of NO production by L-carnitine in monocyte macrophage cells.13 L-carnitine has been shown to scavenge pro-inflammatory and neurotoxic factors such as ROS in vitro.14 L-carnitine can prevent free radical formation by inhibiting activity of enzymes responsible for their gener-
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ation and also by turning on antioxidant activity (neutralize free radicals).
Figure 1. The morphological changes associated with microglia activation by α-syn, from the quiescent, surveying, ramified state, to amoeboid, activated state, secreting neurotoxic cytokines and ROS. The neuroprotective effect of L-carnitine has been demonstrated in the brains of older rats (24-months) by Rani et al.15 Older rats exhibited a higher level of lipid peroxidation compared to younger rats (4-months) in the cortex, hippocampus, striatum and cerebellum, but not the hypothalamus.15 However, after intraperitoneally administering Lcarnitine lipid peroxidation was reduced in the cortex, hippocampus, striatum and cerebellum of the older rats, but was ineffective in the brains of the younger rats (the hypothalamus was unaffected in both cases).15 Both L-carnitine and its acetyl derivative are of interest to researchers investigating neuroprotection. L-carnitine was administered to rat pups (7days) that had sustained hypoxic-ischemic brain injuries, and was shown to improve there long-term functional outcomes.16 Furthermore, L-carnitine has been demonstrated to be deficient in patients with immune and inflammatory disorders (HIV, sepsis syndrome, chronic fatigue syndrome, etc).17 More recently, it is emerging that L-carnitines could play an important role in neurodegenerative diseases such as PD and Alzheimer’s disease.18 Petersen et al. report significant differences in total L-carnitine levels between PD patients and healthy controls.19 However, interestingly they were unable to report a relationship between these low Lcarnitine levels and deficiencies in the organic cation transporter N2 (OCTN2) protein, due to dysregulation of the SLC22A5 gene. L-carnitine is an endogenous antioxidant, transported intracellularly and across the blood brain barrier (BBB) by the OCTN2, and has minimal affinity for the organic cation transporter N1 (OCTN1).20 Microglia cells are highly activated in PD patients and ultimately elevate neurodegenerative activity.21 Regulation of these microglia cells, such that the favorable anti-inflammatory state is favored may improve patient outcomes, related to disease progression.
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Results and Discussion L-carnitine Treatment of Activated Microglia. To simulate microglial activation in vitro, a common neurotoxin such as lipopolysaccharide (LPS) is added in culture, which induces morphological change, accompanied by NO release, which can be quantitatively measured using the Greiss assay (measures nitrites (NO2-) release).22 Both NO2and, nitrates (NO3-) are the final product of NO oxidation pathways and, therefore monitoring NO2- levels is indicative of NO release by the microglia. Here we show the ability of L-carnitine at different concentrations to suppress NO levels in SIM-A9 microglia cells, after a 24-hour pre-incubation period, and LPS stimulation for a further 24-hours. Prior to investigating L-carnitine and its effect on LPS stimulated microglia, the cytotoxicity of L-carnitine was determined (See Figure S-1 of the Supporting Information). The NO produced from microglia incubated with different Lcarnitine concentrations (1, 5, 10 and 15 mM) in comparisons to cells only (no treatment) when incubated for 48hours, was shown to be positively regulated. This data shows that L-carnitine does not inhibit, nor promote excessive NO production in the SIM-A9 microglia cells. L-carnitine has a protective effect on activated SIM A9 microglia following pre-treatment. However, when SIMA9 cells are exposed to 2.5 µg/mL LPS treated with Lcarnitine at 1, 5, 10 and 15 mM without a pre-incubation period, there was an undesirable effect on NO production (See Figure S-2 of the Supporting Information). It is likely that pretreatment with L-carnitine is required in order to activate the necessary antioxidant pathways before a beneficial effect can be observed. The nuclear factor erythroid factor 2 (NRF2), is a key regulator of antioxidants in the body, and is found within most brain cell types including microglia. NRF2 binds the antioxidant response element in the nucleus thereby promoting enzymes such as glutathione peroxidase and glutathione-s-transferase, in an effort to reduce oxidative stress.23 Insufficient NRF2 activation in humans has been linked to both PD and Alzheimer’s disease.24,25 Li et al. show that L-carnitine pretreatment activates the NRF2 pathway in hydrogen peroxide treated liver cells.26 The need for a pre-incubation period may therefore be dictated by the enzymatic neuroprotective mechanism. Based on experience and previous work by Koc et al. we chose to incorporate a 24-hour pre-incubation period into our protocol. This followed a further 24-hours incubation for LPS stimulation. Our results were replicated four times and show promising results with higher concentrations of L-carnitine significantly suppressing NO output from activated microglia cells (Figure 2).
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ACS Chemical Neuroscience
Figure 2. LPS stimulation of SIM A9 cells 48-hours postplating. LPS-induced NO release into the media as determined by the Greiss reaction, and was significantly inhibited by pre-incubation with 10mM and 15mM L-Carnitine, * P
