Background
Parkinson's disease (PD) is the most prevalent neurodegenerative movement disorder [
1]; genetic mutations are found in rare cases of familial PD, however more than 90% of cases are sporadic without known cause [
2]. The role of microglia, the resident immune cells of the Central Nervous System (CNS), has rapidly gained attention from researchers interested in understanding the pathogenesis of PD. Chronic microglial activation, subsequent neuroinflammation and accumulation of Reactive Oxygen Species (ROS) are now accepted as significant contributors in the disease progression [
3,
4]. The importance of the role played by microglia in inducing neuroinflammation in PD is highlighted by the fact that the substantia nigra, a region that shows extensive degeneration of dopaminergic neurons in PD, is also the region most densely populated with microglial cells [
5,
6]. Dopaminergic neurons also show a unique vulnerability to the factors released by activated microglia. It is therefore important to fully understand the interaction between microglia and neurons and the precise mechanisms by which microglia influence dopaminergic neuronal cell loss through the use of
in-vitro and
in-vivo models of PD.
Neurodegenerative diseases are difficult to model. A number of epidemiological studies have identified a strong correlation between exposure to pesticides and increased incidence of PD [
7‐
11], suggesting pesticides as the accelerators/promoters of PD. Some of the pesticides in question were found to inhibit complex I enzyme activity [
8,
12]. Based on these experimental and clinical findings, PD was the first neurological disease to be modelled. Chemicals such as reserpine, methamphetamine, 6-hydroxydopamine, 1-methyl-4-phenyl-1,2,3,6-tetrahydroxypyridine (MPTP), paraquat and rotenone have all been used to make PD models [
13,
14]. Studies by Betarbet and colleagues [
12,
15,
16], reported that chronic treatment with the herbicide rotenone led to selective destruction of nigrostriatal dopaminergic neurons, formation of cytoplasmic inclusions in nigral neurons and induction of hypokinesia and rigidity in rats. These studies have drawn a lot of attention highlighting rotenone's potential use as a model for toxin-induced Parkinsonism.
Rotenone is commonly used as a natural pesticide, an insecticide and to kill 'nuisance' fish in lakes. Rotenone is a lipophilic compound and can easily cross the blood-brain barrier [
13,
14]. Though chronic exposure to low doses of rotenone results in uniform inhibition of complex I throughout the brain, rotenone has been shown to induce selective degeneration of the nigrostriatal pathway [
12]. Although some researchers [
17,
18] have reported non-specific central nervous system and systemic toxicity in rats upon rotenone treatment, recently Cannon et al., (2009) [
2] have reproduced consistently many features of PD in rats following chronic intraparitoneal administration of rotenone. Features include systemic mitochondrial impairment, oxidative damage, microglial activation, selective nigrostriatal dopaminergic degeneration, L-Dopa responsive motor deficit, gastrointestinal deficit and α-synuclein and ubiquitin positive lewy body-like inclusion bodies within TH-positive neurons. Rotenone has also been used successfully to reproduce PD characteristics in
in-vitro slice culture models [
19], primary cultures [
20] and recently we have shown accumulation of α-synuclein positive inclusions within four weeks of chronic low dose treatment of SH-SY5Y dopaminergic cells of human origin [
21].
Microglial activation and neuroinflammation have been identified as important contributors in human PD pathogenesis, and several rotenone models have demonstrated microglial activation in the striatum of the treated animals [
2,
12]. Recent studies have reported that microglia contribute significantly in mediating and potentiating the neurodegenerative effects of rotenone [
22,
23]. There is however, very little or no information available on the direct effects of rotenone on microglial cells. Some authors have reported microglial activation upon rotenone treatment in the microglia of rodent origin [
2,
12]. There are however some important differences between human and rodent microglia in the way they release superoxide in response to the same activating agents. Furthermore, and in contrast to rodent microglia, human microglia are thought to produce very little or no inducible nitric oxide synthase (iNOS) [
24,
25]. In this study, we report the direct effects of chronic low dose rotenone treatment on human microglial cells.
Discussion
Exposure to environmental toxins is considered to be a significant contributor to the pathogenesis of PD. Since the first report by Betarbet et al. in 2000 [
12] that chronic treatment with the herbicide rotenone leads to selective destruction of nigrostriatal dopaminergic neurons in rodents, rotenone has been used widely for establishing
in-vivo and
in-vitro models of PD. We and others have also shown that chronic, low dose treatment of human dopaminergic neuroblastoma SH-SY5Y cells induces α-synuclein positive intraneuronal inclusion bodies [
15,
21]. Rotenone-induced dopaminergic neurodegeneration is attributed to both the mitochondrial impairment within neurons and enhancement of microglial activation [
22]. Using transgenic mice and primary mesencephalic cultures, researchers have shown that activated microglia greatly enhance the rotenone induced neurotoxicity through the production of pro-inflammatory cytokines and NADPH oxidase derived superoxide [
22,
23]. The effect of rotenone on dopaminergic neurons and the contribution of microglia to neuronal death have been extensively studied. There is however little information available on the effect of rotenone treatment on microglia although some researchers have reported microglial activation upon exposure to rotenone in microglia of animal origin using rodent
in-vivo models and primary mesencephalic cultures [
2,
12,
29]. There are some differences between microglia of animal and human origin especially in the way they produce the superoxide anion and iNOS (inducible nitric oxide synthase) in response to the same activating agent [
24,
25]. Therefore, it is important to determine the effect of rotenone on microglia of human origin. Due to limited availability of human tissue, low yield of pure human microglial cultures and ethical issues on the use of human tissue, we used the transformed human microglial cell line (CHME-5). To the best of our knowledge this is the first study investigating the effect of chronic low dose treatment of rotenone on human microglial (CHME-5) cells.
In the present study we have shown that human microglia are activated in response to treatment with rotenone; this was in agreement with other studies using animal models [
2,
12,
22,
23]. Interestingly, and in contrast to dopaminergic SH-SY5Y cells, CHME-5 cells do not show the formation of inclusions/aggregates at the end of week 4. The presence/absence of inclusion bodies was confirmed using both H & E staining to identify any eosinophilic aggregates and immunocytochemistry to detect any α-synuclein and ubiquitin positive aggregates. FLICA assay detects apoptosis in the very early stages if cells are committed to cell death. We did not find any difference in the level of apoptosis between rotenone treated and untreated CHME-5 cells in contrast to the levels of apoptosis in SH-SY5Y cells suggesting an increased level of tolerance in the CHME-5 cells for chronic low dose rotenone. The main components identified in the inclusion bodies include α-synuclein and ubiquitin [
12,
16,
21]. Although microglia have been shown to express both α-synuclein [
30] and ubiquitin [
31], the results of this study indicated that microglia do not form intracellular α-synuclein and ubiquitin positive, eosinophilic protein aggregates, and are resistant to apoptosis and damage induced by rotenone. Understanding the mechanisms by which microglia are protected from the toxic effects of rotenone as well as pathways/factors by which microglia accelerate neuronal death is the next important step.
Studies have shown that rotenone treatment induces mitochondrial impairment leading to excessive generation and accumulation of ROS (Reactive Oxygen Species) [
12,
15,
21,
32] and that intracellular ROS can accelerate protein cross-linking within cells [
21,
29]. We have previously shown a 48% increase in the intracellular ROS in SH-SY5Y cells within one week of treatment with rotenone [
21]. These cells showed protein aggregates and intracellular inclusions within four weeks, with the accumulation of ROS preceding the process of inclusion body formation in these cells [
21]. Surprisingly, in the present study, we did not find any difference in the accumulation of intracellular ROS in rotenone treated CHME-5 cells compared to controls. An interesting observation of the present study was that the rotenone treated SH-SY5Y cells accumulate large amounts of ROS intracellularly and do not release significant amounts into the extracellular environment. However rotenone treated CHME-5 cells do not accumulate substantial amounts of ROS within cells but release large amounts of ROS into the extracellular environment (medium). This may well be one of the reasons why these human microglia do not form protein aggregates, and in addition this release of extracellular ROS may cause damage to neurons.
The major ROS generated by rotenone treatment is superoxide; superoxide dismutase (SOD) is an anti-oxidant and superoxide scavenger enzyme known to protect cells against the toxicity of superoxide [
33]. There are two types of SOD: copper/zinc (Cu/Zn) SOD and manganese (Mn) SOD. Each type of SOD plays a different role in maintaing the health of cells. Cu/Zn SOD (SOD1) protects the cell's cytoplasm, while Mn SOD (SOD2) protects mitochondria from free radical damage [
33,
34]. We speculated that there would be a difference in the levels of SODs in the two types of cells used in this study and that this might be responsible for the different levels of ROS observed. We therefore investigated both SOD1 and SOD2 in SH-SY5Y and CHME-5 cells after treatment with rotenone. Interestingly the basal levels of both SOD1 and SOD2 were higher in SH-SY5Y cells compared to CHME-5 cells. SOD1 levels in CHME-5 cells increased approximately two fold upon exposure to rotenone, however the increase in SOD1 in SH-SY5Y cells was more dramatic. These results suggested that microglia do not accumulate ROS intracellularly and therefore the increase in SOD1 was significant but not dramatic. SH-SY5Y cells show abundant amounts of intracellular accumulation of ROS within cells [
21]; therefore there is an increase in both SOD1 and SOD2 in these cells. However the dramatic increase in SOD1 affords protection against large amounts of intracellular ROS.
Microglia express genes encoding all components of phaogocytic and non phagocytic NADPH oxidases (Noxs); NOX1, P47 phox, P40 phox, P67 phox, NOXO1, NoxA1 and Rac1 and a transmembrane flavocytochrome heterodimer consisting of P22
phox and the gp91
phox (Nox2) catalytic subunit [
33,
35,
36]. Nox2 generates superoxide ions and both Nox1 and 2 are important in the generation of cytotoxic nitrite species [
37]. Several studies have reported that activated microglia generate reactive oxygen and nitrogen species [
36,
38]. In brains from Alzhiemers disease patients, cytocolic Nox components are observed to be markedly translocated to the membranes of microglial cells [
39]. In PD brains, Nox2 has been shown to be increased in microglial cells [
40] and in Central Nervous System (CNS) Nox has been shown to be predominanatly present in microglial cells when compared to other cell types [
41]. Surprisingly we did not observe the intracellular accumulation of ROS (mainly superoxide) within microglia. Cheret et al, [
35] observed intracellular accumulation of superoxide only in Zymosan engulfing microglia and only within phagosomes. We did not use zymosan or any other agent that microglia can engulf, so this may explain in part why we did not see intracellular ROS accumulation. The results from Cheret et al, [
35] and from our study suggest that ROS accumulate in microglia only when they are engulfing an agent and ROS are generally compartmentalised within phagosomes.
Some researchers believe that superoxide diffusion through the phospholipid bilayer is poor and that cytoplasmic superoxide anions do not cause neurotoxicity directly [
33,
42]. However Gao et al, [
22,
23] and others [
43] have attributed rotenone induced neurotoxicity to the release of NADPH oxidase derived superoxide. The main question that remains is that if superoxide ions do not diffuse across the microglial membrane, then how do microglia accelerate rotenone induced neurotoxicity? It has been suggested that superoxide is converted into membrane permeable hydrogen peroxide (H
2O
2) and other down stream products such as single oxygen species and very aggressive hydroxyl radicals (OH
-); these products are released into the extracellular environment and can cross the neuronal membranes or can cause direct neurotoxicity. In our study, we have observed excessive amounts of ROS in the extracellular culture medium of the rotenone treated CHME-5 cells. Dopaminergic neurons are inherently prone to oxidative stress due to their characteristics of accumulation of high amounts of iron and dopamine [
44,
45]. In the presence of redox active iron, highly reactive and toxic hydroxyl radicals (OH
-) are generated from H
2O
2 and superoxide [
46]. Most importantly, extracellular ROS appear to promote mitochondrial generation of intracellular ROS in dopaminergic cells [
47]. Therefore microglia originated superoxide induced by rotenone might damage dopaminergic neurons directly via formation of membrane permeable downstream products or may promote neuronal mitochondrial ROS production that is a result of complex I enzyme inhibition.
Different treatments and signalling pathways lead to the production of either superoxide or nitric oxide (NO) using Nox or iNOS pathways respectively [
33]. Since human microglia produce little or no iNOS [
24,
25], the main pathway in human microglia for superoxide production appears to be via Nox. Activation of the Nox system results in the generation of superoxide within minutes [
36]. While the ROS generated by microglial cells have the potential to harm the producing cells themselves, these do not appear to cause substantial damage to microglial cells [
33], suggesting that microglial cells are well equipped with a strong antioxidative defense mechanisms to protect against oxidative and nitrosative damage. The first line of defence against ROS is the small molecular weight components such as glutathione, ascorbate and α-tocopherol. These compounds react easily with radicals [
33,
48], and additionally, the antioxidant enzymes such as SODs, catalase, glutathione peroxidase (Gpx) and glutathione reductase (GR) also play an important role in clearing the ROS [
33]. Microglia have been shown to contain the highest levels of glutathione (GSH) compared to astrocytes and oligodendrocytes in rat brain cell cultures and in the rat mixed primary cell cultures [
49,
50]. Depending on the type of stimulus, the microglial content of GSH can be increased by increased GSH synthesis or decreased by increased consumption of GSH [
49,
51]. In addition, the presence of several antioxidant enzymes such as SOD1, GPx, and catalase has been reported in brain sections of rat and human origin. Interestingly MnSOD (SOD2) was not detected in unstressed conditions [
33,
34,
52] and peroxyredoxin 1, an antioxidant protein was observed only in activated microglia [
53]. It is thought that as soon as superoxide is produced by Nox, it is quickly converted to O
2 and H
2O
2 in microglia by SODs. H
2O
2 reacts with GSH or with NO to form peroxynitrite. H
2O
2 and peroxynitirite are membrane permeable and can directly damage neurons. In addition H
2O
2 and ROS also facilitate phagocytosis and stimulate expression of microglial Tumour Necrosis Factor-α (TNF-α) [
54]. In relation to anti-oxidative mechanisms, it could be said that microglial cells are well equipped with a strong GSH system and other anti-oxidative enzymes and can therefore adapt easily to stress conditions.
Competing interests
The authors declare that they have no competing interests.
Authors' contributions
SBS conceived of the study, carried out experimental work, analysed and interpreted results and prepared the manuscript. LFBN critically reviewed the manuscript. Both authors have read and approved the final manuscript.