Background
Several studies in rodent models have indicated that Paraquat (1, 1-dimethyl-4, 4-bipyridium dichloride; PQ) an environmental herbicide/pesticide, causes neurotoxicity through the generation of reactive oxygen species (ROS) and formation of apoptosis-related molecules. PQ promotes intracellular generation of ROS via three distinct pathways: (1) reduction of PQ by NADPH-cytochrome P450 reductase and a subsequent redox cycle with involvement of super oxide dismutase (SOD) and glutathione pools, (2) inhibition of mitochondrial electron transport chain, and (3) interaction with other enzymes such as nitric oxide synthase (cytosolic), NADPH oxidase (plasma membrane), thioredoxin reductase (cytosolic form, Trx 1), and xanthine oxidase [
1]. PQ-induced oxidative stress has been reported to be linked to endoplasmic reticulum stress-signaling pathways and subsequent formation of caspase-dependent apoptosis-related molecules [
2,
3]. PQ has also been shown to induce neuronal oxidative stress through activation of glial cells [
4]. However the exact mechanism of neuronal cell death after PQ administration in rodent models is far from clear. Although carrier-mediated (neutral amino acid transporter carriers, such as LAT-1, which transports L-valine and L-phenylalanine) transport of PQ across the blood-brain barrier (BBB) has been reported in rodent studies [
5,
6], there is controversy regarding the entry of PQ through BBB, the cellular metabolism of PQ, and the mechanism of its toxicity in brain of non-human primates and human beings [
4,
7].
Because of its close structural similarity to 1-methyl-4-phenylpyridinium (MPP+, the active metabolite form of MPTP), Paraquat has been suggested to be a risk factor for PD. Systemic administration of Paraquat to adult mice results in a significant decrease in substantia nigra dopaminergic neurons, a decline in striatal dopamine nerve terminal density, and a neurobehavioral syndrome characterized by reduced ambulatory activity. Prolonged exposure to paraquat leads to a remarkable accumulation of
α-synuclein-like aggregates in neurons of the substantia nigra pars compacta in mice [
8].
PQ-induced dopaminergic neuronal cell death in the substantia nigra (SN) has been found to be linked with aggregation of α-synuclein, in addition to mitochondrial dysfunction and oxidative stress. PQ induces α-synuclein aggregation through protein up-regulation [
9,
10]. PQ-induced oxidative stress could facilitate α-synuclein association by altering the biophysical properties of the protein, by proteosomal dysfunction, and/or by impairing mechanisms of protein degradation within neurons [
4,
9,
11]. In the Paraquat-induced mouse model of PD, microglial activation and pesticide exposure act synergistically, and the susceptibility of dopaminergic neurons to toxic injury is dramatically exacerbated by underlying inflammatory processes [
12]. PQ induces neuroinflammation and microglial activation indirectly through factors released from neurons or astrocytes [
13]. PQ induces nigral astrocytosis and microgliosis, the latter showing a reactive phenotype with increased numbers of macrophage antigen complex-1-immunoreactive cells (a marker for activated microglial cells) [
14,
15]. Dopaminergic neurons in the substantia nigra and ventral tegmental area have different susceptibilities to damage by PQ toxicity [
16], and major unanswered questions include whether the protein aggregates cause the selective loss of dopaminergic neurons in the substantia nigra that underlies the clinical symptoms and whether neuroinflammation is a consequence or a cause of nigral cell loss [
17].
Apart from SN, PQ can also damage hippocampal neurons of mouse brain through oxidative stress-induced mitochondrial dysfunction [
18]. PQ also induces cell loss in locus coeruleus, in the area in which catecholaminergic neurons are located [
19].
In vitro studies have shown that PQ induces apoptosis of cultured rat cortical cells [
20]. It is not clear whether PQ-induced dopaminergic cell death is selective or if other cell types are similarly affected [
18,
4,
21] in other regions of brain such as frontal cortex (which is primarily responsible for cognitive and motor responses) and hippocampus (which is primarily responsible for learning, cognition and memory).
Studies with rodent models have suggested that PQ is a potential risk factor for Parkinson's disease (PD). PQ-induced neurotoxicity and PD pathology show molecular similarities including protein aggregation [
22], neuroinflammation [
23], oxidative stress [
24], mitochondrial dysfunction [
25]and caspase activation [
26]. However, a role for PQ in causing Parkinsonism in non-human primates and human beings is uncertain due to a lack of experimental and clinical evidence.
The exact mechanism of PQ-induced neurotoxicity is, therefore, still in debate. An understanding of the molecular basis of PQ-induced neurotoxicity could provide valuable insights into neurodegenerative processes in mammalian brain. In the present study, we sought to define PQ-induced changes in molecular events associated with dopaminergic neurodegeneration in three regions of brain: SN, hippocampus, and frontal cortex (FC). Although PQ toxicity causes dopaminergic cell death in SN, the site of origin of dopaminergic innervation in brain, an effect of PQ on dopaminergic neuronal processes in hippocampus and FC has not been established. PQ toxicity and PD-like motor dysfunction/cognitive impairment both are accompanied by neuronal damage in hippocampus and frontal cortex. The main objective of our present study was to assess possible molecular links between PQ-induced dopaminergic neurotoxicity, alteration of α-synuclein status, and microglial activation in three regions of brain. Dopaminergic neurotoxicity was determined using the neuronal markers FOX-3, tyrosine hydroxylase (TH; the rate-limiting enzyme of DOPA synthesis) and DOPA decarboxylase (the enzyme that catalyzes decarboxylation of L-dopa to dopamine). The microglial marker Iba-1, the microglial activation marker Mac-1, the histological feature of microglial aggregation, and microglial expression of the cytokines interlukin-1ß (IL-1ß) and tumor necrosis factor- α (TNF-α) were determined in the three brain regions. The study was extended with tocopherol (an ROS scavenger) supplementation followed by PQ treatment to assess PQ-induced ROS generation and its possible impact on alterations in α-synuclein status and microglial activation.
Methods
Materials
Paraquat dichloride (PQ; 1,1'-Dimethyl-4,4'-bipyridinium dichloride hydrate) and α-tocopherol were purchased from Sigma Aldrich, Inc. (St. Louis, MO). Among the primary antibodies used, anti-tyrosine hydroxylase mouse monoclonal antibody was purchased from Calbiochem (EMD4Biosciences, NJ, USA); rabbit polyclonal anti-TNF-α and anti-IL-1β, and mouse monoclonal anti-α-synuclein were procured from Cell Signaling Technology, Inc. (Danvers, MA, USA); mouse monoclonal anti-FOX3, anti-DOPA decarboxylase, goat polyclonal anti-Iba 1, and rabbit polyclonal anti-Mac1 were procured from Abcam plc (Cambridge, UK). The secondary antibodies goat anti-rabbit IgG--HRP (horseradish peroxidase) and rabbit anti-mouse IgG--HRP, and a DAB developing system (for immunohistochemistry) were purchased from Bangalore GeNei Pvt. Ltd. (Bangalore, India). FITC-conjugated secondary mouse anti-goat antibody was purchased from Santa Cruz biotechnologies (DA, USA). Hematoxyline and eosin were obtained from Merck Specialties Private Limited (Mumbai, India) and the remaining chemicals were purchased in analytical grade of highest purity (India).
Animals
Adult male Swiss albino mice weighing ~28 gm each (22-24 weeks of age; five mice in each group) were obtained from the National Institute of Nutrition (Hyderabad, India). All animals were housed individually for at least one week prior to experiments in an animal facility (maintained at 25 [ ± 2]°C with 55 [ ± 5]% relative humidity and 12-hr light/dark cycle) located at the Animal Housing Unit in the Department of Zoology, University of Calcutta. All animals were provided rodent chow obtained from National Institute of Nutrition (Hyderabad, India) and filtered water ad libitum. All animal experiments were performed following "Principles of laboratory animal care" (NIH publication No. 85- 23, revised in 1985) as well as specific Indian laws on "Protection of Animals" under the provision of authorized investigators. The protocols were approved by the Institutional Animal Ethics Committee at the University of Calcutta.
PQ administration and supplementation with α-tocopherol
A total of 50 mice received intraperitoneal (i.p) injections of PQ at different concentrations [5, 10, 20, 40, 80 mg/kg body weight (b.w.)] in a total volume of 0.2 ml, twice a week for four consecutive weeks to determine the LD50 dose (n = 10 for each dose of PQ). Thereafter, based on this data, new sets of mice were provided with sublethal doses (10 mg PQ/kg b.w.) twice a week for 4 weeks. Mice were randomly divided into 4 groups comprising (A) vehicle/saline (0.9% NaCl)-treated control (n = 6), (B) PQ-treated (n = 6), (C) α-tocopherol--supplemented. PQ-treated (n = 6), and (D) α-tocopherol-supplemented, vehicle/saline-treated controls (n = 6) for the experiments to be performed.
α-Tocopherol was supplemented to mice that had been treated previously with PQ, 10 mg/kg b.w. for four weeks. α-Tocopherol was injected intraperitonealy at 20 mg/kg b.w. for five consecutive days after the last dose of PQ (up to day six) and sacrificed thereafter on day seven.
General health and gross motor functions assessment
Mice were observed twice daily during the first week of injections (10 mg PQ/kg b.w. for each mouse) and daily thereafter. General health and gross motor functions were assessed by observing in-cage behavior and during brief gentle handling to check for rigidity (hunched posture and increased tail tone), bradykinesia (slowed movement and/or absence of rearing), dystonia (clenched paws), autonomic signs (piloerection) and stopped movement (akynesia). Additionally, body weights were checked prior to and each week after PQ administration. Simple behavioral tests in control and PQ-treated mice were performed three days after the last injection. Asymmetry in body posture and gait abnormalities were tested with the curling test and the footprint test, respectively. The curling test evaluates any asymmetry in body posture [
27]. The mice were lifted gently 2-3 cm above the bedding for 5 seconds and any ipsilateral deviation from its vertical body axis of 10° or greater was recorded. The test was repeated three times for each animal.
For the footprint test mice were placed in a 5-cm-wide, 55-cm-long corridor. The floor of this corridor was covered with white absorbant paper. The animals were first trained to pass straight forward through the corridor. After this training, the paws were colored with different colors (red for the forepaws and violet for the hindpaws), and the mice were then placed into the corridor. Step frequency and stride length were determined with the program Footprints version 1.22 [
27].
Tissue handling
On the 7th day after the final dosing with PQ, mice in each group were euthanized by over-dose of sodium thiopentone (Mancure Drugs Private Ltd., Mumbai, India) and brain tissues harvested for analyses as described in the assays below. The animals used in behavioral studies were same animals that were sacrificed thereafter for brain tissue analysis.
Histological analysis
Animals were deeply anesthetized by overdose of sodium thiopentone (Mancure Drugs Private Ltd., Mumbai, India) and were sacrificed by decapitation. Brains were removed immediately and washed in ice-cold phosphate-buffered saline (PBS, pH 7.4). Then, the tissues were cut into two equal halves along the longitudinal fissure. The tissues were fixed for 24 hours in buffered formaldehyde solution (10% in PBS) at room temperature, dehydrated by graded ethanols (50-100%) and embedded in paraffin (Merck, solidification point 60--62°C). Tissue sections (thickness 5 μm) were then deparaffinized with xylene, rehydrated with graded alcohols (100%-50% ethanol), stained with eosin/haematoxylin (Merck, Mumbai, India) and mounted in DPX resin (Merck, Mumbai, India). Images were captured using an Olympus BX51 microscope attached to an Olympus DP70 camera (U-TVO 63 × C; Olympus Corp., Tokyo, Japan) having both 40× and 100× (wide zoom) lenses.
Immunohistochemistry
Sagittal brain sections (5 µm thick) were cut from paraffin-embedded brain tissue and mounted on positively-charged Super frost slides (Export Mengel CF, Menzel, Braunschweig, Germany). Tissues were deparaffinized, dehydrated through graded alcohols, and then endogenous peroxidase was quenched in a 3% hydrogen peroxide solution for 20 minute at room temperature. Background staining was then inhibited with 5% bovine serum albumin [BSA] (Sisco Research Laboratories Pvt. Ltd. [SRL], Mumbai, India) for 30 minutes at room temperature to avoid nonspecific binding of IgG. Excess liquid was drained and the sections were incubated in a humid chamber overnight at 4°C with primary antibodies (diluted 1:50 in solution containing 5% BSA). The following specific primary antibodies were used for separate cases:,anti-IL-1β (mouse polyclonal), anti-TNF-α (mouse polyclonal), and anti-α-synuclein (mouse monoclonal) (Cell Signaling Technology, Inc. [Danvers, MA, USA]), as well as anti-tyrosine hydroxylase (mouse monoclonal) (Calbiochem [EMD4Biosciences, NJ, USA]) for positive controls (data not shown). After three washes in PBS-T, sections were sequentially incubated in horseradish peroxidase (HRP)-conjugated anti-sera specific for those antigens and were diluted at a 1:30 ratio in Tris-buffered saline containing 0.3% Triton-X and 0.5% blocking agent for 2 hours at room temperature. Immunoreactive complexes were then detected using a DAB system (Merck Specialties). Sections were then counterstained briefly in hematoxylin, dehydrated through graded alcohols (70%, 95%, 100%), cleared in xylenes, and coverslipped with DPX mounting medium. Slides that received no primary antibody served as negative controls. Images were captured using a U-TVO 63 × C microscope (Olympus Corp., Tokyo, Japan) having both 40× and 100× (wide zoom) lenses.
Preparation of cell lysates
Different brain regions -- hippocampus, frontal cortex, and SN -- were dissected out immediately after dissection of sagittal sections of whole brain [
28]. Tissues were homogenized in ice-cold RIPA lysis buffer (150 mM sodium chloride, 1.0% TritonX-100, 50 mM Tris pH 8.0, 0.01% SDS, 0.5% sodium deoxycholate) containing 1 mM PMSF (phenylmethanesulfonylfluoride or phenylmethylsulfonyl fluoride) (SRL, India), 1 μg/ml aprotinin, 1 μg/ml leupeptin and 1 μg/ml pepstatin (Sigma-Aldrich Inc., USA) that were added fresh prior to cell lysis. The samples were sonicated and incubated on ice for 30 min, and centrifuged 3 times at 14,000 rpm for 15 min at 4°C. A portion of the supernatant was reserved for protein determination using the Bradford reagent (Sigma-Aldrich Inc., USA) and subsequent measurement of absorbance was done at 595 nm in a UV-1700 PharmaSpec, Shimadzu spectropho-tometer (Shimadzu Scientific Instruments, Columbia, MD). The remaining supernatant was stored at -20°C.
Western blot analysis
Tissue lysates were diluted in sample buffer (0.312 mM Tris-HCl [pH 6.8], 50% glycerol, 10% SDS, 25% β-mercaptoethanol, and 0.25% bromophenol blue) at a final protein concentration of 5 μg/μl, and were then boiled at 100°C for 5 minutes. Aliquots of each sample (10 μl containing 50 μg protein) were loaded into dedicated wells of 9-12% polyacrylamide gels and separated by electrophoresis for 3 h at 100 V. Proteins were transferred to polyvinylidene difluoride membrane (Amersham Biosciences, Piscataway, NJ) for 1.5 h at 300 mA. After blocking of nonspecific binding with 5% nonfat dry milk in TBST, the membranes were then probed with the following primary antibodies: anti-tyrosine hydroxylase (mouse monoclonal; 1: 2000 dilution), anti-TNF-α, anti-IL-1β (rabbit polyclonal; 1: 1000 dilution), anti-α-synuclein (mouse monoclonal; 1: 1500 dilution) anti-FOX3, anti-DOPA decarboxylase (mouse monoclonal; 1: 2000 dilution), anti-Iba1 (goat polyclonal; 1: 2500 dilution), anti-Mac1 (rabbit polyclonal; 1: 2000 dilution) and incubated overnight at 4°C. The membranes were washed 3 times with Tris-buffered saline-0.01% (v/v) containing Tween-20 at room temperature for 15 minutes and then incubated with alkaline phosphatase (AP)-conjugated secondary antibodies (anti-rabbit, anti-goat and anti-mouse IgG; diluted 1:1000) with TBST for 2 h at room temperature. The membranes were then developed with NBT/BCIP (nitroblue tetrazolium chloride/5-bromo-4-chloro-3-indolyl-phosphate; Hi-Media, Mumbai, India). Band intensity of the detected protein was measured by densitometry (Gel Doc™ XR+ System, Bio-Rad Laboratories, USA). β-Actin was also analyzed on each membrane for confirmation of gel sample loading (i.e., based on constitutive expression).
Immunofluorescence
The immunofluorescence procedure was carried out on sections incubated in blocking buffer (0.3-0.5% Triton X-100, 5% BSA in TBS) for 30 min at room temp followed by an overnight incubation with the primary antibody Iba1 (goat polyclonal; 1: 50 dilution) in blocking buffer at 4°C,. After three washes in TBS-T, primary antibody was revealed by incubating the sections in FITC-conjugated anti-sera specific for those antigens and diluted at a 1:30 ratio in Tris-buffered saline containing 0.3% Triton-X and 0.5% blocking agent for 2 hours at room temperature followed by TBS washing. The tissue sections were counterstained with a nuclear counterstain (DAPI by Vector Laboratories Inc. Burlingame, CA, USA) and mounted with DPX resin. Images were captured in a U-TVO 63 × C microscope (Olympus Corp., Tokyo, Japan) having both 40× and 100× (wide zoom) lenses.
Stereological cell counting
Stereological methods were used for quantification of cells present in stained tissues as previously described [
29]. Briefly, tissue was visualized with an Olympus U-TVO 63 × C microscope and Micro Bright Field CX 9000 camera. Cell counting was done for both side of the brain for each animal, and then right and left values were added to generate a total DA substantia nigra neuron count, in a total of five animals per experimental group. The tissue was quantified using optical fractionators from MicroBrightField, with the software Stereo Investigator (Ver.8). Estimated volumes (μm
3) of TH-negative zones were quantified using the cavalieri method of unbiased stereology in the substantia nigra of every third section. Also, expression of FOX 3-positive cells was quantified in substantia nigra using the cavalieri method. Both immunostains were quantified with a grid spacing of 200 μm using a 2×/0.06 objective. TH-positive cells were also quantified within the area of the substantia nigra pars compacta. The sampling site was customized to count 200 cells per brain, and sampling was done with error coefficients less than 0.07. For counting TH-positive cells the counting frame (CF) was 70 × 70 with a virtual counting grid (CG) of 140 × 140. For FOX3-positive cells CF and CG were the same as for TH-positive cells.
Microglia-specific silver staining
Microglia-specific silver staining was performed as previously described [
30]. Briefly, paraffin-embedded mouse brain sections were deparaffinized with xyline followed by rehydration in a series of graded (100%-50%) ethanols. After washing in distilled water, sections were transferred for silver impregnation in 10% ammoniacal silver nitrate solution for 3-4 seconds. After that, sections were transferred to a 10% formalin solution and then washed with distilled water. The slides were then fixed in 5% sodium thiosulfate solution for 2-5 minutes. Sections were washed and then dehydrated in graded alcohols and finally mounted in DPX (MERCK) for microscopical studies. Images were captured in an Olympus BX51 microscope attached with Olympus DP70 camera (U-TVO 63 × C) (Olympus, USA).
Preparation of homogenatse for ROS scavenging enzyme assay
A 10% tissue homogenate was prepared in 0.1 M phosphate buffer (pH 7.4) containing 0.1 M KCl. Enzyme assays were performed in the supernatant obtained following centrifugation of the homogenate at 9000 × g for 10 min at 4°C.
Discussion
The cellular and molecular mechanisms underlying PQ-induced neurodegeneration are unclear. Several studies have indicated that PQ toxicity causes dopaminergic neuronal cell loss in SN and expression of α-synuclein in SN as well as in FC through formation of superoxide radicals. In this study, PQ-treated animals showed several symptoms including impairment of motor performances, which developed after two weeks of treatment. These physiological changes are indicative of PQ-mediated neurotoxicity. Dopaminergic neurons are more vulnerable to PQ-induced oxidative stress than are other neuronal populations because they are ill equipped to endure oxidative stress [
37,
38]and because PQ promotes dopaminergic neuronal death via a c-Jun-N-terminal kinase 3 (JNK3)-mediated cell signaling pathway [
39]. SN is rich in dopaminergic cells. Therefore, during PQ exposure, SN is the most affected region of brain.
The hippocampal formation receives projections from the midbrain's dopaminergic cell groups and contains mRNA for dopamine receptors [
40], and FC receives output from SN via the thalamus [
41]. Previous studies have shown that PQ deposition is found in hippocampus as well as in FC [
42,
43]. Therefore, apart from SN, PQ-mediated mitochondrial injury by oxidative stress has a toxic influence on hippocampus and FC of mice [
18]. Therefore, along with SN, FC and hippocampus were major areas of concern during PQ-mediated neurotoxicity in the present study.
Multiple but sufficiently low doses of PQ are well tolerated by peripheral organs in rats without apparent oxidative stress [
44]. In the present study, ROS levels increased (as reflected by high activity of ROS-scavenging enzymes) significantly in three regions of brain with PQ doses of 10 mg/kg b.w. or greater. However, ROS levels in peripheral organs increased significantly with all lethal doses (20 mg/kg b.w. to 80 mg/kg b.w.) but not with a sublethal dose (10 mg/kg b.w.) in our treatment regimen. Therefore, in our animal model, impaired motor function appeared to be due to PQ-induced, brain-specific ROS generation, which promotes neurodegeneration, but not due to ROS generation in peripheral organs. Hence, peripheral levels of ROS at a PQ dose of 10 mg/kg b.w. may not be sufficient to increase ROS levels in brain. We may consider that the rise in ROS levels in brain with a PQ dose of 10 mg/kg b.w. is a local effect, and that ROS may be involved in alteration of other parameters in brain. PQ needs time to cross the blood-brain barrier to reach brain tissue and generate ROS generation. As with other, higher, lethal doses, the availability of PQ in brain may not be sufficient to produce maximum toxicity. Such higher doses of PQ may produce acute peripheral toxicity that causes more severe peripheral tissue (e.g., lung, kidney, etc.) damage (Bhattacharyya, unpublished) and our cell counting data support this idea. Numbers of dopaminergic cells decreased most significantly in SN at a sublethal dose of PQ (10 mg/kg b.w.) compared to other lower or lethal doses and to controls. Therefore, we consider a dose of 10 mg/kg b.w. PQ as a sublethal dose that may produce high levels of ROS locally in brain. α-Tocopherol supplementation decreased ROS levels in all three regions of brain.
Increased ROS levels induced differential changes in cellular morphology in all three regions of brain in the present study. Pyknotic nuclei appeared in SN, in FC and in hippocampus of the treatment group, indicating that, apart from SN, hippocampus and FC are also affected by PQ-mediated neurotoxicity. Many Lewy body-like structures in FC indicate that there might be involvement of differential α-synuclein expression level not only in FC but also in SN and hippocampus. PQ exposure induced the formation of α-synuclein-containing deposits. This effect, however, was seen in both control and α-synuclein-overexpressing mice, thus suggesting that neuroprotection is not a mere consequence of lack of protein deposition. α-Synuclein itself may possess properties that counteract toxic injury, and its expression could affect specific stress signaling pathways linked to neuronal survival. For example, Hashimoto and colleagues [
45]have suggested that α-synuclein expression can confer resistance to
in vitro hydrogen peroxide toxicity via inactivation of c-Jun N-terminal kinase, a member of the mitogen-activated protein kinase family. This indicates that α-synuclein overexpression protects against Paraquat-induced neurodegeneration. α-Synuclein is not only a component of Lewy bodies and synapses but also of axons, and aggregated α-synuclein might interfere with axonal transport and lead to cell death [
46]. Microscopical observations show that increased immunoreactivity for α-synuclein is predominant in FC of PQ-treated brain in our studies. However, α-synuclein protein expression levels do not change significantly in FC of PQ-treated mouse brain. Expression of α-synuclein did increase significantly in hippocampus, whereas expression levels of α-synuclein decreased in SN of our treated group. As in SN, cell death was more pronounced, suggesting that decreased α-synuclein levels indicate earlier degeneration in SN than in the other two regions of brain. In hippocampus, for example, α-synuclein levels increased to protect neuronal cells. Previous reports have stated that methylation of human α-synuclein gene intron 1 decreases that gene's expression, while inhibition of DNA methylation activates α-synuclein gene expression. On the other hand, extensive neuronal expression of α-synuclein and disruption of α-synuclein function or abnormal aggregation may have similarly widespread consequences [
47]. Therefore, the reduced expression of α-synuclein in SN, increased expression in hippocampus, and aggregated forms in FC found in our present study might correlate with the α-synuclein gene polymorphism associated with PQ-mediated neurotoxicity in this mouse model and the differential time frames necessary to initiate neurodegeneration in these different regions.
In relation to the hypothesis that differential α-synuclein expression may modulate TH expression levels, we evaluated TH-positive neuronal vulnerability in all three regions of PQ-treated mouse brain. We observed that expression levels of TH decreased in all three regions of PQ-treated mouse brain. The reduction of TH expression may indicate a reordering of protein biosynthesis favoring production of protein required for axonal regeneration at the expense of those involved in neurotransmission [
48]. The most interesting observation is that the distribution of TH immunoreactivity changes from nucleated areas to non-nucleated areas in hippocampus and FC, but not in SN. Hence, we may predict that TH, which is present in axons (non-nucleated areas) shows greater immunoreactivity in non-nucleated areas compared to nucleated areas in hippocampus and FC under our treatment conditions. The presence of pyknotic nuclei in SN may explain in part the total reduction of TH in SN. However, the exact cause of such differential patterns of TH expression in three brain regions under our treatment conditions needs further investigation. Dopaminergic immunoreactivity decreased primarily in SN and FC, while in hippocampus no significant changes were observed. At the same time, there might be neuronal compensatory mechanisms involved to regenerate new dopaminergic neurons such that overall levels of DOPA decarboxylase and FOX3 expression level do not change.
In the present study, differential expression patterns of α-synuclein and TH due to PQ treatment elicited high expression levels of proinflammatory cytokines such as TNF-α in the three regions of mouse brain, and of IL-1β in FC and hippocampus of mouse brain. There might be involvement of activated microglial cells in promoting neuroinflammation. Microglial cells in a resting state continuously maintain homoeostatic activity in the CNS, and in a fully activated phagocytic state microglial cells scavenge neurotoxins, remove dying cells and cellular debris, and secrete trophic factors that promote neuronal survival, reorganization of neuronal circuits and repair [
49,
50]. Insufficient clearance by microglia is prevalent in several neurodegenerative diseases and in normal ageing [
51]. Over-activation of microglia may cause alterations in immunophenotypic expression and inflammatory profile (promoting microglia senescence), and that condition may switch microglial function from neuroprotective to neurotoxic effects [
52]. Increased expression of Mac1 (microglial activation marker) in SN indicates chronic neuroinflammation. However decreased expression of Iba1 (a microglial marker) and Mac 1 with increased cytokine levels in FC might reflect the peripheral supply of cytokines without local production by microglial cells in brain.
Long-standing activation of microglia during chronic neuroinflammation causes sustained release of inflammatory mediators that promote activation of additional microglial proliferation, and further release of inflammatory factors [
53]. In search of involvement of microglial cells in PQ-mediated neurotoxicity; we have found aggregated microglial cells in SN of PQ-treated mouse brain, while microglia-specific staining is less positive in FC. Although there are aggregated microglial cells in hippocampus of treated mouse brain, microglial cell-specific staining decreases compared to controls in hippocampus. At this point it is not clear whether microglial cells degenerate or migrate to other areas with pathogenic lesioning. Chemokines regulate rapid migration of microglia to injury sites in CNS and amplify neuroinflammation [
54]. If microglial cells degenerate, then this degeneration presumably relates to a failure of neuroprotective functions and subsequent contributions to neurodegeneration [
55]. Recent studies indicate that death of microglial cells may occur as a consequence of overproduction of immuno-inflammatory molecules along with production of anti-inflammatory molecules such as IL-13, activation of
Fas-mediated apoptotic signaling, and/or toxins produced by over-activated microglial cells themselves [
56,
57]. Although microglial phenotypic shifting, as seen in our study, occurs in frontal cortex and hippocampus, we found significant high levels of inflammatory molecules in all three regions of brain. Proinflammatory cytokines released from over-activated microglia may act on the endothelium of BBB cells to stimulate upregulation of adhesion molecules for passage of T cells and monocytes that then go on to release more cytokines. This indicates that chronic inflammation may cause an increase in permeability of the BBB [
58‐
61]. From this point of view, we may predict that the high levels of pro-inflammatory/inflammatory molecules in the three regions of brain found in our present study may participate in increasing a peripheral supply of inflammatory responses to those areas of brain. Further investigation is needed to explore these phenomena. As α-tocopherol supplementation decreased TNF-α levels in all three regions of PQ-treated mouse brain, ROS-induced TNF-α production might initiate neuroinflammation with or without involvement of microglial cells during PQ treatment. Other parameters, such as α-synuclein, dopaminergic neuronal status and microglial status, were not altered with α-tocopherol supplementation in PQ-treated brain. To maintain the same time frame of sacrifice as PQ-treated mice (day 7 after the last dose of PQ), we supplemented different sets of PQ-treated mice with five doses of α-tocopherol only. Therefore, further study is needed to evaluate the effects of α-tocopherol supplementation in PQ-treated mice brain for prolonged time spans.
Competing interests
The authors declare that they have no competing interests.
Authors' contributions
SM, NC and AB developed the concept, designed the experiments, and contributed in the data analysis and writing of manuscript. SM carried out all experiments. NC assembled and interpreted all results. AB evaluated and coordinated the whole work. All authors read and approved the final manuscript.