Lipoxygenase (LOX) isozymes can generate products that may increase or decrease neuroinflammation and neurodegeneration. A gene-environment interaction study could identify LOX isozyme-specific effects. Using the 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP) model of nigrostriatal damage in two LOX isozyme-deficient transgenic lines allows for comparison of the contribution of LOX isozymes on dopaminergic integrity and inflammation.
Lipoxygenase (LOX) activity has been implicated in neurodegenerative disorders such as Alzheimer's disease, but its effects in Parkinson's disease (PD) pathogenesis are less understood. Gene-environment interaction models have utility in unmasking the impact of specific cellular pathways in toxicity that may not be observed using a solely genetic or toxicant disease model alone. To evaluate if distinct LOX isozymes selectively contribute to PD-related neurodegeneration, transgenic (i.e. 5-LOX and 12/15-LOX deficient) mice can be challenged with a toxin that mimics cell injury and death in the disorder. Here we describe the use of a neurotoxin, 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP), which produces a nigrostriatal lesion to elucidate the distinct contributions of LOX isozymes to neurodegeneration related to PD. The use of MPTP in mouse, and nonhuman primate, is well-established to recapitulate the nigrostriatal damage in PD. The extent of MPTP-induced lesioning is measured by HPLC analysis of dopamine and its metabolites and semi-quantitative Western blot analysis of striatum for tyrosine hydroxylase (TH), the rate-limiting enzyme for the synthesis of dopamine. To assess inflammatory markers, which may demonstrate LOX isozyme-selective sensitivity, glial fibrillary acidic protein (GFAP) and Iba-1 immunohistochemistry are performed on brain sections containing substantia nigra, and GFAP Western blot analysis is performed on striatal homogenates. This experimental approach can provide novel insights into gene-environment interactions underlying nigrostriatal degeneration and PD.
Use of gene-environment interaction models provides an approach to mimic risk factors that likely influence idiopathic Parkinson’s disease (PD) and affords an opportunity to discern mechanistic insights that are unlikely to be elucidated by use of a genetic or toxicant system alone1,2. Here we illustrate this point and describe application of the 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP) mouse model of nigrostriatal degeneration3 to better understand the selectivity of lipoxygenase (LOX) isozyme activity on neuroinflammation and toxicity4. While a role for LOX isozymes has been widely evaluated in peripheral disorders5,6 as well as CNS disease including stroke7 and Alzheimer’s disease8,9, the role of the family of isozymes in nigrostriatal function and degeneration related to PD is not well understood and warrants study. The MPTP neurotoxin demonstrates preferential degeneration of the nigrostriatal pathway and recapitulates the striatal dopamine depletion and nigral dopaminergic cell loss that underlie motoric impairments in PD patients10. While this model does not reproduce the full cadre of nonmotor and motor PD behaviors and frank α-synuclein-positive Lewy body pathology, it has been useful to elucidate novel mechanistic targets that contribute to nigrostriatal damage and for early-stage translational testing as it is the best characterized noninvasive model available to reliably produce nigral cell death accompanied by striatal dopamine loss11-15. Wide use of the MPTP mouse, with paradigms ranging from acute, subacute to chronic16-18, has allowed for standardization of dosing to result in mild to severe nigrostriatal damage19,20 with activation of different mechanisms of toxicity depending on the treatment regimen18,21,22. Consequently, this permits a ‘window of lesioning’ to be targeted that may result in enhanced or reduced nigrostriatal injury depending on the therapeutic agent or transgenic model utilized23-25.
Also essential for translational and discovery biology studies are the techniques used to assess damage and the evidence such methods provide. For the MPTP mouse model, established metrics to evaluate lesioning are measurement of markers of striatal dopaminergic tone, including dopamine and its metabolites by HPLC, and Western blot analysis of tyrosine hydroxylase (TH), the rate-limiting enzyme in dopamine synthesis, and indicators of degenerative events such as glial activation using Western blot analysis and immunohistochemistry4. Although these are classical neurochemical, biochemical, and histological procedures, the techniques provide critical and reproducible readouts on the extent of damage within the nigrostriatal dopaminergic pathway, indicate mechanisms of toxicity, and have proven to be valuable tools in understanding degenerative events in PD.
Note: All animal procedures and animal care methods should be approved by the institution’s Institutional Animal Care and Usage Committee (IACUC). Study described here was performed in accordance to the guidelines established by SRI International’s IACUC.
1. Acquisition and maintenance of LOX-deficient mice
2. MPTP precautions, storage, preparation, decontamination and disposal
Note: MPTP intoxication from intravenous exposure in humans has been shown to cause parkinsonism10; MPTP is highly lipophilic and can readily cross the blood-brain barrier26. Precautionary measures should be taken to ensure safe handling, detoxification and disposal. Its metabolism involves multiple steps including conversion to 1-methyl-4-phenyl-2,3-dihydropyridinium by the enzyme monoamine oxidase B (MAO-B)27. MAO-B inhibitors may be utilized in case of accidental human intoxication.
3. MPTP administration
4. Tissue harvesting
5. Tissue processing
6. Immunoblotting
7. Neurochemistry
8. Immunohistochemistry
9. Statistics
This toxin exposure paradigm can produce a significant and detectable 20% striatal dopamine depletion in MPTP- vs. saline-injected animals. It is important to note that different lots of MPTP may yield slightly more or less lesioning; thus, for better precision, a preliminary experiment in wildtype mice is recommended prior to use in transgenics when a new lot of neurotoxin is utilized. The use of mild-to-moderate lesioning allows for impact of the transgene to be observed; a severe lesion may produce a 'floor effect' with injury too robust to attenuate or so damaging that it eclipses the effect of a deleterious genetic alteration. The effects of MPTP on striatal dopamine were significantly different in 5-LOX isozyme-deficient mice, but not the 12/15-LOX isozyme-deficient mice (Figure 1). Furthermore, with these methods, we were able to discern a significant difference in dopamine levels due to the 5-LOX deficiency in saline-treated mice (Figure 1).
Immunoblotting for TH and GFAP allowed for confirmation of damage and neuroinflammation, respectively, at the level of the striatum in wildtype mice, an effect that was diminished in the 5-LOX isozyme-deficient striatum (Figures 3A and 3B). The lesion is also discernible at the level of the substantia nigra (Figure 2A and 2B). In the same mice, the depletion of TH-positive neurons and increased GFAP immunoreactivity is observable using dual-label immunofluorescent labeling (Figure 2A). Furthermore, markedly elevated microglial activation (i.e. Iba1 immunoreactivity) in wildtype, but not 5-LOX isozyme-deficient, mice is apparent in the substantia nigra following MPTP exposure (Figure 2B). Thus, the MPTP model can provide a useful tool to assess the impact of genetic predisposition to nigral degeneration and inflammation.
Figure 1. LOX isozyme-selective effects on striatal dopamine after MPTP challenge. (A) Striatal homogenates were used to measure dopamine (DA) by HPLC from WT and 5-LOX-/- littermates given saline or MPTP (n=6-8/group). ‡, marks a significant effect due to genotype; p<0.05. *, marks a significant effect due to genotype and treatment; p<0.05. No significant difference due to treatment was noted in 5-LOX-/- mice (n.s.) indicating that MPTP did not produce striatal DA depletion in this transgenic line. (B) Striatal homogenates were used to measure DA in WT and 12/15-LOX-/- littermates given saline or MPTP (n=6-9/group). No significant difference in DA levels due to genotype was observed. A significant, and similar, reduction due MPTP treatment was noted in both genotypes. *, p<0.01. Data are shown as mean ± SEM.
Figure 2. 5-LOX isozyme effects on nigral TH and astroglia following MPTP challenge. (A) Immunofluorescence staining for TH (FITC; green) and GFAP (568; red) immunoreactivities was performed in nigral brain sections from WT and 5-LOX-/- littermates given saline or MPTP. Fewer TH-positive cell bodies (*) and enhanced GFAP immunoreactivity are apparent in the WT-MPTP group. Bar = 25 µm. (B) Immunohistochemical histology for the Iba-1 on microglia was detected using DAB (brown chromatgen); sections were counterstained by Cresyl violet. Nigral sections from WT and 5-LOX-/- littermates treated with saline or MPTP were assessed. Microglia with ramified cell bodies and long, branching processes are observed in substantia nigra from saline-treated mice (arrow heads). Activated microglia with rounded cell bodies and short, thickened processes, was observed in substantia nigra from MPTP-treated mice (arrows). Bar = 10 µm.
Figure 3. 5-LOX isozyme effects on striatal TH and inflammatory following toxic insult. (A) Striatal TH protein levels were semi-quantitatively measured by Western blot analyses of homogenate from WT and 5-LOX-/- littermates given saline or MPTP (n=6-8/group). Immunoreactivity, measured by optical density, was normalized to β-actin. *, p<0.05. (B) Similarly, GFAP was semi-quantitatively measured by Western blot analyses of striatal homogenate from WT and 5-LOX-/- littermates given with saline or MPTP (n=6-8/group) and normalized to β-actin. *, p<0.05. Data are noted as mean ± SEM.
The design of this gene-environment interaction study allowed us to gain new information regarding the dual nature of the 5-LOX isozyme in the nigrostriatal pathway. By performing HPLC to measure striatal monoamines after saline or MPTP treatment in transgenics lacking the 5-LOX isozyme and their wildtype littermates, we were able to note that its deficiency appears to be protective under toxic conditions (Figure 1), but under normal conditions, lack of the enzyme reduces striatal dopamine levels and may be deleterious. Thus, we are able to demonstrate that the 5-LOX isozyme contributes to striatal dopaminergic tone under normal conditions, but can contribute to damage following toxicant challenge4.
While further evaluation should lend novel mechanistic insights into the role of LOX isozymes in nigrostriatal toxicity, Western blot analyses (Figure 3) as well as immunohistochemical studies (Figure 2) revealed that neuroinflammation markers were, at least in part, attenuated in the 5-LOX isozyme-deficient cohort exposed to MPTP. These findings, using classical biochemical and histological techniques, indicate a critical role of 5-LOX products in potentiation of micro- and astro-glial activation4.
Depending on gene-environment interaction investigated, pathological readouts in addition to glial activation may be analyzed. Of particular importance in PD is loss of dopaminergic neurons in the substantia nigra and potentially pathological accumulation and aggregation of α-synuclein. Along this line, the impact of toxicant exposure in transgenic mice with α-synuclein overexpression has been monitored by evaluation of nigral cell death (i.e. using unbiased stereological cell counting) and insoluble α-synuclein deposition32-35.
The MPTP dose used in the paradigm described here produces a mild lesion with modest, but significant, striatal injury (Figure 1) and glial activation in both striatum and substantia nigra (Figures 2 and 3). Typically, higher doses of the toxicant are used to produce robust nigral dopaminergic cell loss and striatal dopamine depletion23,24,32,36-38,39. It is important to note that toxicity of MPTP can vary between vendors and lots; consequently doses may need to be adjusted to produce the desired lesion. Furthermore, other factors which must be considered are the strain and sex of animals utilized for the studies. Selective sensitivity to the neurotoxin has been demonstrated in distinct background strains of mice, a phenomenon due, at least in part, to differences in activation of subcellular pathways that mediate degeneration, including JNK and c-Jun36-39. Sex-dependent differences in MPTP toxicity have also been reported40,41, and may contribute to variability in studies using transgenic mice in which both sexes are used for poor-breeding lines. In such an instance, use of sex-matched wildtype controls is critical4. Such sex-related effects may account for the difference in lesioning observed in the WT mice between experiments testing the impact of 5- and 12/15-LOX-deficient mice in which one sex was used for one line and both sexes for the other (Figure 1). For gene-environment interaction studies, an MPTP challenge that produces severe injury (e.g. >80% reduction in striatal dopamine) is not recommended as this may mask a possible genetic effect.
While MPTP exposure produces nigral cell death3,42, striatal dopamine depletion3, complex I inhibition43-45, and glial activation46,47 that have been reported in human PD, in mouse, a slowly progressive degeneration that produces stable parkinsonism (i.e. motor deficits) and frank α-synuclein pathology (i.e. Lewy bodies and neurites) fully recapitulating hallmark features of the disease does not occur in the model. However, it is important to note that the MPTP mouse has played a critical role in understanding subcellular pathways that contribute to PD-related neurodegeneration. Variation in exposure paradigms, for example, has revealed activation of distinct mechanisms of toxicity: low-dose, subacute exposure promotes apoptotic cell death48 with a limited immune response49 whereas acute treatment with higher doses produces marked microglial activation50. Indeed, such factors should be considered when utilizing the model for efficacy studies and, relevant to the current study, to unmask the impact of a potential genetic risk factor.
The authors have nothing to disclose.
This work was funded by the National Institutes of Health NIGMS 056062.
1-Methyl-4-phenyl-1,2,3,6-tetra-hydropyridine hydrochloride (MPTP-HCL) | Sigma-Aldrich | M0896 | for PD modeling | |
4% Formaldehyde (paraformaldehyde) solution, phosphate-buffered (PFA) | American MasterTech Scientific | BUP0157 | for immersion fixation | |
Perchloric acid ACS reagent, 70% (PCA) | Sigma-Aldrich | 244252 | for HPLC acid extraction | |
Tris Base | Sigma-Aldrich | T1503 | for tissue homogenization | |
Ethylenediaminotetraacetic acid disodium salt dihydrate (EDTA) | Sigma-Aldrich | E1644 | for tissue homogenization | |
Protease inhibitor cocktail | Sigma-Aldrich | P8340 | for tissue homogenization | |
Phosphatase inhibitor cocktail | Sigma-Aldrich | P5726 | for tissue homogenization | |
Sodium Hydroxide (NaOH) | Sigma-Aldrich | S5881 | for Lowry protein assay | |
Sucrose, molecular biology, ≥99.5% (GC) | Sigma-Aldrich | S0389 | for cryoprotection | |
Phosphate buffered saline, powder, pH 7.4 (for 0.01 M PBS) | Sigma-Aldrich | P3813 | for IHC | |
BCA Protein Assay Kit | Pierce/Thermo | 23225 | for protein determination | |
Novex 12% Tris-Glycine Mini Gels 1.0 mm, 12-well | Invitrogen/Life Technologies | EC60052BOX | for SDS-PAGE | |
NuPAGE LDS Sample Buffer (4x) | Invitrogen/Life Technologies | NP0007 | for SDS-PAGE | |
Novex Sharp Prestained Protein Standard | Invitrogen/Life Technologies | LC5800 | protein ladder | |
Glycine | Sigma-Aldrich | G7126 | for SDS-PAGE | |
Sodium dodecyl sulfate, electrophoresis, 98.5% (SDS) | Sigma-Aldrich | L3771 | for SDS-PAGE | |
Methyl Alcohol, Anhydrous, Reagent | American MasterTech Scientific | SPM1057C | methanol for transfer | |
Sodium chloride (NaCl), ACS reagent | Sigma-Aldrich | S9888 | saline and buffers | |
Nonfat dry milk powder | Carnation | n/a | for immunoblotting | |
Ponceau S solution in 5% acetic acid | Sigma-Aldrich | P7170 | for immunoblotting | |
Anti-Tyrosine Hydroxylase (TH), sheep polyclonal | Chemicon/Millipore | AB1542 | for immunofluorescence | |
Anti-Tyrosine Hydroxylase (TH), rabbit polyclonal | Pel-Freez Biologicals | P40101-0 | for immunoblotting | |
Anti-β Actin, rabbit | Sigma-Aldrich | A2066 | for immunoblotting | |
Anti-Glial Fibrillary Acidic Protein (GFAP), rabbit polyclonal | Chemicon/Millipore | AB5804 | for immunofluorescence | |
Anti-Glial Fibrillary Acidic Protein (GFAP), mouse monoclonal | Covance Inc. | SMI-22R | for immunoblotting | |
Tween-20 | Sigma-Aldrich | P1379 | for immunoblotting | |
Goat Anti-Rabbit IgG (H+L), Peroxidase Conjugated | Fisher Scientific | 31462 | for immunofluorescence | |
goat anti-sheep, peroxidase conjugated | Pierce/Thermo | 31480 | for immunofluorescence | |
goat anti-mouse, peroxidase conjugated | Pierce/Thermo | 31430 | for immunofluorescence | |
SuperSignal West Pico Chemiluminescent Substrate | Pierce/Thermo | 34078 | for immunoblotting | |
CL-XPosure Film 7 in x 9.5 in | Pierce/Thermo | 34089 | for immunoblotting | |
Restore Western Blot Stripping Buffer | Pierce/Thermo | 21059 | for immunoblotting | |
Citric acid monohydrate, ACS reagent, ≥99.0% | Sigma-Aldrich | C1909 | for IHC | |
Normal Donkey Serum | Millipore | S30-100ML | for IHC | |
Polyvinylpyrrolidone (PVP) | Sigma-Aldrich | P5288 | for IHC | |
Bovine Serum Albumin (BSA), lyophilized | Sigma-Aldrich | A3294 | for IHC | |
Triton X-100 | Fisher Scientific | BP151-01 | for IHC | |
Donkey anti-Rabbit IgG, Alexa Fluor 568-labeled | Invitrogen/Life Technologies | A10042 | for IHC | |
Donkey Anti-Sheep IgG (H+L), FITC | Jackson ImmunoResearch | 713-095-147 | for IHC | |
VECTASHIELD Hard-Set Mounting Medium with DAPI | Vector Laboratories | H-1500 | for IHC | |
Normal Goat Serum | Millipore | S26-100ML | for IHC | |
VECTASTAIN ABC Kit (Rabbit IgG ) | Vector Laboratories | PK-4001 | for IHC; 10 µl each of solutions A and B per 1 ml PBS (per instructions ) | |
DAB Peroxidase Substrate Kit, 3,3’-diaminobenzidine | Vector Laboratories | SK-4100 | for IHC; per 5 ml cold ddH2O, add 2 drops buffer stock solution, 2 drops DAB, and 1 drop H2O2 (H2O2 is added immediately before use) | |
Hydrogen peroxide, 30% | Sigma-Aldrich | 216763 | for quench step in IHC | |
Rabbit anti-Iba1 | Biocare Medicals | CP290A | for IHC | |
Cresyl Violet Solution, Regular Strength | FD Neurotechnologies | PS102-01 | counterstain for Iba1 IHC | |
95% Ethanol, reagent alcohol | Sigma-Aldrich | R8382 | dehydration for IHC | |
100% Absolute ethanol | Mallinckrodt | 7019-10 | dehydration for IHC | |
Acetic acid | Sigma-Aldrich | A6283 | destaining for IHC | |
Xylene | Sigma-Aldrich | 534056 | clearing agent for IHC | |
DPX Mountant | Sigma-Aldrich | 06522 | mounting medium for DAB IHC | |
O.C.T. Compound – Frozen Section Embedding Medium | American MasterTech Scientific | EMOCTCS | embeddium medium for cryostat cutting | |
Potassium permanganate | Sigma-Aldrich | 223468 | to decontaminate DAB solution | |
Dopamine hydrochloride | Sigma-Aldrich | H8502 | for HPLC | |
3,4-Dihydroxyphenylacetic acid (DOPAC) | Sigma-Aldrich | 850217 | for HPLC | |
Homovanillic acid (HVA) | Sigma-Aldrich | H1252 | for HPLC | |
Perchloric acid (PCA) – 70% | Sigma-Aldrich | 244252 | for HPLC | |
Sodium dihydrogen phosphate monohydrate | Sigma-Aldrich | 71504 | for HPLC | |
Citric acid monohydrate | Sigma-Aldrich | C1909 | for HPLC | |
1-Octanesulfonic acid sodium salt (OSA) | Sigma-Aldrich | O8380 | for HPLC | |
EDTA | Sigma-Aldrich | E1644 | for HPLC | |
Acetonitrile | EMD | AX0145-1 | for HPLC | |
HPLC-grade distilled deionized water (ddH2O) | Millipore | for HPLC | ||
0.22 µm GSTF membrane | Millipore | for filtration | ||
Corning Netwells | Sigma-Aldrich | CLS3477 | polystyrene insert with polyester mesh bottom, for IHC | |
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Ultrasonic cell disrupter (Soniprep 150) | MSE | MSE.41371.274 | ||
Microcentrifuge | Eppendorf | 5414R | ||
ESA MD-150 reverse-phase column | ESA | |||
HPLC Pump (Ultimate 3000) | Dionex | ISO-3100BM | ||
HPLC Autosampler (Ultimate 3000) | Dionex | WPS-3000TSL | ||
Electrochemical detector | ESA | Coulochem III | ||
Guard Cell | ESA | 5020 | ||
Analytical Cell | ESA | 5011A | ||
Chromeleon software | Dionex | |||
Eclipse E400 | Nikon | E400 | light/fluorescent microscope | |
Disposable mouse cage | Ancare | N10HT | ||
Microfilter top | Ancare | N10MBT | ||
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5-LOX- deficient mice | The Jackson Laboratory | 004155 | ||
12/15-LOX-deficient mice | The Jackson Laboratory | 002778 |