Gene-environment Interaction Models to Unmask Susceptibility Mechanisms in Parkinson's Disease

Summary

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.

Abstract

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.

Introduction

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 alone^1,2. 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 disord....

Protocol

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

1. Purchase 5-LOX-deficient or 12/15-LOX-deficient mice and respective strain and sex-matched controls at age 7-8 weeks and allow several days for acclimation to facility after arrival.
2. Maintain mice in group housing on a 12-h light-dark cycle and give ....

Results

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' wi.......

Discussion

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 an.......

Materials

List of materials used in this article

Name	Company	Catalog Number	Comments
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
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
5-LOX- deficient mice	The Jackson Laboratory	004155
12/15-LOX-deficient mice	The Jackson Laboratory	002778