Systems analysis of genetic variation in MPTP neurotoxicity in mice

Highlights

• Genetic variation in the neurotoxicity of MPTP.

• On multiple endpoints related to dopamine neurobiology.

• In 10 recombinant inbred mouse strains.

• From a systems biology/genetics perspective.

Abstract

We analyzed genetic variation in severity of neuronal damage using the known dopaminergic neurotoxicant, MPTP, as a prototypical chemical denervation agent. Male mice from ten members of the BXD family of recombinant inbred strains received 12.5 mg/kg MPTP s.c. (vs. saline) and 48 h later brains were taken for multiple related biochemical analyses. Striatal dopamine (DA) and its metabolites, DOPAC and HVA, and serotonin and its metabolite, 5-HIAAA, were analyzed by HPLC. DA turnover was assessed using DOPAC/DA and HVA/DA ratios. Striatal tyrosine hydroxylase (TH), glial fibrilary acidic protein (GFAP), and iron content in ventral midbrain were quantified. All dopamine measures, as well as TH and GFAP, demonstrated wide, genotype-dependent differences in response to MPTP. Serotonin was largely unaffected. Principal components analysis (PC) on difference values, saline minus MPTP, for DA, DOPAC, HVA, and TH, yielded a dominant principal component. The PC trait residuals for each genotype were compared against complementary expression data for striatum of the same strains. Three transcripts representing Mtap2, Lancl 1, and Kansl1l were highly correlated with the PC, as was the difference score, MPTP minus saline for GFAP. This systems approach to the study of environmental neurotoxicants holds promise to define individual genetic differences that contribute to variability in susceptibility to risk factors for diseases such as Parkinson's disease.

Introduction

Exposure to various industrial and agricultural chemicals, especially pesticides, has been implicated as conferring risk for multiple diseases, including neurodegenerative disorders. Several of the substances implicated include rotenone, maneb, paraquat, carbamate and organophosphorus insecticides.

Interactions among multiple risk factors, including gene variants and environmental exposure (e.g., Kitada et al., 2012) are thought to underlie differential vulnerability to neurological disease. Although these factors and their interactions have not been defined, exposure to pesticides—usually associated with rural living—has been implicated as one key environmental cofactor. Epidemiological studies have been equivocal, and some have failed to find consistent associations between pesticide exposure and idiopathic or sporadic Parkinson's disease (sPD) (e.g., Li et al., 2005, van der Mark et al., 2012). If gene–environment interactions (GXE) are fundamental to understanding the etiology of diseases such as sPD, then ascertaining the right set of informative probands becomes problematic. Through the use of reference families of genetically diverse lines of mice, we can address the problems of the complex etiology of sPD and other environmentally related neurodegenerative diseases. This approach addresses the gene–environment interaction framework in which to assess models of disease sensitivity and severity. As proof of concept, the administration of model toxicants to cases with precisely defined genomes has proved to be highly useful (Taylor et al., 1973). One example is recombinant inbred (RI) rodents. RI strains typically are derived from two parental inbred strains by first making an F₁ cross and then inbreeding their offspring by many completely independent full sibling matings for 20 or more generations. This process redistributes (randomly segregates) allelic differences between the parents among a potentially large number of independent but genetically related progeny lines (Peirce et al., 2004). The aim is to develop a genetically diverse group of animals that can be used to model individual differences in genomes seen in genetically segregating populations, such as humans, and then to relate these genomic differences to phenotypic differences observed in nearly any normal or pathological bio-behavioral domain. The use of such a genetic reference population confers several advantages. First, in contrast to single gene mutant studies, the range of the phenotype of interest is revealed and thus some indication of individual differences. Second, the use of multiple strains together with multiple measures in one or more domains provides a powerful tool to examine experimental treatments from a systems biology perspective. Third, when genotyped, the reference population of inbred strains can be queried for polymorphisms that are associated with the phenotypes of interest and may lead to the identification of candidate genes that influence the trait.

As a model neurotoxicant, the proneurotoxin, 1-methyl-4-phenyl-1,2,3,6-tetrahydro-pyridine (MPTP) is a useful denervation tool because it damages the nigro-striatal dopaminergic pathway uniquely and selective antagonists are available to manipulate its neurotoxic effects. This has led to the use of MPTP in animal models of Parkinson's disease (PD); for our purposes, MPTP can be used to reproducibly damage a specific neuronal pathway, absent the influence of other factors, such as blood borne cytokines entering through a damaged blood–brain barrier (BBB) (O’Callaghan et al., 1990). MPTP is readily distributed to the brain without damaging the BBB where it is metabolized by monoamine oxidase-B by glial cells to 1-methyl-4-phenylpyridinium (MPP+) taken up into dopamine (DA) neurons via the DA transporter. MPP+ then disrupts the mitochondrial complex I of the electron transport chain, leading to the accumulation of free radicals which in turn destroy the neuron.

The primary objective of this research was to determine differences in susceptibility on a genetic basis, specifically, based in gene–environment interaction. There is evidence in mice for genetically based differences in MPTP neurotoxicity (Cook et al., 2003) and these authors reported a significantly associated marker (QTL) on chromosome 1 near the telomere. Others have identified QTL related to genetic differences in MPTP toxicity in mice on chromosomes 13 and 15 (Sedelis et al., 2003). In this study, we report genetic differences in the effect of MPTP on multiple neurochemical indices related to dopamine in the caudate–putamen in a random sample of 10 of the BXD family of RI strains derived from C57BL/6J and DBA/2J parental inbred strains. This is the first study of its kind to investigate strain-related differences in MPTP neurotoxicity in a panel of RI strains and using multiple outcome measures to begin to assemble a systems level perspective of MPTP neurotoxicity. Previously mentioned studies report effects on single measures and used F₂ or backcross techniques.