Alterations in glutathione S-transferase pi expression following exposure to MPP+-induced oxidative stress in the blood of Parkinson’s disease patients

Parkinson’s disease (PD) is a progressive neurodegenerative disorder characterized by progressive loss of the pigmented dopaminergic (DA) neurons located in the substantia nigra pars compacta (SNpc) that results in a reduction in the number of its afferent projections to the striatum. PD motor symptoms first manifest when approximately 60% of the SNpc DA neurons have already died, and striatal dopamine is reduced by approximately 80% (esupp ref 1). Because the progression of cell loss is thought to occur over a somewhat protracted period of time in a defined spatiotemporal manner, the onset of PD symptoms is typically insidious. Biological models suggest that the progression of PD includes a long presymptomatic period, thus there is significant potential for interventions that could slow or even arrest PD at this stage of the disease. However, there is currently no appropriate diagnostic test or biomarker that can identify this presymptomatic population. One of the proposed etiologies of PD is that an increase in oxidative stress that overwhelms cellular protective mechanisms in the basal ganglia leads to SNpc DA neuron death. This is supported by postmortem studies showing increased production of reactive oxygen species (ROS) and deficient antioxidant capacity in Parkinson’s disease patients [1]. In the brain, the primary scavenger of free radicals is the endogenous antioxidant glutathione. The function of glutathione depends on two enzymes, glutathione peroxidase (GPx, EC 1.11.1.9) and glutathione S-transferase (GST, EC 2.5.1.18), each of which is responsible for the conjugation and removal of both H2O2 and products of lipid and dopamine oxidation (esuppl ref 2). Glutathione S-Transferases (GSTs) are a class of abundant proteins in eukaryotes that appear to function as xenobiotic metabolizing proteins. This class of enzymes may be viewed as a cellular defense against numerous man-made and naturally occurring environmental agents. Structurally, active GST is composed of homodimers containing one of 7 cytosolic subunit classes: alpha, mu, pi, theta, sigma, zeta and omega, 3 microsomal isoforms and 1 mitochondrial form [2]. Functionally, GSTs catalyze the reduction of free radicals by adding glutathione to electrophiles within any number of chemical structures including numerous by-products of oxidative stress, thereby rendering them more soluble and facilitating their elimination from the cell. In the basal ganglia, a double immunofluorescence study has shown that GSTpi is present in the DA neurons of the SNpc and that changes in its expression mediates sensitivity to MPTP [3] and rotenone [4]; thus any change in its activity may influence the ability of these neurons to handle oxidative stress. In addition to GSTpi’s ability to bind and detoxify electrophiles, the C-terminus of this transferase contains a motif that inhibits cJUN N-terminal kinase (JNK) activated signaling, blocking the phosphorylation of cJUN and apoptosis of the cell [5]. In this regard, MPTP induced activation of JNK and c-jun in mice has been shown to be strain dependant, with a sensitive strain showing earlier and more prolonged activation of cJUN and JNK compared to an MPTP- resistant mouse strain [6]. Together, these studies suggest that any change in the activity of GSTpi may underlie the ability of neurons in the SNpc to handle oxidative stress. Previous studies have reported that GSTpi levels are increased in substantia nigra of PD patients [4, 7], as well as in alpha-synuclein overexpressing neuroblastoma cells [8]. Polymorphisms in GSTpi have been associated with PD in a Drosophila model expressing mutant parkin [9] as well as having been linked to age of PD onset in individuals of Italian or Greek origin with the alpha-synuclein A53T (PARK1) mutation [10]. Furthermore, polymorphisms in GSTpi have been correlated with increased risk of PD after exposure to pesticides [esupp ref 3]. In addition to its expression in brain, GSTpi is expressed in numerous other tissues including blood [esupp ref4]. To determine if measurement of GSTpi in blood has the potential to be a biomarker for PD, we treated blood taken from PD patients and that of unaffected environmentally-matched cohorts (generally the spouse of the PD patient) with 1-methyl-4-phenylpyridinium (MPP+), an agent that has been shown to induce oxidative stress through blockade of mitochondrial Complex I following its uptake through the dopamine transporter (DAT) [esupp ref 5]. DAT is present in erythrocytes (esupp ref 6) and lymphocytes (esupp ref 7, 8), and thus this tissue has the potential to be affected by MPP+. GSTpi protein levels were measured in whole blood as well as isolated red blood cells (RBC), white blood cells (WBC), and plasma. We found no differences in GSTpi in plasma, whole blood or RBCs. However, we did find a significant change in GSTpi protein levels in the WBCs of PD patients. This suggests that GSTpi and potentially other members of this and other anti-oxidant families may be viable biomarkers for PD.