Gene transfer of a reserpine-sensitive mechanism of resistance to N-methyl-4-phenylpyridinium.

Abstract

The toxin N-methyl-1,2,3,6-tetrahydropyridine produces a model of neural degeneration very similar to idiopathic Parkinson disease. To understand the cellular mechanisms that modulate susceptibility to its active metabolite N-methyl-4-phenylpyridinium (MPP+), we have transfected a cDNA expression library from the relatively MPP+-resistant rat pheochromocytoma PC12 cells into MPP+-sensitive Chinese hamster ovary (CHO) fibroblasts. Selection of the stable transformants in high concentrations of MPP+ has yielded a clone extremely resistant to the toxin. Reserpine reverses the resistance to MPP+, suggesting that a transport activity protects against this form of toxicity, perhaps by sequestering the toxin within an intracellular compartment. In support of this hypothesis, dopamine loaded into the CHO transformant shows a localized distribution that is distinct from the pattern observed in wild-type cells and is also reversed by reserpine.Systemic administration of N-methyl-1,2,3,6-tetrahydropyridine (MPTP) results in the death of dopaminergic neurons in the substantia nigra (1). Since MPTP reproduces the pattern of neuronal degeneration observed in Parkinson disease (PD), this form of toxicity has been used as a model system to dissect the cellular components responsible for selective neuronal vulnerability in the idiopathic human disease. Toxicity requires conversion of MPTP to the active metabolite N-methyl-4-phenylpyridinium (MPP+) by monoamine oxidase B (2-4). Dopaminergic cells of the substantia nigra then accumulate high levels of MPP+ by uptake through the high-affinity plasma membrane catecholamine transporter (5,6). Inside the cell, MPP+ enters mitochondria (7) and inhibits respiration at the level of complex I, apparently by binding near the site of action of the other mitochondrial toxins rotenone and piericidin (8,9).Studies of patients with PD support the utility of MPTP as a model for idiopathic PD. Both brain tissue and circulating platelets from patients with PD show a selective reduction in the same mitochondrial component affected by MPTP, respiratory complex I (10-12). Deprenyl, which inhibits the enzyme that activates MPTP, monoamine oxidase, also appears to slow the rate of progression in idiopathic PD (13). However, parkinsonism induced by MPTP develops over days to weeks, whereas idiopathic PD develops over years. Explanations for the relatively slow rate of progression in idiopathic PD include chronic, low levels of exposure to an environmental toxin similar to MPTP, oxidative stress related to the cytoplasmic accumulation of dopamine, or the trapping of free radicals by deposited dopamine and lipofuscin that predisposes these cells to mitochondrial injury (14).The MPTP model has suggested multiple pathogenetic factors that may also contribute to idiopathic PD. The amelioration of toxicity with an excitatory amino acid antagonist implies that neural excitation plays a role in the injury produced by MPP+ in vivo (15). In addition, neurotrophic factors such as brain-derived neurotrophic...

Increased vesicular monoamine transporter enhances dopamine release and opposes Parkinson disease-related neurodegeneration in vivo

“…VMAT2 protects against the parkinsonisminducing neurotoxicant MPTP by sequestering its active metabolite, MPP + , into the vesicular lumen away from its site of action at complex I of the mitochondria (52-54). VMAT itself was first identified due to its protective effects against MPP + toxicity in culture (10,52). Our results show that increased VMAT2 confers significant protection from damage to the nigrostriatal dopamine system following MPTP dosing even in the presence of elevated dopamine levels.…”

“…The vesicular monoamine transporter 2 (VMAT2, SLC18A2) is responsible for the packaging of neurotransmitter into vesicles for subsequent release from monoaminergic neurons. VMAT2 is an H + -ATPase antiporter, which uses the vesicular electrochemical gradient to drive the packaging of cytosolic transmitter into small synaptic and dense core vesicles (8)(9)(10). VMAT2 is also essential for survival of dopamine neurons as cytosolic dopamine is neurotoxic (11,12).…”

Toxin Models of Mitochondrial Dysfunction in Parkinson's Disease

“…Once inside the neuron and during the acute phase of MPTP-induced death, there are a few routes that MPP + can take: it can enter the vesicular pathway, bind to vesicular monoamine transporters and translocate into synaptosomal vesicles (61), it can remain in the cytosol and interact with various cytosolic enzymes (53), or it can be concentrated within mitochondria via a mechanism dependent on mitochondrial transmembrane potential (80,84). Within the mitochondria, MPP + binds to complex 1, uncoupling the oxidation of NADH-linked substrates and consequently disrupting the flow of electrons along the ETC, resulting in decreased ATP production and increased generation of ROS (72).…”

Charged Residues in Transmembrane Domains II and XI of a Vesicular Monoamine Transporter Form a Charge Pair That Promotes High Affinity Substrate Recognition

“…This transport activity recognizes multiple monoamine neurotransmitters (dopamine, norepinephrine, and serotonin) with similar affinity and uses a proton electrochemical gradient (⌬ H ϩ, interior positive or acid) across the vesicle membrane to drive uptake, exchanging two protons inside the vesicle for one cytoplasmic amine (3,4). Despite the suggestion that a single protein is responsible for transport in both the brain and the periphery, two vesicular monoamine transporters (VMATs) 1 have recently been identified by expression cloning (5)(6)(7). VMAT1 occurs in the adrenal gland, whereas VMAT2 is expressed in the central nervous system (8,9).…”