An in vitro model of 1‐methyl‐4‐phenyl‐pyridinium (MPP+) toxicity: incubation of rabbit caudate nucleus slices with MPP+ followed by biochemical and functional analysis

Abstract

1 Slices of rabbit caudate nucleus were preincubated for up to 24h in vitro in the presence of the neurotoxic compound 1-methyl-4-phenyl-pyridinium (MPP+). Subsequently the levels of endogenous monoamines in the slices were determined by h.p.l.c. with electrochemical detection. MPP +, in concentrations higher than 32 nm significantly diminished the dopamine levels within the slices in a concentration-and time-dependent manner; at 32yM the depletion was more than 95%. The concentration of the major metabolite of dopamine, dihydroxyphenyl acetic acid (DOPAC) was decreased at concentrations of MPP+ that did not alter dopamine levels. Thus, MPP+ increased the dopamine/DOPAC ratio. 2 In contrast, both 5-hydroxytryptamine (5-HT) levels and 5-HT/5-hydroxyindolacetic acid (5-HIAA) ratios were increased at nanomolar concentrations of MPP +. 5-HT was significantly reduced only at 32 pM.3 The dopamine uptake inhibitor nomifensine reduced the depletory effect of MPP + on dopamine and DOPAC content. 4 Following 24 h pretreatment with MPP +, the uptake of [3H]-dopamine into rabbit caudate nucleus slices was either enhanced (at 0.32 pM, 1 pM and 3.2 pM MPP +) or reduced (at 32 JM MPP+).5 Preincubation of slices with 1OpM MPP + for only 1 h increased their 3H-labelling (in contrast to 24 h pretreatment) whereas after 9 h no net increase was detectable. After 1 and 9 h MPP + pretreatment, much less deaminated metabolites of [3H]-dopamine were found in the incubation medium of MPP+ treated slices than in the medium of control slices. These findings suggest that MPP + strongly inhibits the enzyme monoamine oxidase (MAO) within dopaminergic (and 5-hydroxytryptaminergic) (Snyder & D'Amato, 1986). In favour of the role of the substantia nigra in the pathogenesis it was assumed that within pigmented cell bodies of nigral neurones MPP+ was bound to neuromelanin and gradually released in a depot-like fashion, maintaining a toxic intracellular concentration of MPP +. However, since monoamine uptake is more prominent in nerve endings than in cell bodies (Snyder & D'Amato, 1986), the dopaminergic terminal field in the striatum rather than the cell bodies of the substantia nigra could be a primary target for MPP + toxicity.In the present investigation we studied the effects of long term incubation with MPP+ in caudate tissue of the rabbit in vitro in order to establish clear concentration-effect relationships and to provide a more accessible and less expensive experimental model than that of MPTP-induced Parkinsonism in primates in vivo. Acute dopamine releasing effects of MPTP and of MPP + in rat CNS tissue have already been described (Schmidt et al., 1984;Markstein & Lahaye, 1985;Rollema et al., 1986a). However, acute effects may not reflect adequately the toxic effects of MPP + on the dopaminergic system in vivo since, ultimately, the destruction of dopaminergic cells leads to MPTP-induced Parkinsonism.The analysis of subacute effects of MPTP or MPP + on cultured neuronal cells (Mytilineou & Cohen, 1984) Ci mmol-1) in a volume of 4ml...

The competitive NMDA antagonist CPP protects substantia nigra neurons from MPTP-induced degeneration in primates

“…However, the combination of MPTP and CPP caused a slight reduction in DOPAC and a significant decrease in HVA. The reduction of metabolite concentrations could be related to the fact the MPP + has MAO blocking properties (Feuerstein et al 1988). Taken together, these results indicate that CPP provided no protection against MPTP-induced dopamine depletion in the putamen.…”

A new technique for modeling neuronal connectivity using human pluripotent stem cells

“…For example, hPSC-derived mDA neurons have been used as a model of MPP+ toxicity and GDNF protection (Zeng et al, 2006), but MPP+ toxicity is known to involve the fine terminal arborization of mDA terminals in the striatum, which occur only in the presence of appropriate synaptic targets of mDA neurons as well (Feuerstetin et al, 1988), and thus such studies might be enhanced. Co-cultures of mDA neurons and striatal neurons for use in studies of MPP+ toxicity can be created from rodent fetal material (Koutsilieri et al, 1993), but this allows for similar studies using human material.…”

MPP+-induced toxicity in the presence of dopamine is mediated by COX-2 through oxidative stress

“…Thus, identification of the cellular factor that could facilitate or induce oxidation of dopamine would provide an attractive strategy in the understanding of the pathogenesis of dopaminergic degeneration in PD. MPP + possesses two opposing effects, on one hand it leads to an extensive release of dopamine and on the other hand MPP + inhibits monamine oxidase (MAO)-A (Feuerstein et al 1988 ), with MAO-B only slightly inhibited (Fritz et al 1985 ), thereby counteracting the oxidation of dopamine. A process by which dopamine oxidation still could occur is via COX-2, as COX-2 itself can lead to the generation of ROS (Smith et al 1991 ) and has been shown to react with dopamine to form dopamine-quinone (Teismann et al 2003a ).…”