Differential effects of l‐DOPA on monoamine metabolism, cell survival and glutathione production in midbrain neuronal‐enriched cultures from parkin knockout and wild‐type mice

Abstract

L-DOPA is the most effective treatment for Parkinson's disease but in isolated neuronal cultures it is neurotoxic for dopamine (DA) neurones. Experiments in vivo and clinical studies have failed to show toxicity of L-DOPA in animals or patients but that does not exclude the possibility of a toxic effect of L-DOPA on patients with certain genetic risk factors. Mutations of the parkin gene are the most frequent cause of hereditary parkinsonism. Parkin null mice have a mild phenotype that could be modified by different neurotoxins. The aim of this study was to investigate whether the toxic effects of L-DOPA on DA neurones are amplified in parkin null mice. We have measured the effects of L-DOPA on cell viability, tyrosine hydroxylase (TH) expression, DA metabolism and glutathione levels of parkin knockout (PK-KO) midbrain cultures. Neuronal-enriched cultures from PK-KO mice have similar proportions of the different cell types with the exception of a significant increment of microglial cells. L-DOPA (400 lM for 24 h) reduced the number of TH-immunoreactive cells to 50% of baseline and increased twofold the percentage of apoptotic cells in cultures of wild-type (WT) animals. The PK-KO mice, however, are not only resistant to the L-DOPA-induced pro-apoptotic effects but they have an increased number of TH-immunoreactive neurones after treatment with L-DOPA, suggesting that L-DOPA is toxic for neurones of WT mice but not those of parkin null mice. MAPK and phosphatidylinositol-3 kinase signalling pathways are not involved in the differential L-DOPA effects in WT and PK-KO cultures. Intracellular levels of L-DOPA were not different in WT and parkin null mice but the intracellular and extracellular levels of DA and 3-4-dihydroxyphenylacetic acid, however, were significantly increased in parkin null animals. Furthermore, monoamine oxidase activity was significantly increased in parkin null mice, suggesting that these animals have an increased metabolism of DA. The levels of glutathione were further increased in parkin null mice than in controls both with and without treatment with L-DOPA, suggesting that a compensatory mechanism may protect DA neurones from neuronal death. This study opens new avenues for understanding the mechanisms of action of L-DOPA on DA neurones in patients with Park-2 mutations.

MPTP and DSP-4 susceptibility of substantia nigra and locus coeruleus catecholaminergic neurons in mice is independent of parkin activity

“…Studies from parkin-deficient mouse models indicate small, but significant abnormalities specific to catecholaminergic neuronal systems. These include mitochondrial dysfunction and oxidative damage (Goldberg et al, 2003,Itier et al, 2003,Palacino et al, 2004,Casarejos et al, 2005, impairment in dopaminergic neurotransmission (Goldberg et al, 2003), increases in striatal dopamine turnover due to an increase in DOPAC/DA and DOPAC/ 3-MT levels, significant decreases in levels of striatal dopamine transporter (DAT) and vesicular monoamine transporter-2 (VMAT-2 (Itier et al, 2003), and loss of locus coeruleus neurons and deficits in noradrenergic-dependent acoustic startle response (Von Coelln et al, 2004b). Based on these findings we expected that challenging with the appropriate environmental trigger that specifically targets pathways already impaired in parkin-deficient mice would reveal and recapitulate robust behavioral and pathological features cardinal to PD.…”

Glial Dysfunction in Parkin Null Mice: Effects of Aging

“…PK-KO mice have higher MAO-B activity that their corresponding WT (Itier et al, 2003;Casarejos et al, 2005).…”

“…These mice present abnormal release and metabolism of dopamine and increased susceptibility to rotenone in early adulthood and severe dopamine cell loss while aging (Itier et al, 2003;Casarejos et al, 2005Casarejos et al, , 2006 For glial cultures, the ventral mesencephalon was removed from embryonic tissue (day 13 of gestation), diced in small fragments, and incubated in trypsin-EDTA (0.5% in HBSS) at 37°C for 15 min. Trypsinization was stopped by adding culture medium, and the tissue was gently centrifuged.…”

NP7 protects from cell death induced by oxidative stress in neuronal and glial midbrain cultures from parkin null mice

“…Parkin null mice, while young, are able to compensate the excessive free radical production, due to their abnormal metabolism of DA, with an increased production of glutathione (GSH) [10] and, therefore, are more resistant than wild-type (WT) to L-DOPA toxicity [12], which is also mediated by increased free radical production through the metabolism of catecholamines, or to other free radical producing neurotoxins such as nitric oxide [13]. But parkin null cells are more susceptible to additional insults such as treatment with rotenone, a mitochondrial toxin [14], calcium channel antagonists, such as cinnarizine [15] or to the effects of aging, when the supportive function of glia is reduced and the increased production of GSH collapses [16].…”