Parkinson's disease is a neurodegenerative disorder which is in most cases of unknown etiology. Mutations of the Park-2 gene are the most frequent cause of familial parkinsonism and parkin knockout (PK-KO) mice have abnormalities that resemble the clinical syndrome. We investigated the interaction of genetic and environmental factors, treating midbrain neuronal cultures from PK-KO and wild-type (WT) mice with rotenone (ROT). ROT (0.025-0.1 lM) produced a dosedependent selective reduction of tyrosine hydroxylase-immunoreactive cells and of other neurons, as shown by the immunoreactivity to microtubule-associated protein 2 in PK-KO cultures, suggesting that the toxic effect of ROT involved dopamine and other types of neurons. Neuronal death was mainly apoptotic and suppressible by the caspase inhibitor t-butoxycarbonyl-Asp(OMe)-fluoromethyl ketone (Boc-D-FMK). PK-KO cultures were more susceptible to apoptosis induced by low doses of ROT than those from WT. ROT increased the proportion of astroglia and microglia more in PK-KO than in WT cultures. Indomethacin, a cyclo-oxygenase inhibitor, worsened the effects of ROT on tyrosine hydroxylase cells, apoptosis and astroglial (glial fibrillary acidic protein) cells. N-nitro-L-arginine methyl ester, an inhibitor of nitric oxide synthase, increased ROT-induced apoptosis but did not change tyrosine hydroxylase-immunoreactive or glial fibrillary acidic protein area. Neither indomethacin nor N-nitro-L-arginine methyl ester had any effect on the reduction by ROT of the mitochondrial potential as measured by 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide. Microglial NADPH oxidase inhibition, however, protected against ROT. The roles of p38 MAPK and extracellular signalregulated kinase signaling pathways were tested by treatment with SB20358 and PD98059, respectively. These compounds were inactive in ROT-naive cultures but PD98059 slightly increased cellular necrosis, as measured by lactate dehydrogenase levels, caused by ROT, without changing mitochondrial activity. SB20358 increased the mitochondrial failure and lactate dehydrogenase elevation induced by ROT. Minocycline, an inhibitor of microglia, prevented the dropout of tyrosine hydroxylase and apoptosis by ROT; the addition of microglia from PK-KO to WT neuronal cultures increased the sensitivity of dopaminergic neurons to ROT. PK-KO mice were more susceptible than WT to ROT and the combined effects of Park-2 suppression and ROT reproduced the cellular events observed in Parkinson's disease. These events were prevented by minocycline.

Young parkin null (pk)/)) mice have subtle abnormalities of behaviour, dopamine (DA) neurotransmission and free radical production, but no massive loss of DA neurons. We investigated whether these findings are maintained while ageing. Pk)/) mice have reduced life span and age-related reduced exploratory behaviour, abnormal walking and posture, and behaviours similar to those of early Parkinson's disease (PD), reduced number of nigrostriatal DA neurons and proapoptotic shifts in the survival/death proteins in midbrain and striatum. Contrary to young pk)/) animals 24-month-old pk)/) mice do not have compensatory elevation of GSH in striatum, glutathione reductase (GR) and glutathione peroxidase (GPx) activities are increased and catalase unchanged. Aged pk)/) mice accumulate high levels of tau and fail to up-regulate CHIP and HSP70. Our results suggest that aged pk)/) mice lack of the compensatory mechanisms that maintain a relatively normal DA function in early adulthood. This study could help to explain the effects of ageing in patients with genetic risks for Parkinson's disease.

Abnormal deposition of protein tau takes place in the brain of patients with several neurodegenerative diseases. Few of these patients present frontotemporal dementia with parkinsonism and amyotrophy (FTDPA-17), an autosomal dominant tauopathy related to mutations of the gene that codes for protein tau, localized in chromosome 17. The great majority of patients with tauopathies such as Alzheimer's disease, sporadic frontotemporal dementia or progressive supranuclear palsy do not show a Mendelian pattern of inheritance. We have occasionally seen tauopathies in patients with parkin mutations and, therefore, hypothesized that the protein tau interacts with parkin. We have tested that hypothesis in mice with combined genetic modifications of tau (over-expression of human tau with three mutations known to produce FTDPA-17) and parkin (deleted) proteins. Homozygote parkin null or over-expressing mutated-human tau mice have subtle behavioral and molecular abnormalities but do not express a clinical phenotype of neurodegenerative disease. Mice with combined homozygous mutations of these two genes show progressively abnormal walking already noticeable at 3 months of age, loss of dopamine and dopamine markers in striatum, nuclear tau immunoreactive deposits in motor neurons of the spinal cord, abnormal expression of glial markers and enhanced levels of pro-apoptotic proteins; findings that were absent or less pronounced in homozygote animals with deletions of parkin or over-expression of tau. The double transgenic mice do not express normal mechanisms of adaptation to stress such as increased levels of GSH and Hsp-70. In addition, they have reduced levels of CHIP-Hsc70, a complex known to attenuate aggregation of tau and to enhance ubiquitination of phosphorylated tau. We have found high levels of phosphorylated tau in parkin-/-+tau(VLW) mice and a relative decrease of the inactivated pSer9 to total GSK-3 levels. Our data reveal that there are interactions between tau and parkin that could be relevant for the pathogenesis and treatment of tauopathies. Similarly, we hope that the double transgenic parkin-/-+tau(VLW) mice could be useful for testing of compounds with putative therapeutic value in human tauopathies.

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.

To date, glutathione (GSH) depletion is the earliest biochemical alteration shown in brains of Parkinson's disease patients, but the role of GSH in dopamine cell survival is debated. In this study we show that GSH depletion, produced with GSH synthesis inhibitor, L-buthionine-(S,R)-sulfoximine (BSO), induces selectively neuronal cell death in neuron/glia, but not in neuronal-enriched midbrain cultures and that cell death occurs with characteristics of necrosis and apoptosis. BSO produces a dose-and time-dependent generation of reactive oxygen species (ROS) in neurons. BSO activates extracellular signal-regulated kinases (ERK-1/2), 4 and 6 h after treatment. MEK-1/2 and lipoxygenase (LOX) inhibitors, as well as ascorbic acid, prevent ERK-1/2 activation and neuronal loss, but the inhibition of nitric oxide sintase (NOS), cyclo-oxygenase (COX), c-Jun N-terminal kinase (JNK) and An early and highly selective decrease in glutathione (GSH) in the substantia nigra is present in Parkinson's disease (PD) (Riederer et al. 1989), and low levels of GSH lead to the degeneration of cultured dopaminergic neurons (Jenner and Olanow 1996;Canals et al. 2001aCanals et al. , 2003a. Reactive oxygen species (ROS) have been implicated in a number of neurological disorders including PD (Coyle and Puttfarcken 1993; Di Monte 2001). Glutathione depletion induces accumulation of ROS, leading to cell death (Murphy et al. 1989).Astrocytes play a very important function in the differentiation, survival, pharmacological properties and resistance to injury of dopamine (DA) neurons. The role of glia is mediated, at least in part, by the release of chemical substances to the media (Mena et al. 2002). Several studies suggest that glial cells may be important in the pathogenesis of PD, a common neurodegenerative disorder characterised by degeneration of the nigrostriatal DA system. In this disease, the role of glia could be because of excessive production of toxic products, such as nitric oxide (NO) or cytokines, characteristic of an inflammatory process (Hirsch Address correspondence and reprint requests to Dr M. A. Mena, Dpto. Investigació n, Hospital Ramó n y Cajal, Ctra. de Colmenar, Km. 9, Madrid 28034, Spain. E-mail: maria.a.mena@hrc.esAbbreviations used: BSO, L-buthionine-(S,R)-sulfoximine; COX, cyclo-oxygenase; DA, dopamine; DMEM, Dulbecco's modified Eagle's medium; DTNB, 5,5¢-dithio-bis2-nitrobenzoic acid; EMEM, Eagle's minimal essential medium; ERK, extracellular signal-regulated kinase; FCS, fetal calf serum; FITC, Fluorescein isothiocyanate; GSH, glutathione; iNOS, inducible nitric oxide sintase; JNK, c-Jun N-terminal kinase; LDH, lactate dehydrogenase standard; L-NAME, N-nitro-L-arginine methyl ester; LOX, lipoxygenase; MAP-2, anti-microtubuleassociated protein 2a + 2b; NDGA, nordihydroguaiaretic acid; NGS, normal goat serum; 7NI, 7-nitroindazole; NOS, nitric oxide sintase; p38 MAPK, p38 mitogen-activated protein kinase; PD, Parkinson's disease; ROS, reactive oxygen species; SDS, sodium dodecyl sulfate; TBS, Trisbuffered saline...