Susceptibility to rotenone is increased in neurons from parkin null mice and is reduced by minocycline

Abstract

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.

Reactive oxygen species generation by the ethylene-bis-dithiocarbamate (EBDC) fungicide mancozeb and its contribution to neuronal toxicity in mesencephalic cells

“…APO, which needs activation by peroxidases, inhibits the association of the NADPH oxidase cytosolic subunits (Stolk et al, 1994). It is the most commonly used NADPH oxidase inhibitor and has been reported to decrease NADPH oxidasemediated ROS in previous studies (Gao et al, 2002;Casarejos et al, 2006;Wang et al, 2006;Abramov et al, 2007). AEBSF, a non-specific serine protease inhibitor, deters NADPH oxidase activation by inhibiting the subunit p47 phox (Megyeri et al, 1995;Diatchuk et al, 1997).…”

Cytotoxicity of paraquat in microglial cells: Involvement of PKCδ- and ERK1/2-dependent NADPH oxidase

“…Minocycline, an antibiotic known to inhibit microglial proliferation, attenuated the neurotoxicity due to 6-hydroxydopamine, MPTP and rotenone (Casarejos et al, 2006;Croisier et al, 2005). These studies support the important role of microglia in the initiation and progression of PD.…”

“…Recent studies have provided strong evidence for microglia to produce ROS through activation of NADPH oxidase (Casarejos et al, 2006;Choi et al, 2005) and involvement of this enzyme in a number of neurodegenerative diseases (Sun et al, 2007). This enzyme is comprised of multiple subunits in the cytosol and plasma membrane and assembly of the subunits is initiated by phosphorylation of the cytosolic subunits, e.g., p47phox, p67phox, p40phox, and translocation to the membrane subunits, e.g., gp91phox, p22phox (Choi et al, 2005;Sumimoto et al, 2004).…”

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

“…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].…”

“…Parkin enhances the survival of the dopamine neurons [14,[17][18][19][20][21]. Parkin dysfunction impairs astrocytic function and contributes to the pathogenesis of parkin-linked AR-PD [22].…”