Fluoxetine prevents MPTP-induced loss of dopaminergic neurons by inhibiting microglial activation.

Parkinson's disease (PD) is characterized by degeneration of nigrostriatal dopaminergic (DA) neurons. Mice treated with MPTP (1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine) exhibit microglial activation-induced oxidative stress and inflammation, and nigrostriatal DA neuronal damage, and thus serve as an experimental model of PD. Here, we report that fluoxetine, one of the most commonly prescribed antidepressants, prevents MPTP-induced degeneration of nigrostriatal DA neurons and increases striatal dopamine levels with the partial motor recovery. This was accompanied by inhibiting transient expression of proinflammatory cytokines and inducible nitric oxide synthase; and attenuating microglial NADPH oxidase activation, reactive oxygen species/reactive nitrogen species production, and consequent oxidative damage. Interestingly, fluoxetine was found to protect DA neuronal damage from 1-methyl-4-phenyl-pyridinium (MPP(+)) neurotoxicity in co-cultures of mesencephalic neurons and microglia but not in neuron-enriched mesencephalic cultures devoid of microglia. The present in vivo and in vitro findings show that fluoxetine may possess anti-inflammatory properties and inhibit glial activation-mediated oxidative stress. Therefore, we carefully propose that neuroprotection of fluoxetine might be associated with its anti-inflammatory properties and could be employed as novel therapeutic agents for PD and other disorders associated with neuroinflammation and microglia-derived oxidative damage.

Fluoxetine prevents LPS-induced degeneration of nigral dopaminergic neurons by inhibiting microglia-mediated oxidative stress.

Lipopolysaccharide (LPS)-induced microglial activation causes degeneration of nigral dopaminergic (DA) neurons. Here, we examined whether fluoxetine prevents LPS-induced degeneration of DA in the rat substantia nigra (SN) in vivo. Seven days after LPS injection into the SN, immunostaining for tyrosine hydroxylase (TH) revealed a significant loss of nigral DA neurons. Parallel activation of microglia (visualized by OX-42 and ED1 immunohistochemistry), production of reactive oxygen species (ROS) (assessed by hydroethidine histochemistry), and degeneration of nigral DA neurons were also observed in the SN. Western blot analyses and double-label immunohistochemistry showed an increase in the expression of inducible nitric oxide synthase (iNOS) within activated microglia. LPS also induced translocation of p67(phox), the cytosolic component of NADPH oxidase, to the membrane of SN microglia, indicating activation of NADPH oxidase. The LPS-induced loss of nigral DA neurons was partially inhibited by fluoxetine, and the observed neuroprotective effects were associated with fluoxetine-mediated suppression of microglial NADPH oxidase activation and iNOS upregulation, and decreased ROS generation and oxidative stress. These results suggest that fluoxetine and analogs thereof may be beneficial for the treatment of neurodegenerative diseases, such as PD, that are associated with microglia-derived oxidative damage.

GT1b-induced neurotoxicity is mediated by the Akt/GSK-3/tau signaling pathway but not caspase-3 in mesencephalic dopaminergic neurons.

BACKGROUND: Gangliosides, sialic acid-containing glycosphingolipids exist in mammalian cell membranes particularly neuronal membranes. The trisialoganglioside (GT1b) is one of the major brain gangliosides and acts as an endogenous regulator in the brain. We previously showed GT1b induces mesencephalic dopaminergic (DA) neuronal death, both in vivo and in vitro. We further investigate the underlying mechanisms of GT1b neurotoxicity. RESULTS: Consistent with earlier findings, GT1b attenuated the DA neuron number and dopamine uptake level in mesencephalic cultures. Morphological evidence revealed GT1b-induced chromatin condensation and nuclear fragmentation as well as an increased number of TUNEL-positive cells, compared to control cultures. Interestingly, while GT1b enhanced caspase-3 activity, DEVD, a caspase-3 inhibitor, failed to rescue DA neuronal death. Immunoblot analysis revealed that GT1b inactivates Akt through dephosphorylation at both Ser473 and Thr308, subsequent dephosphorylation of GSK-3beta, a substrate of Akt, and hyperphosphorylation of tau, downstream of GSK-3beta. Moreover, a GSK-3beta specific inhibitor, L803-mt, attenuated tau phosphorylation and rescued DA neurons from cell death in mesencephalic cultures. CONCLUSION: Our data provide novel evidence that a Akt/GSK-3beta/tau-dependent, but not caspase-3 signaling pathway plays a pivotal role in GT1b-mediated neurotoxic actions on mesencephalic DA neurons.

Roles of transient receptor potential vanilloid subtype 1 and cannabinoid type 1 receptors in the brain: neuroprotection versus neurotoxicity.

Transient receptor potential vanilloid subtype 1 (TRPV1), also known as vanilloid receptor 1 (VR1), is a nonselective cation channel that is activated by a variety of ligands, such as exogenous capsaicin (CAP) or endogenous anandamide (AEA), as well as products of lipoxygenases. Cannabinoid type 1 (CB1) receptor belongs to the G protein-coupled receptor superfamily and is activated by cannabinoids such as AEA and exogenous Delta-9-tetrahydrocannabinol (THC). TRPV1 and CB1 receptors are widely expressed in the brain and play many significant roles in various brain regions; however, the issue of whether TRPV1 or CB1 receptors mediate neuroprotection or neurotoxicity remains controversial. Furthermore, functional crosstalk between these two receptors has been recently reported. It is therefore timely to review current knowledge regarding the functions of these two receptors and to consider new directions of investigation on their roles in the brain.

Neuroprotective effect of nicotine on dopaminergic neurons by anti-inflammatory action.

Epidemiological studies have reported that smoking is associated with a lower incidence of Parkinson's disease (PD), leading to theories that smoking in general and nicotine in particular might be neuroprotective. Recent studies suggested cholinergic anti-inflammatory pathway-regulating microglial activation through alpha7 nicotinic receptors. In the present study, we used lipopolysaccharide (LPS)-induced in vitro and in vivo inflammation models to investigate whether nicotine has a protective effect on the dopaminergic system through an anti-inflammatory mechanism. Nicotine pretreatment considerably decreased microglial activation with significant reduction of tumour necrosis factor (TNF)-alpha mRNA expression and TNF-alpha release induced by LPS stimulation. In co-cultures of microglia and mesencephalic neurons, nicotine pretreatment significantly decreased the loss of tyrosine hydroxylase-immunopositive (TH-ip) cells, approximately twice more than the LPS-only treatment. alpha-Bungarotoxin, an alpha7 nicotinic acetylcholine receptor subunit-selective blocker, considerably blocked the inhibitory effects of nicotine on microglial activation and TH-ip neuronal loss. Chronic nicotine pretreatment in rats showed that TH-ip neuronal loss induced by LPS stimulation in the substantia nigra was dramatically decreased, which was clearly accompanied by a reduction in the formation of TNF-alpha. The present study demonstrated that nicotine has a neuroprotective effect on dopaminergic neurons via an anti-inflammatory mechanism mediated by the modulation of microglial activation. Along with various neuroprotective effects of nicotine, the anti-inflammatory mechanism of nicotine could have a major therapeutic implication in the preventive treatment of PD.

Inhibition of thrombin-induced microglial activation and NADPH oxidase by minocycline protects dopaminergic neurons in the substantia nigra in vivo.

The present study shows that activation of microglial NADPH oxidase and production of reactive oxygen species (ROS) is associated with thrombin-induced degeneration of nigral dopaminergic neurons in vivo. Seven days after thrombin injection in the rat substantia nigra (SN), tyrosine hydroxylase immunocytochemistry showed a significant loss of nigral dopaminergic neurons. This cell death was accompanied by localization of terminal deoxynucleotidyl transferase-mediated fluorecein UTP nick-end labelling (TUNEL) staining within dopaminergic neurons. This neurotoxicity was antagonized by the semisynthetic tetracycline derivative, minocycline, and the observed neuroprotective effects were associated with the ability of minocycline to suppress NADPH oxidase-derived ROS production and pro-inflammatory cytokine expression, including interleukin-1beta and inducible nitric oxide synthase, from activated microglia. These results suggest that microglial NADPH oxidase may be a viable target for neuroprotection against oxidative damage.

Transient receptor potential vanilloid subtype 1 mediates cell death of mesencephalic dopaminergic neurons in vivo and in vitro.

Intranigral injection of the transient receptor potential vanilloid subtype 1 (TRPV1; also known as VR1) agonist capsaicin (CAP) into the rat brain, or treatment of rat mesencephalic cultures with CAP, resulted in cell death of dopaminergic (DA) neurons, as visualized by immunocytochemistry. This in vivo and in vitro effect was ameliorated by the TRPV1 antagonist capsazepine (CZP) or iodo-resiniferatoxin, suggesting the direct involvement of TRPV1 in neurotoxicity. In cultures, both CAP and anandamide (AEA), an endogenous ligand for both TRPV1 and cannabinoid type 1 (CB1) receptors, induced degeneration of DA neurons, increases in intracellular Ca2+ ([Ca2+]i), and mitochondrial damage, which were inhibited by CZP, the CB1 antagonist N-(piperidin-1-yl)-5-(4-iodophenyl)-1-(2,4-dichlorophenyl)-4-methyl-1H-pyrazole-3-carboxamide (AM251) or the intracellular Ca2+ chelator BAPTA/AM. We also found that CAP or AEA increased mitochondrial cytochrome c release as well as immunoreactivity to cleaved caspase-3 and that the caspase-3 inhibitor z-Asp-Glu-Val-Asp-fmk protected DA neurons from CAP- or AEA-induced neurotoxicity. Additional studies demonstrated that treatment of mesencephalic cultures with CB1 receptor agonist (6aR)-trans 3-(1,1-dimethylheptyl)-6a,7,10,10a-tetrahydro-1-hydroxy-6,6-dimethyl-6H-dibenzo[b,d] pyran-9-methanol (HU210) also produced degeneration of DA neurons and increases in [Ca2+]i, which were inhibited by AM251 and BAPTA/AM. The CAP-, AEA-, or HU210-induced increases in [Ca2+]i were dependent on extracellular Ca2+, with significantly different patterns of Ca2+ influx. Surprisingly, CZP and AM251 reversed HU210- or CAP-induced neurotoxicity by inhibiting Ca2+ influx, respectively, suggesting the existence of functional cross talk between TRPV1 and CB1 receptors. To our knowledge, this study is the first to demonstrate that the activation of TRPV1 and/or CB1 receptors mediates cell death of DA neurons. Our findings suggest that these two types of receptors, TRPV1 and CB1, may contribute to neurodegeneration in response to endogenous ligands such as AEA.