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.

Thrombin induces nigral dopaminergic neurodegeneration in vivo by altering expression of death-related proteins.

One week after intranigral injection of thrombin resulted in a dose-dependent loss of dopaminergic neurons (20-78%) in the rat substantia nigra (SN), as evidenced by tyrosine hydroxylase (TH) immunohistochemistry. This cell death was accompanied by localization of terminal deoxynucleotidyl transferase-mediated fluorecein UTP nick end labeling (TUNEL) staining within dopaminergic neurons, activation of caspase-3 and attenuation of dopaminergic neuronal cell death in the SN by the caspase inhibitor (zVAD-fmk), indicative of apoptosis. Furthermore, Western blot analyses and double-immunofluorescent staining showed activation of c-Jun N-terminal kinase (JNK) and p53, and a localization of p53 in the dopaminergic neurons in the SN after thrombin, respectively. Intriguingly, Western blot analyses demonstrated significant down-regulation of Bcl-2 protein, but no alteration in Bax protein expression in the SN after thrombin. Consistent with in vivo data, degeneration of dopaminergic neurons and colocalization of TUNEL and TH were observed in mesencephalic cultures, following treatment with thrombin. Cell death was almost completely abolished by the thrombin-specific inhibitor, hirudin. Thrombin receptor-activating peptides (TRAP-6 and-14) did not mimic the effects of thrombin, even at much higher (1,000 to 2,000-fold) concentrations, although expression of protease-activated receptor-1 (PAR-1) mRNA was detected using RT-PCR. Morphological evidence and molecular events in vivo and in vitro collectively suggest that thrombin induces apoptosis in dopaminergic neurons via non-PAR-1 receptors.