CB2 receptor activation prevents glial-derived neurotoxic mediator production, BBB leakage and peripheral immune cell infiltration and rescues dopamine neurons in the MPTP model of Parkinson's disease.

The cannabinoid (CB2) receptor type 2 has been proposed to prevent the degeneration of dopamine neurons in 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP)-treated mice. However, the mechanisms underlying CB2 receptor-mediated neuroprotection in MPTP mice have not been elucidated. The mechanisms underlying CB2 receptor-mediated neuroprotection of dopamine neurons in the substantia nigra (SN) were evaluated in the MPTP mouse model of Parkinson's disease (PD) by immunohistochemical staining (tyrosine hydroxylase, macrophage Ag complex-1, glial fibrillary acidic protein, myeloperoxidase (MPO), and CD3 and CD68), real-time PCR and a fluorescein isothiocyanate-labeled albumin assay. Treatment with the selective CB2 receptor agonist JWH-133 (10 µg kg(-1), intraperitoneal (i.p.)) prevented MPTP-induced degeneration of dopamine neurons in the SN and of their fibers in the striatum. This JWH-133-mediated neuroprotection was associated with the suppression of blood-brain barrier (BBB) damage, astroglial MPO expression, infiltration of peripheral immune cells and production of inducible nitric oxide synthase, proinflammatory cytokines and chemokines by activated microglia. The effects of JWH-133 were mimicked by the non-selective cannabinoid receptor WIN55,212 (10 µg kg(-1), i.p.). The observed neuroprotection and inhibition of glial-mediated neurotoxic events were reversed upon treatment with the selective CB2 receptor antagonist AM630, confirming the involvement of the CB2 receptor. Our results suggest that targeting the cannabinoid system may be beneficial for the treatment of neurodegenerative diseases, such as PD, that are associated with glial activation, BBB disruption and peripheral immune cell infiltration.

Inhibition of ROS-induced p38MAPK and ERK activation in microglia by acupuncture relieves neuropathic pain after spinal cord injury in rats.

Acupuncture (AP) is currently used worldwide to relieve pain. However, little is known about its mechanisms of action. We found that after spinal cord injury (SCI), AP inhibited the production of superoxide anion (O(2)·), which acted as a modulator for microglial activation, and the analgesic effect of AP was attributed to its anti-microglial activating action. Direct injection of a ROS scavenger inhibited SCI-induced NP. After contusion injury which induces the below-level neuropathic pain (NP), Shuigou and Yanglingquan acupoints were applied. AP relieved mechanical allodynia and thermal hyperalgesia, while vehicle and simulated AP did not. AP also decreased the proportion of activated microglia, and inhibited both p38MAPK and ERK activation in microglia at the L4-5. Also, the level of prostaglandin E(2) (PGE2), which is produced via ERK signaling and mediates the below-level pain through PGE2 receptor, was reduced by AP. Injection of p38MAPK or ERK inhibitors attenuated NP and decreased PGE2 production. Furthermore, ROS produced after injury-induced p38MAPK and ERK activation in microglia, and mediated mechanical allodynia and thermal hyperalgesia, which were inhibited by AP or a ROS scavenger. AP also inhibited the expression of inflammatory mediators. Therefore, our results suggest that the analgesic effect of AP may be partly mediated by inhibiting ROS-induced microglial activation and inflammatory responses after SCI and provide the possibility that AP can be used effectively as a non-pharmacological intervention for SCI-induced chronic NP in patients.

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.

IL-13-induced oxidative stress via microglial NADPH oxidase contributes to death of hippocampal neurons in vivo.

In the present study, we investigated the effects of IL-13, a well-known anti-inflammatory cytokine, on the thrombin-treated hippocampus in vivo. NeuN immunohistochemistry and Nissl staining revealed significant loss of hippocampal CA1 neurons upon intrahippocampal injection of thrombin. This neurotoxicity was accompanied by substantial microglial activation, as evident from OX-42 immunohistochemistry results. In parallel, Western blot analysis and hydroethidine histochemistry disclosed activation of NADPH oxidase, generation of reactive oxygen species, and oxidative damage in the hippocampal CA1 area showing hippocampal neuron degeneration. Interestingly, immunohistochemical and biochemical experiments showed that intrahippocampal injection of thrombin increased IL-13 immunoreactivity and IL-13 levels as early as 8 h after thrombin, reaching a peak at 7 days, which was maintained up to 14 days. Moreover, double-label immunohistochemistry revealed IL-13 immunoreactivity exclusively in activated microglia. IL-13-neutralizing Abs significantly rescued CA1 hippocampal neurons from thrombin neurotoxicity. In parallel, neutralization of IL-13 inhibited activation of NADPH oxidase, reactive oxygen species production, and oxidative damage. Additionally, IL-13 neutralization suppressed the expression of inducible NO synthase and several proinflammatory cytokines. To our knowledge, the present study is the first to show that IL-13 triggers microglial NADPH oxidase-derived oxidative stress, leading to the degeneration of hippocampal neurons in vivo, as occurs in cases of Alzheimer's disease.

Interleukin-4-induced oxidative stress via microglial NADPH oxidase contributes to the death of hippocampal neurons in vivo.

We investigated the effects of interleukin-4 (IL-4), a well-known anti-inflammatory cytokine, on thrombin-treated rat hippocampi in vivo. Intrahippocampal injection of thrombin resulted in a significant loss of hippocampal CA1 neurons, as determined by Nissl staining and NeuN immunohistochemistry. Thrombin-induced neurotoxicity was accompanied by substantial microglial activation, as demonstrated by OX-42 immunohistochemistry. In parallel, Western blot analysis and hydroethidine histochemistry revealed activation of NADPH oxidase (as demonstrated by increased translocation of the cytosolic proteins p67(phox) and p47(phox)), generation of reactive oxygen species (ROS), and oxidative damage in the hippocampal CA1 area, where degeneration of hippocampal neurons was evident. Interestingly, immunohistochemical and biochemical analysis demonstrated that intrahippocampal injection of thrombin increased immunoreactivity and levels of IL-4 as early as 8 h post-treatment, reaching a peak at 7 days that was maintained for up to 14 days. Moreover, double-label immunohistochemistry detected IL-4 immunoreactivity solely in activated microglia. In experiments to explore the involvement of IL-4 in neurotoxicity, IL-4-neutralizing antibodies significantly increased the survival of CA1 hippocampal neurons at 7 days post-thrombin treatment. Consistent with these results, IL-4 neutralization inhibited activation of NADPH oxidase, ROS production and oxidative damage. Thus, the present study is the first to demonstrate that IL-4 generates microglial NADPH oxidase-derived oxidative stress and leads to the degeneration of hippocampal neurons in vivo, as occurs in Alzheimer's disease.