Eugene Bok

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

Parkinsons 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.

Cannabinoid Receptor Type 1 Protects Nigrostriatal Dopaminergic Neurons against MPTP Neurotoxicity by Inhibiting Microglial Activation

This study examined whether the cannabinoid receptor type 1 (CB1) receptor contributes to the survival of nigrostriatal dopaminergic (DA) neurons in the 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP) mouse model of Parkinson’s disease. MPTP induced significant loss of nigrostriatal DA neurons and microglial activation in the substantia nigra (SN), visualized with tyrosine hydroxylase or macrophage Ag complex-1 immunohistochemistry. Real-time PCR, ELISA, Western blotting, and immunohistochemistry disclosed upregulation of proinflammatory cytokines, activation of microglial NADPH oxidase, and subsequent reactive oxygen species production and oxidative damage of DNA and proteins in MPTP-treated SN, resulting in degeneration of DA neurons. Conversely, treatment with nonselective cannabinoid receptor agonists (WIN55,212-2 and HU210) led to increased survival of DA neurons in the SN, their fibers and dopamine levels in the striatum, and improved motor function. This neuroprotection by cannabinoids was accompanied by suppression of NADPH oxidase reactive oxygen species production and reduced expression of proinflammatory cytokines from activated microglia. Interestingly, cannabinoids protected DA neurons against 1-methyl-4-phenyl-pyridinium neurotoxicity in cocultures of mesencephalic neurons and microglia, but not in neuron-enriched mesencephalic cultures devoid of microglia. The observed neuroprotection and inhibition of microglial activation were reversed upon treatment with CB1 receptor selective antagonists AM251 and/or SR14,716A, confirming the involvement of the CB1 receptor. The present in vivo and in vitro findings clearly indicate that the CB1 receptor possesses anti-inflammatory properties and inhibits microglia-mediated oxidative stress. Our results collectively suggest that the cannabinoid system is beneficial for the treatment of Parkinson’s disease 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.

NADPH Oxidase 1 Mediates α-Synucleinopathy in Parkinson's Disease

Accumulation of misfolded α-synuclein is the pathological hallmark of Parkinsons disease (PD). Nevertheless, little is known about the mechanism contributing to α-synuclein aggregation and its further toxicity to dopaminergic neurons. Since oxidative stress can increase the expression and aggregation levels of α-synuclein, NADPH oxidases (Noxs), which are responsible for reactive oxygen species generation, could be major players in α-synucleinopathy. Previously, we demonstrated that Nox1 is expressed in dopaminergic neurons of the PD animal models as well as postmortem brain tissue of PD patients, and is responsible for oxidative stress and subsequent neuronal degeneration. Here, using paraquat (PQ)-based in vitro and in vivo PD models, we show that Nox1 has a crucial role in modulating the behavior of α-synuclein expression and aggregation in dopaminergic neurons. We observed in differentiated human dopaminergic cells that Nox1 and α-synuclein expressions are increased under PQ exposure. Nox1 knockdown significantly reduced both α-synuclein expression and aggregation, supporting the role of Nox1 in this process. Furthermore, in rats exposed to PQ, the selective knockdown of Nox1 in the substantia nigra, using adeno-associated virus encoding Nox1-specific shRNA, largely attenuated the PQ-mediated increase of α-synuclein and ubiquitin expression levels as well as α-synuclein aggregates (proteinase K resistant) and A11 oligomers. Significant reductions in oxidative stress level and dopaminergic neuronal loss were also observed. Our data reveal a new mechanism by which α-synuclein becomes a neuropathologic protein through Nox1-mediated oxidative stress. This finding may be used to generate new therapeutic interventions that slower the rate of α-synuclein aggregation and the progression of PD pathogenesis.

MMP-3 Contributes to Nigrostriatal Dopaminergic Neuronal Loss, BBB Damage, and Neuroinflammation in an MPTP Mouse Model of Parkinson’s Disease

The present study examined whether matrix metalloproteinase-3 (MMP-3) participates in the loss of dopaminergic (DA) neurons in the nigrostriatal pathway in a 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP) mouse model of Parkinsons disease with blood brain barrier (BBB) damage and infiltration of peripheral immune cells. Tyrosine hydroxylase (TH) immunostaining of brain sections from MPTP-treated mice showed that MPTP induced significant degeneration of nigrostriatal DA neurons. Moreover, FITC-labeled albumin detection and immunostaining revealed that MPTP caused damage to the BBB and increased the number of ED-1- and CD-3-immunopositive cells in the substantia nigra (SN). Genetic ablation of MMP-3 reduced the nigrostriatal DA neuron loss and improved motor function. This neuroprotective effect afforded by MMP-3 deletion was associated with the suppression of BBB disruption and a decrease in the number of ED-1- and CD-3-immunopositive cells in the SN. These data suggest that MMP-3 could play a crucial role in neurodegenerative diseases such as PD in which BBB damage and neuroinflammation are implicated.

Cannabinoids prevent lipopolysaccharide-induced neurodegeneration in the rat substantia nigra in vivo through inhibition of microglial activation and NADPH oxidase

We investigated the effects of synthetic cannabinoids, WIN55,212-2 and HU210, on LPS-injected rat substantia nigra in vivo. Intranigral injection of LPS resulted in a significant loss of nigral dopaminergic (DA) neurons, as determined by Nissl staining and TH immunohistochemistry. LPS-induced neurotoxicity was accompanied by microglial activation, as demonstrated by OX-42 immunohistochemistry. In parallel, Western blot analysis, ELISA assay and hydroethidine histochemistry revealed activation of NADPH oxidase, as demonstrated by increased translocation of the cytosolic proteins p47(phox) and p67(phox), generation of reactive oxygen species (ROS) and increased level of proinflammatory cytokines (TNF-α and IL-1β), where degeneration of nigral DA neurons was evident. Interestingly, WIN55,212-2 and HU210 increased the survival of nigral DA neurons at 7days post-LPS treatment. Consistent with these results, cannabinoids inhibited activation of NADPH oxidase, ROS production and production of proinflammatory cytokines in the rat SN. The present data suggest that cannabinoids may be beneficial for the treatment of neurodegenerative diseases, such as PD, that are associated with microglial activation.

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 Δ-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.

Interactions between CB1 receptors and TRPV1 channels mediated by 12-HPETE are cytotoxic to mesencephalic dopaminergic neurons

We recently proposed the existence of neurotoxic interactions between the cannabinoid type 1 (CB1) receptor and transient receptor potential vanilloid 1 (TRPV1) channels in rat mesencephalic cultures. This study seeks evidence for the mediator(s) and mechanisms underlying the neurotoxic interactions between CB1 receptors and TRPV1 in vitro and in vivo.

Prothrombin kringle-2 induces death of mesencephalic dopaminergic neurons in vivo and in vitro via microglial activation

We have shown that prothrombin kringle‐2 (pKr‐2), a domain of human prothrombin distinct from thrombin could activate cultured rat brain microglia in vitro. However, little is known whether pKr‐2‐induced microglial activation could cause neurotoxicity on dopaminergic (DA) neurons in vivo. To address this question, pKr‐2 was injected into the rat substantia nigra (SN). Tyrosine hydroxylase (TH) immunohistochemistry experiments demonstrate significant loss of DA neurons seven days after injection of pKr‐2. In parallel, pKr‐2‐activated microglia were detected in the SN with OX‐42 and OX‐6 immunohistochemistry. Reverse transcription PCR and double‐label immunohistochemistry revealed that activated microglia in vivo exhibit early and transient expression of inducible nitric oxide synthase (iNOS), cyclooxygenase‐2 (COX‐2) and several proinflammatory cytokines. The pKr‐2‐induced loss of SN DA neurons was partially inhibited by the NOS inhibitor NG‐nitro‐L‐arginine methyl ester hydrochloride, and the COX‐2 inhibitor DuP‐697. Extracellular signal‐regulated kinase 1/2, c‐Jun N‐terminal kinase and p38 mitogen‐activated protein kinase were activated in the SN as early as 1 hr after pKr‐2 injection, and localized within microglia. Inhibition of these kinases led to attenuation of mRNA expression of iNOS, COX‐2 and several proinflammatory cytokines, and rescue of DA neurons in the SN. Intriguingly, following treatment with pKr‐2 in vitro, neurotoxicity was detected exclusively in co‐cultures of mesencephalic neurons and microglia, but not microglia‐free neuron‐enriched mesencephalic cultures, indicating that microglia are required for pKr‐2 neurotoxicity. Our results strongly suggest that microglia activated by endogenous compound(s), such as pKr‐2, are implicated in the DA neuronal cell death in the SN.

Tnfaip8 l1/Oxi-β binds to FBXW5, increasing autophagy through activation of TSC2 in a Parkinson's disease model.

Abnormal autophagy may contribute to neurodegeneration in Parkinsons disease (PD). However, it is largely unknown how autophagy is dysregulated by oxidative stress (OS), one of major pathogenic causes of PD. We recently discovered the potential autophagy regulator gene family including Tnfaip8/Oxi‐α, which is a mammalian target of rapamycin (mTOR) activator down‐regulated by OS in dopaminergic neurons (J. Neurochem., 112, 2010, 366). Here, we demonstrate that the OS‐induced Tnfaip8 l1/Oxi‐β could increase autophagy by a unique mechanism that increases the stability of tuberous sclerosis complex 2 (TSC2), a critical negative regulator of mTOR. Tnfaip8 l1/Oxi‐β and Tnfaip8/Oxi‐α are the novel regulators of mTOR acting in opposition in dopaminergic (DA) neurons. Specifically, 6‐hydroxydopamine (6‐OHDA) treatment up‐regulated Tnfaip8 l1/Oxi‐β in DA neurons, thus inducing autophagy, while knockdown of Tnfaip8 l1/Oxi‐β prevented significantly activation of autophagic markers by 6‐OHDA. FBXW5 was identified as a novel binding protein for Tnfaip8 l1/Oxi‐β. FBXW5 is a TSC2 binding receptor within CUL4 E3 ligase complex, and it promotes proteasomal degradation of TSC2. Thus, Tnfaip8 l1/Oxi‐β competes with TSC2 to bind FBXW5, increasing TSC2 stability by preventing its ubiquitination. Our data show that the OS‐induced Tnfaip8 l1/Oxi‐β stabilizes TSC2 protein, decreases mTOR phosphorylation, and enhances autophagy. Therefore, altered regulation of Tnfaip8 l1/Oxi‐β may contribute significantly to dysregulated autophagy observed in dopaminergic neurons under pathogenic OS condition.