Inflachromene ameliorates Parkinson's disease by targeting Nrf2-binding Keap1.

Parkinson's disease (PD) is the most common neurodegenerative disease characterized by movement disorder. Despite current therapeutic efforts, PD progression and the loss of dopaminergic neurons in the substantia nigra remain challenging to prevent due to the complex and unclear molecular mechanism involved. We adopted a phenotype-based drug screening approach with neuronal cells to overcome these limitations. In this study, we successfully identified a small molecule with a promising therapeutic effect for PD treatment, called inflachromene (ICM), through our phenotypic screening strategy. Subsequent target identification using fluorescence difference in two-dimensional gel electrophoresis (FITGE) revealed that ICM ameliorates PD by targeting a specific form of Keap1. This interaction led to upregulating various antioxidants, including HO-1, NQO1, and glutathione, ultimately alleviating PD symptoms. Furthermore, ICM exhibited remarkable efficacy in inhibiting the loss of dopaminergic neurons and the activation of astrocytes and microglia, which are critical factors in PD pathology. Our findings suggest that the phenotypic approach employed in this study identified that ICM has potential for PD treatment, offering new hope for more effective therapeutic interventions in the future.

p21-activated kinase 4 controls the aggregation of α-synuclein by reducing the monomeric and aggregated forms of α-synuclein: involvement of the E3 ubiquitin ligase NEDD4-1.

Aggregation of misfolded alpha-synuclein (α-synuclein) is a central player in the pathogenesis of neurodegenerative diseases. Therefore, the regulatory mechanism underlying α-synuclein aggregation has been intensively studied in Parkinson's disease (PD) but remains poorly understood. Here, we report p21-activated kinase 4 (PAK4) as a key regulator of α-synuclein aggregation. Immunohistochemical analysis of human PD brain tissues revealed an inverse correlation between PAK4 activity and α-synuclein aggregation. To investigate their causal relationship, we performed loss-of-function and gain-of-function studies using conditional PAK4 depletion in nigral dopaminergic neurons and the introduction of lentivirus expressing a constitutively active form of PAK4 (caPAK4; PAK4S445N/S474E), respectively. For therapeutic relevance in the latter setup, we injected lentivirus into the striatum following the development of motor impairment and analyzed the effects 6 weeks later. In the loss-of-function study, Cre-driven PAK4 depletion in dopaminergic neurons enhanced α-synuclein aggregation, intracytoplasmic Lewy body-like inclusions and Lewy-like neurites, and reduced dopamine levels in PAK4DAT-CreER mice compared to controls. Conversely, caPAK4 reduced α-synuclein aggregation, as assessed by a marked decrease in both proteinase K-resistant and Triton X100-insoluble forms of α-synuclein in the AAV-α-synuclein-induced PD model. Mechanistically, PAK4 specifically interacted with the NEDD4-1 E3 ligase, whose pharmacological inhibition and knockdown suppressed the PAK4-mediated downregulation of α-synuclein. Collectively, these results provide new insights into the pathogenesis of PD and suggest PAK4-based gene therapy as a potential disease-modifying therapy in PD.

Interleukin 13 on Microglia is Neurotoxic in Lipopolysaccharide-injected Striatum in vivo.

To explore the potential function of interleukin-13 (IL-13), lipopolysaccharide (LPS) or PBS as a control was unilaterally microinjected into striatum of rat brain. Seven days after LPS injection, there was a significant loss of neurons and microglial activation in the striatum, visualized by immunohistochemical staining against neuronal nuclei (NeuN) and the OX-42 (complement receptor type 3, CR3), respectively. In parallel, IL-13 immunoreactivity was increased as early as 3 days and sustained up to 7 days post LPS injection, compared to PBS-injected control and detected exclusively within microglia. Moreover, GFAP immunostaining and blood brain barrier (BBB) permeability evaluation showed the loss of astrocytes and disruption of BBB, respectively. By contrast, treatment with IL-13 neutralizing antibody (IL-13NA) protects NeuN+ neurons against LPS-induced neurotoxicity in vivo . Accompanying neuroprotection, IL-13NA reduced loss of GFAP+ astrocytes and damage of BBB in LPS-injected striatum. Intriguingly, treatment with IL-13NA produced neurotrophic factors (NTFs) on survived astrocytes in LPS-injected rat striatum. Taken together, the present study suggests that LPS induces expression of IL-13 on microglia, which contributes to neurodegeneration via damage on astrocytes and BBB disruption in the striatum in vivo.

Control of hippocampal prothrombin kringle-2 (pKr-2) expression reduces neurotoxic symptoms in five familial Alzheimer's disease mice.

BACKGROUND AND PURPOSE: There is a scarcity of information regarding the role of prothrombin kringle-2 (pKr-2), which can be generated by active thrombin, in hippocampal neurodegeneration and Alzheimer's disease (AD). EXPERIMENTAL APPROACH: To assess the role of pKr-2 in association with the neurotoxic symptoms of AD, we determined pKr-2 protein levels in post-mortem hippocampal tissues of patients with AD and the hippocampi of five familial AD (5XFAD) mice compared with those of age-matched controls and wild-type (WT) mice, respectively. In addition, we investigated whether the hippocampal neurodegeneration and object memory impairments shown in 5XFAD mice were mediated by changes to pKr-2 up-regulation. KEY RESULTS: Our results demonstrated that pKr-2 was up-regulated in the hippocampi of patients with AD and 5XFAD mice, but was not associated with amyloid-ß aggregation in 5XFAD mice. The up-regulation of pKr-2 expression was inhibited by preservation of the blood-brain barrier (BBB) via addition of caffeine to their water supply or by treatment with rivaroxaban, an inhibitor of factor Xa that is associated with thrombin production. Moreover, the prevention of up-regulation of pKr-2 expression reduced neurotoxic symptoms, such as hippocampal neurodegeneration and object recognition decline due to neurotoxic inflammatory responses in 5XFAD mice. CONCLUSION AND IMPLICATIONS: We identified a novel pathological mechanism of AD mediated by abnormal accumulation of pKr-2, which functions as an important pathogenic factor in the adult brain via blood brain barrier (BBB) breakdown. Thus, pKr-2 represents a novel target for AD therapeutic strategies and those for related conditions.

p70S6K on astrocytes protects dopamine neurons from 1-methyl-4-phenylpyridinium neurotoxicity.

Our recent finding has demonstrated that astrocytes confer neuroprotection by endogenously producing ciliary neurotrophic factor (CNTF) via transient receptor potential vanilloid 1 (TRPV1) in Parkinson's disease (PD). In this study, the possible molecular target for TRPV1-mediated CNTF production and its neuroprotective effects on dopamine neurons were further investigated. For comparison, glial cell-line derived neurotrophic factor (GDNF) was also examined. The results show that TRPV1-ribosomal protein 70 S6 kinase (p70S6K) signaling on astrocytes produces endogenous CNTF in the SN of MPP+ -lesioned rat. By marked contrast, the expression of GDNF on astrocytes is independent of TRPV1-p70S6K signaling. Administration of a TRPV1 agonist, capsaicin, increases levels of phosphorylated p70S6K (p-p70S6K; activation of p70S6K) on astrocytes, resulting in the survival of dopamine neurons and behavioral recovery through endogenous production of CNTF in the MPP+ -lesioned rat model of PD. Immunohistochemical analysis reveals expression of p-p70S6K on astrocytes in the SN of PD patients, indicating relevance to human PD. The present in vivo data is the first to demonstrate that astrocytic TRPV1-p70S6K signaling plays a pivotal role as endogenous neuroprotective, and it may constitute a novel therapeutic target for treating PD.

Interleukin-13 Propagates Prothrombin Kringle-2-Induced Neurotoxicity in Hippocampi In Vivo via Oxidative Stress.

The present study investigated expression of endogenous interleukin-13 (IL-13) and its possible function in the hippocampus of prothrombin kringle-2 (pKr-2)-lesioned rats. Here we report that intrahippocampal injection of pKr-2 revealed a significant loss of NeuN-immunopositive (NeuN+) and Nissl+ cells in the hippocampus at 7 days after pKr-2. In parallel, pKr-2 increased IL-13 levels, which reached a peak at 3 days post pKr-2 and sustained up to 7 days post pKr-2. IL-13 immunoreactivity was seen exclusively in activated microglia/macrophages and neutrophils, but not in neurons or astrocytes. In experiments designed to explore the involvement of IL-13 in neurodegeneration, IL-13 neutralizing antibody (IL-13Nab) significantly increased survival of NeuN+ and Nissl+ cells. Accompanying neuroprotection, immunohistochemical analysis indicated that IL-13Nab inhibited pKr-2-induced expression of inducible nitric oxide synthase and myeloperoxidase within activated microglia/macrophages and neutrophils, possibly resulting in attenuation of reactive oxygen species (ROS) generation and oxidative damage of DNA and protein. The current findings suggest that the endogenous IL-13 expressed in pKr-2 activated microglia/macrophages and neutrophils might be harmful to hippocampal neurons via oxidative stress.

Dynamics of T Lymphocyte between the Periphery and the Brain from the Acute to the Chronic Phase Following Ischemic Stroke in Mice.

Stroke causes systemic immunosuppression. T lymphocytes are involved in infarct size in the early stages of stroke. However, the phenotypes of T lymphocytes and their functions in peripheral immune organs and the brain have not been well analyzed in the acute and chronic phases of stroke. Here, we investigated pathological phenotypic alterations in the systemic immune response, especially changes in T lymphocytes, from one day to six months after ischemic stroke in mice. Impairment in thymocyte numbers, development, proliferation, and apoptosis were observed for up to two weeks. The number of mature T cells in the spleen and blood decreased and showed reduced interferon-γ production. Increased numbers of CD4-CD8-CD3+ double-negative T cells were observed in the mouse brain during the early stages of stroke, whereas interleukin (IL)-10+Foxp3+ regulatory T lymphocytes increased from two weeks during the chronic phase. These phenotypes correlated with body weight and neurological severity scores. The recovery of T lymphocyte numbers and increases in IL-10+Foxp3+ regulatory T lymphocytes may be important for long-term neurological outcomes. Dynamic changes in T lymphocytes between the acute and chronic phases may play different roles in pathogenesis and recovery. This study provides fundamental information regarding the T lymphocyte alterations from the brain to the peripheral immune organs following stroke.

Prunus cerasoides Extract and Its Component Compounds Upregulate Neuronal Neuroglobin Levels, Mediate Antioxidant Effects, and Ameliorate Functional Losses in the Mouse Model of Cerebral Ischemia.

Prunus cerasoides (PC) has been reported to have antimicrobial and anti-inflammatory properties, but its potential as a neuroprotective agent in a mouse model of cerebral ischemia has not been explored. Considering neuroglobin (Ngb), an endogenous neuroprotective factor, as a novel approach to neuroprotection, in this study, Ngb promoter activity, Ngb expression changes, and antioxidant protection by PC extract (PCE) and PC component compounds (PCCs) were analyzed in oxygen-glucose deprivation (OGD)-treated neurons. In vivo analysis involved transient middle cerebral artery occlusion (tMCAO) in mice with pre- and post-treatment exposure to PCE. Following ischemic stroke induction, neurological behavior scores were obtained, and cellular function-related signals were evaluated in the ischemic infarct areas. In addition to PCE, certain component compounds from PCE also significantly increased Ngb levels and attenuated the intracellular ROS production and cytotoxicity seen with OGD in primary neurons. Administration of PCE reduced the infarct volume and improved neurological deficit scores in ischemic stroke mice compared with the vehicle treatment. Increased Ngb levels in infarct penumbra with PCE treatment were also accompanied by decreased markers of apoptosis (activated p38 and cleaved caspase-3). Our findings point to the benefits of Ngb-mediated neuroprotection via PCE and its antioxidant activity in an ischemic stroke model.

Interleukin-4-Mediated Oxidative Stress Is Harmful to Hippocampal Neurons of Prothrombin Kringle-2-Lesioned Rat In Vivo.

The present study investigated the effects of reactive microglia/macrophages-derived interleukin-4 (IL-4) on hippocampal neurons in prothrombin kringle-2 (pKr-2)-lesioned rats. pKr-2 was unilaterally injected into hippocampus in the absence or presence of IL-4 neutralizing antibody (IL-4Nab). Immunohistochemical analysis showed a significant loss of Nissl+ and NeuN+ cells and activation of microglia/macrophages (increase in reactive OX-42+ and OX-6+ cells) in the hippocampus at 7 days after pKr-2 injection. The levels of IL-4 expression were upregulated in the reactive OX-42+ microglia/macrophages as early as 1 day, maximal at 3 days and maintained up to 7 days after pKr-2 injection. Treatment with IL-4Nab significantly increased neuronal survival in pKr-2-treated CA1 layer of hippocampus in vivo. Accompanying neuroprotection, IL-4 neutralization inhibited activation of microglia/macrophages, reactive oxygen species-derived oxidative damages, production of myeloperoxidase- and inducible nitric oxide synthase-derived reactive nitrogen species and nitrosative damages as analyzed by immunohistochemistry and hydroethidine histochemistry. These results suggest that endogenous IL-4 expressed on reactive microglia/macrophages mediates oxidative/nitrosative stress and play a critical role on neurodegeneration of hippocampal CA1 layer in vivo.

Functional Crosstalk between CB and TRPV1 Receptors Protects Nigrostriatal Dopaminergic Neurons in the MPTP Model of Parkinson's Disease.

The present study examined whether crosstalk between cannabinoid (CB) and transient potential receptor vanilloid type 1 (TRPV1) could contribute to the survival of nigrostriatal dopamine neurons in the 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP) mouse model of Parkinson's disease (PD). MPTP induced a significant loss of nigrostriatal dopamine neurons and glial activation in the substantia nigra (SN) and striatum (STR) as visualized by tyrosine hydroxylase (TH) or macrophage antigen complex-1 (MAC-1) or glial fibrillary acidic protein (GFAP) immunocytochemistry, respectively. RT-PCR analysis shows the upregulation of inducible nitric oxide synthase, interleukin-1ß, and tumor necrosis factor-α in microglia in the SN in vivo, indicating the activation of the inflammatory system. By contrast, treatment with capsaicin (a specific TRPV1 agonist) increased the survival of dopamine neurons in the SN and their fibers and dopamine levels in the STR in MPTP mice. Capsaicin neuroprotection is accompanied by inhibiting MPTP-induced glial activation and production of inflammatory cytokines. Treatment with AM251 and AM630 (CB1/2 antagonists) abolished capsaicin-induced beneficial effects, indicating the existence of a functional crosstalk between CB and TRPV1. Moreover, treatment with anandamide (an endogenous agonist for both CB and TRVP1) rescued nigrostriatal dopamine neurons and reduced gliosis-derived neuroinflammatory responses in MPTP mice. These results suggest that the cannabinoid and vanilloid system may be beneficial for the treatment of neurodegenerative diseases, such as PD, that are associated with neuroinflammation.

Delayed Treatment of Capsaicin Produces Partial Motor Recovery by Enhancing Dopamine Function in MPP+-lesioned Rats via Ciliary Neurotrophic Factor.

Transient receptor potential vanilloid subtype 1 (TRPV1) on astrocytes prevents ongoing degeneration of nigrostriatal dopamine (DA) neurons in MPP+-lesioned rats via ciliary neurotrophic factor (CNTF). The present study determined whether such a beneficial effect of astrocytic TRPV1 could be achieved after completion of injury of DA neurons, rather than ongoing injury, which seems more relevant to therapeutics. To test this, the MPP+-lesioned rat model utilized here exhibited approximately 70~80% degeneration of nigrostriatal DA neurons that was completed at 2 weeks post medial forebrain bundle injection of MPP+. TRPV1 agonist, capsaicin (CAP), was intraperitoneally administered. CNTF receptor alpha neutralizing antibody (CNTFRαNAb) was nigral injected to evaluate the role of CNTF endogenously produced by astrocyte through TRPV1 activation on DA neurons. Delayed treatment of CAP produced a significant reduction in amphetamine-induced rotational asymmetry. Accompanying this behavioral recovery, CAP treatment increased CNTF levels and tyrosine hydroxylase (TH) activity in the substantia nigra pars compacta (SNpc), and levels of DA and its metabolites in the striatum compared to controls. Interestingly, behavioral recovery and increases in biochemical indices were not reflected in trophic changes of the DA system. Instead, behavioral recovery was temporal and dependent on the continuous presence of CAP treatment. The results suggest that delayed treatment of CAP increases nigral TH enzyme activity and striatal levels of DA and its metabolites by CNTF endogenously derived from CAP-activated astrocytes through TRPV1, leading to functional recovery. Consequently, these findings may be useful in the treatment of DA imbalances associated with Parkinson's disease.

Interleukin-4 and Interleukin-13 Exacerbate Neurotoxicity of Prothrombin Kringle-2 in Cortex In Vivo via Oxidative Stress.

The present study investigated the effects of activated microglia-derived interleukin-4 (IL-4) and IL-13 on neurodegeneration in prothrombin kringle-2 (pKr-2)-treated rat cortex. pKr-2 was unilaterally injected into the Sprague-Dawley rat cerebral cortex and IL-4 and IL-13 neutralizing antibody was used to block the function of IL-4 and IL-13. Immunohistochemical analysis showed a significant loss of NeuN+ and Nissl+ cells and an increase of OX-42+ cells in the cortex at seven days post pKr-2. The levels of IL-4 and IL-13 expression were upregulated in the activated microglia as early as 12 hours post pKr-2 and sustained up to seven days post pKr-2. Neutralization by IL-4 or IL-13 antibodies (NA) significantly increased neuronal survival in pKr-2-treated rat cortex in vivo by suppressing microglial activation and the production of reactive oxygen species, as analyzed by immunohisotochemistry and hydroethidine histochemistry. These results suggest that IL-4 and IL-13 that were endogenously expressed from reactive microglia may play a critical role on neuronal death by regulating oxidative stress during the neurodegenerative diseases, such as Alzheimer's disease and dementia.

Effects of Silibinin Against Prothrombin Kringle-2-Induced Neurotoxicity in the Nigrostriatal Dopaminergic System In Vivo.

Parkinson's disease (PD) and Alzheimer's disease exhibit common features of neurodegenerative diseases and can be caused by numerous factors. A common feature of these diseases is neurotoxic inflammation by activated microglia, indicating that regulation of microglial activation is a potential mechanism for preserving neurons in the adult brain. Recently, we reported that upregulation of prothrombin kringle-2 (pKr-2), one of the domains that make up prothrombin and which is cleaved and generated by active thrombin, induces nigral dopaminergic (DA) neuronal death through neurotoxic microglial activation in the adult brain. In this study, we show that silibinin, a flavonoid found in milk thistle, can suppress the production of inducible nitric oxide synthase and neurotoxic inflammatory cytokines, such as interleukin-1ß and tumor necrosis factor-α, after pKr-2 treatment by downregulating the extracellular signal-regulated kinase signaling pathway in the mouse substantia nigra. Moreover, as demonstrated by immunohistochemical staining, measurements of the dopamine and metabolite levels, and open-field behavioral tests, silibinin treatment protected the nigrostriatal DA system resulting from the occurrence of pKr-2-triggered neurotoxic inflammation in vivo. Thus, we conclude that silibinin may be beneficial as a natural compound with anti-inflammatory effects against pKr-2-triggered neurotoxicity to protect the nigrostriatal DA pathway and its properties, and thus, may be applicable for PD therapy.

Inhibition of Microglia-Derived Oxidative Stress by Ciliary Neurotrophic Factor Protects Dopamine Neurons In Vivo from MPP⁺ Neurotoxicity.

We demonstrated that capsaicin (CAP), an agonist of transient receptor potential vanilloid subtype 1 (TRPV1), inhibits microglia activation and microglia-derived oxidative stress in the substantia nigra (SN) of MPP⁺-lesioned rat. However, the detailed mechanisms how microglia-derived oxidative stress is regulated by CAP remain to be determined. Here we report that ciliary neurotrophic factor (CNTF) endogenously produced by CAP-activated astrocytes through TRPV1, but not microglia, inhibits microglial activation and microglia-derived oxidative stress, as assessed by OX-6 and OX-42 immunostaining and hydroethidine staining, respectively, resulting in neuroprotection. The significant increase in levels of CNTF receptor alpha (CNTFRα) expression was evident on microglia in the MPP⁺-lesioned rat SN and the observed beneficial effects of CNTF was abolished by treatment with CNTF receptor neutralizing antibody. It is therefore likely that CNTF can exert its effect via CNTFRα on microglia, which rescues dopamine neurons in the SN of MPP⁺-lesioned rats and ameliorates amphetamine-induced rotations. Immunohistochemical analysis revealed also a significantly increased expression of CNTFRα on microglia in the SN from human Parkinson's disease patients compared with age-matched controls, indicating that these findings may have relevance to the disease. These data suggest that CNTF originated from TRPV1 activated astrocytes may be beneficial to treat neurodegenerative disease associated with neuro-inflammation such as Parkinson's disease.

Correction to: Release of overexpressed CypB activates ERK signaling through CD147 binding for hepatoma cell resistance to oxidative stress.

The original version of this article contained a mistake. The bands for HA Tag and t-ERK in Figs. 2d, 2h, 3d are incorrect. The author informs that these errors had no influence in the scientific content of the paper. The corrected figures (Figs. 2 and 3) are given below.

Interleukin-4 Contributes to Degeneration of Dopamine Neurons in the Lipopolysaccharide-treated Substantia Nigra in vivo.

The present study investigated the effects of interleukin (IL)-4 on dopamine (DA) neurons in the substantia nigra (SN) in vivo of lipopolysaccharide (LPS)-treated rat. Tyrosine hydroxylase immunohistochemistry showed a significant loss of nigral DA neurons at 3 and 7 day post-LPS. In parallel, IL-4 immunoreactivity was upregulated as early as 1 day, reached a peak at 3 day and remained elevated at 7 day post-LPS. IL-4 immunoreactivity was detected exclusively in microglia. IL-4 neutralizing antibody (NA) significantly increased survival of DA neurons in LPS-treated SN in vivo by inhibiting microglial activation and production of proinflammatory mediator such as IL-1ß as assessed by immunihistochemical, RT-PCR and ELISA analysis, respectively. Accompanying neuroprotection are IL-4NA effects on decreased disruption of blood-brain barrier and astrocytes. The present data suggest that endogenously expressed IL-4 from reactive microglia may be involved in the neuropathological processes of degeneration of DA neurons occurring in Parkinson's disease.

Modulation of M1/M2 polarization by capsaicin contributes to the survival of dopaminergic neurons in the lipopolysaccharide-lesioned substantia nigra in vivo.

The present study examined the neuroprotective effects of capsaicin (CAP) and explored their underlying mechanisms in a lipopolysaccharide (LPS)-lesioned inflammatory rat model of Parkinson's dieases (PD). LPS was unilaterally injected into the substantia nigra (SN) in the absence or presence of CAP or capsazepine (CZP, a TRPV1 antagonist). The SN tissues were prepared for immunohistochemical staining, reverse transcriptase-polymerase chain reaction (RT-PCR) analysis, western blot analysis, blood-brain barrier (BBB) permeability evaluation, and reactive oxygen species (ROS) detection. We found that CAP prevented the degeneration of nigral dopamine neurons in a dose-dependent manner and inhibited the expression of proinflammatory mediators in the LPS-lesioned SN. CAP shifted the proinflammatory M1 microglia/macrophage population to an anti-inflammatory M2 state as demonstrated by decreased expression of M1 markers (i.e., inducible nitric oxide synthase; iNOS and interleukin-6) and elevated expression of M2 markers (i.e., arginase 1 and CD206) in the SN. RT-PCR, western blotting, and immunohistochemical analysis demonstrated decreased iNOS expression and increased arginase 1 expression in the CAP-treated LPS-lesioned SN. Peroxynitrate production, reactive oxygen species levels and oxidative damage were reduced in the CAP-treated LPS-lesioned SN. The beneficial effects of CAP were blocked by CZP, indicating TRPV1 involvement. The present data indicate that CAP regulated the M1 and M2 activation states of microglia/macrophage in the LPS-lesioned SN, which resulted in the survival of dopamine neurons. It is therefore likely that TRPV1 activation by CAP has therapeutic potential for treating neurodegenerative diseases, that are associated with neuroinflammation and oxidative stress, such as PD.

Upregulation of neuronal astrocyte elevated gene-1 protects nigral dopaminergic neurons in vivo.

The role of astrocyte elevated gene-1 (AEG-1) in nigral dopaminergic (DA) neurons has not been studied. Here we report that the expression of AEG-1 was significantly lower in DA neurons in the postmortem substantia nigra of patients with Parkinson's disease (PD) compared to age-matched controls. Similarly, decreased AEG-1 levels were found in the 6-hydroxydopamine (6-OHDA) mouse model of PD. An adeno-associated virus-induced increase in the expression of AEG-1 attenuated the 6-OHDA-triggered apoptotic death of nigral DA neurons. Moreover, the neuroprotection conferred by the AEG-1 upregulation significantly intensified the neurorestorative effects of the constitutively active ras homolog enriched in the brain [Rheb(S16H)]. Collectively, these results demonstrated that the sustained level of AEG-1 as an important anti-apoptotic factor in nigral DA neurons might potentiate the therapeutic effects of treatments, such as Rheb(S16H) administration, on the degeneration of the DA pathway that characterizes PD.

Protection of nigral dopaminergic neurons by AAV1 transduction with Rheb(S16H) against neurotoxic inflammation in vivo.

We recently reported that adeno-associated virus serotype 1 (AAV1) transduction of murine nigral dopaminergic (DA) neurons with constitutively active ras homolog enriched in brain with a mutation of serine to histidine at position 16 [Rheb(S16H)] induced the production of neurotrophic factors, resulting in neuroprotective effects on the nigrostriatal DA system in animal models of Parkinson's disease (PD). To further investigate whether AAV1-Rheb(S16H) transduction has neuroprotective potential against neurotoxic inflammation, which is known to be a potential event related to PD pathogenesis, we examined the effects of Rheb(S16H) expression in nigral DA neurons under a neurotoxic inflammatory environment induced by the endogenous microglial activator prothrombin kringle-2 (pKr-2). Our observations showed that Rheb(S16H) transduction played a role in the neuroprotection of the nigrostriatal DA system against pKr-2-induced neurotoxic inflammation, even though there were similar levels of pro-inflammatory cytokines, such as tumor necrosis factor-alpha (TNF-α) and interleukin-1-beta (IL-1ß), in the AAV1-Rheb(S16H)-treated substantia nigra (SN) compared to the SN treated with pKr-2 alone; the neuroprotective effects may be mediated by the activation of neurotrophic signaling pathways following Rheb(S16H) transduction of nigral DA neurons. We conclude that AAV1-Rheb(S16H) transduction of neuronal populations to activate the production of neurotrophic factors and intracellular neurotrophic signaling pathways may offer promise for protecting adult neurons from extracellular neurotoxic inflammation.

Norfluoxetine Prevents Degeneration of Dopamine Neurons by Inhibiting Microglia-Derived Oxidative Stress in an MPTP Mouse Model of Parkinson's Disease.

Neuroinflammation is the neuropathological feature of Parkinson's disease (PD) and causes microglial activation and activated microglia-derived oxidative stress in the PD patients and PD animal models, resulting in neurodegeneration. The present study examined whether norfluoxetine (a metabolite of fluoxetine) could regulate neuroinflammation in the 1-methyl-4-phenyl-1,2,3,6-tetrahydropypridine (MPTP) mouse model of PD and rescue dopamine neurons. Analysis by tyrosine hydroxylase (TH) immunohistochemistry demonstrated that norfluoxetine prevents degeneration of nigrostriatal dopamine neurons in vivo in MPTP-lesioned mice compared to vehicle-treated MPTP-lesioned control mice. MAC-1 immunostaining and hydroethidine histochemical staining showed that norfluoxetine neuroprotection is accompanied by inhibiting MPTP-induced microglial activation and activated microglia-derived reactive oxygen species production in vivo, respectively. In the separate experiments, treatment with norfluoxetine inhibited NADPH oxidase activation and nitrate production in LPS-treated cortical microglial cultures in vitro. Collectively, these in vivo and in vitro results suggest that norfluoxetine could be employed as a novel therapeutic agent for treating PD, which is associated with neuroinflammation and microglia-derived oxidative stress.