Jau Shyong Hong

Aggregated α-synuclein activates microglia: a process leading to disease progression in Parkinson’s disease

A growing body of evidence indicates that an inflammatory process in the substantia nigra, characterized by activation of resident microglia, likely either initiates or aggravates nigral neurodegeneration in Parkinsons disease (PD). To study the mechanisms by which nigral microglia are activated in PD, the potential role of α‐synuclein (a major component of Lewy bodies that can cause neurodegeneration when aggregated) in microglial activation was investigated. The results demonstrated that in a primary mesencephalic neuron‐glia culture system, extracellular aggregated human α‐synuclein indeed activated microglia; microglial activation enhanced dopaminergic neurodegeneration induced by aggregated α‐synuclein. Furthermore, microglial enhancement of α‐synuclein‐mediated neurotoxicity depended on phagocytosis of α‐synuclein and activation of NADPH oxidase with production of reactive oxygen species. These results suggest that nigral neuronal damage, regardless of etiology, may release aggregated α‐synuclein into substantia nigra, which activates microglia with production of proinflammatory mediators, thereby leading to persistent and progressive nigral neurodegeneration in PD. Finally, NADPH oxidase could be an ideal target for potential pharmaceutical intervention, given that it plays a critical role in α‐synuclein‐mediated microglial activation and associated neurotoxicity.—Zhang, W., Wang, T., Pei, Z., Miller, D. S., Wu, X., Block, M. L., Wilson, B., Zhang, W., Zhou, Y., Hong, J. S., Zhang, J. Aggregated α‐synuclein activates microglia: a process leading to disease progression in Parkinsons disease. FASEB J. 19, 533–542 (2005)

Histone deacetylase inhibitors up-regulate astrocyte GDNF and BDNF gene transcription and protect dopaminergic neurons

Parkinsons disease (PD) is characterized by the selective and progressive loss of dopaminergic (DA) neurons in the midbrain substantia nigra. Currently, available treatment is unable to alter PD progression. Previously, we demonstrated that valproic acid (VPA), a mood stabilizer, anticonvulsant and histone deacetylase (HDAC) inhibitor, increases the expression of glial cell line-derived neurotrophic factor (GDNF) and brain-derived neurotrophic factor (BDNF) in astrocytes to protect DA neurons in midbrain neuron-glia cultures. The present study investigated whether these effects are due to HDAC inhibition and histone acetylation. Here, we show that two additional HDAC inhibitors, sodium butyrate (SB) and trichostatin A (TSA), mimic the survival-promoting and protective effects of VPA on DA neurons in neuron-glia cultures. Similar to VPA, both SB and TSA increased GDNF and BDNF transcripts in astrocytes in a time-dependent manner. Furthermore, marked increases in GDNF promoter activity and promoter-associated histone H3 acetylation were noted in astrocytes treated with all three compounds, where the time-course for acetylation was similar to that for gene transcription. Taken together, our results indicate that HDAC inhibitors up-regulate GDNF and BDNF expression in astrocytes and protect DA neurons, at least in part, through HDAC inhibition. This study indicates that astrocytes may be a critical neuroprotective mechanism of HDAC inhibitors, revealing a novel target for the treatment of psychiatric and neurodegenerative diseases.

Diesel exhaust particles induce oxidative stress, proinflammatory signaling, and P-glycoprotein up-regulation at the blood-brain barrier

Here, we report that diesel exhaust particles (DEPs), a major constituent of urban air pollution, affect blood‐brain barrier function at the tissue, cellular, and molecular levels. Isolated rat brain capillaries exposed to DEPs showed increased expression and transport activity of the key drug efflux transporter, P‐glycoprotein (6 h EC50 was ~5 μg/ml). Upregulation of P‐glycoprotein was abolished by blocking transcription or protein synthesis. Inhibition of NADPH oxidase or pretreatment of capillaries with radical scavengers ameliorated DEP‐induced P‐glycoprotein up‐regulation, indicating a role for reactive oxygen species in signaling. DEP exposure also increased brain capillary tumor necrosis factor‐α (TNF‐α) levels. DEP‐induced P‐glycoprotein up‐regulation was abolished when TNF‐receptor 1 (TNF‐R1) was blocked and was not evident in experiments with capillaries from TNF‐R1 knockout mice. Inhibition of JNK, but not NF‐κB, blocked DEP‐induced P‐glycoprotein up‐regulation, indicating a role for AP‐1 in the signaling pathway. Consistent with this, DEPs increased phosphorylation of c‐jun. Together, our results show for the first time that a component of air pollution, DEPs, alters blood‐brain barrier function through oxidative stress and proinflammatory cytokine production. These experiments disclose a novel blood‐brain barrier signaling pathway, with clear implications for environmental toxicology, CNS pathology, and the pharmacotherapy of CNS disorders.—Hartz, A. M. S., Bauer, B., Block, M. L., Hong, J.‐S., Miller, D.‐S. Diesel exhaust particles induce oxidative stress, proinflammatory signaling, and P‐glycoprotein up‐regulation at the blood‐brain barrier. FASEB J. 22, 2723–2733 (2008)

Role of oxidative stress in epileptic seizures

Oxidative stress resulting from excessive free-radical release is likely implicated in the initiation and progression of epilepsy. Therefore, antioxidant therapies aimed at reducing oxidative stress have received considerable attention in epilepsy treatment. However, much evidence suggests that oxidative stress does not always have the same pattern in all seizures models. Thus, this review provides an overview aimed at achieving a better understanding of this issue. We summarize work regarding seizure models (i.e., genetic rat models, kainic acid, pilocarpine, pentylenetetrazol, and trimethyltin), oxidative stress as an etiologic factor in epileptic seizures (i.e., impairment of antioxidant systems, mitochondrial dysfunction, involvement of redox-active metals, arachidonic acid pathway activation, and aging), and antioxidant strategies for seizure treatment. Combined, this review highlights pharmacological mechanisms associated with oxidative stress in epileptic seizures and the potential for neuroprotection in epilepsy that targets oxidative stress and is supported by effective antioxidant treatment.

Interactive role of the toll-like receptor 4 and reactive oxygen species in LPS-induced microglia activation

Microglia are activated by lipopolysaccharide (LPS) to produce neurotoxic pro‐inflammatory factors and reactive oxygen species (ROS). While a multitude of LPS receptors and corresponding pathways have been identified, the detailed mechanisms mediating the microglial response to LPS are unclear. Using mice lacking a functional toll‐like receptor 4 (TLR4), we demonstrate that TLR4 and ROS work in concert to mediate microglia activation, where the contribution from each pathway is dependent on the concentration of LPS. Immunocytochemical staining of microglia in neuron–glia cultures with antibodies against F4/80 revealed that while TLR4+/+ microglia were activated the low concentration of 1 ng/ml of LPS, TLR4−/− microglia exhibit activated morphology in response to LPS only at higher concentrations (100–1,000 ng/ml). Additionally, tumor necrosis factor‐α (TNF‐α) was only produced from higher concentrations (100–1,000 ng/ml) of LPS in TLR4−/− enriched microglia cultures. Diphenylene iodonium (DPI), an inhibitor of NADPH oxidase, reduced TNF‐α production from TLR4−/− microglia. The influence of TLR4 on LPS‐induced superoxide production was tested in rat enriched microglia cultures, where the presence or absence of serum failed to show any effect on the superoxide production. Further, both TLR4−/− and TLR4+/+ microglia showed a similar increase in extracellular superoxide production when exposed to LPS (1–1,000 ng/ml). These data indicate that LPS‐induced superoxide production in microglia is independent of TLR4 and that ROS derived from the production of extracellular superoxide in microglia mediates the LPS‐induced TNF‐α response of both the TLR4‐dependent and independent pathway.

Femtomolar concentrations of dextromethorphan protect mesencephalic dopaminergic neurons from inflammatory damage.

Inflammation in the brain has increasingly been recognized to play an important role in the pathogenesis of several neurodegenerative disorders, including Parkinsons disease (PD). Progress in the search for effective therapeutic strategies that can halt this degenerative process remains limited. We previously showed that micromolar concentrations of dextromethorphan (DM), a major ingredient of widely used antitussive remedies, reduced the inflammation‐mediated degeneration of dopaminergic neurons through the inhibition of microglial activation. In this study, we report that femto‐ and micromolar concentrations of DM (both pre‐ and post‐treatment) showed equal efficacy in protecting lipopolysaccharide (LPS)‐induced dopaminergic neuron death in midbrain neuron‐glia cultures. Both concentrations of DM decreased LPS‐induced release of nitric oxide, tumor necrosis factor‐α, prostaglandin E2 and superoxide from microglia in comparable degrees. The important role of superoxide was demonstrated by DMs failure to show a neuroprotective effect in neuron‐glia cultures from NADPH oxidase‐deficient mice. These results suggest that the neuroprotective effect elicited by femtomolar concentrations of DM is mediated through the inhibition of LPS‐induced proinflammatory factors, especially superoxide. These findings suggest a novel therapeutic concept of using “ultra‐low” drug concentrations for the intervention of inflammation‐related neurodegenerative diseases.—Li, G., Cui, G., Tzeng, N.‐S., Wei, S.‐J., Wang, T., Block, M. L., Hong, J.‐S. Femtomolar concentrations of dextromethorphan protect mesencephalic dopaminergic neurons from inflammatory damage. FASEB J. 19, 489–496 (2005)

Microglial NADPH oxidase is a novel target for femtomolar neuroprotection against oxidative stress

Inflammation has been increasingly recognized to contribute to the pathogenesis of Parkinsons disease. Several compounds are neuroprotective at femtomolar concentrations through the inhibition of inflammation. However, the mechanisms mediating femtomolar‐acting compounds are poorly understood. Here we show that both gly‐gly‐phe (GGF), a tri‐peptide contained in the dynorphin opioid peptide, and naloxone are neuroprotective at femtomolar concentrations against LPS‐induced dopaminergic neurotoxicity through the reduction of microglial activation. Mechanistic studies demonstrated the critical role of NADPH oxidase in the GGF and naloxone inhibition of microglial activation and associated DA neurotoxicity. Pharmacophore analysis of the neuroprotective dynorphin peptides and naloxone revealed common chemical properties (hydrogen bond acceptor, hydrogen bond donor, positive ionizable, hydrophobic) of these femtomolar‐acting compounds. These results support a common high‐affinity site of action for several femtomolar‐acting compounds, where NADPH oxidase is the critical mechanism governing neuroprotection, suggesting a novel avenue of anti‐inflammatory and neuroprotective therapy.—Qin, L., Block, M. L., Liu, Y., Bienstock, R. J., Pei, Z., Zhang, W., Wu, X., Wilson, B., Burka, T., Hong, J.‐S. Microglial NADPH oxidase is a novel target for femtomolar neuroprotection against oxidative stress. FASEB J. 19, 550–557 (2005)

Reactive microgliosis: extracellular μ-calpain and microglia-mediated dopaminergic neurotoxicity

Microglia, the innate immune cells in the brain, can become chronically activated in response to dopaminergic neuron death, fuelling a self-renewing cycle of microglial activation followed by further neuron damage (reactive microgliosis), which is implicated in the progressive nature of Parkinsons disease. Here, we use an in vitro approach to separate neuron injury factors from the cellular actors of reactive microgliosis and discover molecular signals responsible for chronic and toxic microglial activation. Upon injury with the dopaminergic neurotoxin 1-methyl-4-phenylpyridinium, N27 cells (dopaminergic neuron cell line) released soluble neuron injury factors that activated microglia and were selectively toxic to dopaminergic neurons in mixed mesencephalic neuron-glia cultures through nicotinamide adenine dinucleotide phosphate oxidase. mu-Calpain was identified as a key signal released from damaged neurons, causing selective dopaminergic neuron death through activation of microglial nicotinamide adenine dinucleotide phosphate oxidase and superoxide production. These findings suggest that dopaminergic neurons may be inherently susceptible to the pro-inflammatory effects of neuron damage, i.e. reactive microgliosis, providing much needed insight into the chronic nature of Parkinsons disease.

MAC1 mediates LPS-induced production of superoxide by microglia: The role of pattern recognition receptors in dopaminergic neurotoxicity

Microglia‐derived superoxide is critical for the inflammation‐induced selective loss of dopaminergic (DA) neurons, but the underlying mechanisms of microglial activation remain poorly defined. Using neuron‐glia and microglia‐enriched cultures from mice deficient in the MAC1 receptor (MAC1−/−), we demonstrate that lipopolysaccharide (LPS) treatment results in lower TNFα response, attenuated loss of DA neurons, and absence of extracellular superoxide production in MAC1−/− cultures. Microglia accumulated fluorescently labeled LPS in punctate compartments associated with the plasma membrane, intracellular vesicles, and the Golgi apparatus. Cytochalasin D (CD), an inhibitor of phagocytosis, blocked LPS internalization. However, microglia derived from Toll‐like receptor 4 deficient mice and MAC1−/− mice failed to show a significant decrease in intracellular accumulation of labeled LPS, when compared with controls. Pretreatment with the scavenger receptor inhibitor, fucoidan, inhibited 79% of LPS accumulation in microglia without affecting superoxide, indicating that LPS internalization and superoxide production are mediated by separate phagocytosis receptors. Together, these data demonstrate that MAC1 is essential for LPS‐induced superoxide from microglia, implicating MAC1 as a critical trigger of microglial‐derived oxidative stress during inflammation‐mediated neurodegeneration.

Pituitary Adenylate Cyclase-Activating Polypeptide (PACAP) 38 and PACAP4–6 Are Neuroprotective through Inhibition of NADPH Oxidase: Potent Regulators of Microglia-Mediated Oxidative Stress

Microglial activation is implicated in the progressive nature of numerous neurodegenerative diseases, including Parkinsons disease. Using primary rat mesencephalic neuron-glia cultures, we found that pituitary adenylate cyclase-activating polypeptide (PACAP) 38, PACAP27, and its internal peptide, Gly-Ile-Phe (GIF; PACAP4–6), are neuroprotective at 10–13 M against lipopolysaccharide (LPS)-induced dopaminergic (DA) neurotoxicity, as determined by [3H]DA uptake and the number of tyrosine hydroxylase-immunoreactive neurons. PACAP38 and GIF also protected against 1-methyl-4-phenylpyridinium+-induced neurotoxicity but only in cultures containing microglia. PACAP38 and GIF ameliorated the production of microglia-derived reactive oxygen species (ROS), where both LPS- and phorbol 12-myristate 13-acetate-induced superoxide and intracellular ROS were inhibited. The critical role of NADPH oxidase for GIF and PACAP38 neuroprotection against LPS-induced DA neurotoxicity was demonstrated using neuron-glia cultures from mice deficient in NADPH oxidase (PHOX–/–), where PACAP38 and GIF reduced tumor necrosis factor α production and were neuroprotective only in PHOX+/+ cultures and not in PHOX–/– cultures. Pretreatment with PACAP6–38 (3 μM; PACAP-specific receptor antagonist) was unable to attenuate PACAP38, PACAP27, or GIF (10–13 M) neuroprotection. PACAP38 and GIF (10–13 M) failed to induce cAMP in neuronglia cultures, supporting that the neuroprotective effect was independent of traditional high-affinity PACAP receptors. Pharmacophore analysis revealed that GIF shares common chemical properties (hydrogen bond acceptor, positive ionizable, and hydrophobic regions) with other subpicomolar-acting compounds known to inhibit NADPH oxidase: naloxone, dextromethorphan, and Gly-Gly-Phe. These results indicate a common high-affinity site of action across numerous diverse peptides and compounds, revealing a basic neuropeptide regulatory mechanism that inhibits microglia-derived oxidative stress and promotes neuron survival.