Sang Myun Park

Differential SUMOylation of LXRα and LXRβ Mediates Transrepression of STAT1 Inflammatory Signaling in IFN-γ-Stimulated Brain Astrocytes

To unravel the roles of LXRs in inflammation and immunity, we examined the function of LXRs in development of IFN-gamma-mediated inflammation using cultured rat brain astrocytes. LXR ligands inhibit neither STAT1 phosphorylation nor STAT1 translocation to the nucleus but, rather, inhibit STAT1 binding to promoters and the expression of IRF1, TNFalpha, and IL-6, downstream effectors of STAT1 action. Immunoprecipitation data revealed that LXRbeta formed a trimer with PIAS1-pSTAT1, whereas LXRalpha formed a trimer with HDAC4-pSTAT1, mediated by direct ligand binding to the LXR proteins. In line with the fact that both PIAS1 and HDAC4 belong to the SUMO E3 ligase family, LXRbeta and LXRalpha were SUMO-conjugated by PIAS1 or HDAC4, respectively, and SUMOylation was blocked by transient transfection of appropriate individual siRNAs, reversing LXR-induced suppression of IRF1 and TNFalpha expression. Together, our data show that SUMOylation is required for the suppression of STAT1-dependent inflammatory responses by LXRs in IFN-gamma-stimulated brain astrocytes.

Microglial phagocytosis is enhanced by monomeric alpha-synuclein, not aggregated alpha-synuclein: implications for Parkinson's disease.

Gathering evidence has associated activation of microglia with the pathogenesis of numerous neurodegenerative diseases of the central nervous system (CNS) such as Alzheimers disease and Parkinsons disease. Microglia are the resident macrophages of the CNS whose functions include chemotaxis, phagocytosis, and secretion of a variety of cytokines and proteases. In this study, we examined the possibility that α‐synuclein (α‐syn), which is associated with the pathogenesis of Parkinsons disease, may affect the phagocytic function of microglia. We found that extracellular monomeric α‐syn enhanced microglial phagocytosis in both a dose‐ and time‐dependent manner, but β‐ and γ‐ syn did not. We also found that the N‐terminal and NAC region of α‐syn, especially the NAC region, might be responsible for the effect of α‐syn on microglial phagocytosis. In contrast to monomeric α‐syn, aggregated α‐syn actually inhibited microglial phagocytosis. The different effects of monomeric and aggregated α‐syn on phagocytosis might be related to their localization in cells. This study indicates that α‐syn can modulate the function of microglia and influence inflammatory changes such as those seen in neurodegenerative disorders.

On the mechanism of internalization of α‐synuclein into microglia: roles of ganglioside GM1 and lipid raft

α‐Synuclein (α‐syn) has been known to be a key player of the pathogenesis of Parkinson’s disease and has recently been detected in extracellular biological fluids and shown to be rapidly secreted from cells. The penetration of α‐syn into cells has also been observed. In this study, we observed that dl‐threo‐1‐phenyl‐2‐decanoylamino‐3‐morpholino‐1‐propanol, a glucosyltransferase inhibitor, and proteinase K inhibited the internalization of extracellular monomeric α‐syn into BV‐2 cells, and the addition of monosialoganglioside GM1 ameliorated the inhibition of α‐syn internalization in dl‐threo‐1‐phenyl‐2‐decanoylamino‐3‐morpholino‐1‐propanol‐treated BV‐2 cells. Furthermore, inhibition of clathrin‐, caveolae‐, and dynamin‐dependent endocytosis did not prevent the internalization of α‐syn, but disruption of lipid raft inhibited it. Inhibition of macropinocytosis and disruption of actin and microtubule structures also did not inhibit the internalization of α‐syn. In addition, we further confirmed these observations by co‐culture system of BV‐2 cells and α‐syn‐over‐expressing SH‐SY5Y cells. These findings suggest that extracellular α‐syn is internalized into microglia via GM1 as well as hitherto‐unknown protein receptors in clathrin‐, caveolae‐, and dynamin‐independent, but lipid raft‐dependent manner. Elucidation of the mechanism involved in internalization of α‐syn should be greatly helpful in the development of new treatments of α‐syn‐related neurodegenerative diseases.

Oxidative stress induces lipid-raft-mediated activation of Src homology 2 domain-containing protein-tyrosine phosphatase 2 in astrocytes.

Several protein phosphatases are involved in neuroprotection in response to ischemic brain injury. Here, we report that reactive oxygen species (ROS)-mediated oxidative stress promotes phosphorylation of endogenous SHP-2 through lipid rafts in rat primary astrocytes. SHP-2 was transiently phosphorylated during hypoxia/reoxygenation, an effect abrogated by a ROS scavenger and an NADPH oxidase inhibitor. Additionally, exogenous treatment with hydrogen peroxide (H(2)O(2)) triggered SHP-2 phosphorylation in a time- and dose-dependent manner and led to its translocation into lipid rafts. H(2)O(2)-mediated SHP-2 phosphorylation and translocation were inhibited by filipin III and methyl-beta-cyclodextrin (MCD), lipid-raft-disrupting agents. In the presence of H(2)O(2), SHP-2 formed a complex with STAT-3 and reduced the steady-state STAT-3 phosphorylation level. Interestingly, the effect of H(2)O(2) on SHP-2 phosphorylation was cell-type specific. Remarkably, SHP-2 phosphorylation was induced strongly by H(2)O(2) in astrocytes, but barely detectable in microglia. Our results collectively indicate that SHP-2 is activated by ROS-mediated oxidative stress in astrocytes and functions as a component of the raft-mediated signaling pathway that acts through dephosphorylation and inactivation of other phosphotyrosine proteins, such as STAT-3.

Pink1 deficiency attenuates astrocyte proliferation through mitochondrial dysfunction, reduced akt and increased p38 mapk activation, and downregulation of egfr

PINK1 (PTEN induced putative kinase 1), a familial Parkinsons disease (PD)‐related gene, is expressed in astrocytes, but little is known about its role in this cell type. Here, we found that astrocytes cultured from PINK1‐knockout (KO) mice exhibit defective proliferative responses to epidermal growth factor (EGF) and fetal bovine serum. In PINK1‐KO astrocytes, basal and EGF‐induced p38 activation (phosphorylation) were increased whereas EGF receptor (EGFR) expression and AKT activation were decreased. p38 inhibition (SB203580) or knockdown with small interfering RNA (siRNA) rescued EGFR expression and AKT activation in PINK1‐KO astrocytes. Proliferation defects in PINK1‐KO astrocytes appeared to be linked to mitochondrial defects, manifesting as decreased mitochondrial mass and membrane potential, increased intracellular reactive oxygen species level, decreased glucose‐uptake capacity, and decreased ATP production. Mitochondrial toxin (oligomycin) and a glucose‐uptake inhibitor (phloretin) mimicked the PINK1‐deficiency phenotype, decreasing astrocyte proliferation, EGFR expression and AKT activation, and increasing p38 activation. In addition, the proliferation defect in PINK1‐KO astrocytes resulted in a delay in the wound healing process. Taken together, these results suggest that PINK1 deficiency causes astrocytes dysfunction, which may contribute to the development of PD due to delayed astrocytes‐mediated repair of microenvironment in the brain.

DJ-1 Associates with lipid rafts by palmitoylation and regulates lipid rafts-dependent endocytosis in astrocytes

Parkinsons disease (PD) is the second most common progressive neurodegenerative disease. Several genes have been associated with familial type PD, providing tremendous insights into the pathogenesis of PD. Gathering evidence supports the view that these gene products may operate through common molecular pathways. Recent reports suggest that many PD-associated gene products, such as α-synuclein, LRRK2, parkin and PINK1, associate with lipid rafts and lipid rafts may be associated with neurodegeneration. Here, we observed that DJ-1 protein also associated with lipid rafts. Palmitoylation of three cysteine residues (C46/53/106) and C-terminal region of DJ-1 were required for this association. Lipopolysaccharide (LPS) induced the localization of DJ-1 into lipid rafts in astrocytes. The LPS-TLR4 signaling was more augmented in DJ-1 knock-out astrocytes by the impairment of TLR4 endocytosis. Furthermore, lipid rafts-dependent endocytosis including the endocytosis of CD14, which play a major role in regulating TLR4 endocytosis was also impaired, but clathrin-dependent endocytosis was not. This study provides a novel function of DJ-1 in lipid rafts, which may contribute the pathogenesis of PD. Moreover, it also provides the possibility that many PD-related proteins may operate through common molecular pathways in lipid rafts.

The 15-Deoxy-Δ12,14-Prostaglandin J2 Suppresses Monocyte Chemoattractant Protein-1 Expression in IFN-γ-Stimulated Astrocytes through Induction of MAPK Phosphatase-1

The 15-deoxy-Δ12,14-PGJ2 (15d-PGJ2) is a cyclopentene PG generated from PGD2. It is an endogenous ligand of the peroxisome proliferator-activated receptor-γ that is primarily involved in adipocyte differentiation and lipid metabolism. Its anti-inflammatory actions have recently attracted considerable research attention, although the precise role and underlying mechanisms of these actions are largely unknown. In the present study, we focused on the inhibitory action of 15d-PGJ2 on the chemokine MCP-1, which plays a key role in the initiation and progression of inflammation by recruiting inflammatory cells to lesion sites. We found that 15d-PGJ2 suppressed MCP-1 transcription and protein secretion in IFN-γ-stimulated brain astrocytes. The inhibitory effects of 15d-PGJ2 on MCP-1 resulted from its actions on the transcription factors, AP-1 and specificity protein-1, which play key roles in IFN-γ-induced MCP-1 expression in astrocytes. Of interest, the negative effects of 15d-PGJ2 on AP-1/specificity protein-1 signaling and the resulting inhibition of MCP-1 expression were mediated by MAPK phosphatase (MKP)-1 activity, which was induced by 15d-PGJ2 in a peroxisome proliferator-activated receptor-independent manner. Thus, our data demonstrate a novel anti-inflammatory mechanism of 15d-PGJ2 involving MKP-1. Considering the importance of MCP-1 in inflammatory processes, our results suggest that 15d-PGJ2 analogues may have therapeutic potential to attenuate inflammatory brain diseases by inducing MKP-1 expression.

LRRK2 G2019S mutation attenuates microglial motility by inhibiting focal adhesion kinase

In response to brain injury, microglia rapidly extend processes that isolate lesion sites and protect the brain from further injury. Here we report that microglia carrying a pathogenic mutation in the Parkinsons disease (PD)-associated gene, G2019S-LRRK2 (GS-Tg microglia), show retarded ADP-induced motility and delayed isolation of injury, compared with non-Tg microglia. Conversely, LRRK2 knockdown microglia are highly motile compared with control cells. In our functional assays, LRRK2 binds to focal adhesion kinase (FAK) and phosphorylates its Thr–X–Arg/Lys (TXR/K) motif(s), eventually attenuating FAK activity marked by decreased pY397 phosphorylation (pY397). GS-LRRK2 decreases the levels of pY397 in the brain, microglia and HEK cells. In addition, treatment with an inhibitor of LRRK2 kinase restores pY397 levels, decreased pTXR levels and rescued motility of GS-Tg microglia. These results collectively suggest that G2019S mutation of LRRK2 may contribute to the development of PD by inhibiting microglial response to brain injury.

Identification of the amino acid sequence motif of α-synuclein responsible for macrophage activation

Alpha-synuclein (Syn) is implicated in the pathogenesis of PD and related neurodegenerative disorders. Recent studies have also shown that alpha-synuclein can activate microglia and enhance dopaminergic neurodegeneration. The mechanisms of microglia activation by alpha-synuclein, however, are not well understood. In this study, we found that not only alpha-synuclein but also beta- and gamma-synucleins activated macrophages (RAW 264.7) in vitro. Macrophages treated with synuclein proteins secreted TNF-alpha in a dose-dependent manner. Synuclein family proteins also increased mRNA transcription of COX-2 and iNOS. Two alpha-synuclein deletion mutants, SynDeltaNAC and Syn61-140, activated macrophages, while deletion mutants Syn1-60 and Syn96-140 did not significantly activate them. Finally, we demonstrated that macrophage activation by alpha-synuclein was accompanied by phosphorylation of ERK. These results suggest that synuclein family proteins can activate macrophages, and that macrophage activation needs both the N-terminal and C-terminal domains of alpha-synuclein, but not the central NAC region.

Astrocytes, but not microglia, rapidly sense H₂O₂via STAT6 phosphorylation, resulting in cyclooxygenase-2 expression and prostaglandin release

Emerging evidence has established that astrocytes, once considered passive supporting cells that maintained extracellular ion levels and served as a component of the blood–brain barrier, play active regulatory roles during neurogenesis and in brain pathology. In the current study, we demonstrated that astrocytes sense H2O2 by rapidly phosphorylating the transcription factor STAT6, a response not observed in microglia. STAT6 phosphorylation was induced by generators of other reactive oxygen species (ROS) and reactive nitrogen species, as well as in the reoxygenation phase of hypoxia/reoxygenation, during which ROS are generated. Src–JAK pathways mediated STAT6 phosphorylation upstream. Experiments using lipid raft disruptors and analyses of detergent-fractionated cells demonstrated that H2O2-induced STAT6 phosphorylation occurred in lipid rafts. Under experimental conditions in which H2O2 did not affect astrocyte viability, H2O2-induced STAT6 phosphorylation resulted in STAT6-dependent cyclooxygenase-2 expression and subsequent release of PGE2 and prostacyclin, an effect also observed in hypoxia/reoxygenation. Finally, PGs released from H2O2-stimulated astrocytes inhibited microglial TNF-α expression. Accordingly, our results indicate that ROS-induced STAT6 phosphorylation in astrocytes can modulate the functions of neighboring cells, including microglia, through cyclooxygenase-2 induction and subsequent release of PGs. Differences in the sensitivity of STAT6 in astrocytes (highly sensitive) and microglia (insensitive) to phosphorylation following brief exposure to H2O2 suggest that astrocytes can act as sentinels for certain stimuli, including H2O2 and ROS, refining the canonical notion that microglia are the first line of defense against external stimuli.