Jong-hyeon Kim

Impaired inflammatory responses in murine Lrrk2-knockdown brain microglia.

LRRK2, a Parkinsons disease associated gene, is highly expressed in microglia in addition to neurons; however, its function in microglia has not been evaluated. Using Lrrk2 knockdown (Lrrk2-KD) murine microglia prepared by lentiviral-mediated transfer of Lrrk2-specific small inhibitory hairpin RNA (shRNA), we found that Lrrk2 deficiency attenuated lipopolysaccharide (LPS)-induced mRNA and/or protein expression of inducible nitric oxide synthase, TNF-α, IL-1β and IL-6. LPS-induced phosphorylation of p38 mitogen-activated protein kinase and stimulation of NF-κB-responsive luciferase reporter activity was also decreased in Lrrk2-KD cells. Interestingly, the decrease in NF-κB transcriptional activity measured by luciferase assays appeared to reflect increased binding of the inhibitory NF-κB homodimer, p50/p50, to DNA. In LPS-responsive HEK293T cells, overexpression of the human LRRK2 pathologic, kinase-active mutant G2019S increased basal and LPS-induced levels of phosphorylated p38 and JNK, whereas wild-type and other pathologic (R1441C and G2385R) or artificial kinase-dead (D1994A) LRRK2 mutants either enhanced or did not change basal and LPS-induced p38 and JNK phosphorylation levels. However, wild-type LRRK2 and all LRRK2 mutant variants equally enhanced NF-κB transcriptional activity. Taken together, these results suggest that LRRK2 is a positive regulator of inflammation in murine microglia, and LRRK2 mutations may alter the microenvironment of the brain to favor neuroinflammation.

Uridine 5′-Diphosphate Induces Chemokine Expression in Microglia and Astrocytes through Activation of the P2Y6 Receptor

Chemokines play critical roles in inflammation by recruiting inflammatory cells to injury sites. In this study, we found that UDP induced expression of chemokines CCL2 (MCP-1) and CCL3 (MIP-1α) in microglia, astrocytes, and slice cultures by activation of P2Y6. Interestingly, CCL2 was more highly expressed than CCL3. However, CCL2 synthesis kinetics in response to UDP differed in microglia and astrocytes; microglia rapidly produced small amounts of CCL2, whereas astrocytes continuously synthesized large amounts of CCL2, resulting in a high ultimate level of the chemokine. UDP-induced chemokine expression was reduced in the presence of a specific antagonist of P2Y6 (MRS2578) or small interfering RNA directed against the P2Y6 gene. Inhibition of phospholipase C and calcium increase, downstream signaling pathways of Gq-coupled P2Y6, reduced UDP-induced chemokine expression. UDP activated two calcium-activated transcription factors, NFATc1 and c2. Furthermore, inhibitors of calcineurin (a phosphatase activating NFAT) and NFAT reduced UDP-induced chemokine synthesis. We also found, using a transmigration assay, that UDP-treated astrocytes recruited monocytes. These results suggest that UDP induces chemokine expression in microglia and astrocytes of the injured brain by activation of P2Y6 receptors.

Astrocytes in injury states rapidly produce anti‐inflammatory factors and attenuate microglial inflammatory responses

J. Neurochem. (2010) 115, 1161–1171.

DJ-1 facilitates the interaction between STAT1 and its phosphatase, SHP-1, in brain microglia and astrocytes: A novel anti-inflammatory function of DJ-1.

Parkinsons disease (PD) is a progressive neurodegenerative movement disorder caused by the death of dopaminergic neurons in the substantia nigra. Importantly, altered astrocyte and microglial functions could contribute to neuronal death in PD. In this study, we demonstrate a novel mechanism by which DJ-1 (PARK7), an early onset autosomal-recessive PD gene, negatively regulates inflammatory responses of astrocytes and microglia by facilitating the interaction between STAT1 and its phosphatase, SHP-1 (Src-homology 2-domain containing protein tyrosine phosphatase-1). Astrocytes and microglia cultured from DJ-1-knockout (KO) mice exhibited increased expression of inflammatory mediators and phosphorylation levels of STAT1 (p-STAT1) in response to interferon-gamma (IFN-γ) compared to cells from wild-type (WT) mice. DJ-1 deficiency also attenuated IFN-γ-induced interactions of SHP-1 with p-STAT1 and STAT1, measured 1 and 12h after IFN-γ treatment, respectively. Subsequent experiments showed that DJ-1 directly interacts with SHP-1, p-STAT1, and STAT1. Notably, DJ-1 bound to SHP-1 independently of IFN-γ, whereas the interactions of DJ-1 with p-STAT1 and STAT1 were dependent on IFN-γ. Similar results were obtained in brain slice cultures, where IFN-γ induced much stronger STAT1 phosphorylation and inflammatory responses in KO slices than in WT slices. Moreover, IFN-γ treatment induced neuronal damage in KO slices. Collectively, these findings suggest that DJ-1 may function as a scaffold protein that facilitates SHP-1 interactions with p-STAT1 and STAT1, thereby preventing extensive and prolonged STAT1 activation. Thus, the loss of DJ-1 function may increase the risk of PD by enhancing brain inflammation.

Adenosine induces hemeoxygenase‐1 expression in microglia through the activation of phosphatidylinositol 3‐kinase and nuclear factor E2‐related factor 2

Adenosine, a purine nucleoside, has been reported to suppress the inflammatory responses of microglia in the brain. However, the underlying mechanisms of its anti‐inflammatory action are unclear at present. Here we show that adenosine reduces the increase in intracellular reactive oxygen species (ROS) through expression of an antioxidant enzyme, hemeoxygenase‐1 (HO‐1). The H2O2‐induced intracellular ROS level was significantly low in microglia pretreated with adenosine for 3–6 h, compared with that in untreated cells. Adenosine induced HO‐1 mRNA and protein expression within 3 h, which was maintained for up to 12 h. Nuclear factor E2‐related factor 2 (Nrf2), a transcription factor, and phosphatidylinositol 3‐kinase (PI3K) and Akt pathways appear to mediate HO‐1 expression. In response to adenosine, Nrf2 translocated from the cytosol to nuclei, and bound to the antioxidant response element (ARE). Adenosine enhanced HO‐1 promoter activity in an ARE‐dependent manner. Moreover, the nucleoside stimulated Akt phosphorylation, and suppressors of PI3K (LY294002 and wortmannin) reduced adenosine‐induced HO‐1 expression. However, we propose that the effects of adenosine are independent of adenosine receptors, since agonists and antagonists of A1, A2a, and A3 had little effect on the regulation of intracellular ROS and HO‐1 expression. Our results collectively suggest that adenosine acts as an endogenous regulator of brain inflammation via modulation of microglial ROS production.

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.

Spatial and temporal correlation in progressive degeneration of neurons and astrocytes in contusion-induced spinal cord injury

BackgroundTraumatic spinal cord injury (SCI) causes acute neuronal death followed by delayed secondary neuronal damage. However, little is known about how microenvironment regulating cells such as microglia, astrocytes, and blood inflammatory cells behave in early SCI states and how they contribute to delayed neuronal death.MethodsWe analyzed the behavior of neurons and microenvironment regulating cells using a contusion-induced SCI model, examining early (3–6 h) to late times (14 d) after the injury.ResultsAt the penumbra region close to the damaged core (P1) neurons and astrocytes underwent death in a similar spatial and temporal pattern: both neurons and astrocytes died in the medial and ventral regions of the gray matter between 12 to 24 h after SCI. Furthermore, mRNA and protein levels of transporters of glutamate (GLT-1) and potassium (Kir4.1), functional markers of astrocytes, decreased at about the times that delayed neuronal death occurred. However, at P1 region, ramified Iba-1+ resident microglia died earlier (3 to 6 h) than neurons (12 to 24 h), and at the penumbra region farther from the damaged core (P2), neurons were healthy where microglia were morphologically activated. In addition, round Iba-1/CD45-double positive monocyte-like cells appeared after neurons had died, and expressed phagocytic markers, including mannose receptors, but rarely expressed proinflammatory mediators.ConclusionLoss of astrocyte function may be more critical for delayed neuronal death than microglial activation and monocyte infiltration.

Anaesthesia in patients with Brugada syndrome

Brugada syndrome is characterized by right bundle branch block, ST segment elevation in the precordial leads and sudden death caused by ventricular fibrillation. We present two successful anaesthetic management cases in patients with Brugada syndrome.

Prevention of propofol-induced pain in children: combination of alfentanil and lidocaine vs alfentanil or lidocaine alone

BACKGROUND Pain from a propofol injection is a common side-effect in paediatric patients. This prospective, randomized, double-blind study evaluated the efficacy of a combined pretreatment of alfentanil with lidocaine on the incidence and severity of propofol injection pain in children. METHODS After obtaining parental consent, 120 paediatric patients were allocated randomly into one of the three groups (n=40, in each). The patients in the alfentanil group received alfentanil 15 microg kg(-1) 90 s before the propofol injection. The patients in the lidocaine group received propofol 3 mg kg(-1) premixed with lidocaine 0.1% over a 15 s period. The patients in the combination group received both alfentanil and lidocaine. RESULTS The incidence of propofol injection pain (severity 2 or more) in the combination group (2.6%) was significantly lower than that in the alfentanil and lidocaine groups (30% and 38.5%, respectively) (P=0.001 and <0.001, respectively). No patient in the combination group complained of moderate or severe pain from propofol injection. CONCLUSIONS Our study demonstrated that the combination treatment of two different analgesic modalities, alfentanil and lidocaine, could prevent the moderate and severe pain on propofol injection, and reduce the incidence of mild pain compared with each drug alone.

Suppression of miR-155 Expression in IFN-γ-Treated Astrocytes and Microglia by DJ-1: A Possible Mechanism for Maintaining SOCS1 Expression

Previously, we reported that DJ-1, encoded by a Parkinsons disease (PD)-associated gene, inhibits expression of proinflammatory mediators in interferon-gamma (IFN-γ)-treated astrocytes and microglia through inhibition of STAT1 activation. Here, using microglia and astrocytes cultured from wild-type (WT) and DJ-1-knockout (KO) mouse brains, we examined how DJ-1 regulates suppressor of cytokine signaling 1 (SOCS1), a negative feedback regulator of STAT1 (signal transducer and activator of transcription) that is also induced by STAT1. We found that IFN-γ significantly increased SOCS1 mRNA expression in WT microglia and astrocytes, but not in KO cells, although STAT1 was highly activated in these latter cells. We further found that SOCS mRNA stability was decreased in DJ-1-KO cells, an effect that appeared to be mediated by the microRNA, miR-155. IFN-γ increased the levels of miR-155 in DJ-1-KO cells but not in WT cells. In addition, an miR-155 inhibitor rescued SOCS1 expression and decreased STAT1 activation in DJ-1-KO cells. Taken together, these results suggest that DJ-1 efficiently regulates inflammation by maintaining SOCS1 expression through regulation of miR-155 levels, even under conditions in which STAT1 activation is decreased.