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The brain has an intrinsic capacity to repair injury, but the specific mechanisms are largely unknown. In this study, we found that, despite their incipient death, damaged neurons play a key repair role with the help of monocytes infiltrated from blood. Monocytes phagocytosed damaged and/or dying neurons that expressed osteopontin (OPN), with possible subsequent activation of their inflammasome pathway, resulting in pyroptosis. During this process, monocytes released CD63-positive exosome-like vesicles containing OPN. Importantly, following the exosome-like vesicles, neuron and astrocyte processes elongated toward the injury core. In addition, exosomes prepared from the injured brain contained OPN, and enhanced neurite outgrowth of cultured neurons in an OPN-dependent manner. Thus, our results introduce the concept that neurons in the injured brain that are destined to die perceive the stressful condition and begin the regeneration processes through induction of OPN, ultimately executing the repair process with the help of monocytes recruited from the circulation.

Defects in repair of damaged brain accumulate injury and contribute to slow-developing neurodegeneration. Here, we report that a deficiency of DJ-1, a Parkinson's disease (PD) gene, delays repair of brain injury due to destabilization of Sox9, a positive regulator of astrogliosis. Stereotaxic injection of ATP into the brain striatum produces similar size of acute injury in wild-type and DJ-1-knockout (KO) mice. However, recovery of the injury is delayed in KO mice, which is confirmed by 9.4T magnetic resonance imaging and tyrosine hydroxylase immunostaining. DJ-1 regulates neurite outgrowth from damaged neurons in a non-cell autonomous manner. In DJ-1 KO brains and astrocytes, Sox9 protein levels are decreased due to enhanced ubiquitination, resulting in defects in astrogliosis and glial cell-derived neurotrophic factor/ brain-derived neurotrophic factor expression in injured brain and astrocytes. These results indicate that DJ-1 deficiency causes defects in astrocyte-mediated repair of brain damage, which may contribute to the development of PD.

PINK1 (PTEN induced putative kinase 1), a familial Parkinson's 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.

PINK1 (PTEN induced putative kinase 1), a familial Parkinson's 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.

Gathering evidence has associated activation of microglia with the pathogenesis of numerous neurodegenerative diseases of the central nervous system (CNS) such as Alzheimer's disease and Parkinson's 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 alpha-synuclein (alpha-syn), which is associated with the pathogenesis of Parkinson's disease, may affect the phagocytic function of microglia. We found that extracellular monomeric alpha-syn enhanced microglial phagocytosis in both a dose- and time-dependent manner, but beta- and gamma- syn did not. We also found that the N-terminal and NAC region of alpha-syn, especially the NAC region, might be responsible for the effect of alpha-syn on microglial phagocytosis. In contrast to monomeric alpha-syn, aggregated alpha-syn actually inhibited microglial phagocytosis. The different effects of monomeric and aggregated alpha-syn on phagocytosis might be related to their localization in cells. This study indicates that alpha-syn can modulate the function of microglia and influence inflammatory changes such as those seen in neurodegenerative disorders.

Brain inflammation is a suggested risk factor for neurodegenerative disease. Interestingly, severe inflammation in the substantia nigra pars compacta (SNpc) accelerates the onset and progression of Parkinson's disease. In this study, we examined the underlying mechanisms of severe inflammation in the SNpc by comparing the inflammatory process with that in the cortex. In intact brain, the densities of CD11b(+) microglia were similar in the SNpc and cortex. However, lipopolysaccharide injection enhanced the CD11b(+) cell number in the SNpc, but not in the cortex. Previously, we reported that CD11b and myeloperoxidase (MPO) double-positive neutrophils infiltrate the SNpc following LPS injection (GLIA 55:1577-88). Notably, the MPO(+) neutrophil number increased dramatically in the SNpc, but only slightly in the cortex. The extent of neutrophil infiltration appeared to correlate with neuronal damage. We confirmed that loss of neurons in the SNpc was significantly reduced in neutropenic rats versus normal rats following LPS injection. In addition, the densities of astrocytes were much lower in the intact SNpc, compared with the cortex. Furthermore, after LPS injection, damage of endothelial cells and astrocytes, and blood-brain barrier (BBB) permeability was more pronounced in the SNpc. These results collectively suggest that excessive neutrophil infiltration and environmental factors, such as lower astrocyte density and higher BBB permeability, contribute to severe inflammation and neuronal death in the SNpc.

When the brain suffers injury, microglia migrate to the damaged sites and become activated. These activated microglia are not detected several days later and the mechanisms underlying their disappearance are not well characterized. In this study, we demonstrate that interleukin (IL)-13, an anti-inflammatory cytokine, selectively induces cell death of activated microglia in vitro. Cell death was detected 4 days after the coaddition of IL-13 with any one of the microglial activators, lipopolysaccharide (LPS), ganglioside, or thrombin. This cell death occurred in a time-dependent manner. LPS, ganglioside, thrombin, or IL-13 alone did not induce cell death. Among anti-inflammatory cytokines, IL-4 mimicked the effect of IL-13, while TGF-beta did not. Cells treated with IL-13 plus LPS, or IL-13 plus ganglioside, showed the characteristics of apoptosis when analyzed by electron microscopy and terminal deoxynucleotidyl transferase-mediated dUTP nick end labeling staining. Electron micrographs also showed microglia engulfing neighboring dead cells. We propose that IL-13 and IL-4 induce death of activated microglia, and that this process is important for prevention of chronic inflammation that can cause tissue damage.

We examined the expression of outward rectifier K+ channels in activated microglia in vivo. For this purpose, lipopolysaccharide (LPS, 2 μg) was injected into the cortex near the hippocampal region of rat brains, and K+ channel expression was examined using antibodies against shaker-type K+ channels, Kv1.5 and Kv1.3. OX-42-positive microglia were found around the injection sites from 8 h after the LPS injection and remained there for 3 days. The OX-42-positive microglia expressed Kv1.5 immunoreactivity, and the time course of Kv1.5 expression was closely correlated with that of OX-42. In saline-injected brains, OX-42-positive cells also expressed Kv1.5 immunoreactivity even though far fewer OX-42-positive cells were found. Increase of Kv1.5 expression after LPS injection was also demonstrated by immunoblot analysis. On the other hand, Kv1.3 immunoreactivity was barely detected in OX-42-positive cells over the entire experimental period. The expression of Kv1.5 preceded that of inducible nitric oxide synthase (iNOS), which is a prominent indication of microglial activation. iNOS was not detectable until 12 h, and thereafter it was maintained for 3 days together with Kv1.5 and OX-42. These results suggest that in vivo as well as in vitro activated microglia expressed outward K+ channels and that some of the channels at least are Kv1.5. GLIA 24:408–414, 1998. © 1998 Wiley-Liss, Inc.

Gathering evidence has associated activation of microglia with the pathogenesis of numerous neurodegenerative diseases of the central nervous system (CNS) such as Alzheimer's disease and Parkinson's 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 alpha-synuclein (alpha-syn), which is associated with the pathogenesis of Parkinson's disease, may affect the phagocytic function of microglia. We found that extracellular monomeric alpha-syn enhanced microglial phagocytosis in both a dose- and time-dependent manner, but beta- and gamma- syn did not. We also found that the N-terminal and NAC region of alpha-syn, especially the NAC region, might be responsible for the effect of alpha-syn on microglial phagocytosis. In contrast to monomeric alpha-syn, aggregated alpha-syn actually inhibited microglial phagocytosis. The different effects of monomeric and aggregated alpha-syn on phagocytosis might be related to their localization in cells. This study indicates that alpha-syn can modulate the function of microglia and influence inflammatory changes such as those seen in neurodegenerative disorders.

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. © 2008 Wiley-Liss, Inc.

Generally, it has been accepted that microglia play important roles in brain inflammation. However, recently several studies suggested possible infiltration of blood neutrophils and monocytes into the brain. To understand contribution of microglia and blood inflammatory cells to brain inflammation, the behavior of microglia, neutrophils, and monocytes was investigated in LPS (lipopolysaccharide)-injected substantia nigra pars compacta, cortex, and hippocampus of normal and/or leukopenic rats using specific markers of neutrophils (myeloperoxidase, MPO), and microglia and monocytes (ionized calcium binding adaptor molecule-1, Iba-1), as well as a general marker for these inflammatory cells (CD11b). CD11b-immunopositive (CD11b(+)) cells and Iba-1(+) cells displayed similar behavior in intact and LPS-injected brain at 6 h after the injection. Interestingly, however, CD11b(+) cells and Iba-1(+) cells displayed significantly different behavior at 12 h: Iba-1(+) cells disappeared while CD11b(+) cells became round in shape. We found that CD11b/Iba-1-double positive (CD11b(+)/Iba-1(+)) ramified microglia died within 6 h after LPS injection. The round CD11b(+) cells detected at 12 h were MPO(+). These CD11b(+)/MPO(+) cells were not found in leukopenic rats, suggestive of neutrophil infiltration. MPO(+) neutrophils expressed inducible nitric oxide synthase, interleukin-1beta, cyclooxygenase-2, and monocyte chemoattractant protein-1, but died within 18 h. CD11b(+) cells detected at 24 h appeared to be infiltrated monocytes, since these cells were once labeled with Iba-1 and were not found in leukopenic rats. Furthermore, transplanted monocytes were detectable in LPS-injected brain. These results suggest that at least a part of neutrophils and monocytes could have been misinterpreted as activated microglia in inflamed brain.

PKR, the double-stranded RNA (dsRNA)-activated serine/threonine kinase, has been implicated as an important component of host responses to infection and various situations of cellular stress. The involvement of PKR in signal transduction and regulation of transcription suggested to us that it may play an important role in lipopolysaccharide (LPS)-induced activation of STAT1 in rat brain immune cells. We found that LPS rapidly stimulated the phosphorylation of PKR within 5 min, followed by phosphorylation of STAT1 at 2 h in rat primary microglia and astrocyte. Using 2-aminopurine (2-AP), a pharmacological inhibitor of PKR, and PKR-specific short interfering RNA (siRNA), we demonstrated that activation of PKR was essential for LPS-induced activation of STAT1. Inhibition of PKR activity by 2-AP resulted in suppression not only of STAT1 phosphorylation, but also of nuclear factors binding activity to GAS/ISRE elements. 2-AP also significantly suppressed the downstream events of LPS-stimulated STAT1 phosphorylation, including STAT-mediated transcriptional responses and generation of nitric oxide, a hallmark of brain inflammation. Consistent with these results, transfection of PKR-specific siRNA markedly attenuated all the STAT1 dependent inflammatory signaling responses tested. We further revealed that activation of PKR by LPS led to the induction of IFN-beta through activation of NF-kappaB, triggering the phosphorylation of STAT1 in rat brain glial cells. Taken together, these findings indicate that PKR functions as an essential modulator in LPS-induced STAT inflammatory signaling events, and provides new insight into endotoxin-induced CNS diseases following infection.

Microglia, the major immune effector cells in the central nervous system, are activated when the brain suffers injury. A number of studies indicate that gangliosides activate microglia. However, the signaling mechanisms involved in microglial activation are not yet to be elucidated. Our results show that gangliosides induce the expression of interleukin (IL)-1beta, tumor necrosis factor-alpha (TNF-alpha), and inducible nitric oxide synthase (iNOS) in rat brain microglia and BV2 murine microglia via protein kinase C (PKC) and NADPH oxidase. Expression of IL-1beta, TNF-alpha, and iNOS in ganglioside-treated cells was significantly reduced in the presence of inhibitors of PKC (GF109203X, Go6976, Ro31-8220, and rottlerin) and NADPH oxidase (diphenyleneiodonium chloride [DPI]). In response to gangliosides, PKC-alpha, betaII, and delta and NADPH oxidase p67(phox) translocated from the cytosol to the membrane. ROS generation was also activated within 5 min of ganglioside treatment. Ganglioside-induced ROS generation was blocked by PKC inhibitors. Furthermore, ganglioside-induced activation of NF-kappaB, an essential transcription factor that mediates the expression of IL-1beta, TNF-alpha, and iNOS, was reduced in the presence of GF109203X and DPI. Our results collectively suggest that gangliosides activate microglia via PKC and NADPH oxidase, which regulate activation of NF-kappaB.

Previously we have reported that thrombin induces inflammatory mediators in brain glial cells (Ryu et al. 2000. J Biol Chem 275:29955). In the present study, we found that thrombin induced a negative regulator of a cytokine signaling molecule, cytokine-induced SH2 protein (CIS), in rat brain astrocytes. In response to thrombin, CIS expression was increased at both the mRNA and protein levels. Although STAT5 is known to regulate CIS expression, thrombin did not activate STAT5, and inhibitors of JAK2 (AG490) and JAK3 (WHI-P97 and WHI-P154) had little effect on thrombin-induced CIS expression. In contrast, cytosolic phospholipase A(2) (cPLA(2)), cyclooxygenase (COX), and lipoxygenase (LO) play a role in CIS expression, since inhibitors of cPLA(2), cyclooxygenase (COX), and LO significantly reduced CIS expression. Reactive oxygen species (ROS) scavengers (N-acetyl-cysteine [NAC] and trolox) reduced thrombin-induced CIS expression, and inhibitors of COX and LO reduced ROS produced by thrombin. Furthermore, prostaglandin E(2) (PGE(2)) and leukotriene B(4) (LTB(4)), products of COX and LO, respectively, potentiated thrombin-induced CIS expression, indicating that ROS, and PGE(2) and LTB(4) generated by COX and LO, mediate CIS expression. Since interferon-gamma (IFN-gamma)-induced GAS-luciferase activity and tyrosine phosphorylation of STAT1 and STAT3 were lower in CIS-transfected cells compared to control vector-transfected cells, CIS could have anti-inflammatory activity. These data suggest that thrombin-stimulation of ROS and prostaglandin and leukotriene production via the cPLA(2), COX and LO pathways results in CIS expression. More importantly, CIS expression may be a negative feedback mechanism that prevents prolonged inflammatory responses.