Hey-Kyeong Jeong

Resident microglia die and infiltrated neutrophils and monocytes become major inflammatory cells in lipopolysaccharide-injected brain

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‐1β, 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.

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.

Brain Inflammation and Microglia: Facts and Misconceptions

The inflammation that accompanies acute injury has dual functions: bactericidal action and repair. Bactericidal functions protect damaged tissue from infection, and repair functions are initiated to aid in the recovery of damaged tissue. Brain injury is somewhat different from injuries in other tissues in two respects. First, many cases of brain injury are not accompanied by infection: there is no chance of pathogens to enter in ischemia or even in traumatic injury if the skull is intact. Second, neurons are rarely regenerated once damaged. This raises the question of whether bactericidal inflammation really occurs in the injured brain; if so, how is this type of inflammation controlled? Many brain inflammation studies have been conducted using cultured microglia (brain macrophages). Even where animal models have been used, the behavior of microglia and neurons has typically been analyzed at or after the time of neuronal death, a time window that excludes the inflammatory response, which begins immediately after the injury. Therefore, to understand the patterns and roles of brain inflammation in the injured brain, it is necessary to analyze the behavior of all cell types in the injured brain immediately after the onset of injury. Based on our experience with both in vitro and in vivo experimental models of brain inflammation, we concluded that not only microglia, but also astrocytes, blood inflammatory cells, and even neurons participate and/or regulate brain inflammation in the injured brain. Furthermore, brain inflammation played by these cells protects neurons and repairs damaged microenvironment but not induces neuronal damage.

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.

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.

Inflammatory responses are not sufficient to cause delayed neuronal death in ATP-induced acute brain injury.

Background Brain inflammation is accompanied by brain injury. However, it is controversial whether inflammatory responses are harmful or beneficial to neurons. Because many studies have been performed using cultured microglia and neurons, it has not been possible to assess the influence of multiple cell types and diverse factors that dynamically and continuously change in vivo. Furthermore, behavior of microglia and other inflammatory cells could have been overlooked since most studies have focused on neuronal death. Therefore, it is essential to analyze the precise roles of microglia and brain inflammation in the injured brain, and determine their contribution to neuronal damage in vivo from the onset of injury. Methods and Findings Acute neuronal damage was induced by stereotaxic injection of ATP into the substantia nigra pars compacta (SNpc) and the cortex of the rat brain. Inflammatory responses and their effects on neuronal damage were investigated by immunohistochemistry, electron microscopy, quantitative RT-PCR, and stereological counting, etc. ATP acutely caused death of microglia as well as neurons in a similar area within 3 h. We defined as the core region the area where both TH+ and Iba-1+ cells acutely died, and as the penumbra the area surrounding the core where Iba-1+ cells showed activated morphology. In the penumbra region, morphologically activated microglia arranged around the injury sites. Monocytes filled the damaged core after neurons and microglia died. Interestingly, neither activated microglia nor monocytes expressed iNOS, a major neurotoxic inflammatory mediator. Monocytes rather expressed CD68, a marker of phagocytic activity. Importantly, the total number of dopaminergic neurons in the SNpc at 3 h (∼80% of that in the contralateral side) did not decrease further at 7 d. Similarly, in the cortex, ATP-induced neuron-damage area detected at 3 h did not increase for up to 7 d. Conclusions Different cellular components (microglia, astrocytes, monocytes, and neutrophils) and different factors (proinflammatory and neurotrophic) could be produced in inflammatory processes depending on the nature of the injury. The results in this study suggest that the inflammatory responses of microglia and monocytes in response to ATP-induced acute injury could not be neurotoxic.

PINK1 Deficiency Enhances Inflammatory Cytokine Release from Acutely Prepared Brain Slices

Parkinsons disease (PD) is the second most common neurodegenerative motor disease caused by degeneration of dopaminergic neurons in the substantia nigra. Because brain inflammation has been considered a risk factor for PD, we analyzed whether PTEN induced putative kinase 1 (PINK1), an autosomal recessive familial PD gene, regulates brain inflammation during injury states. Using acutely prepared cortical slices to mimic injury, we analyzed expression of the pro-inflammatory cytokines tumor necrosis factor-α, interleukin (IL)-1β, and IL-6 at the mRNA and protein levels. Both mRNA and protein expression of these cytokines was higher at 6-24 h after slicing in PINK1 knockout (KO) slices compared to that in wild-type (WT) slices. In serial experiments to understand the signaling pathways that increase inflammatory responses in KO slices, we found that IκB degradation was enhanced but Akt phosphorylation decreased in KO slices compared to those in WT slices. In further experiments, an inhibitor of PI3K (LY294002) upstream of Akt increased expression of pro-inflammatory cytokines. Taken together, these results suggest that PINK1 deficiency enhance brain inflammation through reduced Akt activation and enhanced IκB degradation in 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.

Repair of astrocytes, blood vessels, and myelin in the injured brain: possible roles of blood monocytes

Inflammation in injured tissue has both repair functions and cytotoxic consequences. However, the issue of whether brain inflammation has a repair function has received little attention. Previously, we demonstrated monocyte infiltration and death of neurons and resident microglia in LPS-injected brains (Glia. 2007. 55:1577; Glia. 2008. 56:1039). Here, we found that astrocytes, oligodendrocytes, myelin, and endothelial cells disappeared in the damage core within 1–3 d and then re-appeared at 7–14 d, providing evidence of repair of the brain microenvironment. Since round Iba-1+/CD45+ monocytes infiltrated before the repair, we examined whether these cells were involved in the repair process. Analysis of mRNA expression profiles showed significant upregulation of repair/resolution-related genes, whereas proinflammatory-related genes were barely detectable at 3 d, a time when monocytes filled injury sites. Moreover, Iba-1+/CD45+ cells highly expressed phagocytic activity markers (e.g., the mannose receptors, CD68 and LAMP2), but not proinflammatory mediators (e.g., iNOS and IL1β). In addition, the distribution of round Iba-1+/CD45+ cells was spatially and temporally correlated with astrocyte recovery. We further found that monocytes in culture attracted astrocytes by releasing soluble factor(s). Together, these results suggest that brain inflammation mediated by monocytes functions to repair the microenvironment of the injured brain.

Astrogliosis Is a Possible Player in Preventing Delayed Neuronal Death

Mitigating secondary delayed neuronal injury has been a therapeutic strategy for minimizing neurological symptoms after several types of brain injury. Interestingly, secondary neuronal loss appeared to be closely related to functional loss and/or death of astrocytes. In the brain damage induced by agonists of two glutamate receptors, N-ethyl-D-aspartic acid (NMDA) and kainic acid (KA), NMDA induced neuronal death within 3 h, but did not increase further thereafter. However, in the KA-injected brain, neuronal death was not obviously detectable even at injection sites at 3 h, but extensively increased to encompass the entire hemisphere at 7 days. Brain inflammation, a possible cause of secondary neuronal damage, showed little differences between the two models. Importantly, however, astrocyte behavior was completely different. In the NMDA-injected cortex, the loss of glial fibrillary acidic protein-expressing (GFAP+) astrocytes was confined to the injection site until 7 days after the injection, and astrocytes around the damage sites showed extensive gliosis and appeared to isolate the damage sites. In contrast, in the KA-injected brain, GFAP+ astrocytes, like neurons, slowly, but progressively, disappeared across the entire hemisphere. Other markers of astrocytes, including S100β, glutamate transporter EAAT2, the potassium channel Kir4.1 and glutamine synthase, showed patterns similar to that of GFAP in both NMDA- and KA-injected cortexes. More importantly, astrocyte disappearance and/or functional loss preceded neuronal death in the KA-injected brain. Taken together, these results suggest that loss of astrocyte support to neurons may be a critical cause of delayed neuronal death in the injured brain.