Tomomi Sanagi

P2Y12 receptor‐mediated integrin‐β1 activation regulates microglial process extension induced by ATP

Microglia are the primary immune surveillance cells in the brain, and when activated they play critical roles in inflammatory reactions and tissue repair in the damaged brain. Microglia rapidly extend their processes toward the damaged areas in response to stimulation of the metabotropic ATP receptor P2Y12 by ATP released from damaged tissue. This chemotactic response is a highly important step that enables microglia to function properly at normal and pathological sites in the brain. To investigate the molecular pathways that underlie microglial process extension, we developed a novel method of modeling microglial process extension that uses transwell chambers in which the insert membrane is coated with collagen gel. In this study, we showed that ATP increased microglial adhesion to collagen gel, and that the ATP‐induced process extension and increase in microglial adhesion were inhibited by integrin blocking peptides, RGD, and a functional blocking antibody against integrin‐β1. An immunoprecipitation analysis with an antibody against the active form of integrin‐β1 showed that P2Y12 mediated the integrin‐β1 activation by ATP. In addition, time‐lapse imaging of EGFP‐labeled microglia in mice hippocampal slices showed that RGD inhibited the directional process extension toward the nucleotide source, and immunohistochemical staining showed that integrin‐β1 accumulated in the tips of the microglial processes in rat hippocampal slices stimulated with ADP. These findings indicate that ATP induces the integrin‐β1 activation in microglia through P2Y12 and suggest that the integrin‐β1 activation is involved in the directional process extension by microglia in brain tissue.

FERULIC ACID INDUCES NEURAL PROGENITOR CELL PROLIFERATION IN VITRO AND IN VIVO

Ferulic acid (4-hydroxy-3-methoxycinnamic acid; FA) is a plant constituent and is contained in several medicinal plants for clinical use. In this paper, we investigated the effects of FA on the proliferation of neural stem/progenitor cells (NSC/NPCs) in vitro and in vivo. FA significantly increased the proliferation of NSC/NPCs cultured from the telencephalon of embryonic day-14 rats, and increased the number and size of secondary formed neurospheres. An in vitro differentiation assay showed that FA did not affect the percentage of either neuron-specific class III beta-tubulin (Tuj-1)-positive cells or glial fibrillary acidic protein (GFAP)-positive cells in the total cell population. Oral administration of FA increased the number of newly generated cells in the dentate gyrus (DG) of the hippocampus of corticosterone (CORT)-treated mice, indicating that FA enhances the proliferation of adult NSC/NPCs in vivo. We also found that oral administration of FA increased cAMP response element binding protein (CREB) phosphorylation and brain-derived neurotrophic factor (BDNF) mRNA level in the hippocampus of CORT-treated mice, and ameliorated the stress-induced depression-like behavior of mice. These novel pharmacological effects of FA may be useful for the treatment of mood disorders such as depression.

Pigment epithelium‐derived factor induces pro‐inflammatory genes in neonatal astrocytes through activation of NF‐κB and CREB

Pigment epithelium‐derived factor (PEDF) is a potent and broadly acting neurotrophic factor that protects neurons in various types of cultured neurons against glutamate excitotoxicity and induced‐apoptosis. Some of the effects of PEDF reflect specific changes in gene expression, mediated via activation of the transcription factor NF‐κB in neurons. To investigate whether PEDF also modulates gene expression in astrocytes, we employed the use of RT‐PCR to analyze the gene expression of certain pro‐inflammatory genes and found that genes such as IL‐1β, IL‐6, TNF‐α, MIP1α, and MIP3α were induced in PEDF‐treated cultured neonatal astrocytes, but not in adult astrocytes. Electrophoresis mobility shift assay (EMSA) revealed that a time‐ and dose‐dependent increase of NF‐κB‐ and AP‐1‐DNA binding activity was observed in PEDF‐treated neonatal astrocytes. Furthermore, rapid phosphorylation of CREB protein had occurred in PEDF‐treated neonatal astrocytes. Upregulation of pro‐inflammatory and AP‐1‐related genes by PEDF was blocked by overexpression of dominant negative CREB or a mutated form of IκBα. These results suggest that the induction of pro‐inflammatory genes is mediated via activation of NF‐κB, AP‐1, and CREB in neonatal astrocytes. Taken together, these results demonstrate that PEDF is a multipotent factor, capable of affecting not only neurons, but also neonatal astrocytes, and suggests that it may act as a neuroimmune modulator in the developmental brain.

Adenosine A3 receptor is involved in ADP-induced microglial process extension and migration

J. Neurochem. (2012) 121, 217–227.

Appearance of Phagocytic Microglia Adjacent to Motoneurons in Spinal Cord Tissue From a Presymptomatic Transgenic Rat Model of Amyotrophic Lateral Sclerosis

Microglial activation occurs early during the pathogenesis of amyotrophic lateral sclerosis (ALS). Recent evidence indicates that the expression of mutant Cu2+/Zn2+ superoxide dismutase 1 (SOD1) in microglia contributes to the late disease progression of ALS. However, the mechanism by which microglia influence the neurodegenerative process and disease progression in ALS remains unclear. In this study, we revealed that activated microglia aggregated in the lumbar spinal cord of presymptomatic mutant SOD1H46R transgenic rats, an animal model of familial ALS. The aggregated microglia expressed a marker of proliferating cell, Ki67, and phagocytic marker proteins ED1 and major histocompatibility complex (MHC) class II. The motoneurons near the microglial aggregates showed weak choline acetyltransferase (ChAT) immunoreactivity and contained reduced granular endoplasmic reticulum and altered nucleus electron microscopically. Furthermore, immunopositive signals for tumor necrosis factor‐α (TNFα) and monocyte chemoattractant protein‐1 (MCP‐1) were localized in the aggregated microglia. These results suggest that the activated and aggregated microglia represent phagocytic features in response to early changes in motoneurons and possibly play an important role in ALS disease progression during the presymptomatic stage.

The Neuroprotective Role of PEDF: Implication for the Therapy of Neurological Disorders

Neuronal degeneration is closely associated with cognitive, motor and visual dysfunctions. Neuroprotective strategies have been investigated with the view to being employed as potential therapy for patients with these disabilities. Pigment epithelium-derived factor (PEDF) is a 50-kDa secreted glycoprotein and a non-inhibitory member of the serine protease inhibitor (SERPIN) gene family. PEDF is detected in a broad range of human tissues, including almost all brain areas, and has been shown to have strong neuroprotective properties for various types of neurons including cerebellar granule neurons, hippocampal neurons, striatal neurons, retinal neurons and spinal cord motor neurons. These observations raise the possibility that application of PEDF may be helpful in designing new therapeutic strategies for neurodegenerative diseases such as amyotrophic lateral sclerosis, Parkinsons disease, Huntingtons disease, Alzheimers disease and brain ischemia.

Pigment epithelium-derived factor induces pro-survival genes through cyclic AMP-responsive element binding protein and nuclear factor kappa B activation in rat cultured cerebellar granule cells: Implication for its neuroprotective effect.

Pigment epithelium-derived factor (PEDF) protects immature cerebellar granule cell neurons (CGCs) against apoptosis induced by K+ and serum deprivation. However, the precise mechanism of this protection remains unknown. We recently reported that the transcription factor nuclear factor kappa B (NF-kappaB) is activated in PEDF-treated CGCs. Although it is well known that NF-kappaB blocks apoptotic cell death through the induction of pro-survival factors, the effects of PEDF on the expression of these factors are not fully understood. In this study, we employed the use of reverse transcriptase-polymerase chain reaction to analyze the gene expression of certain pro-survival genes and found that genes such as c-IAP1, c-IAP2, FLIPs, A1/Bfl-1 and Mn-SOD were induced in PEDF-treated neurons. On the other hand, no induction was observed of the pro-apoptotic Bcl-2 family members Bax and Bid at any time from 3 to 24 h following PEDF addition. Furthermore, phosphorylation of cyclic AMP-responsive element binding protein (CREB) and increment of nuclear cyclic AMP-response element (CRE)-like DNA binding were observed in PEDF-treated CGCs. The anti-apoptotic effect of PEDF was blocked by overexpression of dominant negative CREB or a mutated form of IkappaBalpha. These results suggested that induction of both CRE- and NF-kappaB-dependent genes is required for the observed neuroprotective effects of PEDF on CGCs.

Gene transfer of PEDF attenuates ischemic brain damage in the rat middle cerebral artery occlusion model

Pigment epithelium‐derived factor (PEDF) is a 50‐kDa glycoprotein that protects various types of cultured neurons against neurotoxic stimuli, but its precise role in the CNS is not fully understood. In this study, we used rats whose brains were transfected to over‐express human PEDF in order to elucidate the neuroprotective effect of PEDF following transient middle cerebral artery occlusion (MCAO). A replication‐defective adenoviral vector containing the human PEDF gene (Ad.PEDF) or E. coliβ‐galactosidase (Ad.LacZ) was directly injected into the right striatum at 7 days prior to 70 min of MCAO in rats. Infarct volume and degree of edema of the Ad.PEDF‐treated group were significantly reduced compared to the Ad.LacZ‐treated group 24 h after MCAO. Degeneration of neurons, astrocytes, and oligodendrocytes caused by MCAO were attenuated by over‐expression of PEDF. The up‐regulation of pro‐inflammatory genes (TNFα, IL‐1β, IL‐6, COX‐2, and iNOS) and water channel aquaporin 4 after MCAO was significantly reduced in Ad.PEDF‐injected striatum. In conclusion, the results from this study provide the first in vivo evidence that PEDF is effective in protecting CNS neurons from ischemic insult, suggesting that PEDF may have a role as an endogenous neuroprotectant in the CNS.

The regulation of pro-inflammatory gene expression induced by pigment epithelium-derived factor in rat cultured microglial cells.

Pigment epithelium-derived factor (PEDF) is a potent and broadly acting neurotrophic factor that protects various cultured neurons against apoptotic stimuli. To investigate whether PEDF acts not only on neurons, but also glial cells, we analyzed the effects of recombinant human PEDF (rhPEDF) on cytokine mRNA levels, transcription factors, and signal transduction pathways in cultured microglial cells. RT-PCR analysis revealed that pro-inflammatory genes such as IL-1beta, IL-6, and TNFalpha were induced in rhPEDF-treated cultured microglial cells. Furthermore, rapid phosphorylation of CREB protein had occurred in rhPEDF-treated neonatal astrocytes. Up-regulation of pro-inflammatory genes by rhPEDF was blocked by overexpression of dominant negative CREB or a mutated form of IkappaBalpha. These results suggest that the induction of pro-inflammatory genes by rhPEDF is mediated via activation of NF-kappaB or CREB in microglial cells. Taken together, these results demonstrate that PEDF is a multipotent factor, capable of affecting not only neurons, but also microglial cells, and suggests that it may act as a neuro-immune modulator in the rodent brain.

Adenoviral Gene Delivery of Pigment Epithelium-Derived Factor Protects Striatal Neurons from Quinolinic Acid-Induced Excitotoxicity

The 50-kDa secreted glycoprotein pigment epithelium-derived factor (PEDF) is neuroprotective for various types of cultured neurons, but whether it is neuroprotective for neurons in vivo is not known. We examined the effects of adenovirus-mediated gene transfer of PEDF on quinolinic acid (QA)-induced neurotoxicity in rats. Adenoviral vector containing the human PEDF gene (Ad.PEDF) or Escherichia coli &bgr;-galactosidase gene (Ad.LacZ) was directly injected into the right striatum 7 days before the injection of QA. Immunohistochemical analysis using antibodies specific for the neuronal markers dopamine and cyclic adenosine monophosphate-regulated phosphoprotein of 32 kDa, neuronal nuclei, and choline acetyltransferase revealed that the QA-induced striatal damage was significantly reduced in Ad.PEDF-treated rats. Overexpression of PEDF also reduced the expression of the inflammation-related genes for interleukin 1&bgr;, tumor necrosis factor&agr;, and macrophage inflammatory protein 1&agr; 1 day after QA injection. Deletion analysis of human PEDF protein demonstrated that overexpression of PEDF&Dgr;44-121 failed to protect neurons against QA-induced excitotoxicity, whereas PEDF&Dgr;78-121 retained the neuroprotective activity, suggesting that amino acid residues 44-77 of the PEDF sequence are essential for PEDF-mediated neuroprotection in vivo. These results provide the first evidence that PEDF and its deletion mutant PEDF&Dgr;78-121 are effective in protecting CNS neurons against excitotoxicity in vivo.