Jae K. Ryu

Fractalkine and fractalkine receptors in human neurons and glial cells

Fractalkine has been identified as a novel chemokine that exhibits cell adhesion and chemoattractive properties in the central nervous system (CNS), and the fractalkine receptors, CX3CR1, are also expressed in the CNS. In the present study, the expression of fractalkine and fractalkine receptors was investigated in enriched populations of human CNS neurons, astrocytes, and microglia. In addition, the regulatory role played by protein kinase C (PKC) in fractalkine secretion in neurons was determined in A1 human hybrid neuronal cell line produced between a human cerebral neuron and a human neuroblastoma cell. Human neurons and astrocytes expressed fractalkine mRNA as determined by the revserse transcriptase‐polymerase chain reaction (RT‐PCR) analysis, while human microglia preparation did not express the fractalkine message. Human neurons and microglia expressed CX3CR1 mRNA, but astrocytes did not. These results suggest that fractalkine secreted by CNS neurons and astrocytes produce biological effects in neurons and microglia. Although phorbol ester did not change the expression of fractalkine mRNA level in A1 hybrid neurons, it did upregulate fractalkine secretion over unstimulated controls. This upregulation of fractalkine production was suppressed by the treatment with Ro32‐0432, a PKC inhibitor. These results indicate that intracellular signals transduced by PKC play an important role in the regulation of soluble fractalkine at the post‐transcriptional level in human neurons. As for the biological function of fractalkine, extracellularly applied fractalkine increased the number of bromodeoxyuridine‐labeled microglia 3‐fold over the untreated controls, indicating fractalkine induces proliferation of human microglia. These observations suggest that fractalkine released by injured neurons could induce proliferation, activation and/or migration of microglia at the injured brain sites.

A leaky blood–brain barrier, fibrinogen infiltration and microglial reactivity in inflamed Alzheimer’s disease brain

This study has used immunohistochemical examination of tissue obtained from Alzheimer’s disease (AD) brains and rat hippocampus injected with Aβ1‐42 peptide to determine effects of induced inflammatory reactivity on integrity of blood–brain barrier (BBB) and viability of neurons. Tissue from AD, but not non‐demented, brains exhibited a diffuse pattern of staining for fibrinogen and immunoglobulin (IgG) indicative of BBB leakiness with considerable fibrinogen immunoreactivity (ir) appearing in association with Aβ deposits. Immunostaining for the endothelial cell specific glycoprotein, von Willebrand factor, showed morphological evidence for altered blood vessels in AD tissue. AD brains also demonstrated extensive areas of fibrinogen ir in association with microglial reactivity. In vivo, intra‐hippocampal injection of Aβ1‐42 caused time‐dependent (1–7 days after injection) increases in double staining of fibrinogen with areas of microgliosis. Two independent pharmacological strategies were employed to examine how Aβ1‐42 stimulation (7 days injection) may be linked to neurodegeneration. The defibrinogenating compound, ancrod, reduced inflammatory reactivity, levels of parenchymal fibrinogen and IgG, and was neuroprotective. These results prompted use of Aβ1‐42 plus fibrinogen as a novel in vivo inflammatory stimulus and this combination significantly enhanced inflammatory reactivity, vascular perturbations and neuronal damage compared to Aβ1‐42 alone. A second approach, using anti‐Mac‐1 (antibody for antigen CD11b) to block activation of microglia, was highly effective in attenuating effects of Aβ1‐42 plus fibrinogen amplification of inflammatory and vascular responses and conferred significant neuroprotection. The overall findings from study of AD tissue and in vivo in Aβ1‐42 and Aβ1‐42 plus fibrinogen stimulated rat hippocampus suggest microglial responses to promote increased extravasation of blood protein as a critical component in amplifying inflammatory reactivity and causing neuronal damage in inflamed AD brain.

Metabolic Communication between Astrocytes and Neurons via Bicarbonate-Responsive Soluble Adenylyl Cyclase

Astrocytes are proposed to participate in brain energy metabolism by supplying substrates to neurons from their glycogen stores and from glycolysis. However, the molecules involved in metabolic sensing and the molecular pathways responsible for metabolic coupling between different cell types in the brain are not fully understood. Here we show that a recently cloned bicarbonate (HCO₃⁻) sensor, soluble adenylyl cyclase (sAC), is highly expressed in astrocytes and becomes activated in response to HCO₃⁻ entry via the electrogenic NaHCO₃ cotransporter (NBC). Activated sAC increases intracellular cAMP levels, causing glycogen breakdown, enhanced glycolysis, and the release of lactate into the extracellular space, which is subsequently taken up by neurons for use as an energy substrate. This process is recruited over a broad physiological range of [K⁺](ext) and also during aglycemic episodes, helping to maintain synaptic function. These data reveal a molecular pathway in astrocytes that is responsible for brain metabolic coupling to neurons.

Proactive transplantation of human neural stem cells prevents degeneration of striatal neurons in a rat model of Huntington disease

We have investigated the effectiveness of transplantation of human neural stem cells into adult rat striatum prior to induction of striatal damage with the mitochondrial toxin 3-nitropropionic acid (3-NP). Systemic 3-NP administration caused widespread neuropathological deficits similar to ones found in Huntington disease (HD) including impairment in motor function (rotarod balance test) and extensive degeneration of neuron-specific nuclear antigen (NeuN)(+) neurons, calbindin(+) neurons and glutamic acid decarboxylase (GAD)(+) striatal neurons. Animals receiving intrastriatal implantation of human neural stem cells (hNSCs) 1 week before 3-NP treatments exhibited significantly improved motor performance and reduced damage to striatal neurons compared with control sham injections. In contrast, transplantation of hNSCs at 12 h after the initial 3-NP administration did not lead to any improvement in motor performance or protect striatal neurons from the 3-NP-induced toxicity. These results indicate that the presence of grafted hNSCs before 3-NP treatment is required for host striatal neuronal protection and enhanced motor function. Immunoreactivity of brain-derived neurotrophic factor (BDNF) was found in vitro in cultured hNSCs and in vivo in grafted NSCs with expression and secretion of BDNF demonstrated by RT-PCR, immunocytochemistry, dot-blot, and ELISA analyses. Thus, protective effects of proactive transplantation of hNSCs may be due, in part, to effects mediated by BDNF. The findings in this work have particular relevance to a rat model of HD in that proactive transplanted hNSCs protect host striatal neurons against neuronal injury and improve motor impairment induced by 3-NP toxicity.

Upregulated expression of purinergic P2X7 receptor in Alzheimer disease and amyloid-β peptide-treated microglia and in peptide-injected rat hippocampus

The expression of the purinergic receptor subtype P2X7R, a nonselective cationic channel activated by high levels of adenosine triphosphate (ATP), has been studied in adult microglia obtained from Alzheimer disease (AD) and nondemented (ND) brains, in fetal human microglia exposed to A&bgr;1-42 peptide and in vivo in A&bgr;1-42-injected rat hippocampus. Semiquantitative reverse transcriptase-polymerase chain reaction showed enhanced expression (increase of 70%) of P2X7R in AD microglia compared with ND cells (analysis of 6 AD and 8 ND cases). Immunohistochemical analysis showed prominent P2X7R expression in association with A&bgr; plaques and localized to HLA-DR-immunoreactive microglia. In cultured fetal human microglia, cells exposed to A&bgr;1-42 (5 &mgr;M for 18 hours) had significantly elevated levels of P2X7R (by 106%) compared with untreated cells. Amplitudes of Ca2+ responses in these cells, induced by the selective P2X7R agonist BzATP, were increased by 145% with A&bgr;1-42 pretreatment relative to control (no peptide pretreatment) and were largely blocked if the P2X7R inhibitor-oxidized ATP (oxATP) was added with peptide in pretreatment solution. In vivo, double immunostaining analysis showed considerable P2X7R colocalized with microglia after injection of A&bgr;1-42 (1 nmol) into rat hippocampus. The overall results suggest roles of P2X7R in mediating microglial purinergic inflammatory responses in AD brain.

Modulation of the Purinergic P2X7 Receptor Attenuates Lipopolysaccharide-Mediated Microglial Activation and Neuronal Damage in Inflamed Brain

We investigated the involvement and roles of the ionotropic purinergic receptor P2X7R in microglia in mediating lipopolysaccharide (LPS)-induced inflammatory responses and neuronal damage in rat striatum. A detailed in vivo study showed that LPS injection into striatum markedly increased the expression of P2X7R in microglia compared with control (saline)-injected animals. Additionally, LPS injection upregulated a broad spectrum of proinflammatory mediators, including inducible nitric oxide synthase (nitric oxide production marker), 3-nitrotyrosine (peroxynitrite-mediated nitration marker), 4-hydroxynonenal (lipid peroxidation marker), and 8-hydroxy-2′-deoxyguanosine (oxidative DNA damage marker), and reduced neuronal viability. The P2X7R antagonist oxidized ATP (oxATP) was effective in attenuating expressions of all inflammatory mediators and in addition inhibited LPS-induced activation of the cellular signaling factors p38 mitogen-activated protein kinase and transcriptional factor nuclear factor κB. Most importantly, in vivo, oxATP blockade of P2X7R also reduced numbers of caspase-3-positive neurons and increased neuronal survival in LPS-injected brain. In vitro, LPS stimulation of cultured human microglia enhanced cellular expressions of a host of proinflammatory factors, including cyclooxygenase-2, interleukin-1β (IL-1β), IL-6, IL-12, and tumor necrosis factor-α; all factors were inhibited by oxATP. A novel finding was that LPS potentiated intracellular [Ca2+]i mobilization induced by the P2X7R ligand 2′,3′-O-(4-benzoyl-benzoyl) ATP, which could serve as a mechanistic link for P2X7R amplification of inflammatory responses. Our results suggest critical roles for P2X7R in mediating inflammation and inhibition of this subtype purinergic receptor as a novel therapeutic approach to reduce microglial activation and confer neuroprotection in inflamed and diseased brain.

Minocycline inhibits neuronal death and glial activation induced by β-amyloid peptide in rat hippocampus

Minocycline, a second‐generation tetracycline compound, has been examined as a neuroprotectant in β‐amyloid (Aβ)‐injected rat hippocampus. At 7 days post‐injection, Aβ1‐42 caused a significant loss of granule cell layer neurons (28% reduction) compared to control uninjected hippocampus. Hippocampal injection of Aβ peptide also led to marked gliosis with numbers of microglia (increased by 26‐fold) and immunoreactivity of astrocytes (increased by 11‐fold) relative to control, as determined from immunohistochemical analysis. Intraperitoneal administration of minocycline significantly reduced neuronal loss induced by Aβ1‐42 (by 80%) and also diminished numbers of microglia (by 69%) and astrocytes (by 36%) relative to peptide alone. Peptide injection increased expression of cyclooxygenase‐2 (COX‐2) in most (about 70%) of granule cells, a subset (about 20%) of microglia, but not in astrocytes; in the presence of minocycline, COX‐2 immunostaining was abolished in microglia. The results from this study suggest that minocycline may have efficacy in the treatment of AD.

Adenosine triphosphate induces proliferation of human neural stem cells: Role of calcium and p70 ribosomal protein S6 kinase

Human neural stem cells (NSCs) grown in culture responded to extracellularly applied adenosine triphosphate (ATP), and the rate of proliferation increased as shown by immunocytochemical and RT‐PCR analysis. Activation of P2 purinoceptors by ATP is coupled to the release of intracellular calcium ([Ca2+]i) from thapsigargin‐sensitive intracellular stores. ATP‐induced proliferation was blocked by thapsigargin, an inhibitor of the endoplasmic reticulum Ca2+‐ATPase. Neither EGTA, a calcium chelator, nor caffeine had any effect on ATP‐induced [Ca2+]i increases. Multiblot kinase analysis, by which activation of 24 different kinases could be determined, showed that application of ATP to NSCs predominantly activated p70 ribosomal protein S6 kinase (p70 S6 kinase). As well, rapamycin, a p70 S6 kinase inhibitor, blocked the ATP‐mediated proliferative response in NSCs. After outlining a role for p70 S6 kinase in ATP‐mediated NSC proliferation, we examined the possibility that phosphatidylinositol 3‐kinase (PI3‐kinase) acts upstream of p70 S6 kinase. The application of wortmannin, a PI3‐kinase inhibitor, decreased both ATP‐mediated p70 S6 kinase activation and NSC proliferation. From these results, we conclude that ATP application to NSCs induces release of Ca2+ from intracellular Ca2+ stores and that this increase in intracellular Ca2+ in turn promotes NSC proliferation. The increase in NSC proliferation observed following ATP application can also be mediated by PI3‐kinase‐dependent p70 S6 kinase activation.

Peripheral benzodiazepine receptor ligand PK11195 reduces microglial activation and neuronal death in quinolinic acid-injected rat striatum

The effects of the peripheral benzodiazepine receptor (PBR) ligand, PK11195, were investigated in the rat striatum following the administration of quinolinic acid (QUIN). Intrastriatal QUIN injection caused an increase of PBR expression in the lesioned striatum as demonstrated by immunohistochemical analysis. Double immunofluorescent staining indicated PBR was primarily expressed in ED1-immunoreactive microglia but not in GFAP-immunoreactive astrocytes or NeuN-immunoreactive neurons. PK11195 treatment significantly reduced the level of microglial activation and the expression of pro-inflammatory cytokines and iNOS in QUIN-injected striatum. Oxidative-mediated striatal QUIN damage, characterized by increased expression of markers for lipid peroxidation (4-HNE) and oxidative DNA damage (8-OHdG), was significantly diminished by PK11195 administration. Furthermore, intrastriatal injection of PK11195 with QUIN significantly reduced striatal lesions induced by the excitatory amino acid and diminished QUIN-mediated caspase-3 activation in striatal neurons. These results suggest that inflammatory responses from activated microglia are damaging to striatal neurons and pharmacological targeting of PBR in microglia may be an effective strategy in protecting neurons in neurological disorders such as Huntingtons disease.

ATP-induced in vivo neurotoxicity in the rat striatum via P2 receptors

The present study examined the in vivo effects of ATP on the striatum of Sprague–Dawley rats. Intrastriatal administration of ATP produced dose-dependent striatal lesions as confirmed by cresyl violet staining. Additional immunostaining using neuronal nuclear protein (NeuN), OX-42 and GFAP antibodies revealed that ATP caused death of both neurons and glial cells. The nonmetabolizable ATP analogue ATP&ggr;S and P2X receptor agonist &agr;,&bgr;-methylene ATP (&agr;,&bgr;-MeATP) mimicked ATP effects, whereas either P2Y receptor agonist ADP or P1 receptor agonist adenosine did not. The P2 receptor antagonist reactive blue 2, but not pyridoxal-phosphate-6-azophenyl-2′,4′-disulphonic acid (PPADS) attenuated ATP-induced striatal injury. These results suggest that intrastriatal administration of ATP causes P2X receptor-mediated cell death in the striatum and support the hypothesis that extracellular ATP can be an important mediator of neuropathological events of brain injuries.