James G. McLarnon

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.

Human neural stem cells: electrophysiological properties of voltage-gated ion channels

We have characterized the profile of membrane currents in an immortalized human neural stem cell line, HB1.F3 cells, using whole-cell patch clamp technique. Human neural stem cell line generated from primary cell cultures of embryonic human telencephalon using a replication-incompetent retroviral vector containing v-myc expresses nestin, a cell type-specific marker for neural stem cells. The human neural stem cells expressed both outward and inward K+ currents with no evidence for Na+ currents. The density of the outward, delayed rectifying type K+ current was 1.8 ± 0.015 nA/pF, and that of the inwardly rectifying K+ current was 0.37 ± 0.012 nA/pF (at 30 mM of [K+]o). In order to induce neuronal differentiation of the neural stem cells, a full-length coding region of NeuroD, a neurogenic transcription factor, was transfected into HB1.F3 cells. Introduction of NeuroD induced expression of Na+ currents with the current density of 0.042 ± 0.011 nA/pF. The presence of two types of K+ currents and expression of Na+ currents induced by NeuroD appear to reflect the characteristic physiological features of human neural stem cells.

Generation and characterization of immortalized human microglial cell lines: Expression of cytokines and chemokines

Microglia are a major glial component of the central nervous system (CNS), play a critical role as resident immunocompetent and phagocytic cells in the CNS, and serve as scavenger cells in the event of infection, inflammation, trauma, ischemia, and neurodegeneration in the CNS. Studies of human microglia have been hampered by the difficulty of obtaining sufficient numbers of human microglia. One way to circumvent this difficulty is to establish permanent cell lines of human microglia. In the present study we report the generation of immortalized human microglial cell line, HMO6, from human embryonic telencephalon tissue using a retroviral vector encoding myc oncogene. The HMO6 cells exhibited cell type-specific antigens for microglia-macrophage lineage cells including CD11b (Mac-1), CD68, CD86 (B7-2), HLA-ABC, HLA-DR, and ricinus communis agglutinin lectin-1 (RCA), and actively phagocytosed latex beads. In addition, HMO6 cells showed ATP-induced responses similar to human primary microglia in Ca2+ influx spectroscopy. Both human primary microglia and HMO6 cells showed the similar cytokine gene expression in IL-1beta, IL-6, IL-8, IL-10, IL-12, IL-15, and TNF-alpha. Using HMO6 cells, we investigated whether activation was induced by Amyloid-beta fragments or lipopolysaccharide (LPS). Treatment of HMO6 cells with Amyloid-beta 25-35 fragment (Abeta(25-35)) or Amyloid-beta 1-42 fragment (Abeta(1-42)) led to increased expression of mRNA levels of cytokine/chemokine IL-8, IL-10, IL-12, MIP-1beta MIP-1, and MCP-1, and treatment with LPS produced same results. Expression of TNF-alpha and MIP1-alpha was not detected in unstimulated HMO6 cells, but their expression was later induced by long-term exposure to Abeta(25-35) or Abeta(1-42.) ELISA assays of spent culture media showed increased protein levels of TNF-alpha and IL-8 in HMO6 cells following treatment with Abeta(25-35) or LPS. Taken together, our results demonstrate that treatment of human primary microglia and HMO6 immortalized human microglia cell line with Abeta(25-35), Abeta(1-42) and LPS upregulate gene expression and protein production of proinflammatory cytokines and chemokines in these cells. The human microglial cell line HMO6 exhibits similar properties to those documented in human microglia and should have considerable utility as an in vitro model for the studies of human microglia in health and disease.

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.