Daniel L. Gilbert

Production of superoxide anions by a CNS macrophage, the microglia.

Microglia have been implicated in both physiological and pathological processes of the brain. Their possible roles have been compared to those of macrophages and granulocytes. Here we demonstrate the specific ability of microglia to secrete the superoxide radical ion in response to a complement activated agent, opsonized zymosan, and to phorbol myristate acetate. As in other organs, this endogenously produced reactive oxygen intermediate could have both beneficial and deleterious effects.

Effect of Divalent Cations on Potassium Conductance of Squid Axons: Determination of Surface Charge

Potassium conductance-voltage curves have been determined for a squid axon in high external potassium solution for a wide range of divalent cation concentrations. A decrease in divalent ion concentration shifts the conductance-voltage curve along the voltage axis in the direction of more hyperpolarized voltages by as much as 9 mv for an e-fold change in concentration. When the divalent ion concentration is less than about 5 mM, a further decrease does not cause a significant shift of the conductance-voltage curve. These results can be explained by assuming that on the outer surface of the membrane there is a negative fixed charge which can bind calcium ions, and that the axon is sensitive to the resulting double-layer potential. From our data, the best value for charge density was found to be one electronic charge per 120 square angstroms, and a lower limit to be one electronic charge per 280 square angstroms.

Slow Changes of Potassium Permeability in the Squid Giant Axon

A slow potassium inactivation i.e. decrease of conductance when the inside of the membrane is made more positive with respect to the outside, has been observed for the squid axon. The conductance-potential curve is sigmoid shaped, and the ratio between maximum and minimum potassium conductance is at least 3. The time constant for the change of potassium conductance with potential is independent of the concentration of potassium in the external solution, but dependent upon potential and temperature. At 9 degrees C and at the normal sea water resting potential, the time constant is 11 sec. For lower temperature or more depolarizing potentials, the time constant is greater. The inactivation can be described by modifying the Hodgkin-Huxley equation for potassium current, using one additional parameter. The modified equation is similar in form to the Hodgkin-Huxley equation for sodium current, suggesting that the mechanism for the passive transport of potassium through the axon membrane is similar to that for sodium.

Induction of Superoxide Anion and Nitric Oxide Production in Cultured Microgliaa

Although it is clear that the central nervous system (CNS) can undergo damage by exposure to reactive oxidant species (ROS), the source of the damaging molecules is not always apparent. The CNS, like other tissues, contains a variety of cellular mechanisms that are capable of generating both reactive oxygen and reactive nitrogen species, including autooxidation of molecules such as nor-epinephrine and dopamine as well as the action of specific enzymes such as xanthine oxidase or nitric oxide ~ynthase .~J .~-~ However, one potentially important source of ROS has been generally overlooked, that is, the production of ROS by the CNS macrophage, the microglia. Microglia were originally described by del Rio Hortega6 in 1932 and were correctly identified at that time as a CNS-specific component of the reticule endothelial system of the body. Like other tissue macrophages, the microglia generate superoxide anion and nitric oxide as part of their normal function. Nitric oxide is produced by the action of a cytosolic enzyme known as the inducible nitric oxide synthase (iNOS), while superoxide anion is produced by a membrane-bound NADPH o x i d a ~ e . ~ * ~ . ~ ~ The release of these reactive oxidants from microglia can be important to the CNS for two primary reasons: (1) microglia are distributed in a matrixlike arrangement throughout all regions of the CNS,9.l0 and (2) the generation of reactive oxidants by microglia can be induced under a variety of conditions, including injury and i n f l a m m a t i ~ n . ~ ~ ~ ~ Microglia, therefore, not only generate significant quantities of reactive oxidants but are also in an ideal position to promote oxidative damage to neurons and other glia. In order to understand how microglia contribute to changes in the oxidative environment of the CNS, we have examined the regulation of superoxide anion and nitric oxide production by cultured microglia. Microglia were prepared from a mixed glial culture of neonatal rat cerebral cortices as previously de~cr ibed .~ lJ~ Cells were grown at 37°C in Dulbeccos Modified Eagle Medium (DMEM) supplemented with 10% fetal calf serum, 1 mM glutamine, and 25 pg/ml gentamycin for 14 days. On day 14, the microglia were separated from the mixed glial culture by shaking, the

Microglial Contribution to Oxidative Stress in Alzheimer's Disease

Abstract: Microglia are the CNS macrophage and are a primary cellular component of plaques in Alzheimers disease (AD) that may contribute to the oxidative stress associated with chronic neurodegeneration. We now report that superoxide anion production in microglia or macrophages from 3 different species is increased by long term exposure (24 hours) to Aβ peptides. Since Aβ competes for the uptake of opsonized latex beads and for the production of superoxide anion by opsonized zymosan, a likely site of action are membrane receptors associated with the uptake of opsonized particles or fibers. The neurotoxic fibrillar peptides Aβ (1–42) and human amylin increase radical production whereas a non‐toxic, non‐fibrillar peptide, rat amylin, does not. We also report that the effect of Aβ peptides on superoxide anion production is not associated with a concomitant increase in nitric oxide (NO) production in either human monocyte derived macrophages (MDM) or hamster microglia from primary cultures. Since NO is known to protect membrane lipids and scavenge superoxide anion, the lack of Aβ‐mediated induction of NO production in human microglia and macrophages may be as deleterious as the over‐production of superoxide anion induced by chronic exposure to Aβ peptides.

Protection from oxidation enhances the survival of cultured mesencephalic neurons

Oxidative stress has been linked to the destruction of dopaminergic neurons in the substantia nigra and may be a significant factor in both Parkinsons disease and MPTP toxicity. Using primary cultures of embryonic rat mesencephalon and standard immunocytochemical techniques, we have examined the survival of tyrosine hydroxylase-containing (TH+) neurons cultured in the presence of antioxidants and/or in an environment of low oxygen partial pressure. The number of TH+ neurons increased approximately twofold if superoxide dismutase, glutathione peroxidase (GP), or N-acetyl cysteine (NAC) were added to the culture media. Exposure of the neurons to a 5% oxygen environment (38 torr, i.e., 38 mm Hg) also increased the survival of TH+ neurons by about twofold. A dramatic enhancement of survival, however, was seen when NAC was used in combination with the 5% oxygen environment. In this case, the number of TH+ neurons increased fourfold from nontreated controls. Morphological changes were also noted. GP increased the average neurite length while NAC increased the average area of the cell body in the TH+ neuron. These results suggest that manipulation of oxidative conditions by changing the ambient O2 tension or the level of antioxidants promotes survival of TH+ neurons in culture and may have implications for transplantation therapies in Parkinsons disease.

Fifty Years of Radical Ideas

Abstract: My role in the free radical theory of oxygen toxicity is discussed. Rebeca Gerschman and I published several papers on this subject. This sparked my interest in geochemistry and I developed the idea that oxygen was the best qualified biological potential energy source for the following reasons: great abundance, easily accessible, possession of a high thermodynamic potential, and its slow reaction rate. Ionization radiation can be viewed as a catalyst for reactive oxygen species since a killing dose imparts an infinitesimal small amount of energy. Next, Carol A. Colton and I showed that in the mammalian brain that stimulated microglia produce the superoxide radical anion and its implications in Alzheimers disease is discussed. More recently, I have become interested in the role of sulfhydryl groups in transcription factors.

Regulation of microglial function by interferons

Cultured neonatal rat microglia were pretreated with varying doses of either purified interferon (IFN) alpha/beta or recombinant IFN gamma for 24 or 48 h and the following functional parameters examined; superoxide anion production, interleukin-1 secretion and chemotaxis. IFN gamma produced a marked increase in superoxide anion levels when PMA was used to initiate superoxide anion production but had no effect in OPZ-stimulated microglia. Treatment with IFN alpha/beta potentiated superoxide anion production in both PMA and OPZ-stimulated cells. Interleukin-1 activity was decreased by treatment with IFN gamma for 24 h while IFN alpha/beta increased IL-1 activity at 48 h. Removal of serum from the treatment media prevented the action of IFN alpha/beta on IL-1 production. Treatment with IFN alpha/beta or gamma decreased chemotaxis of microglia in response to zymosan activated serum. The data indicate that IFN gamma and alpha/beta can regulate microglial function and that this regulation may differ from that seen for other monocytically derived macrophages.

Polyribonucleotides induce nitric oxide production by human monocyte-derived macrophages.

Cytokine‐mediated activation of inducible nitric oxide synthase (iNOS) in monocytes or macrophages is species specific. In contrast to rat or mouse, human macrophages do not produce measurable levels of nitric oxide (NO) when induced by inflammatory mediators. Exposure to noncytokine mediators such as tumor cells or viruses, however, has recently been shown to activate human iNOS. NO production in response to these mediators is much lower than that seen for rat or mouse cells and often requires several days of stimulation. We have found that the synthetic, double‐stranded polyribonucleotide polyinosinic‐polycytidilic acid (Poly I:C), commonly used to mimic viral exposure, activated iNOS in human monocyte‐derived macrophages (MDM). The production of NO, measured by nitrite accumulation, was detected after 24 h of stimulation with Poly I:C. The single‐stranded polyribonucleotide Poly I, but not Poly C, also increased NO production. Nitrite production was enhanced when the MDM were primed (pretreated) with γ or α interferon or other immune mediators such as IL‐4 and was reduced by the iNOS inhibitor, N‐methyl‐L‐arginine (L‐NMMA). The use of Poly I:C to initiate NO production in human macrophages provides a useful tool to study the differences between the commonly used animal models and human cells and may provide insight into the pathophysiological significance of these differences. J. Leukoc. Biol. 62: 369–373; 1997.

Enhanced production of superoxide anion by microglia from trisomy 16 mice

Disruption of normal oxygen radical metabolism in the CNS may contribute to the neuropathological changes associated with Down syndrome (trisomy 21) and its mouse counterpart, the trisomy 16 (Ts16) mouse. One potent source of oxyradicals is the CNS-specific macrophage, the microglial cell. We prepared primary glial cultures from the cerebral cortices of Ts16 and normal littermate mice taken at day 15 of gestation. Microglia were isolated from confluent cultures after 14 days in vitro and assayed for superoxide anion production using a cytochrome C reduction assay. Stimulation by either opsonized zymosan (OPZ) or phorbol myristate acetate (PMA), produced significantly higher levels (2.8-20 fold) of superoxide per mg protein in Ts16 microglial cultures. Resting, i.e. unstimulated secretion, was not significantly different from littermate controls. Astrocyte enriched cultures, stimulated by OPZ, exhibited low levels of superoxide production which was higher in Ts16 mice than normal littermates. Microglial enriched cultures from rat neonatal cerebral cortices were exposed for 24 h to medium from the Ts16 glial cultures. Superoxide production in the Ts16 media treated rat microglia was significantly higher than in those treated with littermate conditioned media.