Nobuji Maeda

Effects of norepinephrine on rat cultured microglial cells that express α1, α2, β1 and β2 adrenergic receptors

Microglial cells rapidly become activated in response to even minor damage of neurons, suggestive of the intimate interactions between neurons and microglial cells. Although mediators for microglia-neuron interactions have not been well identified, neurotransmitters are possible candidates transmitting signals from neurons to microglial cells. Among the neurotransmitters, we focused on the effects of norepinephrine and other adrenergic agonists on the functions of rat cultured microglial cells. Reverse transcriptase polymerase chain reaction studies revealed that microglial cells expressed mRNAs encoding alpha1A, alpha2A, beta1 and beta2 receptors. Norepinephrine and a beta2 adrenergic agonist terbutaline elevated intracellular cAMP level of microglial cells. Norepinephrine, an alpha1 agonist phenylephrine, a beta1 agonist dobutamine and terbutaline suppressed the expressions of mRNAs encoding pro-inflammatory cytokines, interleukin-6 and tumor necrosis factor alpha. Release of tumor necrosis factor alpha and nitric oxide was suppressed by norepinephrine, phenylephrine, dobutamine and terbutaline. An alpha2 agonist clonidine and dobutamine upregulated the expression of mRNA encoding catechol-O-methyl transferase, an important enzyme to degrade norepinephrine. Norepinephrine, dobutamine and terbutaline upregulated the expressions of mRNA encoding 3-phospshoglycerate dehydrogenase, an essential enzyme for synthesis of L-serine and glycine, which are amino acids necessary for neuronal survival. Clonidine upregulated the expression of mRNA encoding an anti-apoptotic factor Bcl-xL. These results suggest that norepinephrine participates in the regulation of brain function at least partly by modulating the functions of microglia.

Apolipoprotein E and Reelin ligands modulate tau phosphorylation through an Apolipoprotein E receptor/disabled-1/glycogen synthase kinase-3β cascade

Neurofibrillary tangles comprised of highly phosphorylated tau proteins are a key component of Alzheimers disease pathology. Mice lacking Reelin (Reln), double‐knockouts lacking the VLDL receptor (VLDLR) and ApoE receptor2 (ApoER2), and mice lacking disabled‐1 (Dab1) display increased levels of phosphorylated tau. Because Reln binds to recombinant ApoE receptors, assembly of a Reln/ApoE‐receptor/Dab1 (RAD) complex may initiate a signal transduction cascade that controls tau phosphorylation. Conversely, disruption of this RAD complex may increase tau phosphorylation and lead to neurodegeneration. To substantiate this concept, we mated Reln‐deficient mice to ApoE‐deficient mice and found that in the absence of Reln, tau phosphorylation increased as the amount of ApoE decreased. Paralleling the change in tau phosphorylation levels, we found that GSK‐3β activity increased in Reln‐deficient mice and further increased in mice lacking both Reln and ApoE. CDK‐5 activity was similar in mice lacking Reln, ApoE, or both. GSK‐3β and CDK‐5 activity increased in Dab1‐deficient mice, independent of ApoE levels. Further supporting the idea that increased tau phosphorylation results primarily from increased kinase activity, the activity of two phosphatases was similar in all conditions tested. These data support a novel, ligand‐mediated signal transduction cascade— initiated by the assembly of a RAD complex that suppresses kinase activity and controls tau phosphorylation.

Suppressive effects of phosphodiesterase type IV inhibitors on rat cultured microglial cells: comparison with other types of cAMP-elevating agents.

We investigated the effects of inhibitors of cAMP-specific phosphodiesterase type IV (PDE IV) on cultured rat microglial cells. Microglial cells expressed mRNA encoding PDE IV. Rolipram and RO-20-1724, specific inhibitors of PDE IV, elevated the intracellular cAMP level much higher than the other types of PDE inhibitors. cAMP in astrocytes but not in cerebrocortical neurons was similarly increased in response to treatment with PDE IV inhibitors examined. The PDE IV inhibitors, a beta-adrenergic agonist isoproterenol and an adenylyl cyclase stimulant forskolin suppressed the proliferation of microglial cells as revealed by PCNA-immunocytochemical staining. The PDE IV inhibitors suppressed release of TNF alpha and nitric oxide (NO) from lipopolysaccharide-activated microglial cells in pure culture, while they did not affect NO release from microglial cells in neuron-microglia coculture. The PDE IV inhibitors also suppressed superoxide anion production by phorbol ester-treated microglial cells. Isoproterenol and forskolin similarly suppressed the macrophage-like functions of activated microglial cells. However, the PDE IV inhibitors displayed novel effects distinct from those of isoproterenol, forskolin and 8Br-cAMP, regarding expression of mRNAs encoding PDE IV, metallothionein-1 and hemeoxigenase-1. The present data showed that the PDE IV inhibitors can be available to control microglial function and that their effects on glial cells should be taken into account when PDE IV inhibitors are used for treatment of brain diseases, such as multiple sclerosis.

Gamma‐Ray‐Irradiated Red Blood Cells Stored in Mannitol‐Adenine‐Phosphate Medium: Rheological Evaluation and Susceptibility to Oxidative Stress

Background and Objectives: To evaluate the rheological properties and the oxidative susceptibility of γ-ray-irradiated red blood cells (RBCs). Materials and Methods: RBCs in mannitol-adenine-phosphate (MAP) medium were irradiated with 35 Gy and stored at 4°C for 4 weeks. The deformability of the RBCs was examined under shear flow in relation to the morphological and biochemical changes. The RBCs were further exposed to 1 mM FeSO4 and 5 mM ascorbate to examine the oxidative susceptibility. Results: The RBC deformability was decreased during storage, and the impairment was further enhanced by the irradiation, which promoted cell shrinkage and intracellular hemoglobin condensation accompanying potassium loss. Lipid peroxidation and protein aggregation of the RBC membrane as well as echinocytosis were not enhanced by the irradiation. The exposure to free iron did not stimulate the oxidation of the irradiated RBC membrane. Conclusion: The decreased deformability of γ-ray-irradiated RBCs in MAP medium was mainly induced by dehydration due to potassium loss, and the membrane lipids and proteins were stably preserved against oxidative stress.

Cytochrome c release from mitochondria to the cytosol was suppressed in the ischemia-tolerance-induced hippocampal CA1 region after 5-min forebrain ischemia in gerbils

Cytochrome c was detected by immunoblotting in the cytosolic fraction 3 h after 5-min ischemia in the non-ischemia-tolerant CA1 region in which about 96% of neurons had developed delayed neuronal death, while less cytosolic cytochrome c was detected in the ischemia-tolerance-induced CA1 region where many more neurons survived. In the immunohistochemical study using anti-non-native cytochrome c monoclonal antibody, immunoreactivity was observed throughout the cytoplasm in the non-ischemia-tolerant CA1 neurons, but not in the normal and ischemia-tolerant CA1 neurons. Then we determined whether Bcl-2, Bax, Bcl-xL and Bcl-xS, which regulate the release of cytochrome c from mitochondria, were altered in the ischemia-tolerant CA1 region. Bcl-2 and Bax were up-regulated in the ischemia-tolerant group, but Bcl-xL and Bcl-xS showed no apparent difference in their expression. These results suggest that cytochrome c release is prevented in CA1 neurons in gerbils in which ischemia-tolerance had been induced and that the altered ratio of Bcl-2 to Bax may play a part in this mechanism.

Reduced oxygen release from erythrocytes by the acceleration-induced flow shift, observed in an oxygen-permeable narrow tube.

The oxygen release from flowing erythrocytes under accelerational force (0-4 g) was examined using an oxygen-permeable, fluorinated ethylenepropylene copolymer tube (25 microm in inner diameter). The narrow tube was fixed vertically on the rotating disk of a new centrifuge apparatus, and erythrocyte suspension was perfused in the direction of Earth gravity. The accelerational force was applied perpendicularly to the flow direction of cells by centrifugation. The microscopic images of the flowing cells obtained at five different wavelengths were analyzed, and marginal cell-free layer and oxygen saturation of the cells were measured. By lowering oxygen tension around the narrow tube, erythrocytes were deoxygenated in proportion to their traveling distance, and the deoxygenation was enhanced with decreasing flow velocity and hematocrit. With increase of the g-value, the shift of flowing erythrocyte column to the centrifugal side was increased, the column was compressed, and the oxygen release from the cells was suppressed. Qualitatively, similar results were obtained by inducing erythrocyte aggregation with Dextran T-70 (MW = 70,400), without accelerational force. These results conclude that both the accumulation of erythrocytes under accelerational force and the enhancement of erythrocyte aggregation by macromolecules lead to the reduction of oxygen release from the flowing cells.

L-Serine regulates the activities of microglial cells that express very low level of 3-phosphoglycerate dehydrogenase, an enzyme for L-Serine biosynthesis.

Microglia are well known to become activated during various kinds of neuropathological events. The factors that are responsible for the activation, however, are not fully determined. In the present study, L‐Ser was shown to enhance production of nitric oxide (NO), interleukin‐6 (IL‐6) and tumor necrosis factor α (TNF α) by lipopolysaccharide (LPS)‐stimulated cultured rat microglial cells. L‐Ser, however, did not enhance the expression of mRNAs encoding inducible NO synthase, IL‐6 and TNF α. On the other hand, astrocytes did not depend on L‐Ser for release of IL‐6 and TNF α. The expression of an enzyme 3‐phosphoglycerate dehydrogenase (3PGDH), which is essential for L‐Ser biosynthesis from a glycolytic intermediate 3‐phosphoglycerate, was investigated. As revealed by Western blotting and immunocytochemical staining, 3PGDH‐protein expression in vitro was the highest in astrocytes, intermediate in neurons and the lowest in microglial cells. Semiquantitative RT‐PCR showed that microglial cells expressed 3PGDH‐mRNA at a lower level than astrocytes. In frozen sections from rat forebrain, only astrocytes were immunoreactive for 3PGDH. The present study suggested that L‐Ser is able to modulate microglial function mainly at the translation level because microglial cells cannot synthesize sufficient amount of L‐Ser due to the scarce expression of 3PGDH. J. Neurosci. Res. 64:392–401, 2001.

Improvement of the viability of cultured rat neurons by the non-essential amino acids L-serine and glycine that upregulates expression of the anti-apoptotic gene product Bcl-w.

The non-essential amino acids L-serine (Ser) and glycine (Gly) have recently been shown to exhibit specific actions in the nervous system. In the present study, L-Ser and Gly promoted the survival of cultured rat cerebrocortical neurons in a concentration-dependent manner as revealed by Alamar blue assay and microtubule-associated protein-2 (MAP2) immunoblotting. The maximum effects of the amino acids were detected at the concentrations of 30-100 microM. L-Ser was more effective than Gly. D-Ser failed to promote neuronal survival. L-Ser and Gly upregulated expression of the anti-apoptotic gene product Bcl-w, while they did not affect the expression of Bcl-xL. The promotion of neuronal survival by L-Ser and Gly may be, at least in part, attributable to the upregulated Bcl-w.

Histochemical cytochrome c oxidase activity and caspase-3 in gerbil hippocampal CA1 neurons after transient forebrain ischemia.

We examined the cytochrome c oxidase (COX) activity in gerbil hippocampal CA1 neurons after 5-min ischemia by a histochemical method in the presence or absence of exogenous cytochrome c. In the CA1 neurons, COX activity without exogenous cytochrome c decreased from 1 h after ischemia, but was restored by the addition of exogenous cytochrome c in the following 6 h after ischemia. These results suggest that it is not COX activity but endogenous cytochrome c that is changed in the early phase after ischemia, and that COX activity begins to decrease 9 h after ischemia. We examined caspase-3 in the CA1 region by immunoblotting, as caspase-3 is known to take part in the cell-death cascade downstream from cytochrome c. Although pro-caspase-3 was strongly detected, active caspase-3 was not detected before and until 84 h after 5-min ischemia. Our data suggested that delayed neuronal death is likely to progress via cytochrome c-release but not via caspase-3 activation.