Tadanori Ogata

Cascading glia reactions: a common pathomechanism and its differentiated control by cyclic nucleotide signaling.

Abstract: A pathological glia activation, stimulated by inflammatory proteins, β‐amyloid, or brain ischemia, is discussed as a common pathogenic factor for progressive nerve cell damage in vascular and Alzheimer dementia. A critical point seems to be reached, if the cytokine‐controlled microglial upregulation causes a secondary activation of astrocytes which loose the negative feedback control, are forced to give up their physiological buffering function, and may add to neuronal damage by the release of nitric oxide (NO) and by promoting toxic β‐amyloid formation. A strengthening of the cyclic adenosine‐5′,3′‐monophosphate (cAMP) signaling exerted a differential inhibition of the stimulatory cytokines tumor necrosis factor‐α (TNF‐α and interleukin‐1β (IL‐1β) released from cultured rat microglia, but maintained the negative feedback signal IL‐6; cAMP inhibited also the release of free oxygen radicals (OR) but not of NO. Reinforcement of the NO‐induced cyclic guanosine monophosphate (cGMP) increase by blockade of the phosphodiesterase (PDE) subtype‐5 with propentofylline counterbalanced the toxic NO action that causes with OR neuronal damage by peroxynitrate formation. In rat cultured astrocytes, a prolonged cAMP elevation favored cell differentiation, the expression of a mature ion channel patter, and an improvement of the extracellular glutamate uptake. Cyclic AMP signaling could be strengthened by PDE blockade and by raising extracellular adenosine, which stimulates A2 receptor‐mediated cAMP synthesis. Via an A1 receptor‐mediated effect, elevated adenosine was found to overcome a deficient intracellular calcium mobilization resulting from an impaired muscarinic signaling at pathologically decreased acetylcholine concentrations. We suggest that pharmaca, which elevate extracellular adenosine and/or block the degradation of cyclic nucleotides, may be used to counteract glia‐related neuronal damage in dementing processes.

Steroid hormones protect spinal cord neurons from glutamate toxicity

The effects of steroid hormones on glutamate neurotoxicity were examined in cultured spinal cord neurons. The extent of neuronal damage, produced by glutamate exposure for 15 min, was estimated based on the activity of lactate dehydrogenase released from degenerated neurons to the media during 24 h of post-exposure incubation. This damage was dependent on the glutamate concentrations used. The addition of dexamethasone, a synthetic steroid, in post-exposure media remarkably reduced the extent of damage in a dose-dependent manner. The half effective concentration for the steroid was approximately 0.7 microM, which was in the range of pharmacological concentration. Dexamethasone was effective even when it was added 2 h after glutamate exposure. Some endogenous steroid hormones--aldosterone, progesterone and testosterone--also showed similar neuroprotective effects. However, cholesterol, a precursor of these steroid hormones, had no effect on glutamate neurotoxicity. This direct protective effect on neurons against glutamate neurotoxicity may explain, at least partly, the mechanisms of beneficial effects of steroid hormones on in vivo spinal cord injury.

Differential regulation of microglial activation by propentofylline via cAMP signaling

A pathological microglial activation is believed to contribute to progressive neuronal damage in neurodegenerative diseases by the release of potentially toxic agents and by triggering reactive astrocytic changes. Using cultured microglia from neonatal rat brains, we investigated the mode of propentofylline action in strengthening cAMP-dependent intracellular signaling. We compared this action with the effects of dibutyryl-cAMP, a cell-permeable cAMP analog. Propentofylline inhibited lipopolysaccharide (LPS)-induced release of both tumor necrosis factor (TNF)-alpha and interleukin (IL)-1beta in a dose-dependent manner within the therapeutic low micromolar range. However, LPS-induced release of IL-6 and NO were not affected by propentofylline. All these differential effects of propentofylline on LPS-induced microglial release were mimicked by the addition of dibutyryl-cAMP. Microglial proliferation and phorbol myristate acetate (PMA)-induced O2- release were also dose-dependently inhibited by propentofylline as well as dibutyryl-cAMP. These results suggest that propentofylline, probably via reinforcement of cAMP intracellular signaling, alters the profile of the newly adopted immune properties in a way that it inhibits potentially neurotoxic functions while maintaining beneficial functions. This differential regulation of microglial activation may explain the neuroprotective mechanism exerted by propentofylline.

Glia-related pathomechanisms in Alzheimer's disease: a therapeutic target?

Reactive glial cell properties could contribute to pathomechanisms underlying Alzheimers disease by favoring oxidative neuronal damage and beta-amyloid toxicity. A critical step is apparently reached when pathological glia activation is no longer restricted to microglia and includes astrocytes. By giving up their differentiated state, astrocytes may lose their physiological negative feed-back control on microglial NO production and even contribute to neurotoxic peroxynitrate formation. Another consequence is the impairment of the astrocyte-maintained extracellular ion homeostasis favoring excitotoxic damage. By the production of apolipoprotein-E, triggered by the microglial cytokine interleukine-1beta, reactive astrocytes could promote the transformation of beta-amyloid into the toxic form. A pharmacologically reinforced cAMP signaling in rat glial cell cultures depressed oxygen radical formation in microglia and their release of TNF-alpha and interleukine-1beta, feed-forward signals which mediate oxidative damage and secondary astrocyte activation. Cyclic AMP also favored differentiation and expression of a mature ion channel pattern in astrocytes improving their glutamate buffering. A deficient cholinergic signaling that increases the risk of pathological APP processing was compensated by an adenosine-mediated reinforcement of the second messenger calcium. A combination therapy with acetylcholine-esterase inhibitors together with adenosine raising pharmaca, therefore, may be used to treat cholinergic deficiency in Alzheimers disease.

Release of Excitatory Amino Acids from Cultured Hippocampal Astrocytes Induced by a Hypoxic‐Hypoglycemic Stimulation

Abstract: An excess release of excitatory amino acids (EAA) is an important factor for postischemic brain damage. In the present communication, we demonstrate that cultured hippocampal cells release EAA after hypoxic‐hypoglycemic treatment. The amounts of EAA released from astrocytes were appreciably above those released from neurons. Furthermore, the amount of aspartate released from astrocytes was comparable to that of glutamate, although the endogenous content of aspartate was one‐fifth that of glutamate. The endogenous content of aspartate in astrocytes increased even after hypoxic‐hypoglycemic treatment. These results suggests that ischemic neuronal death is due, at least in part, to the excitotoxicity of aspartate and glutamate‐derived from surrounding astrocytes.

Adenosine enhances intracellular Ca2+ mobilization in conjunction with metabotropic glutamate receptor activation by t-ACPD in cultured hippocampal astrocytes

2Cl-Adenosine, a non-metabolized adenosine agonist, enhanced the increase in intracellular Ca2+ concentration ([Ca2+]i) in cultured hippocampal astrocytes induced by (+-)-1-aminocyclopentane-trans-1,3-dicarboxylic acid (t-ACPD), a metabotropic glutamate agonist. In the absence of 2Cl-adenosine, the half effective concentration (EC50) of t-ACPD was about 80 microM. On the other hand, in the presence of 1 microM 2Cl-adenosine, the EC50 of t-ACPD shifted to about 5 microM, although the maximum [Ca2+]i did not change. The synergistic effect of 2Cl-adenosine with t-ACPD on [Ca2+]i elevation was not inhibited by the elimination of extracellular Ca2+, but was inhibited by A1-specific adenosine antagonists. These results indicate that adenosine can act via the A1 receptor as an endogenous co-activator of the metabolic processes induced by metabotropic glutamate receptor activation.

Adenosine triphosphate inhibits cytokine release from lipopolysaccharide-activated microglia via P2y receptors.

Microglial proliferation and activation have been reported to occur after several central nervous system injuries. In this study, we tested the effects of adenosine triphosphate (ATP) on cultured microglia obtained from the spinal cord of rat embryos. The amounts of tumor necrosis factor alpha (TNF-alpha), interleukin 1beta and interleukin 6 released from the microglia, which were stimulated by lipopolysaccharide (LPS; 100 ng/ml), were inhibited by the simultaneous addition of ATP in a dose dependent manner (10-300 microM). We examined the effect of several endogenous purines (ATP, ADP, CTP, UDP, UTP) and P(2)y receptor agonists (ADPbetaS and 2-methylthio-ATP) on LPS-induced TNF-alpha release. The rank order of inhibitory potency of endogenous purines on TNF-alpha release was: ATP>ADP>>UTP>UDP>CTP. The latter three were much less potent than the former two. The addition of 10 microM 2-methylthio-ATP showed a potency similar to 100 microM ATP. The addition of ADPbetaS, however, showed less effect. These endogenous purines and selective ATP receptor agonists showed a similar inhibitory effect in their rank order on LPS-induced interleukin 6 release. We demonstrate that ATP inhibits cytokine release from LPS-activated microglia via metabotropic receptors. The application of P(2)y receptor agonists might be considered as a pharmacological treatment of several pathological conditions of the spinal cord, including toxic immunoreactions.

Potentiated cAMP rise in metabotropically stimulated rat cultured astrocytes by a Ca2+-related A1/A2 adenosine receptor cooperation.

Adenosine agonists favoured an intracellular Ca2+ rise in cultured type 1 astrocytes if the metabotropic glutamate receptors were concomitantly stimulated by (2S,1′s, 2′s)‐2‐(carboxycyclopropyl) glycine (l‐CCG‐I; group II agonist), quisqualate (group I agonist) or 1 ‐aminocyclopentane‐trans‐1,3‐dicarboxylic acid (t‐ACPD; group VII agonist). Since the generation of a Ca2+ signal reflected a newly adopted adenosine A1 receptor action, we tested the possible consequence that the established opposing control of the cellular cAMP content by inhibitory A1 and stimulatory A2 receptor activation was also altered. During metabotropic receptor stimulation by L‐CCG‐I, quisqualate or t‐ACPD, the non‐selective adenosine agonist 2‐chloroadenosine (CI‐adenosine) caused a potentiated cAMP increase which markedly exceeded that produced by CI‐adenosine alone. This cAMP potentiation resulted from altered and Ca2+‐dependent A1/A2 receptor cooperation. It was abolished by A1 receptor blockade and could not be achieved in the presence of t‐ACPD by the A1 agonist R(‐)N6‐(2‐phenylisopropyl)‐adenosine or by the A2 agonist 5′‐N‐ethyl carboxyamidoadenosine alone, but was obtained using their combination. The cAMP potentiation was blocked by intracellular Ca2+ chelation and the required A1 receptor action could be mimicked by a Ca2+ signal generated by the P2y receptor agonist adenosine 5β‐(β‐thio) diphosphate. The results support the conclusion that nanomolar concentrations of adenosine may influence astrocyte reactions by stimulating the Ca2+ and cAMP‐dependent signalling cascade.

Pathological immuno-reactions of glial cells in Alzheimer's disease and possible sites of interference

A significant role of a pathological glial cell activation in the pathogenesis of Alzheimers disease is supported by the growing evidence that inflammatory proteins, which are produced by reactive astrocytes, promote the transformation of diffuse beta-amyloid deposits into the filamentous, neurotoxic form. A number of vicious circles, driven by the release of TNF-a and free oxygen radicals from microglial cells, may cause an upregulated microglial activation and their production of interleukin-1 which triggers, secondarily, the crucial activation of astrocytes. Reactive functional changes of glial cells seem to be controlled by an altered balance of the second messengers Ca2+ and cAMP and can be counterregulated by the endogenous cell modulator adenosine which strengthens the cAMP-dependent signalling chain. A further reinforcement of the homeostatic adenosine effects on glial cells by pharmaca, such as propentofylline, may add to neuroprotection in Alzheimers disease.

Programmed cell death in rat microglia is controlled by extracellular adenosine

The induction of programmed cell death by adenosine was investigated in cultured rat microglial cells using the enzyme-linked immunosorbent assay (ELISA) for determining DNA fragmentation. Twelve hours exposure to micromolar levels of the unselective adenosine receptor agonist 2-chloro-adenosine led to the appearance of DNA fragments in the cytosolic fraction preceding damage of the plasma membrane. This effect was still seen in the presence of an adenosine uptake blocker. Conventional A1, A2 or A3 agonists and antagonists were rather ineffective, suggesting mediation via an atypical adenosine receptor subtype. Microglial DNA fragmentation was inhibited by H-7 and staurosporine but not by dibutyryl-cyclic AMP, pointing to a protein kinase C linked mechanism. Such an induction of programmed cell death by an elevation of the extracellular adenosine concentration may provide an endogenous control mechanism to limit the function of activated microglial cells.