Stephan Miller

Roles of Metabotropic Glutamate Receptors in Brain Plasticity and Pathology

In summary, the mGluRs are a large family of receptor subtypes with diverse properties in terms of transduction coupling, pharmacology, and anatomical distribution. Many divergent studies have demonstrated that activation of these receptors can result in either neuroprotection or neuropathology. We hypothesized that the mGluRs of astrocytes may have a role in determining the response following administration of mGluR agonists in vivo, and we have defined a suitable in vitro model for the study of these receptors. The experimental plasticity demonstrated in the astrocyte culture model may represent a more general principle that conditions in the microenvironment may differentially alter mGluR subtype expression as part of development, functional specialization, or pathology. This astrocyte model of receptor regulation provides a system suitable for studying the effects of specific growth factors, neurotrophins, cytokines, and other substances released by neurons and glia that may act in both autocrine and paracrine fashions. Alteration in the ratios of receptors by such variables could then modify future signaling properties and neuroglial interactions, a form of conditioning of the astrocytic response that would alter the physiological output following glutamate release. One measure of the value of this model will be its usefulness in stimulating the generation of hypotheses that can be tested in vivo. For example, the morphology of the astrocytes when cultured in the defined medium has similarities to the morphology of astrocytes undergoing reactive gliosis in pathological states. It is also interesting to note that treatments that have been reported to increase excitatory amino acid-stimulated PI hydrolysis in ex vivo brain slices (lesions, ischemia, and kindling) are accompanied by reactive gliosis. Those findings combined with the present in vitro results lead us to speculate that mGluR5 expression may also be altered in vivo during reactive gliosis. If so, it will be important to examine the functional consequences of such a change with regard to the astrocytic response to injury and maintaining the balance between excitatory transmission and excitotoxicity.

Metabotropic Glutamate Receptor Agonists Potentiate Cyclic AMP Formation Induced by Forskolin or β‐Adrenergic Receptor Activation in Cerebral Cortical Astrocytes in Culture

Abstract: The metabotropic glutamate receptor (mGluR) agonist 1‐aminocyclopentane‐1S,3R‐dicarboxylic acid (ACPD) potentiated the accumulation of cyclic AMP induced by either β‐adrenergic receptor stimulation (isoproterenol) or direct activation of adenylyl cyclase (AC) with forskolin in rat cerebral cortical astrocytes grown in a defined medium. In contrast, ACPD inhibits the cyclic AMP response in astrocytes cultured in a serum‐containing medium. Pharmacological characterization indicated that a group I mGluR, of which only mGluR5 is detectable in these cells, is involved in the potentiation of cyclic AMP accumulation. Potentiation was elicited by mGluR I agonists [e.g., (R,S)‐3,5‐dihydroxyphenylglycine (DHPG)], but not by mGluR II or III agonists; it was pertussis toxin resistant and abolished by procedures suppressing mGluR5 function (phorbol ester pretreatment or DHPG‐induced receptor down‐regulation). Nevertheless, it appears that products generated through the mGluR5 transduction pathway, such as elevated [Ca2+]i or activated protein kinase C (PKC), are not involved in the potentiation as it was not influenced by either the intracellular calcium chelator BAPTA‐AM or the PKC inhibitor Ro 31‐8220. An inhibitor of phospholipase C, U‐73122, markedly attenuated mGluR5‐activated phosphoinositide hydrolysis but did not significantly affect the DHPG potentiation of the cyclic AMP response. A mechanism is proposed in which the potentiating effect on AC could be mediated by free βγ complex that is liberated after the agonist‐bound mGluR5 interacts with its coupled G protein.

Exposure of Astrocytes to Thrombin Reduces Levels of the Metabotropic Glutamate Receptor mGluR5

Abstract: Thrombin is one of the first regulatory molecules present at sites of CNS trauma or injury. Exposure of neuronal and glial cells to thrombin produces potent morphological as well as cytoprotective and cytotoxic effects, but little is known about how this important modulator affects neurotransmitter signaling. In astrocyte cultures that have been morphologically differentiated by exposure to transforming growth factor‐α, addition of thrombin induced a retraction of astrocytic processes and suppressed the stimulation of phosphoinositide hydrolysis by the selective metabotropic glutamate receptor (mGluR) agonist 1‐aminocyclopentane‐1S,3R‐dicarboxylic acid. In addition to the suppression of phosphoinositide hydrolysis, thrombin treatment produced a corresponding reduction in level of mGluR5 mRNA as demonstrated with ribonuclease protection assay and reduced content of mGluR5 receptor protein as seen with western blotting. In contrast, thrombin exposure up‐regulated astrocyte β‐actin mRNA levels. A synthetic hexapeptide with a sequence corresponding to the amino‐terminus of the thrombin receptors tethered ligand also mimicked the ability of thrombin to suppress mGluR5 levels and to increase β‐actin mRNA content, suggesting that these effects of thrombin are mediated by proteolytically activated cell surface thrombin receptors. Thrombins suppressive effect on mGluR5 was resistant to pretreatment with pertussis toxin or various protein kinase and protein phosphatase inhibitors. However, the serine/threonine protein kinase inhibitor H‐7 did prevent thrombin‐induced reversal of astrocyte stellation and induction of β‐actin mRNA levels, indicating that these effects of thrombin involve a signaling pathway distinct from the one that mediates the suppressive effects of thrombin on mGluR5.

Pharmacological dissociation of glutamatergic metabotropic signal transduction pathways in cortical astrocytes

Using cultured cortical astrocytes we demonstrate differential activation of metabotropic signal transduction pathways with 1-aminocyclopentane-trans-1S3R-dicarboxylic acid (1S3R-ACPD) and the glutamate transport inhibitor trans-2,4-pyrrolidine dicarboxylic acid (trans-2,4-PDC). Phosphoinositide hydrolysis was more potently stimulated by 1S3R-ACPD than by L-trans-2,4-PDC; however, L-trans-2,4-PDC was far more efficacious than 1S3R-ACPD at inhibiting cyclic AMP accumulation. The metabotropic receptor antagonist (+)-alpha-methyl-4-carboxyphenylglycine ((+)-MCPG) inhibited 1S3R-ACPD stimulation of phosphoinositide hydrolysis but not its ability to inhibit cyclic AMP accumulation thereby demonstrating a means to pharmacologically dissociate these two metabotropic signal transduction pathways in astrocytes. (+)-MCPG produced similar antagonism of the metabotropic agonist properties of L-trans-2,4-PDC. The metabotropic effects of L-trans-2,4-PDC could not be reduced with enzymatic treatment of the cultures to remove extracellular glutamate, suggesting that these effects are not secondary to the ability of this compound to inhibit glutamate uptake. Taken together the findings indicate the presence of multiple glutamatergic signal transduction pathways in astrocytes and suggest a similarity in the pharmacophores for metabotropic receptors and glutamate transporters.

Stimulation of phosphoinositide hydrolysis by trans-(±)-ACPD is greatly enhanced when astrocytes are cultured in a serum-free defined medium

Recent studies have demonstrated that astrocytes have much greater abilities to produce and respond to signalling molecules in the CNS than had been previously estimated. We now report a dramatic enhancement in the ability of a glutamate metabotropic receptor agonist, 1-aminocyclopentane-trans-(+/-)-1,3-dicarboxylic acid (trans-(+/-)-ACPD, to stimulate phosphoinositide hydrolysis in astrocytes cultured in a serum-free defined medium compared with astrocytes cultured in conventional serum-containing medium (43.2 +/- 3.6 vs. 3.2 +/- 0.48-fold of basal, respectively). This enhancement was selective to trans-(+/-)-ACPD as little or no difference in the response to carbachol or norepinephrine was seen between the two culture conditions. These results indicate a great potential for the phosphoinositide pathway in astrocyte glutamatergic signal transduction.

1-Aminocyclopentane-trans-1,3-Dicarboxylic Acid Induces Glutamine Synthetase Activity in Cultured Astrocytes

Abstract: In this study we have investigated the effect of excitatory amino acids on the activity of glutamine synthetase, a glial‐specific enzyme that plays a key role in the regulation of glutamate concentration in the CNS. We found that of L‐glutamate, N‐methyl‐D‐aspartate, α‐amino‐3‐hydroxy‐5‐methylisoxazole‐4‐propionate, kainate, and 1‐aminocyclopentane‐trans‐1,3‐dicarboxylic acid (trans‐ACPD), only the metabotropic glutamate receptor agonist trans‐ACPD had an effect on glutamine synthetase specific activity in cultures of rat type I cortical astrocytes. Exposure of astrocytes to 1.0 mMtrans‐ACPD for 24 h resulted in an increase in glutamine synthetase activity to 149 ± 11% of that in control cultures. This effect was concentration dependent, stereoselective, and blocked by cycloheximide. In addition, the increase in glutamine synthetase activity occurred at lower concentrations of trans‐ACPD that did not produce morphological alterations or lysis of the astrocytes as measured by the lactate dehydrogenase content. These findings are consistent with the hypothesis that activation of the metabotropic excitatory amino acid receptor in astrocytes is coupled to the regulation of an enzyme essential to the metabolism and recycling of the excitatory transmitter L‐glutamate.

β-Amyloid Increases Enzyme Activity and Protein Levels of Glutamine Synthetase in Cultured Astrocytes

Previous studies have reported that beta-amyloid peptides induce properties of reactivity in cultured astrocytes. We report here that aggregated A beta peptides increase expression of the enzyme glutamine synthetase in cultured astrocytes, as assessed by enzyme assay, Western blot analysis, and immunocytochemistry. The enhanced enzyme levels occur gradually over a period of 4 days after A beta exposure and maintain peak values for at least several days thereafter. These data suggest that A beta-related reactive astrocytosis in Alzheimers disease brain may benefit local neurons by enhancing glial capacity to regulate levels of the excitotoxin glutamate.

Biochemical quantitation and histochemical localization of glucose-6-phosphate dehydrogenase activity in the olfactory system of adult and aged rats.

The activity of glucose-6-phosphate dehydrogenase, the rate-limiting enzyme of the hexose monophosphate shunt, was examined in olfactory epithelium, respiratory epithelium, olfactory bulb, and occipital cortex in Fisher 344 rats aged 4 and 24 months. Marked differences in this enzyme were found in olfactory compared to nonolfactory tissues. Olfactory epithelium and olfactory bulb have much greater glucose-6-phosphate dehydrogenase activity than respiratory epithelium and occipital cortex at both ages. Glucose-6-phosphate dehydrogenase remains fairly constant between adulthood and senescence in respiratory epithelium and occipital cortex. However, glucose-6-phosphate dehydrogenase activity decreases during the same time in both of the olfactory tissues examined. Previous studies of changes in this enzyme with aging have shown increases in enzyme activity in some brain regions, but never the decreases that we describe in olfactory tissues. Glucose-6-phosphate dehydrogenase histochemistry revealed intense staining of both the apical layer of olfactory epithelium and of Bowmans glands along with their ducts. Histochemistry of the olfactory bulb showed strongest staining in the nerve and glomerular layers of the bulb. The functional implications of these findings are discussed.

Receptor-coupled phospholipase C and adenylyl cyclase function with different calcium pools in astrocytes

ASTROCYTES express phospholipase C (PLC)-coupled metabotropic glutamate receptors (only mGluR5 is detectable) and adenylyl cyclase (AC)-linked β-adrenergic receptors. Calcium-sensitive effector enzymes are associated with these signal transduction pathways, but the relevant calcium compartments involved were found to be different. mGluR5-linked PLC responded primarily to extracellular Ca2+, suggesting a close spatial relation between the enzyme and Ca2+ entry channels. On the other hand, the calcium-inhibited AC associated with β-adrenergic receptors was sensitive to intracellular Ca2+ selectively accessible to intracellular Ca2+ chelation. Furthermore, cAMP formation induced by direct activation of AC by forskolin was less responsive to intra-cellular Ca2+ chelation than that evoked by the receptor-activated AC, raising the possibility of selective access of the receptor to a pool of calcium-inhibited AC and/or the calcium modulation of some components of the coupling pathway.