Graham P. Wilkin

Inflammatory regulators in Parkinson's disease : iNOS, lipocortin-1, and cyclooxygenases-1 and -2

Degeneration of dopaminergic neurons and focal gliosis are pathological hallmarks of Parkinsons disease and although the brain is described as immune-privileged focal immune reactions surround failing nigral neurons. We examined the cellular distribution of pro- and anti-inflammatory molecules in human parkinsonian and neurologically normal substantia nigra and caudate-putamen postmortem. An up-regulation of nitric oxide synthase- and cyclo-oxygenase-1- and -2-containing amoeboid microglia was found in parkinsonian but not control nigra. Astroglia contained low levels of these molecules in both groups. Lipocortin-1-immunoreactive amoeboid microglia were present within the astrocytic envelope of neurons adjacent to or within glial scars in parkinsonian nigra only. Lipocortin-1 is known to have neuroprotective and anti-inflammatory properties. Up-regulation of nitric oxide synthase is generally associated with neurodestruction whereas prostaglandin synthesis may be either neurodestructive or protective. The balance of these molecules is likely to be decisive in determining neuronal survival or demise.

Glial heterogeneity in expression of the inwardly rectifying K(+) channel, Kir4.1, in adult rat CNS.

Previous electrophysiological evidence has indicated that astrocytes and oligodendrocytes express inwardly rectifying K+ channels both in vitro and in vivo. Here, for the first time, we have undertaken light microscopic immunohistochemical studies demonstrating the location of one such channel, Kir4.1, in both cell types in regions of the rat CNS. Some astrocytes such as those in the deep cerebellar nuclei, Bergmann glia, retinal Müller cells, and a subset in hippocampus express Kir4.1 immunoreactivity, but not others including those in white matter. Oligodendrocytes also express this protein, strongly in perikarya and to a lesser extent in their processes. Expression of Kir4.1 in astrocytes and oligodendrocytes would enable these cells to clear extracellular K+ through this channel, whereas nonexpressors might use other mechanisms. GLIA 30:362–372, 2000.

Substance P-induced release of prostaglandins from astrocytes : regional specialisation and correlation with phosphoinositol metabolism

Abstract: Addition of substance P (SP) to astrocytes cultured from rat neonatal spinal cord evoked a time‐and concentration‐dependent increase in the accumulation of phosphoinositol and the release of prostaglandin (PG) D2 and PGE2. Both basal and stimulated releases were reduced to similar levels by indomethacin. In contrast, astrocytes cultured from cerebral cortex and cerebellum showed no SP‐stimulated increase in phosphoinositol accumulation or release of PGs. Release of PGD2 and PGE2 was, however, stimulated by the calcium ionophore A23187, and both phosphoinositol accumulation and PG release were stimulated from cortical astrocytes incubated in the presence of serum. The results from this study suggest that SP‐stimulated phosphoinositol accumulation and release of PGs from cultured rat neonatal astrocytes are regionally specialised in favour of cells derived from spinal cord.

Expression of COX-2 by normal and reactive astrocytes in the adult rat central nervous system.

We have used a previously characterized antiserum against cycloxygenase-2 (COX-2) together with cold methanol fixation to immunohistochemically locate the protein in astrocytes in rat brain. Although in cerebral cortex most enzyme was located in neuronal perikarya as previously described, a number of glial fibrillary acidic protein (GFAP)-positive astrocytes were also labeled. No COX-2-positive neurons were seen in the cerebellum, but here also a subset of GFAP+ astrocytes was present which contained the enzyme. The number of COX-2-positive astrocytes increased considerably after injection of the neurotoxin kainate into the cerebellum. These immunohistochemical data were supported by semiquantitative RT-PCR results, which were used to assess the levels of COX-2 mRNA relative to the housekeeping gene hypoxanthine phosphoribosyl transferase. PGE2 levels were measured in contralateral and lesioned cerebellum to correlate changes in COX-2 immunoreactivity and mRNA with physiological events. PGE2 levels increased by 230% in the lesioned cerebellar hemispheres in comparison to the contralateral ones. We discuss the possibility that the targets for astrocytic prostaglandins might include both autocrine effects and paracrine responses of neurons, lymphocytes and capillary endothelial cells.

Identification of 5-hydroxytryptamine receptors positively coupled to adenylyl cyclase in rat cultured astrocytes

1 5‐Hydroxytryptamine (5‐HT) elicited a dose‐dependent stimulation of intracellular adenosine 3′: 5′‐cyclic monophosphate (cyclic AMP) accumulation in cultured astrocytes derived from neonatal rat (Sprague Dawley) thalamic/hypothalamic area with a potency (pEC50) of 6.68 ± 0.08 (mean ± s.e.mean). 2 In order to characterize the 5‐HT receptor responsible for the cyclic AMP accumulation the effects of a variety of compounds were investigated on basal cyclic AMP levels (agonists) and 5‐carboxamidotryptamine (5‐CT) stimulated cyclic AMP levels (antagonists). The rank order of potency for the agonists investigated was 5‐CT (pEC50 = 7.81 ±+ 0.09)>5‐methoxytryptamine (5‐MeOT) (pEC50 = 6.86 ±0.36) > 5‐HT (pEC50 = 6.68± 0.08). The following compounds, at concentrations up to 10 μm, did not affect basal cyclic AMP levels 8‐hydroxy‐2‐(di‐n‐propylamino)tetralin (8‐OH‐DPAT), cisapride, sumatriptan, DOI and RU 24969. The rank order of potency of antagonists was meth‐iothepin (p Ki = 7.98 ±0.25)>mesulergine (p Ki = 7.58 ± 0.18)>ritanserin (p Ki = 7.20 ±0.24) >clozapine (p Ki = 7.03±0.19)>mianserin (p Ki= 6.41 ±0.19). The following compounds, at concentrations up to 10 μM, were inactive: ketanserin, WAY100635, GR127935. This pharmacological profile is consistent with that of 5‐HT7 receptor subtype‐mediated effects. 3 The cultured astrocytes exhibited regional heterogeneity in the magnitude of cyclic AMP accumulation (Emax). Cells cultured from the thalamic/hypothalamic area had significantly higher Emax values (588 ± 75% and 572 ± 63% of basal levels for 5‐CT and 5‐HT, respectively) compared to brainstem (274 ± 51% and 318 ± 46%, respectively) and colliculus astrocytes (244 ± 15% and 301±24%, respectively). No significant differences in pEC50 (for either 5‐HT or 5‐CT) values were observed. 4 Reverse transcriptase‐polymerase chain reaction (RT‐PCR) with primers specific for the 5‐HT7 receptor confirmed expression of messenger RNA for this receptor subtype by the cultured astrocytes derived from all regions investigated. Primers specific for the 5‐HT6 receptor also amplified a cDNA fragment from the same samples. 5 From these findings, we conclude that astrocytes cultured from a number of brain regions express functional 5‐HT receptors positively coupled to adenylyl cyclase and that the level of receptor expression or the efficiency of receptor coupling is regionally‐dependent. The pharmacological profile of the receptor on thalamic/hypothalamic astrocytes suggests that the 5‐HT7 receptor is the dominant receptor that is functionally expressed even though astrocyte cultures have the capacity to express both 5‐HT6 and 5‐HT7 receptor messenger RNA.

Down‐regulation of GAP‐43 During Oligodendrocyte Development and Lack of Expression by Astrocytes In Vivo: Implications for Macroglial Differentiation

The discovery of molecular markers which are selectively expressed during the development of specific classes of rat central nervous system macroglia has greatly advanced our understanding of how these cells are related. In particular, it has been shown in tissue culture that oligodendrocytes and some astrocytes (type‐2) may be derived from a common progenitor cell (O‐2A progenitor). However, the existence of type‐2 astrocytes in vivo has yet to be unequivocally established. Recently, it has been reported that the neural‐specific growth‐associated protein‐43 (GAP‐43, otherwise known as 8–50, F1, pp46 and neuromodulin) may be expressed by cells of the O‐2A lineage in vitro. We set out to examine the cellular specificity of GAP‐43 in O‐2A progenitors and their descendants in vitro and in vivo. Using a polyclonal antiserum against a GAP‐43 fusion protein we have shown the presence of immunoreactive GAP‐43 in the membranes of bipotential O‐2A glial progenitor cells and type‐2 astrocytes by Western blotting and immunocytochemistry of cells in culture. In contrast to previous studies, double labelling with mature oligodendrocyte markers showed that GAP‐43 is down‐regulated during oligodendrocyte differentiation in vitro. Immunohistochemical staining of sections of developing rat brain demonstrated the same developmental regulation of GAP‐43, suggesting that oligodendrocytes only express GAP‐43 at immature stages. In addition, normal and reactive astrocytes in tissue sections were not labelled with GAP‐43.

Cultured astrocytes express messenger RNA for multiple serotonin receptor subtypes, without functional coupling of 5-HT1 receptor subtypes to adenylyl cyclase

The literature describing the expression of 5-HT receptor subtypes by astrocytes is controversial and incomplete. It is clear that primary cultures of astrocytes express receptors of the 5-HT2 family coupled to phospholipase C and of the 5-HT7 receptor family positively coupled to adenylyl cyclase. Cultured astrocytes have also been reported to express receptors of the 5-HT1 family, although the exact subtypes present are unknown. In the present study we have investigated which of the known rat G-protein coupled 5-HT receptor mRNAs are expressed by cultured astrocytes. Reverse transcriptase-polymerase chain reaction (RT-PCR) revealed expression of 5-HT1A, 5-HT1B, 5-HT1D, 5-HT1F, 5-HT2A, 5-HT2B, 5-HT2C, 5-HT5B, 5-HT6 and 5-HT7 receptor mRNAs in astrocytes derived from 2-day old rats and cultured for 10-12 days. Messenger RNAs for 5-HT4 and 5-HT5A receptors were not detected. The functional expression of 5-HT1 receptor subtypes was investigated by measuring the ability of 5-HT1 receptor agonists: 8-OH-DPAT (5-HT1A receptors), RU24969 (5-HT1A, 5-HT1B, 5-HT1D, and 5-HT1F receptors) or sumatriptan (5-HT1B, 5-HT1D, and 5-HT1F receptors) to modulate forskolin or isoproterenol stimulated cAMP production. These compounds, at concentrations up to 10 microM, did not significantly attenuate cAMP production. These results indicate that although astrocytes express mRNA for each of the five 5-HT1 receptor subtypes which have been isolated from the rat, these receptors are not coupled to the inhibition of adenylyl cyclase.

Astrocytes from Forebrain, Cerebellum, and Spinal Cord Differ in Their Responses to Vasoactive Intestinal Peptide

Abstract: Astrocytes from cortex, cerebellum, and spinal cord responded to isoproterenol and vasoactive intestinal peptide (VIP) with increases in intracellular cyclic AMP levels. The response to VIP was as great as that to isoproterenol in cortical astrocytes (180–fold and 185–fold, respectively), and the effect of VIP in combination with isoproterenol was partially additive. Spinal cord astrocytes also responded to VIP and isoproterenol with equal potency (seven‐ to ninefold and eight‐to 13–fold, respectively), but the level of response was much smaller than in cortex. Spinal cord astrocytes were synergistic in their response to VIP and isoproterenol. The response to VIP was lowest in cerebellar astrocytes (only threefold), and no additivity was observed when VIP was added together with isoproterenol. A small response to α‐melanocyte stimulating hormone (α‐MSH) was also observed in cortex and cerebellum, but not in spinal cord. Somatostatin inhibited the response to isoproterenol in cortex and cerebellum, but had no effect in spinal cord. The results from the above study show that astrocytes obtained from these three regions of the rat CNS express quite different responses to NIP and α‐MSH and further point to possible astrocyte heterogeneity.

Elevated glial brain-derived neurotrophic factor in Parkinson's diseased nigra

We show the cellular distribution of immunoreactivity (IR) for brain-derived-neurotrophic-factor (BDNF), neurotrophin-3 (NT-3) and tyrosine kinase receptors TRKB and TRKC in idiopathic Parkinsons disease (IPD) and controls at post-mortem. In both groups, nigral neurons, astrocytes, ramified and amoeboid microglia expressed all antigens. Caudate-putamen neurons expressed all antigens except BDNF with similar distribution between groups. In IPD nigra, increased numbers of BDNF-IR and, less frequently, NT-3-IR ramified glia surrounded fragmented neurons, accompanied by BDNF-IR in surrounding neuropil. Amoeboid microglia were abundant only in IPD nigral scars. In IPD, glia might up-regulate neurotrophins in response to signals released from failing nigral neurons.

Identification of B2 Bradykinin Binding Sites on Cultured Cortical Astrocytes

Bradykinin was found to bind to specific high‐affinity sites in cultured cortical astrocytes from rat brain, and this binding appeared to be specific for the B2 bradykinin receptor subtype. Nonlinear regression analysis of saturation experiments using a computer programme revealed a single KD of 16.6 ± 2.6 nM and a Bmax of 352.2 ±30.7 fmol/mg of protein. These results indicate that astrocytes possess bradykinin receptors and that these are predominantly of the B2 subtype.