Ann K. Snyder

Astroglial plasticity and glutamate function in a chronic mouse model of Parkinson's disease.

Astrocytes play a major role in maintaining low levels of synaptically released glutamate, and in many neurodegenerative diseases, astrocytes become reactive and lose their ability to regulate glutamate levels, through a malfunction of the glial glutamate transporter-1. However, in Parkinsons disease, there are few data on these glial cells or their regulation of glutamate transport although glutamate cytotoxicity has been blamed for the morphological and functional decline of striatal neurons. In the present study, we use a chronic mouse model of Parkinsons disease to investigate astrocytes and their relationship to glutamate, its extracellular level, synaptic localization, and transport. C57/bl mice were treated chronically with 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine and probenecid (MPTP/p). From 4 to 8 weeks after treatment, these mice show a significant loss of dopaminergic terminals in the striatum and a significant increase in the size and number of GFAP-immunopositive astrocytes. However, no change in extracellular glutamate, its synaptic localization, or transport kinetics was detected. Nevertheless, the density of transporters per astrocyte is significantly reduced in the MPTP/p-treated mice when compared to controls. These results support reactive gliosis as a means of striatal compensation for dopamine loss. The reduction in transporter complement on individual cells, however, suggests that astrocytic function may be compromised. Although reactive astrocytes are important for maintaining homeostasis, changes in their ability to regulate glutamate and its associated synaptic functions could be important for the progressive nature of the pathophysiology associated with Parkinsons disease.

Functional expression of TREK-2 K+ channel in cultured rat brain astrocytes.

Background K+ channels whose subunit contains four transmembrane segments and two pore-forming domains (4TM/2P) have been cloned recently. We studied whether 4TM/2P K+ channels are functionally expressed in astrocytes that are known to have a large background (resting) K+ conductance and a large resting membrane potential. Reverse transcriptase-PCR analysis showed that, among five 4TM/2P K+ channels examined, TASK-1, TASK-3 and TREK-2 mRNAs were expressed in cultured astrocytes from rat cortex. In cell-attached patches, we mainly observed three K+ channels with single-channel conductances of 30, 117 and 176 pS (-40 mV) in symmetrical 140 mM KCl. The 30 pS channel was the inward rectifying K+ channel that has been previously described in astrocytes. The 117 pS K+ channel also showed inward rectification and was insensitive to 1 mM tetraethylammonium and 1 mM 4-aminopyridine. The 176 pS channel was the Ca2+-activated K+ channel. The 117 pS K+ channel was determined to be TREK-2, as judged by its electrophysiological properties and activation by membrane stretch, free fatty acids and intracellular acidosis. In approximately 50% of astrocytes in culture, whole-cell K+ current increased markedly following application of arachidonic acid. The number of TREK-2 channels in these cells was estimated to be approximately 500-1000/cell. Our results show that TREK-2 is functionally expressed in cortical astrocytes in culture, and suggest that TREK-2 may be involved in K+ homeostasis of astrocytes during pathological states.

Flow Cytometric Analysis of Glucose Transport by Rat Brain Cells

The fluorescent, non-metabolizable glucose analog 6-[N-(7-nitrobenz-2-oxa-1,3-diazol-4-yl)amino]-6-deoxyglucose (NBDG) was used to measure rates of hexose transport by dissociated brain cells from developing and adult rats. Flow cytometric analysis of glucose uptake and expression of glucose transporters was performed by mapping on size by granularity, which discriminated between neurons and astrocytes in a suspension of mixed brain cells. These mapped cell populations were identified by immunofluorescent staining with antisera to neuron-specific enolase (NSE) and glial fibrillary acidic protein (GFAP). Specific uptake of the analog by membrane glucose transporters was confirmed by its inhibition by D-glucose and by cytochalasin B. Both neurons and astrocytes expressed the GLUT1 and GLUT3 transporter isoforms. This was confirmed by the additive inhibition of NBDG uptake by antibodies to these transporter isoforms in both cell types. The advantages of flow cytometric analysis of glucose transport include continuous monitoring over extremely short periods of time, increased precision of cell-by-cell flow cytometric measurements versus average uptake rates obtained with radioisotopes, and simultaneous analysis of uptake by different cell populations. Moreover, both uptake rates and the abundance of specific transporters can be determined directly and rapidly on the same cell suspension.

Ethanol-Induced Changes in Insulin-Like Growth Factors and IGF Gene Expression in the Fetal Brain

Abstract Brain growth retardation is a major feature of the fetal alcohol syndrome (FAS). Insulin-like growth factors (IGF-I and IGF-II) have been shown to exert significant metabolic and growth-promoting effects. Previously, we showed that circulating levels of IGF-I as well as hepatic gene expression of both IGFs were decreased in newborn offspring of rats fed ethanol during pregnancy. This study investigated the effects of maternal ethanol ingestion on fetal rat brain growth and on levels of IGF-I and IGF-II, as well as their mRNAs, in fetal brain. IGF-binding protein (IGFBP) levels also were determined. Rats were fed 5% w/v ethanol in a liquid diet during gestation (EF group). Weight-matched animals were pair-fed equicaloric control diet (PF group) or were fed ad libitum (AF group). The mean fetal brain weight of EF offspring was 13% and 16% lower (P < 0.01) than that of PF and AF offspring, respectively. Body weight of EF pups was decreased to a greater extent, resulting in higher brain to body weight ratios in EF pups than in either control group (P < 0.05). IGF-I levels in EF pups decreased by 33% and 41% compared with the corresponding PF and AF values (P < 0.01). IGF-l mRNA levels decreased by 27% and 40% compared with PF and AF values, respectively. A positive correlation was observed between brain IGF-I level and brain weight (r = 0.561, P < 0.01). IGF-II levels were not affected despite a 50% decrease in IGF-II expression. In PF animals, the fetal brain IGF-I and IGF-II mRNA levels were reduced by 28% and 21%, apparently in response to undernutrition. IGF-binding proteins levels were low in the EF group but not statistically significant compared with control values. The diminished fetal brain concentration of IGF-I and decreased gene expression of IGFs may play a role in brain growth retardation associated with FAS.

Decreased glucose transporter 1 gene expression and glucose uptake in fetal brain exposed to ethanol

Using pregnant rats fed equicaloric liquid diets (AF, and libitum-fed controls; PF, pair-fed controls; EF, ethanol-fed), we have previously shown that maternal alcoholism produces a specific and significant decrease of glucose in the fetal brain, which is accompanied by growth retardation. To further define the mechanisms of ethanol-induced perturbations in fetal fuel supply, we have examined (i) the uptake of 2-deoxyglucose (2-DG) by dissociated brain cells from fetal rats that were exposed to ethanol in utero and (ii) the steady-state levels of the glucose transporter-1 (GT-1) mRNA. A 9% decrease in brain weight (P less than 0.001) and a 54.8% reduction in 2-DG uptake into brain cells (P less than 0.02) were found in offspring of EF mothers compared to the AF group. Brain weight correlated with the rate of 2-DG uptake (P less than 0.05). Northern blot analysis showed a 50% reduction of GT-1 mRNA in EF brain relative to that in the AF and PF groups. We conclude that glucose transport into the brain is an important parameter altered by maternal ethanol ingestion.

Omega-Oxidation of Monocarboxylic Acids in Rat Brain

The accumulation of dicarboxylic acids is a prominent feature of inborn and toxin induced disorders of fatty acid metabolism which are characterized by impaired mental status. The formation of dicarboxylic acids is also a critical step in liver in the induction of intracellular fatty acid binding proteins and the proliferation of peroxisomes. In order to understand what potential roles dicarboxylic acids have in brain, we examined the extent of omega-oxidation in rat brain. Homogenates of rat brain catalyze the omega-oxidation of monocarboxylic acids with a specific activity of between 0.87 and 5.23 nmol/mg of post-mitochondrial protein/h, depending on the substrate. The activity is remarkably high, between one-fourth and 4 times the activity found in rat liver, depending on the chain length of the substrate. Specific activity increases with increasing chain length of the substrate. The omega-oxidation of palmitic acid is linear over a range of 0.125–3.0 mg of protein and 5–50 μM substrate for up to 45 minutes of incubation. The product of omega-oxidation in brain is almost exclusively dicarboxylic acid. Cultured rat neurons, astrocytes, and oligodendrocytes all contain omega-oxidation activity. Western blots of rat brain homogenate demonstrate a protein that is recognized by antibody to rat liver CYP4A omega-hydroxylase. These results demonstrate that the omega-oxidative pathway is prominent in brain and could play a role in brain fatty acid metabolism.

Effects of ethanol ingestion on glucose transporter-1 protein and MRNA levels in rat brain

In the normal adult brain, glucose provides 90% of the energy requirement, as well as substrate for nucleic acid and lipid synthesis. We have previously observed that ethanol impairs hexose uptake by rat astrocytes in culture. In the present study, male Sprague-Dawley rats, 200-250 g, were fed liquid diet in which 36% of the calories were derived from ethanol (EF) for 4 weeks. Controls were fed ad libitum (AF) or pair-fed (PF) an equicaloric diet without ethanol. Blood glucose levels did not differ between the groups at the time of study. Glucose transport by brain plasma membranes was characterized by cytochalasin B binding and showed a slight increase in transporter number (mean +/- SEM of 4 experiments = 76.4 +/- 2.5 pmoles/mg protein in EF vs. 69.5 +/- 1.0 in PF) with no change in affinity (1.8 +/- 0.1 nM-1 in EF and 1.6 +/- 0.1 in PF). Glucose transporter, GLUT-1, was increased on Western blots. In contrast, Northern analysis of cortical tissue, using a rat brain glucose transporter cDNA insert (1.59 kb Bgl II fragment of pSPGT-1), showed a 23 to 35% decrease in steady-state levels of glucose transporter mRNA. GLUT-1 mRNA, localized in brain sections by in situ hybridization histochemistry, showed marked reductions in choroid plexus and hippocampus following ethanol treatment. Ethanol appears to have multiple effects on brain GLUT-1.

Effects of ethanol on GLUT1 protein and gene expression in rat astrocytes

Effects of ethanol on glucose transporter gene expression were examined in cultured rat astrocytes. Exposure to 50 or 100 mM ethanol for 18 hours significantly inhibited hexose uptake and reduced the number of glucose transporters, as indicated by binding studies with cytochalasin B. Indirect immunofluorescence and immunoperoxidase staining showed marked reduction of the GLUT1 glucose transporter by exposure to 100 mM ethanol for 5 or 18 hours, but no obvious change in response to 50 mM ethanol. Western blot analysis showed GLUT1 protein levels to be decreased by 52±12% (p<0.05) after exposure to 100 mM ethanol for 18 hours.In situ hybridization histochemistry indicated an increase in steady-state GLUT1 mRNA in astrocytes exposed to 50 or 100 mM ethanol for 5 or 18 hours. Quantitation of GLUT1 mRNA levels by northern blot analysis showed that GLUT1 mRNA levels were increased by 59 and 112% in cells treated for 5 h with 50 and 100 mM ethanol, respectively. A similar effect was observed after treatment for 18 hours, but ethanol did not alter actin gene expression. Experiments using actinomycin D to block RNA synthesis suggest that this increase in steady-state mRNA level results from increased message stability. These results suggest that ethanol acts on GLUT1 gene expression at the post-transcriptional level.

Ethanol and glucose-deprivation neurotoxicity in cortical cell cultures☆

The toxic effects of ethanol on rat cortical cell cultures were compared with neuronal damage induced by glucose deprivation. Exposure to decreased glucose concentrations produced dose-dependent neuronal injury, as indicated by the release of lactate dehydrogenase (LDH) into the culture medium. Complete glucose deprivation resulted in mean LDH release that was more than 60% greater than that from sister cultures incubated in the presence of 5.5 mmol/L glucose. Exposure to ethanol (25, 50, or 100 mmol/L) similarly resulted in dose-related LDH release. The degree of injury resulting from complete glucose deprivation or 100 mmol/L ethanol approximated that produced by exposure to 100 mmol/L glutamic acid. Ethanol did not significantly alter LDH release from cultures consisting of only astrocytes. Both effects were inhibited by the N-methyl-D-aspartate (NMDA) receptor antagonist, D,L-2-amino-5-phosphonovaleric acid (APV). Glutamate levels were increased in the culture medium to 191% +/- 8% of the control value after glucose deprivation (P < .001) and to 186% +/- 16% after exposure to 100 mmol/L ethanol (P < .01). 3H-glutamate uptake by cultured astrocytes was reduced by glucose deprivation and by ethanol. This range of ethanol concentrations has previously been shown to inhibit hexose uptake by cultured astrocytes. The present results suggest that decreased glucose uptake by astrocytes in the presence of ethanol impairs their uptake of glutamate, which contributes to excitotoxic neuronal injury.

Differential removal of insulin-like growth factor binding proteins in rat serum by solvent extraction procedures.

Solvent extraction of serum and other biological fluids at an acidic pH is a convenient method to remove the insulin-like growth factor binding proteins (IGFBPs); however, an incomplete removal of IGFBPs can occur and this can potentially interfere with the radioimmunoassay of insulin-like growth factors (IGFs). This study compared the removal of IGFBPs from normal adult rat serum and 5-day old neonatal rat serum by acid-gel filtration, and three solvent extraction methods, i.e., acid-ethanol (AE), acid-cryo-ethanol (ACE) and formic acid-acetone (FAA) treatments by western ligand blotting and slot-blotting analysis. In adult rat serum all three extraction methods removed nearly 75% of total IGFBPs present. For the neonatal serum, AE and FAA were very inefficient in eliminating the IGFBPs, while ACE was somewhat better, as it removed nearly 30% of IGFBPs. Ligand blots of extracted samples showed that IGFBPs of lower size range, 24 to 32 kDa (IGFBP-4, IGFBPs-1 and-2), were resistant to solvent extraction. Acid-gel filtration, in contrast, eliminated >95% of IGF-binding components in both sera. Determination of IGF-I concentrations in samples after gel filtration and extraction methods revealed lower IGF-I values in neonatal serum in acid extracted samples. These data caution against using solvent extractions for IGFBP removal in fetal/neonatal serum.