Carol L. Zielke

Global up-regulation of chromosome 21 gene expression in the developing Down syndrome brain

Down syndrome (DS) results from complete or partial triplication of human chromosome 21. It is assumed that the neurological and other symptoms are caused by the overexpression of genes on chromosome 21, but this hypothesis has not yet been assessed on a chromosome-wide basis. Here we show that expression of genes localized to chromosome 21 is globally up-regulated in human fetal trisomy 21 cases, both in cerebral cortex extracts and in astrocytic cell lines cultured from cerebral cortex. This abnormal regulation of gene expression is specific to chromosome 21. Our data describe transcriptional changes that are specific to many genes assigned to chromosome 21 and do not directly measure the clinical phenotype of DS. However, it is possible that these gene expression changes ultimately relate to the phenotypic variability of DS.

Sulforaphane Protects Astrocytes Against Oxidative Stress and Delayed Death Caused by Oxygen and Glucose Deprivation

Oxidative stress is an important molecular mechanism of astrocyte injury and death following ischemia/reperfusion and may be an effective target of intervention. One therapeutic strategy for detoxifying the many different reactive oxygen and nitrogen species that are produced under these conditions is induction of the Phase II gene response by the use of chemicals or conditions that promote the translocation of the transcriptional activating factor NRF2 from the cytosol to the nucleus, where it binds to genomic antioxidant response elements. This study tested the hypothesis that pre‐ or post‐treatment of cultured cortical astrocytes with sulforaphane, an alkylating agent known to activate the NRF2 pathway of gene expression protects against death of astrocytes caused by transient exposure to O2 and glucose deprivation (OGD). Rat cortical astrocytes were exposed to 5 μM sulforaphane either 48 h prior to, or for 48 h after a 4‐h period of OGD. Both pre‐ and post‐treatments significantly reduced cell death at 48 h after OGD. Immunostaining for 8‐hydroxy‐2‐deoxyguanosine, a marker of DNA/RNA oxidation, was reduced at 4 h reoxygenation with sulforaphane pretreatment. Sulforaphane exposure was followed by an increase in cellular and nuclear NRF2 immunoreactivity. Moreover, sulforaphane also increased the mRNA, protein level, and enzyme activity of NAD(P)H/Quinone Oxidoreductase1, a known target of NRF2 transcriptional activation. We conclude that sulforaphane stimulates the NRF2 pathway of antioxidant gene expression in astrocytes and protects them from cell death in an in vitro model of ischemia/reperfusion.

Mitochondrial malic enzyme activity is much higher in mitochondria from cortical synaptic terminals compared with mitochondria from primary cultures of cortical neurons or cerebellar granule cells.

Most of the malic enzyme activity in the brain is found in the mitochondria. This isozyme may have a key role in the pyruvate recycling pathway which utilizes dicarboxylic acids and substrates such as glutamine to provide pyruvate to maintain TCA cycle activity when glucose and lactate are low. In the present study we determined the activity and kinetics of malic enzyme in two subfractions of mitochondria isolated from cortical synaptic terminals, as well as the activity and kinetics in mitochondria isolated from primary cultures of cortical neurons and cerebellar granule cells. The synaptic mitochondrial fractions had very high mitochondrial malic enzyme (mME) activity with a Km and a Vmax of 0.37 mM and 32.6 nmol/min/mg protein and 0.29 mM and 22.4 nmol/min mg protein, for the SM2 and SM1 fractions, respectively. The Km and Vmax for malic enzyme activity in mitochondria isolated from cortical neurons was 0.10 mM and 1.4 nmol/min/mg protein and from cerebellar granule cells was 0.16 mM and 5.2 nmol/min/mg protein. These data show that mME activity is highly enriched in cortical synaptic mitochondria compared to mitochondria from cultured cortical neurons. The activity of mME in cerebellar granule cells is of the same magnitude as astrocyte mitochondria. The extremely high activity of mME in synaptic mitochondria is consistent with a role for mME in the pyruvate recycling pathway, and a function in maintaining the intramitochondrial reduced glutathione in synaptic terminals.

Direct measurement of oxidative metabolism in the living brain by microdialysis: a review

This review summarizes microdialysis studies that address the question of which compounds serve as energy sources in the brain. Microdialysis was used to introduce 14C‐labeled glucose, lactate, pyruvate, glutamate, glutamine, and acetate into the interstitial fluid of the brain to observe their metabolism to 14CO2. Although glucose uptake from the systemic system supplies the carbon source for these compounds, compounds synthesized from glucose by the brain are subject to recycling including complete metabolism to CO2. Therefore, the brain utilizes multiple compounds in its domain to provide the energy needed to fulfill its function. The physiological conditions controlling metabolism and the contribution of compartmentation into different brain regions, cell types, and subcellular spaces are still unresolved. The aconitase inhibitor fluorocitrate, with a lower inhibition threshold in glial cells, was used to identify the proportion of lactate and glucose that was oxidized in glial cells versus neurons. The fluorocitrate data suggest that glial and neuronal cells are capable of utilizing both lactate and glucose for energy metabolism.

Elevation of Amino Acids in the Interstitial Space of the Rat Brain following Infusion of Large Neutral Amino and Keto Acids by Microdialysis: Leucine Infusion

A microenvironment similar to that found in maple syrup urine disease was created in the brain of free-moving, awake rats by the infusion of leucine into the brain using microdialysis. To determine the effects on amino acid homeostasis the eluate of the probe was analyzed. Perfusion with leucine elevated the interstitial levels of large neutral amino acids suggesting hetero exchange of large neutral amino acids from neuronal cells into the interstitial space. The data also demonstrated the inter relationship of leucine and glutamine, both of which may be nitrogen sinks in the brain. Elevation of large neutral amino acids in the interstitial space suggests a decreased concentration in neurons which might have an effect on the synthesis of serotonin and catecholamines and suggests a mechanism by which elevated leucine may affect neuronal function in maple syrup urine disease.

Effect of fluorocitrate on cerebral oxidation of lactate and glucose in freely moving rats.

Glucose is the primary carbon source to enter the adult brain for catabolic and anabolic reactions. Some studies suggest that astrocytes may metabolize glucose to lactate; the latter serving as a preferential substrate for neurons, especially during neuronal activation. The current study utilizes the aconitase inhibitor fluorocitrate to differentially inhibit oxidative metabolism in glial cells in vivo. Oxidative metabolism of 14C‐lactate and14C‐glucose was monitored in vivo using microdialysis and quantitating 14CO2 in the microdialysis eluate following pulse labeling of the interstitial glucose or lactate pool. After establishing a baseline oxidation rate, fluorocitrate was added to the perfusate. Neither lactate nor glucose oxidation was affected by 5 μmol/L fluorocitrate. However, 20 and 100 μmol/L fluorocitrate reduced lactate oxidation by 55 ± 20% and 68 ± 12%, respectively (p < 0.05 for both). Twenty and 100 μmol/L fluorocitrate reduced 14C‐glucose oxidation by 50 ± 14% (p < 0.05) and 24 ± 19% (ns), respectively. Addition of non‐radioactive lactate to 14C‐glucose plus fluorocitrate decreased 14C‐glucose oxidation by an additional 29% and 38%, respectively. These results indicate that astrocytes oxidize about 50% of the interstitial lactate and about 35% of the glucose. By subtraction, neurons metabolize a maximum of 50% of the interstitial lactate and 65% of the interstitial glucose.

Comparison of the oxidation of glutamine, glucose, ketone bodies and fatty acids by human diploid fibroblasts

The contribution of glutamine, glucose, ketone bodies and fatty acids to the oxidative energy metabolism of human diploid fibroblasts ws studied. The rate of glutamine oxidation by fibroblasts was 98 nmol/h per mg cell protein compared to 2 nmol/h per mg cell protein or less for glucose, acetoacetate, D-3-hydroxybutyrate, octanoic acid and palmitic acid. Glucose inhibited glutamine oxidation by 85%, while the other substrates had no effect. Therefore, these cells meet their energy requirement almost solely by anaerobic glycolysis and glutamine oxidation.

Compartmentation of [14C]Glutamate and [14C]Glutamine Oxidative Metabolism in the Rat Hippocampus as Determined by Microdialysis

Abstract: Metabolic compartmentation of amino acid metabolism in brain is exemplified by the differential synthesis of glutamate and glutamine from the identical precursor and by the localization of the enzyme glutamine synthetase in glial cells. In the current study, we determined if the oxidative metabolism of glutamate and glutamine was also compartmentalized. The relative oxidation rates of glutamate and glutamine in the hippocampus of free‐moving rats was determined by using microdialysis both to infuse the radioactive substrate and to collect 14CO2 generated during their oxidation. At the end of the oxidation experiment, the radioactive substrate was replaced by artificial CSF, 2 min‐fractions were collected, and the specific activities of glutamate and glutamine were determined. Extrapolation of the specific activity back to the time that artificial CSF replaced 14C‐amino acids in the microdialysis probe yielded an approximation of the interstitial specific activity during the oxidation. The extrapolated interstitial specific activities for [14C]glutamate and [14C]glutamine were 59 ± 18 and 2.1 ± 0.5 dpm/pmol, respectively. The initial infused specific activities for [U‐14C]glutamate and [U‐14C]glutamine were 408 ± 8 and 387 ± 1 dpm/pmol, respectively. The dilution of glutamine was greater than that of glutamate, consistent with the difference in concentrations of these amino acids in the interstitial space. Based on the extrapolated interstitial specific activities, the rate of glutamine oxidation exceeds that of glutamate oxidation by a factor of 5.3. These data indicate compartmentation of either uptake and/or oxidative metabolism of these two amino acids. The presence of [14C]glutamine in the interstitial space when [14C]glutamate was perfused into the brain provided further evidence for the glutamate/glutamine cycle in brain.

Effect of α-Ketoisocaproate and Leucine on the in Vivo Oxidation of Glutamate and Glutamine in the Rat Brain

Leucine and α-ketoisocaproate (α-KIC) were perfused at increasing concentrations into rat brain hippocampus by microdialysis to mimic the conditions of maple syrup urine disease. The effects of elevated leucine or α-KIC on the oxidation of L-[U-14C]glutamate and L-[U-14C]glutamine in the brain were determined in the non-anesthetized rat. 14CO2 generated by the metabolic oxidation of [l4C]glutamate and [14C]glutamine in brain was measured following its diffusion into the eluant during the microdialysis. Leucine and α-KIC exhibited differential effects on 14CO2 generation from radioactive glutamate or glutamine. Infusion of 0.5 mM α-KIC increased [l4C]glutamate oxidation approximately 2-fold; higher concentrations of α-KIC did not further stimulate [14C]glutamate oxidation. The enhanced oxidation of [14C]glutamate may be attributed to the function of α-KIC as a nitrogen acceptor from [14C]glutamate yielding [14C]α-ketoglutarate, an intermediate of the tricarboxylic acid cycle. [14C-]glutamine oxidation was not stimulated as much as [14C-]glutamate oxidation and only increased at 10 mM α-KIC reflecting the extra metabolic step required for its oxidative metabolism. In contrast, leucine had no effect on the oxidation of either [14C]glutamate or [14C]glutamine. In maple syrup urine disease elevated α-KIC may play a significant role in altered energy metabolism in brain while leucine may contribute to clinical manifestations of this disease in other ways.

In vivo glutamine hydrolysis in the formation of extracellular glutamate in the injured rat brain

Hydrolysis of extracellular glutamine as a potential source of increased extracellular glutamate in the quinolinic acid (QUIN)‐injured brain of the unanesthetized, free‐moving rat was examined by microdialysis and HPLC analysis. Injury was initiated by injection of 100 nmoles of QUIN into the hippocampus. Immediately postinjury or 24 hr postinjury, the injection site was perfused with artificial cerebrospinal fluid + 14C‐glutamine to measure its conversion to 14C‐glutamate. L‐trans‐pyrrolidine‐2,4‐dicarboxylate (L‐PDC), a glutamate uptake inhibitor, was added to the perfusate to enhance the detection of extracellular 14C‐glutamate. QUIN injury was followed by an immediate increase in extracellular glutamate that persisted 24 hr later. When 14C‐glutamine was added to the perfusate, a significant amount of 14C‐glutamate was recovered, and it was greater following QUIN injury than in control animals (P < 0.001). Up to 32% of the extracellular 14C‐glutamine was converted to 14C‐glutamate following QUIN injury. Considering the high concentration of glutamine normally present in the extracellular fluid, glutamine hydrolysis is a potential and important source for the increase in extracellular glutamate after neuronal injury in vivo. J. Neurosci. Res. 60:632–641, 2000