Stephan Verleysdonk

Comparison of lactate transport in astroglial cells and monocarboxylate transporter 1 (MCT 1) expressing Xenopus laevis oocytes: Expression of two different monocarboxylate transporters in astroglial cells and neurons

The transport of lactate is an essential part of the concept of metabolic coupling between neurons and glia. Lactate transport in primary cultures of astroglial cells was shown to be mediated by a single saturable transport system with aK m value for lactate of 7.7 mm and aV max value of 250 nmol/(min × mg of protein). Transport was inhibited by a variety of monocarboxylates and by compounds known to inhibit monocarboxylate transport in other cell types, such as α-cyano-4-hydroxycinnamate andp-chloromercurbenzenesulfonate. Using reverse transcriptase-polymerase chain reaction and Northern blotting, the presence of mRNA coding for the monocarboxylate transporter 1 (MCT1) was demonstrated in primary cultures of astroglial cells. In contrast, neuron-rich primary cultures were found to contain the mRNA coding for the monocarboxylate transporter 2 (MCT2). MCT1 was cloned and expressed in Xenopus laevis oocytes. Comparison of lactate transport in MCT1 expressing oocytes with lactate transport in glial cells revealed that MCT1 can account for all characteristics of lactate transport in glial cells. These data provide further molecular support for the existence of a lactate shuttle between astrocytes and neurons.

Intranasal delivery of cells to the brain

The safety and efficacy of cell-based therapies for neurodegenerative diseases depends on the mode of cell administration. We hypothesized that intranasally administered cells could bypass the blood-brain barrier by migrating from the nasal mucosa through the cribriform plate along the olfactory neural pathway into the brain and cerebrospinal fluid (CSF). This would minimize or eliminate the distribution of cellular grafts to peripheral organs and will help to dispense with neurosurgical cell implantation. Here we demonstrate transnasal delivery of cells to the brain following intranasal application of fluorescently labeled rat mesenchymal stem cells (MSC) or human glioma cells to naive mice and rats. After cells crossed the cribriform plate, two migration routes were identified: (1) migration into the olfactory bulb and to other parts of the brain; (2) entry into the CSF with movement along the surface of the cortex followed by entrance into the brain parenchyma. The delivery of cells was enhanced by hyaluronidase treatment applied intranasally 30 min prior to the application of cells. Intranasal delivery provides a new non-invasive method for cell delivery to the CNS.

Metabolism of Glycine in Primary Astroglial Cells: Synthesis of Creatine, Serine, and Glutathione

Abstract: The metabolism of [2‐13C]glycine in astrogliarich primary cultures obtained from brains of neonatal Wistar rats was investigated using 13C NMR spectroscopy. After a 24‐h incubation of the cells in a medium containing glucose, glutamate, cysteine, and [2‐13C]glycine, cell extracts and incubation media were analyzed for 13C‐labeled compounds. Labeled creatine, serine, and glutathione were identified in the cell extracts. If arginine and methionine were present during the incubation with [2‐13C]glycine, the amount of de novo synthesized [2‐13C]creatine was two‐fold increased, and in addition, 13C‐labeled guanidinoacetate was found in cell extracts and in the media after 24 h of incubation. A major part of the [2‐13C]glycine was utilized for the synthesis of glutathione in astroglial cells. 13C‐labeled glutathione was found in the cell extracts as well as in the incubation medium. The presence of newly synthesized [2‐13C]serine, [3‐13C]serine, and [2,3‐13C]serine in the cell extracts and the incubation medium proves the capability of astroglial cells to synthesize serine out of glycine and to release serine. Therefore, astroglial cells are able to utilize glycine as a precursor for the synthesis of creatine and serine. This proves that at least one cell type of the brain is able to synthesize creatine. In addition, guanidinoacetate, the intermediate of creatine synthesis, is released by astrocytes and may be used for creatine synthesis by other cells, i.e., neurons.

Rapid uptake and degradation of glycine by astroglial cells in culture: Synthesis and release of serine and lactate

Free glycine is known to have vital functions in the mammalian brain, where it serves mainly as both neurotransmitter and neuromodulator. Despite its importance, little is known about the metabolic pathways of glycine synthesis and degradation in the central nervous system. In this study, the pathway of glycine metabolism in astroglia‐rich primary cultures from rat brain was examined. The cells were allowed to degrade glycine in the presence of [U‐14C]glycine, [U‐13C]glycine or [15N]glycine. The resulting intra‐ and extracellular metabolites were analyzed both by high‐performance liquid chromatography and by 13C/15N nuclear magnetic resonance spectroscopy. Glycine was rapidly consumed in a process obeying first‐order kinetics. The initial glycine consumption rate was 0.47 nmol per mg protein. The half‐life of glycine radiolabel in the incubation medium was shorter than that of glycine mass. This suggests that glycine is produced from endogenous sources and released simultaneously with glycine uptake and metabolism. As the main metabolites of the glycine carbon skeleton in astroglia‐rich primary cultures from rat brain, serine and lactate were released during glycine consumption. The main metabolite containing the glycine amino nitrogen was glutamine. To establish a metabolic pathway from glycine to serine in neural tissue, homogenates of rat brain and of neural primary cultures were assayed for their content of serine hydroxymethyltransferase (SHMT) and glycine cleavage system (GCS). SHMT activity was present in homogenates of rat brain as well as of astroglia‐rich and neuron‐rich primary cultures, whereas GCS activity was detectable only in homogenates of rat brain and astroglia‐rich primary culture. Of the two known SHMT isoenzymes, only the mitochondrial form was found in rat brain homogenate. It is proposed that, in neural tissue, glycine is metabolized by the combined action of SHMT and the GCS. Owing to the absence of the GCS from neurons, astrocytes appear to be the only site of this part of glycine metabolism in brain. However, neurons are able to utilize as energy source the lactate formed by astroglial cells in this metabolic pathway. GLIA 27:239–248, 1999.

Primary cultures as a model for studying ependymal functions: glycogen metabolism in ependymal cells

Ependymal cells form a single-layered, ciliated epithelium at the interface between the cerebrospinal fluid and the brain parenchyma. Although their morphology has been studied in detail, ependymal functions remain largely speculative. We have established and characterized a previously described cell culture model to investigate ependymal glycogen metabolism. During growth in minimal medium lacking many non-essential amino acids including L-glutamate, but containing glucose at physiological concentration, the cells contained negligible amounts of glycogen (7+/-3 nmol glucosyl residues/mg protein) despite the presence of insulin. However, during a period of 24 h, the cells accumulated glycogen to very high levels after transferal to a medium containing insulin, glucose at a 5-fold higher concentration, and all proteinogenic amino acids except L-asparagine and L-serine (990+/-112 nmol glucosyl residues/mg protein). Omission of insulin resulted in a 50% reduction in glycogen accumulation. Upon glucose deprivation, glycogen was degraded with a half-life of 21 min. The ependymal primary cultures contained 80+/-5 mU glycogen phosphorylase (Pho)/mg protein and stained positively with antibodies raised against this enzyme. Astroglial cultures built up less glycogen and had less Pho activity under identical conditions. Ependymal glycogen was mobilized by noradrenaline and serotonin. Our results indicate that ependymal cells maintain glycogen as a functional energy store, subject to rapid turnover dependent on the availability of energy substrates and the presence of appropriate signal molecules. Thus ependymocytes appear to be active players in the multitude of processes resulting in normal brain function, and ependymal primary cultures are suggested as a suitable model for studying the role of ependymal cells in these processes.

Synthesis and release of L-serine by rat astroglia-rich primary cultures

L‐serine is known to have important functions in the mammalian CNS other than being a constituent of proteins. It is the metabolic precursor of the neuroactive substances D‐serine and glycine, serves as a building block for phospholipid biosynthesis and is likely to be a neurotrophic factor. Based on the observation that rat astroglia‐rich primary cultures release L‐serine into their culture medium, the biosynthesis and release of L‐serine in these cultures has been investigated. Release of L‐serine is due to both biosynthesis from glucose and to proteolysis. Amino groups for L‐serine synthesis originate from transamination of amino acids that are either taken up from the extracellular space or produced intracellularly by proteolysis. The enzymes of the “phosphorylated pathway” of serine biosynthesis, i.e., 3‐phosphoglycerate dehydrogenase, phosphoserine aminotransferase and phosphoserine phosphatase are present in rat brain as well as in rat astroglia‐rich primary cultures and their specific activities have been determined. The presence of these enzymes indicates the operation of the “phosphorylated pathway” of L‐serine biosynthesis in brain. GLIA 30:19–26, 2000.

Distribution of secretory pathway Ca2+ ATPase (SPCA1) in neuronal and glial cell cultures.

1. Secretory pathway Ca2+ ATPase type 1 (SPCA1) is a newly recognized Ca2+/Mn2+-transporting pump localized in membranes of the Golgi apparatus.2. The expression level of SPCA1 in brain tissue is relatively high in comparison with other tissues.3. With the aim to determine the expression of SPCA1 within the different types of neural cells, we investigated the distribution of SPCA1 in neuronal, astroglial, oligodendroglial, ependymal, and microglial cell cultures derived from rat brains.4. Western Blot analysis with rabbit anti-SPCA1 antibodies revealed the presence of SPCA1 in homogenates derived from neuronal, astroglial, ependymal, and oligodendroglial, but not from microglial cells.5. Cell cultures that gave rise to positive signal in the immunoblot analysis were also examined immunocytochemically.6. Immunocytochemical double-labeling experiments with anti-SPCA1 serum in combination with antibodies against cell-type specific proteins showed a localization of the SPCA1signal within cells stained positively also for GFAP, α-tubulin or MBP.7. These results definitely established the expression of SPCA1 in astroglial, ependymal, and oligodendroglial cells.8. In addition, the evaluation of neuronal cultures for the presence of SPCA1 revealed an SPCA1-specific immunofluorescence signal in cells identified as neurons.

Expression of 3‐hydroxyisobutyrate dehydrogenase in cultured neural cells

The branched‐chain amino acids (BCAAs) – isoleucine, leucine, and valine – belong to the limited group of substances transported through the blood–brain barrier. One of the functions they are thought to have in brain is to serve as substrates for meeting parenchymal energy demands. Previous studies have shown the ubiquitous expression of a branched‐chain alpha‐keto acid dehydrogenase among neural cells. This enzyme catalyzes the initial and rate‐limiting step in the irreversible degradative pathway for the carbon skeleton of valine and the other two branched‐chain amino acids. Unlike the acyl‐CoA derivates in the irreversible part of valine catabolism, 3‐hydroxyisobutyrate could be expected to be released from cells by transport across the mitochondrial and plasma membranes. This could indeed be demonstrated for cultured astroglial cells. Therefore, to assess the ability of neural cells to make use of this valine‐derived carbon skeleton as a metabolic substrate for the generation of energy, we investigated the expression in cultured neural cells of the enzyme processing this hydroxy acid, 3‐hydroxyisobutyrate dehydrogenase (HIBDH). To achieve this, HIBDH was purified from bovine liver to serve as antigen for the production of an antiserum. Affinity‐purified antibodies against HIBDH specifically recognized the enzyme in liver and brain homogenates. Immunocytochemistry demonstrated the ubiquitous expression of HIBDH among cultured glial (astroglial, oligodendroglial, microglial, and ependymal cells) and neuronal cells. Using an RT‐PCR technique, these findings were corroborated by the detection of HIBDH mRNA in these cells. Furthermore, immunofluorescence double‐labeling of astroglial cells with antisera against HIBDH and the mitochondrial marker pyruvate dehydrogenase localized HIBDH to mitochondria. The expression of HIBDH in neural cells demonstrates their potential to utilize valine imported into the brain for the generation of energy.

Studies on Fructose Metabolism in Cultured Astroglial Cells and Control Hepatocytes: Lack of Fructokinase Activity and Imrrunoreactivity in Astrocytes

Astroglia-rich primary cultures derived from the brains of newborn rats can be grown in the presence of sorbitol or fructose. In the present study, evidence was obtained by enzymatic analysis and immunocytochemistry that fructose is further metabolized to fructose-6-phosphate and that fructokinase is lacking in the astrocytes. In contrast, fructose-1-phosphate as well as fructokinase immunoreactivity could be detected in cultured hepatocytes. Considerable amounts of astroglial glycogen were synthesized from fructose. Lactate release in fructose-fed cultures was still 30% that of glucose-fed cells and was abolished in the presence of 2-deoxyglucose. No glycogen was synthesized when sorbitol, which is converted intracellularly to fructose, replaced glucose in the incubation medium. However, lactate release from sorbitol-fed cultures was still significant and was not abolished by 2-deoxyglucose. The results are compatible with the idea of astroglial glycogen being a store of lactate rather than glucose. Furthermore, the results demonstrate that sorbitol is an adequate substrate for astroglial glycolysis but, in contrast to fructose, cannot be utilized for the buildup of glycogen stores.

Expression of pyruvate carboxylase in cultured oligodendroglial, microglial and ependymal cells.

The mitochondrial enzyme, pyruvate carboxylase (PC; EC 6.4.1.1) is considered to play a significant role in the intermediary metabolism of neural tissue. PC-catalyzed carboxylation of pyruvate to oxaloacetate is a major anaplerotic reaction in brain. Anaplerosis is essential for homeostasis of the members of the tricarboxylic acid (TCA) cycle. Several biochemical pathways rely on withdrawing TCA cycle members. Prominent among these are biosynthesis of fatty acids and of non-essential amino acids such as aspartate, asparagine, glutamate and glutamine, gluconeogenesis, glycogen synthesis, and regeneration of NADPH. The expression of PC in brain has already been described and assigned to astrocytes. Since pyruvate carboxylase deficiency is associated with malformations of the brain, e.g., inadequate development of the corpus callosum and the lack of myelination, one can hypothesize that PC may be expressed also in glial cells other than astrocytes. Therefore, the expression of PC was investigated in cultured oligodendroglial, microglial, and ependymal cells. As assessed by RT-PCR, all these cultures contain PC mRNA. This mRNA is generated in a transcription process that is regulated by the “distal class” of promoters of the PC gene. The expression of PC among cultured glial cells was studied with a rabbit antiserum by immunoblotting and immunocytochemistry. The results indicate that PC is not only expressed in cultured astroglial cells but also in cultured oligodendrocytes, microglial cells, and ependymocytes. It appears that the intermediary metabolism of these cells includes the anaplerotic action of PC as well as possibly also functions of the enzyme in biosynthetic pathways and the provision of NADPH for defense against reactive oxygen species.