Jacques Borg

HIGH AFFINITY UPTAKE OF L-GLUTAMATE AND L-ASPARTATE BY GLIAL CELLS

The presence of an efficient, high affinity uptake system specific for L‐glutamate and L‐aspartate has been demonstrated in cultured glial cells originating from syrian hamster astroblasts (line NN). The system was found to be temperature dependent, to require sodium ions and its structural specificity is similar to systems studied previously in brain slices and synaptosomes. Co‐culturing of the glial cells with neuroblastoma cells brought about only minor changes in structural specificity while values of kinetic parameters remained virtually unchanged. The temperature dependence and the susceptibility to metabolic inhibitors suggest that the uptake is mediated by an active transport system.

Progressive Motor Neuron Impairment in an Animal Model of Familial Amyotrophic Lateral Sclerosis

Mutations of Cu,Zn superoxide dismutase cause an autosomal dominant form of familial amyotrophic lateral sclerosis. An animal model of the disease has been produced by expressing mutant human SOD1 in transgenic mice (G93A). In order to quantify the dysfunction of the motor unit in transgenic mice, electromyographic recordings were performed during the course of the disease. The first alterations in neuromuscular function appeared between P63 and P90. The deficits became even more striking after P100; compound muscle action potentials in the hindlimb decreased by 80% of initial value. Spontaneous fibrillation potentials were measured in more than 50% of transgenic mice. The number of motor units in the gastrocnemius muscle was progressively reduced over time, down to 18% of the control value at P130. Moreover, distal motor latencies increased after P120. These data suggest that the initial dysfunctions of motor unit are related to a severe motor axonal degeneration, which is followed at later periods by myelin alteration.

Glutamine and glutamate transport in cultured neuronal and glial cells.

The uptake of L-glutamine in neuronal and glial cultures derived from rat cerebral hemispheres was found to be mediated by a low affinity-high capacity mechanism which was concentrative in both cell types; the calculated Km and Vmax were twice as high in glial than in neuronal cultures. In contrast L-glutamate was taken up by a high affinity system which was particularly efficient and concentrative in the glial cells. Different transport mechanisms for L-glutamine appeared to operate in the two cell types: L-glutamine uptake in neurons was sodium-dependent, specifically inhibited by L-glutamine but not affected by high potassium concentrations in the external medium; on the other hand, glial glutamine transport was decreased when potassium concentration increased, was sodium-independent and significantly inhibited by 3 structurally related amino acids. No significant contribution of homoexchange could be detected in either cell type. After [14C]glutamine preincubation, the radioactivity released into the superfusion medium by neuronal cells was increased in the presence of a high potassium concentration; no such effect could be seen in the case of glial cultures. A regulatory mechanism is suggested where astrocyte depolarization and repolarization would channel a flux of glutamine toward the neurons, subsequent to a glutamate flux in the opposite direction.

Changes in the Uptake of GABA and Taurine During Neuronal and Glial Maturation

Abstract: The influence of the time of culture on GABA and taurine uptake was investigated in spontaneously matured cultures of glial and neuronal origins and in cultures treated with cyclic nucleotides. In the spontaneously matured cultures the capacity of the high‐affinity neuronal GABA transport system increased with time in culture. Essentially opposite results were found for the uptake of GABA by glial cultures. In contrast with the neuronal uptake of GABA, the capacity of the taurine transport system was significantly decreased. Uptake of taurine into glia, however, exhibited a progressive increase with the period of culture. The values of Km, for the high‐affinity systems were always found to range around 10 μM. It is suggested that, in mature cells, neuronal uptake sites are of prime importance for GABA transport, while taurine uptake may be more specifically directed towards glial cells. When cultures were treated with cyclic nucleotide derivatives, a morphological differentiation was induced, which could not be linked to a stimulation of GABA or taurine uptake systems as compared with the non‐treated cultures.

Uptake of l‐Glutamate and Taurine in Neuroblastoma Cells with Altered Fatty Acid Composition of Membrane Phospholipids

Abstract: Effects of altered composition of membrane lipids on high‐affinity uptake of l‐glutamate and taurine were studied in an established neuroblastoma cell line M1. Increase in participation of certain fatty acids (20: 5ω3 and 22: 5ω3) as constituents of membrane lipids resulted in a fourfold increase in the maximum initial rate of l‐glutamate uptake (Vmax) while increase in Vmax of taurine uptake was much smaller. Neither structural requirements of l‐glutamate uptake nor passive permeability of the membrane to l‐glutamate or taurine seemed to be influenced by the alterations in the lipid composition. Since increased proportions of 20: 5ω3 and 22: 5ω3 in the membrane phosphatides caused no dramatic changes in kinetic parameters of taurine uptake and incorporation of either linoleic or linolenic acid alone into the phosphatides had only a relatively small effect on some of the measured parameters, the possibility of a specific relationship between 22: 5ω3 and/or 20: 5ω3 and l‐glutamate uptake is discussed. Unlike l‐glutamate uptake systems in other preparations, the high‐affinity uptake system of l‐glutamate in neuroblastoma cells did not readily accept l‐aspartate as a substrate.

CHARACTERIZATION OF TAURINE UPTAKE BY NEURONAL AND GLIAL CELLS IN CULTURE

Uptake of [35S]taurine was studied in parallel on glial and neuronal cells maintained in continuous culture, including transformed neuronal cells. Both glial and neuronal taurine uptake systems were concentrative, highly sodium‐dependent and inhibited by closely related structural analogues such as hypotaurine, β‐alanine and GABA. Strychnine was found to be a potent inhibitor of taurine uptake, especially in the glial cells, while parachloromercuriphenylsulphonate was more efficient on the neuronal clones. In contrast with uptake by neuroblastoma cells, the glial transport was dependent on the presence of calcium in the incubation medium.

Prevention of mutant SOD1 motoneuron degeneration by copper chelators in vitro.

An animal model of familial amyotrophic lateral sclerosis (FALS) has been generated by overexpression of human CuZn superoxide dismutase (SOD1) containing a substitution of glycine to alanine at position 93 in transgenic G93A mice. The loss of motoneurons shown in this model has been attributed to a dominant gain of function of this mutated enzyme, which might be due to copper toxicity. This hypothesis was tested in purified spinal motoneurons cultures originating from G93A transgenic embryos. Spinal motoneurons were isolated from E13 embryos by several steps including density gradient centrifugation. The effect of copper chelators on survival and neurite growth of motoneurons was investigated. Survival of G93A motoneurons was decreased by 46% as compared to wild-type motoneurons. Moreover, G93A motoneurons showed reduced neurite outgrowth. Copper chelators strikingly increased viability of G93A motoneurons (by over 200%) but had no effect on wild-type cells. Presence of DDC in the medium increases the length of neurites from G93A motoneurons. The present results suggest the capacity of copper chelators to reduce the effect of reverse function of mutated SOD1 on motoneurons.

High-affinity uptake of γ-aminobutyric acid in cultured glial and neuronal cells

Both glial and neuronal cells maintained in primary culture were found to accumulate [3H]GABA by an efficient “high-affinity” uptake system (apparentKm=9 μM,Vmax=0.018 and 0.584 nmol/mg/min, respectively) which required sodium ions and was inhibited by 1 mM ouabain. Strychnine and parachloromercuriphenylsulfonate (pCS) (both at 1 mM) also strongly inhibited uptake of [3H]GABA, but metabolic inhibitors (2,4-dinitrophenol, potassium cyanide, and malonate) were without effect. Only three structural analogs of GABA (nipecotate, β-alanine, and 2,4-diaminobutyrate) inhibited uptake of [3H]GABA, while several other compounds with structural similarities to GABA (e.g. glycine,l-proline, and taurine) did not interact with the system. The kinetic studies indicated presence of a second uptake (Km=92 μM,Vmax=0.124 nmol/mg/min) in the primary cultures containing predominantly glioblasts. On the other hand, only one of the neuronal cell lines transformed by simian virus SV40 appeared to accumulate [3H]GABA against a concentration gradient. ApparentKm of this uptake was relatively high (819 μM), and it was only weakly inhibited by 1 mM ouabain and 1 mM pCS. The structural specificity also differed from that of the uptake observed in the primary cultures. Significantly, none of the nontransformed continuous cell lines of either tumoral (glioma, C6; neuroblastoma, Ml; MINN) or normal (NN; I6) origin actively accumulated [3H]GABA. It is suggested that for the neurochemical studies related to GABA and requiring homogeneous cell populations, the primary cultures offer a better experimental model than the continuous cell lines.

Selective changes in mitochondria respiratory properties in oxidative or glycolytic muscle fibers isolated from G93AhumanSOD1 transgenic mice

Cases of familial amyotrophic lateral sclerosis (FALS) are associated with mutations in cytosolic copper, zinc superoxide dismutase (SOD1). Total SOD activity and functional mitochondrial properties were studied in muscles and nervous tissues of control and transgenic mice mimicking the disease. It was found that total SOD activity was lower in nervous tissues than in muscles in both transgenic and control mice. In addition SOD activity increased during progression of disease in muscle but not in nervous tissue of transgenic mice. Maximal oxygen consumption and apparent Km for ADP were decreased in mitochondria from transgenic soleus (an oxidative muscle). However there was no difference between control and transgenic mice in respiratory parameters of mitochondria in the EDL muscle (a glycolytic muscle). These findings indicate that oxidative stress due to SOD1 mutations could alter energy metabolism in FALS mice, thereby affecting primarily oxidative muscle of the limbs, independently of motoneuron loss.

Peripheral administration of a long-chain fatty alcohol promotes septal cholinergic neurons survival after fimbria-fornix transection

Peripherally administered n-hexacosanol, a long-chain fatty alcohol, strongly attenuated the degeneration of cholinergic neurons in the medial septum and the vertical limb of the diagonal band of Broca following transection of their dorsal projection to the hippocampus by unilateral fimbria-fornix aspiration. The maintenance of AChE-positive neurons was particularly striking in the vertical limb of the diagonal band of Broca, where 83% of the neurons survived after two weeks of n-hexacosanol treatment compared to 51% in the non-treated animals. This compound has already been found to possess some neurotrophic activities on cultured neurons from rat cerebral cortex. But the present study is the first report of an in vivo neurotrophic influence exerted by an exogenously administered long-chain fatty alcohol. The low dosage needed and the peripheral administration of this compound may be of great advantage in the reduction of cell loss in some neurodegenerative disorders like Alzheimers disease or stroke.