CDP-choline protects motor neurons against apoptotic changes in a model of chronic glutamate excitotoxicity in vitro

Introduction
A glutamate-mediated mechanism is accepted to be involved in progressive motor neuron (MN) loss in amyotrophic lateral sclerosis (ALS) [36,40,41]. Glutamate receptor overactivation evokes excitotoxic neuronal injury via various pathways, including necrotic and apoptotic mode of cell death [49]. The in vitro model of chronic glutamate excitotoxicity, originally introduced by Rothstein et al. [37], has been widely used for the study of the mechanism responsible for progressive neuronal injury and neuroprotection. Citicoline, also known as CDP-choline (cytidine-5-diphosphocholine), is an endogenous nucleoside that exhibits neuroprotective abilities in certain central nervous system (CNS) injury models [8,11,50]. The neuroprotective properties of CDP-choline seem to be related to its action on glutamate-mediated cell death, but the precise mechanism remains not fully understood [1,21]. Citicoline might decrease the extracellular level of glutamate by inhibition of neuronal glutamate efflux and increased astrocytic glutamate uptake. It has been suggested that the neuroprotective effect of this compound is related to inhibition of the glutamate-induced apoptotic pathway of cell injury [31]. The present study was performed to determine the neuroprotective efficacy of CDP-choline on development of MN neurodegeneration in organotypic cultures of rat lumbar spinal cord chronically exposed to DL-threo-b- hydroxyaspartate (THA) – a specific glutamate uptake blocker.
Material and Methods
The study was performed on organotypic cultures prepared from lumbar spinal cord obtained from 8-day-old rat pups. The explants were placed on collagen-coated cover glasses with two drops of nutrient medium (consisting of 25% inactivated fetal bovine serum and 75% Dulbeco Modified Eagle’s Medium supplemented with glucose to a final concentration of 600 mg% and with antibiotics), sealed into Maximow double assemblies and kept at 36.6°C. The medium was changed twice a week. On the 10-14th day in vitro (DIV), the well-differentiated cultures were subjected to the specific glutamate uptake blocker DL-threo-beta-hydroxyaspartate (THA, Sigma) in a concentration of 100 µM and to CDP-choline in three experimental groups: 1/ control group incubated with 100 µM CDP-choline, 2/ cultures treated with 100 µM THA, 3/ cultures incubated with medium containing THA but pretreated with CDP-choline in concentrations as before. After 2, 3, 5, 7 and 14 days post treatment the cultures were processed for the electron microscope. They were rinsed in cacodylate buffer (pH 7.2), fixed in a mixture containing 0.8% formaldehyde and 2.5% glutaraldehyde for 1 hour, postfixed in 1% osmium tetroxide, dehydrated in alcohols in graded concentrations and embedded in Epon 812. Ultrathin sections were counterstained with uranyl acetate and lead citrate and examined in a JEOL 1200EX electron microscope.
Results
The rat spinal cord cultures exposed to CDP-choline alone exhibited quite well preserved motor neurons with a large nucleus with dispersed chromatin and abundant cytoplasm containing numerous organelles (Fig. 1). Normal astroglial cells and neuropil filled with neuronal and glial processes were observed (Figs. 2, 3). Chronic THA exposure resulted in a distinct type of MN injury including necrosis with total destruction of cytoorganelles (Fig. 4), apoptotic changes with more or less condensed chromatin and apoptotic bodies (Fig. 5) and autophagocytosis with the presence of numerous autophagic vacuoles containing destroyed organelles (Figs. 6, 7). Moreover, a few completely damaged neurons that shared apoptotic, autophagic and necrotic characteristics were observed. In the spinal cord cultures treated with CDP-choline together with THA, evidence of inhibition of progressive MN neurodegeneration, especially limitation of apoptotic changes, was documented. Typical apoptotic changes, characterized by peripheral condensation and margination of nuclear chromatin or aggregation of nuclear chromatin in dense masses beneath the margin of the nucleus, could be seen only sporadically. Numerous large motor neurons exhibited quite well-preserved organelles (Fig. 8) or displayed only subtle mitochondrial swelling. Membrane-bound apoptotic profiles with fragments of compact chromatin and/or cytoplasmic structures were not seen in the cultures treated with CDP-choline. Nevertheless, some neurons showed various degrees of cytoplasmic vacuolization and contained numerous vesicles and/or vacuoles of various size and damaged mitochondria (Fig. 9). Totally destroyed, necrotic cells appeared occasionally (Fig. 10). More often MNs exhibited characteristics typical for autophagocytosis. These changes were observed especially after a longer time (7, 14 days) of the experiment. The cytoplasm of these neurons displayed many autophagic vacuoles of various size and shape, containing damaged organelles, i.e. ribosomes, mitochondria and/or unidentified vacuoles (Figs. 11, 12). In some large MNs, numerous secondary lysosomes and small dark bodies or autophagosomes were observed. A striking feature is that most MNs with advanced autophagocytic changes of the cytoplasm exhibited a well-preserved nucleus.
Discussion
Glutamate-induced excitotoxicity is widely accepted to underlie the neuronal injury in certain acute or chronic neurodegenerative processes [9,12,30]. Both neurons and astroglial cells are involved in glutamate transmission [5,19,23,32,33]. It has been documented that astroglia express functional ionotropic (iGluRs) and metabotropic (mGluRs) Glu receptors and their dysfunction might result in ineffective Glu uptake and increase of its extracellular level [13,24,38]. Recently, the glutamate-mediated mechanism via defective glial and/or neuronal glutamate transport is widely accepted as responsible for progressive MN loss [36,38,40,41]. In ALS patients, elevated levels of glutamate in cerebrospinal fluid and selective reduction of EAAT2 glutamate astrocytic transporter have been documented [39,43]. The decrease or loss of GLT-1 glutamate transporter has also been observed in various animal models of ALS, including SOD-1 transgenic mice and a mutant SOD1 G93A rat model [7,45]. Morphological studies demonstrated the progression of morphological changes within CNS in a transgenic rat model of familial amyotrophic lateral sclerosis [17,20,35]. It seems that loss of glutamate transporters in ALS might be secondary to astrocytic activation as alterations in glutamate transporters are accompanied by marked astrogliosis. Thus, cell death in amyotrophic lateral sclerosis results from the interplay between neuronal and glial cells [15]. It could be suggested that manipulation of glutamate transporters might be useful in therapeutic treatments in MN degeneration. Organotypic cultures of spinal cord, that maintain neuron-astrocyte structural and metabolic interactions, are especially useful to study the mechanism of progressive MN degeneration. Our previous ultrastructural studies performed on organotypic cultures of rat lumbar spinal cord chronically exposed to specific glutamate uptake blockers DL-threo-b-hydroxyaspartate (THA) and L-trans-pyrrolidine-2, 4-dicarboxylate (PDC) evidenced different modes of cell death, i.e. necrotic, apoptotic and/or autophagic degeneration [27,28], accompanied by distinct glial changes [29]. There is increasing evidence that both programmed cell death [48] and so-called “autophagic cell death” participate in cell degeneration in certain pathological conditions [25]. In the present study we tested the neuroprotective ability of citicoline – a naturally endogenous nucleoside that play an important role in formation and repair of cell membranes [52]. It has been documented that citicoline activates the biosynthesis of phosphatidylcholine (PtdCho) – a phospholipid essential for the maintenance of intra- and extracellular membrane [26,46]. Moreover, the hydrolytic products of citicoline (cytidine and choline) are involved in the formation of nucleic acids, proteins and acetylcholine [3,47]. Such properties allow citicoline to be used in clinical trials in certain central nervous system (CNS) disorders [50]. The neuroprotective efficacy of this agent was documented in patients with severe stroke, Alzheimer’s disease, glaucoma and in various experimental models of CNS injury in vivo and in vitro [4,6,10,16,18,21,22,34]. It has been suggested that the neuroprotective action of citicoline might be related to a decrease of extracellular glutamate level. The protective effect of CDP-choline might also be associated with its actions on cell membrane stability [1,2,42]. The activation of phospholipases and degradation of membrane phospholipids have been reported in various neurodegenerative disorders [14,42,44,51]. The present study demonstrated that CDP-choline exerts neuroprotection in progressive MN injury in a model of chronic excitotoxicity in vitro. It inhibited mainly neuronal apoptotic changes, whereas necrotic and autophagocytic abnormalities were not reduced. This confirms the suggestion that citicoline might protect neurons against the glutamate-induced apoptotic pathway [31], probably via a negative effect on activation of the caspase cell death pathway.
Acknowledgements
The study was supported by the Ministry of Science and Education, grant No. 2 PO5A17429. The authors wish to thank Jolanta Gebarowska, Elzbieta Grzywaczewska and Mariola Zielinska for their skilful technical assistance.
References
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