Abstract
Extracellular accumulation of toxic concentrations of glutamate (Glu) is a hallmark of many neurodegenerative diseases, often accompanied by hypoxia and impaired metabolism of this neuromediator. To address the question whether the multifunctional neuroprotective action of erythropoietin (EPO) extends to the regulation of extracellular Glu-level and is age-related, young and culture-aged rat astroglial primary cells (APC) were simultaneously treated with 1mM Glu and/or human recombinant EPO under normoxic and hypoxic conditions (NC and HC). EPO increased the Glu uptake by astrocytes under both NC and especially upon HC in culture-aged APC (by 60%). Moreover, treatment with EPO up-regulated the activity of glutamine synthetase (GS), the expression of glutamate-aspartate transporter (GLAST) and the level of EPO mRNA. EPO alleviated the Glu- and hypoxia-induced LDH release from astrocytes. These protective EPO effects were concentration-dependent and they were strongly intensified with age in culture. More than a 4-fold increase in apoptosis and a 2-fold decrease in GS enzyme activity was observed in APC transfected with EPO receptor (EPOR)-siRNA. Our in vivo data show decreased expression of EPO and a strong increase of EPOR in brain homogenates of APP/PS1 mice and their wild type controls during aging. Comparison of APP/PS1 and age-matched WT control mice revealed a stronger expression of EPOR but a weaker one of EPO in the Alzheimer’s disease (AD) model mice. Here we show for the first time the direct correlation between the extent of differentiation (age) of astrocytes and the efficacy of EPO in balancing extracellular glutamate clearance and metabolism in an in-vitro model of hypoxia and Glu-induced astroglial injury. The clinical relevance of EPO and EPOR as markers of brain cells vulnerability during aging and neurodegeneration is evidenced by remarkable changes in their expression levels in a transgenic model of AD and their WT controls.
Highlights
Glutamate (Glu), the major excitatory neurotransmitter in the mammalian central nervous system (CNS), is crucial for brain functions such as learning and memory
In aged cultures (DIV21) glutamine synthetase (GS) was strongly decreased when compared to young astroglial primary cells (APC)
6A-F, cf. +Glu with +Glu+1U E or with +Glu+5U E) with an exception of 21-day old APC exposed to 1U/ml EPO upon hypoxic condition and 1mM Glu (Figure 5F). These results show that generally the GS activity in APCs was more effectively increased by 5U/ml than by 1U/ml EPO, in 14 and 21-day old APC upon hypoxia when compared with the control exposed to Glu only
Summary
Glutamate (Glu), the major excitatory neurotransmitter in the mammalian central nervous system (CNS), is crucial for brain functions such as learning and memory (for review, see 1). Increased extracellular Glu concentration is known to be a common factor in the pathologies of Alzheimer’s disease (AD), Parkinson’s disease (PD), multiple sclerosis (MS) and stroke [3]. These pathological conditions are accompanied by local hypoxia, which is believed to exacerbate disease progression (for review, see 4). Aging leads to increased extracellular Glu levels in the CNS, which in turn cause excitotoxicity and a reduction in the number of glutamatergic synapses [5,6]. Advanced age impairs the delivery of oxygen to cells and tissues, rendering neurons more susceptible to damage [4]
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