Erythropoietin protects primary motor neuron cultures from apoptotic but not necrotic death in vitro

Erythropoietin (EPO), in addition to promoting hematopoiesis, has protective effects in different in vitro and in vivo models of neurodegeneration. Although the molecular mechanisms involved in EPO activity are still not fully understood, several studies have shown that EPO acts by protecting neurons that are committed to die by apoptosis. In this study we tested the effect of EPO in primary cultures of spinal motor neurons under two different stimuli postulated to be responsible for motor neuron death in amyotrophic lateral sclerosis (ALS): excitotoxicity and lack of neurotrophic factors. Methodology: Spinal cords were obtained from 15-day Sprague Dawley rat embryos. Primary cultures of motor neurons, either purified by immunopanning or co-cultured with glial cells and other neuronal cells were used to evaluate the effect of EPO after serum/BDNF deprivation or upon exposure of motor neuron cultures for 48 h to equipotent concentration of two glutamate agonists (kainate or AMPA) inducing about 50% cell death. Immunocitochemical and pharmacological experiments were assessed to evaluate the type of death induced by various detrimental stimuli and to determine the mechanism of protection played by EPO. Results: Recombinant human EPO (2.5 pmol/ml) protected motor neurons by reducing spontaneous apoptosis even in basal condition. EPO protected against the apoptosis induced by 5 μM kainate or by serum/BDNF deprivation, as demonstrated by nuclear morphology and caspase activation and cytochrome-c release from mitochondria, but was ineffective against non apoptotic cell death induced by 1 μM AMPA. The target of EPO protection seems to be the mithochondria permeability transition pore, since EPO reduced the kainate-induced mitochondria depolarisation. The role of mitochondrial depolarisation in inducing apoptosis after kainate treatment was further confirmed by the loss of mitochondrial energisation state, as assessed by the fluorescent probe JC-1 and the selective effect of cyclosporinA2 on motor neuron survival in cultures exposed to kainate. Conclusion: Our study indicates that: I) EPO prevents apoptotic cell death induced by kainate or serum deprivation either in mixed cultures and purified motor neurons; II) EPO protection is selective against cell death associated to the mitochondrion-dependent apoptosis; III) this effect of EPO is due to a direct action on motor neurons, as confirmed by the presence of EPO receptor on purified motor neuron