Astrocyte mediate neuroprotection in a model of status epilepticus, modulating protease calpain activity in neurons

LAURA MONTROULL; ANDREA BEATRÍZ CRAGNOLINI; VICTOR DANELON; DANIEL MASCÒ

Bertolino

Taller; International Astrocyte School; 2016

Epilepsy is a neurodegenerative disease that affects between 1 and 2% of the world population, characterized by recurrent seizures. This causes permanent damage, resulting in cognitive dysfunction and other serious neurological conditions, including neuronal death. Several strategies have been proposed to protect neurons once the pathology has occurred. Between them, is the Preconditioning stimulus (PC), a preceding sub lethal seizure insult, an intrinsic phenomenon that protects neurons from subsequent injuries. The molecular mechanisms underlying this neuroprotection have been extensively studied, but almost all studies have focused on neurons, without taking astrocyte role account. In this study, an in vitro model of status epilepticus (SE), using pure hippocampal neurons cultures and a coculture system of neuron-astrocyte, we tested the hypothesis that astrocytes has an essential role in mediating neuroprotection. We showed that in pure hippocampal neuron culture, 9hr after SE was induced, we found a significant neuronal (50%) death and a PC could not induce neuroprotection. But, when SE was induced in a neuron-astrocytes coculture system, astrocytes slowed down neuronal death, being 40% 24hr after completition of SE. Interestingly, when a PC was induce in this coculture system, there was a complete neuroprotection. Importantly, inhibiting astrocytes methabolism activity in PC with fluorocitrate, abolished the induction of neuroprotection. To study the underlying mechanisms, we focused on calpain, a family of Ca2+ -dependent non-lysosomal cysteine proteases as a key molecule in astrocyte mediated neuroprotection. As a feature of SE is the increase in intracellular ccalcium level, this leads to the pathological activation of calpain. It has been demonstrated that a PC, when astrocyte are present, can modulate neuron calcium response. Taking this into account is probably that a PC can modulate neuron calpain activity, only when astrocytes are present. To test this, we used the principal calpain substrates (alpha-spectrin, NF-M and TrkB) to assess if PC could modulate calpain activity. In pure hippocampal neuron culture, a PC could not modulate calpain activity, and the degradation patterns of the principal calpain substrates in SE were similar as in PC/SE groups. Interestingly, in the coculture system, when astrocytes were present, a PC could modulate neuron calpain activity were aberrantly activated. These results indicate that a PC could protect neurons from seizure injury, only when astrocyte are present, by modulating calpain activity, providing new insights into potential seizure therapies.