Astrocytes mediate neuroprotection in a model of hyperactivation, modulating calpain protease activity in neurons

CRAGNOLINI, ANDREA BEATRIZ; MASCÓ, DANIEL HUGO; VICTOR DANELON

Congreso; Glia in health & disease; 2020

Cold Spring Harbor Laboratory

The recurrent seizures in Epilepsy, particularly in the condition called Status Epilepticus (SE), are characterized by aberrant intracellular calcium levels ([Ca2+]i) causing neuronal death and leading to permanent neurological damage. The severity of the neurological damage depends, among other causes, on the duration of the stimulus which is related to the [Ca2+]i. Preconditioning stimulus (PC) is one of many strategies that have been proposed to protect neurons. It consists of an injurious stimuli, including pharmaceutical drugs, brief episodes of ischemia or seizures, applied at doses close to but below the threshold of cell injury, which confers endogenous neuroprotection against a subsequent harmful insult. It has been shown that PC can exert neuroprotection through modulation of neuronal [Ca2+]I. While most of these studies were mainly focused on neurons, recent findings show that astrocytes might have an active role on the neuroprotection induced by PC. However, little is known about the underlying neuroprotective mechanisms triggered by the PC and astrocytes. Seizure-associated inflow of Ca2+ induces changes in the expression levels of several genes and in the activation of proteases, such as calpains. Calpains are a family of Ca2+-dependent non-lysosomal cysteine proteases, that can exert their functions under physiological and pathological conditions. In this study we tested the hypothesis that astrocytes have an essential role mediating the neuroprotective effect of PC by modulating neuronal [Ca2+]I, thus avoiding the abnormal activation of Calpain. Using a neuron-astrocyte co-culture in vitro model of SE¸ we found that the incubation in a Mg2+-free buffer during 3h hyperactivated neurons and caused cell death 24h later. Interestingly, when neurons were hyperactivated for 30min, 24h prior to the SE, preconditioning, there was a complete neuroprotection. Importantly, inhibiting astrocytes metabolic activity with fluorocitrate during the PC, the neuroprotection was completely abolished. We found that PC modulated neuronal calpain activity since the calpain-substrates-degradation patterns in the PC+SE cultures were similar to the control cultures and significantly lower to the SE cultures. When the astrocytes were metabolically inhibited during the PC, neuronal calpain activity was abnormally activated and the PC could not induce neuroprotection. These results indicate that a PC could protect neurons from seizure-induced injury when astrocytes are metabolically active, mainly by modulating neuronal calpain activity, which provides new insights into potential neuroprotective therapies.