Excitotoxicity and NMDA receptor-mediated acquired neuroprotection in human iPSC-derived brain organoids

Excitotoxic cell death, resulting from excess brain glutamate and mediated predominantly through NMDA receptors (NMDARs), is implicated in various acute and chronic pathological conditions of the human brain, such as stroke, traumatic brain injury and neurodegenerative diseases. In contrast, physiological NMDAR activation renders rodent neurons more resistant to potentially toxic stressors - a mechanism referred to as acquired neuroprotection. It is, however, not known whether such a dual action of NMDAR-signalling also exists human neurons. This study employed human induced pluripotent stem cell-derived forebrain organoids to investigate NMDAR-mediated pro-death and pro-survival signalling in human neurons. Exposure of forebrain organoids to high concentrations of NMDA caused an excitotoxic cascade involving loss of plasma membrane potential, cessation of synaptic activity, shut-off of the transcription factor CREB, induction of death-promoting P53 expression, structural disintegration and ultimately cell death. In contrast, treatment of brain organoids with low doses of NMDA triggered enhanced synaptic activity and survival-promoting signalling including the CREB phosphorylation and the expression of neuroprotective genes. This condition, if applied as a pre-treatment, protected organoids against excitotoxic insults within a defined time window. Surprisingly, the protective mechanism relied solely on the activation of NMDARs per se and occurred independently of the concomitant synaptic activation. Moreover, application of established as well as newly developed drugs to combat excitotoxicity protected human forebrain organoids against NMDA-induced cell death. The findings presented here show that many aspects of the dual action of NMDAR-signalling, including NMDAR-mediated acquired neuroprotection, are preserved in human neurons. The forebrain organoid methodology established here may serve as a platform for the development and testing of drugs counteracting excitotoxic cell death arising, for example, from acute cerebrovascular ischaemia and neurodegenerative diseases in humans