In vitro evidence for post-insult neuroprotective activity of an evolutionarily conserved motif against excitotoxic neuronal cell death

Abstract

In vitro excitotoxic cell death experiments can be considered a screening model of stroke to evaluate the neuroprotective property of specific compounds. Survival of neurons following excitotoxicity is influenced by the neurotrophic factors (nerve growth factor and brain-derived neurotrophic factor). Here, a novel 12 amino-acid peptide [AYKSYVRALPLL (TUF1)] with a high level of evolutionary conservation was assessed for its neuroprotective property in an in vitro model of glutamate-induced N-methyl-D-aspartic acid receptor hyperactivation and excitotoxicity. This peptide shares 100% homology to the conserved motif (SYVRAL) of the neurotrophic factors, which is found in numerous US patents. Following exposure to toxic levels of glutamate (500\u2009µM), cultured primary rat forebrain neurons treated with TUF1 showed a dose-dependent survival rate compared with untreated neurons. The neuroprotective effect was blocked by p75 neurotrophic receptor (p75NTR) inhibitor (MC192), but not by tyrosine kinase receptor inhibitor (K252a) or N-methyl-D-aspartic acid receptor antagonists (MK801 and D-amino-5-phosphonovaleric acid). Serine to alanine substitution that abolishes p75NTR interaction showed a loss of neuroprotective effect. Collectively, the findings showed that TUF1 can protect cultured primary cortical neurons from excitotoxic cell death through the p75NTR-dependent pathway. Given that TUF1 is derived from TMEM35 (NACHO), which is required for the assembly and expression of nicotinic acetylcholine receptors, mechanism of TUF1 action may involve organization of nicotinic acetylcholine receptor and p75 neurotrophin receptor to modulate neuronal responses, including Ca2+ signaling, to cytotoxic events. Unlike nerve growth factor, which requires a pre-insult exposure, TUF1 has neuroprotective properties even with post-insult administration, making it a potential target for therapeutic development in mitigating neuronal damage due to stroke and brain injury.