Abstract
The neuronal loss caused by excessive glutamate release, or ‘excitotoxicity’, leads to several pathological conditions, including cerebral ischemia, epilepsy, and neurodegenerative diseases. Over-stimulation of presynaptic N-methyl-D-aspartate (NMDA) receptors is known to trigger and support glutamate spillover, while postsynaptic NMDA receptors are responsible for the subsequent apoptotic cascade. Almost all molecules developed so far are unable to selectively block presynaptic or postsynaptic NMDA receptors, therefore a deeper knowledge about intracellular NMDA pathways is required to design more specific inhibitors. Our previous work showed that presynaptic c-Jun N-terminal kinase 2 (JNK2) specifically regulates NMDA-evoked glutamate release and here we demonstrate that an interaction between Syntaxin-1a and JNK2 is fundamental to this mechanism. Based on this evidence, a new cell permeable peptide (CPP), “JGRi1”, has been developed to disrupt the JNK2/STX1a interaction to indirectly, but specifically, inhibit presynaptic NMDA receptor signaling. JGRi1 reduces the NMDA-evoked release of glutamate both in in-vitro and ex-vivo experiments while also being able to widely diffuse throughout brain tissue via intraperitoneal administration. In conclusion, the JNK2/STX1 interaction is involved in presynaptic NMDA-evoked glutamate release and the novel CPP, JGRi1, acts as a pharmacological tool that promotes neuroprotection.
Highlights
The excess of glutamate release is recognized as a major cause for the massive neuronal death occurring in several pathologies including stroke, traumatic brain injury, spinal cord injury, epilepsy, and neurodegenerative conditions such as Amyotrophic Lateral Sclerosis (ALS), Alzheimer’s and Huntington’s diseases[1]
Very little is known about the role of JNK in the presynaptic compartment, especially in relation to the activity of NMDA receptors, it has been reported that the contribution of presynaptic NMDA receptors to spontaneous release of glutamate requires activation of the JNK pathway[20], and we have recently demonstrated that Jun N-terminal kinase 2 (JNK2) modulates the NMDA-evoked glutamate release, presumably through its interaction with JIP-113,21
Through the use of a series of phosphomimetic and phospho-null mutations of STX1a, it has previously been shown that the phosphorylation of STX1a (Ser[14] p-STX1a) regulates the N-terminal interaction with Munc18123, which is a fundamental step for the SNARE complex assembly during neurotransmitter release[24,25]
Summary
The excess of glutamate release is recognized as a major cause for the massive neuronal death occurring in several pathologies including stroke, traumatic brain injury, spinal cord injury, epilepsy, and neurodegenerative conditions such as Amyotrophic Lateral Sclerosis (ALS), Alzheimer’s and Huntington’s diseases[1]. A more efficient and less toxic strategy would be to selectively block NMDA receptors by targeting the intracellular signalling pathways that mostly contribute to the excessive glutamate overflow. A promising approach using Tat-linked peptides was successful in reducing neuronal toxicity supported by disrupting the interaction of postsynaptic NMDA receptors with postsynaptic protein PSD-956–9. The administration of JNK inhibitor, D-JNKi1, which blocks the interaction of JNK to target proteins carrying the JNK binding domain (JBD) such us JIP-1, is able to prevent NMDA receptor-mediated neuronal death[10,16]. In this study we propose Syntaxin-1a (STX1a) as a key JNK2 interactor within this cellular mechanism further study is required to fully describe it To elucidate this pathway further, a new Tat-based, cell-penetrating peptide, JGRi1, has been designed to disrupt the interaction between JNK2/STX1a. The JNK2/STX1a interaction has proved to be an interesting new pharmacological target to modulate glutamate overflow, and JGRi1 an attractive compound for further study in models of neuroexcitotoxicity as a neuroprotective agent
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