Novel Small Molecule Positive Allosteric Modulator SPAM1 Triggers the Nuclear Translocation of PAC1-R to Exert Neuroprotective Effects through Neuron-Restrictive Silencer Factor

Abstract

The neuropeptide pituitary adenylate cyclase-activating polypeptide (PACAP) exerts effective neuroprotective activity through its specific receptor, PAC1-R. We accidentally discovered that as a positive allosteric modulator (PAM) of PAC1-R, the small-molecule PAM (SPAM1) has a hydrazide-like structure, but different binding characteristics, from hydrazide for the N-terminal extracellular domain of PAC1-R (PAC1-R-EC1). SPAM1 had a significant neuroprotective effect against oxidative stress, both in a cell model treated with hydrogen peroxide (H2O2) and an aging mouse model induced by D-galactose (D-gal). SPAM1 was found to block the decrease in PACAP levels in brain tissues induced by D-gal and significantly induced the nuclear translocation of PAC1-R in PAC1R-CHO cells and mouse retinal ganglion cells. Nuclear PAC1-R was subjected to fragmentation and the nuclear 35 kDa, but not the 15 kDa fragments, of PAC1-R interacted with SP1 to upregulate the expression of Huntingtin (Htt), which then exerted a neuroprotective effect by attenuating the binding availability of the neuron-restrictive silencer factor (NRSF) to the neuron-restrictive silencer element (NRSE). This resulted in an upregulation of the expression of NRSF-related neuropeptides, including PACAP, the brain-derived neurotrophic factor (BDNF), tyrosine hydroxylase (TH), and synapsin-1 (SYN1). The novel mechanism reported in this study indicates that SPAM1 has potential use as a drug, as it exerts a neuroprotective effect by regulating NRSF.

Integrated Transcriptomic and Proteomic Study of the Mechanism of Action of the Novel Small-Molecule Positive Allosteric Modulator 1 in Targeting PAC1-R for the Treatment of D-Gal-Induced Aging Mice

“…Proteome sequencing confirmed that these proteins, which have been validated to be associated with PACAP and play neuroprotective and nerve-regenerative roles in the nervous system, also exhibit increased expression following treatment with SPAM1. Since our previous report confirmed that SPAM1 can re-set the PACAP-PAC1R system by up-regulating the expression of both PACAP and PAC1-R [9], the results of our multi-omic study not only demonstrate the positive activities of SPAM1 in the nervous system, such as neuroprotection and nerve regeneration, but also help to confirm the positive regulating effect of SPAM1 on re-setting the PACAP-PAC1R system. Several corresponding signal pathways were also revealed by our multi-omic analysis, including the neurotrophin signaling pathway (which is consistent with our SPAM1 data in RGC-5 cells [9]), circadian entrainment, olfactory transduction, and GnRH signaling, which is regulated by PACAP [51].…”

“…GPCRs can regulate gene transcription through an unconventional mode of signal transduction, whereby the GPCR cleaves and relies on its carboxyl-terminal structural domain to translocate to the nucleus [20]. In a previous study, we demonstrated that PAC1-R also has such a transduction pattern [9,21,22]. PAC1-R has a high affinity for PACAP due to a specific region of its N-terminal extracellular domain 1 (EC1) [1].…”

“…It was demonstrated that SPAM1 can re-set the PACAP-PAC1R system by up-regulating PACAP and PAC-1R expression [9], and the results of the whole-transcriptome and proteome analyses indicate that SPAM1 is indeed associated with the PACAP-PAC1R-related pathways; for example, SPAM1 works through the cAMP signaling pathway, calcium signaling pathway, and neurotrophin signaling pathway.…”

“…Additionally, SPAM1 targeted PAC1-R with significantly higher affinity than DOX, and SPAM1 had more effective cytoprotective activity [8]. We showed in our latest study that SPAM1 induces the nuclear translocation of PAC1-R, which exerts neuroprotective effects by regulating neuronal restriction silencing factor (NRSF) [9].…”