Blue light induces the nuclear translocation of neuropeptide receptor PAC1-R associated with the up-regulation of PAC1-R its own in reactive oxygen species associated way.

PAC1-R is neuropeptide PACAP (pituitary adenylate cyclase activating polypeptide) preferring receptor mediates the antioxidant and cytoprotective effects of PACAP. It was found in this research that in both PAC1R-CHO cells with high expression of PAC1R-eGFP and retinal ganglion cells (RGC-5) with natural expression of PAC1-R, blue light and hydrogen peroxide (H2O2) trigger the significant nuclear translocation of PAC1-R, and the nuclear translocation of PAC1-R was positive correlation with the up-regulation of expression level and promoter activity of PAC1-R its own, while red light worked much less efficiently than blue light. Reactive oxygen species (ROS) scavenger NAC (N-acetyl-L-cysteine) and palmitoylation inhibitor 2-bromopalmitate (2-BP) disturbed the nuclear shifting associated with the correlative up-regulation of PAC1 significantly, and PAC1-R mutant (M-PAC1-R) on Cys25/Ala25 displayed the significant decreased nuclear trafficking efficiency. Furthermore, the Western Blot results with the antibody raised against the C-terminal of PAC1-R showing PAC1-R in the nucleus was fragmentation hinting that C-terminal of PAC1-R with theoretical nuclear location signal (NLS) may be involved in activation of PAC1-R promoter in the nucleus. All above results indicated that PAC1-R makes the nuclear translocation to trigger the activation of PAC1-R itself promoter resulting into the up-regulation of of PAC1-R in response to the oxidative stress induced by blue light and ROS such as H2O2 .

TAT-tagging of VIP exerts positive allosteric modulation of the PAC1 receptor and enhances VIP neuroprotective effect in the MPTP mouse model of Parkinson's disease.

BACKGROUND: The cationic Arginine-rich peptide (CARP) TAT had been tagged at the C-terminal end of the vasoactive intestinal peptide (VIP) to construct VIP-TAT in order to improve traversing ability. Interestingly, it was found that TAT may bind the positive allosteric modulation (PAM) site of the N-terminal extracellular domain of neuropeptide receptor PAC1 (PAC1-EC1), imitating the C-terminus part of pituitary adenylate cyclase-activating polypeptide (PACAP) PACAP(28-38) fragment. METHODS: To test this hypothesis, we addressed the neuroprotective effects of VIP, VIP-TAT and PACAP38 in Parkinson's Disease (PD) cellular and mouse models. We also analyzed the peptides affinity for PAC1 and their ability to activate it. RESULTS: VIP-TAT had in vitro and in vivo neuroprotective effects much efficient than VIP in PD cellular and mouse models. The isothermal titration calorimetry (ITC) and competition binding bioassays confirmed that TAT binds PAC1-EC1 at the same site as PACAP(28-38). The cAMP experiments showed TAT-VIP results in a higher activation potency of PAC1 than VIP alone. CONCLUSIONS: The correlation of the peptides cationic properties with their affinity for PAC1 and their ability to activate the receptor, indicated that electrostatic interactions mediate the binding of TAT to the PAM domain of the PAC1-EC1, which induces the conformational changes of PAC1-EC1 required to promote the subsequent structural interaction and activation of the receptor with VIP. GENERAL SIGNIFICANCE: VIP-TAT has some potency for the development of a novel drug targeting neurodegenerative diseases.

Hormesis effect of hydrogen peroxide on the promoter activity of neuropeptide receptor PAC1-R.

Pituitary adenylate cyclase-activating polypeptide (PACAP) receptor 1 (PAC1-R) is the neuropeptide PACAP-preferring receptor-mediating neuroprotective activity. In order to clarify the biological mechanism of its expression, we cloned the 2,526 bp promoter fragment from -2,500 to +26 of the transcription initiation site of human ADCYAP1R1 gene and constructed the novel promotor reporter system named pYr-PromDetect-PAC1p. It was found in SH-SY5Y cells low concentration (<10 nM) of hydrogen peroxide (H2 O2 ) significantly promoted the activity of PAC1-R promoter in dose-dependent way, which was significantly inhibited by the transcription factor specificity protein 1 (SP1) inhibitor mithramycin A and was further confirmed in the deletion mutation of the predicted SP1 binding sites. Moreover, higher concentration of H2 O2 (>10 nM) inhibited the activity of PAC1-R in dose-dependent way. The hormesis effect of H2 O2 on PAC1-R promoter would help to further clarify the physiological effect of low-dose reactive oxygen on nervous system. PRACTICAL APPLICATIONS: PAC1-R mediates well-known neuroprotective, neurotrophic, and neurogenesis effects, which is an important drug target for neurodegenerative diseases. The hormesis effects of oxidative stress on PAC1-R expression not only help to explain the hormesis effects of oxidative stress on nerve system, but also offer a novel strategy to increase the expression of PAC1-R for the nerve protection or nerve generation. For example, taking advantage of low degree of oxidative stress to increases the expression of PAC1-R might help prevent subsequent surgical serious injury on the nervous system. The activation of PAC1-R promoter by low concentration of H2 O2 would help to further clarify the physiological effect of low-dose reactive oxygen on nervous system.