The Neuroprotective Peptide PACAP1-38 Contributes to Horizontal Cell Development in Postnatal Rat Retina.

Purpose: PACAP1-38, a member of the secretin/glucagon superfamily, is expressed in the developing retina with documented neuroprotective effects. However, its function in retinal cell differentiation has yet to be elucidated. Our goals, therefore, were to identify PAC1 expressing cells morphologically, investigate the PACAP1-38 action functionally, and establish PACAP1-38 regulated events developmentally during the first postnatal week in rat retina. Methods: P1 retinal sections or whole mounts of Wistar rats were used to reveal PAC1 and calbindin immunoreactive structures. P1, P3, or P7 pups were injected intravitreally with 100 pmol PACAP1-38. Tissues were harvested 24 hours post-treatment, then processed for calbindin immunohistochemistry to determine horizontal cell number, or 6, 12, 24 hours post-treatment for real-time PCR and immunoblots to detect PCNA expression. To localize proliferating cells, anti-PCNA antibody was applied. Results: We showed various PAC1 expressing cells in RPE, NBL, and GCL in P1 retina including calbindin positive horizontal cells. We found that PACAP1-38 induced a marked cell number increase at P3 and P7 and showed upregulated cell proliferation as its mechanism; however, it was ineffective at P1. PACAP1-38 induced proliferative cells localized in the NBL, and double-marker studies demonstrated that the induced proliferative cells were horizontal cells. Conclusions: PACAP1-38 appears to act in retinal differentiation by inducing mitosis selectively in a time and cell specific manner through PAC1. The control of horizontal cell proliferation raises the novel possibilities that (1) PACAP1-38 may be a major player in retinal patterning and (2) PACAP signaling may be critical in retinoblastoma.

Pituitary Adenylate Cyclase Activating Polypeptide (PACAP1-38) Exerts Both Pro and Anti-Apoptotic Effects on Postnatal Retinal Development in Rat.

PACAP1-38, a ubiquitous and multifunctional regulator has been in the focus of neurotoxicity research due to its impressive neuroprotective potential. Although the literature extensively demonstrated its repressive effect on the apoptotic machinery in neurodegenerative models, there is a striking absence of analysis on its role in normal development. We performed quantitative analyses on caspase activity in developing retina upon 100, 50, 25 or 1 pmol intravitreal PACAP1-38 injection from postnatal day 1 (P1) through P7 in Wistar rats. Retinas were harvested at 6, 12, 18, 24 or 48 h post-injection. Apoptotic activity was revealed using fluorescent caspase 3/7 enzyme assay, western blots and TUNEL assay. Unexpectedly, we found that 100 pmol PACAP1-38 increased the activity of caspase 3/7 at P1 and P5 whereas it had no effect at P7. At P3, as a biphasic effect, PACAP1-38 repressed active caspase 3/7 at 18 h post-injection while increased their activity in 24 h post-injection. Amounts, smaller than 100 pmol, could not inhibit apoptosis whereas 50, 25 or 1 pmol PACAP1-38 could evoke significant elevation in caspase 3/7 activity. TUNEL-positive cells appeared in the proximal part of inner nuclear as well as ganglion cell layers in response to PACAP1-38 treatment. The fundamental novelty of these results is that PACAP1-38 induces apoptosis during early postnatal retinogenesis. The dose as well as stage-dependent response suggests that PACAP1-38 has a Janus face in apoptosis regulation. It not only inhibits development-related apoptosis, but as a long-term effect, facilitates it.

Pituitary Adenylate Cyclase-Activating Peptide (PACAP), a Novel Secretagogue, Regulates Secreted Morphogens in Newborn Rat Retina.

Purpose: Pituitary adenylate cyclase-activating peptide (PACAP)1-38 has been reported to be responsible for regulation of a disparate array of developmental processes in the central nervous system, and its antiapoptotic effect has been revealed in numerous models, pointing to its relevance in the etiology of neurodegenerative disorders. However, its function in retinal development remains unclear. Here, we aimed to point out that versatility can be achieved through interaction with other regulators, in which PACAP can act indirectly on the retinal microenvironment. Methods: Wistar rats at age postnatal day 1 were injected intravitreally with PACAP or PAC1 receptor antagonist (PACAP6-38, M65) or VPAC1 antagonist (PG97-269) alone or in combination. Retinas were removed at 3, 6, 12, or 24 hours after injection. Changes in mRNA level were assessed using quantitative PCR, whereas changes in protein levels were measured by Western blot. Results: Intravitreal injection of PACAP or PAC1 receptor antagonists or the VPAC1 antagonist showed that PACAP receptors regulate the expression of five key secreted molecules (i.e., Fgf1, Bmp4, Wnt1, Gdf3, and Ihh), wherease other crucial morphogens (i.e., Fgf2, Fgf4, Fgf8, Fgf9, Shh, and Bmp9) were not affected. Pharmacologic dissection revealed that both PAC1 and VPAC1 induced downstream signaling and could cause upregulation of Fgf1, Bmp4, and Wnt1, whereas expression of Gdf3 might be mediated through the VPAC2 receptor. Conclusions: Our data are the first to shed light on PACAP as a secretagogue regulating a sustained production of morphogens, which in turn could enable PACAP to serve as a mitogen for retinal cells, to induce ganglion cell differentiation, and to contribute to RPE development.