The Neuroprotective Effects of Pituitary Adenylate-Cyclase Activating Polypeptide (PACAP) in Focal Cerebral Ischemia

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A homolog of the vertebrate pituitary adenylate cyclase-activating polypeptide is both necessary and instructive for the rapid formation of associative memory in an invertebrate

Similar to other invertebrate and vertebrate animals, cAMP dependent signaling cascades are key components of long-term memory (LTM) formation in the snail Lymnaea stagnalis, an established experimental model for studying evolutionarily conserved molecular mechanisms of long-term associative memory. Although a great deal is already known about the signaling cascades activated by cAMP, the molecules involved in the learning-induced activation of adenylate cyclase (AC) in Lymnaea remained unknown. Using Matrix-Assisted Laser Desorption/Ionization Time-of-flight (MALDI-TOF) mass spectroscopy in combination with biochemical and immunohistochemical methods, recently we have obtained evidence for the existence of a Lymnaea homologue of the vertebrate pituitary adenylate cyclase activating polypeptide (PACAP) and for the AC activating effect of PACAP in the Lymnaea nervous system. Here we first tested the hypothesis that PACAP plays an important role in the formation of robust LTM after single-trial classical food-reward conditioning. Application of the PACAP receptor antagonist PACAP6-38 around the time of single-trial training with amyl acetate and sucrose blocked associative LTM, suggesting that in this strong food-reward conditioning paradigm the activation of AC by PACAP was necessary for LTM to form. We found that in a weak multi-trial food-reward conditioning paradigm, lip-touch paired with sucrose, memory formation was also dependent on PACAP. Significantly, systemic application of PACAP at the beginning of multi-trial tactile conditioning accelerated the formation of transcription dependent memory.Our findings provide the first evidence to show that in the same nervous system PACAP is both necessary and instructive for fast and robust memory formation after reward classical conditioning

Stability Test of PACAP in Eye Drops

PACAP is a neuropeptide with widespread distribution and diverse biological functions. It has strong cytoprotective effects mediated mainly through specific PAC1 receptors. Experimental data show protective effects of PACAP in the retina and cornea in several pathological conditions. Although intravitreal injections are a common practice in some ocular diseases, delivery of therapeutic agents in the form of eye drops would be more convenient and would lead to fewer side effects. We have previously shown that PACAP, in the form of eye drops, is able to pass through the ocular barriers and can exert retinoprotective effects. As eye drops represent a promising form of administration of PACAP in ocular diseases, it is important to investigate the stability of PACAP in solutions used in eye drops. In this study, the stability of PACAP1-27 and PACAP1-38 in eye drops was measured in four common media and a commercially available artificial tear solution at both room temperature and +4 °C. Mass spectrometry results show that the highest stability was gained with PACAP1-38 in water and 0.9% saline solution at +4 °C, representing 80–90% drug persistence after 2 weeks. PACAP1-38 in the artificial tear showed very fast degradation at room temperature, but was stable at +4 °C. In summary, PACAP1-38 has higher stability than PACAP1-27, with highest stability at +4 °C in water solution, but both peptides in each medium can be stored for relatively longer periods without significant degradation. These data can provide reference for future therapeutic use of PACAP in eye drops

PACAP neuroprotektív hatása Parkinson-kór és újszülöttkori neuronális károsodás patkány modellekben = The neuroprotective effects of PACAP in rat models of Parkinson's disease and neonatal neuronal damage

A pályázat szerves folytatása volt a korábbi T034491 projektnek. A jelen pályázatban PACAP neuroprotektív hatásainak vizsgálatát folytattuk, az eredeti terv szerint két fő téma köré csoportosítva: a PACAP hatása bazális ganglionok károsodásában és újszülöttkori agykárosodásban. Kimutattuk, hogy a PACAP csökkenti a dopaminerg sejtpusztulást a substantia nigrában és jelentősen javítja a neurológiai tüneteket patkány Parkinson kór modellben. Leírtuk az unilaterális substantia nigra roncsolás modell és ebben a PACAP protektív hatásainak kor és nemfüggését. Igazoltuk a PACAP védő hatását Huntington chorea, neurotrauma modellekben, valamint toxicus és ischemiás retinadegenerációban. Igazoltuk, hogy a PACAP nemcsak neuroprotektív, hanem általános citoprotektív tulajdonságú: védő hatást fejt ki szívizom-és endothelsejtekben, vese és bélischemiában. A hatásmechanizmust tekintve igazoltuk, hogy ezen rendszerekben a PACAP antiapoptotikus tulajdonságú, in vitro és in vivo gátolja a proapoptotikus és aktiválja az antiapoptotikus jelátviteli útvonalakat. Eddig nem ismert jelátviteli útvonalak igazolásán kívül leírtuk, hogy a PACAP hatása jelentős különbségeket mutat a kor, nem, napi ritmus és sejttípustól függően. Újszülöttkori léziókban (toxicus, hypoxiás) leírtuk a kora postnatalis neuronális fejlődést, és kimutattuk a PACAP védő hatását bizonyos reflexfejlődési mintázatra. A támogatás segítségével született közlemények összesített impakt faktora 139,807 (absztraktok nélkül). | We continued the investigation on the neuroprotective effects of PACAP. The focus of our research was to examine the neuroprotective effects in models of basal ganglia diseases and neonatal brain injuries. We showed that PACAP treatment ameliorates the substantia nigra dopaminergic cell loss and improves the neurological deficits in a model of Parkinson`s disease. We examined how this unilateral substantia nigra degeneration model and the effects of PACAP depend on age and gender. We have provided evidence that PACAP is also protective in models of Huntington chorea, brain trauma, toxic and ischemic retinal degeneration. We have also shown that PACAP is not only neuroprotective, but is a general cytoprotective peptide: it has protective effects in cardiomyocytes, endothelial cells, kidney and intestinal ischemia. Considering the neuroprotective mechanism, we showed that PACAP has antiapoptotic effects in these models, it activates the antiapoptotic and inhibits the proapoptotic signaling pathways both in vitro and in vivo. In addition to providing evidence for new neuroprotective signaling mechanisms, we also showed that the protective effects of PACAP depend on age, gender, diurnal cycle and cell type.We described the early postnatal neurobehavioral development in toxic and hypoxic injuries and the protective effects of PACAP on some neurological reflexes. The impact factor of the publications with the help of this grant is 139.807 (without abstracts)

Influence of Terminal Differentiation and PACAP on the Cytokine, Chemokine, and Growth Factor Secretion of Mammary Epithelial Cells.

Pituitary adenylate cyclase-activating polypeptide (PACAP), a neuropeptide with trophic and cytoprotective effects, has been shown to affect cell survival, proliferation, and also differentiation of various cell types. The high PACAP level in the milk and its changes during lactation suggest a possible effect of PACAP on differentiation of mammary epithelial cells. Mammary cell differentiation is regulated by hormones, growth factors, cytokines/chemokines and angiogenic proteins. In this study, differentiation was hormonally induced by lactogenic hormones in confluent cultures of HC11 mouse mammary epithelial cells. We investigated the effect of PACAP on mammary cell differentiation as well as release of cytokines, chemokines and growth factors. Differentiation was assessed by expression analysis of the milk protein β-casein. Differentiation significantly decreased the secretion of IP-10, RANTES and the EGFR-ligands EGF and amphiregulin. The changes in the levels of IP-10 and RANTES may be relevant for the alterations in homing of T cells and B cells at different stages of mammary gland development, while the changes of the EGFR-ligands may facilitate the switch from proliferative to lactating stage. PACAP did not modulate the expression of β-casein or the activity of hormone induced pathways as determined by analysis of phosphorylation of Akt, STAT5 and p38 MAPK. However, PACAP decreased the release of EGF and amphiregulin from non-differentiated cells. This may influence the extracellular signal related transactivation of EGFR in the non-differentiated mammary epithelium and is considered to have an impact on the modulation of oncogenic EGFR signaling in breast cancer

Pituitary adenylate cyclase activating polypeptide concentrations in the sheep mammary gland, milk, and in the lamb blood plasma after suckling

Pituitary adenylate cyclase activating polypeptide (PACAP) is involved in development and reproduction. We previously described elevated PACAP levels in the milk compared to the plasma, and the presence of its specific PAC1 receptor in the mammary gland. This study aimed to determine PACAP and vasoactive intestinal peptide (VIP) levels in female suckling lambs compared to ewe plasma and mammary gland, as well as their age-dependent alterations. mRNA expressions of PACAP, VIP, PAC1 receptor and brain-derived neurotrophic factor (BDNF) were quantified in the milk whey and mammary gland. PACAP38-like immunoreactivity (PACAP38-LI) was measured in plasma, milk whey and mammary gland by radioimmunoassay, VIP-LI by enzyme-linked immunoassay. PACAP38-LI was 5, 6 times higher in the milk compared to the plasma of lactating sheep. It significantly increased in the lamb plasma 1 h, but returned to basal level 2 h after suckling. However, VIP mRNA was not present in the mammary gland, we detected the VIP protein in the milk whey. BDNF mRNA significantly decreased with age to approximately 60% and 25% in the 3- and 10-year-old sheep respectively, compared to the 3-month-old lambs. No differences were found between mammary and jugular vein plasma PACAP and VIP concentrations, or during the daily cycle. We propose a rapid absorption of PACAP38 from the milk and/or its release in suckling lambs. PACAP accumulated in the milk might be synthesized in the mammary gland or secreted from the plasma of the mothers. PACAP is suggested to have differentiation/proliferation promoting and immunomodulatory effects in the newborns and/or a local function in the mammary gland

PACAP is Protective Against Cellular Stress in Retinal Pigment Epithelial Cells

The integrity of the innermost, pigment epithelial layer of the retina is crucial for the photoreceptor survival and for maintaining the outer blood–retina barrier. In several ocular degenerations, such as diabetic retinopathy or macular edema, the stress caused by various harmful stimuli (hypoxia, oxidative stress, hyperosmosis) lead to severe molecular biological changes in this layer, promoting neovascularization of the retina. Pituitary adenylate cyclase activating polypeptide (PACAP) occurs throughout the whole body, including the eye. It has numerous functions in the retina, including the previously described anti-apoptotic and anti-angiogenic effects in retinal pigment epithelial cells. The aim of this present study was to investigate the influence of PACAP on different stress factors. In accordance with previous findings, PACAP significantly ameliorated the increased Hif1-α levels in hypoxic conditions. In H2O2-induced oxidative stress PACAP had an anti-apoptotic effect, it could decrease the expression of cytochrome-c and p53, while it upregulated the concentration of three antioxidants, namely SOD2, PON2 and thioredoxin. In conclusion, we provided new information on the molecular biological background of the retinoprotective effect of PACAP