Der Einfluss von PACAP (Pituitary Adenylate Cyclase Activating Peptide)- und PAC1-Defizienz bei ApoE-defizienten Mäusen auf das cholinerge Neurotransmittersystem des Gehirns unter Standard und unter hoher Cholesterol-/ Fett-Diät

Background: Atherosclerosis, stroke and Alzheimer's disease are common age-related diseases in which systemic inflammation or neuroinflammation play an important role. Pituitary adenylate cyclase activating peptide (PACAP) is a neuroprotective, immunomodulatory and vasoregulatory peptide that interacts with the PAC1, VPAC1, and VPAC2 receptors. PACAP's many functions are displayed by its influence on hypertension and its interaction with neurotransmitters. Moreover, PACAP shows protective effects in neurodegeneration and leads to recovery after brain damage. There is current evidence in the literature that apolipoprotein E (ApoE) affects the cholinergic transmitter system. ApoE deficiency is characterized by elevated plasma lipid levels and represents a well-established mouse model for studying aging processes. Currently, it is unknown how the knockout of PACAP, PAC1 and ApoE together with a high-cholesterol diet affects the hippocampal cholinergic neurotransmitter system. This is the first time to be investigated in a research work. It remains to be seen if the hypotheses, that a high-fat diet and genetic knockout lead to changed cholinergic innervation in the hippocampus, will prove true. Methods and Materials: In order to investigate the influence of PACAP on the cholinergic neurotransmitter system, the brains of single or double gene deficient mice (ApoE-/-, PACAP-/- ApoE -/- and PAC1-/- ApoE-/-) were examined and compared to wild-type mice. These mice were fed either standard chow for 30 weeks or standard chow for 10 weeks followed by high cholesterol chow (`Western Diet`) for 20 weeks. The analysis was realised with the help of sagittal paraffin sections of the brains and immunohistochemical experiments with nickel-enhanced streptavidin-biotin-peroxidase as well as following microscopy. Since the hippocampus is considered the center of learning and memory and there is high cholinergic activity and a high occurrence of PACAP and PAC1, different hippocampal regions (CA1, CA3 and dentate gyrus) were examined. The acetylcholine-synthesizing cholineacetyltransferase (ChAT) and the vesicular acetylcholine transporter (VAChT) were used as markers for the cholinergic neurotransmitter system. Results: In the final ChAT analysis, all groups and feedings were compared. Significance (p value 0.0062) was determined when performing the Two Way ANOVA (analysis of variance). The subsequently performed Tukey`s multiple comparison test showed a significant difference (p value 0.0471) (PACAP-/- ApoE -/- > PAC1-/- ApoE-/-). Moreover, this test revealed a significant difference (p value 0.0059) between PACAP-/- ApoE -/- feeding Standard 30 and PAC1-/- ApoE-/- feeding 10+20 (PACAP-/- ApoE -/- > PAC1-/- ApoE-/-). The final VAChT examination, in which all groups and feedings were compared, could not prove any significance. Conclusion: The hypotheses that high-fat diet and genetic knockout lead to changed cholinergic innervation in the hippocampus could not be confirmed to a significant extent. It is noted that in the ChAT group, knockout of PAC1 in both feedings resulted in a significantly bigger impact compared to PACAP knockout. The loss of PACAP specific PAC1 receptor under high-fat diet leads to a reduction of cholinergic hippocampal innervation. There is no signaling on PAC1 because of the PAC1 knockout. The decreased cholinergic activity can cause a loss of anti-inflammatory cerebral functions. In reverse conclusion this means that PAC1 could be protective. The protection of the PAC1-gene could result in less neuroinflammation and neurodegeneration. As PACAP offers great potential for research due to its diverse properties and functions, future research will also deal with this topic. The therapeutic possibilities of PACAP can be expected with special excitement.