Katja Gehrig-Burger

The neuropeptide PACAP promotes the α-secretase pathway for processing the Alzheimer amyloid precursor protein

The neuropeptide pituitary adenylate cyclase‐activating polypeptide (PACAP) has neurotrophic as well as anti‐apoptotic properties and is involved in learning and memory processes. Its specific G protein‐coupled receptor PAC1 is expressed in several central nervous system (CNS) regions, including the hippocampal formation. Here we examined the effect of PAC1 receptor activation on ?‐secretase cleavage of the amyloid precursor protein (APP) and the production of secreted APP (APPs?). Stimulation of endogenously expressed PAC1 receptors with PACAP in human neuroblastoma cells increased APPs? secretion, which was completely inhibited by the PAC1 receptor specific antagonist PACAP‐(6–38). In HEK cells, stably overexpressing the functional PAC1 receptors PACAP‐27 and PACAP‐38 strongly stimulated ?‐secretase cleavage of APP. The PACAP‐induced APPs? production was dose dependent and saturable. This increase of ?‐secretase activity was completely abolished by hydroxamate‐based metalloproteinase inhibitors, including a preferential ADAM 10 inhibitor. By using several specific protein kinase inhibitors, we show that the MAP‐kinase pathway [including extracellular‐regulated kinase (ERK) 1 and ERK2] and phosphatidylinositol 3‐kinase mediate the PACAP‐induced ?‐secretase activation. Our findings provide evidence for a role of the neuropeptide PACAP in stimulation of the nonamyloidogenic pathway, which might be related to its neuroprotective properties

Probes for studying cholesterol binding and cell biology

Cholesterol is a multifunctional lipid in eukaryotic cells. It regulates the physical state of the phospholipid bilayer, is crucially involved in the formation of membrane microdomains, affects the activity of many membrane proteins, and is the precursor for steroid hormones and bile acids. Thus, cholesterol plays a profound role in the physiology and pathophysiology of eukaryotic cells. The cholesterol molecule has achieved evolutionary perfection to fulfill its different functions in membrane organization. Here, we review basic approaches to explore the interaction of cholesterol with proteins, with a particular focus on the high diversity of fluorescent and photoreactive cholesterol probes available today.

Cholesterol interaction with the related steroidogenic acute regulatory lipid‐transfer (START) domains of StAR (STARD1) and MLN64 (STARD3)

The steroidogenic acute regulatory (StAR)‐related lipid transfer (START) domains are found in a wide range of proteins involved in intracellular trafficking of cholesterol and other lipids. Among the START proteins are the StAR protein itself (STARD1) and the closely related MLN64 protein (STARD3), which both function in cholesterol movement. We compared the cholesterol‐binding properties of these two START domain proteins. Cholesterol stabilized STARD3‐START against trypsin‐catalyzed degradation, whereas cholesterol had no protective effect on STARD1‐START. [3H]Azocholestanol predominantly labeled a 6.2 kDa fragment of STARD1‐START comprising amino acids 83–140, which contains residues proposed to interact with cholesterol in a hydrophobic cavity. Photoaffinity labeling studies suggest that cholesterol preferentially interacts with one side wall of this cavity. In contrast, [3H]azocholestanol was distributed more or less equally among the polypeptides of STARD3‐START. Overall, our results provide evidence for differential cholesterol binding of the two most closely related START domain proteins STARD1 and STARD3.

DHEA-Bodipy—A functional fluorescent DHEA analog for live cell imaging

The androgen dehydroepiandrosterone (DHEA) has been reported to protect neuronal cells against dysfunction and apoptosis. Several signaling pathways involved in these effects have been described but little is known about the intracellular trafficking of DHEA. We describe design, synthesis and characterization of DHEA-Bodipy, a novel fluorescent DHEA analog. DHEA-Bodipy proved to be a functional DHEA derivative: DHEA-Bodipy (i) induced estrogen receptor alpha-mediated gene activation, (ii) protected PC12 rat pheochromocytoma cells against serum-deprivation-induced apoptosis, and (iii) induced stress fibers and focal adhesion contacts in SH-SY5Y human neuroblastoma cells. DHEA-Bodipy bound rapidly and specifically to plasma membranes of living PC12 cells. We analyzed metabolism and trafficking of DHEA-Bodipy in human neuroblastoma cells. DHEA-Bodipy is the first functional fluorescent DHEA derivative suitable for live cell imaging of intracellular DHEA transport and localization.

Depletion of calcium stores contributes to progesterone-induced attenuation of calcium signaling of G protein-coupled receptors

Progesterone non-genomically attenuates the calcium signaling of the human oxytocin receptor and several other Gαq protein-coupled receptors. High progesterone concentrations are found in the endometrium during pregnancy opposing the responsiveness of the underlying myometrium to labor-inducing hormones. Here, we demonstrate that within minutes, progesterone inhibits oxytocin- and bradykinin-induced contractions of rat uteri, calcium responses induced by platelet-activating factor in the human endometrial cell line MFE-280, and oxytocin-induced calcium signals in PHM1-31 immortalized pregnant human myometrial cells. Using human embryonic kidney (HEK293) cells as model system, we analyzed the molecular mechanisms underlying these effects. Our data indicate that progesterone rapidly depletes intracellular calcium stores. The resulting desensitization of the cells might contribute to the quiescence of the uterus during pregnancy.

A novel cholesterol‐based detergent

Design, synthesis and characterization of CHAPSTEROL, a novel cholesterol‐based detergent developed for functional solubilization of cholesterol‐dependent membrane proteins are described. To validate CHAPSTEROL, we employed the oxytocin receptor, a G protein‐coupled receptor requiring cholesterol for its high‐affinity binding state. Using the photoactivatable cholesterol analogue [3H]6,6‐azocholestan‐3β‐ol[3αH], we demonstrate that solubilization by CHAPSTEROL leads to an enrichment of cholesterol‐binding proteins whereas the widely used bile acid derivative CHAPSO leads to a significant depletion of cholesterol‐binding proteins. Similar to Triton X‐100 and CHAPS, CHAPSTEROL maintains the localization of caveolin as well as cholesterol and sphingomyelin to lipid rafts, i.e. detergent‐insoluble microdomains of the plasma membrane. The data suggest that CHAPSTEROL is an appropriate detergent for the solubilization of cholesterol‐dependent membrane proteins and isolation of rafts.

CHAPSTEROL. A novel cholesterol-based detergent.

Design, synthesis and characterization of CHAPSTEROL, a novel cholesterol‐based detergent developed for functional solubilization of cholesterol‐dependent membrane proteins are described. To validate CHAPSTEROL, we employed the oxytocin receptor, a G protein‐coupled receptor requiring cholesterol for its high‐affinity binding state. Using the photoactivatable cholesterol analogue [3H]6,6‐azocholestan‐3β‐ol[3αH], we demonstrate that solubilization by CHAPSTEROL leads to an enrichment of cholesterol‐binding proteins whereas the widely used bile acid derivative CHAPSO leads to a significant depletion of cholesterol‐binding proteins. Similar to Triton X‐100 and CHAPS, CHAPSTEROL maintains the localization of caveolin as well as cholesterol and sphingomyelin to lipid rafts, i.e. detergent‐insoluble microdomains of the plasma membrane. The data suggest that CHAPSTEROL is an appropriate detergent for the solubilization of cholesterol‐dependent membrane proteins and isolation of rafts.

P4-256: PACAP peptides as new α-secretase activators

Proteolytic cleavage of the amyloid precursor protein (APP) by -secretase within the A sequence precludes formation of amyloidogenic peptides and leads to a release of soluble APPs , which has neuroprotective, anti-apoptotic and growth promoting properties. Therefore, pharmacological up-regulation of the -secretase could be a possible approach for treatment of Alzheimer disease (AD). The neuropeptide pituitary adenylate cyclase-activating polypeptide (PACAP) has neurotrophic, neuroprotective as well as anti-apoptotic properties and is involved in learning and memory processes. Its specific G protein-coupled receptor PAC1 is expressed in several CNS regions including the hippocampal formation. Here we examined the effect of PAC1 receptor activation on -secretase cleavage of the amyloid precursor protein (APP) and the production of secreted APP (APPs ). Stimulation of endogenously expressed PAC1 receptors with PACAP in human neuroblastoma cells increased APPs secretion which was completely inhibited by the PAC1 receptor specific antagonist PACAP (6-38). In HEK cells stably overexpressing functional PAC1 receptors PACAP-27 and PACAP-38 strongly stimulated -secretase cleavage of APP. The PACAP-induced APPs production was dose-dependent and saturable. This increase of -secretase activity was completely abolished by hydroxamate-based metalloproteinase inhibitors including a preferential ADAM 10 inhibitor. By using several specific protein kinase inhibitors we show that the MAP-kinase pathway (including ERK1 and ERK2), and PI3-kinase mediate the PACAP-induced -secretase activation. PACAP treatment had no influence on expression level of -secretases (ADAM 10, ADAM 17 and ADAM 9) and on the substrate APP. According to our results, the neuroprotective effects of PACAP may be mediated by activation of the -secretase pathway. PACAP neuropeptides are able to cross the blood brain barrier and might be of therapeutic value for the treatment of Alzheimer disease. Therefore, we are currently testing PACAP peptides in an APP mouse model.

A novel cholesterol-based detergent: A novel cholesterol-based detergent

Design, synthesis and characterization of CHAPSTEROL, a novel cholesterol‐based detergent developed for functional solubilization of cholesterol‐dependent membrane proteins are described. To validate CHAPSTEROL, we employed the oxytocin receptor, a G protein‐coupled receptor requiring cholesterol for its high‐affinity binding state. Using the photoactivatable cholesterol analogue [3H]6,6‐azocholestan‐3β‐ol[3αH], we demonstrate that solubilization by CHAPSTEROL leads to an enrichment of cholesterol‐binding proteins whereas the widely used bile acid derivative CHAPSO leads to a significant depletion of cholesterol‐binding proteins. Similar to Triton X‐100 and CHAPS, CHAPSTEROL maintains the localization of caveolin as well as cholesterol and sphingomyelin to lipid rafts, i.e. detergent‐insoluble microdomains of the plasma membrane. The data suggest that CHAPSTEROL is an appropriate detergent for the solubilization of cholesterol‐dependent membrane proteins and isolation of rafts.