No abstract

Living cells respond to various environmental cues and process them into a series of spatially and temporally regulated signaling events, which can be tracked in real time with an expanding repertoire of genetically encodable FRET-based biosensors. A series of these biosensors, designed to track dynamic activities of signaling enzymes such as protein kinases and small GTPases, have yielded invaluable information regarding the spatiotemporal regulation of these enzymes, shedding light on the orchestration of signaling pathways within the native cellular context. In this chapter, we first review the generalizable modular designs of FRET-based biosensors, followed by a detailed discussion about biosensors for reporting protein kinase activities and GTPase activation. Two general designs, uni- and bimolecular reporters, will be discussed with an analysis of their strengths and limitations. Finally, an example of using both uni- and bimolecular kinase activity reporters to visualize PKA activity in living cells will be presented to provide practical tips for using these biosensors to explore specific biological systems.

Senile plaques comprised of Aβ peptides are a hallmark of Alzheimer’s disease (AD) brain, as are activated glia that release inflammatory molecules, including eicosanoids. Previous studies have demonstrated that amyloid precursor protein (APP) and Aβ levels can be increased through activation of thromboxane A2-prostanoid (TP) receptors on neurons. We demonstrate that TP receptor regulation of APP expression depends on Gαq-signaling and conventional protein kinase C isoforms. Importantly, we discovered that Gαq-linked prostaglandin E2 and leukotriene D4 receptors also regulate APP expression. Prostaglandin E2 and thromboxane A2, as well as total APP levels, were found to be elevated in the brains of aged 5XFAD transgenic mice harboring Aβ plaques and activated glia, suggesting that increased APP expression resulted from eicosanoid binding to Gαq-linked neuronal receptors. Notably, inhibition of eicosanoid synthesis significantly lowered brain APP protein levels in aged 5XFAD mice. These results provide new insights into potential AD therapeutic strategies.

A hallmark pathological feature of the Alzheimer's disease (AD) brain is the presence of senile plaques, which comprise amyloid β (Aβ) peptides that are derived from the amyloid precursor protein (APP). The plaque-containing AD brain is thought to be under oxidative stress, as evidenced by increased lipid oxidation products that include isoprostane-F2αIII (iPF2αIII). IPF2αIII can bind to and activate the thromboxane A2-prostanoid (TP) receptor, and TP receptor activation causes increased Aβ production through enhancement of APP mRNA stability. Moreover, TP receptor antagonists have been shown to block iPF2αIII-induced increases of Aβ secretion. Thus, the TP receptor may be a potential drug target for AD therapy. However, here we show that existing TP receptor antagonists have poor blood-brain barrier (BBB) permeability, likely due to the presence of a carboxylic acid moiety that is believed to be important for receptor interaction, but which may hamper passive diffusion across the BBB. We now report selected analogues of a known tetrahydronaphthalene TP receptor antagonist, wherein the carboxylic acid moiety has been replaced by heterocyclic bioisosteres. These heterocyclic analogues retained relatively high affinity for the mouse and human TP receptors, and, unlike the parent carboxylic acid compound, several examples freely diffused across the BBB into the brain upon administration to mice. These results reveal that brain-penetrant tetrahydronaphthalene TP receptor antagonists can be developed by substituting the carboxylic acid moiety with a suitable nonacidic bioisostere. Compounds of this type hold promise as potential lead structures to develop drug candidates for the treatment of AD. KEYWORDS: Alzheimer's disease, amyloid precursor protein, antagonist, blood-brain barrier, plaques, thromboxane receptor A lzheimer's disease (AD) is the most common cause of dementia in elderly populations, affecting approximately 10% of individuals over age 65. 1,2 The AD brain is characterized by overt neurodegeneration 3 and the presence of senile plaques comprising insoluble fibrils of Aβ peptides which are produced through sequential proteolytic cleavage of the amyloid precursor protein (APP). 4 A second pathological hallmark of the AD brain is the occurrence of intracellular inclusions composed of hyperphosphorylated tau proteins. 5,6 Familial forms of AD can be caused by mutations in APP that result in increased proteolytic generation of amyloidogenic Aβ peptides, as well as by mutations in the presenilin proteins that are required for the proteolytic processing of APP to yield Aβ. 7−10 These genetic data provide compelling support for the "amyloid" hypothesis, which postulates that it is the production of Aβ that drives disease pathogenesis in AD. Accordingly, there is significant interest in identifying therapeutic strategies that will lead to decreased Aβ production, and there are ongoing efforts to identify inhibitors of the enzymes that generate Aβ. 11,12 A consequence of Aβ plaque deposition in the AD ...