More so in the lab than on water, successful fishing expeditions are prized rarities. Today’s issue of Science describes one such catch—a high-throughput functional genomics screen that has reeled in what might become a novel therapeutic target for Alzheimer disease. Led by Bart de Strooper at K.U. Leuven in Belgium, with colleagues there and at the Belgian drug discovery company Galapagos, the new study suggests that G protein-coupled receptor 3 (GPR3) stimulates Aβ production through an unusual mechanism that promotes assembly and trafficking of the γ-secretase complex.

Looking beyond the β- and γ-secretases for other modulators of Aβ production, first author Amantha Thathiah and colleagues performed a broad screen using an adenovirus expression library of 1,905 unique genes encoding druggable targets. Among the handful of candidate genes that emerged from the screening data to endure the typical gauntlet of confirmational expression and knockdown studies, GPR3 piqued the researchers’ interest because it resides in a chromosome locus linked to AD. Furthermore, immunohistochemistry in postmortem human brain tissue revealed GPR3 expression in AD-affected regions such as the hippocampus and cortex. What’s more, the researchers found higher GPR3 protein expression in a subset of sporadic AD brain samples, compared with tissue from age-matched control patients.

To get a handle on how GPR3 might regulate Aβ production, de Strooper’s team examined whether the orphan receptor modulates the activity of β- or γ-secretase, the proteases that deliver the first and final cuts releasing Aβ from its parent molecule APP (amyloid precursor protein). Overexpression of GPR3 in several human cell lines did not influence β-secretase expression or activity, as assessed by immunoprecipitation and mass spectrometry analysis, suggesting that GPR3’s effects on Aβ lay downstream of β-secretase.

The researchers eventually showed that GPR3 acts by modulating γ-secretase activity. Here’s how they came to that conclusion. When they expressed GPR3 and APP, a direct γ-secretase substrate, in primary mouse hippocampal neurons, they saw a substantial increase in Aβ1-40 and Aβ1-42 secretion that vanished when the cultures were treated with a selective γ-secretase inhibitor.

So what is GPR3 doing to γ-secretase? This question might first be addressed in terms of what GPR3 is not doing. It did not appear to affect cleavage of another γ-secretase substrate, Notch. It did not seem to regulate Aβ through cyclic adenosine monophosphate (cAMP) signaling or G protein-coupling. Furthermore, overexpression of GPR3 had no measurable effect on expression of individual γ-secretase subunits.

What the researchers did find, however, using gel electrophoresis that separates proteins in their native state, was enhanced expression of mature γ-secretase complex in GPR3-overexpressing cells. They also saw that transduction of GPR3 boosted cell-surface expression of γ-secretase components. “In the absence of ligand stimulation, GPR3 displays an extremely high level of basal activity, which potentially has an effect on the trafficking and assembly of the γ-secretase complex,” de Strooper explained in an e-mail to ARF.

The researchers were able to confirm these effects in vivo. Hippocampal injections of purified GPR3 adenoviral vector drove up Aβ1-40 and Aβ1-42 production in AD mice (APP/PS1) without affecting expression of γ-secretase subunits. Conversely, cultured primary hippocampal neurons from Gpr3+/- and Gpr3-/- mice generated less of these Aβ peptides relative to wild-type cells, whereas transducing the Gpr3-deficient cells with GPR3 adenoviral vector restored normal Aβ production. To drive home the effects of GPR3 shortage on Aβ generation in living animals, the researchers crossed APP/PS1 mice with Gpr3 knockout mice and showed reduced Aβ1-40 and Aβ1-42 production in triple transgenic offspring lacking one or both copies of Gpr3.

“The key question now is to understand exactly how GPR3 affects the assembly of the γ-secretase complex, so we need to explore the cell biology of this novel control mechanism,” de Strooper suggested. “Once this is clarified, we would like to screen for compounds that selectively decrease GPR3 expression and/or regulate the activation mechanism.” (One small-molecule agonist of a different GPR, which appears to work by boosting cholinergic transmission, is in Phase 2 testing as a potential treatment for AD [see ARF related news story].)

However, Philip Wong of Johns Hopkins University in Baltimore, Maryland, noted via e-mail that before finding drugs to selectively target GPR3, further studies are needed to evaluate GPR3 function—particularly in learning and memory—since the receptor appears to be highly expressed in hippocampal neurons (see full comment below). Other scientists contacted for this story cautioned that yet another fundamental way of clarifying the importance of GPR3 for AD would be to examine in more detail its expression in cells of the brain regions affected in this disease.—Esther Landhuis.

Reference:
Thathiah A, Spittaels K, Hoffmann M, Staes M, Cohen A, Horre K, Vanbrabant M, Coun F, Baekelandt V, Delacourte A, Fischer DF, Pollet D, de Strooper B, Merchiers P. The Orphan G Protein-Coupled Receptor 3 Modulates Amyloidbeta Peptide Generation in Neurons. Science. 2009 Feb 13;323:946-951. Abstract

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Comments on News and Primary Papers

  1. The identification by Thathiah and coworkers of the orphan G protein-coupled receptor 3 (GPR3) as a regulator of amyloid-β production is very interesting. Particularly intriguing is the idea that this protein apparently affects formation and cell-surface localization of γ-secretase to influence processing of APP, but not of Notch. While these observations suggest that GPR3 may be a useful target for development of anti-amyloid therapy for Alzheimer disease (AD), further evaluations are needed to examine its in-vivo impact on amyloid deposition in AD mouse models as well as its functional role(s), including in learning and memory as GPR3 is highly expressed in neurons of the hippocampus. Although Notch signaling may not be influenced by GPR3, the observation that GPR3 knockout mice exhibit premature ovarian aging indicates that other important pathways are regulated by GPR3 and that targeting GPR3 may lead to adverse affects in the brain. Thus, careful examination of GPR3’s therapeutic potential should point us in the right direction regarding development of drugs that selectively target GPR3 for AD.

  2. This paper is on the identification of a novel receptor that can regulate Aβ production. The receptor is a GPCR coupled to adenylate cyclase, but its natural ligand is unknown, and cAMP elevation by other means did not alter Aβ production. siRNA knockdown of the GPCR3 lowers Aβ production, but it is unclear whether this means an antagonist would do the same. Other evidence suggests that GPCR3 enhances the formation of mature γ-secretase complexes with increased Aβ production, but no effect on Notch processing. This evidence, along with a previous report that knockout of GPCR3 has only a mild phenotype, without any apparent Notch loss-of-function, suggests this orphan receptor might be a worthy target. In my view, the jury is still out, especially since it is hard to see how facilitating γ-secretase maturation leads to a selective elevation of APP processing over Notch.

    View all comments by Michael Wolfe

References

News Citations

  1. Keystone Drug News: Agonists for M1, Serotonin Receptors Prime Cholinergic Pump

Paper Citations

  1. . The orphan G protein-coupled receptor 3 modulates amyloid-beta peptide generation in neurons. Science. 2009 Feb 13;323(5916):946-51. PubMed.

Other Citations

  1. APP/PS1

Further Reading

Papers

  1. . The orphan G protein-coupled receptor 3 modulates amyloid-beta peptide generation in neurons. Science. 2009 Feb 13;323(5916):946-51. PubMed.

Primary Papers

  1. . The orphan G protein-coupled receptor 3 modulates amyloid-beta peptide generation in neurons. Science. 2009 Feb 13;323(5916):946-51. PubMed.