G-Protein Receptor Knockout Rescues Several Models of Alzheimer’s

The G-protein coupled receptor Gpr3 helps γ-secretase pump out Aβ, and targeting the receptor may present a therapeutic strategy, according to a study published in Science Translational Medicine on October 14. Reducing or eliminating the expression of Gpr3 dampened Aβ production in four different mouse models of Alzheimer’s disease, including APP knock-in mice. The researchers, led by Amantha Thathiah and Bart De Strooper at KU Leuven in Belgium, found that AD mice lacking the receptor fared better on memory tests. They also reported that in people, elevated levels of the protein correlated with progression of the disease. The researchers plan to test a suite of potential Gpr3 inhibitors in mice as potential therapeutics.

The specific biological roles played by G-protein coupled receptors makes them attractive targets for small-molecule therapeutics. Roughly half of all marketed drugs bind G-protein coupled receptors (GPCRs) (see Ma and Zemmel, 2002). When triggered, these 7-transmembrane receptors touch base with various G proteins in the cytoplasm, which then modulate myriad signaling pathways. Many GPCRs have no known endogenous ligand, yet still have important known functions. Gpr3 is one of these “orphan” receptors. In 2009, researchers in De Strooper’s lab fingered Gpr3 for triggering Aβ production by γ-secretase, and reported that APP/PS1 mice deficient in the receptor had a reduced amyloid burden (see Feb 2009 news). A subsequent study from the group reported revealed a twist—the receptor promoted APP processing through recruitment and interaction with β-arrestin 2, an intracellular scaffolding protein, rather than through coupling with G-proteins (see Dec 2012 news). The researchers found that following its recruitment to the membrane by Gpr3, β-arrestin 2 also interacted directly with the Aph-1α subunit of γ-secretase, and promoted localization of the complex to lipid rafts in the plasma membrane, Thathiah told Alzforum. The researchers speculated that this may somehow ramp up the APP processing activity of the secretase.

First author Yunhong Huang and colleagues wanted to make certain that Gpr3’s effects on Aβ production would hold up in multiple models of AD. The recent advent of APP-KI mice, which express human APP with familial AD mutations at physiological levels, motivated the researchers to conduct such a comparison study, Thathiah said.

The researchers started off by analyzing Aβ production in their tried-and-true model, the APP/PS1 mouse. At 9 to 12 months of age, APP/PS1 mice lacking one or both copies of Gpr3 had less than half as much soluble and insoluble Aβ40 and Aβ42 in the hippocampus and cortex as control animals expressing two copies of the gene. Amyloid plaque burden, as measured by confocal imaging of brain slices, also dropped by half in mice lacking Gpr3.

Roughly the same pattern emerged when the researchers created APPDutch mice lacking Gpr3. This mouse strain overexpresses human APP harboring the E693Q mutation, which causes a form of familial cerebral amyloid angiopathy and does not develop parenchymal amyloid plaques. APPDutch mice lacking Gpr3 had less Aβ40 and Aβ42 in the hippocampus and cortex than those expressing Gpr3. Wild-type mice, which express only endogenous mouse APP, also had a moderate reduction in mouse Aβ40 and Aβ42 when crossed onto the Gpr3 knockout background.

Finally, the researchers turned to two strains of APP-KI mice: the APPNL strain, which expresses physiological levels of APP with the Swedish mutation, and APPNL-F mice, which also harbors the Beyreuther/Iberian APP mutation. The Swedish mutation increases the total amount of Aβ40 and Aβ42, while the Beyreuther/Iberian mutation raises the A42/40 ratio. When crossed to a Gpr3 KO background, both strains of APP-KI mice had reduced levels of Aβ40 and Aβ42. Interestingly, the Aβ42/40 ratio fell in the APPNL-F mice compared to their counterparts expressing Gpr3.

Amyloid Clarity.

Clarified brain (top and bottom) from APPNL-F mouse lacking Gpr3 reveals a reduced amyloid burden (bottom panel, Thioflavin S, green). Blood vessels stained with lectin appear as magenta (bottom panel). [Courtesy of Huang et al., Science Translational Medicine, 2015.]

To carefully scrutinize the effects of Gpr3 loss on amyloid plaque deposition in APPNL-F mice, the researchers rendered the animals’ brains transparent using a technique similar to 3DISCO. A flurry of optical clearing methods have come on the scene in recent years (for examples, see Jul 2014 news and Sep 2015 news), and the researchers chose this one, which uses an organic solvent to dissolve lipids. They injected the mice intravenously with fluorescently labeled lectin to label the brain vasculature, then clarified the brains and stained them with Thioflavin S to label amyloid plaques. Fluorescence microscopy of the clear brain allowed the researchers to accurately calculate the total volume of amyloid plaques in the animals, a feat that would not have been possible with two-dimensional imaging. They found that APPNL-F mice lacking Gpr3 had roughly half the number of plaques and only a third the total amyloid volume as those expressing Gpr3.

Could ablating Gpr3 also reduce cognitive deficits? To find out, the researchers looked to APP/PS1 mice, which develop memory problems at a young age. Though not as smart as wild-type mice, APP/PS1 mice more quickly learned the location of a submerged platform in the Morris water maze when they lacked the Gpr3 gene. They were also less anxious and willing to spend more time in an exposed area than APP/PS1 mice. Thathiah said they plan to assess cognition in the knock-in mice as well, but those animals take at least a year to develop cognitive symptoms.

Given the role of Gpr3 in Aβ production and memory in mice, the researchers wondered if it tied into AD pathology in people. They measured Grp3 in brain samples from 131 people of various ages, and found no correlation between protein level and age. However, Gpr3 was elevated in brains from 18 AD patients. In two other sample sets totaling 40 brains, Grp3 levels in the brain correlated with the disease severity measured through Braak staging.

“This study provides an important validation of Gpr3 as a therapeutic target for AD,” commented Michael Wolfe of Brigham and Women’s Hospital in Boston, who called the results impressive. “It will be critical to show that these effects occur upon pharmacological inhibition in adult mice, rather than on genetic deletion from conception onward,” he said.

A handful of commercially available Gpr3 inhibitors exist, Thathiah said, but they have not been tested for their ability to block the recruitment of β-arrestin 2, a requisite for Gpr3’s effect on γ-secretase activity. Thathiah told Alzforum that her lab, which will soon move to the University of Pittsburg in Pennsylvania, plans to screen for new inhibitors and test commercially available ones.

Klaus Heese of Hanyang University in Seoul noted that the relationship between GPCRs and APP processing has a long history, as previous studies pinpointed interactions between other GPCRs and γ-secretase function. “Huang and colleagues add further evidence about the significance of GPCRs and APP processing,” he wrote. Targeting Gpr3 might offer a promising therapeutic approach, Heese added.—Jessica Shugart

Comments

  1. This study provides an important validation of Gpr3 as a therapeutic target for AD. The results are impressive, with reduced amyloid and improved cognitive function in multiple AD mouse models. However, as the authors themselves point out, it will be critical to show that these effects occur upon pharmacological inhibition in adult mice, rather than on genetic deletion from conception onward.

    More fundamentally, there must be more convincing evidence from clinical trials that targeting Aβ or Aβ production will truly slow disease progression or delay disease onset. How early must anti-amyloid therapeutics be given in order to provide clear benefit? If AD becomes a tau-driven process years before disease onset, then targeting Aβ, either directly with antibodies or indirectly through Gpr3, will not be effective. We all keenly await the results of the various ongoing AD prevention trials.

    View all comments by Michael Wolfe

  2. As a co-author, I would like to comment that BBB-permeable Gpr3-specific antagonist(s) without any side effect can become ideal anti-Aβ medication(s). They could be less expensive than therapeutic antibodies. A failure of Aβ immunotherapy could generate a serious negative social impact on the Aβ hypothesis, which, in my view, is already scientifically proven. We should realize that there are many options, such as GPCR agonists and antagonists, for the anti-Aβ strategy in addition to immunotherapy, to which the concern about the preventive timing raised by Michael Wolfe in the previous comments also applies.

    View all comments by Takaomi Saido

  3. To clarify my comments, I agree with Takaomi Saido that the Aβ hypothesis is essentially proven scientifically by the FAD mutations in APP and the presenilins.

    My point is that Aβ may be an impractical target, because it may need to be reduced or blocked so many years in advance before the pathogenic process becomes primarily tau-driven. While ongoing trials may ultimately show clear disease modification (and I certainly hope that they do), the possibility remains that they may not. The failure of these trials, especially if there are no major safety concerns and clear evidence for target engagement, would make it very difficult to convince pharma, venture capitalists, and funding agencies that they should pour more money, resources, and time into targeting Aβ. Presently this remains an unresolved issue, and we must await the results of the various prevention trials.

    In the meantime, pursuing other means of targeting Aβ, such as via Gpr3, is certainly worthwhile, but we should be prepared for blow-back and major obstacles to continuing this avenue of translational investigation in the event anti-Aβ agents currently in the pipeline fail in the clinic.

    View all comments by Michael Wolfe

  4. The current study by the groups of De Strooper and Thathiah adds further details about the potential mechanism involved in regulation of Aβ precursor protein (APP) processing by G protein-coupled receptors (GPCRs) and is based on their previous findings showing that the orphan GPCR 3 (GPR3) modulates Aβ peptide generation in neurons (Thathiah and De Strooper, 2009; Thathiah et al., 2009; Thathiah and De Strooper, 2011; Thathiah et al., 2013).

    In fact, this is not a completely new idea, as Nishimoto and colleagues had shown in 1993 a clear link between APP and GPCR signaling pathways and that the cytosolic part of APP can bind to and activate Go (a major GTP-binding protein in brain) in a ligand-dependent manner (Nishimoto et al., 1993; Okamoto et al., 1995,1996). However, the connections between APP and GPCRs had been neglected for a long time and re-emerged only recently with the accumulation of new evidence that linked APP with GPCRs and the addition of further details on APP processing affected by GPCR systems. It is thus of utmost interest for the pharmaceutical industry due to the relative easy access of these receptors to pharmaceuticals (see reviews: Thathiah and De Strooper, 2009; Heese, 2013; Thathiah and De Strooper, 2011).

    Indeed, the current data by Huang et al. add further evidence about the significance of GPCRs and APP processing. Here, they demonstrate that genetic deletion of GPR3 in AD mouse model reduces amyloid plaques and alleviates cognitive deficits in these mice. Furthermore, human AD postmortem brain-tissue samples showed a correlation between elevated GPR3 and AD progression, which is in line with reduced p60TRP (another pivotal GPCR-modulating protein) expression in AD brains (Heese et al., 2004).

    Similarly, Pei and colleagues showed a few years ago that a GPCR/secretase complex regulates β- and γ-secretase specificity for Aβ production and contributes to AD pathogenesis (Teng et al., 2010). Specifically, they demonstrated that the δ-opioid receptor (OPRD1, otherwise known as DOR) promotes the β- and γ-secretase-mediated processing of APP and that knockdown of OPRD1 reduces the secretase activities and ameliorates Aβ pathology and Aβ-dependent behavioral deficits, without affecting the processing of Notch, N-cadherin, or APLP in AD model mice (Teng et al., 2010), and thus suggested that intervention in either the formation or trafficking of the GPCR/secretase complex could eventually lead to a new strategy for the treatment of AD, potentially with fewer side effects than current treatments.

    Further extensive research has revealed numerous interacting partners of GPCRs, including the GPCR-associated sorting protein (GPRASP) family members (Abu-Helo and Simonin, 2010; Moser et al., 2010). p60TRP (also known as GPRASP3, GASP3, or BHLHB9) is a member of this GPRASP family (Heese et al., 2004) that also regulates the endocytic recycling of OPRD1 (Mishra and Heese, 2011). A transgenic p60TRP in vivo imaging mouse model has been used recently to show that p60TRP can mediate a reduction in the phosphorylation of APP, a reduction in the activities of β- and γ-secretases (Bace1/Psen2), and an increase in the activity of protein phosphatase PP2A, while increasing synaptic connections in the brain and enhancing cognitive functions in these mice (Mishra and Heese, 2011; Manavalan et al., 2013).

    Taken together, the integration of various data obtained thus far may open new avenues for the treatment of AD via an APP-GPCR-linkage pathway. Since a silver bullet might not soon be found for the treatment of AD, it is time to think about alternative approaches that may help treat patients suffering from this devastating disease. The paper by Huang et al. provides promising data that supports this idea.

    References:

    Abu-Helo A, Simonin F. Identification and biological significance of G protein-coupled receptor associated sorting proteins (GASPs). Pharmacol Ther. 2010 Jun;126(3):244-50. PubMed.

    Heese K, Yamada T, Akatsu H, Yamamoto T, Kosaka K, Nagai Y, Sawada T. Characterizing the new transcription regulator protein p60TRP. J Cell Biochem. 2004 Apr 1;91(5):1030-42. PubMed.

    Heese K. G Proteins, p60TRP, and Neurodegenerative Diseases. Mol Neurobiol. 2013 Jan 24; PubMed.

    Manavalan A, Mishra M, Sze SK, Heese K. Brain-site-specific proteome changes induced by neuronal P60TRP expression. Neurosignals. 2013;21(3-4):129-49. Epub 2013 Feb 6 PubMed.

    Mishra M, Heese K. P60TRP interferes with the GPCR/secretase pathway to mediate neuronal survival and synaptogenesis. J Cell Mol Med. 2011 Nov;15(11):2462-77. PubMed.

    Moser E, Kargl J, Whistler JL, Waldhoer M, Tschische P. G protein-coupled receptor-associated sorting protein 1 regulates the postendocytic sorting of seven-transmembrane-spanning G protein-coupled receptors. Pharmacology. 2010;86(1):22-9. Epub 2010 Jun 26 PubMed.

    Nishimoto I, Okamoto T, Matsuura Y, Takahashi S, Murayama Y, Ogata E. Alzheimer amyloid protein precursor complexes with brain GTP-binding protein G(o). Nature. 1993 Mar 4;362(6415):75-9. PubMed.

    Okamoto T, Takeda S, Giambarella U, Murayama Y, Matsui T, Katada T, Matsuura Y, Nishimoto I. Intrinsic signaling function of APP as a novel target of three V642 mutations linked to familial Alzheimer's disease. EMBO J. 1996 Aug 1;15(15):3769-77. PubMed.

    Okamoto T, Takeda S, Murayama Y, Ogata E, Nishimoto I. Ligand-dependent G protein coupling function of amyloid transmembrane precursor. J Biol Chem. 1995 Mar 3;270(9):4205-8. PubMed.

    Teng L, Zhao J, Wang F, Ma L, Pei G. A GPCR/secretase complex regulates beta- and gamma-secretase specificity for Abeta production and contributes to AD pathogenesis. Cell Res. 2010 Feb;20(2):138-53. PubMed.

    Thathiah A, De Strooper B. G protein-coupled receptors, cholinergic dysfunction, and Abeta toxicity in Alzheimer's disease. Sci Signal. 2009;2(93):re8. PubMed.

    Thathiah A, De Strooper B. The role of G protein-coupled receptors in the pathology of Alzheimer's disease. Nat Rev Neurosci. 2011 Feb;12(2):73-87. PubMed.

    Thathiah A, Horré K, Snellinx A, Vandewyer E, Huang Y, Ciesielska M, De Kloe G, Munck S, De Strooper B. β-arrestin 2 regulates Aβ generation and γ-secretase activity in Alzheimer's disease. Nat Med. 2013 Jan;19(1):43-9. PubMed.

    Thathiah A, Spittaels K, Hoffmann M, Staes M, Cohen A, Horré 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 amyloid-beta peptide generation in neurons. Science. 2009 Feb 13;323(5916):946-51. PubMed.

    View all comments by Klaus Heese

Make a Comment

To make a comment you must login or register.

References

News Citations

  1. Big Haul? A G Protein-coupled Receptor Regulates Aβ Production
  2. Could β-Arrestin Provide New Way to Halt Aβ Accumulation?
  3. Transparent Bodies Allow Neural Networks to ‘Apparate’
  4. With ScaleS, You Can See Through the Brain to Behold Synapses

Research Models Citations

  1. APP NL-F Knock-in
  2. APPPS1
  3. APPDutch

Paper Citations

  1. Ma P, Zemmel R. Value of novelty?. Nat Rev Drug Discov. 2002 Aug;1(8):571-2. PubMed.

Further Reading

Papers

  1. Huang Y, Thathiah A. Regulation of neuronal communication by G protein-coupled receptors. FEBS Lett. 2015 Jun 22;589(14):1607-19. Epub 2015 May 14 PubMed.
  2. Heese K. G Proteins, p60TRP, and Neurodegenerative Diseases. Mol Neurobiol. 2013 Jan 24; PubMed.
  3. Thathiah A, De Strooper B. The role of G protein-coupled receptors in the pathology of Alzheimer's disease. Nat Rev Neurosci. 2011 Feb;12(2):73-87. PubMed.

Primary Papers

  1. Huang Y, Skwarek-Maruszewska A, Horré K, Vandewyer E, Wolfs L, Snellinx A, Saito T, Radaelli E, Corthout N, Colombelli J, Lo AC, Van Aerschot L, Callaerts-Vegh Z, Trabzuni D, Bossers K, Verhaagen J, Ryten M, Munck S, D'Hooge R, Swaab DF, Hardy J, Saido TC, De Strooper B, Thathiah A. Loss of GPR3 reduces the amyloid plaque burden and improves memory in Alzheimer's disease mouse models. Sci Transl Med. 2015 Oct 14;7(309):309ra164. PubMed.

Annotate

To make an annotation you must Login or Register.