In Alzheimer’s research, Aβ usually hogs the attention from its parent protein, amyloid precursor protein (APP). Yet evidence is accumulating that other APP fragments, particularly those produced by β-secretase (BACE1) cleavage, such as β-CTF and sAPPβ, may play crucial roles both in neuronal health and in AD. In the February 1 Journal of Neuroscience, researchers led by Angèle Parent at the University of Chicago, Illinois, put the spotlight on membrane-bound APP cleavage fragments (β-CTF and α-CTF). The authors describe a signaling pathway by which these fragments might stimulate neurite outgrowth and promote synaptic health.

Dale Bredesen at the Buck Institute, Novato, California, noted that the paper provides further evidence that APP functions as a single-pass transmembrane G protein-coupled receptor, and also lays out a detailed signaling mechanism. “I think this opens up a whole new way to evaluate the effects mediated by APP, and also provides an additional suggestion that APP signaling could be quite important in normal plasticity,” he told ARF. Bredesen was not involved in the work.

In contrast, in the December 14, 2011, European Molecular Biology Organization (EMBO) Molecular Medicine, researchers led by Luciano D’Adamio at the Albert Einstein College of Medicine, Bronx, New York, reported that inhibiting BACE1, but not γ-secretase, rescues synaptic plasticity and memory in a mouse model of dementia. Together, these studies underscore how APP fragments may appear to have varied effects. They also imply that researchers should look beyond Aβ for the causes of AD pathology and for therapeutic targets.

Membrane-bound APP can be cut on the extracellular side of the cell membrane by either α- or β-secretase, creating a soluble shed portion (sAPPα or sAPPβ) and a membrane-bound fragment with an intracellular tail (α-CTF or β-CTF). The β-CTF fragment is then snipped by γ-secretase to generate Aβ and an APP intracellular domain (AICD) that may travel to the nucleus. It is not yet clear what all these fragments do, although there are clues. For example, numerous studies have shown that APP plays a physiological role in modulating neurite outgrowth (see, e.g., ARF related news story; ARF news story; Small et al., 1999; and Young-Pearse et al., 2008), but the exact mechanisms are unknown. Since synapses form on dendritic spines, neurite growth is intimately linked to synaptic health, making this pathway particularly pertinent to AD.

Parent and colleagues focused on intracellular signaling by membrane-bound APP fragments. First author Carole Deyts designed a fusion protein, mAICD, by joining the intracellular portion of human APP to membrane-bound motifs. Deyts and colleagues transfected various cell cultures (including neuroblastoma cells, primary mouse cortical neurons, and immortalized hippocampal neurons) with the construct, and found that transfected cells grew longer and more complex neurites. The findings contrast with previous studies that highlighted axon and tau toxicity by BACE1-cleaved APP products, including N-terminal (see, e.g., ARF related news story) and C-terminal fragments (see ARF related news story and ARF news story).

Looking for a mechanism, the authors saw higher levels of cyclic AMP (cAMP) and activated protein kinase A (PKA) in transfected cells. Membrane-bound adenylate cyclase produces cAMP, which is known to activate PKA. Inhibiting either adenylate cyclase or PKA in these cultures abolished the neurite outgrowth, the authors report, indicating that this pathway is crucial for mediating mAICD’s effects. PKA modifies numerous downstream proteins. It activates cAMP-response element binding protein-1 (CREB), a key protein for learning and memory, and inactivates the kinase GSK-3β. Researchers have associated active GSK-3β with neurite retraction (see Hur and Zhou, 2010), and it also phosphorylates tau. The authors confirmed that CREB activity increased, and GSK-3β activity decreased, in cells overexpressing mAICD.

Deyts and colleagues next explored how mAICD might activate adenylate cyclase. They showed by co-immunoprecipitation that mAICD binds the G protein GαS, which is known to stimulate cAMP signaling. The authors identified a G protein-binding site in the intracellular portion of APP, and showed that mutating one amino acid in this motif prevented mAICD from interacting with GαS, and also blocked neurite growth. Expression of a dominant negative GαS protein likewise inhibited mAICD-induced neurite extension. The results imply that membrane-bound APP fragments act as G protein-coupled receptors to promote neurite growth, suggest the authors.

To show that physiological membrane-bound APP fragments have the same effect as mAICD, the authors treated neuroblastoma cultures with a γ-secretase inhibitor, which causes APP-CTF fragments to build up. Neurite length increased in treated cells, and again the effect could be blocked by inhibiting adenylate cyclase, indicating the same pathway was at work as in mAICD-laced cells. Finally, in conjunction with γ-secretase inhibition, the authors blocked either β or α cleavage to allow either α-CTF or β-CTF to accumulate, but found that neurites grew equally well in either condition, suggesting both fragments can stimulate outgrowth.

Parent told ARF she is interested in the therapeutic possibilities of targeting G proteins in AD, pointing out that many such drugs are available from cancer research (see Thathiah and De Strooper, 2011). She is currently investigating whether stimulating signaling through membrane-bound APP fragments can help restore memory in animal models of AD. “People have been paying a lot of attention to Aβ production, and thinking APP is bad for you. I think APP is actually very good [for you] if it works well,” Parent suggested.

Intriguingly, APP was previously shown by a Japanese group to bind to a different G protein, Go (see Nishimoto et al., 1993). In opposition to GαS, Go signaling decreases cAMP levels and can lead to cell death. Nishimoto and colleagues showed that three AD-associated mutations in APP all increased Go-mediated cell death. Bredesen noted that the new paper complements Nishimoto’s work. “There has been the suggestion for years that APP can mediate both neurite retraction and neurite extension. We’ve called it a molecular switch,” he said. These papers add to the evidence that APP can have either beneficial or harmful effects, depending on which G proteins are present. The next step will be to show which ligands activate the positive and negative effects of APP, Bredesen added.

In the second paper, D’Adamio and colleagues provide more evidence for the harmful effects of signaling by BACE1 cleavage products, using a mouse model of familial Danish dementia (FDDKI). Danish dementia is caused by loss of the Bri2 protein, which normally binds to APP and prevents its cleavage. To make the mouse, the researchers inserted the Danish mutation into the endogenous Bri2 mouse gene. D’Adamio noted that, unlike many AD mouse models, this animal does not overexpress APP; instead, it builds up APP cleavage products. Joint first authors Robert Tamayev and Shuji Matsuda found they were able to rescue synaptic and memory deficits in FDDKI mice by inhibiting BACE1, but not by inhibiting γ-secretase. This implies that a BACE1 cleavage fragment (β-CTF or secreted sAPPβ) is the toxic entity, rather than Aβ.

The data also hint that inhibiting γ-secretase could be harmful, as that would cause BACE1 cleavage products to build up. Intriguingly, some previous papers have suggested that AD-causing presenilin mutations might be loss-of-function mutations, fitting with the idea that lack of γ-secretase could lead to memory problems (see, e.g., Shen and Kelleher, 2007; ARF related news story on Qiang et al., 2011). A clinical trial of the γ-secretase inhibitor semagacestat was halted because patients on the drug did worse than those on placebo (see ARF related news story and ARF news story).

The authors developed a short peptide, called modulator of β-cleavage of APP (MoBA), that binds to APP and prevents BACE1 cleavage. MoBA does not prevent α-secretase cleavage, nor does it bind to β-CTF, the authors report. MoBA had comparable effects to BACE1 inhibition in the FDDKI mice, restoring memory and synaptic plasticity. This peptide might be a therapeutic alternative to BACE1 inhibitors, D’Adamio suggested, as it binds only to APP and does not affect other BACE1 substrates. In ongoing work, preliminary data indicate that MoBA is as effective in restoring synaptic plasticity in APP/PS1 mice as it is in the FDDKI mice, D’Adamio added.—Madolyn Bowman Rogers

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  1. This is an excellent study. Not only is the novel interaction of APP/APLP C-terminal fragments with GαS shown to trigger neurite outgrowth, but also the involvement of cAMP/PKA activation and GSK-3β inhibition is in agreement with published evidence showing a role for these signaling events in neurite formation. The authors provide a thoughtful consideration of the implications of APP-CTF-mediated signaling for normal APP function and Alzheimer's disease therapeutics.

    View all comments by Suzanne Guenette
  2. The paper by Deyts and colleagues is of great interest. It shows that the C-terminal stubs derived from α- and β-secretase cleavages of APP, when accumulating, could alter various signaling pathways involving protein kinase A or GSK-3β kinases, and have functional consequences on neurite growth and dendritic arborization. Although the empirical observation of APP-CTF fragment-linked toxicity has already been documented both in vitro and in vivo, the present work’s "added value" stands in the delineation of downstream cascades underlying this toxicity, and, noticeably, by the demonstration that an identified sequence domain located within the sequence of the APP intracellular domain (AICD) could interact with a heterotrimeric G protein subunit.

    Another interesting aspect of the paper is the demonstration that the above-described phenotype could be mimicked by an AICD-based construct in which extracellular and transmembrane domains of APP have been replaced by a MyrPalm domain of the Lyn kinase. This is interesting, but also raises some questions. AICD is theoretically released from α- and β-secretase-derived CTF stubs. Therefore, there exists an equilibrium between CTF stubs and their proteolytic fragment AICD that is tightly regulated. Most of the work examines the consequences of overexpressing CTF or an artificial AICD construct tethered to the membrane, but does not really establish whether endogenous CTF or AICD could interfere with PKA or GSK-3β pathways. This would be particularly important with respect to the fact that α-CTF-derived AICD appears to be very short lived and undergoes rapid cytosolic degradation, while β-CTF-derived AICD appears to be produced in the endocytic pathway that protects it from exacerbated degradation. The β path allows AICD to signal to the nucleus where it can modulate gene transcription (for a review, see 1). Therefore, the source of endogenous AICDs, i.e., derived from α-CTF or β-CTF stubs, and their respective abilities to modulate PKA and GSK-3β pathways, could better help us understand which of the fragments, besides Aβ or Aβ-related species, can contribute to neuronal morphological alterations.

    It remains that the paper by Deyts and colleagues unravels a dual mechanism by which membrane-embedded APP-CTF and APP-CTF-derived AICD could, in a complementary manner, trigger deleterious effects via distinct cellular pathways in neuronal cells.

    References:

    . The physiology of the β-amyloid precursor protein intracellular domain AICD. J Neurochem. 2012 Jan;120 Suppl 1:109-24. PubMed.

    View all comments by Frédéric Checler

References

News Citations

  1. <i>Drosophila</i> Brain APPeaLs for Protection after Trauma
  2. Another Take on APP and Neurite Outgrowth—The Role of Reelin
  3. Keystone: Death Receptor Ligand—New Role for APP, New Model for AD?
  4. Induced Neurons From AD Patients Hint at Disease Mechanisms
  5. Paper Alert: γ-Secretase Modulators Trump Inhibitors
  6. Alzheimer’s Neurons Made to Order: Direct Conversion From Skin Cells
  7. Lilly Halts IDENTITY Trials as Patients Worsen on Secretase Inhibitor
  8. Paris: Semagacestat Autopsy and Other News of Trial Tribulations

Paper Citations

  1. . Neurite-outgrowth regulating functions of the amyloid protein precursor of Alzheimer's disease. J Alzheimers Dis. 1999 Nov;1(4-5):275-85. PubMed.
  2. . Secreted APP regulates the function of full-length APP in neurite outgrowth through interaction with integrin beta1. Neural Dev. 2008;3:15. PubMed.
  3. . GSK3 signalling in neural development. Nat Rev Neurosci. 2010 Aug;11(8):539-51. PubMed.
  4. . The role of G protein-coupled receptors in the pathology of Alzheimer's disease. Nat Rev Neurosci. 2011 Feb;12(2):73-87. PubMed.
  5. . Alzheimer amyloid protein precursor complexes with brain GTP-binding protein G(o). Nature. 1993 Mar 4;362(6415):75-9. PubMed.
  6. . The presenilin hypothesis of Alzheimer's disease: evidence for a loss-of-function pathogenic mechanism. Proc Natl Acad Sci U S A. 2007 Jan 9;104(2):403-9. PubMed.
  7. . Directed conversion of Alzheimer's disease patient skin fibroblasts into functional neurons. Cell. 2011 Aug 5;146(3):359-71. PubMed.

Further Reading

Primary Papers

  1. . Novel GαS-protein signaling associated with membrane-tethered amyloid precursor protein intracellular domain. J Neurosci. 2012 Feb 1;32(5):1714-29. PubMed.
  2. . β- but not γ-secretase proteolysis of APP causes synaptic and memory deficits in a mouse model of dementia. EMBO Mol Med. 2012 Mar;4(3):171-9. PubMed.