The prevailing theory that Aβ peptides are a major culprit in Alzheimer’s disease offers no shortage of possible therapeutic approaches—two of which make the pages of this week’s Journal of Neuroscience. Patricia Salinas and colleagues at University College London in the U.K. bolster the connection between Wnt signaling and AD by showing that synapse loss induced by toxic Aβ peptides requires the Wnt antagonist Dickkopf-1 (Dkk1). And in another study, researchers led by Takashi Horiguchi at Takeda Pharmaceutical Company Ltd., Kanagawa, Japan, report that a component of the ubiquitin-proteasome pathway curbs amyloid-β production by promoting ubiquitination and degradation of its parent molecule, amyloid precursor protein (APP). Though still a way from clinical testing, the research provides new avenues for amyloid-based therapies in early-stage AD.

Before neurons die and cognition fades, synapses wither and disappear—and amyloid-β peptides can trigger this synaptic breakdown. It therefore stands to reason that Aβ’s dirty work would involve factors that promote synapse formation or maintenance. Wnt signaling proteins function as such, and prior work has made a case for this pathway’s involvement in AD. A genomewide association study linked sporadic AD to a variant of a Wnt co-receptor (low-density lipoprotein receptor-related protein 6, or LRP6) that tones down Wnt signaling (ARF related news story on De Ferrari et al., 2007). Other research suggested that brain levels of Dkk1, which inhibits Wnt signaling, are elevated in AD patient biopsies and in AD mouse models, and that Aβ peptides turn on Dkk1 expression in cultured mouse cortical neurons (Caricasole et al., 2004; Rosi et al., 2010). Furthermore, excitatory neurotransmission in rat hippocampal neuronal cultures rose with exposure to Wnt3a and dropped in the presence of Dkk1 (Avila et al., 2010).

Now, joint first authors Silvia Purro and Ellen Dickins and colleagues at University College London confirm that Aβ induces Dkk1 expression, and they link Dkk1 directly with loss of synapses. Incubating mouse hippocampal slices with small Aβ oligomers prepared in vitro (Klein, 2002), the researchers saw the expected boost in Dkk1 mRNA and protein levels, along with synaptic loss marked by decreased colocalization of vGlut1 and PSD95 pre- and post-synaptic markers. Treatment with Dkk1 antibodies wiped out these effects, suggesting that Aβ cannot harm synapses without the Wnt pathway modulator. “We expected only a partial protection, thinking maybe other molecules are involved,” Salinas told ARF. On the contrary, “the data suggest Dkk1 is quite critical for Aβ’s effects on synapses.”

Dkk1 mimicked some of Aβ’s effects. When the researchers added Dkk1 to cultures of rat hippocampal neurons, fewer cells stained for presynaptic (VAMP2, synapsin-1, Bassoon, Cask) and postsynaptic (PSD-95, Grip) than in control cultures. The synaptic sites re-formed when Dkk1 was removed. However, Dkk1 did not induce cell death, as assessed by TUNEL staining, nor did it influence total levels of synaptic proteins on Western blots. These data suggest that Dkk1 decreases the number of synaptic sites by causing synaptic proteins to disperse rather than to degrade. The scientists further demonstrated Dkk1’s effect on synaptic disassembly through electron microscopy studies that measured shrinkage of synapses, and time-lapse recordings showing dispersal of synaptic proteins within a half-hour of Dkk1 treatment.

Whereas prior work indicated that Aβ upregulates Dkk1 and ablates synapses, this study examines whether Dkk1 directly triggers synaptic damage. “Apparently, it does. When you incubate neurons in the presence of Dkk1, synapses start to disassemble rapidly—very rapidly. That’s the main contribution,” said Giancarlo De Ferrari of Universidad Andres Bello in Santiago, Chile. De Ferrari hypothesized years ago that sustained loss of Wnt signaling function could lead to AD (see review by De Ferrari and Inestrosa, 2000).

Because Dkk1 goes up early in AD when neurons have not yet begun dying, it may be possible to use Dkk1 as a biomarker, Salinas said. And, “if we develop [drugs] targeting Dkk1, the hope is that we could stop disease progression at the early stages before loss of memory.” Her lab is collaborating with a small biotechnology company to find small brain-penetrant molecules that interfere with Dkk1 function. In the future, the scientists hope to test whether such molecules could slow disease in AD mouse models, Salinas said.

In the second paper, first author Tomomichi Watanabe and colleagues investigated an earlier stage of amyloidosis—production of the Aβ peptides themselves. They focused on F-box and leucine-rich repeat protein 2 (FBL2), a component of the ubiquitin-proteasome system that helps cells dispose of damaged or misfolded proteins. Malfunction of the ubiquitin-proteasome pathway is linked with neurodegenerative disease, and a microarray study found abnormally low FBL2 expression in AD brains (Blalock et al., 2004). Presuming FBL2 would influence hallmark protein aggregates in AD, and having discovered early on that it neither binds tau nor influences tau phosphorylation, the team turned its attention toward Aβ, Watanabe noted.

Overexpression of FBL2 lowered Aβ secretion—and FBL2 knockdown raised it—in several cell culture systems, including human embryonic kidney cells and mouse neuronal cell lines and primary cortical neurons. FBL2-expressing cells also had considerably less intracellular Aβ. Levels of β- and γ-secretases or Aβ-degrading enzymes in those in-vitro experiments did not change, though, suggesting that FBL2 should not influence rates of APP processing or Aβ clearance. Could FBL2 regulate the amount of APP? Further experiments in FBL2-transfected cells showed that FBL2 binds APP and promotes its ubiquitination and degradation. In addition, a greater proportion of APP lingers on the cell surface, and this membrane APP becomes internalized more slowly in FBL2-overexpressing cells relative to control cells. This suggests that ubiquitination by FBL2 impedes APP endocytosis as well. FBL2 ubiquitination may function as a dual regulator, the authors propose, sending intracellular APP on its way for proteasomal degradation while keeping cell-surface APP from getting endocytosed for β-secretase cleavage. The net effect would be less Aβ production, the authors suggest.

That scenario appears to be true in at least one animal model as well. AD transgenic mice overexpressing FBL2 had more ubiquitinated APP, fewer plaques, and less insoluble brain Aβ compared to control AD mice. The FBL2-overexpressing AD strain was generated by crossing AD mice that express mutant APP and presenilin-1 (Willuweit et al., 2009) with transgenic mice that overexpress human FBL2 ~16-fold in the brain. The researchers plan to test behavior and cognition in these mice, Watanabe noted.

“This is a really interesting and exciting paper,” M. Paul Murphy of the University of Kentucky, Lexington, wrote in an e-mail to ARF (see full comment below). “Although we’ve known for some time that the ubiquitin pathway is intimately involved with the development of AD at several levels, this is probably the most significant step forward in our understanding of the underlying mechanism in a long while.” Gunnar Gouras of Lund University, Sweden, said the data are “quite thorough and provide important new insights into APP metabolism.” (See full comment below.)

Activation of the FBL2 pathway may hold promise as a new therapeutic target because it slows APP processing into Aβ in two ways—speeding degradation of intracellular APP and keeping membrane APP from reaching endosomes, suggest the authors. However, because augmenting protein function is generally harder than blocking it, “targeting this pathway will be challenging,” Murphy said. But since it could potentially treat other diseases that involve defective protein turnover, not just AD, “the payoff could be huge,” he said.—Esther Landhuis

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  1. This is a really interesting and exciting paper. Although we’ve known for some time that the ubiquitin pathway is intimately involved with the development of Alzheimer’s disease at several levels, this is probably the most significant step forward in our understanding of the underlying mechanism in a long while. Although this is an important discovery, there are still some crucial unknowns. For instance, although Watanabe and colleagues do a great deal to advance our knowledge of how FBL2 controls APP processing, we still don’t know very much about its broader role in the degradation of other proteins. In all likelihood, FBL2 probably affects the turnover of multiple targets, and we’re going to need to know more of these to evaluate its real potential as an novel avenue for therapeutic development. Also, their data show that you would likely have to augment the expression or improve the function of FBL2 to tap into this potential, and upregulating something is a harder thing to do than knocking it down or blocking its activity. So targeting this pathway as a therapeutic strategy will be challenging. On the upside, even if it’s a hard pathway to tap into for a therapy, there could be some real potential here for treating other diseases that involve defective protein turnover, in addition to AD. So the payoff could be huge.

    View all comments by M. Paul Murphy
  2. This paper by Watanabe and colleagues is of considerable interest; it provides evidence that Aβ precursor protein (APP) is ubiquitinated and that FBL2, a component of the SCF E3 ubiquitin ligase complex, is important in regulating APP trafficking, processing, and degradation. A better understanding of the regulated processing and degradation of APP is important, given the focus on Aβ in both diagnostic and therapeutic AD research. (For an excellent recent review on the cell biology of APP, see Rajendran and Annaert, 2012.) Although transmembrane proteins at the plasma membrane are ubiquitinated to induce internalization and degradation in the endosomal-lysosomal system (e.g., the epidermal growth factor receptor), the authors provide evidence that FBL2-induced ubiquitination of APP is more analogous to that of protease-activated receptor-1 PAR1, where ubiquitination inhibits internalization (Wolfe et al., 2007). They show that FBL2 interacts with the C-terminus of APP and, when overexpressed, FBL2 reduces (and when knocked down, increases) both extra- and intracellular Aβ. Their data are strengthened by showing that overexpression of FBL2 reduces Aβ in the brains of transgenic mice.

    It is not quite clear how they envision Aβ or APP, which should be associated with endocytic vesicles, to be degraded by the proteasome in the cytoplasm; endosome-associated proteasome might be considered (Almeida et al., 2006). Immunofluorescence microscopy could have been made use of as a complementary method to, for example, confirm the subcellular localization of FBL2, or cell surface dynamics of APP. And using the Aβ/APP antibody 6E10 to detect intraneuronal Aβ in the brain is not optimal, given the controversy about this highlighted by last summer’s ARF Webinar. Overall, the data presented here are quite thorough and provide important new insights into APP metabolism. FBL2 might also provide a new target for experimental therapy.

    References:

    . Membrane trafficking pathways in Alzheimer's disease. Traffic. 2012 Jun;13(6):759-70. PubMed.

    . Ubiquitination differentially regulates clathrin-dependent internalization of protease-activated receptor-1. J Cell Biol. 2007 Jun 4;177(5):905-16. PubMed.

    . Beta-amyloid accumulation impairs multivesicular body sorting by inhibiting the ubiquitin-proteasome system. J Neurosci. 2006 Apr 19;26(16):4277-88. PubMed.

    View all comments by Gunnar Gouras

References

News Citations

  1. Genetics Link Late-onset AD to Chromosome 12 and Wnt Signaling

Paper Citations

  1. . Common genetic variation within the low-density lipoprotein receptor-related protein 6 and late-onset Alzheimer's disease. Proc Natl Acad Sci U S A. 2007 May 29;104(22):9434-9. PubMed.
  2. . Induction of Dickkopf-1, a negative modulator of the Wnt pathway, is associated with neuronal degeneration in Alzheimer's brain. J Neurosci. 2004 Jun 30;24(26):6021-7. PubMed.
  3. . Increased Dickkopf-1 expression in transgenic mouse models of neurodegenerative disease. J Neurochem. 2010 Mar;112(6):1539-51. PubMed.
  4. . Canonical Wnt3a modulates intracellular calcium and enhances excitatory neurotransmission in hippocampal neurons. J Biol Chem. 2010 Jun 11;285(24):18939-47. PubMed.
  5. . Abeta toxicity in Alzheimer's disease: globular oligomers (ADDLs) as new vaccine and drug targets. Neurochem Int. 2002 Nov;41(5):345-52. PubMed.
  6. . Wnt signaling function in Alzheimer's disease. Brain Res Brain Res Rev. 2000 Aug;33(1):1-12. PubMed.
  7. . Incipient Alzheimer's disease: microarray correlation analyses reveal major transcriptional and tumor suppressor responses. Proc Natl Acad Sci U S A. 2004 Feb 17;101(7):2173-8. PubMed.
  8. . Early-onset and robust amyloid pathology in a new homozygous mouse model of Alzheimer's disease. PLoS One. 2009;4(11):e7931. PubMed.

Further Reading

Papers

  1. . Induction of Dickkopf-1, a negative modulator of the Wnt pathway, is associated with neuronal degeneration in Alzheimer's brain. J Neurosci. 2004 Jun 30;24(26):6021-7. PubMed.
  2. . Wnt signaling function in Alzheimer's disease. Brain Res Brain Res Rev. 2000 Aug;33(1):1-12. PubMed.
  3. . Common genetic variation within the low-density lipoprotein receptor-related protein 6 and late-onset Alzheimer's disease. Proc Natl Acad Sci U S A. 2007 May 29;104(22):9434-9. PubMed.
  4. . Increased Dickkopf-1 expression in transgenic mouse models of neurodegenerative disease. J Neurochem. 2010 Mar;112(6):1539-51. PubMed.
  5. . Incipient Alzheimer's disease: microarray correlation analyses reveal major transcriptional and tumor suppressor responses. Proc Natl Acad Sci U S A. 2004 Feb 17;101(7):2173-8. PubMed.

Primary Papers

  1. . The secreted Wnt antagonist Dickkopf-1 is required for amyloid β-mediated synaptic loss. J Neurosci. 2012 Mar 7;32(10):3492-8. PubMed.
  2. . FBL2 regulates amyloid precursor protein (APP) metabolism by promoting ubiquitination-dependent APP degradation and inhibition of APP endocytosis. J Neurosci. 2012 Mar 7;32(10):3352-65. PubMed.