One possible road to Alzheimer’s disease starts with amyloid-β and passes through tau. What lies in between? New research led by Simon Lovestone, King’s College London, U.K., adds a few more molecular waypoints. As reported November 20 in Molecular Psychiatry, the scientists propose that clusterin (CLU)—a top risk gene for sporadic AD—mediates Aβ toxicity. In addition, they identify several targets downstream of Aβ and upstream of tau. The genes are also activated by an antagonist of Wnt, a cell surface signaling molecule implicated in cell proliferation and differentiation, fleshing out connections between AD and the Wnt signal transduction pathway.

Lovestone’s initial foray into the amyloid cascade began at the tau end. Given that glycogen synthase kinase 3 (GSK-3) phosphorylates tau, “I was looking for anything that might explain how GSK-3 activity is altered in AD,” said Lovestone, who also has a longstanding interest in Wnt signaling because it blocks GSK-3 activity. He was intrigued by a paper reporting that Aβ25-35 induces expression of Dickkopf1 (Dkk1), a Wnt antagonist. Silencing Dkk1 blocked Aβ toxicity in neuronal cultures (Caricasole et al., 2004). In the current study, Lovestone’s team confirmed that finding, and showed Aβ1-42 oligomers do the same.

Meanwhile, clusterin, also known as apolipoprotein J, came into view as a potential component of this amyloid/Dkk1 pathway, Lovestone said. Both Dkk1 and clusterin are regulated by Wnt signaling (Gonzalez-Sancho et al., 2005; Schepeler et al., 2007) and are elevated in the brains of AD mouse models (Rosi et al., 2010). Moreover, after clusterin was discovered as a risk gene for late-onset AD (Harold et al., 2009; see also Lambert et al., 2009), Lovestone and others found that its levels in plasma track with AD severity (see ARF related news story; ARF news story). First author Richard Killick and colleagues wondered if Dkk1 and clusterin might act in a common pathway to mediate Aβ toxicity through tau.

In support of this idea, the researchers found that exposing cultured rat neurons to Aβ25-35 not only turned on Dkk1, but also affected clusterin. Aβ treatment changed the protein’s distribution, driving up intracellular clusterin 3.5-fold while halving the amount that was secreted. This seemed consistent with prior work suggesting intracellular clusterin is pro-apoptotic, whereas secreted clusterin is protective (Trougakos et al., 2004; for review, see Bertram and Tanzi, 2010). Silencing clusterin with small interfering RNAs (siRNA) protected the cultured neurons against Aβ-induced Dkk1 expression and cell death, the authors report.

To further explore the neurotoxic signaling pathway, the scientists turned to whole-genome expression analyses of Aβ25-35-treated mouse neurons and Dkk1-treated rat neurons. They compared Aβ- and Dkk1-responsive genes using microarrays, and found 2,061 shared genes. Moreover, five of the top eight genes on each list were the same. “We saw an extraordinarily significant overlap,” Lovestone said. “That told us that the pathway downstream of Dkk1 is the Aβ pathway.”

Of the five shared genes, only one—cyclin D1, a protein that regulates the cell cycle—is a known Wnt target. The other four are transcription factors: early growth response 1 (EGR1), Ngfi-A-binding protein 2 (NAB2), Krueppel-like factor 10 (KLF10), and FOS. The researchers confirmed by quantitative reverse-transcriptase PCR that Aβ and Dkk1 induced the five target genes in cultured rodent neurons. They saw similar results using synthetic oligomers of Aβ42 (see Tizon et al., 2010). Oligomeric Aβ is now widely believed to be the more neurotoxic.

What about in people? The researchers found the Dkk1-responsive genes were enriched in postmortem brains of AD patients and Down's syndrome patients (who have three copies of amyloid precursor protein), but not in healthy elderly or in frontotemporal dementia patients (who have tau pathology but no plaques). Consistent with the human data, expression of the five common Aβ/Dkk1-responsive genes was unusually high in an amyloid-based AD mouse model (Tg2576), but looked normal in tauopathy mice (hTau). The findings add further evidence that Aβ is responsible for inducing the Dkk1-responsive genes, and suggest the target genes lie between amyloid and tau, the scientists reported.

To examine how the five target genes affect cell survival, Killick and colleagues individually suppressed them in cultured rodent neurons. Silencing EGR1 or KLF10 substantially protected the cells from Aβ-induced death, whereas cyclin D1, FOS, or NAB2 knockdown had no effect. These data suggest that EGR1 and KLF10 are needed for Aβ-induced toxicity. Knocking down EGR1 and NAB2 reduced tau phosphorylation, as seen on anti-PHF-1 immunoblots.

To see if Dkk1 is an important mediator of Aβ toxicity in vivo, Killick and colleagues generated transgenic mice overexpressing murine Dkk1 in neurons. All five Aβ/Dkk1 target genes were elevated—as were phospho-tau levels—in the neonatal temporal cortex, and older transgenic mice developed problems with hippocampal-dependent memory recall.

Given that four of the five Aβ/Dkk1 target genes are not involved in Wnt signaling, the scientists checked if Aβ toxicity in cultured neurons is mediated by the non-canonical Wnt-planar cell polarity (PCP) pathway, which induces gene expression through c-Jun N-terminal kinase (JNK). Sure enough, Western blots showed increased levels of activated JNK1 in Aβ- and Dkk1-exposed cells. Moreover, treatment with a JNK inhibitor blocked target gene induction by Dkk1.

All told, the data reveal a “novel understanding of how Aβ mediates its action, which is via Dkk1 and non-canonical Wnt signaling,” Lovestone told Alzforum. The results build on recent research showing that Aβ-induced synapse loss requires Dkk1 (ARF related news story on Purro et al., 2012), and that clusterin binds and stabilizes Aβ oligomers (ARF related news story on Narayan et al., 2012).

At first glance, the current report of reduced Aβ toxicity with clusterin knockdown may appear at odds with brain expression GWAS by two independent groups showing that the protective clusterin allele associates with increased clusterin expression (Allen et al., 2012; Ling et al., 2012). However, Lovestone said these papers cannot be directly compared. “We are measuring clusterin distribution at a subcellular level in response to Aβ, whereas Allen et al. are measuring RNA expression of clusterin at an organ level.”

In a follow-up to their current study, Lovestone and colleagues will try to better understand how Aβ alters clusterin trafficking and how the Aβ Dkk1 target genes mediate cell death.—Esther Landhuis.

Reference:
Killick R, Ribe EM, Al-Shawi R, Malik B, Hooper C, Fernandes C, Dobson R, Nolan PM, Lourdusamy A, Furney S, Lin K, Breen G, Wroe R, To AW, Leroy K, Causevic M, Usardi A, Robinson M, Noble W, Williamson R, Lunnon K, Kellie S, Reynolds CH, Bazenet C, Hodges A, Brion JP, Stephenson J, Paul Simons J, Lovestone S. Clusterin regulates β-amyloid toxicity via Dickkopf-1-driven induction of the wnt-PCP-JNK pathway. Mol Psychiatry. 2012 Nov 20. Abstract

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References

News Citations

  1. Research Brief: Clusterin Grabs Spotlight Among Elite Few LOAD Genes
  2. Research Brief: Link Tightens Between Plasma Clusterin and AD
  3. Boost APP Ubiquitination, Wnt Signaling to Stave Off AD?
  4. Keeping It Under Wraps: Clusterin Encases Aβ Oligomers In Vitro

Paper Citations

  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. . The Wnt antagonist DICKKOPF-1 gene is a downstream target of beta-catenin/TCF and is downregulated in human colon cancer. Oncogene. 2005 Feb 3;24(6):1098-103. PubMed.
  3. . Clusterin expression can be modulated by changes in TCF1-mediated Wnt signaling. J Mol Signal. 2007;2:6. PubMed.
  4. . Increased Dickkopf-1 expression in transgenic mouse models of neurodegenerative disease. J Neurochem. 2010 Mar;112(6):1539-51. PubMed.
  5. . Genome-wide association study identifies variants at CLU and PICALM associated with Alzheimer's disease. Nat Genet. 2009 Oct;41(10):1088-93. PubMed.
  6. . Genome-wide association study identifies variants at CLU and CR1 associated with Alzheimer's disease. Nat Genet. 2009 Oct;41(10):1094-9. PubMed.
  7. . Silencing expression of the clusterin/apolipoprotein j gene in human cancer cells using small interfering RNA induces spontaneous apoptosis, reduced growth ability, and cell sensitization to genotoxic and oxidative stress. Cancer Res. 2004 Mar 1;64(5):1834-42. PubMed.
  8. . Alzheimer disease: New light on an old CLU. Nat Rev Neurol. 2010 Jan;6(1):11-3. PubMed.
  9. . Cystatin C protects neuronal cells from amyloid-beta-induced toxicity. J Alzheimers Dis. 2010;19(3):885-94. PubMed.
  10. . The secreted Wnt antagonist Dickkopf-1 is required for amyloid β-mediated synaptic loss. J Neurosci. 2012 Mar 7;32(10):3492-8. PubMed.
  11. . The extracellular chaperone clusterin sequesters oligomeric forms of the amyloid-β(1-40) peptide. Nat Struct Mol Biol. 2012 Jan;19(1):79-83. PubMed.
  12. . Novel late-onset Alzheimer disease loci variants associate with brain gene expression. Neurology. 2012 Jul 17;79(3):221-8. PubMed.
  13. . Genetics of clusterin isoform expression and Alzheimer's disease risk. PLoS One. 2012;7(4):e33923. PubMed.
  14. . Clusterin regulates β-amyloid toxicity via Dickkopf-1-driven induction of the wnt-PCP-JNK pathway. Mol Psychiatry. 2012 Nov 20; PubMed.

Other Citations

  1. Tg2576

External Citations

  1. top risk gene
  2. hTau

Further Reading

Papers

  1. . Clusterin regulates β-amyloid toxicity via Dickkopf-1-driven induction of the wnt-PCP-JNK pathway. Mol Psychiatry. 2012 Nov 20; 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. . The extracellular chaperone clusterin sequesters oligomeric forms of the amyloid-β(1-40) peptide. Nat Struct Mol Biol. 2012 Jan;19(1):79-83. PubMed.
  4. . Novel late-onset Alzheimer disease loci variants associate with brain gene expression. Neurology. 2012 Jul 17;79(3):221-8. PubMed.
  5. . Genetics of clusterin isoform expression and Alzheimer's disease risk. PLoS One. 2012;7(4):e33923. PubMed.
  6. . The secreted Wnt antagonist Dickkopf-1 is required for amyloid β-mediated synaptic loss. J Neurosci. 2012 Mar 7;32(10):3492-8. PubMed.

Primary Papers

  1. . Clusterin regulates β-amyloid toxicity via Dickkopf-1-driven induction of the wnt-PCP-JNK pathway. Mol Psychiatry. 2012 Nov 20; PubMed.