Another Take on APP and Neurite Outgrowth—The Role of Reelin

A new study strengthens the idea that reelin, an extracellular matrix protein essential for brain development, works, at least in part, by binding amyloid precursor protein (APP). In the June 10 Journal of Neuroscience, researchers led by Bill Rebeck at Georgetown University, Washington, DC, report that, together, reelin and APP support neurite outgrowth in vitro and in vivo. “I think the larger story here is the idea that APP is a synaptic protein and its presence is necessary for either the formation or stability of synaptic connections, and these connections rely on extracellular matrix proteins, of which reelin is one,” Rebeck told ARF.

In addition to helping the field settle on a role for APP, for which consensus remains elusive, the findings could also have implications for Alzheimer disease because the researchers report that reelin reduces endocytosis of APP and promotes the non-amyloidogenic processing of the precursor. “I think for me it introduces a new element to the mechanism of APP signaling and cleavage, exposing the involvement of extracellular ligands, and may contribute to new therapeutic approaches for AD,” suggested Bernadette Allinquant, INSERM, France. Allinquant was not involved in the work but has studied the reelin/APP interaction.

Reelin is perhaps best known because of the reeler mouse—a reelin knockout that has stark neurodevelopmental problems. Reelin is predominantly produced by the Cajal-Retzius cells during development, and by interacting with members of the low-density lipoprotein receptor family, it activates the cytoplasmic protein disabled (Dab-1) and thereby regulates neuronal migration. “That whole pathway has been beautifully worked out, but what was not so clear is what happens when reelin binds APP,” said Rebeck. For one, knocking out APP has a much milder effect than reelin knockout (see Zheng et al., 1995), which suggests the APP/reelin interaction is only part of the neurodevelopmental picture.

Rebeck said he was drawn to study reelin because it shares some of the same protein partners as APP, including F-spondin (see ARF related news story), ApoE receptors (for a review, see Hoe and Rebeck, 2008), and Dab-1 (see Hoareau et al., 2008). Work from his lab showed that both Dab-1 and reelin reduce β cleavage of APP (see Hoe et al., 2006) and this current paper delves into the reelin/APP interaction in more detail.

First author Hyang-Sook Hoe and colleagues report that reelin and APP interact with each other through specific domains—the E1 extracellular domain on APP, and domains 3-6 of reelin. The two proteins co-immunoprecipitate from mouse brain lysates, primary hippocampal neurons, and COS7 cells expressing both proteins. Interestingly, reelin expression is increased by about one-third in the brain of APP-negative mice and decreased in Tg2576 transgenic mice, which overexpress human APP. “The findings suggest an interaction between APP and reelin is important for maintaining normal reelin levels in the brain,” write the authors.

In turn, reelin also influences APP dynamics. The researchers found that in several different systems (COS7 cells, primary cortical and hippocampal neurons, and Neuro2A cells), reelin treatment led to increased APP on the cell surface. The authors reasoned that might be due to reduced endocytosis, and were able to show, using green fluorescent protein tagged APP, that ~40 percent less surface APP was taken up by hippocampal neurons treated with reelin. The treatment also increased α-secretase processing of APP, decreasing release of Aβ40/42.

What is the biological significance of the reelin/APP interaction? Both have been shown to promote neurite outgrowth (see Qiu et al., 1995 and Niu et al., 2004) and here Rebeck and colleagues show that they cooperate toward that end. In cultured hippocampal neurons, overexpression of APP increased dendritic neurite complexity in response to reelin, while knocking down APP with interfering RNAs had the opposite effect, abolishing the reelin response. “These data support the hypothesis that a reelin-APP interaction is critical for induction of neurite outgrowth in primary hippocampal neurons,” write the authors. That interaction seems critical in vivo, too, because Hoe and colleagues found that in APP-overexpressing transgenic mice dendritic arborization was significantly increased, while it was significantly decreased in APP knockout mice.

Finally, the authors pieced together exactly how reelin and APP are held together. Work from Dennis Selkoe’s group at Harvard Medical School showed that APP colocalizes with β1 integrin, a cell surface protein that interacts with extracellular matrix proteins (see Yamazaki et al., 1997), and that β1 integrin mediates neurite outgrowth induced by sAPPα, which is shed by α-secretase (see Young-Pearse et al., 2008). Hoe and colleagues confirmed the APP/β1 integrin colocalization in hippocampal neurons, and they also found that APP colocalizes with the α3 integrin, which forms a complex with its β1 partner. They also showed that APP, reelin, and α3β1 integrin co-immunoprecipitate from mouse brain lysates, indicating that the three form a complex. In fact, the three proteins may be co-dependent because overexpression or loss of APP reduced and increased, respectively, the expression of α3β1 integrin, much like it did for reelin. Furthermore, the researchers found that β1 integrin and reelin synergistically cooperate to reduce endocytosis of APP and retain the protein in the cell surface. Lastly, Hoe and colleagues showed that an antibody against the α3β1 integrin prevents neurite outgrowth by both reelin and APP.

All told, the work paints a triptych, suggesting that reelin, APP, and α3β1 integrin work together to promote neurite outgrowth. This may not be entirely crucial for development, given the milder phenotype of the APP knockout, admits Rebeck, but he agreed there could be some redundancy between APP and its homologs, APLP1 and APLP2. Knocking out all three leads to lissencephaly, a severe developmental problem.

The complex may also have a role in the adult brain, which would be more germane to Alzheimer disease research. “We are used to thinking about proteins in development as being purely developmental, and reelin fits into that category, but then they stick around your whole life,” said Rebeck. “The question is, What do they do all that time?” He suggested that while reelin is involved in migration and growth of neuronal processes, it might help maintain those processed in the adult brain. “One of the things we always talk about is plasticity, which we depend on every day,” said Rebeck. “That has to be driven by the strengthening or weakening or elimination of synapses, or the formation of new synapses. That, of course, goes on a lot during development but also as long as we’re alive.” One way to address the role of reelin in adulthood, he suggested, would be to make a conditional knockout, which has not yet been done.

Whether reelin is involved in pathogenic mechanisms that lead to AD remains to be seen. But one interesting outcome from this work is that overexpressing APP can attenuate reelin expression and lead to dendritic abnormalities in young mice, which lead the authors to suggest that some models of AD might exhibit behavioral problems that are independent of Aβ accumulation.—Tom Fagan

Comments

  1. This very interesting paper provides another link in our understanding of the role of APP in synaptogenesis and Alzheimer's disease. Understanding the function of APP and its molecular partners (e.g., reelin) will undoubtedly provide key insights into the mechanisms of synaptic loss and disease progression, the role of ApoE, and the potential roles of reelin and the LDL receptor family in pathogenesis.

    Just to set the record straight, while the paper by Qiu et al. in J Neuroscience is undoubtedly important, there were several seminal papers from our group prior to the Qiu paper, which provided both in vitro and in vivo evidence that APP was involved in neurite outgrowth (Milward et al., 1992; Small et al., 1994; Williamson et al., 1995; Clarris et al., 1995).

    References:

    Milward EA, Papadopoulos R, Fuller SJ, Moir RD, Small D, Beyreuther K, Masters CL. The amyloid protein precursor of Alzheimer's disease is a mediator of the effects of nerve growth factor on neurite outgrowth. Neuron. 1992 Jul;9(1):129-37. PubMed.

    Small DH, Nurcombe V, Reed G, Clarris H, Moir R, Beyreuther K, Masters CL. A heparin-binding domain in the amyloid protein precursor of Alzheimer's disease is involved in the regulation of neurite outgrowth. J Neurosci. 1994 Apr;14(4):2117-27. PubMed.

    Williamson TG, Nurcombe V, Beyreuther K, Masters CL, Small DH. Affinity purification of proteoglycans that bind to the amyloid protein precursor of Alzheimer's disease. J Neurochem. 1995 Nov;65(5):2201-8. PubMed.

    Clarris HJ, Key B, Beyreuther K, Masters CL, Small DH. Expression of the amyloid protein precursor of Alzheimer's disease in the developing rat olfactory system. Brain Res Dev Brain Res. 1995 Aug 28;88(1):87-95. PubMed.

    View all comments by David Small

  2. Work from several labs, including our own, has shown functions for APP in the developing nervous system, including roles in migration, neurite outgrowth, and synaptogenesis. Each of these functions implicates APP in mediating differential adhesion to extracellular factors. Our lab has shown that full-length APP acts through DAB1 to affect neuronal precursor cell migration into the cortical plate (Young-Pearse et al., 2007). In addition, we have addressed the mechanism by which APP affects neurite outgrowth by showing that APP and integrin β1 biochemically and functionally interact in embryonic rodent neurons (Young-Pearse et al., 2008). However, the extracellular factor(s) that APP is acting with to execute these functions previously had not been elucidated. This recent work by Hoe et al. begins to answer this key question by showing that Reelin interacts with APP to regulate neurite outgrowth. These data nicely complement the authors' previous studies showing an interaction between APP/ApoER2 and F-spondin (Hoe et al., 2005), another secreted factor expressed in the developing brain initially identified by the Südhof lab to biochemically interact with APP (Ho and Südhof, 2004). Together, these studies support a (speculative) model whereby the function of APP is determined by both its extracellular binding partner as well as by its dimerization state with itself or other type-I transmembrane domain proteins (ApoER2, ITGβ1, etc.).

    In addition to aiding in elucidating the mechanism by which APP functions during development, the authors also provide data that could suggest that the APP-Reelin interaction has some role in AD pathogenesis. The authors show that exogenously applied Reelin increases α-secretase cleavage while decreasing β-secretase cleavage and Aβ generation. In addition, the authors point out that Reelin protein and mRNA levels are decreased in AD brains. Based upon these data, the speculation would be that a loss of Reelin in aged brains would lead to an increase in Aβ production, and that this would lead to pathology. To address this possibility, it would be critical to examine whether a loss of endogenous Reelin (in knockout animals) significantly increases Aβ generation and plaque formation, and/or enhances the behavioral defects observed in APP transgenic animals.

    References:

    Ho A, Südhof TC. Binding of F-spondin to amyloid-beta precursor protein: a candidate amyloid-beta precursor protein ligand that modulates amyloid-beta precursor protein cleavage. Proc Natl Acad Sci U S A. 2004 Feb 24;101(8):2548-53. PubMed.

    Hoe HS, Wessner D, Beffert U, Becker AG, Matsuoka Y, Rebeck GW. F-spondin interaction with the apolipoprotein E receptor ApoEr2 affects processing of amyloid precursor protein. Mol Cell Biol. 2005 Nov;25(21):9259-68. PubMed.

    Young-Pearse TL, Chen AC, Chang R, Marquez C, Selkoe DJ. Secreted APP regulates the function of full-length APP in neurite outgrowth through interaction with integrin beta1. Neural Dev. 2008;3:15. PubMed.

    Young-Pearse TL, Bai J, Chang R, Zheng JB, LoTurco JJ, Selkoe DJ. A critical function for beta-amyloid precursor protein in neuronal migration revealed by in utero RNA interference. J Neurosci. 2007 Dec 26;27(52):14459-69. PubMed.

    View all comments by Tracy Young-Pearse

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References

News Citations

  1. AβPP Ligand Found? Neurons Respond to F-Spondin

Paper Citations

  1. Zheng H, Jiang M, Trumbauer ME, Sirinathsinghji DJ, Hopkins R, Smith DW, Heavens RP, Dawson GR, Boyce S, Conner MW, Stevens KA, Slunt HH, Sisoda SS, Chen HY, Van der Ploeg LH. beta-Amyloid precursor protein-deficient mice show reactive gliosis and decreased locomotor activity. Cell. 1995 May 19;81(4):525-31. PubMed.
  2. Hoe HS, Rebeck GW. Functional interactions of APP with the apoE receptor family. J Neurochem. 2008 Sep;106(6):2263-71. PubMed.
  3. Hoareau C, Borrell V, Soriano E, Krebs MO, Prochiantz A, Allinquant B. Amyloid precursor protein cytoplasmic domain antagonizes reelin neurite outgrowth inhibition of hippocampal neurons. Neurobiol Aging. 2008 Apr;29(4):542-53. PubMed.
  4. Hoe HS, Tran TS, Matsuoka Y, Howell BW, Rebeck GW. DAB1 and Reelin effects on amyloid precursor protein and ApoE receptor 2 trafficking and processing. J Biol Chem. 2006 Nov 17;281(46):35176-85. PubMed.
  5. Qiu WQ, Ferreira A, Miller C, Koo EH, Selkoe DJ. Cell-surface beta-amyloid precursor protein stimulates neurite outgrowth of hippocampal neurons in an isoform-dependent manner. J Neurosci. 1995 Mar;15(3 Pt 2):2157-67. PubMed.
  6. Niu S, Renfro A, Quattrocchi CC, Sheldon M, D'Arcangelo G. Reelin promotes hippocampal dendrite development through the VLDLR/ApoER2-Dab1 pathway. Neuron. 2004 Jan 8;41(1):71-84. PubMed.
  7. Yamazaki T, Koo EH, Selkoe DJ. Cell surface amyloid beta-protein precursor colocalizes with beta 1 integrins at substrate contact sites in neural cells. J Neurosci. 1997 Feb 1;17(3):1004-10. PubMed.
  8. Young-Pearse TL, Chen AC, Chang R, Marquez C, Selkoe DJ. Secreted APP regulates the function of full-length APP in neurite outgrowth through interaction with integrin beta1. Neural Dev. 2008;3:15. PubMed.

Further Reading

Primary Papers

  1. Hoe HS, Lee KJ, Carney RS, Lee J, Markova A, Lee JY, Howell BW, Hyman BT, Pak DT, Bu G, Rebeck GW. Interaction of reelin with amyloid precursor protein promotes neurite outgrowth. J Neurosci. 2009 Jun 10;29(23):7459-73. PubMed.

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