. Presenilin/gamma-secretase-dependent processing of beta-amyloid precursor protein regulates EGF receptor expression. Proc Natl Acad Sci U S A. 2007 Jun 19;104(25):10613-8. PubMed.

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  1. This interesting work by Zhang and colleagues contributes to the current debate in the field as to whether regulated intramembrane proteolysis of APP, like that of Notch, results in the activation of a biologically relevant nuclear signaling pathway. Here, the authors provide evidence that EGFR, a type 1 transmembrane protein of the ErbB family, is a bona fide AICD target gene. This adds to the growing list of candidate AICD target genes (e.g., KAI1/CD82, neprilysin, APP, etc.). The result presented herein could explain, at least in part, the observed tumorigenesis in PS haplo-insufficient mice (note that this phenotype is not observed in APP/APLPs KO mice).

    Work performed by our group (1), and others (2-4), have raised concerns about technical issues and the physiological relevance related to AICD-mediating gene transcription regulation. As mentioned in this paper, future research should take into account the current knowledge and limitations associated with the study of AICD signaling.

    The authors present nice data showing that loss of PS in vivo leads to an upregulation of full-length EGFR. This effect could be rescued in immortalized PS dKO fibroblasts by stably transfecting human wild-type PS1 (one technical note: PS dKO MEFs cannot be derived from PS WT littermates, as stated in the text). Interestingly, steady-state EGFR levels are as well increased in APP/APLP2 dKO fibroblasts; this effect is fully rescued with full-length APP and partially rescued with a membrane-bound form of AICD (C57).

    Hence, we can likely exclude a role for the brain-specific APLP1 family member in EGFR expression regulation. A (weak) effect upon AICD overexpression is observed in luciferase assays under the control of an isolated EGFR promoter region. Physiological support for AICD-mediated signaling comes from ChIP experiments performed with wild-type (C57/BL) and APP/APLP2 dKO mice. Whether Fe65 and/or Tip60 binds to the EGFR promoter in the dKO mice is unfortunately not shown. To my knowledge, these are the first data showing ChIP experiments performed at the endogenous level using an APP antibody.

    Overall, this paper brings new evidence for a role for AICD in mediating gene transcription regulation under physiological conditions. Of course, independent confirmation and much more work are required to understand the physiological relevance of AICD/EGFR signaling.

    References:

    . Regulated intramembrane proteolysis of amyloid precursor protein and regulation of expression of putative target genes. EMBO Rep. 2006 Jul;7(7):739-45. PubMed.

    . A dominant role for FE65 (APBB1) in nuclear signaling. J Biol Chem. 2006 Feb 17;281(7):4207-14. PubMed.

    . A {gamma}-secretase-independent mechanism of signal transduction by the amyloid precursor protein. J Biol Chem. 2005 Nov 4;280(44):36895-904. PubMed.

    . Response to: Pardossi-Piquard et al., "Presenilin-Dependent Transcriptional Control of the Abeta-Degrading Enzyme Neprilysin by Intracellular Domains of betaAPP and APLP." Neuron 46, 541-554. Neuron. 2007 Feb 15;53(4):479-83. PubMed.

  2. The β amyloid precursor protein (βAPP) undergoes several physiopathological cleavages among which the one generated by γ-secretase liberates the C-terminal end of the Aβ peptides, thereby conditioning its 40- or 42-amino acid-long species. An additional ε cleavage on βAPP located 8-9 residues downstream of the Aβ40 cleavage site had been reported, which added complexity to the overall scheme of βAPP proteolysis. The putative physiological significance of this C-terminal βAPP fragment (first named AID for βAPP intracellular domain [1]) was questionable. Was AID a byproduct of γ-secretase action devoted to be rapidly eliminated (as its high cellular lability could suggest), or could it be a βAPP derivative responsible for unknown intracellular signaling? D’Adamio and his group were the first to suggest that the cytosolic release of AID could be of physiological importance. Thus, this fragment was shown to participate in the control of cell death (1).

    This initial observation was the starting point of a series of studies aimed at understanding AID function. Based on both empirical and experimental considerations, it was rapidly suggested that AID could behave as a transcription factor. First, it was noteworthy that the ε cleavage occurring on βAPP resembled the one occurring on Notch. This cleavage liberates a Notch Intracellular Domain (NICD) which has been unequivocally characterized as a transcription factor (2). Based on this analogy, the AID fragment was renamed AICD for APP Intracellular Domain. This first clue of a putative role of AID/AICD in the transcriptional control of yet unknown targets was more objectively supported by the fact that numerous independent studies (too many to be listed here) all led to the description of AID-like immunoreactivity in the nucleus and that this could be potentiated by stabilization of AID by the intracellular adaptor protein Fe65. Subsequently, several independent laboratories identified transcriptional targets of AICD including KAI1, βAPP, GSK3β, neprilysin, p53, and BACE.

    The paper by Zhang in the laboratory of Huaxi Xu is an excellent study evidencing another AICD transcriptional target. Both cellular (APP- or PS1/PS2-deficient fibroblasts) and in vivo models led to the conclusion that altering endogenous levels of AICD affects the expression of the EGF receptor. First, the article indeed shows the influence of the pharmacological (γ-secretase inhibitors) or genetic manipulation on AICD levels. This is crucial as AICD is labile and several controversies due to unreproduced data could have come from the fact that AICD expression was too low to trigger an associated phenotype.

    The second major point of the article is the observation that the phenotype occurs in vivo, in agreement with studies showing that GSK3β and neprilysin levels were indeed increased in AICD/Fe65 double transgenic mice (3,4). Finally, of most importance, this study reports for the first time CHIP experiments in which in vivo physiological levels of AICD physically interact with EGFR promoter.

    It is interesting to note that AICD represses EGFR promoter transactivation. It is to my knowledge the first target that is downregulated by AICD, ruling out the hypothesis of an aspecific stimulatory/enhancing effect that would have been related to AICD overexpression. Furthermore, it is worth noting that EGFR is the third target of AICD that could act as tumor suppressor, besides p53 and KAI1 (5,6). This is an important aspect that correlates well with the observation of skin tumors in mice altered for their presenilin expression. With this respect, the present study is certainly of utmost interest for understanding the putative link between AD pathology and cancer development and adds another level of concern for the design of safe PS-interacting inhibitors.

    References:

    . Generation of an apoptotic intracellular peptide by gamma-secretase cleavage of Alzheimer's amyloid beta protein precursor. J Alzheimers Dis. 2000 Nov;2(3-4):289-301. PubMed.

    . Notch: a membrane-bound transcription factor. J Cell Sci. 2002 Mar 15;115(Pt 6):1095-7. PubMed.

    . Response to correspondence: Pardossi-Piquard et al., "Presenilin-dependent transcriptional control of the Abeta-degrading enzyme neprilysin by intracellular domains of betAAPP and APLP." Neuron 46, 541-554. Neuron. 2007 Feb 15;53(4):483-6. PubMed.

    . Activation of GSK-3 and phosphorylation of CRMP2 in transgenic mice expressing APP intracellular domain. J Cell Biol. 2005 Oct 24;171(2):327-35. PubMed.

    . Presenilin-dependent gamma-secretase-mediated control of p53-associated cell death in Alzheimer's disease. J Neurosci. 2006 Jun 7;26(23):6377-85. PubMed.

    . Exchange of N-CoR corepressor and Tip60 coactivator complexes links gene expression by NF-kappaB and beta-amyloid precursor protein. Cell. 2002 Jul 12;110(1):55-67. PubMed.

  3. Whether or not AICD plays a transcriptional regulatory role has been much debated since the initial studies on AICD were published in 2001 (1,2). Several studies identifying AICD gene targets in tissue culture cells (3,4) have appeared since then amidst the reports that failed to see AICD playing a role in gene transcription (see comments by Sebastian Hebert). This important paper from Huaxi Xu's group shows that EGFR is an AICD gene target. They provide multiple lines of evidence to support their conclusion and, more importantly, confirm AICD's association with EGFR promoter by chromatin immunoprecipitation under physiological conditions. Obviously, the debate about the transcriptional role of AICD is likely to linger on until more studies confirm these results. However, the ultimate verification of AICD's gene targets will have to come from in vivo studies using AICD transgenic mouse models (5).

    Yet there remain two important issues that escaped attention in this paper. First, how does EGFR downregulation by AICD relate to APP function? APP and AICD levels are shown to alter during neuronal differentiation (6). Does EGFR play a role in this process? Secondly, what are the implications of these findings in Alzheimer disease? Zhang et al. chose, perhaps wisely, not to comment on these aspects, but a complete understanding of APP biology will require satisfactory answers to these questions. In any case, the skeptics will now find it more difficult to shrug off the importance of AICD in APP function.

    References:

    . A transcriptionally [correction of transcriptively] active complex of APP with Fe65 and histone acetyltransferase Tip60. Science. 2001 Jul 6;293(5527):115-20. PubMed.

    . The gamma -secretase-cleaved C-terminal fragment of amyloid precursor protein mediates signaling to the nucleus. Proc Natl Acad Sci U S A. 2001 Dec 18;98(26):14979-84. PubMed.

    . The APP intracellular domain forms nuclear multiprotein complexes and regulates the transcription of its own precursor. J Cell Sci. 2004 Sep 1;117(Pt 19):4435-48. PubMed.

    . Modulation of gene expression and cytoskeletal dynamics by the amyloid precursor protein intracellular domain (AICD). Mol Biol Cell. 2007 Jan;18(1):201-10. PubMed.

    . Activation of GSK-3 and phosphorylation of CRMP2 in transgenic mice expressing APP intracellular domain. J Cell Biol. 2005 Oct 24;171(2):327-35. PubMed.

    . Physiological regulation of the beta-amyloid precursor protein signaling domain by c-Jun N-terminal kinase JNK3 during neuronal differentiation. J Neurosci. 2005 Jun 8;25(23):5533-43. PubMed.

  4. The work by Zhang et al. provides compelling evidence that APP, through presenilin-dependent γ-secretase processing and AICD production, regulates EGFR expression. While other studies have supported a similar role of APP/AICD, and several putative targets have been identified, the authors showed here that AICD can bind to the EGFR promoter under physiological conditions. This is a novel and important finding.

    Whether the APP-EGFR pathway is solely responsible for the skin tumor phenotype seen in presenilin hypomorphic mice remains to be tested, but this study goes beyond skin and may have implications for a physiological function for APP in CNS through EGFR regulation. It is also tempting to speculate that deregulation of EGFR, or other APP-mediated signaling pathways, may contribute to AD pathogenesis.

    Although AD is unlikely a disease of APP gross loss of function, it is important to note that disease-causing mutations in APP (and presenilins) not only affect Aβ production, but probably also other APP processing products including AICD and, by extension, AICD-mediated signaling.