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APP Binds to the EGFR Ligands HB-EGF and EGF, Acting Synergistically with EGF to Promote ERK Signaling and Neuritogenesis

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Abstract

The amyloid precursor protein (APP) is a transmembrane glycoprotein central to Alzheimer’s disease (AD) with functions in brain development and plasticity, including in neurogenesis and neurite outgrowth. Epidermal growth factor (EGF) and heparin-binding EGF-like growth factor (HB-EGF) are well-described neurotrophic and neuromodulator EGFR ligands, both implicated in neurological disorders, including AD. Pro-HB-EGF arose as a putative novel APP interactor in a human brain cDNA library yeast two-hybrid screen. Based on their structural and functional similarities, we first aimed to verify if APP could bind to (HB-)EGF proforms. Here, we show that APP interacts with these two EGFR ligands, and further characterized the effects of APP-EGF interaction in ERK activation and neuritogenesis. Yeast co-transformation and co-immunoprecipitation assays confirmed APP interaction with HB-EGF. Co-immunoprecipitation also revealed that APP binds to cellular pro-EGF. Overexpression of HB-EGF in HeLa cells, or exposure of SH-SY5Y cells to EGF, both resulted in increased APP protein levels. EGF and APP were observed to synergistically activate the ERK pathway, crucial for neuronal differentiation. Immunofluorescence analysis of cellular neuritogenesis in APP overexpression and EGF exposure conditions confirmed a synergistic effect in promoting the number and the mean length of neurite-like processes. Synergistic ERK activation and neuritogenic effects were completely blocked by the EGFR inhibitor PD 168393, implying APP/EGF-induced activation of EGFR as part of the mechanism. This work shows novel APP protein interactors and provides a major insight into the APP/EGF-driven mechanisms underlying neurite outgrowth and neuronal differentiation, with potential relevance for AD and for adult neuroregeneration.

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Funding

This work was supported by Fundação para a Ciência e a Tecnologia (FCT), Centro 2020 and Portugal 2020, the COMPETE program, QREN, and the European Union (FEDER program), via funding for the ongoing GoBack project (PTDC/CVT-CVT/32261/2017), the finished PTDC/SAU-NMC/111980/2009 project and JR SFRH/BD/78507/2011 PhD grant, and the support to the IBiMED Research Unit strategic program (UID/BIM/04501/2013; UID/BIM/04501/2019) and the LiM Facility via the Portuguese Platform of BioImaging (PPBI-POCI-01-0145-FEDER-022122). The authors also acknowledge the Swiss National Science Foundation (SNF 31003A_166177 grant). Microphotographs were acquired in the LiM facility of iBiMED/UA, a member of the Portuguese Platform of BioImaging (PPBI; POCI-01-0145-FEDER-022122).

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Author notes

  1. Luísa Bastos

    Present address: Roche Sistemas de Diagnósticos, Lda, 2720-413, Amadora, Portugal

  2. Odete A. B. da Cruz e Silva and Sandra I. Vieira contributed equally to this work.

Authors and Affiliations

  1. Institute of Biomedicine (iBiMED), Department of Medical Sciences, University of Aveiro, Agra do Crasto, 3810-193, Aveiro, Portugal

    Joana F. da Rocha, Luísa Bastos, Sara C. Domingues, Ana R. Bento, Odete A. B. da Cruz e Silva & Sandra I. Vieira

  2. Institute for Regenerative Medicine (IREM), University of Zurich, Zurich, Switzerland

    Uwe Konietzko

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  1. Joana F. da Rocha
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Contributions

JR performed the EGF experiments, LB built the MycHB-EGF construct and performed the HB-EGF assays, SD performed the YTH screens and helped on the yeast co-transformation assays, and ARB performed the real-time PCR assays, under SIV supervision, with the help of OAB, and of UK (rodents’ brains IPs). JR, LB, ARB, and SIV analyzed and interpreted all data, and wrote the manuscript, which all authors have revised.

Corresponding author

Correspondence to Sandra I. Vieira.

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The authors declare that they have no conflict of interest.

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All authors have approved the final version of the manuscript and give their consent to be published.

Ethics Approval

The experiments including rodent brain lysates have followed the European legislation for animal experimentation, namely the 2010/63/EU and Council Directive 86/609/EEC. The experiments were approved and supervised by our Institutional Animal Care and Use Committee (IACUC): Comissão Responsável pela Experimentação e Bem-Estar Animal (CREBEA), and followed the 3R’s (Replacement, Reduction and Refinement) guidelines. The number of animals used and the suffering was minimized. The animals were housed under a controlled environment and their health status and well-being monitored daily. Under the EU guidelines, no specific ethics approval was required for this project since the rats were not manipulated and only euthanized by cervical stretching followed by decapitation, for brain removal.

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Online resource 1.

Subcellular distribution and co-localization of exogenous Myc-HB-EGF with endogenous HB-EGF. HeLa cells transiently overexpressing the Myc-HB-EGF cDNA were immunolabeled with the anti-Myc tag antibody (green) and with the anti-HB-EGF antibody (red). Bar = 10 μm (PNG 2682 kb)

High resolution image (TIFF 7683 kb)

Online resource 2.

Effects of EGF treatment and APP overexpression on SH-SY5Y cell proliferation and survival. a SH-SY5Y cells were either transfected (‘Tf’) with the GFP empty vector (‘V’) or APP695-GFP (‘APP’) cDNAs, and stimulated or not for 3 min with 100 ng/mL human recombinant EGF (‘V+EGF’ and ‘APP+EGF’) immediately before cell media change. Fresh media included the Click-iT® EdU, a nucleotide analog incorporated in proliferating cells, and cells were fixed processed for analysis after 12h. b Representative epifluorescence microphotographs of cells tested with the Click-iT® EdU Alexa Fluor® imaging kit. Edu is labeled with a red fluorescing dye, and stains proliferating cells. Blue fluorescing Hoechst 33342 was used to assed the total number of cells. Bar = 50 μm. c Percentage of cell proliferation was calculated by counting the number of EdU red fluorescing cells versus the total number of Hoechst blue fluorescing cells (scored in 20 fields of view). d Plot of the total number of Hoechst positive cells per well in each experimental condition (scored in 20 fields of view). n = 3. Statistically non-significant results by the one-way ANOVA. Data is presented as mean ± standard error of the mean. (PNG 5360 kb)

High resolution image (TIFF 15363 kb)

Online resource 3.

APP and EGF/EGFR interplay in SH-SY5Y neuronal-like differentiation. a APP and EGF combined treatment increases βIII-tubulin protein levels. SH-SY5Y cells, either transfected with the GFP empty vector (‘V’) or APP695-GFP (‘APP’) cDNAs, were stimulated or not for 3 min with 100 ng/mL of human recombinant EGF (‘V+EGF’ and ‘APP+EGF’) immediately before cell media change (16h before fixation/harvesting). When indicated, this EGF stimulation was performed under EGFR inhibition with 10 μM PD168393 (‘INIB’), added 1h before EGF. Cells were collected for analysis after 24h of cell transfection. Immunoblots of cell lysates were probed with the βIII-tubulin antibody, and βIII-tubulin protein levels were plotted as fold increases of the non-treated GFP-expressing cells (‘V’). n = 4-5. *p < 0.05, **p < 0.01 by one-way ANOVA followed by the Tukey’s multicomparison post-test. b EGFR inhibition reduces APP levels and APP-induced ERK activation, even in the absence of EGF. SH-SY5Y cells overexpressing APP-GFP were treated with 10 μM of the EGFR inhibitor PD168393 for 6 hours before transfection medium change, when the cells were incubated for more 16h with fresh culture medium. Immunoblots of cell lysates were probed with the APP C-terminal, phospho-ERK1/2 (‘pERK’), and total ERK 1/2 (‘tERK’) antibodies. Total APP full-length protein levels and the pERK/ERK ratio were plotted as fold increases of the respective non-treated APP-GFP control cells. n = 3. *p<0.05, **p<0.01 using the two-tailed unpaired t-test. Of note, in a and b, the lanes are from the same blots and were rearranged into the present order. Ponceau-S staining of total proteins bands was used as loading control for all the data, and data are presented as mean ± standard error of the mean (PNG 4166 kb)

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da Rocha, J.F., Bastos, L., Domingues, S.C. et al. APP Binds to the EGFR Ligands HB-EGF and EGF, Acting Synergistically with EGF to Promote ERK Signaling and Neuritogenesis. Mol Neurobiol 58, 668–688 (2021). https://doi.org/10.1007/s12035-020-02139-2

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