Ras signal triggers β-Amyloid Precursor Protein (APP) expression

Abstract

It has recently been discovered that the Drosophila β-amyloid protein precursor like (Appl) gene, the ortholog of the human β-Amyloid Precursor Protein (APP) gene, is transcriptionally activated by receptor tyrosine kinase activity that involves Ras/MAPK signaling in vivo. This regulation is specifically controlled in photoreceptor neurons of the Drosophila retina. This suggests that some cases of Alzheimer disease, those which have been associated with high expression of the APP gene, may involve Ras signal transduction.

Drosophila has been a classic model for the discovery of genes that underlie development, behavior or physiology. The advantages of this model are multiple, but recent advances in genome-wide analysis are providing an enormous amount of information on genes that, once validated in flies, can be tested in vertebrates for conserved functions.

Several receptor tyrosine kinase (RTK) signaling transduction cascades that involve the small GTPase Ras are evolutionary conserved and have been shown to be crucial for the control of cell differentiation and proliferation in different tissues and organisms.¹ The function of RTK signals has been thoroughly studied in model tissues from several organisms, in particular in the Drosophila compound eye.² For example, Ras in the fly retina, which is decisive for the activation of genes involved in the differentiation of photoreceptor neurons, responds to two different transmembrane RTK: the epidermal growth factor receptor (Egfr) and Sevenless.

The retina consists of many hundreds of units called ommatidia that are arranged in an extremely regular pattern. Each ommatidium contains four cone cells that act as lenses, eight photoreceptor neurons R1-R8, six pigmented cells and a bristle. The specification of the eight photoreceptors occurs gradually in the eye imaginal disc and requires the exquisite interplay of signal transduction inputs. This results in the appropriate recruitment of cells from the undifferentiated pluripotential imaginal disc epithelium to give rise to the typical arrangement of differentiated ommatidia. The gradual recruitment of cells to form each ommatidium occurs via a network of signals and interactions between several precursors. Most photoreceptors require Egfr signaling for their specification.³ However, only the R7 photoreceptor requires additional signaling from Sevenless RTK for its specification.^4-6 Sevenless is expressed in many cells in the ommatidium but only the precursor of R7 can activate it through the action of the ligand bride of sevenless, which is expressed in the adjacent R8 precursor cell.⁷ This signal triggers high and sustained levels of Ras in the R7 precursor cell, which ends with the differentiation of the R7 neuron.⁸ This interaction is precisely regulated to ensure that only one cell per ommatidium becomes an R7.^9,10

Thus, the high specificity of Sevenless and the exclusive requirement of the signal for R7 differentiation is a scenario within which to study the transcriptional response triggered by Ras. Activated Ras in R7 triggers Raf serine-threonine kinase which phosphorylates mitogen-activated protein kinase (MAPK).¹¹ In Drosophila, few transcriptional regulators can be phosphorylated by the MAPK, but they include the transcriptional activator pointed (pnt).^12-15 Upon phosphorylation by MAPK, the ETS domain protein encoded by the P2 transcript of pnt binds to the DNA.¹⁴

The R7 genetic program of differentiation has recently been used to study the transcriptional signature that responds specifically to Ras and that is driven by the Sevenless/Ras/MAPK/pnt signaling cascade.¹⁶ To this end, the transcriptome of eye imaginal discs carrying either gain- or loss-of-function of sevenless has been analyzed and compared with wild type.¹⁶ The first mutant condition reflects an excess of R7 cells and the second mutant condition a lack of R7 formation. The ultimate goal was to discover which genes respond to Ras in R7 neurons. Many of the genes recovered from the transcriptome contained ETS binding sites, and therefore are putative direct targets of the Sevenless/Ras/MAPK/pnt signaling.¹⁶ They include the β-amyloid precursor protein-like (Appl) gene, which is the ortholog of the human β-Amyloid Precursor Protein (APP) gene.¹⁷ In humans, APP is the precursor molecule that, upon proteolysis, generates β-amyloid peptide, which forms the amyloid plaques found in the brains of Alzheimer’s disease patients.¹⁸ Remarkably, different ligands of the membrane receptors with intrinsic tyrosine kinase activity modulate APP expression in mammalian cell cultures^19-22 and APP regulatory sequences respond to Ras/MAPK in mammalian PC12 cells.²³

In Drosophila, Appl is specifically expressed in developing neurons^24-26 and has been associated with axonal function.^27-32 Mora et al., (2012) describes two main phenotypes in photoreceptors for the Appl loss of function mutants: (1) R7 photoreceptor axonal targeting was disrupted and (2) UV light discrimination ability, which is the wavelength captured by R7 neurons, was altered. Abnormal light discrimination was strongly enhanced in individuals that were mutant for Appl and also heterozygous for loss of neurotactin function; this latter gene is involved in axon guidance.¹⁶ Thus, Appl cooperates with other guidance receptors for axonal targeting. It is worth mentioning that APP proteins are conserved, not only structurally but also functionally, as human APP rescues the phenotype of the Drosophila Appl loss-of-function mutant when ectopically activated.³³

Several pieces of evidence suggest that Ras acts directly upstream on Appl in vivo. In a gene expression analysis, Appl mRNA and protein have been localized in the eye photoreceptors at very early stages of specification.¹⁶ Ectopic activation of the constitutively activated form of Ras^V12 resulted in additional Appl expression.¹⁶ Similarly, inhibition of Ras, resulted in the blocking of Appl expression.¹⁶ Moreover, Appl contains the consensus ETS binding sites and when pnt was removed from clones, their neuronal precursors lacked Appl.¹⁶ Thus, it can be concluded that Pnt mediates Ras1/MAPK activation of Appl which is consistent with putative direct regulation of Appl through Pnt binding to specific ETS regulatory sequences. Interestingly, enhancer-lacZ reporters from two ETS sites found in regulatory regions of the Appl gene were activated after induction of Ras^V12.¹⁶ Moreover, chromatin immunoprecipitation (ChIP)-PCR assays of eye imaginal discs using HA-tagged pnt under the UAS inducible promoter showed enrichment of one of the Appl ETS sites.¹⁶ These results demonstrate that Appl is a direct target of Ras/MAPK signaling through direct binding of Pnt-P2 to Appl ETS sites.

The role of RTK signaling in the regulation of the expression of the APP gene family has also been analyzed in vertebrates. It has been found that appb, a fish ortholog of Appl, is strongly expressed in the nervous system after constitutive expression of the fibroblast growth factor receptor 1 (fgfr1).¹⁶ This demonstrates an evolutionarily conserved mechanism of appb transcriptional regulation by RTK in neural tissues.

In conclusion, the RTK/Ras/MAPK cascade is a strong candidate as a transcriptional regulator of APP genes. However, the relation of this cascade with Alzheimer disease remains speculative. It is widely accepted that the pathogenic origin of Alzheimer disease is a neurotoxic environment created by β-amyloid accumulation in oligomer aggregates, which results from APP protein cleavage by the β- and γ-secretases.^34,35 However, in some cases of familial Alzheimer patients increased transcriptional activity of the APP gene has been reported.³⁶ Thus it is tempting to speculate that this increased APP transcription is RTK/Ras/MAPK-mediated, as found in Drosophila photoreceptors and zebrafish brain in vivo (Mora et al., 2013) and also in mammalian cell cultures.^20,23 Further in vivo mammalian studies are required to establish whether transcriptional misregulation of RTK/Ras causes increased APP, and whether that control can account for cases of Alzheimer disease.

Mora N, Almudi I, Alsina B, Corominas M, Serras F. β amyloid protein precursor-like (Appl) is a Ras1/MAPK-regulated gene required for axonal targeting in Drosophila photoreceptor neurons. J Cell Sci. 2013;126:53–9. doi: 10.1242/jcs.114785.

Disclosure of Potential Conflicts of Interest

No potential conflicts of interest were disclosed.