Collin RW and Martens GJ (2006), The coding sequence of amyloid-beta precursor p...

XB-ART-428

Dev Biol 2006 May 04;10871:41-51. doi: 10.1016/j.brainres.2006.02.101.

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The coding sequence of amyloid-beta precursor protein APP contains a neural-specific promoter element.

Collin RW , Martens GJ .

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The amyloid-beta precursor protein APP is generally accepted to be involved in the pathology of Alzheimer's disease. Since its physiological role is still unclear, we decided to study the function of APP via stable transgenesis in the amphibian Xenopus laevis. However, the application of constructs encoding (mutant) APP fused to the C-terminus of the green fluorescent protein GFP (GFP-APP), and harboring a tissue-specific or an inducible gene promoter did not result in transgene expression of APP in neuronal and neuroendocrine cells. Surprisingly, a construct encoding either Xenopus or human APP fused to the N-terminus of GFP (APP-GFP) gave fluorescence throughout the whole brain of the tadpole, despite the fact that a proopiomelanocortin gene promoter was used to target transgene expression specifically to the intermediate pituitary cells. Detailed analysis with deletion mutants revealed the presence of a neural-specific, transcriptionally active DNA element within the 3'-end of the APP-coding sequence that gave rise to an aberrant transcript and protein in the APP-GFP transgenic animals. The DNA element appears to prevent proper APP transgene expression in Xenopus neuronal and neuroendocrine cells. Thus, the coding sequences of Xenopus and human APP contain a neural-specific promoter element, the physiological significance of which is at present unclear.

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Species referenced: Xenopus laevis
Genes referenced: app pomc

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	Fig. 1 – Overview of the generated GFP-APP and APP-GFP fusion constructs. Overview of the constructs encoding N-terminal (A) or C-terminal (B) GFP-fusion proteins of wild-type and mutant APP. Arrows indicate the β-, α- or γ-secretase processing sites within APP. SP: signal peptide sequence; delC: mutant lacking the cytoplasmic tail of APP; ICAM TM/CT: mutant containing the transmembrane domain and cytoplasmic tail of the intracellular adhesion molecule. C99: mutant containing only the C-terminal part of APP remaining after β-secretase cleavage; AICD: mutant containing only the C-terminal part of APP remaining after γ-secretase cleavage. The transmembrane domain of APP is indicated by a dashed box.
	Fig. 2 – Tissue-specific targeting of GFP-APP fusion proteins in transgenic Xenopus. (A) Fluorescent images of tadpoles microinjected in the first-cell stage with the constructs depicted below the images. No fluorescence was observed using the neural tubulin gene promoter (pNtub: upper panel), while tail muscle fluorescence was found when the EF1α gene promoter (pEF1α: middle panel) was used and tail and jaw muscle fluorescence using the cardiac actin promoter (pCac: lower panel). (B) Western blot analysis of tail lysates of wild-type tadpoles and tadpoles transgenic for GFP-APP and expressing the fusion protein in muscle cells. Proteins were resolved on a 10% SDS-PAGE gel and the anti-APP antibody C87 was used. Molecular weight markers (Mr) are indicated on the right. SP: signal peptide sequence; TM: transmembrane domain; pA: poly-adenylation signal.
	Fig. 3 – Inducible expression of GFP-APP in transgenic Xenopus. Fluorescent images of tadpoles microinjected in the first-cell stage with the two constructs depicted on the left of the images. Gene expression was induced by incubating the animals in the presence of doxycyclin for 12 h. No fluorescence was observed after doxycyclin treatment using the construct encoding GFP-APP (upper right panel). Using the construct encoding GFP alone, fluorescence was observed in neuronal cells of the transgenic tadpoles after doxycyclin treatment (lower right panel). The transmembrane domain of APP is indicated by a dashed box. Tet: Tet activator protein; pTRE: promoter containing Tet-responsive elements. SP: signal peptide sequence; pA: poly-adenylation signal.
	Fig. 4 – Tissue-specific targeting of APP-GFP fusion proteins in transgenic Xenopus. Fluorescent images of tadpoles microinjected in the first cell-stage with the constructs depicted below the respective images. Fluorescence in the intermediate pituitary (IP) was observed when the AICD-GFP construct was targeted with the POMC gene promoter (pPOMC; upper panel), while fluorescence in neuronal cells was found when the pPOMC APP-GFP construct was used (middle panel). For comparison, GFP expression driven by the neural tubulin gene promoter (pNtub: lower panel) is shown. The transmembrane domain of APP is indicated by a dashed box. AICD: APP intracellular domain; SP: signal peptide sequence; pA: poly-adenylation signal.
	Fig. 5 – Identification of the region within Xenopus APP cDNA responsible for its aberrant promoter activity. (A) 5′-end deletion mutants of the APP-GFP construct were used in transgenesis and the generated animals were screened for fluorescence (fluo) in the brain (+: fluorescence, −: no fluorescence). The linear fragments depicted were produced via digestions with the indicated restriction enzymes. The region within APP cDNA encoding the transmembrane domain is indicated by a dashed box. pA: poly-adenylation signal. (B) Sequence analysis of the 5′-RACE PCR products to determine the start site of the aberrant transcript (indicated by an asterisk). The sequence of the nested adapter oligo used to perform the 5′-RACE PCR is underlined. (C) Western blot analysis of brain lysates of wild-type tadpoles (lane 1; background bands), tadpoles transgenic for 5′-deleted (microinjected with NaeI/PauI digested APP-GFP construct) ΔAPP-GFP (lane 2; presence of a 32-kDa product not present in lane 1) or animals expressing GFP alone (lane 3; 30-kDa GFP-product). Proteins were separated on a 12.5% SDS-PAGE gel and an anti-GFP antibody was used. Molecular weight markers (Mr) are indicated on the left.
	Fig. 6 – Comparative analysis of the region in the Xenopus and human APP cDNAs responsible for the aberrant promoter activity. Identical nucleotides are white on a black background. The transcription start site inXenopus APP cDNA is indicated by an asterisk and the direction of transcription by an arrow. The codon representing the start methionine is underlined. The N-terminal region of the aberrant APP-GFP protein fragment is presented below the alignment. The positions of exons 16, 17 and 18 of the APP gene are indicated by vertical lines.