A role for SC35 and hnRNPA1 in the determination of amyloid precursor protein isoforms

Abstract

The beta-amyloid peptide (Abeta) that accumulates in senile plaques in Alzheimer's disease is formed by cleavage of the amyloid precursor protein (APP). The APP gene has several intronic Alu elements inserted in either the sense or antisense orientation. In this study, we demonstrate that binding of SC35 and hnRNPA1 to Alu elements on either side of exon 7 in the transcribed pre-mRNA is involved in alternative splicing of APP exons 7 and 8. Neuronal cells transfected with the full-length form of APP secrete higher levels of Abeta than cells transfected with the APP695 isoform lacking exons 7 and 8. Finally, we show that treatment of neuronal cells with estradiol results in increased expression of APP695, SC35 and hnRNPA1, and lowers the level of secreted Abeta. An understanding of the regulation of splicing of APP may lead to the identification of new targets for treating Alzheimer's disease.

Figure 1

Schematic representation of part of the APP gene. (a) Exons 6 to 9 (black boxes) are shown in relation to introns containing Alu elements. There are fourteen Alu elements in intron 6 designated by arrows. Eight of them are in the sense (S) and six are in antisense (A) orientation. Intron 7 contains a single Alu sequence in the antisense orientation and intron 8 has four Alu sequences. (b) An alignment of the 35bp hnRNPA1 binding motif (hnRNPA1B) with Alu elements present in introns 6, 7 and 8 of the APP gene. Matches to hnRNPA1B are underlined (italicized for the degenerative code). The B-box consensus of the promoter for polymerase III is also shown with the invariant bases underlined. The four HREs are superimposed. (c) Position of the SC35 binding motif within the Alu element from intron 7, inserted in antisense orientation. The 35bp hnRNPA1 binding sequence in dsDNA is underlined.

Figure 2

EMSA analysis of hnRNP-A1 binding to RNA. RNA probes 1 (transcribed from the 35bp hnRNPA1 binding motif in dsDNA), 2 (transcribed from the same template as probe 1 but with deleted B-box from DNA template) and 3 (transcribed from template with deleted HREs) labelled with ³²P (lanes a), probes incubated with 100, 200 or 300ng hnRNP-A1 in the presence of 0.1 μg/μl poly-d(IC) (lanes b, d, f respectively), probes incubated in the presence of 0.1 μg/μl poly-d(IC) and 1 μg/μl sonicated salmon sperm DNA (lanes c, e, g respectively) were separated in a polyacrylamide gel, and detected by autoradiography. A representative image from three experiments is shown.

Figure 3

Deletion of the SC35 and hnRNPA1 binding motifs from the APP pre-mRNA suppresses alternative skipping of exons 7 and 8. (a) Diagram of the APP minigene containing exons 7 and 8 with the designated SC35 binding site in pre-mRNA (SC35bs, striped box). Alu elements in sense (S) and antisense (A) orientation in introns 6 and 8 of the minigene are marked. Black boxes show segments of the pcDNA/HisMax-TOPO vector including the T7 promoter (left box) and the BGH polyadenylation sequence (right box). RT-PCR experiments using Xpress forward and BGH reverse primers (black arrows) reveal four products as shown, one containing both exons 7 and 8, one containing only exon 7, one containing only exon 8, and one containing neither exons 7 nor 8. Similar minigenes as in a were prepared but with either the inversed SC35 binding site (b) or Alu elements: inversed sense Alu in intron 6 (c), sense Alu in intron 8 (d), or antisense Alu in intron 8 (e). RT-PCR experiments using Xpress forward and BGH reverse primers reveal one fragment only in b, containing both exons 7 and 8, while in c and d a minor product with skipped exon 8 was detected as well. Sequences of all RT-PCR products were confirmed by sequencing. Representative images of at least three experiments are shown.

Figure 4

Role of hnRNPA1 and SC35 in splicing of the APP pre-mRNA in living cells. (a) Either siRNA against hnRNPA1 mRNA (A1 siRNA), against SC35, or a control siRNA (ctr siRNA) were cotransfected in HEK293 with the minigene encoding the SC35 binding motif. HnRNPA1 and SC35 expression was analyzed by Western blot 48h after transfection. (b) Cytoplasmic RNA was isolated from cells described in a and analyzed by RT-PCR and sequencing for splice variants of the pre-mRNA transcribed from the APP minigene. (c) Knockdown efficiency for hnRNPA1 and SC35 in the above experiments, quantified as a ratio of expression of the proteins in cells transfected with either SC35 or hnRNPA1 specific siRNA (white bars) and with control siRNA (black bars). Expression in the control cells is set as 100%. Values are shown as means ±SD for two independent experiments, each analyzed in duplicate. Representative images of three experiments are given in (a) and (b).

Figure 5

Cotransfection of HEK293 Tet On cells with APP770, APP751 and APP695. (a) Western blot of equal amounts of soluble subcellular proteins fractionated either from non-transfected cells or from cells co-transfected with the three protein isoforms, with and without doxycyclin (dox) induction, detected with anti-APP, N-terminal antibody. (b) Western analysis for the secreted Aβ peptide by HEK293 Tet On cells, under the same conditions as described in a. (c) Quantification of secreted Aβ. Values are mean ±SD of three independent experiments (*p < 0.01, **p < 0.001). The amount of Aβ secreted by non-transfected cells was set as 1.

Figure 6

Transfection of NT2N neuronal cells with either APP770 or APP695. (a) Western blot of soluble subcellular proteins (equal amounts) fractionated from non-transfected cells or from cells transfected with either APP770 or APP695, with anti-APP, N-terminal antibody. (b) Western analysis for the secreted by NT2N Aβ peptide under the same conditions as described in a. (c) Quantification of secreted Aβ. Data shown are mean values ±SD of three independent experiments (*p < 0.01, **p < 0.001). The amount of Aβ secreted by non-transfected cells was set as 1.

Figure 7

Treatment of neuronal cells by E₂ upregulates expression of SC35 and hnRNPA1 and reduces secretion of Aβ. Western analysis for soluble subcellular proteins (equal amounts) fractionated from NT2N cells and NT2N treated with 10nM of E₂ for 96 hours, using anti-APP, N-terminal antibody (a), anti-Aβ (b), or antibodies against SC35 and hnRNPA1 (c). Expression of β-actin was detected as a control. Representative images are shown of at least three experiments.