Translational regulation of BACE-1 expression in neuronal and non-neuronal cells

Abstract

As the main beta-secretase of the central nervous system, BACE-1 is a key protein in the pathogenesis of Alzheimer's disease. Excessive expression of the protein might cause an overproduction of the neurotoxic beta-amyloid peptide. Therefore, a tight regulation of BACE-1 expression is expected in vivo. In addition to a possible transcriptional control, the BACE-1 transcript leader contains features that might constitute mechanisms of translational regulation of protein expression. Moreover, recent work has revealed an increase of BACE-1 protein and beta-secretase activity in some Alzheimer's disease patients, although a corresponding increase of transcript has not been reported. Here we show that BACE-1 translation could be modulated at multiple stages. The presence of several upstream ATGs strongly reduces the translation of the main open reading frame. This inhibition could be overcome with conditions that favour skipping of upstream ATGs. We also report an alternative splicing of the BACE-1 transcript leader that reduces the number of upstream ATGs. Finally, we show that translation driven by the BACE-1 transcript leader is increased in activated astrocytes independently of the splicing event, indicating yet another mechanism of translational control. Our findings might explain why increases in BACE-1 protein or activity are reported in the brain of Alzheimer's disease patients even in the absence of changes in transcript levels.

Figure 1

Scheme of the proposed alternative splicing in BACE-1 transcript leader. (A) Alignment of full-length BACE-1 transcript leaders from human (Hs), pig (Ss), rat (Rn) and mouse (Mm) cDNAs. Arrowheads mark proposed donor–acceptor splice sites for the new variant of the BACE-1 transcript leader. Conserved nucleotides are highlighted in grey. Upstream ATGs are written in bold and boxed sequences represent the putative uORFs. (B) Scheme of the alternative splicing within the first exon of human BACE-1. Dotted line and arrows delimit the sequence missing in the shorter variant. Arrowheads highlight the upstream ATGs, while the black and the striped boxes, the uORF and the first codons of the main ORF, respectively.

Figure 2

RNase protection assay for the BACE-1 transcript leader. (A) Scheme for the synthesis of the riboprobe for BACE-1. The plasmid pGEMD-BACE1-5′x with the full-length BACE-1 transcript leader was linearized with SpeI and used as template for the synthesis of the ³²P-labelled antisense riboprobe performed with the T7 RNA polymerase (probe length 484 nt). (B) Scheme of RNase protection assay. The protected fragments deriving from the full-length and the short BACE-1 leaders are 435 and 240 nt, respectively. The short variant produces an additional fragment of 26 nt that remains undetectable. (C) Result of RNase protection. 15 µg of total RNA from either human exocrine pancreas (Pc) or brain (Br) were incubated with 55 000 c.p.m. of ³²P-labelled riboprobe. The longer fragment of 435 nt is visible below the undigested probe, while the shorter fragment of 240 nt is present in the sample from pancreas only. Size markers of 316 and 240 nt were prepared as described in Materials and Methods.

Figure 3

Translation driven by the BACE-1 transcript leaders. (A) Scheme of bi-monocistronic constructs. The plasmid pBRm2L-Nco drives expression of the two reporter genes, Rluc and Fluc, under the control of separate T7 promoters and in an optimal context for translation initiation. The long and short BACE-1 transcript leaders were cloned in front of the Fluc ORF to generate pBRm2L-B1x and pBRm2L-B1y, respectively. (B) Transient transfection of pBRm2L-B1x or pBRm2L-B1y in SK-N-BE, U-373-MG, neurons, astrocytes and HeLa cells. To account for differences in transient transfection efficiencies, the activity (relative luminescence units, RLU) of Fluc was normalized to that of Rluc (Fluc/Rluc). The corresponding RLU ratio values for the empty vector pBRm2L-Nco are also shown for comparison. All the results are the mean of at least three independent experiments. Measurements of luciferase activity were performed in triplicate as described in Materials and Methods. The inset shows an RT–PCR experiment (with two different total RNA loadings) to evaluate the relative amount of Fluc transcripts in transfected HeLa cells. The efficiency of transfection was monitored by Rluc activity and proved to be comparable in all samples. (C) Cytoplasmic distribution of Fluc mRNA by sucrose gradients. Cytoplasmic extracts of transfected HeLa cells were lysed and resolved on 7–50% w/v sucrose gradients. Fractions were analysed by RT–PCR to reveal the localization of the transcript deriving from the transfected DNA (pBRm2L-Nco, pBRm2L-B1x or pBRm2L-B1y). The trace shows the continuous absorbance profile monitored during the collection of the fractions. Fractions 1–3 contain mainly mRNPs and the ribosomal subunits; the monosome region peaks at fraction 4; fractions 5–10 contain the polyribosomes.

Figure 4

In vitro translation driven by the BACE-1 transcript leader. (A) Activity of Fluc translated under the control of the long (B1x) or the short (B1y) BACE-1 transcript leaders. The cRNA produced from the empty vector pZac-Luc+ gives the 100% value. (B) Decay of the corresponding cRNAs during the translation reactions. cRNA from pZac-Luc+ is the control and radioactive input is considered to be 100%.

Figure 5

Effect of upstream ATGs in BACE-1 transcript leader on translation efficiency. (A) Scheme of the long BACE-1 transcript leader in the bi-monocistronic plasmid. The four upstream ATGs are marked with arrowheads. The black box represents the upstream ORF. The upstream ATGs were mutated to TTG, generating pBRm2L-B1x-mut1, pBRm2L-B1x-mut2, pBRm2L-B1x-mut3. (B) Transient transfection of bi-monocistronic plasmids in SK-N-BE cells. Results are normalized to the value of the long BACE-1 leader (pBRm2L-B1x).

Figure 6

Translation driven by BACE-1 transcript leader in resting and activated astrocytes. Astrocytes were transfected in normal culture conditions (resting) or after activation with IL-1β and TNF-α (activated). Transfection was performed with bi-monocistronic plasmids containing either the long (pBRm2L-B1x) or short (pBRm2L-B1y) BACE-1 transcript leader. The increase in Fluc translation between resting and activated astrocytes is also reported (fold increase).