Alternative splicing of BACE1 pre-mRNA and its implications for Alzheimer disease

Proteolysis of the β-amyloid precursor protein (APP) to produce the amyloid β-peptide (Aβ) is implicated in the pathogenesis of Alzheimer disease (AD). Initial cleavage of APP is executed by β -site A PP c leaving e nzyme 1 (BACE1), and thus reducing BACE1 activity is an attractive therapeutic strategy. Mechanisms that regulate BACE1 mRNA have the potential to affect BACE1 protein and activity levels. BACE1 pre-mRNA undergoes alternative splicing, but the significance of these splicing events is unclear. This dissertation investigates regulation of BACE1 alternative splicing and examines its role in Aβ generation and AD pathogenesis. BACE1 alternative splicing was characterized by quantitatively measuring relative levels of known BACE1 transcripts, and a novel BACE1 transcript was identified and quantified. A subtle difference in BACE1 alternative splicing between brain and pancreas was detected, suggesting the cellular environment can affect BACE1 splicing. BACE1 protein isoforms resulting from pre-mRNA alternative splicing exhibited severely impaired activity, and increasing BACE1 alternative splicing reduced Aβ generation. Therefore, BACE1 alternative splicing can affect Aβ production, and targeting BACE1 splicing is a potential strategy for lowering BACE1 activity. To identify potential regulators of BACE1 alternative splicing, a BACE1 minigene was constructed. The sequence from exon 3 through exon 5 of BACE1 was required for the minigene to splice properly. Minigene splicing was validated by showing specific splicing inhibition upon splice site mutation. Furthermore, mutating the BACE1 minigene at a predicted enhancer sequence significantly altered minigene splicing. This observation provides evidence of a regulatory site involved in BACE1 alternative splicing, and indicates minor sequence changes can dramatically affect BACE1 splicing. To examine whether variations in BACE1 splicing are implicated in AD, BACE1 transcripts in AD and control brains were measured by real-time PCR. No significant changes in BACE1 splicing appeared to correlate with AD. Mutating the BACE1 minigene at sites corresponding to single nucleotide polymorphisms (SNPs) to the minor allele did not affect minigene splicing, suggesting these SNPs are not associated with changes in BACE1 splicing. Although BACE1 alternative splicing may not have a profound impact on AD pathogenesis, understanding the process and its regulation could lead to identification of new therapeutic targets.