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Tau exon 10 splicing is altered by autosomal dominant mutations that cause frontotemporal dementia with parkinsonism chromosome 17-type and by unknown mechanisms in other related neurodegenerative disorders. Identifying cis- and trans-regulators of tau exon 10 splicing is therefore crucial for understanding disease mechanisms. We previously identified several splicing enhancers and silencers within exon 10 and intron 10. Here, we show that splicing factors SF2/ASF, Tra2beta, and a 50-kDa nuclear protein bind in vitro to the polypurine enhancer at the 5' end of exon 10. Disease splicing mutations N279K and Delta280K disrupt the enhancer and alter associations with these factors. N279K targets robustly bind Tra2beta compared with the normal enhancer, which may explain why N279K enhances exon 10 splicing in vivo. In contrast, factor associations with Delta280K targets are nearly undetectable, explaining why Delta280K almost abolishes exon 10 splicing in vivo. Small interfering RNA-mediated suppression of endogenous SF2/ASF and Tra2beta significantly reduces exon 10 splicing. Exogenous SF2/ASF dramatically enhances normal exon 10 splicing and efficiently rescues the Delta280K splicing defect. Domain deletion analyses show that the C-terminal RS domains of SF2/ASF and Tra2beta are required for normal exon 10 splicing in vivo. In contrast to Tra2beta, the SF2/ASF RS domain remains essential in the presence of a strengthened enhancer or when either weak splice site is strengthened. The data suggest that SF2/ASF has both essential and regulatory roles, whereas Tra2beta has a supporting role in exon 10 splicing.

Sequential proteolysis of the amyloid precursor protein (APP) by beta- and gamma-secretase activities yields the amyloid beta peptide that is widely deposited in the brains of individuals with Alzheimer's disease. The membrane-anchored aspartyl protease beta-site APP-cleaving enzyme (BACE) exhibits all of the characteristics of a beta-secretase and has been shown to cleave APP at its beta-site in vitro and in vivo. We found that BACE undergoes cleavage on a surface-exposed alpha-helix between amino acid residues Leu-228 and Ala-229, generating stable N- and C-terminal fragments that remain covalently associated via a disulfide bond. The efficiency of BACE endoproteolysis was observed to depend heavily on cell and tissue type. In contrast to brain where holoprotein was predominant, BACE was found primarily as endoproteolyzed fragments in pancreas, liver, and muscle. In addition, we observed a marked up-regulation of BACE endoproteolysis in C2 myoblasts upon differentiation into multinucleated myotubes, a well established model system of muscle tissue specification. As in liver, BACE exists as endoproteolyzed fragments in the hepatic cell line, HepG2. We found that HepG2 cells are capable of generating amyloid beta peptide, suggesting that endoproteolyzed BACE retains measurable beta-secretase activity. We also found that BACE endoproteolysis occurs only after export from the endoplasmic reticulum, is enhanced in the trans-Golgi network, and is sensitive to inhibitors of vesicular acidification. The membrane-bound proteases tumor necrosis factor alpha-converting enzyme and furin were not found to be responsible for this cleavage nor was BACE observed to mediate its own endoproteolysis by an autocatalytic mechanism. Thus, we characterize a specific processing event that may serve to regulate the enzymatic activity of BACE on a post-translational level.

tau mutations that deregulate alternative exon 10 (E10) splicing cause frontotemporal dementia with parkinsonism chromosome 17-type by several mechanisms. Previously we showed that E10 splicing involved exon splicing enhancer sequences at the 5' and 3' ends of E10, an exon splicing silencer, a weak 5' splice site, and an intron splicing silencer (ISS) within intron 10 (I10). Here, we identify additional regulatory sequences in I10 using both non-neuronal and neuronal cells. The ISS sequence extends from I10 nucleotides 11-18, which is sufficient to inhibit use of a weakened 5' splice site of a heterologous exon. Furthermore, ISS function is location-independent but requires proximity to a weak 5' splice site. Thus, the ISS functions as a linear sequence. A new cis-acting element, the intron splicing modulator (ISM), was identified immediately downstream of the ISS at I10 positions 19-26. The ISM and ISS form a bipartite regulatory element, within which the ISM functions when the ISS is present, mitigating E10 repression by the ISS. Additionally, the 3' splice site of E10 is weak and requires exon splicing enhancer elements for efficient E10 inclusion. Thus far, tau FTDP-17 splicing mutations affect six predicted cis-regulatory sequences.

Mutations in the tau gene are pathogenic causing autosomal dominant frontotemporal dementia with Parkinsonism-chromosome 17 type (FTDP-17). Some mutations in tau exon 10 (E10) and immediately adjacent sequences cause disease by altering E10 splicing. To determine the mechanism of normal E10 splicing regulation and how FTDP-17 mutations alter splicing, mutational analysis of E10 was performed. The results show that E10 contains a complex array of both enhancer and inhibitor cis-acting elements that modulate usage of a weak 5' splice site. The 5' end of E10 contains a previously unrecognized multipartite exon splicing enhancer (ESE) composed of an SC35-like binding sequence, a purine-rich sequence, and an AC-rich element. Downstream of this ESE is a purine-rich exon splicing inhibitor. Intronic sequences immediately downstream of E10 also are inhibitory. The results support an alternative model in which I10 inhibitory sequences appear to function as a linear sequence. The cis-elements described are not redundant, and all appear required for normal E10 splicing. Results with double mutations demonstrate that the ESE and the intronic inhibitory element collaborate to regulate splicing. Thus splicing of tau E10 is regulated by a complex set of cis-acting elements that span nearly the entire exon and also include intronic sequences.