As drug discovery in AD research is shifting from inhibiting the γ-secretase to inhibiting the β-secretase (BACE), interest in the entire life cycle of BACE has grown, concomitantly. In the current online Early Edition of PNAS, Vincent Mauro, George Rogers, and Gerald Edelman at the Scripps Research Institute in La Jolla, California, shine a light on structural features of the BACE1 mRNA that might help explain why levels of the enzyme increase in Alzheimer's disease.
Most evidence to date suggests that the increases in BACE seen in the brains of AD patients arise neither from genetic mutations or polymorphisms, nor from transcriptional changes. (However, see ARF concurrent news story on Li et al., who report increased BACE mRNA levels in AD brain.) Researchers have thus begun to hunt for mechanisms that could increase translation of the protein from its mRNA, or that extend the lifespan or activity of the protein.
Mauro and colleagues have helped to open this new chapter in AD research by focusing on the leader region of the BACE mRNA. For those who haven't cracked their molecular biology texts for a few years, the leader sequence follows the promoter and operator sequences at the 5' end (i.e., the beginning) of the mRNA. By definition, the leader ends at the start codon (AUG), which signals the translational machinery that the next open reading frame (ORF) contains the code for the actual protein. A potential problem is the presence of any upstream AUGs in the leader sequence; the BACE1 mRNA, for example, contains four of the imposter AUGs (though only three ORFS, because one of the AUGs is immediately followed by a stop codon).
Theoretically, these uAUGs have the capacity to trick the ribosome and its associated cogs and gears into starting translation ahead of the actual gene, and such a false start can block translation. (This need not always be a bad thing, as the current study will demonstrate.) However, the translational engine has its own tricks to avoid false starts. Rogers et al. set out to discover what might be at work here to allow increased production of BACE in Alzheimer's disease.
When the researchers transfected the BACE1 leader—in a construct with a reporter gene—into two different cell types (rat B104 neuroblastoma and rat PC12), they found that the gene was relatively efficiently translated, though there were clear differences in the amounts translated in the two cell types. They also demonstrated that the translation was 5' cap-dependent; this indicates that the translation machinery is not assembled at an internal ribosome entry site (IRES), one of the tricks that the translation engine can use to avoid uAUGs. Deleting or mutating some or all of the uAUGs also led to differential changes in translation between the two cell types. These changes were small (two- to fourfold), but they reinforced the notion that certain cellular conditions enable these uAUGs to inhibit BACE1 translation.
Another trick that ribosomes can use to avoid getting snagged on uAUGs is "leaky scanning," whereby the ribosome surveys the nucleotides immediately before and after the AUG, and bypasses sites that look troublesome. The authors found that this was unlikely in the case of BACE, as all four uAUGs were found individually to be efficient initiators of translation when attached to a gene other than BACE1.
How, then, does BACE ever get translated if these uAUGs can hijack the translational engine before it ever reaches the BACE1 AUG? The authors argue that the most likely way is by "shunting," wherein ribosomes simply jump over large stretches of the mRNA. Shunting often occurs in areas where the molecule makes hairpin turns or loops, and could be at work in AD, the scientists propose. "For example, the translation of a uORF might to some extent inhibit BACE1 translation in the normal brain, whereas during Alzheimer’s disease, translation might increase because of a shunting mechanism that enables ribosomes to bypass the upstream AUGs," they write.
One possible mechanism for this shift is that the relative accessibility of the uORFs and the BACE ORF may change in AD, or even during aging, as a function of changes in the three-dimensional structure of the BACE1 mRNA. This hypothesis could be tested in neurons cultured from brain tissue removed from AD patients during surgery for epilepsy, the authors suggest.—Hakon Heimer
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