Regulated intramembranous proteolysis, RIP for short, denotes an unusual way of cutting proteins lodged in a membrane right inside their membrane-spanning parts. This process of proteolysis, smack in the middle of a lipid bilayer, was considered extremely rare, with only one prior example in the cholesterol biosynthesis pathway, until recently, when the workings of γ-secretase became more clear. Now, RIP is receiving a large amount of attention in the Alzheimer’s field, as researchers bear down on the details of how this complex process occurs and is controlled by multiple proteins. The hope is that a deeper understanding of the regulatory events and players involved in APP proteolysis will yield additional drug targets to pursue should current efforts to develop direct γ-secretase inhibitors fail.

In a symposium on the topic, Li-Huei Tsai of Harvard Medical School presented a new aspect of APP proteolysis regulation. She said that one way in which APP proteolysis might be controlled inside the cell lies in phosphorylation of APP. She detailed a series of studies that found eight phosphorylation sites on the intracellular part of APP, which, once cleaved in the reaction that also frees Aβ, translocates to the nucleus and is thought to affect gene expression there.

Asking which phosphorylation event could influence Aβ production, Tsai’s team identified threonine 668, and found, with Cindy Lemere at Brigham and Women’s hospital, that it is selectively phosphorylated in the hippocampal neuropil of postmortem sections of human AD brain and APP-transgenic mice, but not various controls. Two structures carrying this phosphorylation stood out: dystrophic neurites and granular vesicles.

Since the same area also had robust neurofibrillary pathology, Tsai et al. looked for possible interactions and found that APP phosphorylated on threonine 668 occurs in those neurons that also carry hyperphosphorylated tau. Biochemical and mass spectrometry experiments indicated that as many as seven of the eight phosphorylation sites of the cytoplasmic APP tail may be phosphorylated in AD samples; some were hyperphosphorylated.

In cultured hippocampal neurons, phosphorylated APP did not co-localize strongly with overall APP, but phosphorylated APP and BACE1 both appeared to reside in the growth cones, and they were on endosomal vesicles, not in the lysosomal pathway, Tsai reported. With Rachael Neve at McLean Hospital in Belmont, Massachusetts, Tsai’s group conducted further experiments with rat primary neurons infected with various viral APP constructs to ask whether threonine 668 phosphorylation affects Aβ production, and indeed, preliminary experiments suggest it may do so.

Taken together, the data led Tsai to suggest a working hypothesis. As APP matures and moves through the secretory pathway of consecutive membrane compartments, hyperphosphorylation of its cytoplasmic domain might influence the decision point where APP will either undergo cleavage by α-secretase and become inserted into the cell membrane, or will be cleaved by BACE1 in endosomal vesicles, leading to Aβ generation. Tsai invited the audience to consider whether, perhaps, phosphorylation on threonine 668 somehow directs APP toward the endosomal compartment, favoring cleavage by BACE.-Gabrielle Strobel.

Addendum, added 12 November 2002.
A look in the literature revealed that the above news story, written on-site right after the presentation, omitted mention of previous literature about APP phosphorylation. In fact, Tsai had pointed out that New York researchers including Paul Greengard, Joseph Buxbaum, Sam Gandy, and others have studied this question for the past 12 years, and other labs are pursuing it, as well.—Gabrielle Strobel

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