The amyloid precursor protein (APP) travels widely in neurons. After synthesis in the ER/Golgi, the protein makes its way to the cell surface and back, and out to axon terminals. Along the way, it meets up with the β- and γ-secretases and gets cleaved into a number of fragments, including Aβ and others with a plethora of proposed functions. Full-length APP has been suggested to be an adaptor protein that links vesicles to the kinesin motors that power microtubule-mediated transport out to axons (see ARF related news story), but a new study may be cause to revise that model. In a report out in the March 18 issue of Journal of Neuroscience, Zoia and Virgil Muresan at the University of Medicine and Dentistry of New Jersey in Newark, with Nicholas Varvel and Bruce Lamb at Case Western Reserve University in Cleveland, Ohio, look at the distribution of different APP epitopes in cultured neurons and in mouse brain in situ using specific antibodies. They conclude that cleavage of APP occurs for the most part before the protein reaches axons, and that the different fragments segregate into separate transport vesicles and are delivered to distinct destinations in the axon terminal. Only when APP is overexpressed in cultured neurons do many vesicles appear to contain much full-length APP. The results suggest that in normal conditions, APP cleavage occurs early and often, and that its fragments have their own functional roles in axons.
A second report on APP transit, from Huaxi Xu and colleagues at the Burnham Institute in La Jolla, California, looks at the role of APP in trafficking of the γ-secretase to the cell surface. The γ-secretase subunit presenilin-1 regulates APP trafficking, and the data, published online March 10 in the Journal of Biological Chemistry, suggest that the reciprocal is also true. Cells that lack APP show altered accumulation of presenilin-1 on the surface, which results in changes in the processing of another substrate, the Notch protein. Taken together, the two studies reveal more details of the complex interactions between APP’s movements, its processing, and the potential roles of the derived fragments.
To get an accurate picture of the location and timing of APP cleavage in cells, Virgil Muresan and coworkers immunostained endogenous APP in mouse neurons in culture and in situ with antibodies to three different epitopes. They compared staining patterns of the N-terminal antibody 22C11, the C-terminal antibody 2452, and 4G8, which recognizes the central (Aβ) domain. In cultured neurons, they found most of the APP C-terminal reactivity in the cell body, with lesser amounts in neurites and nerve terminals, while the N-terminal reactivity was evenly spread throughout the three regions. Aβ was mostly in the cell body and neurites, with little in terminals. Surprisingly, double labeling showed that the N- and C-terminal labels rarely appeared in the same place, suggesting that the cells contained relatively little full-length APP. They got the same results in primary neurons and under several conditions of cell fixation.
Taking a closer look, the investigators found that the three antibodies labeled distinct sets of transport vesicles along neurites. They also found many vesicles that carried a phospho-C-terminal APP epitope, but not the N-terminal epitope, suggesting that many carried phospho-CTFs (C-terminal fragments) or phospho-AICD (APP intracellular domain), rather than full-length phospho-APP. Separation of the different fragments was also seen in growth cones in the neurites of the cultured cells, with the pAPP antibody detected in the peripheral zones while the N-terminal antibody picked up vesicles in the central part of the structure. Only pAPP C-terminal fragments were detected in lamellipodium and fillipodium of growth cones and were concentrated in regions of advancement and turning, a finding which may indicate a role for the pCTFs in regulating growth cone mobility and neurite extension.
In brain tissue, the researchers found a similar segregated pattern of epitope staining when they used tissue from a transgenic mouse expressing low levels of human (Swedish mutant) APP. In cells expressing higher levels of APP, the epitopes were more often seen together, with 93 percent of vesicles appearing to carry full-length APP, compared to less than a quarter when only endogenously expressed APP was present. The authors conclude that the processing and transport of APP into neurites depends on the level of expression of APP.
“Overall, our results overturn the long-held view according to which transport of APP within neurites occurs, under normal conditions, mostly as full-length protein,” the authors write. All the same, Muresan pointed out in a e-mail to ARF that the CTFs should be capable of carrying out the same function of recruitment of kinesin-1 to vesicles as full-length APP, as they are still transmembrane proteins. “Moreover, even the AICD (i.e., C-γ, the product of γ-secretase cleavage) may not fall off the vesicle, and could thus still carry kinesin-1,” he wrote. “We (and others) have found that the AICD, when expressed in neurons, becomes associated with what appear to be vesicles. We have mentioned in the Discussion section of our paper that this association may involve the positively charged lysines in the AICD, which could easily interact with membrane acidic phospholipids. It may very well be that the release of the AICD from the vesicle is a regulated event, and does not just occur automatically after γ-secretase cleavage. Of course, this is only speculation at this time.”
How could the early cleavage of APP bear on the events that take place in Alzheimer disease? Muresan says, “From a conceptual point of view, our data suggest that the different APP fragments have a life of their own, independent of full-length APP, and that they are transported to different locations within the neuron, where they perform independent functions. If for some reason the processing of APP or the transport of the processed fragments is altered—which may happen in AD conditions—the normal function of these fragments may be perturbed. From this point of view, it is the alteration of the normal function of APP that may somehow trigger the neuronal pathology. More and more studies suggest that AD may start with a perturbation of the synapse function. Our paper suggests that some of the APP fragments (e.g., the phosphorylated CTFs) may indeed play crucial roles at the neurite terminals.”
The study also suggests that Aβ is present normally within neurons, where it could have deleterious effects on axonal transport (see ARF related news story). “Our results allow for the interpretation that the Aβ that is normally produced inside neurons may somehow form oligomers under still unknown conditions relevant to AD,” Muresan said.
The processing of APP falls to enzymes, the secretases, whose activity also depends on their location in cells. The γ-secretase controls APP trafficking, and the paper from Xu and colleagues shows that this relationship goes both ways. First authors Yun Liu and Yun-Wu Zhang find cells deficient in APP show a faster transit of presenilin-1 (PS1) from the trans-Golgi network to the cell surface, where it accumulates along with other components of the γ-secretase complex. This results in more active cleavage of the cell surface substrate, Notch. Restoring APP brings presenilin and other γ-secretase proteins back to normal levels. The researchers conclude that interaction between APP and PS1 may be required for the proper retention of APP/PS1 in the trans-Golgi network and subsequent delivery to the cell surface. The researchers also find that phospholipase D1 promotes cell surface accumulation of presenilin-1 independent of the effects of APP. Neither APP nor PLD1 affected trafficking of the β-secretase.—Pat McCaffrey
- Muresan V, Varvel NH, Lamb BT, Muresan Z. The cleavage products of amyloid-beta precursor protein are sorted to distinct carrier vesicles that are independently transported within neurites. J Neurosci. 2009 Mar 18;29(11):3565-78. PubMed.
- Liu Y, Zhang YW, Wang X, Zhang H, You X, Liao FF, Xu H. Intracellular trafficking of presenilin 1 is regulated by beta-amyloid precursor protein and phospholipase D1. J Biol Chem. 2009 May 1;284(18):12145-52. PubMed.