. APP anterograde transport requires Rab3A GTPase activity for assembly of the transport vesicle. J Neurosci. 2009 Nov 18;29(46):14534-44. PubMed.

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  1. We read this paper by Szodorai et al. and found it interesting, especially with regard to the possibility that the small GTPase, Rab3A, could regulate sorting of APP into the cargo vesicle, and—possibly transport by kinesin-1. In fact, the idea that kinesins are somehow regulated by small GTPases has gained support over the past decade; an older paper even proposed that a Rab protein (Rab6) serves as anchor on the cargo vesicle for a kinesin motor (1).

    We also found it interesting—and very important—that APP not only can be, but is indeed processed by a secretase within the transport vesicle. This idea was first proposed by Larry Goldstein’s group (2), although, in that case, the secretases that were reported to process APP were those of the amyloidogenic pathway, not α-secretase, as reported here by Stefan Kins’s group. The new findings by Szodorai et al. certainly reopen the possibility that, under specific circumstances, APP might be also processed via β- and γ-secretase within the transport vesicle, to generate sAPPβ and Aβ, as Kamal et al. (2) initially proposed. So far, such conditions have not been identified, but they may very well be related to some neuronal pathology. While the extent of APP processing within the transport vesicle detected by Szodorai et al. is small, yet to be identified conditions may increase the extent of this processing.

    A third point made by this paper is that APP can be transported in cargo vesicles that recruit kinesin-1 (specifically, kinesin-1C) by a mechanism that does not involve APP, in other words, that APP can be simply a cargo protein, not a receptor for kinesin-1 on the vesicle. This may be true. However, this does not mean that all APP is transported in this way in vivo. We ourselves have published extensively on the transport of APP (3-6), and emphasized our view that transport of exogenously expressed proteins is not necessarily relevant for the transport of the endogenous APP; for example, we showed that the level of expression of APP determines how the protein is processed and transported (4). Unless the level of expression is close to the level of the endogenous APP, a tagged APP may not be a reliable reporter of what is happening with the endogenous protein. This may be true for most proteins, but is especially true for proteins whose functions rely on their interaction with other proteins, such as APP. In such cases, overexpression will certainly alter the equilibrium of interactions, and the endogenous interactions will certainly be abnormal, particularly if normal binding partners are in limited supply. More or less, an overexpressed protein will start behaving like a dominant negative construct, if its concentration rises above a critical level.

    Overexpression of proteins is a great tool, mostly when one observes the effect of overexpression on the behavior of endogenous proteins. Otherwise, the spirit of Heisenberg’s uncertainty principle applies nowhere better than in the study of cells using exogenous expression of tagged proteins. One needs to be aware that the tagged proteins report the behavior of the protein in a perturbed environment (perturbed by the tool: the expressed protein itself). Why is this important for APP transport? It is because the sorting of APP into transport vesicles may become non-operational by overwhelming the sorting machinery. At some level of expression, it is likely that APP will enter by default many transport vesicles that form in the trans-Golgi network (TGN), not only those where it is normally sorted. We are not saying that this is happening in the experiments reported by Szodorai et al. We just want to draw attention that what they see (i.e., that the APP cytoplasmic domain is not important in transport) may not be completely true in endogenous conditions.

    Szodorai et al. make a meritorious effort to isolate transport vesicles that contain APP, and thoroughly characterize the purified membranes in terms of protein content. This is how they identified Rab3A. However, in our view, this vesicle population may represent only a fraction of the vesicles that carry APP. This is because the immunoprecipitations have been done from non-denatured membrane fractions (likely preserving the endogenous protein-protein interactions), using an antibody to the APP cytoplasmic domain (CT20). This domain is most likely involved in protein-protein interactions, and may not be always accessible to the CT20 antibody, being blocked by interacting proteins. Thus, it is likely that their immuno-isolation procedure was selected for those vesicles where the cytoplasmic domain of APP was freely available to the antibody, and not masked by interactions with other cellular proteins.

    Another point that we would like to make is that transport of APP certainly uses several pathways, not only one. We showed in 2005 that phosphorylated APP is transported into neurites by recruiting kinesin-1 via JIP-1 (6). This mechanism is certainly not true for non-phosphorylated APP, which comprises most of the APP within a neuron (6). Also, we recently published that a fraction of APP could be, in fact, transported into neurites after APP is processed into fragments, and that these fragments are transported with different vesicle populations. Cleavage of APP in cultured cells occurs largely in the endoplasmic reticulum-Golgi intermediate compartment, the Golgi/TGN, and recycling endosomes (7-9), and most APP in the brain, in vivo, is present as fragments (7-10). There is no doubt that a fraction of APP is transported to the plasma membrane as full-length protein (this fraction is ~10 percent of the APP synthesized in cultured cells (9). As Szodorai et al. now show, this APP may be sorted in a Rab3A-dependent manner into cargo vesicles that recruit kinesin-1C via a “receptor” that is not APP. As we recently pointed out, the problem of transport of APP—a protein that has such complex cell biology—is still far from being solved (3), but progress in elucidating the potentially many mechanisms of APP transport in neurons are being made. The paper by Szodorai et al. is an important step in this direction.

    References:

    . Interaction of a Golgi-associated kinesin-like protein with Rab6. Science. 1998 Jan 23;279(5350):580-5. PubMed.

    . Kinesin-mediated axonal transport of a membrane compartment containing beta-secretase and presenilin-1 requires APP. Nature. 2001 Dec 6;414(6864):643-8. PubMed.

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    . 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.

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    . Alzheimer's A beta(1-42) is generated in the endoplasmic reticulum/intermediate compartment of NT2N cells. Nat Med. 1997 Sep;3(9):1021-3. PubMed.

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