APP is subject to two alternative cleavages: a potentially amyloidogenic pathway and a non-amyloidogenic pathway involving α-site APP cleaving enzymes. The amyloidogenic pathway is initiated by the β-site APP cleaving enzyme (BACE), which generates the C-terminal fragment β-CTF. The membrane-bound β-CTF is further cleaved by the γ-secretase complex in a sequential mode generating Aβ peptides of varying lengths and the APP intracellular domain AICD. Aβ42 represents an intermediate product and is by far the predominant species deposited in senile plaques. It is regarded as the key factor in the development of AD.

The regulatory mechanism of intramembrane cleavage at γ-cleavage sites is a pivotal issue for understanding the mechanism leading to the disease. APP processing by BACE was repeatedly reported to occur in cholesterol- and sphingolipid-rich detergent-resistant membrane domains, which are also called lipid rafts. The γ-complex has also been found associated with lipid rafts, implying that Aβ may be a product of intra-raft reactions.

To further address the...  Read more

APP is subject to two alternative cleavages: a potentially amyloidogenic pathway and a non-amyloidogenic pathway involving α-site APP cleaving enzymes. The amyloidogenic pathway is initiated by the β-site APP cleaving enzyme (BACE), which generates the C-terminal fragment β-CTF. The membrane-bound β-CTF is further cleaved by the γ-secretase complex in a sequential mode generating Aβ peptides of varying lengths and the APP intracellular domain AICD. Aβ42 represents an intermediate product and is by far the predominant species deposited in senile plaques. It is regarded as the key factor in the development of AD.

The regulatory mechanism of intramembrane cleavage at γ-cleavage sites is a pivotal issue for understanding the mechanism leading to the disease. APP processing by BACE was repeatedly reported to occur in cholesterol- and sphingolipid-rich detergent-resistant membrane domains, which are also called lipid rafts. The γ-complex has also been found associated with lipid rafts, implying that Aβ may be a product of intra-raft reactions.

To further address the question of how the lipid environment can influence APP processing, Anja Schneider and colleagues now provide evidence that flotillins/reggie proteins, which are associated with cholesterol-rich lipid rafts, promote the clustering of APP and thus may stimulate the sequential endocytosis of APP into a specialized clathrin-dependent pathway. The authors propose a model in which flotillin acts as a scaffolding protein and serves as a platform for the clustering of APP, followed by endocytosis as a necessary event for the initial cleavage of APP by BACE. Consequently, the authors report that flotillin modulates BACE–mediated cleavage of APP by regulating its endocytosis.

In N2a cells expressing the Swedish mutant APP, BACE-derived APP cleavage products were found reduced by flotillin-2 knockdown but not by flotillin-1 knockdown. In addition, α-CTF was found reduced by flotillin-2 knockdown, indicating that both alternative pathways of APP, by β- and α-cleavages, were affected. Thus, not only amyloidogenic processing of APP was regulated by flotillin-2 but also α-cleavage and seemingly decreased full-length APP levels (see Figure 1 of the paper).

As is often the case in science, the present results raise several intriguing issues and challenges that definitely will be the subject of further studies. Thus, it remains to be clarified why both alternative processing pathways of APP were affected. Also, it has been known that all components required for the production of Aβ are localized to the cell surface and to lipid rafts, including the γ-secretase complex, which is responsible for the final cleavage event resulting in Aβ generation. This leaves open the question of why the effects should be solely based on endocytotic effects. APP-BACE recognition, cleavage, and the transfer of the product β-CTF to the γ-secretase complex could also be impaired. This view is supported by findings from Urano et al., 2005, who revealed that both cholesterol and protein isoprenylation influence the active γ-secretase complex association with lipid rafts. Hattori et al. reported that flotillin-1 binds to BACE and that overexpression of flotillin-1 results in recruiting BACE into lipid rafts and influenced β-secretase activity in cultured cells (Hattori et al., 2006). This has not been investigated for flotillin-2, although BACE-flotillin interactions cannot be excluded to affect BACE oligomerization (Schmechel et al., 2004; Westmeyer et al., 2004) and thereby indirectly influence APP processing.

Other questions that remain are how APP is recruited into rafts and why flotillin-2 depletion inhibits internalization. Munter et al. showed that shorter forms of Aβ are produced by γ-cleavage when β-CTF dimerization through the transmembrane region is inhibited (Munter et al., 2007) and inhibitors of APP dimerization targeting the ectodomain contact site can directly affect BACE cleavage (Kaden et al., 2008). Therefore, flotillin as well as cholesterol could influence the general tendency of APP to oligomerize and thereby modulate APP processing. Finally, it would be reasonable to examine the effect of flotillin or cholesterol on the production of intracellular soluble Aβ oligomers, such as dimers, tetramers inside, which are causative for the pathogenesis.

Nevertheless, by using state-of-the-art techniques, the authors show that APP endocytosis was not impaired in N2a cells by knockdown of flotillin-1 but by flotillin-2 knockdown. This significantly reduced APP internalization by 44 percent compared to wild-type cells. Supporting findings were presented based on siRNA experiments and with hippocampal neurons, which produce large amounts of flotillin. When the authors compared the endocytosis rates of cholesterol-depleted or untreated cells, they found a significant reduction of APP endocytosis using a biotinylation assay, which was in agreement to cell biological approaches presented. An artificial clustering of APP by using a bivalent secondary antibody dramatically stimulated the endocytosis of APP and thus could partially rescue the inhibitory effect of flotillin-2 knockdown on the endocytosis of APP. This strengthens the authors’ assumption that cholesterol/flotillin-dependent clustering, as well as antibody-mediated clustering, stimulates APP internalization and thereby could promote amyloidogenic APP processing specifically in the endocytotic pathway.

View all comments by Gerd Multhaup

Comment by Charles Duyckaerts, Jack-Christophe Cossec, and Marie-Claude Potier
Accumulation of the Aβ peptide by the neuron is thought to be the initial event that induces the cascade of reactions that leads to the full blown pathology of Alzheimer disease. A number of studies indicate that the regulation of APP cleavage through the sorting of APP, BACE, and the components of γ-secretase complex and their gathering in the cell membrane is crucial in the much more prevalent sporadic cases, in which there is no evidence of increased APP synthesis.

Schneider et al. have added a new protagonist in this interplay among APP, the secretases, and cholesterol. In this paper, they indeed describe a potentially important link between AD and flotillin. Flotillin-1 and -2 are proteins anchored at the cell membrane. They are associated with lipid rafts, the 50 to 100 nm large microdomains, enriched in cholesterol, which seem to "float" over the membrane glycerophospholipids. In lipid rafts, the diffusion coefficients are smaller than in non-raft domains: molecules are less...  Read more

Comment by Charles Duyckaerts, Jack-Christophe Cossec, and Marie-Claude Potier
Accumulation of the Aβ peptide by the neuron is thought to be the initial event that induces the cascade of reactions that leads to the full blown pathology of Alzheimer disease. A number of studies indicate that the regulation of APP cleavage through the sorting of APP, BACE, and the components of γ-secretase complex and their gathering in the cell membrane is crucial in the much more prevalent sporadic cases, in which there is no evidence of increased APP synthesis.

Schneider et al. have added a new protagonist in this interplay among APP, the secretases, and cholesterol. In this paper, they indeed describe a potentially important link between AD and flotillin. Flotillin-1 and -2 are proteins anchored at the cell membrane. They are associated with lipid rafts, the 50 to 100 nm large microdomains, enriched in cholesterol, which seem to "float" over the membrane glycerophospholipids. In lipid rafts, the diffusion coefficients are smaller than in non-raft domains: molecules are less mobile and can probably more easily interact. This has suggested a new way of regulating enzymatic reactions: β-cleavage of APP, for instance, could be regulated by the movement, and collision of rafts containing either APP or BACE, and by their encounter (Ehehalt et al., 2003).

Schneider et al. show that APP endocytosis is reduced when production of flotillin-2 is inhibited by siRNA. Using the modern tools of fluorescent microscopy, they conclude that flotillin-2 is associated with an increased clustering of APP at the cell surface, which can be mimicked by antibodies: a primary antibody that labels APP promotes clustering of APP when a bivalent secondary antibody is applied, an effect that is not obtained if the secondary antibody is monovalent. The antibody mediated clustering also promotes APP endocytosis. The decrease in APP endocytosis induced by depletion of the cholesterol content of the membrane can be overcome by antibody mediated clustering—an observation that suggests that cholesterol also plays a role in APP clustering. APP endocytosis requires clathrin, as Schneider et al. demonstrate with the use of dominant positive mutants involving proteins of the clathrin pathway, a pathway that could appear more complex than presently thought: transferrin endocytosis, which is also clathrin dependent, is indeed affected by an epsin 1 mutant while APP endocytosis is not.

These results have now to be transferred to human pathology: was the antibody used to detect the flotillin accumulation in tangle-bearing neurons (Girardot et al., 2003) really specific for flotillin-1, or did it also recognize flotillin-2 that, at least in the cell systems used by Schneider et al., seems to be the active molecule? How and why could flotillin accumulate in tangle-bearing neurons? Could this accumulation interfere with Aβ production? Much remains to be done, on the other hand, concerning the clathrin pathway and AD lesions. Literature on these topics is rather old and should be reanalyzed in view of these new results.

The data of Schneider et al. show that the understanding of Alzheimer pathogenesis needs more than enzymatic activities in a test tube: the dynamic topography of the protagonists is crucial, as is the biophysics of cell membrane that allow or prevent the gathering of the protagonists.

We are personally involved in research that aims to understand the relationship between membrane cholesterol and the clathrin mediated pathway. Our current results in HEK293 cell cultures also show that increasing plasma membrane cholesterol promotes APP endocytosis and the production of Aβ through a clathrin dependent pathway. Since enlargement of Rab 5-positive endosomes are the earliest appearing Alzheimer disease’s specific cellular pathology that is also modulated by APP gene dosage (Nixon et al., 2004), is Alzheimer disease a disease of the endocytosis machinery?

References:
Ehehalt R, Keller P, Haass C, Thiele C, Simons K. Amyloidogenic processing of the Alzheimer beta-amyloid precursor protein depends on lipid rafts. J Cell Biol. 2003 Jan 6;160(1):113-23. Abstract

Girardot N, Allinquant B, Langui D, Laquerrière A, Dubois B, Hauw JJ, Duyckaerts C. Accumulation of flotillin-1 in tangle-bearing neurones of Alzheimer's disease. Neuropathol Appl Neurobiol. 2003 Oct;29(5):451-61. Abstract

Nixon RA. Niemann-Pick Type C disease and Alzheimer's disease: the APP-endosome connection fattens up. Am J Pathol. 2004 Mar;164(3):757-61. Abstract

View all comments by Charles Duyckaerts
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