Rajendran and colleagues provide further insights into the biology of Alzheimer disease (AD). They present more evidence for endosomes as important sites of Aβ generation using elegant cell biology experiments. By crosslinking experiments, they show that there was increased colocalization of APP with BACE in early endosomes. Aβ secretion was dramatically reduced by inhibiting endocytosis or reducing recycling. They provide further confirmation for Aβ localization to multivesicular bodies (MVBs) by immunoelectron microscopy and present exciting new data on Aβ in secreted exosomes. It has been described that in some cells MVBs can fuse with the plasma membrane and secrete their inner (luminal) vesicles (exosomes). Interestingly, the authors localize the exosome component Alix to plaques in AD brain, and note that flottilin-1, known to be contained in exosomes, was previously shown to associate with plaques. Yet, they point out that, at least in APP transfected neuroblastoma cells, exosome-associated Aβ appears to account for only about 1 percent of Aβ secreted. They hypothesize...  Read more

Rajendran and colleagues provide further insights into the biology of Alzheimer disease (AD). They present more evidence for endosomes as important sites of Aβ generation using elegant cell biology experiments. By crosslinking experiments, they show that there was increased colocalization of APP with BACE in early endosomes. Aβ secretion was dramatically reduced by inhibiting endocytosis or reducing recycling. They provide further confirmation for Aβ localization to multivesicular bodies (MVBs) by immunoelectron microscopy and present exciting new data on Aβ in secreted exosomes. It has been described that in some cells MVBs can fuse with the plasma membrane and secrete their inner (luminal) vesicles (exosomes). Interestingly, the authors localize the exosome component Alix to plaques in AD brain, and note that flottilin-1, known to be contained in exosomes, was previously shown to associate with plaques. Yet, they point out that, at least in APP transfected neuroblastoma cells, exosome-associated Aβ appears to account for only about 1 percent of Aβ secreted. They hypothesize that in the slowly progressive disease process, secreted exosome-associated Aβ may be important in the progression of the disease. Despite evidence in support of intraneuronal MVB Aβ involvement in plaque formation, the authors did not consider the alternative—that plaque-associated Alix and flotillin-1, which localize to exosomes and MVB inner vesicles, could originate from MVBs without being secreted.

View all comments by Claudia Almeida
View all comments by Gunnar K. Gouras

I strongly recommend this article. But what percent of amyloid-β is bound to exosomes in AD is still shrouded in mystery. Much more experimental data are required to understand this better.

View all comments by K.S. Jagannatha Rao
View all comments by Bharathi Shrikanth Gadad

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
View all comments by Marie-Claude POTIER