Several studies have indicated that both cholesterol and sphingomyelin metabolism can affect the generation of Aβ. In this very elegant paper, Tobias Hartmann’s group has decided to go the opposite way and analyze whether Aβ could affect cholesterol and SM metabolism. They have used several genetic and biochemical approaches to reach the unexpected conclusion that the Aβ peptide can stimulate SM hydrolysis and reduce the biosynthesis of both SM and cholesterol. These effects could potentially be explained by perturbation of the lipid bilayer produced by Aβ. However, the fact that Aβ (in physiological concentrations) can stimulate both a purified neutral SMase (nSMase) activity in vitro and the nSMase activity recovered from cell homogenates suggests a direct effect of the peptide on the enzyme rather than on the lipid environment.

It has long been known that sphingomyelin and cholesterol like to go together (1). Increased biosynthesis of cholesterol is accompanied by increased generation of sphingomyelin. Indeed, the same transcriptional machinery (SREBP) regulates both...  Read more

Several studies have indicated that both cholesterol and sphingomyelin metabolism can affect the generation of Aβ. In this very elegant paper, Tobias Hartmann’s group has decided to go the opposite way and analyze whether Aβ could affect cholesterol and SM metabolism. They have used several genetic and biochemical approaches to reach the unexpected conclusion that the Aβ peptide can stimulate SM hydrolysis and reduce the biosynthesis of both SM and cholesterol. These effects could potentially be explained by perturbation of the lipid bilayer produced by Aβ. However, the fact that Aβ (in physiological concentrations) can stimulate both a purified neutral SMase (nSMase) activity in vitro and the nSMase activity recovered from cell homogenates suggests a direct effect of the peptide on the enzyme rather than on the lipid environment.

It has long been known that sphingomyelin and cholesterol like to go together (1). Increased biosynthesis of cholesterol is accompanied by increased generation of sphingomyelin. Indeed, the same transcriptional machinery (SREBP) regulates both biosynthetic pathways. The ultimate goal is to keep or cluster cholesterol at the plasma membrane (PM). Sphingomyelin is probably the best “cholesterol-binding lipid” and is highly enriched in the PM. Indeed, its stoichiometry of cholesterol binding is 3:1 (cholesterol:SM), which is extremely high considering that phosphatydylcholine (another common “cholesterol-binding lipid”) binds cholesterol with a 1:1 stiochiometric ratio (cholesterol:PC). This close relationship works the other way around, too. Cell surface hydrolysis of SM is accompanied by a fast translocation of cholesterol to the endoplasmic reticulum (ER). The retro-translocation has the ultimate effect of down-regulating cholesterol biosynthesis (through the HMG-CoA reductase) and increasing the storage of cholesterol ester (which, however, is only temporary and limited to certain cell types). In addition to the effects produced by the retro-translocation of PM-cholesterol, ceramide (one of the products of SM hydrolysis) can down-regulate the proteolysis/activation of SREBP and, therefore, reduce both biosynthesis and uptake of cholesterol (2, 3).

Our group has recently shown that normal aging of the brain is accompanied by activation of nSMase and consequent liberation of the second messenger ceramide, which is able to induce Aβ generation (4). This age-associated effect could be blocked by nSMase inhibitors and by genetic disruption of the ligand-binding domain of the neurotrophin receptor p75NTR, which is responsible for the activation of nSMase (in the brain and during the normal process of aging). If we join the results produced by Grimm et al. and our group (4), we can envision a model in which aging activates ceramide production and Aβ generation by acting through nSMase; Aβ can further stimulate nSMase by an apparent direct interaction, fostering an additional production of Aβ. Sphingomyelin hydrolysis would have the additional effect of reducing cholesterol biosynthesis in astrocytes, affecting the secretion of lipoprotein particles required for neurons to generate/sustain their own synapses (5). In conclusion, a vicious circle might operate that leads to abnormal production of Aβ and affects synaptogenesis. Tobias’s group has shown that the nSMase inhibitor GW4869 can block Aβ production in neurons; our group has shown that a different nSMase, manumycin A, can block Aβ production in both primary neurons and mice (4). This strategy seems to work for both the age-dependent and the Aβ-mediated effect, and is predicted to act upstream of the “vicious circle.”

I have so far considered the effects on cholesterol metabolism described by Grimm et al. as a consequence of SM hydrolysis because there is no evidence of a possible direct effect of the Aβ peptide on the HMG-CoA reductase (at the enzymatic/protein level). Indeed, even though Aβ was given to intact cells, the authors observed a decrease in the incorporation of acetate into the mevalonic pathway; a fact that implicates the HMG-CoA reductase, an ER membrane-based protein. However, we could have yet another surprise and discover that Aβ can act directly on the enzyme itself. It would be interesting to look at SREBP processing and HMG-CoA degradation under the above conditions, and at the effects of Aβ on a purified/enriched preparation of HMG-CoA in vitro.

Finally, one can wonder how the lack of presenilins can stimulate the SM-synthase activity. In fact, in contrast to nSMase, SM-synthase is an allosteric enzyme that seems to respond to the levels of one of its own substrates, palmitoyl-CoA. Interestingly enough, both the mevalonic and the fatty acid/palmitoyl-CoA biosynthetic pathways are under the control of the SREBP family of transcription factors (6). Even though we know that the intramembrane proteolysis of SREBP does not depend on presenilins, I still wonder whether SREBPs play any role behind the curtains. Who knows? Maybe Tobias has another ace ready for us.

References:
1. Slotte, J.P. et al. (1994). Flow and distribution of cholesterol-Effects of phospholipids. In Current Topics in Membranes. Cell Lipids (Hoekstra, D, ed.), pp. 483-502, Academic Press, San Diego, CA.

2. Worgall TS, Johnson RA, Seo T, Gierens H, Deckelbaum RJ. Unsaturated fatty acid-mediated decreases in sterol regulatory element-mediated gene transcription are linked to cellular sphingolipid metabolism. J Biol Chem. 2002 Feb 8;277(6):3878-85. Epub 2001 Nov 13. Abstract

3. de Chaves EP, Bussiere M, MacInnis B, Vance DE, Campenot RB, Vance JE. Ceramide inhibits axonal growth and nerve growth factor uptake without compromising the viability of sympathetic neurons. J Biol Chem. 2001 Sep 28;276(39):36207-14. Epub 2001 Jul 13. Abstract

4. Costantini C, Weindruch R, Della Valle G, Puglielli L. A TrkA-to-p75NTR molecular switch activates amyloid beta-peptide generation during aging. Biochem J. 2005 Oct 1;391(Pt 1):59-67. Abstract

5. Mauch DH, Nagler K, Schumacher S, Goritz C, Muller EC, Otto A, Pfrieger FW. CNS synaptogenesis promoted by glia-derived cholesterol. Science. 2001 Nov 9;294(5545):1354-7. Abstract

6. Dobrosotskaya IY, Seegmiller AC, Brown MS, Goldstein JL, Rawson RB. Regulation of SREBP processing and membrane lipid production by phospholipids in Drosophila. Science. 2002 May 3;296(5569):879-83. Abstract

View all comments by Luigi Puglielli

We appreciate the interesting study by Hartmann and colleagues. A decade ago we reported that Aβ peptides modulate the cholesterol esterification rate (1). We later showed that Aβ modulates the metabolism of cholesterol and phospholipids (2-4). We studied Aβ's effects on lipid metabolism in a number of test systems, including hepatic cells (2), cultured nerve cells (3), fetal rat brain model (3), and ex vivo in rat hippocampal slices (4) and found that it is tissue and oxidation level-dependent. This is discussed in detail in our recent publication (5) that explored the effects of Aβ on synaptic plasticity and its interrelation with the neural cholesterol homeostasis modulation by Aβ.

Our early study of Aβ's effect on cholesterol esterification was subsequently confirmed by others (6). In this regard, it is important to note that Aβ is a structure-functional component of lipoproteins (7,8,9). Aβ therefore, can affect the reverse cholesterol transport from neuronal tissue to the periphery in addition to its role in cholesterol synthesis and intracellular dynamics. This is...  Read more

We appreciate the interesting study by Hartmann and colleagues. A decade ago we reported that Aβ peptides modulate the cholesterol esterification rate (1). We later showed that Aβ modulates the metabolism of cholesterol and phospholipids (2-4). We studied Aβ's effects on lipid metabolism in a number of test systems, including hepatic cells (2), cultured nerve cells (3), fetal rat brain model (3), and ex vivo in rat hippocampal slices (4) and found that it is tissue and oxidation level-dependent. This is discussed in detail in our recent publication (5) that explored the effects of Aβ on synaptic plasticity and its interrelation with the neural cholesterol homeostasis modulation by Aβ.

Our early study of Aβ's effect on cholesterol esterification was subsequently confirmed by others (6). In this regard, it is important to note that Aβ is a structure-functional component of lipoproteins (7,8,9). Aβ therefore, can affect the reverse cholesterol transport from neuronal tissue to the periphery in addition to its role in cholesterol synthesis and intracellular dynamics. This is supported by earlier studies by Michikawa et al. (10), Igbavboa et al. (11), and us (4), who reported the effects of Aβ on cellular cholesterol uptake and efflux.

"My belief is that Aβ is involved in this interaction by modulating cellular/membrane cholesterol, so, both cholesterol and Aβ (and APP processing) affect each other," I noted three years ago during the ARF live discussion, "Cholesterol and Alzheimer's—Charging Fast but Still at a Distance from Solid Answers." I am glad Dr. Hartmann's skepticism and willingness to see more experiments "to prove this point" has now materialized in the excellent publication by Dr. Hartmann's group.

References:
1. Koudinov AR, Koudinova NV, Berezov TT. Alzheimer's peptides A beta 1-40 and A beta 1-28 inhibit the plasma cholesterol esterification rate. Biochem Mol Biol Int. 1996 Apr;38(4):747-52. Abstract

2. Koudinova NV, Berezov TT, Koudinov AR Multiple inhibitory effects of Alzheimer's peptide Abeta1-40 on lipid biosynthesis in cultured human HepG2 cells. FEBS Lett. 1996 Oct 21;395(2-3):204-6. Abstract

3. Koudinova NV, Koudinov AR, Yavin E. Alzheimer's Abeta1-40 peptide modulates lipid synthesis in neuronal cultures and intact rat fetal brain under normoxic and oxidative stress conditions. Neurochem Res. 2000 May;25(5):653-60. Abstract

4. Koudinov AR, Koudinova NV. Essential role for cholesterol in synaptic plasticity and neuronal degeneration. FASEB J. 2001 Aug;15(10):1858-60. Freely available at http://www.fasebj.org/cgi/content/short/00-0815fjev1 . PMID: 11481254 ; Koudinov AR, Koudinova NV. Cholesterol's role in synapse formation. Science. 2002 Mar 22;295(5563):2213. No abstract available. Abstract

5. Koudinov AR, Koudinova NV. Amyloid beta protein restores hippocampal long term potentiation: a central role for cholesterol? Neurobiol. Lipids. 2003 Sept; 1:8, Freely available at: http://neurobiologyoflipids.org/content/1/8/.

6. Liu Y, Peterson DA, Schubert D. Amyloid beta peptide alters intracellular vesicle trafficking and cholesterol homeostasis. Proc Natl Acad Sci USA. 1998; 95:13266-71 Abstract

7. Koudinov AR, Koudinova NV, Kumar A, Beavis RC, Ghiso J. Biochemical characterization of Alzheimer's soluble amyloid beta protein in human cerebrospinal fluid: association with high density lipoproteins. Biochem Biophys Res Commun. 1996 Jun 25;223(3):592-7. Abstract

8. Koudinov A, Matsubara E, Frangione B, Ghiso J. The soluble form of Alzheimer's amyloid beta protein is complexed to high density lipoprotein 3 and very high density lipoprotein in normal human plasma. Biochem Biophys Res Commun. 1994 Dec 15;205(2):1164-71. Abstract

9. Koudinov AR, Berezov TT, Koudinova NV. The levels of soluble amyloid beta in different high density lipoprotein subfractions distinguish Alzheimer's and normal aging cerebrospinal fluid: implication for brain cholesterol pathology? Neurosci Lett. 2001 Nov 16;314(3):115-8. Abstract

10. Michikawa M, Gong JS, Fan QW, Sawamura N, Yanagisawa K. A novel action of Alzheimer's amyloid beta-protein (Abeta): oligomeric Abeta promotes lipid release. J Neurosci. 21, 7226-35 (2001) Abstract

11. Igbavboa U, Avdulov NA, Chochina SV, Sun GY, Wood WG. Amyloid beta peptides and cholesterol dynamics. Neurosci Lett. S55, S25 (2000)

View all comments by Alexei R. Koudinov

A wealth of cellular and animal studies indicates that cholesterol regulates Aβ generation. Use of statins is currently being explored as a safe and available strategy that may help protect against Alzheimer disease. While awaiting the outcome of large clinical trials, mechanistic studies are revealing an unexpectedly complex picture of the lipid-Aβ connection. Cholesterol is no longer the only player; cholesteryl-esters, ceramide, sphingomyelin (SM), as well as isoprenoids are among the newest additions to the lipid list. Now, Tobias Hartmann and colleagues add a remarkable twist to the story. Not only do a variety of lipids regulate Aβ generation, but Aβ can also reach back and control cellular cholesterol and SM levels. This provocative conclusion is supported by solid in vitro and in vivo studies, which assign separate functions to Aβ40 (inhibition of HMG-CoA reductase, resulting in decreased cholesterol synthesis) and Aβ42 (activation of SMase, resulting in decreased SM levels). Separate functions of the two peptides are shown in a variety of systems, including in vitro...  Read more

A wealth of cellular and animal studies indicates that cholesterol regulates Aβ generation. Use of statins is currently being explored as a safe and available strategy that may help protect against Alzheimer disease. While awaiting the outcome of large clinical trials, mechanistic studies are revealing an unexpectedly complex picture of the lipid-Aβ connection. Cholesterol is no longer the only player; cholesteryl-esters, ceramide, sphingomyelin (SM), as well as isoprenoids are among the newest additions to the lipid list. Now, Tobias Hartmann and colleagues add a remarkable twist to the story. Not only do a variety of lipids regulate Aβ generation, but Aβ can also reach back and control cellular cholesterol and SM levels. This provocative conclusion is supported by solid in vitro and in vivo studies, which assign separate functions to Aβ40 (inhibition of HMG-CoA reductase, resulting in decreased cholesterol synthesis) and Aβ42 (activation of SMase, resulting in decreased SM levels). Separate functions of the two peptides are shown in a variety of systems, including in vitro activation of nSMase by Aβ42, but much less by Aβ40; down-regulation of high cellular de novo cholesterol synthesis in APP/APLP2-/- MEF cells by exposure to Aβ40, but not Aβ42; and increased cholesterol together with decreased SM in cells expressing PS1 containing FAD mutations, leading to elevated Aβ42/Aβ40 ratios. Given that cholesterol and SM are integral components of lipid rafts, it would be interesting to examine how lipid raft levels and function are separately regulated by the two peptides in cells expressing FAD mutant presenilins.

This study is important not only for Alzheimer disease, but also for basic cholesterol biology, as Aβ may regulate either HMG-CoA reductase or the SREB pathway. Although Aβ42 appears to directly activate SMase in in vitro assays, the exact mechanism for Aβ40 remains to be elucidated. Exposure of intact cells to Aβ40 reduces the activity of HMG-CoA reductase, an enzyme with established ER localization. The intracellular localization of HMG-CoA reductase would suggest an indirect mechanism of action for exogenous Aβ40. However, extracellular Aβ40 could not normalize cholesterol synthesis in APP/APLP2-/- MEF cells, indicating that perhaps small amounts of intracellular Aβ40 or AICD may also regulate HMG-CoA reductase activity in wild-type cells. Interestingly, lack of Aγ-secretase function in PS1/2-/- MEF cells elevates cholesterol and SM levels quite strongly, while in APP/APLP2-/- MEF cells (which are derived from different mice), levels of both lipids increase more moderately. The implication is that the impact of the γ-secretase/APP/Aβ lipid regulatory system might be quite different in strength depending on which specific cells or tissues are analyzed. One can also ask the question whether, if one looks at other tissues, perhaps there are Aγ-secretase substrates in addition to APP and APLP2 which may regulate cellular cholesterol and SM levels. These and other questions raised by Tobias will further define the delicate network of the newly established reciprocal lipid-Aβ connection.

View all comments by Dora M. Kovacs

Researchers have long speculated that the Aβ peptide might have a physiological function. Unfortunately, evidence of a normal role for Aβ in cellular processes has been notoriously difficult to obtain and has led to the prevailing notion that Aβ is merely a toxic byproduct of APP metabolism—nasty “junk,” if you will. Strong evidence for a physiological function of Aβ did not emerge until 2003, when work by Malinow and colleagues suggested that Aβ may act as a negative regulator of excitatory synaptic transmission (Kamenetz et al., 2003). Surprisingly little else has been published about this putative function of Aβ, for reasons that are unclear. Now, the paper by Hartmann and colleagues reports an exciting new role for Aβ in regulating both cholesterol and sphingomyelin biosynthesis, apparently via two complex feedback loops that center on γ-secretase. The evidence they present in favor of this complex feedback regulation is extensive and quite compelling. Adding a Baroque yet intriguing twist, they discovered that the C-terminus of...  Read more

Researchers have long speculated that the Aβ peptide might have a physiological function. Unfortunately, evidence of a normal role for Aβ in cellular processes has been notoriously difficult to obtain and has led to the prevailing notion that Aβ is merely a toxic byproduct of APP metabolism—nasty “junk,” if you will. Strong evidence for a physiological function of Aβ did not emerge until 2003, when work by Malinow and colleagues suggested that Aβ may act as a negative regulator of excitatory synaptic transmission (Kamenetz et al., 2003). Surprisingly little else has been published about this putative function of Aβ, for reasons that are unclear. Now, the paper by Hartmann and colleagues reports an exciting new role for Aβ in regulating both cholesterol and sphingomyelin biosynthesis, apparently via two complex feedback loops that center on γ-secretase. The evidence they present in favor of this complex feedback regulation is extensive and quite compelling. Adding a Baroque yet intriguing twist, they discovered that the C-terminus of Aβ determines which of the two lipid pathways is to be regulated. Aβ40 inhibits HMG CoA reductase and thus lowers cholesterol levels, while Aβ42 directly activates SMase and therefore lowers sphingomyelin levels. Moreover, Aβ42-raising FAD mutations in presenilin cause cholesterol levels to increase (because reduced Aβ40 levels relieve HMG CoA reductase inhibition) and sphingomyelin levels to fall (due to Aβ42-induced stimulation of SMase). In pathology, this feedback loop could lead to a vicious circle of ever-increasing Aβ42 and cholesterol levels, and could provide a plausible explanation for the observed relationships between cholesterol levels, Aβ generation, and AD. Thus, the results of Hartmann and colleagues suggest that the variable C-termini of Aβ are not just mistakes of an indiscriminate γ-secretase, but that the Aβ40/Aβ42 ratio may in fact be physiologically determined for the regulation of lipid homeostasis. This is a fascinating paper that has far-reaching implications for the entire field.

View all comments by Robert Vassar

Aβ generation strongly depends on lipids. First of all, APP is a membrane protein, defining its most proximate neighboring molecules; second, substrate turnover of the secretases is regulated by membrane lipid composition; and third, Aβ peptides are signaling molecules involved in cholesterol and sphingolipid homeostasis. Now the Greengard lab adds a new stone to this mosaic. PLD1, a phospholipase, apparently binds to PS1 and absence of PLD1 increases Aβ generation. The story is complex because at least two independent pathways are involved. The first pathway modifies assembly or stability of γ-secretase and is independent of PLD1 enzymatic activity; the other one strictly depends the phospholipase activity, altering APP trafficking in the presence of PS1 and overexpressed PLD1. Moreover, it changes neurite growth, but only in the presence of PS-FAD.

Interestingly, PLD1 affects Aβ generation as much as it affects Notch cleavage. Gopal Thinakaran recently reported that NICD generation in adult cells, unlike Aβ, is produced outside of rafts. Does this indicate a role of PLD1...  Read more

Aβ generation strongly depends on lipids. First of all, APP is a membrane protein, defining its most proximate neighboring molecules; second, substrate turnover of the secretases is regulated by membrane lipid composition; and third, Aβ peptides are signaling molecules involved in cholesterol and sphingolipid homeostasis. Now the Greengard lab adds a new stone to this mosaic. PLD1, a phospholipase, apparently binds to PS1 and absence of PLD1 increases Aβ generation. The story is complex because at least two independent pathways are involved. The first pathway modifies assembly or stability of γ-secretase and is independent of PLD1 enzymatic activity; the other one strictly depends the phospholipase activity, altering APP trafficking in the presence of PS1 and overexpressed PLD1. Moreover, it changes neurite growth, but only in the presence of PS-FAD.

Interestingly, PLD1 affects Aβ generation as much as it affects Notch cleavage. Gopal Thinakaran recently reported that NICD generation in adult cells, unlike Aβ, is produced outside of rafts. Does this indicate a role of PLD1 for embryonic processing of APP, which appears to take place outside of the raft? Clearly it will be important to see whether this interaction (and the altered trafficking/neurite sprouting) can be found with adult wild-type mice. Alternatively—and the γ-secretase components data may suggest this—PLD1 could act as an (anti?) cofactor during assembly of the γ-secretase complex. In such a case, PLD1 should modify total cellular γ-secretase activity with no selective impact on specific γ-secretase substrates.

It is clear from these publications that PLD1 has functional interactions with PS and protein trafficking, including trafficking of APP. Neurite outgrowth is only affected when PS-FAD is overexpressed, and the authors conclude that this is due to altered APP trafficking. But is this really the only interpretation? The effect of PLD1 on APP trafficking was not assayed in the wild-type situation, and there are many other possible pathways, for example, altered PLD1 localization. Complicating interpretation further is that PS-FAD causes altered membrane fluidity, which could easily impair vesicle budding or trafficking for some cargo proteins. Does PLD1 "restore" this situation because it corrects membrane fluidity, or is there a close functional and mechanistic relation, as the interaction with PS may suggest? The multiple links PLD1 offers to APP biology are truly fascinating, and it will be exciting to see how this story develops over time.

View all comments by Tobias Hartmann

Several lines of evidence suggest that presenilins (PS) could contribute to both AβPP processing and trafficking to the membrane, but whether these two functions were related and intimately linked to the proposed catalytic activity of presenilins remained a matter of question. In these two back-to-back papers, the groups of Dr. Paul Greengard and Dr. Huaxi Xu interestingly suggest that phospholipase D1 (PLD1) could interact physically with PS, promote AβPP trafficking, and modulate Aβ production by apparently distinct mechanisms.

First, the group convincingly demonstrates that endogenous PS1 physically interacts with PLD1 but not with other PLD members, and binds to this phospholipase via its cytoplasmic loop domain. Apparently, PS1 recruits PLD1 in the Golgi/TGN, since PLD1 distributes within both cytosolic and Golgi/TGN compartments in wild-type ES cells, while PS1 deficiency triggers diffuse and only cytosolic localization of PLD1. Interestingly, PLD1 overexpression reduced the levels of both secreted and intracellular Aβ and increased βCTF, while PLD1 reduction by...  Read more

Several lines of evidence suggest that presenilins (PS) could contribute to both AβPP processing and trafficking to the membrane, but whether these two functions were related and intimately linked to the proposed catalytic activity of presenilins remained a matter of question. In these two back-to-back papers, the groups of Dr. Paul Greengard and Dr. Huaxi Xu interestingly suggest that phospholipase D1 (PLD1) could interact physically with PS, promote AβPP trafficking, and modulate Aβ production by apparently distinct mechanisms.

First, the group convincingly demonstrates that endogenous PS1 physically interacts with PLD1 but not with other PLD members, and binds to this phospholipase via its cytoplasmic loop domain. Apparently, PS1 recruits PLD1 in the Golgi/TGN, since PLD1 distributes within both cytosolic and Golgi/TGN compartments in wild-type ES cells, while PS1 deficiency triggers diffuse and only cytosolic localization of PLD1. Interestingly, PLD1 overexpression reduced the levels of both secreted and intracellular Aβ and increased βCTF, while PLD1 reduction by antisense approach led to the opposite phenotype, that is, increase in Aβ levels and redution in βCTF-like immunoreactivity.

By which mechanism could PLD1 trigger Aβ reduction? Greengard and colleagues suggest that this could occur via the disruption of the PS-dependent γ-secretase complex by PLD1. First, they show that PLD1 interacts physically with PS1 but not with Pen-2, another member of the γ-secretase complex. By using the anti-Pen-2 immunoprecipitation approach, it is shown that PLD1 overexpression reduces the interaction of Pen-2 with the other components of the complex, PS, Aph-1, and nicastrin.

These data are interesting since the disruption of the complex is far from complete, but sufficient to trigger a drastic decrease of Aβ production. It had been suggested that perhaps a limited reduction of γ-secretase activity could lead to significant Aβ reduction without eliciting the deleterious effects on the production of other γ-secretase-derived products. Particularly, PLD1 overexpression was a smart way to examine whether one could partly diminish Aβ-production without affecting the Notch pathway. However, Greengard et al. unfortunately show that partial disruption of the γ-secretase complex also leads to the inhibition of NICD, the γ-secretase-derived product of Notch.

The influence of PLD1 on AβPP processing is independent of its catalytic activity. Thus, catalytically inactive PLD1 (K898R) reduces secreted and intracellular Aβ production and apparently disrupts γ-secretase assembly, although to a lesser extent. These data are interesting and somewhat puzzling. Although Pen-2 immunoprecipitation of cells expressing PLD1K898 led to reduced immunoreactivities of the various components compared to control cells, the extent of inhibition appears variable depending on the component examined. While PLD1 and its mutant similarly reduced PS1 NTF and Aph-1 immunoreactivities, PS1-CTF and nicastrin expression were differentially affected. These data may be due to the fact that the stochiometry of the components of the γ-secretase complex is not 1/1/1/1, and that unlike wild-type PLD1, mutant PLD1 might have interacted differently with some of the components, either free or inside the complex.

It remains that while catalytically inactive PLD1 reduces Aβ, this mutant was unable to affect AβPP trafficking. Therefore, this suggests that PLD1 could harbor two distinct functions. First, the promotion of AβPP trafficking, and second, the inhibition of γ-secretase activity.

As do most interesting papers, these raise new questions. First, it was usually admitted that FAD mutations in PS generally lead to similar effects on both AβPP trafficking and γ-secretase processing. Here we are facing a new molecule that displays opposite effects on trafficking of AβPP and Aβ production. Is the increased trafficking to the membrane shortening transit in the Golgi/TGN and thereby, Aβ production? This would mean that one of the main sites of Aβ production indeed occurs intracellularly and not at the plasma membrane. Second, if PLD1 and its mutant both display "γ-secretase disruption" and trigger Aβ reduction while only wild-type PLD1 affects AβPP trafficking, what is the role of the PLD catalytic site? Since both processing and trafficking are affected when PLD1 is overexpressed, that is, when γ-secretase is partly disrupted, does that mean that only a fraction of the γ-secretase complex is necessary for underlying these two functions? Alternatively, is there a γ-secretase subcomplex specifically targeted by PLD1 (that would explain the rather selective effect of mutant PLD1 on certain γ-secretase components in the "disruption/immunoprecipitation" experiments)? Is the catalytic PLD1 interacting with a subcomplex of γ-secretase that participates only in processing?

With cell biology papers, the question stands as to whether the observed cellular phenotype could account for in-vivo physiological mechanisms. In this context, it would be interesting to examine deeply the influence of PLD1 or its mutant in cells overexpressing PS-FAD mutants distinct from ΔE9, particularly because the PS1/PLD1 physical interaction was demonstrated on endogenous PS1, that is, intact protein harboring the integral cytoplasmic loop while the ΔE9-PS1 deletion truncates PS1 from a part of its cytoplasmic domain. Whether ΔE9-PS1 physically interacts with PLD1 has not been included in the article, although functionality of the system argues in favor of such an interaction. Finally, it would be of most interest to examine the effect of PLD1 inhibitors such as 1-Butanol in transgenic mice that overproduce Aβ to examine whether this inhibitor (if it’s not toxic per se) could accelerate amyloidogenesis and Aβ deposits.

Overall, these two papers are very interesting and raise fundamental questions about the roles of presenilins, which, undoubtely, will be adressed very soon.

View all comments by Frédéric Checler