Posted 15 October 2005

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Tom Fagan
Hi, folks. I'm Tom Fagan from Alzforum and I'll be moderating today, if need be. Luigi, do you want to start us off with a brief statement about the role of ceramide and cholesterol and what issues we might address today?

Luigi Puglielli
Well, it depends on the specific interests of each person. I would expect questions on metabolism and molecular/biochemical roles of cholesterol and ceramide, and their role (if any) in aging and AD. Then, we'll take it from there, and go wherever the audience wants to go.

Tom Fagan
Well, let me pose the first question, perhaps. Is it known why ceramide levels rise as we age?

Luigi Puglielli
Good question. We have a paper in press in Biochemical journal (see Constantini et al., 2005) where we show that ceramide levels increase during aging in the brain of wild-type animals but not in p75NTR knockout animals. This would indicate that p75NTR is the most important upstream activator of ceramide production in the brain (at least during the normal/physiologic process of aging). That does not rule out additional events (for example, those occurring during pathology or in very old age). This very old age issue is potentially very interesting because we compared 3- and 22-month-old animals but we are still waiting to see what happens after 22 months of age.

Anne Fagan
Quiet crowd today! I'm here mostly to learn, especially since I could not access the online articles. Luigi, have you been able to look at brain Aß levels in the p75 KOs?

Luigi Puglielli
Yes, Anne. They don't increase during aging (3 to 22 months) and are approximately 15 to 20 percent lower than age-matched controls (WT animals).

Tom Fagan
So the p75 data would fit in with what Elliott Mufson and others have observed regarding AD and neuronal death (see, for example, ARF related news story). I wonder, Luigi, (pardon my ignorance) if there are enzymes in the ceramide synthesis pathway that are inducible?

Anne Fagan
Tom, are you thinking about such enzymes as potential therapeutic targets?

Tom Fagan
Anne, yes, that would be one aspect—sure.

Luigi Puglielli
Tom, as far as I know the enzymes involved with the biosynthesis of ceramide are not inducible. However, we need to keep in mind that ceramide (as a second messenger) is generated by hydrolysis of cell-surface sphingomyelin. So the target would be neutral sphingomyelinase (SM). That enzyme is activated on a need-only basis by upstream events (in our case—the brain—p75NTR).

Tom Fagan
Though I'm not sure how little ceramide we can tolerate, it's essential as far as I remember, correct?

Luigi Puglielli
Well, we treated cells and animals with nSMase inhibitors and they did just fine (no problem with the animals). The inhibitor simple prevented the age-associated increase in ceramide and Aß levels in WT animals.

Barbara Tate
Along those lines, would it be possible to speculate on the potential downside of inhibition of ceramide synthesis or sphingomyelinase?

Luigi Puglielli
Barbara, I can't say anything about blocking ceramide synthesis. However, ceramide, being the backbone of all major glycosphingolipids, it is essential in the brain, so I wouldn't go there. All the "compounds" known to block ceramide synthesis are very toxic (neurotoxins secreted by different fungi or bacteria).

Anne Fagan
Luigi, I should know this but I don't: Has anyone crossed the various AD mouse models (APP Tg) with P75 KOs?

Luigi Puglielli
Anne, there is one group in Italy that has crossed AD11 Tg mice (they have a block in NGF production and resemble some of the AD features) with p75NTR KOs. The plaques observed in AD11 disappear almost completely when they cross them with p75NTR KOs. This confirms our results.

Tom Fagan
Anne, Luigi, all, see our coverage of Antonio Cattaneo's talk in Sorrento on this subject.

Anne Fagan
Thanks for the link, Tom. Impossible to keep up with all the literature without you guys!

Tom Fagan
So Luigi, what happens if you give animals ceramide? Do they produce more Aß?

Luigi Puglielli
Well, you can't just give ceramide to animals (it doesn't work that way). However, if you give it to the cells, Aß goes up. We published this in a JBC paper of 2003. Ceramide treatment increases the steady-state levels of BACE1 and activates ß-cleavage of APP (see ARF related news story; see also ARF related news story on lipid rafts, sphingomyelin synthesis, and AßPP processing).

Anne Fagan
Interesting, Luigi. Is there cell death in those mice?

Luigi Puglielli
Anne, this was a poster in Sorrento. I remember that plaques disappeared; minimal effect of tau (disappeared from the typical AD areas but was still evident in other regions). No apparent death of neurons (they seemed to be better).

Anne Fagan
Looks like I should have gone to Sorrento! :)

Luigi Puglielli
The poster is referenced in our Biochemical journal paper if you need the information.

Tom Fagan
So back to ceramide. What about downstream of sphingomyelinase? How stable is ceramide and might there be ways to accelerate clearance/metabolism?

Luigi Puglielli
I am not aware of mechanisms to regulate clearance. Once it has been activated (after sphingomyelin hydrolysis), ceramide is recycled back to sphingomyelin and the classical inactive-active second messenger cycle goes on.

Anne Fagan
Luigi, are there any links (biochemical, cellular, or other) between ceramide and ApoE?

Luigi Puglielli
Anne, the only one I seem to remember is that ceramide reduces the uptake of lipoproteins (see de Chaves et al., 2001). This was done in peripheral cell lines and primary neurons. The mechanism is receptor-mediated, so no particular effect on the lipoprotein itself. I should also add that there are additional links between ceramide and cholesterol metabolism (other than LDL uptake), but we haven't looked at them yet.

Anne Fagan
All, it's so hard for me to try to pull all of this together into some nice, cohesive hypothesis regarding the role of cholesterol/ApoE/ceramide in AD. There are lots of interesting connections. Guess it keeps us all in business, though!

Tom Fagan
Luigi, do you know if anyone has thought to measure ceramide in vivo using PET or some other imaging technique?

Luigi Puglielli
Tom, with the exception of animals, and postmortem tissue (AD and age-matched control brains)… no.

Tom Fagan
Barbara, at Pfizer do you have a particular interest in ceramide and/or AD? Well, for AD, I assume you do…

Barbara Tate
Every large pharma is interested in AD. If there were good targets that could come out of cardiovascular disease, that would be good.

Tom Fagan
Indeed! Luigi, what are your thoughts on the LAG connection (see background articles: The LAG protein compromises longevity in yeast and is a ceramide synthase)? Do we know how important that gene is in humans and do you think it impacts the aging process in us as well as in yeast?

Luigi Puglielli
The LAG connection is very interesting. As background, I need to remind you that it was generally accepted that the signaling active ceramide could only come from hydrolysis of cell-surface SM. However, some new report seems to suggest that also newly synthesized ceramide could have signaling activity (still needs to be confirmed). Now, LAG1 was identified as a longevity-assurance gene in yeast (references are in our ADJ review; see background text). Genetic disruption of LAG1 increases the lifespan of yeast. LAG1 seems to act as a long-chain (C26) ceramide synthase. A human homolog of LAG1 can correct the phenotype in yeast, so we do have a human LAG1 "activity." If we extrapolate from yeast to human (big jump, though!), you could argue that the increased production of ceramide is involved with the aging of the tissue (in our case, the brain). This conclusion seems to be supported by studies with primary cells in culture, where ceramide induces senescence and activates genetic/biochemical pathways involved with aging. However, we still must be aware that the aging process of the yeast or primary cells in culture does not completely mimic what happens during organismal aging in mammals.

James Semmel
Luigi, how does your research explain why women suffer from Alzheimer disease disproportionately more than men do?

Luigi Puglielli
James, ovariectomy (surgical menopause) in female rats is accompanied by down-regulation of TrkA and up-regulation of p75NTR in the brain. If you give back estrogens to the animals (orally), TrkA goes back up and p75NTR goes back down. So menopause seems to accelerate the TrkA-to-p75NTR switch. That would potentiate what we have described during aging in male mice. This is a nice connection (very exciting).

Anne Fagan
Luigi, you've alluded to your data regarding the effects of ceramide on AßPP processing. Any data looking at the converse: effects of Aß on ceramide metabolism?

Luigi Puglielli
Anne, no we haven't looked at the effect of Aß on ceramide production (see ARF related news story on the role of Aß in driving ceramide synthesis from sphingomyelin).

Tom Fagan
Luigi, has much work been done on human/mammalian LAG? Is it essential, for example? And before people start drifting off, (and without giving away unpublished results!), can you tell us a little about where this research is leading you, what are the questions you feel need to be addressed, etc.? You're obviously excited about the estrogen p75/TrkA connection, but are there other avenues that need to be explored?

Luigi Puglielli
Tom, the yeast KO is fine. It just lives longer (everybody would like that). I am not aware of KO in mice. Bear in mind that there are many ceramide synthases, so I wouldn't expect much "damage" from that. What the yeast story seems to indicate is that aging (in yeast!) is associated with increased production of long-chain ceramide…so maybe we should keep an eye on them. Interestingly, if you look at brain ceramide during aging, the ceramide that goes up is mostly long-chain (C24 and C26). I don't know why Mother Nature seems to go toward long-chain instead of the short-chains during aging. They might have different roles. We just don't know yet.

Tom Fagan
Anne, any interest in looking at ceramide as a biomarker?

Anne Fagan
To be honest, ceramide hasn't been on my radar, Tom. I'll need to catch up on it before deciding whether it may be an attractive candidate to pursue.

Tom Fagan
Anne, that's one of the reasons we have chats!

Anne Fagan
Yes, Tom, I've gotten many ideas from these chats. And collaborations! Thanks for the stimulating chat, everyone. I must be off. Until next time….

Luigi Puglielli
Tom, we are trying to identify all the molecular players of the signaling cascade (upstream and downstream). It is a nice connection between aging and AD.

Tom Fagan
Anne, before you head off, maybe the C24 versus C26 ratio would be the biomarker to follow.

Anne Fagan
Perhaps…I'll have to look into that. Thanks, Tom, Luigi, and everyone.

Barbara Tate
Thank you.

Luigi Puglielli
All the best to you.