Summary

A Live Webinar Discussion was held on 29 October 2008, with presentations from Ilya Bezprozvanny, Beth Stutzmann, Kevin Foskett, Kim Green, and Brian Bacskai and Kishore Kuchibhotla.

This live discussion began with a Webinar featuring a slide talk with audio provided via a telephone line. Following the talk, the audience moved to a chatroom for Q&A and discussion.

View/Listen to Webinar Recording

Note: We regret that the sound quality is low for the first 9 minutes of the presention.

Transcript:

Participants: Tom Fagan (Alzheimer Research Forum), Kevin Foskett (University of Pennsylvania), Brian Bacskai and Kishore Kuchibhotla (Massachusetts General Hospital, Charlestown), Ilya Bezprozvanny (University of Texas Southwestern Medical Center, Dallas), James Moyer, Kim Green (University of California, Irvine), Beth Stutzmann (Rosalind Franklin University/The Chicago Medical School), Carlos Villalobos (IBGM, University of Valladolid and Spanish Research Council [CSIC]), Kinga Michno, Ivan Goussakov (Rosalind Franklin University of Medicine and Science), Craig Atwood (University of Wisconsin, Madison), Volodymyr Rybalchenko (University of North Texas), Shreaya Chakroborty (Rosalind Franklin University).

Note: Transcript has been edited for clarity and accuracy.

Tom Fagan
Hi, everyone. We'll just wait a few minutes until folks have made their way in.

Kevin Foskett
Kishore, why do you think there is no neurodegeneration in the Alzheimer's mouse brain despite chronic high Ca2+ varicosities and blebs?

Brian Bacskai and Kishore Kuchibhotla
Kevin, we have found that the moderate calcium overload can persist for quite some time, suggesting that even though the dendrite might not be functioning properly, it has not completely degenerated. In addition, we have no evidence that this leads to cell body loss in the neurons—a sort of dendritic pruning.

Tom Fagan
We had one question submitted by e-mail: “Doesn't Ca compete with heavy metals such as cadmium or lead, and if so, what would the consequence be in AD pathogenesis?” Anyone want to field that one?

Ilya Bezprozvanny
Cadmium and lead have much higher affinity for calcium channels than calcium (1,000-fold), so Ca is not likely to compete them out.

James Moyer
Kishore, are you able to visualize dendritic degeneration (e.g., in response to challenge) using your in vivo approach? And does this susceptibility differ in transgenics?

Brian Bacskai and Kishore Kuchibhotla
James, are you referring to dendritic degeneration in a non-transgenic mouse or in response to an acute lesion?

James Moyer
Kishore, in response to glutamate challenge or some other acute insult.

Brian Bacskai and Kishore Kuchibhotla
James, we can see dendrites respond rapidly to high glutamate concentrations but have not compared that carefully to transgenic mice.

Beth Stutzmann
Kishore, I think you partially addressed my follow-up question to Kevin's, but have you looked further down into the cell body regions to determine extent of calcium overload?

Brian Bacskai and Kishore Kuchibhotla
Beth, we have not fully characterized the cell bodies in layer 5 just yet.

Tom Fagan
Everyone, a general question that was raised by Kim, I think: what is going on upstream? How do presenilins (PSs) affect so many different calcium channels?

Beth Stutzmann
Tom, I don't think PS affects all calcium channels equally; e.g., I see no effects whatsoever on spike-evoked calcium entry, suggesting that L-type channels are not affected. There is more evidence, although scattered, that Aβ affects calcium channels (e.g., downregulates voltage-gated P/Q calcium channel).

Kim Green
Beth, absolutely, which is why I think that presenilins are mediating their effects at the endoplasmic reticulum (ER) level.

Kevin Foskett
Tom, I think that what will be critical to distinguish is between primary and secondary mechanisms. A primary defect in one pathway (for example, hyperactivation of the IP3 receptor by mutant presenilins) may affect transcriptional events and cell bioenergetics that could lead to secondary aspects of calcium signaling dysregulation. I personally do not think that the multiple mechanisms described will (at the end of the day) be determined to be primary mechanisms.

Tom Fagan
Kevin, do you think PSs are interacting directly with the receptor, then?

Kevin Foskett
Tom, co-immunoprecipitation experiments are consistent with an interaction of both wild-type (WT) and mutant presenilins with the IP3 receptor. The electrophysiological results are most consistent with a direct interaction, but we have not demonstrated that using biochemical approaches, and the molecular basis of the co-IP is still unclear.

Ilya Bezprozvanny
Kevin, I am surprised you see effects of InsP3 R-mediated ER leak in permeabilized DT40 cell preparation. Where does InsP3 come from in these experiments?

Kevin Foskett
Ilya, we believe that IP3 is either retained inside the permeabilized cells or is produced locally. Indeed, unpublished data from our lab suggests that IP3 can be generated in the nuclear envelope. Therefore, in the endoplasmic reticulum, more generally.

James Moyer
Beth, in your triple transgenics, what happens to induction of long-term depression (LTD) in the presence of dantrolene? Is it exaggerated or affected in any way?

Beth Stutzmann
James, we are running those experiments right now, as a matter of fact. Ask me again in a week or so.... We are expecting to see differences between non-transgenic and 3xTg, particularly since LTD requires the recruitment of ER calcium stores.

Tom Fagan
Also, great to see some of our friends from Spain have joined us. Carlos Villalobos also had some interesting questions posed when he logged in. Carlos, do you want to address those?

Carlos Villalobos
Thank you Tom, I'll try. A little bit of Aβ now. I would like to know, according to the opinion of the panel, what is the main suspect pathway for Aβ-induced entry of calcium into neurons?

Brian Bacskai and Kishore Kuchibhotla
Carlos, there are many possible entry points, including: 1) Aβ as a calcium channel, 2) via NMDA channels, 3) via voltage-gated calcium channels. It will be possible to differentially test these possibilities using pharmacology and imaging approaches.

Carlos Villalobos
Brian and everyone in the audience, Nelson Arispe came up more than a decade ago with the amyloid channel, but still there is no consensus on that. What do you think?

Brian Bacskai and Kishore Kuchibhotla
Carlos, the amyloid channel is an intriguing hypothesis, and we think the prep we have is well suited to test it. A key issue is whether we can reliably and selectively block an Aβ channel in vivo.

Ilya Bezprozvanny
Carlos, I think Aβ channels described by Arispe are critical for Aβ-induced Ca elevation. It is also important that these channels form more easily if cells express phosphatidylserine on the surface; please see detailed discussion in TINS review (Bezprozvanny and Mattson, 2008) of this idea.

Brian Bacskai and Kishore Kuchibhotla
Carlos and Ilya, one critical point about Aβ and calcium overload is that not every dendrite responds to Aβ in the same way. What makes a given dendrite susceptible to the Aβ-induced overload compared to another dendritic process?

Ilya Bezprozvanny
Brian and Kishore, if I have to speculate I will argue that dendrites that have more phosphatidylserine on the surface are more susceptible for "amyloid attack" (based on Harvey Pollard’s and Arispe's work; see Lee et al., 2002).

James Moyer
Ilya, is there any evidence that when Aβ forms a calcium pore in the membrane, it is preferentially linked to ER sources of calcium, or other cytoskeletal proteins or kinases, etc.?

Kevin Foskett
Ilya, ER leak function might predict that downstream events from normal ER Ca leak signals would be inhibited, whereas hyperactivity of the IP3r or RyR might be expected to enhance those pathways. These ideas can be tested in in vitro and in vivo models.

Kinga Michno
I'm interested in knowing what the take is on data generated by Cristina Fasolato's group (see, for example, Zatti et al., 2006) demonstrating that the ER calcium (and Golgi) calcium levels are decreased when FAD and in fact WT PS are expressed? It seems to me that a more logical consequence to increased calcium channel activity would be decrements in internal stores. I'd appreciate hearing the panel’s thoughts on this.

Kevin Foskett
Kinga, our results also show a diminished ER Ca2+ store in agreement with Fasolato. We suggest it is due to hyperactivity of the IP3 receptor.

Ilya Bezprozvanny
Kinga, most data point to elevated Ca content in ER stores in FAD cells. From our experience MEF cells tend to "drift" a lot, so the ER Ca content changes a lot with passage number. We now confirmed elevated ER Ca level in primary hippocampal neuronal cultures from PS-FAD mutant mice.

Kinga Michno
Ilya, I'm scrambling to find the reference (unsuccessfully at the moment but I'll keep looking), but perhaps you will be familiar with much earlier work in PSEN1/PSEN2 double knockout cells which demonstrated that these cells actually exhibit decrements in internal stores content (using thapsigargin, I believe). Any comments?

Ilya Bezprozvanny
Kinga, yes, Cell Calcium paper from De Smedt's group (see Kasri et al., 2006). Please see detailed discussion on Alzforum when our Cell paper came out (see Tu et al., 2006 and related comments); both Fasolato and De Smedt posted their comments there. Bottom line is that MEF cells "drift" and different labs use different passage number. Also, thapsigargin is not a good way to determine store content as it depends on the leak.

Kinga Michno
Ilya, one last question for you. In your experimental approach describing PS’s passive calcium channel activity, can you be certain that in your method of isolating PS protein for your experiments you did not also pull down a calcium channel (since at least a couple have been shown to bind to PS).

Ilya Bezprozvanny
Kinga, we affinity purified His-PS1 on Ni column and reconstituted in liposomes and planar lipid bilayers to rule this out (see our paper, Tu et al., 2006).

Ivan Goussakov
Brian and Kishore, how do you really separate cell compartment soma, dendrites, and spines while using recently available time resolution?

Brian Bacskai and Kishore Kuchibhotla
Ivan, you can use the structure of the neuron to differentiate the soma, dendrite, and dendritic spine. What you see is what you can resolve.

Craig Atwood
An open question. I don't think anyone would disagree that Ca plays a role in Alzheimer's and other neurodegenerative diseases. Is Ca a downstream mechanism, induced by changes in APP/presenilin metabolism? And if so, then whatever regulates APP and presenilin metabolism would ultimately be regulating AD biochemical, pathological, and cognitive changes. In this respect, are drugs that target Ca metabolism really only acting as a band-aid for upstream causes of the disease?

Ilya Bezprozvanny
Craig, the question of amyloid and calcium—upstream or downstream—is the central question in my opinion. I do not think we know if it is abnormal calcium driving amyloid pathology or amyloid pathology driving calcium. Data point out on both so far.

Beth Stutzmann
Craig, I think calcium has an equal opportunity role to act as an early initiator of AD pathogenesis (as in FAD cases), and to accelerate existing histopathology and cognitive deficits (as an sporadic)—depending upon where/when in the cycle it enters.

Kevin Foskett
Craig, it is very compelling to me that calcium signaling disruptions are observed in a wide variety of cell types in a wide variety of labs upon expression of mutant presenilins. To me, this suggests a fundamental, proximal defect that could be upstream of pathology in AD.

Craig Atwood
Kevin, I absolutely agree that mutant presenilins can affect calcium metabolism and be a downstream mechanism driving FAD. But what is driving the sporadic forms of the disease? What are the age-related changes regulating Ca metabolism (and amyloid metabolism/PS1)?

Brian Bacskai and Kishore Kuchibhotla
Ilya, with changes that you see in Ca+ leak, how do you think the neuron (or neural system) would respond during intact brain development? Is it possible that the increased leak would be masked by some compensatory mechanisms?

Ilya Bezprozvanny
Brian and Kishore, absolutely. There are many compensation mechanisms in case of reduced ER Ca leak. I think increased expression of RyanR in aging PS1-M146V KI mice as observed by Beth (see Stutzmann et al., 2006) is one of them. Neurons try to reduce their ER Ca levels back to normal range, as ER stress response is activated when ER Ca is too high.

Craig Atwood
Ilya/Beth, irrespective of whether it is Ca or Aβ, would you agree that upstream mechanisms/causes are what regulate amyloid and Ca metabolism in the first place? Amyloid, for example, doesn't just drop out of the sky into the brain! And I suspect the same for Ca. So something upstream has to be regulating Ca and amyloid metabolism, or both. Does Ca metabolism change during cell division? There is a lot of evidence that pyramidal neurons in the AD brain have re-entered the cell division cycle and are attempting to divide.

Beth Stutzmann
Take, for example, the APP/tau mice, which do not show early calcium signaling defects but will generate plaques. I think calcium deficits will be downstream of this. Look at the PS mutations, and calcium far precedes AD pathology. Interestingly, I see similar levels of calcium dysregulation in mPS1 mice and 3xTg mice, up until ~12 months when there is significant plaque/tangle pathology. At this late pathology stage, there are even greater calcium responses in the 3xTg.

Kim Green
Craig, the development of sporadic AD is complex, and risk for the disease can be increased by a wide variety of events, but an aged brain is essential. Calcium may not be initiating the disease in all cases, but may play a role in some.

Ilya Bezprozvanny
Craig, there is ample literature on aging-related changes in neuronal calcium signaling. It seems to be generally consistent with enhanced Ca signaling as neurons age. We cite some of these in TINS review. My general feeling is that increased Ca signaling is a normal result of aging, but in AD it is accelerated (or more damaging to neurons).

Kevin Foskett
Craig, the FAD mutations provide clues that Ca2+ could be involved in sporadic AD. The same molecular mechanisms involving PSs and the IP3 receptor associated with FAD may be different in sporadic AD. For example, different Ca2+ regulatory proteins may contribute to Ca2+ homeostasis dysfunction in sporadic AD. Indeed, identification of CALHM1 as a risk factor for sporadic AD and its role in plasma membrane Ca2+ permeability is consistent with such a notion.

Kim Green
Beth, a transgenic mouse is preprogrammed to develop pathology due to the transgene promoters used. Therefore, they are useful for looking at the downstream effects of pathology, but not so useful for understanding what causes the pathology in the first place. We need models of sporadic AD, in which we can modulate calcium pathways and evaluate the effects on pathology.

Beth Stutzmann
Kim, true, but that was largely my point. I would expect to see downstream calcium effects in the APP/tau mice which can be linked to the histopathology (as I do in the aged 3xTg mice), but make no assumptions about how the histopathology got there. Regarding more sporadic models being needed, I couldn't agree more. There are ApoE4 mice, but as far as I know, there are few calcium studies on them. It is known that ApoE4 expression will result in increases in calcium, though.

Craig Atwood
Ilya/Kevin, I agree. But the question still remains as to what age-related changes are regulating Ca changes in the brain. Yes, they occur, but what is the mechanism? I know, for example, that reproductive hormones (sex steroids, gonadotropins, GnRH, activins) can regulate the cell cycle and also amyloid production and tau phosphorylation. Do these hormones affect Ca metabolism in the brain? Is anything known about how hormones may regulate Ca metabolism in the ovary or testis?

Ilya Bezprozvanny
Craig, we are now getting into the "realm" of Ca changes in aging. Maybe Alzforum can have a different discussion just on that issue. I do not think any of the panelists are experts on this (at least I am definitely not).

Kevin Foskett
Craig, your questions are good ones. However, one could look at it from the opposite point of view, and ask, What is it about aging that may make the brain more susceptible to damage by perhaps normal calcium signaling mechanisms?

Craig Atwood
Kevin, thanks. Yes, we did look at this angle (see Bowen and Atwood, 2004), hence my questions on the cell cycle and how aberrant re-entry into the cell cycle may affect how a cell responds to Ca.

James Moyer
Craig, we have seen similar increases in afterhyperpolarization (AHP) and enhanced frequency accommodation in aged rabbit CA1 neurons; these effects were differentially reduced by nimodipine in aged neurons.

Beth Stutzmann
Craig, I would refer you to Phil Landfield's and Oliver Thibault’s studies on changes in calcium with aging: basically, what they find is an upregulation in L-type calcium channels which are linked to increased Iahp currents, which serve to reduce membrane excitability. [Editor’s note: see, for example, Thibault et al., 2007.]

Craig Atwood
Beth, thanks, will look it up.

Volodymyr Rybalchenko
Oxidative stress is generally increased in cells of aging organisms.

Carlos Villalobos
Kishore, how can calcineurin promote further calcium increases?

Brian Bacskai and Kishore Kuchibhotla
Carlos, great question. This result surprised us, but there is at least a bit of literature supporting this in reduced prep (but still no mechanistic insight).

Tom Fagan
Everyone, do we have any consensus as to which Ca ER channel might be most important from a quantitative viewpoint? I imagine it might be difficult to compare across methodologies, but is there any indication as to which channel response is most robust?

Beth Stutzmann
Tom, the data is still quite preliminary on my end, but I'm finding that when I evoke an IP3 response (with caged IP3) or a RyR response (with caffeine), I am not really getting a “pure” channel response. When attempting to isolate either the RyR or the IP3R, I've found that I get a much larger mixed calcium response in the mPS neurons compared to non-transgenics, so that CICR seems upregulated/sensitized. But, this effect is much larger and extends throughout all neuronal compartments for the RyR activation.

Brian Bacskai and Kishore Kuchibhotla
Tom, I think your question is an important one. What is the magnitude of the calcium alterations based on ER stores refilling or leaking. How does this affect function? The difference between resting and dynamic changes in calcium may start to address this.

Kevin Foskett
Tom, there will not be a consensus regarding the importance of a particular ER Ca permeability until the published studies are reproduced in other labs, and until the very different hypotheses that the various mechanisms predict are tested.

Carlos Villalobos
Brian/Kishore, that is difficult to say. Among multiple things, a chance is that a cytosolic calcium overload depends not only on local calcium entry but also on local calcium uptake by, for instance, surrounding mitochondria.

Craig Atwood
Beth, how does ApoE4 affect calcium? I know ApoE4 carries cholesterol into cells for use in membranes and conversion to sex steroids. Do sex steroids alter Ca channels?

Beth Stutzmann
Craig, I'm going to cut and paste from my 2007 review to save time: The mechanism by which ApoE4 alters intracellular calcium is thought to involve a cell-surface LDL receptor mediated process. The increased calcium influx from extracellular sources most likely reflects activity-dependent activation, and not a steady-state upregulation, which would too quickly become neurotoxic. I believe the mechanism is still unclear, but can again refer you to the primary literature: Tolar et al., 1997; Moulder et al., 1999; Tolar et al., 1999; Veinbergs et al., 2002; Qiu and Gruol, 2003).

Shreaya Chakroborty
Volodymyr, your studies have shown that wild-type PS increases the open probability of RyR channels. Do you know if mutant PS keeps the RyR locked in an open configuration similar to the effect it has on the InsP3R (Kevin's work)?

Volodymyr Rybalchenko
Shreaya, we did not study the effects of mutant PS. Also, we tested the effects of the soluble cytoplasmic terminus of PS. There are almost no mutations found in cytoplasmic terminus related to AD. Our mechanism could probably play a role in the occurrence of sporadic AD.

Tom Fagan
I'd like to throw the perpetual "what's next" question. What are the crucial experiments that need to be done? Are there technical challenges? Funding challenges? In an "anything is possible world," what would you look at next?

Brian Bacskai and Kishore Kuchibhotla
Tom, one "what next" question is, what seeds an individual plaque to deposit where it does? Is calcium related to that pathological event or is it completely downstream?

Craig Atwood
Brian and Kishore, there is a whole literature on this that includes metal ions (Cu, Zn) and pH changes as the nucleating factors. See Huang et al., 2004, and also studies by Drs. Teplow and Landsbury regarding nucleation (see, for example, Roychaudhuri et al., 2008).

Tom Fagan
Brian/Kishore, so how would you get a plaque to form in culture? Has anyone considered that question?

Brian Bacskai and Kishore Kuchibhotla
Tom, why do we need to go to culture? We can watch in vivo.

Tom Fagan
Brian/Kishore, true, but I thought culture would give you more controls over manipulating the system.

Brian Bacskai and Kishore Kuchibhotla
Tom, you can't make a plaque in culture. Yet.

Kevin Foskett
Tom, the approaches taken by Stutzmann, and especially Brian and Kishore, to appropriate animal models will be the way to get at the pathophysiological relevance of the Ca hypothesis generally and any of the proposed mechanisms specifically. This will be expensive because it involves high tech and animals, so the challenge will be for the Alzheimer's Association to raise enough money to fund us well!!

Ilya Bezprozvanny
Tom, I think the most important direction is to sort out upstream/downstream issues for Ca/amyloid. This is a "chicken/egg" problem that needs to be sorted out, which is not easy due to limitations of available AD mouse models that do not have degeneration, only plaques.

Beth Stutzmann
Tom, I think that approaching this calcium “chicken and egg” problem can also be addressed from the opposite side of the fence. If we can normalize the calcium, will that reduce/prevent/have no effect on AD pathology? We can acutely normalize the exaggerated calcium with dantrolene, but what are the chronic effects? Sounds like a trivial experiment, but it's not.

Ilya Bezprozvanny
Beth, the problem is readout. When you say “AD pathology,” what do you plan to look at? Plaques?

Beth Stutzmann
Ilya, yes, very good point. Ultimately, I think people with AD want their memory and minds back, and aren't too interested if plaques are there. That would be the ultimate readout. There is still little consensus if Aβ has anything to do with cognitive impairments clinically. Tau is looking like a better histopathological correlate, as is synapse loss. Mice are already pretty cognitively impaired to start with, so using a well-correlated marker might be a better tool.

Craig Atwood
Brian and Kishore, I believe that someone did make plaques in cell culture a few years back, but the name escapes me.

Tom Fagan
Craig, really! Plaques in culture could be very useful, I'd have thought. Maybe the process is too hit-and-miss.

Brian Bacskai and Kishore Kuchibhotla
Craig, if we could make bona fide plaques in culture, we and almost everyone else would be using them every day.

Craig Atwood
Brian, look up work by Janusz Frackowiak (see Frackowiak et al., 2005).

Tom Fagan
Craig, ah, muscle cells. Wonder if this has been repeated.

Craig Atwood
He has a few papers on this general area, all of which I cannot remember, but this will get Brian headed in the right direction.

Carlos Villalobos
Audience, does anyone know the mechanism of neurotoxicity by Aβ fibrils?

Craig Atwood
Carlos, according to Mark Mattson, by upregulating Ca entry into neurons and leading to apoptosis.

Carlos Villalobos
Craig, do you know if additional mechanisms have been claimed for Aβ fibrils?

Craig Atwood
Carlos, yes, look up Liu et al., 2005 intro and last figure for an overview of p25/35 and tau mechanisms. There are oxidative mechanisms also.

Kim Green
Ilya, while animal models don't have the neuronal loss like human AD, they do have significant cognitive decline. This suggests that the pathology influences cognition (probably at the synaptic level), rather than just through neurodegeneration, and gives us a pathway we can explore and then hopefully block!

Tom Fagan
Thanks all for the thoughts on future directions. Beth, is there an alternative to dantrolene?

Beth Stutzmann
Tom, looking into it....

Ilya Bezprozvanny
Tom, me, too.

Craig Atwood
Beth, you get at the original question I posited, if you can stabilize Ca but still see functional loss, then Ca is not the answer. I guess that is the key Ca experiment to do.

Beth Stutzmann
Craig, that is a fundamental question (that may force some of us to switch jobs....). But yes, it is certainly possible that the calcium dysregulation is an epiphenomenon that has little to do with AD. I don't think this is the case, but perhaps calcium dysregulation is not specific to AD, as similar changes have been reported in PD, HD, and other neurodegenerative diseases.

Carlos Villalobos
Beth, it may be that normalizing calcium release/entry is not enough if intracellular calcium buffers are being lost.

Ivan Goussakov
Brian and Kishore, calcium transient in dendrite, which starts from normal glutamatergic synaptic activation, should be dependent on synaptic density. Ca++ diffusion (even diffusion only) cannot be followed up in time with recent Ca imaging technology. Is it difficult to differentiate between compartments?

Brian Bacskai and Kishore Kuchibhotla
Ivan, yes, there are limitations on spatial and temporal resolution, but the state of the art is advancing rapidly. We can easily resolve spines from parent dendrites and can scan at kHz rates in subfields if necessary.

Ivan Goussakov
Brian and Kishore, so I will be looking for the future when the scan rates can exceed 1 to 5 KHz per line scan and for fluorophores that will be able to give at least 10 photons per 0.3 ms.

Brian Bacskai and Kishore Kuchibhotla
Ivan, I think I'm starting to understand what you're getting at. The limitation for the studies you're proposing is in the dyes themselves. They allow ms time resolution at best. Perhaps voltage sensitive dyes would be more appropriate?

Ivan Goussakov
Brian and Kishore, right spines and dendrites need to be separated not only in space (by frame imaging) but in time in order to see time sequence of Ca++ transients. So can we talk about [Ca++] difference in compartments while working with averaged images (10-30 ms) of branches covered by spines?

Brian Bacskai and Kishore Kuchibhotla
Ivan, we can talk offline, but we haven't tried yet.

Tom Fagan
Brian and Kishore, Ilya mentioned in his talk about a potential Ca influx channel—not sure if that has come up in the discussion here—but do you have thoughts?

Ilya Bezprozvanny
Brian and Kishore, yes, it looks like we all have to start running "memory paradigms" in mice, no way around it.

Brian Bacskai and Kishore Kuchibhotla
Ilya, the behavioral assays can be informative, but have limitations....

Ilya Bezprozvanny
Brian and Kishore, what is a good readout, then? Short of a human clinical trial?

Brian Bacskai and Kishore Kuchibhotla
Ilya, all of the approaches in mice or culture (calcium levels, function, structure, and behavior) are helpful...but you might be right that the only way to really know if any of these are important in AD is if they actually work in the clinic. We have to remind ourselves that we're working with models.

Ilya Bezprozvanny
Brian and Kishore, that is why I tested Dimebon (see Wu et al., 2008), which was claimed to be very effective in human AD Phase 2 trials (see Doody et al., 2008). But effects of Dimebon on Ca signaling required a 50 μM dose—not likely to be physiological. Instead we found that Dimebon hits a number of receptors—histamine, serotonin, adrenergic. Most likely effects on these receptors are responsible for clinical effects on Dimebon as a “cognitive enhancer” in short-term trials.

Shreaya Chakroborty
Volodymyr, how would you test your hypothesis that your WT PS-RyR mechanism could play a role in sporadic AD? Could you use a similar approach as in your 2007 paper?

Volodymyr Rybalchenko
Shreaya, this would be a disclosure of the commercially valuable information. :)

Shreaya Chakroborty
I understand, but I had to try. :)

Kevin Foskett
All, a fundamental issue for those of us studying the role of Ca signaling in the pathogenesis of AD is to prove to the amyloid field that this is a relevant mechanism(s). A key aspect going forward is to establish the relevance of the Ca hypothesis in a convincing manner. Clearly, it seems to me, animal models will be the key. The mouse offers clear benefits, as well as limitations. Is there a prospect for new animal models to complement the mouse?

Kim Green
Kevin, I think that Brian and Kishore’s work has shown that calcium is downstream of pathology, and we can work with that on existing models. We will need new models to prove that calcium can influence pathological progression in vivo, and I think we are still some time away from that.

Beth Stutzmann
Kevin and all, yes, operative word I think is “mechanism,” which is why the single channel studies are so important. Historically, the Aβ group is protein biochemistry based, and has much to learn about channel biophysics and why it's important. I cannot reach that limit of resolution in my studies (nor can Brian and Kishore), but it is critical information that can be integrated with the next level of analysis.

Tom Fagan
All, someone just mentioned the clinic. This might be a very naive question, but are there any known conditions, drugs, etc., that affect calcium homeostasis and could they be worthy of study in this context?

Carlos Villalobos
Tom, a few days ago we saw no response (calcium increase) of vascular smooth muscle cells to Aβ oligomers, but they were a cell line, not primary cells.

Tom Fagan
Carlos, "days ago"; that's hot data!

Carlos Villalobos
Is there a better audience to release it to?

Ilya Bezprozvanny
Tom, I mentioned Memantine and Dimebon in my presentation. These are only two clinical drugs tested in clinical AD trials which have a Ca connection. Do not know if there are any more that are being tested in clinic.

Carlos Villalobos
Although NSAIDs are not generally considered as calcium antagonists, in fact there are: NSAIDs that are mitochondrial calcium antagonists and protect both in vitro and (likely) in vivo.

Tom Fagan
Folks, I hate to break up a wonderful relationship(s), and the discussion is going gangbusters, but I think we do have to wrap up by 2:30. So before we start signing off, I just want to thank all the speakers again for a wonderful job. I was afraid we'd run way over time on the Webinar, but it was really well done. Kudos to all. And thanks to everyone who joined in the typed discussion. I think this was really informative. We will prepare a transcript that will be circulated and eventually posted on the chat background page. The Webinar will also be available for viewing at some point in the near future, I believe. If anyone has any summary statements or points they want to make, go for it!

Brian Bacskai and Kishore Kuchibhotla
Tom and Nico and June, thanks for having us. This was informative and fun.

Ilya Bezprozvanny
Tom, thank you and Alzforum for putting this together. It was really fun!

Kim Green
Thanks to everyone!

Beth Stutzmann
Thanks for the opportunity. Hopefully this will bring more attention to the AD research world regarding the role of calcium.

Tom Fagan
Thanks, all. We look forward to more exciting data soon. We've undoubtedly not exhausted this discussion, so another may be in the cards before long.

Craig Atwood
 Ciao.

Ivan Goussakov
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By Tom Fagan

Calling all confirmed and potential Calcinists. Though the calcium hypothesis of AD has been with us for nearly two decades, it is only within the last few years that the theory has blossomed, mostly through links to the endoplasmic reticulum (ER). Work from Ilya Bezprozvanny’s lab at the University of Texas Southwestern Medical Center, Dallas, suggested that presenilins, best known as a component of the γ-secretase that released amyloid-β, are actually part of the long sought ER calcium leak channel (see ARF related news story). Beth Stutzmann, Rosalind Franklin University, Chicago, Illinois, has a different take on the presenilin-calcium connection. Her work suggests that presenilins aberrantly modulate the ER ryanodine receptor, and that in a mutant presenilin (PS) background, that channel releases more calcium into the cytosol (see ARF related news story).

Very recent data from Kevin Foskett’s lab at the University of Pennsylvania, Philadelphia, showed that PSs interact with the inositol 1,4,5-trisphosphate receptor (InsP3R) calcium release channel, again in the ER, releasing calcium that then leads to enhanced Aβ production (see ARF related news story). And finally, Kim Green at Frank LaFerla’s lab at University of California, Irvine, recently published data linking PSs to the SERCA pump that drives calcium into the ER (see ARF related news story). And if all this presenilin-ER-calcium news isn’t enough to convince you that there is a connection between AD and calcium, recent analysis from Brian Bacskai’s group at Massachusetts General Hospital, Charlestown, has linked Aβ to calcium toxicity in vivo. Kishore Kuchibhotla and colleagues used in vivo imaging to show that cells in the vicinity of Aβ plaques accumulate intracellular calcium to toxic levels, leading to degeneration of dendritic spines (see ARF related news story).

Do these somewhat disparate observations fit into a single calcium hypothesis of AD? Join Alzforum for a Live Webinar Discussion on 29 October, 12:30 p.m., when Bezprozvanny, Stutzmann, Foskett, Green, and Bacskai and Kuchibhotla will give brief presentations to be followed by a group discussion. As always, your comments are welcome.

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  1. Using the exact techniques that we used in the Ito studies to look at ER calcium in fibroblasts bearing presenilin mutations, we also examined ER calcium in lines bearing the APP670/671 lines. (No presenilin mutation; single APP mutation; Gibson et al., 1997.) In the lines bearing APP mutation we found a decrease in release of ER with bradykinnin rather than the increase observed in the PS1 lines. Is the role of ER different in cells with single APP mutations?

    References:
    Gibson GE, Vestling M, Zhang H, Szolosi S, Alkon D, Lannfelt L, Gandy S, Cowburn RF. Abnormalities in Alzheimer's disease fibroblasts bearing the APP670/671 mutation. Neurobiol Aging. 1997 Nov-Dec;18(6):573-80.
    Abstract

  2. Our Ca-sensitive microelectrode measurements show that resting Ca is significantly elevated in adult cultured neurons that express APPswe/β-amyloid in vivo as compared to those from non-transgenic control mice. The elevation appears to be due mainly to excessive influx (through the plasmalemma). In these neurons, ryanodine receptor blockade does not lower internal Ca, but IP3R blockade does (see Lopez et al., 2008).

    References:

    . Increased intraneuronal resting [Ca2+] in adult Alzheimer's disease mice. J Neurochem. 2008 Apr;105(1):262-71. PubMed.

  3. We also have evidence that calcium dynamics are dysregulated in neurons from two-year-old rats (Brewer et al., 2006), so the calcium hypothesis of AD may intersect with the aging in non-familial AD as well.

    References:

    . Prevention of age-related dysregulation of calcium dynamics by estrogen in neurons. Neurobiol Aging. 2006 Feb;27(2):306-17. PubMed.

  4. Several recently appeared papers on the AD-Ca2+ relation have undoubtedly shown that the issue has come to ripeness. The data showing a significant increase of free Ca2+ inside neurons close to amyloid plaques, coupled with neuronal degeneration and functional impairment, provide a convincing milestone around which previous research can be coagulated and new research can start to give further knowledge on related issues. The generic cytotoxic effects of prefibrillar amyloids, and in particular the toxicity of Aβ diffusible ligands, and the role of membrane cholesterol in modulating such toxicity may thus gain new significance. Actually, there is increasing support for the scenario whereby Aβ diffusible ligands exert their cytotoxicity in surrounding neurons by affecting cell membrane permeability to free Ca2+ in a cholesterol-dependent way with subsequent Ca2+ ingress into the mitochondria, triggering off oxidative stress and activation of the apoptotic pathways. The scenario accommodates many recently reported data suggesting that Aβ oligomers would increase Ca2+ by affecting the function of specific Ca2+ channels either in the plasma membrane (as shown by the recently published data on the NMDA and AMPA receptors) and/or by disrupting non-specifically membrane architecture. Both effects, together with APP processing, seem to be modulated by the content of membrane cholesterol as shown by many data, including those focused on the protective role of Seladin-1 against cell damage by soluble Aβ (and other protein) aggregates. Of course, AD is a multifactorial disease even at the molecular and cell biology levels, and other effects must be taken into account including those resulting from glia activation by the plaques.

    References:

    . Abeta plaques lead to aberrant regulation of calcium homeostasis in vivo resulting in structural and functional disruption of neuronal networks. Neuron. 2008 Jul 31;59(2):214-25. PubMed.

    . beta-Amyloid-induced dynamin 1 degradation is mediated by N-methyl-D-aspartate receptors in hippocampal neurons. J Biol Chem. 2006 Sep 22;281(38):28079-89. PubMed.

    . Abeta oligomers induce neuronal oxidative stress through an N-methyl-D-aspartate receptor-dependent mechanism that is blocked by the Alzheimer drug memantine. J Biol Chem. 2007 Apr 13;282(15):11590-601. PubMed.

    . Nonspecific interaction of prefibrillar amyloid aggregates with glutamatergic receptors results in Ca2+ increase in primary neuronal cells. J Biol Chem. 2008 Oct 31;283(44):29950-60. PubMed.

    . The role of seladin-1/DHCR24 in cholesterol biosynthesis, APP processing and Abeta generation in vivo. EMBO J. 2006 Jan 25;25(2):432-43. PubMed.

    . Seladin-1/DHCR24 protects neuroblastoma cells against Abeta toxicity by increasing membrane cholesterol content. J Cell Mol Med. 2008 Oct;12(5B):1990-2002. PubMed.

  5. The best time to attack the dementia of AD is before it begins. Our data concerning abnormal elevation of resting calcium stand in strong distinction to other kinds of neuronal calcium measurements in two critical regards: 1) the mice from which the neurons were taken were young adults (~one year), and 2) in the transgenic strains we used, there are no extracellular amyloid deposits at the ages examined.

    <p>Thus, these data (Lopez et al., 2008) strongly suggest that either very low levels of extracellular amyloid and/or intracellular amyloid are responsible for elevating resting calcium in neurons of young mice. Modest but significant elevation of intracellular calcium in neurons has profound implications for neuronal function, synaptic plasticity, and pathogenesis. These data suggest that resting calcium may be a critical physiological index of early pathogenesis. This may be significant for treatment development for MCI and for presymptomatic and very early stages of AD dementia.

    References:

    . Increased intraneuronal resting [Ca2+] in adult Alzheimer's disease mice. J Neurochem. 2008 Apr;105(1):262-71. PubMed.

References

News Citations

  1. Presenilins Open Escape Hatch for ER Calcium
  2. San Diego: “Calcinists” See Years of Compensation Prior to Alzheimer’s
  3. Channel Surfing—Two Studies Strengthen Calcium-AD Connection
  4. Pump It Up—Presenilins Linked to ER SERCA Activity
  5. More Calcium News: Plaques Cause Dendrite Damage via Ion Overload

Paper Citations

  1. . Neuronal calcium mishandling and the pathogenesis of Alzheimer's disease. Trends Neurosci. 2008 Sep;31(9):454-63. PubMed.
  2. . Annexin 5 and apolipoprotein E2 protect against Alzheimer's amyloid-beta-peptide cytotoxicity by competitive inhibition at a common phosphatidylserine interaction site. Peptides. 2002 Jul;23(7):1249-63. PubMed.
  3. . Presenilin mutations linked to familial Alzheimer's disease reduce endoplasmic reticulum and Golgi apparatus calcium levels. Cell Calcium. 2006 Jun;39(6):539-50. PubMed.
  4. . Up-regulation of inositol 1,4,5-trisphosphate receptor type 1 is responsible for a decreased endoplasmic-reticulum Ca2+ content in presenilin double knock-out cells. Cell Calcium. 2006 Jul;40(1):41-51. PubMed.
  5. . Presenilins form ER Ca2+ leak channels, a function disrupted by familial Alzheimer's disease-linked mutations. Cell. 2006 Sep 8;126(5):981-93. PubMed.
  6. . Enhanced ryanodine receptor recruitment contributes to Ca2+ disruptions in young, adult, and aged Alzheimer's disease mice. J Neurosci. 2006 May 10;26(19):5180-9. PubMed.
  7. . Living and dying for sex. A theory of aging based on the modulation of cell cycle signaling by reproductive hormones. Gerontology. 2004 Sep-Oct;50(5):265-90. PubMed.
  8. . Expansion of the calcium hypothesis of brain aging and Alzheimer's disease: minding the store. Aging Cell. 2007 Jun;6(3):307-17. PubMed.
  9. . Neurotoxicity of the 22 kDa thrombin-cleavage fragment of apolipoprotein E and related synthetic peptides is receptor-mediated. J Neurosci. 1997 Aug 1;17(15):5678-86. PubMed.
  10. . Analysis of a novel mechanism of neuronal toxicity produced by an apolipoprotein E-derived peptide. J Neurochem. 1999 Mar;72(3):1069-80. PubMed.
  11. . Truncated apolipoprotein E (ApoE) causes increased intracellular calcium and may mediate ApoE neurotoxicity. J Neurosci. 1999 Aug 15;19(16):7100-10. PubMed.
  12. . Neurotoxic effects of apolipoprotein E4 are mediated via dysregulation of calcium homeostasis. J Neurosci Res. 2002 Feb 1;67(3):379-87. PubMed.
  13. . Interleukin-6, beta-amyloid peptide and NMDA interactions in rat cortical neurons. J Neuroimmunol. 2003 Jun;139(1-2):51-7. PubMed.
  14. . Trace metal contamination initiates the apparent auto-aggregation, amyloidosis, and oligomerization of Alzheimer's Abeta peptides. J Biol Inorg Chem. 2004 Dec;9(8):954-60. PubMed.
  15. . Amyloid beta-protein assembly and Alzheimer disease. J Biol Chem. 2009 Feb 20;284(8):4749-53. PubMed.
  16. . Extracellular deposits of A beta produced in cultures of Alzheimer disease brain vascular smooth muscle cells. J Neuropathol Exp Neurol. 2005 Jan;64(1):82-90. PubMed.
  17. . Amyloid-beta-induced toxicity of primary neurons is dependent upon differentiation-associated increases in tau and cyclin-dependent kinase 5 expression. J Neurochem. 2004 Feb;88(3):554-63. PubMed.
  18. . Evaluation of Dimebon in cellular model of Huntington's disease. Mol Neurodegener. 2008 Oct 21;3 PubMed.
  19. . Effect of dimebon on cognition, activities of daily living, behaviour, and global function in patients with mild-to-moderate Alzheimer's disease: a randomised, double-blind, placebo-controlled study. Lancet. 2008 Jul 19;372(9634):207-15. PubMed.

Further Reading

Papers

  1. . Neurons regulate extracellular levels of amyloid beta-protein via proteolysis by insulin-degrading enzyme. J Neurosci. 2000 Mar 1;20(5):1657-65. PubMed.