A very clear presentation, from the point of view of a plant ecologist! But I am not clear about the relation (if any) between the oligomers and plaques — are they forerunners?
Judges’ Queries and Presenter’s Replies
Presentation Discussion
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There are probably many forms of oligomers. Some oligomers are “on pathway” to become protofibrils, fibrils, and eventually plaques (forerunners), but some are probably not (ie. in a “dead end”, unable to transition to fibrils). Its thought that the “on pathway” oligomers are the toxic ones. Its not yet clear which of the several species are the most problematic.
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Further posting is closed as the event has ended.
Jon Kellar
Faculty: Project Co-PI
Could you use Fick’s Second Law to determine ABO diffusion rates? Why or why not?
Andrew Russell
Hi Jon! Thanks for the question.
My understanding of Fick’s Second Law leads me to believe that any model of ABO diffusion that is based on Fick’s Laws would be deeply flawed. There would be several factors in the complex system of a living cell that would be hard to properly account for. The crowded environment of the cytosol causing steric, repulsion, and cage effects, the heterogeneous “solvent” through which it passes, and the likelihood that the ABO’s will have some interactions with other molecules/membranes, are all factors should be included in an informative model and would be difficult to account for with only Fickean diffusion models.
Marc Porter
Faculty: Project PI
What are the pore forming assays you plan to use in future work? What is their purpose?
Andrew Russell
Hello Marc! I’d like to start exploring the potential pore-forming abilities of ABOs with liposome flux assays. In these assays, liposomes are generated in one buffer (so that this first buffer is INSIDE the liposome) and then, after ultracentrifugation, put into a different buffer which can then be monitored for leakage of the first buffer. For example, an electrolyte-containing or low pH buffer can be trapped inside vesicles, ABO can be added during the liposome formation or after (since it may or may not be able to insert into an established membrane), and when these vesicles are placed into a solution with less electrolyte or different pH, we will be able to monitor changes in conductance or pH, respectively, to get a sense of the leakage. I admit it is possible that other components of cellular membranes may play roles in ABO pore formation in the disease, but the purpose of an experiment with such a reductionist approach is to determine if ABOs are sufficient to disrupt the integrity of a lipid bilayer.
Adriane Ludwick
Faculty: Project Co-PI
The next direction in your work is very clear…cytotoxicity testing. Should the testing not show toxicity, what would be your next steps? What rationale would you offer for the lack of toxicity?
Andrew Russell
Thanks for your interesting question, Adriane. Since there is quite a bit of evidence testifying to the toxicity of ABOs in AD, I would see a lack of demonstrable cytotoxicity as an indication of one or the other of two possibilities. 1) the ability of the ABO core to damage cells is masked or altered by the split-luciferase tags (in other words, the ABO is relevant but shielded), or 2) this method of ABO production gives non AD-relevant forms of ABOs. In either case, I would interpret it as a fault of my particular approach and not as proof that ABOs are non toxic. If there is no measurable cytotoxicity, I would pursue a ABO expression and purification method that allows for ABO’s to be stabilized by buffer conditions without the potentially confounding additional domains.
Peter Gannett
Faculty: Project Co-PI
Is the Abeta protein you express full length or truncated. If truncated, by how much and might this alter the interpretation of the data.
Andrew Russell
Hello Peter! ABeta peptides are, as you hinted at by your question, ALL truncated. The protein APP (amyloid precursor protein) is a single pass transmembrane protein that is sequntially cleaved Beta- and Gamma-secretase to generate ABeta peptides of different lengths (38, 40, 42, and 43 amino acids, for example). We are focused on ABeta peptides, specifically the 42 amino acid variety, which has been demonstrated to be more toxic, and not the non-truncated APP protein. I’m not sure I’ve answered your question, though, if I misread your intent. Two other ways I might interpret your question are “Is your ABeta construct BEING truncated after expression by endogenous proteases?” or “Will the length of your peptide (specifically 42aa and NOT 43, 40, or 38) change how you interpret your data?”. To answer the first, I’d have to admit that, at the moment, I do not know if bacterial proteases are targeting my construct. As for the second, I think it’s quite possible that ABeta oligomerization in patients IS affected by heterogeneity in the ABeta peptide population, but that this question is outside the scope of this work (but, indeed, is a complication that it would behoove us to look into.)
Antal Jakli
Faculty: Project Co-PI
Are AB oligomers also relevant in real Alzheimer, or only AB?
Andrew Russell
AB oligomers are definitely relevant to real AD and have been observed in patients. Actually, ABeta alone (as a monomer) is not really thought to be toxic.