Participants: Ruth Itzhaki, Keith Crutcher, Frank LaFerla, and June Kinoshita

Friday, January 23, 1998

Keith Crutcher: "Hi Ruth. Sorry I'm late. What is the current status of confirmations of your findings?"

Ruth Itzhaki: "We have examined frontal and temporal cortex from 15 more ADs and 4 more age-matched normals, and the results tally exactly with our previous ones (all done blind)."

Keith Crutcher: "You indicated that E4 is not an independent risk factor in your sample. Does that conflict with other reports?"

Ruth Itzhaki: "Also we've done 25 more cold sore people and 40 more non-cold sore people and again the results agree"

June Kinoshita: "I'm not familiar with the cold-sore study. Could you describe it briefly?"

Ruth Itzhaki: "Don't think so because E4 alone is not essential nor sufficient, in other studies."

June Kinoshita: "It's also my impression that E4 is probably only a risk factor if there is some other as-yet unknown risk factor present."

Ruth Itzhaki: "June, we found that apoE4 is a strong risk factor for cold sores: almost everybody carries latent HSV1 in the peripheral nervous system but only some people get cold sores - for previously unknown reasons. Now we've found e4 is, so in both CNS and PNS, HSV1 and E4 together are very damaging."

June Kinoshita: "That's very interesting. I guess the next question is, have you found a history of cold sores among AD patients?"

Ruth Itzhaki: "Very good question but impossible to find out as medical records re: cold sores don't exist, and patients wouldn't know, and their carers might not remember, especially as the frequency of occurrence usually drops with age"

Keith Crutcher: "I don't know enough about viral infections. Can you determine how acute or chronic the infection is with your methods?"

Ruth Itzhaki: "Yes, we have found that the infection is latent, i.e., at the stage we examine it, only the viral genome is there, not any whole viruses. This is the state of HSV1 usually in the PNS - except when it reactivates and then becomes an acute infection."

June Kinoshita: "Do you think E4 raises the risk that HSV1 will get into the CNS in the first place?"

Ruth Itzhaki: "No, because many aged normals are HSV1-positive in brain but few have an E4 allele"

June Kinoshita: "I see."

Keith Crutcher: "So what is your current view of why they interact?"

June Kinoshita: "What do you think of butyrylcholinesterase (K allele) as a co- risk factor with ApoE4?"

Ruth Itzhaki: "Keith, we still think that either they compete for entry into neuronal cells - with E4 competing less well and so allowing more spread of virus, or perhaps E4 repairs virus-induced damage less well, or HSV1 might interact with expression of apoE, etc"

Frank LaFerla: "Hi all"

Ruth Itzhaki: "Sorry, June, don't know, but isn't the K allele frequency low in the population?"

June Kinoshita: "Is there evidence that HSV1 and ApoE share a binding site?"

Ruth Itzhaki: "Hi, Frank"

June Kinoshita: "Hi frank. Glad to have you aboard!"

Frank LaFerla: "Thanks, took a while to figure out. Hope I am not too late."

Ruth Itzhaki: "Yes, both go for heparan sulphate glycoproteins on the cell surface"

June Kinoshita: "Hi Ruth, re: K allele, I think you're right. Still its presence together with E4 seems to raise the risk of AD very high. It's quite intriguing."

Ruth Itzhaki: "Could then explain only a limited number of AD cases"

Keith Crutcher: "Ruth, I know that HSV is latent in peripheral neurons. Is it also mainly in neurons in the CNS?"

Frank LaFerla: "There is a report by Becker claiming that an HSV protein shares resemblance to apoE, although it is not as apparent as the author claims."

Ruth Itzhaki: "Keith, we don't know - will have to try in situ PCR though will be very hard to detect!"

Ruth Itzhaki: "Frank, I agree. We think the Becker paper is weird"

June Kinoshita: "I have the impression that HSV1 may preferentially damage certain cortical regions (e.g. temporal lobe). Is that true?"

Frank LaFerla: "Although I think there may be something to the fact, that the gB protein (which is the one he claims resembles apoE) is the same that we find beta-amyloid like region.3"

Frank LaFerla: "Ruth, how do you think that HSV acts in AD?"

Frank LaFerla: "Do you think that it is an initiator of the disease, a cofactor for progression, etc.?"

Ruth Itzhaki: "Yes, good point , but if you look at the sequences they hardly share anything"

Ruth Itzhaki: "Your results are more convincing. as to HSV1 action, it damages or kills cells in many ways- takes over protein synthesis , takes over cell membranes etc, as you probably know."

Ruth Itzhaki: "I think it could well be an initiator but we really don't know" Bowden grins evilly.

June Kinoshita: "Do you find HSV1 genes in all types of CNS neurons, or is there any correlation between viral presence and neurons that are affected in AD?"

Ruth Itzhaki: "We don't yet know if it's in neurons or glial cells - will have to try in situ PCR. It would be nice to look for an association with damaged cells."

Frank LaFerla: "But if HSV acts early on, perhaps as an initiator, it may be difficult to find evidence of HSV in damaged cells, don't you think?"

Ruth Itzhaki: "Do you mean because they'll have died?"

Frank LaFerla: " Yes."

Frank LaFerla: "By the way, we have replicated your PCR results with the samples that you sent us and now working on the UCI brain repository samples. The PCR is very technically challenging, unlike other PCR methods."

Ruth Itzhaki: "It could perhaps cause limited damage and not cell death, in some cells"

Frank LaFerla: "I agree. I think its a good experiment to do, especially since it is not clear, when HSV acts in the pathogenesis of AD."

Ruth Itzhaki: "That sounds very interesting. What exactly have you replicated - detection of HSV1 and correlation with apoE4 in ADs?"

Frank LaFerla: "Just the detection of HSV1, now going on to the correlation. We have been experimenting with different protocols to try and increase the signal intensity"

Frank LaFerla: "Doing some goofy things like trying to linearize the DNA first but we found that your exact protocol gives the strongest signal strength."

Ruth Itzhaki: "If you remember I said it's very hard - and this workshop was to be about the problems of accurate detection."

Frank LaFerla: "I remember. Have you increased your sample sizes further?"

Ruth Itzhaki: "yes, we have examined a further 15 ADs and 4 normals, using frontal and temporal in each. The results agreed completely with our Lancet study"

June Kinoshita: "I'm afraid it's 9AM here in California and I need to drive my daughter to school. Please feel free to continue this discussion as long as you like. It's being recorded and the log will be posted on the Forum web site. Thank you all for participating. Thanks especially to Ruth for the workshop."

June Kinoshita applauds Ruth Itzhaki fervently.

Ruth Itzhaki: "Many thanks June. Hope your daughter isn't waiting"

Keith Crutcher: "Thanks Ruth!"

Ruth Itzhaki: "Frank, did you ever receive our emails of a few months ago?"

Ruth Itzhaki: "Keith, it was a pleasure"

Frank LaFerla: "No, there has been a lot of chaos here with moving into the new building. Can you resend? I will email you soon. My email has been recently straighten up since moving into the new neuroscience bldg."

Ruth Itzhaki: "Frank, they were just to ask about your progress. We'd like very much to hear from you. Wish we could get others to try to repeat. Surely people should be interested as it could lead to prevention or treatment."

Frank LaFerla: "I agree. Especially since treatments already exist for HSV1. I think there is alot of negative bias since this hypothesis was considered in the 1980's and nothing much came of it then."

Ruth Itzhaki: "Yes, that's because there was never reproducible detection of virus in pre-PCR days."

Frank LaFerla: "You are right. Now several world-wide laboratories have rendered that a moot point! What has been the reception that you have been accorded regarding your findings especially since it appeared in Lancet?"

Ruth Itzhaki: "Do you mean that they will try to repeat the search for virus, using PCR?"

Frank LaFerla: "I mean, how do you think the field views the hypothesis that HSV may play a role in AD?"

Ruth Itzhaki: "Alas there's much hostility - though not with any scientific arguments, or there's lack of interest. I know of one very well-funded group that just couldn't detect virus reproducibly, and wasn't prepared to spend more time trying."

Frank LaFerla: "Can I ask a few technical questions on here or is that taboo?"

Ruth Itzhaki: "Yes, quite Ok"

Frank LaFerla: "Good. Why do you use CsTFA instead of CsCl to prepare your gradients and do you think it makes a difference? CsTFA is so expensive!"

Ruth Itzhaki: "Because the RNA goes down as a pellet, and we sometimes want to use it, not just DNA"

Frank LaFerla: "I like that as well since we are planning to do RT-PCR on gB. Have you ever looked for expression of HSV genes in AD?"

Ruth Itzhaki: "I agree, but I bet we're more hard up than you!"

Ruth Itzhaki: "Yes, TK, and LAT"

Frank LaFerla: "Our funding situation is ok, but it is still expensive."

Frank LaFerla: "Have you published those findings yet?"

Ruth Itzhaki: "Which? You know our Lancet paper, and the earlier ones from here mention the RNA work. There's also on VZV."

Frank LaFerla: "I know of the Lancet work. But I didn't think you were looking by RT-PCR for TK or LAT in the Lancet, but I may be mistaken."

Ruth Itzhaki: "You're right, they were described in an earlier paper of ours; I'd have to look them up to check which"

Frank LaFerla: "What type of PCR machine are you using?"

Ruth Itzhaki: "Techne"

Frank LaFerla: "Do you think that apoE serves as a receptor for HSV1?"

Ruth Itzhaki: "We wondered if there is a direct interaction but haven't yet examined that; do you mean when apoE has attached to the cell membrane?"

Fmlsaferla: "Yes. Does it serve as a viral receptor to allow entry of HSV1 into cells?"

Ruth Itzhaki: "Don't know any evidence that apoE could bind directly to HSV1. One of our ideas is that the two compete for entry."

Frank LaFerla: "I think there is a study that suggests that apoE can interact with the gB protein (its not the Becker report)"

Frank LaFerla: "You think HSV1 and apo E compete for entry into neurons?"

Ruth Itzhaki: "I'd very much like to know the reference"

Ruth Itzhaki: "Yes"

Frank LaFerla: "I will email it to you, need to look it up."

Ruth Itzhaki: "Thanks"

Frank LaFerla: "Well I need to go teach at 10am in California. Nice chatting with you, Ruth. All the best!"

Ruth Itzhaki: "Same to you. Goodbye"

Ruth Itzhaki: "Keith, do you want to continue, or shall I say goodbye?"

Keith Crutcher: "I actually need to go soon but thanks for taking the time to be here.

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