Well, it’s a great pleasure to be at Lindau, which I visit every year that I can.
But I have a very risky job that I’ve set myself this time because I want to talk about...
give 2 short talks, about 15 minutes.
And they’re 2 topics which seem to have nothing to do with each other.
One of them is applied cancer research and the other is pure neurobiology.
So in fact one of them is the most applied research possible.
And in fact we have formed a new company even, Avelas Biosciences to commercialise the probes.
And I should say right away that the first half of the talk has some commercial involvement.
But the other half is very pure research and may seem to have nothing to do with the first half
though you will see that they have a good deal in common of a subtle nature.
The acting molecules against cancer are activatable cell penetrating peptides.
ACPPs as we call them which we invented that sort of molecule.
And the primary use has been for florescent imaging, matrix metalloproteinase activities, MMPs,
but of course can be reasonably applied to even chemotherapy, delivering chemotherapy.
And complementary technology not involving activatable cell penetrating peptides is the staining of peripheral nerve
which via very humble phage display type procedure.
But it proves to be quite useful in a complementary way.
And as I said there’s a conflict of interest disclosure.
However, the second half is molecules for long term memory storage.
And that has to do with neurobiology of the peripheral net.
And in general the people down the left hand column are in my lab and our outside collaborators are down the right.
Varda Lev-Ram and Sakina Palida here and with mass spec help from outside, John Yates’s lab.
So I have to first describe just very, very briefly the 2008 Nobel Prize in order to dismiss it, ok?
If you had to pick a single photograph to encompass what I did.
I created a palette, or my lab I should say created a palette, of nonoligomerizing fluorescent proteins.
So we greatly improved, the green fluorescent protein and red choral florescent protein.
But the essence is... And by the way this is GFP, the natural colour of the jellyfish protein.
And we also gave some cute names.
Such as mHoneydew, banana, orange, tomato etc.
I am glad to see from yesterday that mCherry came out useful to Steve Chu.
But each fluorescent protein has the common property that it was encoded by a DNA clone.
And it’s not toxic, synthesisable by any aerobic organism in only 100% yield etc.
and links molecular biology and biological processes with imaging.
And that is its essential nature.
So the fluorescent proteins have given us some major advantages.
And of course we welcome the fact that fluorescent proteins have had a major impact on basic biomedical science
because every one of these proteins was clonable.
Most proteins and key biochemical steps can be linked in direct imaging
in live cells or intact model organisms such as yeast, worms, flies, mice.
The unfortunate problem comes when one wants to work on people.
The first problem: Humans are too thick and opaque for whole body fluorescent scanning
and therefore you have to use magnetic resonance imaging such
as we just heard about from Kurt Wüthrich or x-ray CT scans or positron emission tomography or so on.
But the really ugly aspect is that fluorescent proteins have to be introduced
by transfection or genetic engineering of some general variety.
And if you don’t use genetic engineering to introduce them, you’re not using the major power of fluorescent proteins.
And unfortunately on humans gene therapy is not yet sufficiently advanced to do it.
And it’s coming along but not far enough.
And it will be a long time before we make transgenic human beings including the ethical problems
that introduction of a fluorescent protein to make you or some part of you glow green would not directly benefit you as a patient.
So for clinical applications we still felt the need for a synthetic molecules, synthetic small molecules
to light up and treat diseased tissues.
And therefore we turned away from the thing that got me the Nobel Prize and toward this, the first half of the talk.
Most cancer deaths occur due to metastases growing in distant organs.
And they of course all start at the primary tumour stage but really do you die from them.
In order for cells to wander away, to metastasize, they...
Here’s primary massive tumour cells and a lot of people study the tumour biology and so on.
And it’s very, very important.
But finally to break through the basement membrane of the blood vessels or the lymphatics,
also to just carve their way through the healthy extracellular matrix,
the cells have to express and over active proteases, certain proteases.
And 2 of which...
The best known of which are MMP2, that’s matrix metalloproteinase 2, and the same number 9 of about 25 or so total proteases.
And this is so well known that these cancer cells have to activate these proteases
before they can chew their way like with molecular machetes, before they chew their way through and metastasize, is that...
You can even get the cartoon right off the internet.
Of course this was generated by EMD Microsciences.
And they sell reagents for in vitro study of proteases.
But our goal was to study in vivo and to detect the activation of MMP2 and MMP9 in vivo. And to cut a long story short.
The way we did so is this mechanism where a polycation up here is fused first to a payload
because we want to be able to carry other things with it and a polycation has a bunch of positive charges.
And in particular we just use 9 positive charges.
We settled on 9. And they’re just a bunch of arginines in a row.
Very, very simple.
Arginine is a pos charge amino acid. And it’s a diamino acid.
So it’s synthentic now, not natural amino acid.
But that alone is not sufficient.
We have to give this some selectivity.
And the way we do that is to attach the string to 9 negative charges,
matching negative charges here in the polyanion form, also deglutinated by the way.
And they neutralise each other and they’re held together by virtue of a cleavable linker.
So this composite 4 part chimera is made synthetically.
And it does not enter cells and is pretty inactive.
It goes around the blood stream and checks out tissue.
But if it doesn’t find a protease the body just excretes it.
It comes out staining the kidney, through the kidneys and your urine may turn a colour.
But that’s all. However, if the protease is present, it cuts this cleavable linker in situ.
And there snip, snip, snip and the same protease that is used in the tissue then cleaves this linker
and the negative charges are thrown away.
And the positive charge is now unopposed, stick to the outside of the plasma membrane first and absorb very strongly.
And then some gets endocytosed and of that some gets into the cytoplasmic nucleus. So that’s the principle.
Where there is protease you get a local accumulation and retention of the fluorescent dye in this construct.
So let me cut this short, this discussion, and just let you detect, see what it looks like.
Here is a tumour in the ear of a mouse.
Can you see the tumour? I bet you don’t.
And this is the problem that surgeons had for thousands of years.
Tumours are not visible to the naked eye.
But I’m going to start the movie and I’m going to put it into fluorescence mode.
And here I illuminate with a fluorescence light.
And you can see perfectly well which regions are fluorescent
and therefore have the ACPPs being hydrolysed and the red dye retained.
So, that’s good. Except that you can’t see your instruments.
You can’t see the anatomy.
So you don’t... This is not advantageous either.
So what we prefer is this: an overlay mode which is false colouring the fluorescence blue green.
And the reason we false colour blue-green is merely that there’s nothing natural which is blue-green in the mouse.
So here we can easily distinguish between the fluorescence due to the fluorescent dye and the anatomy of the mouse.
So that’s the primary tumour.
And we could have cut it out.
But we’re actually after the metastases.
And here you see the metastases through the skin of the mouse and in the overlay mode.
This is our third mode of display.
Now in this mode the fluorescence is rather diffuse and attenuated because it doesn’t go very well through flesh.
And that’s the problem that I alluded to.
You can’t work with fluorescence through anything deep.
However in a little while, just a few moments my surgical colleague, Doctor Quyen Nguyen, will come and open up the animal.
There.
And there of course you see the cut open tissue
and in this case now the bright fluorescence or the activity of the tissue to retain the fluorescence is shown here.
And here we are going to take apart the tissue and do a little dissection.
And can cut out the tissue, the diseased tissue and proceed with the operation much more easily
than if we had not had the fluorescence to guide us.
And with this sort of method we can do a whole variety of surgeries.
This happens to be pancreatic surgery done with the ACPP versus without.
And it improves tumour free survival.
This is a survival curve measured in weeks since the operation.
And if you operate on a number of animals, we observed this traditional surgery does not save very many animals,
whereas the fluorescence it does a whole lot better.
And also the tumour weight at termination of 8 weeks, when we checked, killed the animals to check how well we had done.
You clearly have a lot less tumour left and very little metastatic burden.
And it’s statistically significant.
Furthermore we can prove that in various tumour models.
And so far we’ve tested something like 26 or 28 or whatever and we have not yet found a tumour model
which, of either mouse or human xenograft which does not work with this type of technique.
They all seem to have this MMP9, combination of MMP9 and MMP2 activity.
And part of the reason it’s relatively universal is that any individual tumour doesn’t actually make all the MMPs itself.
It gets the help of the tumour micro environment which is the host’s contribution.
So the host pretty universally likes to make this when inflamed.
And therefore we think it’s fairly general.
In fact now very recently we have been able, published in 2014,
we’ve been able to hitch this same system, instead of delivering a fluorescent dye,
we just deliver a conventional cancer chemotherapeutic.
In this case it’s so called monomethylauristatin which is a potent microtubule inhibitor.
But it then now can regress orthotopic xenografts in early trials.
And this monomethylauristatin is actually used attached via the same technology
as used currently in antibody directed conjugates.
But this does not use an antibody.
It’s using an ACPP in our technology to deliver the same type of story.
So percent survival, we get cures.
Not every animal was cured but a significant number are cured with the ACPP,
whereas the equal same concentration of drug applied, free or with various controls, is not sufficient to save a single one.
Now how about the nerves?
Counter labelling nerve is a challenging problem.
In surgery you do not want to cut the nerves.
You want to cut the cancer but spare the nerves.
And current methods for finding nerves have great problems.
So, peripheral nerves turn out to have distinctive surface molecules.
You don’t have to do this ACPP complicated mechanism.
You just use phage display.
You get sequences such as NTQTLAKAPEHT.
And eventually fluorescently label them.
And it lights up the nerves.
So let me give you an example here.
So this is the flank, the hind flank here of the mouse.
And you can’t see the tumour or the nerve yet.
I haven’t opened it up.
But you agree you can’t see where the tumour boundaries are.
But we’re going to, that’s the tumour itself.
And here is the boundaries of the tumour in the overlaying mode.
Except that today we chose to use green, a GFP colour but it’s arbitrary.
The computer just puts green colour.
So here now we have naked, exposed a little more nakedly.
We’re doing the cancer surgery and Doctor Nguyen actually doesn’t cut this fast, its speed up about 2-fold.
But you are revealing the nerves.
And you can see that all this region here was a hunk of pseudo coloured green tissue which you want to get rid of.
And here is the sciatic nerve revealed, which you will want to preserve.
But you have...
And you can see it, there happens to be a fork here, you will have to separately cut out, preserve the branch.
Save out the branch and the main trunk right?
Well you just made a mistake.
You’re about to make a mistake.
You will paralyse the animal.
Because, in a moment, that’s the fluorescence of the nerve highlighting peptide.
The actual nerve just does a zigzag and rapidly dives into the tumour tissue here.
And you would have missed it by just reflected light illumination.
Now of course we prefer to combine it in pseudo colour mode.
But using a different wave length to distinguish, today blue is good.
And blue-green is what you want to save.
And the green is what you want to cut out, ok?
So makes it fairly simple.
Even a more practical, clinically practical example is shown here of rat cavernosal nerve.
Now what you think is the cavernosal nerve.
Well at least half of, slightly half, less than half the audience ought to know because it mediates male erections.
And this is the great danger of prostate cancer surgery.
When accidently the prostate is damaged.
Well the prostate must be removed but the nerves running through the prostate can be very readily damaged.
And can you see the nerves.
I suspect it’s rather difficult.
This happens to be a rat.
But here you can see the nerves running through.
Once you turn on the fluorescence illumination.
So here too even a more practical sense, you have a benefit.
So, synthetic contrast mechanism for clinical application currently,
we already have a mechanism of cleavage activated uptake which delivers contrast agents for optical.
I haven’t shown you magnetic resonance imaging but it does for MRI and chemotherapeutic agents.
And though I emphasised here metastatic ability, if you switch to different enzymes such as thrombin for thrombosis,
you can also measure these other biological processes and disease processes.
The nerve homing peptide represents complementary non-amplifying labelling which happens to be complementary,
as you see, but of entirely different mechanism.
Each has some unwanted background tissue.
And honestly if I had GFP at my disposal, a lot would be easier.
I’d have a lot cleaner background, a lot greater specificity.
But I don’t, can’t use GFP so I have to do this not so good...
I genuinely don’t have the technology with GFP.
But earliest clinical application will be in image guided surgery.
Drug delivery will be difficult because there is some background labelling.
And produce showing that the dye goes in the wrong position.
But it is important to work in vivo and directly with clinicians.
Now the second half, I’m sorry I’m running late.
But the hypothesis that I wish to explore and seemingly unrelated to the first half of the talk is
that lifelong memories are stored as the pattern of holes in the perineuronal net,
the PNN which is a rather strange specialised form of extracellular matrix
deposited around selected neurons during critical periods in the brain.
And the PNN is interrupted by holes where synapses occur.
And this is the essence.
And where there are large holes in the PNN naturally you can make a strong synapse
because you’ve got a lot of surface area.
Small holes make weak or even no holes make no connections.
And therefore the PNN sort of serves as a punch card in...
Well I don’t know, how many...
Maybe you younger people don’t even remember punch cards.
But the older guys do.
That’s how the oldest form of computer memory storage was had.
But of course it’s in 3 dimensions.
The stability of synaptic strength in memory states is what actually stores the memory from moment to moment.
But that, I postulate, comes from the stability of the PNN.
And the great advantage of the PNN is that its molecules can last a lifetime with no turnover
whereas the synaptic background molecules,
because of the proteasome, ubiquitin pathways, autosomes, phagasome etc, they turn over every few days.
So there’s now no need to postulate a copying mechanism that preserves the information
which we would have to copy a hundred or thousands of times if we relied on the synaptic macromolecules.
And unlike intracellular proteins, extra cellular matrix lacks an automatic turnover like the ubiquitin system etc. etc.
Of course it can be degraded and then you have to call upon synapsis specialised proteases.
So this is the structure and biochemistry, a little bit of the perineuronal net.
It’s this complicated meshwork made of these specialised constituents, aggrecan, versican, neurocan etc, hyalronan
and all stuck to the plasma membrane in both places except where synapses occur.
And then you have a synapse.
The PNNs are excluded from synapses and where there’s a synapse you don’t have the PNN
as we can see here according to this recent micrograph.
Actually the existence of the PNN was noted as usual in neurobiology all the way back to Ramón y Cajal.
So he always got things first.
Here from the literature is a higher power electron micrograph of a cortical neuron.
And you can sort of see how this makes a punctuated dark deposit...
well stained with a dark deposit diaminobenzidine.
That’s the PNN. And where there in the gaps between the PNN are the synapses as shown here.
This is a pre synaptic axon, a synapse with the post synaptic density surrounded by PNN.
So my metaphor and this is a seductive metaphor is the synapse is like a gateway between the cells. Right?
This cell near here and on the other side another cell.
But the PNN is like the wall in which the gateway is inserted.
And what maintains the exact position of the gateway?
Well, you see all this tremendous lot of activity and people coming in and out of the gateway.
It’s by the way the Damascus Gate in Jerusalem. The evening comes by.
But what maintains is the position and size of the gate.
What remembers where the gate way ought to be? The stone wall.
And we ought to not forget the stone wall.
Of course until modified by masons.
Now, this is some evidence that intercellular proteins don’t last that long.
As if we had to have evidence.
A rat cortical neurons or their synaptic proteins have a half-life all uniformly of a few days.
And very occasionally you get these long lived ones.
And in this sample even these were the extra cellular aggregation proteins.
We have begun to measure the lifetime of the PNN which had never been done before.
And so far we’ve only done 180 days, 6 months.
And the experiments are ongoing to do longer periods.
But if you’re doing an experiment like this, you raise them until they’re young adults on nitrogen 15
and then switch them to nitrogen 14 for a long period like 6 months and then sacrifice them.
What happens?
You see that all the synaptic...
the PNN is still heavy showing that it was laid down early, up to P45.
And then it persists there.
And the exact isotopic ratio until you check the aged animal.
Or you can do the experiment in reverse whereas of course if you check it,
the synaptic proteins the conventional synaptic proteins can’t keep hold of the isotope.
They immediately flush out the isotope in a few days.
It’s not quite as fast as I showed in the previous slide
because of the limitations of working on whole animals and isotope washout in whole animals is a little bit slower.
One will eventually have ability to look at people in the cadaver specimens using the nitrogen...
the carbon 14 left over by nuclear bomb testing in the Test Ban Treaty of 1963.
That was a wonderful natural experiment involving radioactive labelling done on everybody alive on earth.
And it is possible to do the same experiment.
But we have yet to try it.
We’re going to do it soon.
So proposed molecular memory mechanisms, I list here 4 mechanisms.
The 3 current in the literature are from John Lisman, CaM kinase 2.
Todd Sacktor proposed PKMzeta.
Eric Kandel, another Nobel laureate proposed CEPB.
Of course all of the proteins proposed up to now had inside the post synaptic compartment,
whereas I am proposing that we ought to look just outside the synapse.
They all have different synaptic activation.
And I don’t have time to discuss them.
But they all, in order to allow themselves to be copied and copied and copied repetitively
because of the short half-life and yet the long term information storage,
they have to propose these various complicated mechanisms where the essence of the idea is that,
I postulate a protein that does not need re-copying because it doesn’t turn over.
And by the way it explains critical periods because up to the time you lay down the proteins you don’t have anything to memorise.
You cannot memorise until you label and stop turning over.
So literature evidence for proteolytic degradation...
Well we haven’t contributed.
There is already in the literature proteases such as,
well guess what MMP9, how boring, are locally translated and secreted in response to synaptic activity.
Local puffing of MMP9 onto neural spines can mimic the growth of the spines, the enlargement and potentiation.
Here is a bit of new stuff.
Global disruption with chondritinase.
Now you have the use of some different enzymes of the PNN can reopen critical periods.
So that it’s well-known, or at least it was in the literature
that if the ocular dominance period cannot be changed in an adult unless you put on this enzyme for a little while,
chew up the old extracellular matrix and then allow the animal to re-establish synaptic, re-establish the PNN.
During that period you can test their eyes again and re-establish ocular dominance columns.
And finally inhibitors are supposed to block potentiation.
Now, I don’t know that MMP’s are really that essential or primary they have just got the most reagents that you can buy.
Other enzymes may be important but I believe that some sort of combination of enzymes will be necessary.
Now we wish that we could directly implant or delete specific memories
but this requires knowing how the even more difficult problem of in general how synaptic activity encodes experiences.
And that’s too hard a problem for us to solve, a grander problem.
So let me just observe that stable information storage, we know as writing and reading.
That’s what we call storage of information in non-volatile format.
And it’s essentially for stability and civilisation.
That’s why we have a process of writing and reading and do not try to use purely oral transmission from generation to generation.
And I should note, you may not think that making holes is a good form of storage.
But remember the lowest tech permanent media are like clay tablets
or carving in stone or even tearing your ticket when you go to the theatre.
They instantly have to show that you are...
prove that you, in an unforgeable manner, that you’ve used the ticket.
They just tear it.
And that’s... You don’t need a complicated mechanism, don’t have to learn how to write, you don’t need pens and pencils.
You just tear. Of course it matches with the clinical observation that early memories are better preserved,
often better preserved than recently formed ones because of the different system involved.
So, I don’t say that this is the only mechanism,
but I think it should be considered as a complement to the traditional, more traditional intrasynaptic memories.
And I hope you’ve noticed that the extracellular matrix is also a neglected subject which deserves more credit or attention.
And we have tended to think that this is just so boring.
But there may be something there.
Thank you.
Applause.