The Silver Lining Nervgen Playbook. Part 2
Transcribed and Edited for Clarity by Allan Lee, Managing Editor
CLEVELAND–On a cold morning in late November 2018, we walked through snow flurries a few blocks from our hotel to the grand laboratory, replete with scientific instruments and annotated writing boards, of Dr. Jerry Silver near one edge of the Case Western University campus. What follows is an sweeping overture covering Silver’s life and work to date explained as only Silver can explain it. The discussion wound down only when Silver had to leave to give a 10 am lecture to medical students. We think you’ll enjoy his thoughts….and his candor: Silver held back on little. His scientific excellence, dedication, passion and indefatigability are manifest…..as is the fact that he’s clearly a man of principle. Is this an interview with a future Nobel laureate? If Silver’s science leads to gait recovery in patients with spinal cord injury, then likely, yes, that is what you are about to read.
NervGen CEO Ernest Wong, PhD, joined Dr. KSS and Allan Lee for the meet-up in Silver’s lab.
We have left Dr. Silver’s words largely as they emerged from his lips, only mildly blue-penciling them for clarity or conciseness. Silver’s descriptions are rich and adjectival, and as we spoke he alluded in no particular order to various images and publications associated with his work. We grappled with whether to try capturing all those here and marching them out on page as Silver alludes to them, and concluded that Silver’s presentation to us was extemporaneous, and that a complete graphical match-up might prove very challenging if not futile. Instead, we encourage readers to be wooed by the cant of scientific investigation in what he says, by his descriptors, by his scientific thought processes peeking through in his sentences. While rich, his descriptions also are straightforward and without any guile. Take them at face value and recognize he’s accustomed to speaking to audiences that are intelligent but non-specialist. We find Silver’s urban Cleveland argot charming and have not tried to disguise it.
One of the things that really hit me hard when I first read your work is this: I’d come to thinking of chondroitin sulfate proteoglycan molecules as being, you know, amorphous viscous giant blobby frondy things; they leave smears on your polyacrylamide electrophoretic gels. I mean.,…can your enzyme PTPsigma function in a literal lock and key receptor fashion for chondroitin sulfate proteoglycan when you think of that as, you know, goopy and globby?
That’s an interesting question to start our discussion. You know they used to be called mucopolysaccharides but the name changed….muco, cell mucus, cell snot. [Their official term is now glycosaminoglycans, but “proteoglycans” remains in wide use.] But why do we even make this stuff? Because these are molecules that very old in evolution, there’s a lot of sugar, not sugar like glucose, sweet sugar, long chain disaccharide sugars, but more like snail slime….like sialic acid, lots of sulfate, high negative charge. They were evolved to be cell-protective, they’re protective oral molecules, for example, and I think cells probably started making them because they protect against bacterial invasion, viral invasion.
There’s a family of these, the glycosaminoglycans, with chondroitin sulfate’s siblings being things like dermatan sulfate, hyaluronan and both heparan and heparin.
Yeah…the sugars have various configurations. I’m not an expert on sugar chemistry but there’s a protein core and then the sugars stick out in size kind of like bottle brushes and the more sugar, the bigger the molecule. Among the big proteoglycans, one of the major ones is called aggrecan, very prevalent in cartilage. They all end in “an”….so, proteoglycAN. You know that cartilage is kind of slippery, you probably felt it, you cooked it, not very tasty as there’s not much in cartilage. It has no nervous-system intervention, no blood supply because it’s bendable. It’s meant to be bendable but you don’t want to put a lot of blood vessels and nerves and a structure that can be bent like you can do with your ear because that’s not a good thing. It’ll hurt, so it’s well known that proteoglycan and cartilage keep out the innervation. It’s well known that in cartilage, proteoglycans bind a lot of water; that’s why they’re slippery. The end called the linker region binds to a glycosaminoglycan called hyaluronic acid that binds a lot of water. Why bind a lot of water? That adds probably to the umbrella mechanism of these protective molecules, and so I began to study them a long time ago. I think it’s my nature to ask questions that other people don’t or that aren’t popular at the time.
Do glial cells extrude chondroitin sulfate?
Yeah, they make it and no one really knew that these molecules were even in brain, and when I started to study them. It was thought that there was no large extracellular matrix molecule in brain at all, zero. It came from people who study electron microscopy images; shrink the tissue, you don’t see any room between one sulfate and another for any matrix. And so I asked the question a long time ago, if there are such things as barrier molecules, or even barriers at all in the nervous system during development, why would you have them? Well to separate nerve tracts that need to be on one side or another, so they don’t cross each other—– to turn nerve cells, their fibers, away from areas they shouldn’t go or grow. You know, you’re going this way, and you’re supposed to end up there, you have to turn…. might there be a barrier right along there, you know, especially if a lot of axons are turning in the retina. All of our nerve fibers always exit through the back end of the eye through the optic nerve, never through our pupil.
Why not through the pupil? They can, in certain mutations. So we looked in those regions and basically what I saw when you just fix tissue and cut it and stain it properly are a lot of big extracellular spaces. They’re huge! I can show you pictures, I didn’t know how much how much actual detail you want to get into. But I’ll show you a picture that started it all.
Is there a good way to stain them?
Yeah, yeah. that became available right around the 1990s. So I’ll show you the picture that started off my whole my whole career. So this is a one micron plastic section here. You just take tissue….. this happens to be from the embryonic spinal cord and this is the middle of the spinal cord. That’s central canal, that’s where the CSF flows and the spinal cord, the left side, the right side, that’s the top—- and this wedge shaped region right here, that’s called a roof plate, roof on the top, and you see the holes inside.
You know, there’s this big three to four micron diameter opening. I believe they’re real because I don’t see them here, just there. So turns out that if you look at the nerve cells in the spinal cord, these are the nerve fibers; see them in blue? None of them cross the roof. See? it’s blank. All the other ones are on the side. That’s like a embryonic A13 rat spinal cord. This region at the top will become a little like a long shape, and it’s called the dorsal median torso. Middle line septum separates two columns of axons called the dorsal columns, and they’re dorsal columns because they’re columnar, mediate light touch sensation from the extremity, and proprioception, where my arms are in space. You close your eyes you know that….that’s proprioception. The dorsal columns are never crossed in the spinal cord. Even if the nerve fibers from the left side and the right side come right up to each other at the top of the spinal cord, they will never follow one another across the midline, like never…. because you need to know what your left side is to separate perception and action from your right side.
So the dorsal columns are uncrossed projections…. and I always wonder let’s keep them from crossing a little bit. That’s why we looked up there and that’s it. That’s it. This is what we found so there’s the tissue that becomes a dorsal median septum in the adult and it’s full of holes and I thought holes, they’re real. The better you fix this issue the better the holes look. I thought to myself, there must be some kind of a jelly in there, that’s known to be inhibitory, and that binds a lot of water.
So this is like mid80s, and so you can’t take the tissue and grind it up and do RNA analysis, right? You just have to start thinking about what that substance might be….and so you ponder. You just do it. You go through the literature asking yourself, What binds a lot of water? What’s known to be inhibitory? And it points directly to proteoglycans because they bind a lot of water and they’re known to be inhibitory.
One of the most interesting papers I read about the potency of proteoglycan in being inhibitory. It’s a paper by group associated with a researcher named Carson. Carson was interested in placenta….. the placenta and the uterus and placental invasion. So I don’t know if you guys have ever seen a placenta. If you’ve ever been in a delivery room, you’ve seen one, and they’re gross. Placenta is like, this big [makes sweeping hand motions] and it looks like it has invasive potential… like a tumor. Basically, it IS like a tumor and it invades, invades the uterus, but it’s maintained, you know? The body can’t let it go wild, right?
So why doesn’t the placenta eat the uterus of the mother? Because the uterine lining is filled with aggrecan. According to Carson, you get rid of the aggrecan in the in the uterus by digesting it (I’ll tell you how we do that, with an enzyme), and a pregnancy behaves like an invasive cancer. So we owe our existence on earth to aggrecan.
Right around 1989, antibody became available for the first time that allowed us to see proteoglycan wherever it existed. It’s made by Sigma IGN, and it’s called CS- 56. That was the first time. Why did it take so long? The reason is proteoglycan is incredibly ancient. They’ve been around for like, forever, and I don’t know where they first started. Not sure if plants make them, they may, but sharks, slimy, slimy creatures, and cartilaginous fishes make proteoglycan because they’re fully cartilaginous. And because they’re so highly conserved, you don’t make antibodies for them unless the sugars, these long and disaccharide sugars, get clipped.
Say you you have an inflammation ….so if you have like an inflamed joint, and you’re unlucky, and you have a certain disposition genetically and you clip the sugar groups because there’s inflammation in the joint, that exposed sugar can become antigenic, and then you start mounting autoimmune response that’s called arthritis. And one of the reasons this antibody was developed because they used an enzyme chondroitinase to very briefly cut the sugar, and generate antibodies so that antibody became available: that was that picture.
The roof plate is full of proteoglycan and no place else that started the whole story and then we did lots of other experiments afterwards we can talk about but that’s where it all started. So proteoglycans are really prevalent in barriers, it’s not the only place that we saw them, we also saw proteoglycans in the first demonstration ever of the role of proteoglycans in developing central nervous system. So I told you that the spinal cord group plate has a lot of proteoglycan, if you actually cut the roof plate, so undercut it….let me show you another picture.
So this is an experiment I did a collaboration with a group at MIT. So there’s a region of the brain goes farther towards the top that’s called the midbrain, and it’s an area that receives fibers from the retina from the eye and there’s the roof plate….remember, that dorsal median septum I told you, there it is, in the tectum. What it does is to keep the retinal fibers from crossing over the midline, because you need to see things on this side or that side of the world so when that the nerve fibers get to the brain you want to keep left and right eye separate and all the fibers from one eye right now labelled stop abruptly at the roof plate again, but this is a different part of the nervous system is farther forward but that’s still the roof plate and it has proteoglycans in it. There’s the CS-56, there it is…and there’s the little barrier. It’s just like a piece of paper.
Is the barrier glially elaborated?
It is. These are glia, these are real glia, so here’s what we did. This is a hamster newborn…. they’re born precociously. They’re like little embryos when they’re born and visual system is still developing. So in order to test if barriers even existed in the nervous system, they put a little curved knife under the tectum and they pushed it in, and they cut the top off the roof plate. So they basically amputated its top part. So there’s the roof, but when you cut the top off, you can’t reform it properly. So these are the cell bodies of these glial cells, these glia down here. So when you cut the top off, they still survive, but it leaves a gap here. Yeah, so instead of the retinal fibers stopping abruptly like they normally do, if you cut the top the head off the roof plate like this, with this knife, just cut the top off, then the nerve fibers no longer obey the midline. See? They keep on going til they cross it.. There’s the midline and they keep going here. They’re in the wrong part of the brain. So now the hamster grows up, but it’s got nerve fibers in the wrong place. If you wiggle something like a sunflower seed in the visual field of these fibers, that turns the head the wrong direction to try to eat! So they put the seed here, and they keep running away. It’s really bizarre. So we knew, this is like mid90s, that these barriers actually existed. No one had believed that there were such things as barriers, normal barriers to normal nerve growth. It was heretical….it was always thought that the way you get nerves to grow where they want to, is to call them, summon them, lure them with molecules called neurotrophins. And so it was a radical that there were such things as barriers. Don’t believe me? No, I’m telling you they thought I was nuts! Especially they didn’t believe the fact that the molecules that were important in creating this normal barrier are cartilaginous molecules that have been around in, in fish, in their cartilage, for eons…and especially when there is no matrix in the brain! So this can’t be! It’s impossible! [raises hands in bemused mock frustration]
Is there any evidence that the body elaborates its own set of chondroitinases or other proteoglycan-degrading enzymes?
Yeah, it does. So there are matrix metalloproteinases that the body makes, because it’s these kinds of molecules that can be degraded by them.
So I’m talking about the case of the embryo. But these these molecules [proteoglycans] go away, they’re not there forever. Eventually, some nerve fibers will cross the dorsal midline, but then the proteoglycans go away. So there are ways of regulating proteoglycans, and I’ll show you one example, which is the next most important paper. So let’s go back where it all started.
[Silver makes drawing on sketch pad.] So that’s a little stick figure of aggrecan. See, there’s the protein, right in the middle, there’s the link or region that binds hyaluronic acid, you’re on again. So all the water binding, and these are long-chain disaccharide sugars
When you say glycoprotein, you think of a big protein backbone, and “sugar coating.” But here it’s the other way around: a little tiny protein core and massive palm-frond-like glycan groups. Those are the proteoglycans. Glycan dwarfs protein. A proteoglycan is a glycoprotein with the protein vs glyco size allocations reversed.
Yeah, it’s a monster sugar! And so that’s aggrecan. And proteoglycans come in a whole variety of flavors.
So let’s go over here. So this is the most important early paper we published. Now we’ve cut the roof plate with a knife and, as a result, the nerve fibers project to the wrong place. That was the role of a proteoglycan, to be a barrier. We didn’t know that at the time….could have been who knows what else, right?
So we turn to the retina. I had a great student in the lab at the times, P—–, brilliant kid, I can tell you for hours about crazy P—-. He’s a little nuts; actually, he’s a lot nuts. And he’s one of those genius type people who should be locked up in a padded room and given everything they need, so they can just think about stuff. So this is a rat retina and it was alive at one time, obviously; we can keep them alive. Just take the eye out of a rat and it can live as an eyeball for multiple days. In vitro, you just put it in good stuff [nutrient media]. And it does its thing. So in the retina and we asked the question: Well, why don’t your axons grow out of your pupil? I mean, they always turn towards the back of the nerve and exit the optic disk, which becomes the optic nerve. And you can see this is a retinal whole-mount. So the retina, you know, it looks like this. It’s a ball and the pupils up here, but of course, we open it like this, then the pupil ends up outside right, and the nerve is up is here in the back end. So that’s the pupil region and there’s this little bright green thing: that’s the optic nerve head of the developing, eye of a rat. And see this green? These green things are the nerve cells called retinal ganglion cells that send their axons or nerve fibers out of the eye, to form the optic nerve, the output of the retina. Now, notice that the green nerve fibers always turn left away from this orange stuff. Now, guess what the orange stuff is?
Right! So what we found actually the retina develops in a very beautifully organized way. So the very first ganglion cells form right there near the nerve head, and then progressively more peripheral. What we found was that there’s a receding wave of proteoglycan that starts going away near the center, we think actually degraded by the developing nerve fibers themselves because they produce enzymes that degrade the proteoglycan. So proteoglycan is everywhere, right? And then the very first nerve cells form right there start to release a little protease, they degrade the matrix and that leaves their pathway open for them and then they always go away from the orange. See? They’re always turning in this direction. Here’s the nerve cell, they always go in this direction. It’s a big blow-up of the developing nerve head right, actually kind of looks like a kitchen sink. There’s more going on here, push-pull always in biology. So how do we test for the very first time ever in history that proteoglycans play a really important role in patterning nervous system development? Well, we just soaked the eyeball in chondroitinase. It was bathing in something that degrades proteoglycan.So we knew from tissue culture study before that the inhibitory part of the proteoglycan was the glycan, the sugar.
Did you assume at that point it was just electrostatic? Repulsion by glycan negative charge?
Yes, we didn’t know if there’s a receptor, talking 1980s, right? This is 1992, so we soaked the living retina in chondroitinase, very specific, and remove chunks off the sugar chains and then looked at the retina. What happens to the nerve fibers? Well, this is what a normal retina looks like, if you cut it and you stain it with an antibody that sees the nerve cells only, That’s what the nerve cells look like in your in developing retina. They look like that, the lens over here… this area of retina hasn’t developed yet. No nerve fibers yet. Now this is a chondroitinase-treated retina, obviously different from that. I mean there’s lots of things that have happened: first, you get nerve cells over here that shouldn’t be there. They said that these are nerve chains, that’s completely abnormal. Some grow out of the pupil, I mean, it’s really screwed up.
So that was a Science paper….we published in Science 1992, and the world still does not believe this. I was pretty much alone, you know. A few people read the paper and no one was buying it. It took another almost decade, 10 years. I actually proposed a grant to study these mechanism. I had an NIH grant to study this, but I never believed there was one thing that controlled this very important phenomena, getting these nerve fibers to go out the eye. I thought there was push and pull and even structural alignment, geometry, and I wanted to study multiple mechanisms. But they said, No. You can’t do that… you have to study only one thing. It was ridiculous, so I lost my eye grant and I had to find something to do. Frankly, it was the NIH that forced me to study regeneration.
So the next thing we did was to ask if these barrier molecules that nature produces in the embryo reappear after injury, and play a barrier role in preventing nerve growth in the adult. Maybe a mechanism that’s used, that’s evolved, you know. For one thing, proteoglycans are good barriers to nerve growth….good that is unless it’s creating a good barrier where you don’t want one… or you’re making a new barrier in the adult. That’s bad.
We made lesions of the spinal cord and stayed with the same antibody, and you can see the bright stain, this blue staining. So there’s the lesion of spinal cord and these pink things…. they’re reactive astrocytes. The blue cells are glia. It’s the same kind of glia that you saw in the roof plate. So astrocytes produce a protein called GFAP, glial fibrillary acidic protein, and it’s specific for astrocytes and notice there are lots of glia in here. But these that make barriers like to make a lot of GFAP. GFAP inside of an astrocyte gives it more tensile strength, makes it stronger, more resilient. It’s just structural, makes them stiffer, and that’s also part of the barrier property of the cell. So they make a lot of GFAP. See this? It’s just beautiful…..there’s zillions of astrocytes. There’s glia here, but you’re not making GFAP. OK, so again, same thing happens after injury. So now the astrocytes are in the vicinity of the injury lesion, which is right here. It’s a spinal cord with a cut. You cut spinal cord and this way it’s called sagittal, so this is towards the brain This is towards the tail and there’s the lesion and you can see these pink guys, those are the reactive astrocytes. You can see them here…. see the red? There’s the lesion and that’s the scar, those are the reactive astrocytes. What are they trying to do? Wall off the injury. That’s what scars do, that’s a scar’s job. There’s a huge amount of inflammation in here and its job is to make a barrier, a wall, because we don’t have fibroblasts in our central nervous system like we do in our skin. So when we have a scar on our skin: that’s fibroblast cells that make a lot of collagen. Collagen is really dense, like fingernail.
Clinically do you ever think of the proteoglycan as as being markedly anti-inflammatory molecules? I mean even heparin, given for a hot deep vein thrombosis (DVT), cools the DVT off.
Yeah, well they piss macrophages off. When macrophages see proteoglycan they get very, very active. But there’s a fight going on. So the astrocytes respond and one of the ways they respond to this lesion is to start making barrier molecule and the blue stuff, that’s proteoglycan. The same antibody as chondroitin sulfate proteoglycan, as you can see. There’s a lot of blue here and less blue as you get farther away. We now know some of the triggers that the astrocytes see that they don’t like. Brain is protected by blood brain barrier and the brain doesn’t like to see non-brain stuff. You don’t want to go to a Chinese restaurant, eat glutamate, MSG, and not have a blood brain barrier because you’re going to get epilepsy and die! So there’s there’s a reason to keep stuff out of the brain because it can be like a transmitter. So when you cut it you open, you bleed, and stuff from the blood called fibrinogen plus a molecule called TGF beta, which is attached is one of the major triggers that the astrocyte sees, initiate the production of proteoglycan in abundance. So the proteoglycans are there and they clearly play a role in stopping regeneration.
So we did a study. At the time there’s a guy named Martin Schwab telling the world that the major impediment to regeneration is myelin, the fatty wrapping around nerve cells. He had some evidence that myelin proteins, specific myelin proteins, were the bad guys. So this is the lesion and you got some bad guys in there, and we have alligators and we have sharks and these are the macrophages. We know they do some bad things but that badness was thought to be because of molecules that are inhibitory in myelin. He (Martin) had an antibody called IN-1, inhibitor of neuroides 1, that he claimed overcame inhibition by myelin, so the world was really excited about that. Wow! One protein, one antibody, you get regeneration, you fix it! And he called it “no-go.” Really clever marketing, wow that’s easy, genius marketing! I had chondroitin sulfate proteoglycan, he had “no-go.” I wish I came up with a better name [laughter all around]. So he’s winning, my grants are getting killed and my papers are getting murdered. No believes me because Schwab, his way is easy.
So we did this simple experiment. One of my best, a really important paper. So the lesion is here, this area in the spinal cord. You remember the dorsal columns? So there’s the spinal cord here. Yeah, cerebellum, there’s the cerebrum. That’s brainstem, and the dorsal columns because you’re looking at the spinal cord from above they’re big going all the way down. This is all white matter: that’s why it looks white. Schwab said all of this is what’s inhibiting regeneration, “no-go” everywhere and “no-go” is incredibly robustly released when there’s a lesion. Don’t forget the dorsal columns are sending projections. You know even from this height, you can feel your hairs on your skin, it’s all ascending white matter tract going up. If you make a lesion here, you can see the lesion, you cut all the nerve fibers…. all the fatty myelin around those nerve fibers is now released because they’re dying. So this is a swamp of “no-go” alright!
But there’s the lesion, and proteoglycans are right at it. So we did this experiment. What we did was take nerve cells from a green mouse, GFP mouse, purified them. These are adult sensory nerve cells, the same nerve cells that produce axons in the dorsal column. We harvested them, purified them, and very gently stuck them right here into the sea of alligators. Would they regenerate? If we gently took the cell bodies and put them there very gently, through a micro needle? Impossible, people thought; no way! And what we found is that WAY [peals of laughter from all], you can get a huge amount of growth, and it looks like kind of green hair, see it? [Audible gasps] These are the nerve cells we put and there they are and these are their nerve fibers growing. It’s like hair…. like how could that be? And the rate of growth is one millimeter per day, the same rate of growth that you get in the peripheral nervous system. This was IMPOSSIBLE! It was like a paradigm shift.
I’ll tell you a funny story. When we presented this data for the very first time I did it in England at a meeting of a group called the International Spinal Research Trust, which I’m still involved with. At the time there’s a very famous scientist who discovered the gate theory of pain. His name is Pat, was a crusty old kind of a British guy. He used to chainsmoke, and he was so scruffy, right? His hair was like everywhere…. but a brilliant scientist and when I presented this data because it had a had a really great punch line. So these nerve fibers are growing like little Eveready Energizer bunnies in a sea of “no-go”!
But when they get to the lesion, when they get down here where the proteoglycans are, they go into the lesion and stop, see? They get stuck, they can’t come out. So these are the green, see this? These are the ones from the mouse growing, and when they get to the area where the proteoglycans are, they get stuck. Like wow, they can grow amongst the alligators, but they get stuck when they get to the sharks. So this was the best first evidence, from a simple but elegant experiment, that failed nerve regeneration was a consequence likely of scar itself. Perhaps most likely, the proteoglycans within scar that prevented regeneration. So I’m giving this talk, and I presented this simple data. So Pat was in the audience and he was so freaked out. He said, “We have to stop the meeting! And we have to, we have to…. we have to discuss this!” You know how the British are…. they use the word “brilliant”. And “bravo”. It was like a standing ovation…. this was the moment of high drama. I’ll never forget, it was one of the best days of my young life.
So that’s kind of the history of where it all started. Proteoglycans are in great excess because they’re sugary. If they crystallize, they’re called amyloids. It’s one of the main constituents of Alzheimer’s disease. Plaque is proteoglycan. Proteoglycan is everywhere, they’re ubiquitous. So so that’s where it all started. You have any questions about this? Or you want me to keep on talking?
It’s a very clear exposition, I would say! I mean, at what point did you discover protein tyrosine phosphatase? When did that come?
Okay, that comes many years later. Alright, so we’re going along. In 1990, we first published the proteoglycan in the spinal cord. In 1992/93, we started to talk about regeneration and scarring. In around 2002, 12 years later, the group in England, because they were following my work, they started to get interested in it and they started to believe it. So they were doing these kinds of funky experiments that indirectly show these proteoglycans to be involved with regeneration failure, but what we needed to do was to get rid of the proteoglycan in the adult after spinal cord injury or any CNS injury and the system should be able to recover…. regeneration and recovery of behavior.
So I was in competition with the British. After I told them all about it, they started playing around with it. I thought because of my interest in the retina from early development, maybe we could crush the optic nerve and get the optic nerve to regenerate. It’s pretty simple and it’s one nerve tract we focused on and the Brits focused on spinal cord injury. And so we were having problems getting a needle in the optic nerve…. it’s too thin and as soon as it scars it gets really tough. You can’t inject it, and we kept breaking the optic nerve, and they [the British] just made a lesion in the spinal cord, injected into spinal cord and they saw a little bit of regeneration. So in 2002 I get the paper on my desk from the Brits using chondroitinase in the spinal cord injury. But the field was struggling. It was just me. So for 12 years people thinking I’m nuts, killing my grants and all my papers were in second-tier journals except for the Science paper in ‘92. But all the other stuff, always kept down, “can’t possibly be, this this must be artifact…”
So I get this Nature paper to review and they’re saying they’re getting some regeneration and some recovery in spinal cord injury. One author is Elizabeth Bradbury, and her name is synonymous with chondroitinase; she’s still the queen of chondroitinase in England. So 12 years go by between 1990 and 2002 and we still don’t know how proteoglycans are inhibitory. It was thought that it was all about charge. Sulfate is negatively charged, and cells hate negative charge. If you want cells to grow in a dish, you put positively-charged stuff in a dish. Cells love positive charge, hate negative…. that’s what we always thought, that it was electrostatic. Or it was thought that maybe the proteoglycan, because they bind a lot of water, make an umbrella, and that it shields and covers up the good molecules, right?, with this fog. So nerve cells can’t see the good stuff to grow on….that’s another theory.
But okay, I’ll you the story about PTP sigma. So it’s kind of a funny interesting story alright, the PTP segment story. It all starts here with this guy John Flanagan and the reason the two of us work together because neither of us ski [laughter all around]. So the two of us are at winter brain conference, everyone else is skiing but I don’t ski, so I’m in the coffee shop and so was John. He says to me, “Jerry I gotta talk to you, I think I might have a receptor for CSPG.” So this is 2008. So how many years have gone by since 1990? Almost 20 years and I said “Really?!” and he said “Yeah I think.” This guy! He says protein tyrosine phosphatase sigma, which is a member of the so called LAR family of receptor protein tyrosine phosphatases. The LAR family is a group of enzymic phosphatases, and they’re receptor phosphatases. They’re well known to be involved with synaptogenesis, stickiness, adhesion where you need adhesion to get a synapse. They were discovered at the nerve-muscle junction synapse in fruit flies and well known to bind to heparan sulfate proteoglycan, HSPG. HSPG says go, but CSPG says stop. So how can this receptor be both?
Alright, so it’s a little complicated, but he said he thought it’s high affinity binding, and they had some knockout animals. So there’s a PTP sigma knockout and John told me that he needed us to help him because he doesn’t do tissue culture. He doesn’t have any in vivo, spinal cord injury stuff, but I do! So we’re sitting in a coffee shop, he said, “Would you like to collaborate?”
I said “Hell yeah!”
So we’ve got this spot assay and we had an in vivo assay. So John says, “I need you to do this work in two weeks.” Well jeez…. But he’s already talking to the people [editors] at Science. He’s telling them he’s got this receptor and would they be interested? And they said, well yeah, but you got to show some data, other than binding data. So I came back from the winter brain conference. I put my whole lab on to this project, everything else stopped….Fernando, Kevin, Sarah and I.
So we’ve got this spot model, it’s just basically a coffee ring. Remember, I told you that proteoglycans are arranged in a gradient because of the TGF beta? So this is a coffee ring, see the red thing out here? If you make a coffee ring, spilling coffee on linen [analogy], the ring is a beautiful gradient. And the physics of coffee rings has actually worked out in 2011 after like thousands of years of coffee ring. So we make the ring, there it is, and we culture nerve cells on the coffee ring. See this is the ring, nothing can cross because it’s a gradient of proteoglycan; you can’t get across unless you’re a PTP sigma knockout cell. They can cross and that’s where it started. So I mean, it goes pretty quickly. So knockout cells, knockout neurons, from the mouse PTP sigma knockout placed on our coffee rings will cross the ring.
Okay, so that that’s cool. And you can kind of see what’s going on here. So this is the coffee ring, to show you some other stuff because I’ve been telling you that proteoglycans repel things, they’re repulsive. That’s not what happens in vivo. In the scar, the nerves aren’t turning and being repelled; they’re getting entrapped. Studying embryology, roof plates and pupil of the retina, I told you that nerve fibers turn away from the guardrails, a veering phenomena. But that’s not what happens in vivo! Axons: remember, we transplanted them and they came down and they got entrapped, not turning—they’re not turning away from the sky and growing all over the place, they get stuck. So how can you get two different biologies with one molecule and one receptor? It’s all about geometry and up-regulation of the receptor. Okay, so here’s a coffee ring, down here in the red. Red is aggrecan, they bind with sigma. Laminin is green, we see it over here is a growth- promoting molecule cells love, called laminin, and we mix the two together.
Why do we mix the two together? To test if something is inhibitory. You need to test inhibition in the presence of growth promotion; if you want to test the brakes of your car, you’re not going to test them on a flat surface, you want to test them going down hill and going really fast. So you need something positive that you can oppose with your inhibitor to show its potency. So I’ve always mixed aggrecan with something that neurons love, which is laminin, because if this is potently inhibitory, it will block the function of laminin, like your brakes will block your descent on a hill. So that’s what we do in our coffee ring. In making a coffee ring with two molecules, you get inverse gradients in the ring. The physics of that has been worked out. So there’s the ring, it’s these little red lines, thin, blown up; there’s the coffee ring and you get two gradients. Increasing red is proteoglycan. So red getting stronger as you go out. Green, laminin, is decreasing. See? Here’s laminin only green, here’s red, only proteoglycan. A sharp interface like in the retina or in the roof plate—-met by axons and then they turn. So at sharp interfaces, the growth cone is coming along, growing on laminin and over here making integrin receptors for laminin. Then, here’s the proteoglycan, oh whoa!, there’s another molecule. It’s got tons of integrin, so it’s met as an inhibitory molecule right away. So they talk to this, but integrin wins, right? And they turn along the edge. That’s what we think is going on: you get this turning phenomena out here at the sharp edge. But now if you’re a neuron that ends up on the inside of the gradient, right here, where there’s a lot of laminin so they can stick to it, then some of them will send their axons into the gradient, just like when we transplanted the neurons, remember? They were growing along the alligators and they got into the scar, and they get stuck.
Alright, that’s what happens in vivo, they get stuck, and if you get into the proteoglycan, now what happens is the LAR receptors, the sticky synaptic receptors, increase. So now the cell sees the proteoglycan gradually and up goes the LAR family receptors for the proteoglycans, and those are sticky and they make buckets of it. So these cells are completely different…. totally different biology. So here, see that? They grow into that, they grow into the gradient, and they just stop, they get stuck. This is a completely different biology. Here’s a magnified version of time lapse images. See that? Yup, see, stuck like glue, even though it’s trying to pull itself away, it can’t let go. Like look at this guy. See, every process is stuck, they will not let go. It’s even ripping itself off the dish.
So neurons are dumb, glia are smart….. because they tell neurons what to do. But the neurons are so dumb that when they get stuck like this and they start up- regulating these synaptic receptors, they think there’s a post-synaptic membrane and they start making synapses on the dish. Yeah, they’re synapsing on plastic, but they don’t care. If there were neurons there, they’d synapse on that, it doesn’t matter. So they’re trying to make a synapse on the dish. But that’s really great, because this phenomena is exactly like what happens in vivo.
Okay, so neurons get stuck and so there’s an axon growing into the rim, the coffee ring. The ring is here, beyond this dashed line, and you see the receptor going up gets really dense. So the sticky receptor just increases, then they get so stuck they can’t move. We think that’s the basis of regeneration failure, too stuck, too tight, and I won’t go into the cells that they’re stuck on. Actually, there’s a form of stem cell in the lesion that makes a lot of proteoglycan and a mixture of laminins and other goodies, just like we’re making in our dish, and that’s the cell they’re stuck in. They’re actually stem cells in the lesion and they hang out with them for the rest of your life. They’re stuck there forever, and they won’t let go.
Does any adhesiveness of this involve RGD binding? [RGD is arginine-glycine-aspartate, an adhesive protein motif.]
Well, that’s the laminin side. So that’s the sticky adhesive binding domain of laminin, the RGD sequence, but that adhesion is not so sticky. It’s nice kind of sticky. You see what I mean? In order to grow, you don’t want to be walking on ice. It’s an analogy, you want to be walking on carpeting or cement so you don’t slip but that’s not so stuck you can’t move. That’s nice adhesion. Cells need certain optimal amount of adhesion, then they’ll grow on it. No adhesion, they won’t grow at all. They won’t grow on, like, a meniscus, the surface of a dish of culture fluid, so you need an optimal amount of adhesion. Too much, you’re stuck and you make a synapse. So now, you know: there’s a receptor, and it’s sticky; it causes this flypaper effect.
Does it fulfill receptor criteria in all senses? I’m not doubting, just maybe I haven’t heard that yet.
Yeah, we don’t know a lot about the phosphatase activity, what its substrates are. People are now finding what they are, there’s one called cortapin and it’s involved with lysosomal turnover and cytoskeletal rearrangements.
There’s something with cathepsin D in this isn’t there? [Cathepsin D is a proteinase.]
That’s another part of the story, it’s a downstream effect of mucking about. So the receptors are tied to protease, we’ll talk about that at lunch.
I just think this longitudinal sense of continuity in your career is brilliant, beautiful. Echoing the British, bravo!
It doesn’t end…. it doesn’t end ever it seems.
What you’re doing now is a rung on the ladder that you began climbing more than 20 years ago.
Yeah, 30 even…..mid-1980s. I just always believed that this is really important, and the papers are the culmination of 30 years of work because we’ve cured a form of spinal cord injury with the enzyme. Now we want to cure it with our peptide [ISP] because the enzyme has to be injected locally…..we’ll talk more [at lunch]… I have to teach medical students now….
Afterword: Because of the crowds and din in the lunch cafeteria, we elected not to continue recording the conversation there, fearing we could not capture material. Dr. Silver will explain the biology further at an upcoming BioPub conference call. With editing by Dr. KSS for scientific clarity. The authors acknowledge participation in the NervGen IP