Little-known protein might provide key to treatment

Host Amber Smith: Upstate Medical University in Syracuse, New York invites you to be "The Informed Patient" with the podcast that features experts from Central New York's only academic medical center. I'm your host, Amber Smith. Today I'm speaking with a scientist who studies retinal disease at Upstate. William Spencer recently received a grant from the E. Matilda Ziegler Foundation for the Blind. He's an assistant professor of ophthalmology and visual science at Upstate. Welcome to "The Informed Patient," Dr. Spencer.

Will Spencer, PhD: Thank you for having me.

Host Amber Smith: You've done research on progressive rod-cone degeneration. Can you explain what that is and why you're studying it?

Will Spencer, PhD: Yeah, so progressive rod-cone degeneration is a disease that was originally discovered in the 1970s in dogs. It's actually the most common cause of blindness in dogs. And since it was discovered has been discovered in just about every different type of dog you can imagine. So, well over 40 different dog breeds are affected by this disease. And it was studied by Dr. Gustavo Aguirre at University of Pennsylvania for decades. This disease was, in the dog. He's a veterinarian and a scientist.

And he eventually mapped the disease to a single mutation in a gene that he named PRCD, after the disease. So the gene, it codes for a protein, is called PRCD, and each letter of that gene name stands for progressive rod-cone degeneration.

So, it was mapped in 2006. So that's when he found this, discovered this new gene. And at the same time, he found that this exact mutation, and others, is present in humans that are blind. And having the disease in humans called retinitis pigmentosa. And so, I'm studying this because there's very little known about the protein, and that's kind of my area of expertise is understanding the function of a novel protein. What does it do? How does it work? And in the effort that we could understand a little bit more about the photoreceptor cell, and maybe develop new therapies, including for this disease, progressive rod-cone degeneration.

Host Amber Smith: So, this is really pretty recent, at least in humans.

Will Spencer, PhD: Yeah. You mean the discovery of...

Host Amber Smith: ...the discovery of the gene that causes this...

Will Spencer, PhD: So, yes, it was probably one of the most recent. And the reason for that, probably, is that this particular protein and the gene that codes for this protein, is very small. It's extremely small. It's only, 6 kilodaltons. So your average protein, let's say, is closer to 100 kilodaltons. So it's just a very small protein, and that probably made it difficult to detect biochemically in the early days of studying photoreceptor cells, because it was just so small.

Host Amber Smith: Well, can you tell me what the symptoms of PRCD are? And are they the same in humans? Or do we know if they're the same in dogs, as well?

Will Spencer, PhD: So in dogs, it was named progressive rod-cone degeneration because there is the progressive death of rod photoreceptors, followed by cone photoreceptors. And so this is essentially exactly how the disease develops in humans. You have this progressive death of rods, followed by cones. So, in a human, you have 100 million rod photoreceptors and about 5 million cone photoreceptors. And most of the cones are in the center of the retina, in a region called the macula.

And so, because you're kind of losing the rod photoreceptors first, those are primarily on the periphery of the retina, kind of on the edges of the retina. So, the symptoms would be that you get tunnel vision slowly, so you lose your peripheral vision over time. And then eventually you lose your vision completely. And so this is very similar in the dog and in the human, these symptoms.

Host Amber Smith: Is it treatable at this point?

Will Spencer, PhD: No. So there is no treatment whatsoever for retinitis pigmentosa right now, including retinitis pigmentosa that's caused by mutation and PRCD. And so there's no treatment for progressive rod-cone degeneration in the dog or this same disease in the human.

Host Amber Smith: Can you tell us what you've learned about this condition through your research?

Will Spencer, PhD: Yeah. So I mentioned rod and cone photoreceptors. So, maybe some of you have heard of rods and cones. The rods are used for kind of nighttime vision, and cones for daytime vision.

And cones, humans have three different types of cones. You have red, green, and blue. And they're like the pixels of your retina thatenable you to see colors. They're called rods and cones because of the structures that are attached to these cells, called the rod and cone outer segments. Well, the outer segments are these, they're basically antennas. There are essentially gigantic cylinders that are filled with these disc shaped layers of membrane. Membrane, of course, is like the skin of the cell. It's like a lipid skin of the cell. So, you have this big cylinder that has layers and layers of this lipid membranes. And these lipids, this membrane material, serves to contain photopigment protein. So this is protein that absorbs light.

And by putting this photopigment in a big cylinder and packed with layers and layers of this photopigment, this enhances the sensitivity of your vision. So your rod photoreceptors are capable of detecting one photon of light. So that's as sensitive as they could be. And much of that can be attributed to this humongous light sensing structure, this outer segment, and the arrangement of those membrane layers.

And what we found -- sorry to preface that with a long-winded story -- but what we found is that this PRCD protein is specifically residing in that outer segment structure, in that light sensing structure.

So it was previously unknown where this protein was or even what cell type it was expressed in. So we found that it is in the photoreceptor cell, and specifically in the outer segment. And we also found that the mutation in the protein that causes blindness in humans and dogs causes this protein to be mislocalized from the outer segment to other cellular compartments throughout the photoreceptor cell.

So this blindness-causing mutation is preventing the protein from reaching its "home" in the outer segment. And what that really means is that this little protein has some essential function in the outer segment. It's doing something there that's important for vision. So what is it doing?

Well, we made a genetically modified mouse. Mice are a tool that we use to understand how genes work. And there's a lot of kind of tools that we can do with mice, which is why they're a good model system. And so we made a genetically modified mouse that doesn't have PRCD protein at all. This was a great tool for us to look at, "OK, what is PRCD doing? Well, let's just remove it and see what happens to the cell." Well, without PRCD, the light-sensing outer segment cylinder structure starts having some issues.

Now, as I mentioned, the outer segment structure is packed with all of these layers and layers of disc membranes. They're shaped like pancakes. So, imagine a big cylinder that's filled with pancakes. And over time, light is kind of damaging these membranes, the photoreceptor cells continuously replacing those discs. So every day, each of your photoreceptors builds 100 new photoreceptor disc membranes. This is just a very demanding task of the photoreceptor cell. But it's doing it every day for the rest of your life, in each of your photoreceptors.

And these disk membranes are formed at the bottom of the giant cylinder. That's where they're added. So they're each pancake, so to speak, is added to the bottom of the stack. And what happens if you don't have PRCD is that the disks, as they're being added at the bottom, start to partially fragment. So some of that kind of material is shed into the space surrounding the cells. So, instead of going into the cylinder, it's kind of like a leaky faucet where you're spilling some of the guts of this light sensor throughout the extracellular space.

So, this is, perhaps, what we think is causing the death of photoreceptors. There's all this membrane junk that's accumulating around them. And in other neurological diseases, when you have membrane junk accumulating around neurons, this is toxic for the cells, for some reason. It's not really understood why that is, but that's where we are. And so that's what we know about PRCD, in a nutshell.

Host Amber Smith: Well, let me ask you, does our body's immune system help clear out that membrane trash that needs to get removed?

Will Spencer, PhD: Yeah, so that's a good question. So, yes. What we found in the mouse, for example, that doesn't have PRCD -- this was also observed and dogs affected by the disease -- that the retina's resident immune cells, called microglia, kind of migrate to that outer segment layer where all of these light sensors are located in the retina. And they start to kind of engulf and clear and eat those fragments of those disk membranes. Clearly the response of these microglia cells is insufficient. They aren't able to clean up all the junk, because it's accumulating.

But it does appear that the retina does have some capacity to know, "hey, this is bad. We need to do something about it. Let's bring in the garbage trucks, try to clean up the junk." But, it's possible that by doing that, that those cells are causing collateral damage. Those microglia, they're trying to clean up the junk, but they could be damaging the photoreceptors in the process. And that will be a part of my research moving forward.

Host Amber Smith: This is Upstate's "The Informed Patient" podcast. I'm your host, Amber Smith. I'm talking with Dr. Will Spencer, an assistant professor of ophthalmology and visual science at Upstate, and we're talking about his research into progressive rod-cone degeneration.

Now, the grant you received recently is directed at your research into the role ectosomes play in retinal disease. What can you tell us about ectosomes?

Will Spencer, PhD: Ectosomes are small membrane vesicles. They're like little bubbles, like little, as I mentioned, those disc membranes -- they fragment. Well, when you have a membrane structure that breaks into a smaller membrane structure, that's released outside of the cell, it typically will be round, just like if you blew a soap bubble. It's round. It wants to be round. And these small little round membrane structures, if they're released straight out of the cell, they're called ectosomes. And so, actually, in the case of, if you don't have PRCD, for example, in the mouse, that doesn't have that membrane junk that's released coming off those disc membranes -- those are ectosomes.

Now what's really kind of fascinating is that a few years ago, we found that the photoreceptor cell is kind of building its light sensitive structure, adapting machinery that normally produces these vesicles. This is kind of confusing, but let me just explain. So the photoreceptors' outer segment to this giant cylinder is actually a specialized type of antenna that's present on, basically, every cell in your body called a cilia. So just about every cell in your body has this kind of antenna-like structure that can do different sensory things. It just happens to be the light sensor of the photoreceptor. And it's recently been appreciated that cilia have this innate ability to release little ectosomes from their membrane that can serve a range of functions, from removing unwanted protein to cell-to-cell communication.

And what we found is that the photoreceptor cell also has an ability to release these little ectosomes. But the photoreceptor cell has evolved to block that process. So normally, the ectosomes are not released because the photoreceptor cell expresses some very specific machinery that instead of allowing that vesicle to release, that vesicle, it buds, but it's retained at the membrane. So it's kind of like it's hanging on, like a hanging chad or something. And, that is the source of building material for building those disk membranes. So, in other words, the photoreceptor cell is a hair split away from instead of building its light sensor to releasing all of that membrane material in the form of a vesicle, an ectosome.

Host Amber Smith: So, why does the photoreceptor hold on to these ectosomes? Are the ectosomes seen as not being helpful, or are they seen as being harmful?

Will Spencer, PhD: Through evolution, usually when a new mechanism is needed by a cell, it's not going to reinvent the wheel. The cell will usually take some other machinery and adapt it for different function. So the core machinery is ectosome release. Maybe I shouldn't say ectosome release, but ectosome construction, so to speak, building the ectosome.

Host Amber Smith: So, what role do you think ectosomes play in retinal disease?

Will Spencer, PhD: There's actually a number of different cases, not just PRCD, where the photoreceptor cell releases these ectosomes. And, in all cases where these ectosomes start accumulating, this is promoting retinal disease. Well, we don't actually know if it's directly promoting disease or not, but it's associated with disease. And cases that have more ectosome accumulation typically have faster retinal degeneration.

So, probably the ectosomes are toxic to the retina. This is membrane material that's kind of accumulating in the retina. And as I mentioned, when you have membrane material that's accumulating around neurons, this is generally not good for neurons. For example, in multiple sclerosis, when you have your axons that are wrapped with membrane called myelin, when this myelin starts degenerating and fragmenting and accumulating around the axons, this is causing problems and resulting in disease.

So my hunch is that the ectosomes are not exactly a good thing and that this is a defect in building the outer segment. So, when you have certain types of defects are leading to the release of these vesicles. Because, as I mentioned, the photoreceptors are really just hair split away from instead of building that outer segment, it wants to release it all as a vesicle.

And I mean, maybe I can put this in perspective. So each of your outer segment, your light-sensing structures is huge. It's packed with 1,000 disc membranes. If you actually unfolded all of those disc membranes in one of your retinas, and just like laid it flat, it would cover the surface area of about the size of a large beach towel. So that just kind of tells you just how much membrane there is. And it has to be folded and perfectly packed. The whole point of that is to maximize the efficiency of photon capture, to make your vision sensitive. And if it's not packed correctly -- for example, it's being partially or fully released in the form of these vesicles -- this is creating a mess. And as I said, you're building 100 new discs every day. That's the size of a piece of paper, approximately.

So you're generating tons of this membrane constantly. It needs to be a well-oiled machine. And, that membrane has to be packaged nicely. And if it's not, then this is causing disease.

Host Amber Smith: Well, Dr. Spencer, before we wrap up, I want to ask you what attracted you to the field of science in general, and then how you ended up in ophthalmology and visual science.

Will Spencer, PhD: So, I guess maybe I'm a nerd at heart. My, my dad was a chemist and kind of always wanted me to be a chemist, maybe, or, kind of introduced me to it.

But at a young age, I remember we were, I was in grade school and we were at an auction where you could walk by and put down. And there was a microscope, like something you would find in a high school biology lab. And my dad was like, "oh, that's a nice microscope," you know, and he put down $100 thinking that was like, a $500 microscope or something. And turns out that he won the bid, and he was almost, like, grumpy that he won, which I thought was funny.

But when I was in grade school, I got this nice microscope. I mean, for a grade school kid it was a cool one. And I just remember putting pond water on there and looking at little protozoas moving around. Like, I was just captivated at a young age by that.

Even though he wanted me to be a chemist, I guess it's sort of his fault, that I -- I just felt like biology was so interesting, and I had excellent high school biology teachers. They were just superb.

Host Amber Smith: So, as a visual science researcher, do you believe there's a cure for blindness waiting to be discovered?

Will Spencer, PhD: Yes, I think there's some serious progress. I think it's an optimistic future. It's really, there's some amazing breakthroughs that have happened. So, you know, with CRISPR and gene editing, there's a thorough testing process for therapies to make it as a real treatment. And the retina is really at the forefront of gene therapy.

And this, for example, could be a very promising treatment for cases of retinitis pigmentosa, including PRCD. You know PRCD is really about not having that protein in that light-sensing structure. So, that's what the mutation is causing. It's causing the protein to be mislocalized. So, if we just put back the protein that has the normal, the correct sequence, this probably would rescue the degeneration.

It's actually a slow degeneration. So, if this could be recognized early, then if gene therapy could be developed more, and as we get better and better at it, this is something to be excited about.

Host Amber Smith: Do you think that clinical trials would involve dogs before they involve humans, since this is a disease that affects dogs too?

Will Spencer, PhD: It certainly could. Yeah. There's, I've heard estimations of tens of thousands of dogs in the United States that are blind from PRCD. I guess the one challenge is that typically you don't know if the dog has it until it's already kind of severely has a degenerated retina, at which point the gene therapy wouldn't work, really. The photoreceptors are already gone. But this could be tested in the mouse and worked out there very nicely.

Host Amber Smith: Well, thank you so much for your time, Dr. Spencer.

Will Spencer, PhD: Yep. Thank you.

Host Amber Smith: My guest has been Dr. Will Spencer. He's an assistant professor of ophthalmology and visual science at Upstate. "The Informed Patient" is a podcast covering health, science and medicine brought to you by Upstate Medical University in Syracuse, New York and produced by Jim Howe. Find our archive of previous episodes at upstate.edu/informed. This is your host, Amber Smith, thanking you for listening.