Episode 130: Meet the Researcher: Gene Silencing to Prevent and Treat Levodopa-Induced Dyskinesia

Dan Keller 0:02 Welcome to this episode of Substantial Matters: Life and Science of Parkinson's. I'm your host, Dan Keller, at the Parkinson's Foundation. We want all people with Parkinson's and their families to get the care and support they need. Better care starts with better research and leads to better lives. In this podcast series, we highlight the fruits of that research—the treatments and techniques that can help you live a better life now, as well as research that can bring a better tomorrow.

After a few years of treatment with levodopa, people with Parkinson's disease may develop drug-induced dyskinesia characterized by erratic, involuntary twitches, jerks, twisting, or writhing of the face, arms, legs, or trunk. Early on, they can be alleviated by adjusting medications, but long-term exposure to dopaminergic drugs can create changes in the striatum, a part of the brain involved in Parkinson's. Changes in calcium channels, which regulate the flow of calcium into nerve cells.

Having researched the role of these calcium channels in dyskinesia, Dr. Kathy Steece-Collier of Michigan State University is now working on a way to silence the gene for producing one specific calcium channel using a short form of RNA called short hairpin RNA, or shRNA. Her laboratory has packaged the genetic instructions for making the shRNA into a harmless virus and delivered it to the striatum of experimental animals using a one-time injection.

I led off our conversation by asking her to take me stepwise from how levodopa works to what goes wrong to cause dyskinesia after long term treatment with the drug, to how shRNA can be an effective treatment for dyskinesia. Let's talk about how levodopa medication works and what goes wrong to cause the side effect of dyskinesia, and then you can tell me about the approaches your lab is taking to prevent or reverse these problematic behaviors. So, how does levodopa medication work?

Dr. Kathy Steece-Collier 2:33 So, levodopa is a basic ingredient necessary for producing dopamine and is given to boost dopamine in the parkinsonian brain. Dopamine cannot enter our brain, so we give levodopa instead, which does readily enter the brain. A second drug, carbidopa, is given with levodopa to prevent levodopa from being converted to dopamine in the bloodstream. This would cause a lot of side effects and prevent adequate amounts of levodopa to get into the brain. Once levodopa enters the brain, it's taken into the remaining dopamine neurons, those that have not died yet in the disease, and it boosts production of this neurotransmitter. And dopamine is essential for producing smooth, purposeful movements.

Dan Keller 3:20 Is replacement of dopamine using levodopa equivalent to what the brain naturally does, or not? And if it's not, how does it differ?

Dr. Kathy Steece-Collier 3:30 Yeah, unfortunately, it does not produce dopamine the same. So, nigral dopamine neurons, those that die off in Parkinson's disease, actually have what's called pacemaking activity. So like your heart has pacemaking activity, and those cells are constantly active. The dopamine neurons are constantly active in making dopamine and tonically or continuously releasing dopamine, but there are these intermittent increases or decreases in dopamine that help with executing those appropriate purposeful movements.

So levodopa is given generally as a pill, and this results in a pretty significant increase in brain dopamine, as long as the drug is still in your system. Such an abnormally sustained elevation of dopamine following levodopa, though, prevents the neurons in a part of the brain that is receiving this information from hearing or realizing those pattern responses—that slight increase or decrease in dopamine release. So when levodopa is in your brain, you have these sustained elevations, but as the drug wears off, the amount of dopamine decreases. So you take another pill and there's another rise in dopamine, then it decreases. So you have this constant up and down fluctuation in brain dopamine, which is very different than what's normally in the brain. And this, together with these periods of abnormally sustained elevation of dopamine, is quite different from what the brain normally produces itself.

Dan Keller 4:56 So, where do the dyskinesias come from? The dopamine is doing beneficial things for you, but after a while, dyskinesias develop.

Dr. Kathy Steece-Collier 5:05 Absolutely. So, this elevation of dopamine is wonderful, but it's a non-physiological dopamine replacement. So, the brain tries to maintain balance or homeostasis at all times. So, this sustained elevation, together with these up and down fluctuations, is thought to be a key element in disrupting normal brain cell function and circuitry connections, and the way the cells talk to each other. So, it appears that, you know, as the cells are trying to maintain balance, they're changing different aspects of their physiology.

So, over the past couple of decades, research has revealed that there's an abundance of changes that happen in the neurons, both in response to Parkinson's, and then on top of this abnormal replacement of dopamine, there are changes in brain proteins, genes, electrical activity of the brain cells, and even structural changes to the nerve cells themselves. And just like in anything, structure dictates function.

Dan Keller 6:07 Just to set the stage, it sounds like levodopa-induced dyskinesia is still a problem, even though more and more is being learned about changes that occur with PD. How common is it, and when does it occur mostly in the course of the disease?

Dr. Kathy Steece-Collier 6:25 I mean, there's different estimates of how frequently it occurs, but approximately 40% of patients will develop levodopa-induced dyskinesias after about four to six years of levodopa treatment, and the incidence increases to an estimated 90 or 95% of all Parkinson's disease patients after about 10 to 15 years. So even though we've known about dyskinesia side effects since the start of levodopa use back in the 1960s and there have been a wealth of studies examining ways to prevent these abnormal movements, effective therapies remain elusive.

Currently, there's only one FDA-approved drug. It's an extended-release form of amantadine. This was approved in 2017 and there's different formulations that are being tried, but the effect of this single FDA-approved drug is partially effective, and it's not without insignificant side effects. So, additional options are still really needed.

Dan Keller 7:23 I understand you've been a recipient of the Parkinson's Foundation's International Research Grant Award. What has that allowed you to do, and what are you studying to ameliorate this problem?

Dr. Kathy Steece-Collier 7:37 The Parkinson's Foundation grant was seed money that has snowballed into amazing studies. So it allowed us to get some small NIH funding, and now more significant NIH funding. And what I'd like to do is explain a little bit about the mechanism and the approach we're using. It's very innovative, and that comment I made about function follows form, and even when the cells in the brain are changing their structure, it's having a huge impact on the physiology. So, what I'd like to do is tell you a little bit about some of the research we're doing, and explain some of the biology behind it.

Central to the biology of levodopa-induced dyskinesia are changes in what's called synaptic plasticity—so synapse is a junction at which neurons talk to each other. Abnormal synaptic changes occur with levodopa-induced dyskinesia in a brain region called the striatum. So, the striatum is a brain region about in the center of your brain, and it's where the dopamine neurons that die off in Parkinson's disease send their projections to communicate to produce these smooth, purposeful movements.

So brain cells are unique. They talk to each other in different areas of the brain by sending out these long tentacle-like processes called axons. They send them off to different brain regions. The basic structure of a neuron is that it has a cell body, like other cells in your body, and then it has these long tentacle-like processes that reach out to other brain regions to talk to other neurons, but the cell body also has what can be thought of as antennae, and they're called dendrites. The tentacles from one neuron—the dopamine neuron—reach up to the striatum, and those tentacles from the dopamine neurons make connections with other neurons on these antennae or these dendrites within the striatum.

Dopamine neurons make connections with a very special nerve cell called a medium spiny neuron. They're named because they have a cell body that's a medium size, and these antennae that receive input are studded with thousands and thousands of little spines. These spines are very important structural elements onto which brain cells from lots of different brain regions make contact.

When dopamine neurons that project up to the striatum make contact with these medium spiny neurons die off, there is a loss of dopamine up in the striatum, and these medium spiny neurons lose those thousands of tiny spines, and thus they lose their ability to have normal connections with other brain regions. We have known about this, that this occurs in people with Parkinson's disease since the 1980s. We can study the consequences of all of this in animal models of Parkinson's disease, and what we, the research community, have seen is that when levodopa is given under conditions where there are loss of these dendritic spines, and there's the development of levodopa-induced dyskinesia, there's interestingly a restoration of all these little spines and the connections onto them. But if you look really carefully, this reestablished circuitry is miswired.

So, we've been studying ways to prevent the loss of these spines, so we could prevent the subsequent rewiring, speculating that if we could prevent spine loss, we could prevent nerve cell miswiring and prevent levodopa-induced dyskinesias. Well, it wasn't until 2016 when a landmark study came out of Dr. Jim Surmeier's lab at Northwestern University in Chicago, and he and his postdoc, Michelle Day, discovered the mechanism behind spine loss on these medium spiny neurons.

So, it turns out that in the absence of dopamine, there is overactivity of a particular entity that's found on these spines. So the loss of medium spiny neurons is due to abnormal activity of what's called a calcium channel. It can be thought of as a small passageway that allows calcium ions to pass in and out of these tiny spinous structures. The specific calcium channels are called CaV 1.3 channels—so CaV 1.3. In order to protect itself from abnormal calcium signaling at these CaV channels, the neuron retracts its dendritic spine, so there's a loss of these spines and a loss of ability for other neurons to talk to these medium spiny neurons. In order to test the idea that preventing spine loss could prevent levodopa-induced dyskinesias, we needed a way to block or antagonize these CaV channels that become dysfunctional.

Our group, together with another group, undertook a study in Parkinson's rats, where we used drugs that could antagonize or silence the dysfunctional activity at these CaV channels. Both of our studies demonstrated these drugs could provide protection against levodopa-induced dyskinesia, but the drugs are not very potent or specific for the CaV 1.3 channel, so the benefit was only partial and didn't last over time.

So we needed a more powerful way to silence these CaV channels, so we turned to a gene therapy approach. This is what we wrote up as an application asking for research money to test our hypothesis, that if we could find a more potent way to silence these CaV channels, we could prevent levodopa-induced dyskinesias, and this is what the Parkinson's Foundation International grant allowed us to do. So we developed this gene therapy approach in collaboration with a colleague of mine, Dr. Frederick Manfredsson, and it's slightly different than the gene therapy approach that's talked about in one of the other podcasts on the Parkinson's Foundation site. What we're doing is injecting something called an shRNA, and it's an artificial molecule that can be used to actually silence a gene of interest, which in our case is the gene that's responsible for making the CaV 1.3 calcium channels. This results in the brain cells in the injected brain area producing less CaV 1.3 calcium channels, in essence silencing that abnormal signaling, and the results we've seen with this over the last several years has just been astonishing.

Dan Keller 13:45 So, rather than blocking the function of existing calcium channels, this approach actually reduces the number of calcium channels.

Dr. Kathy Steece-Collier 13:56 Yes, so we don't want to completely get rid of these calcium channels, because they're very important in signaling mechanisms, but I'm going to use the term these channels become overactive in the absence of dopamine, and so the cells don't want all that calcium just influxing, because too much calcium inside a cell can be very toxic. So if we can reduce the amount of that calcium influx by silencing or decreasing the number of these calcium channels, we thought we could potentially prevent that spine loss. And again, our group, and another group, which also involves Dr. Surmeier, has shown that if we use these drugs, we can prevent spine loss, but again, the potency of the drugs that are available is not very strong, and so the efficacy on preventing dyskinesias wanes over time, and it's only partial, even at its optimal effect.

So developing this potent effect that allows us continuous high-potency silencing of the CaV channels again has given us real remarkable effects. So what we've seen, and we've only been able to test this gene therapy in animals so far, but it provides some of the strongest preclinical data to date, demonstrating amelioration of levodopa-induced dyskinesia in Parkinsonian rats. What we've done so far is shown that in young adult male rats, this approach can completely prevent induction of levodopa-induced dyskinesia, even when we give levodopa for a prolonged period of time and use a dose escalation paradigm, where we raise the dose of levodopa to quite high doses.

And perhaps more impressive is that we can actually significantly reverse already established severe dyskinesias. More recent data is showing that it looks like this efficacy can be maintained into late middle age. As we're moving toward understanding the potential clinical utility of this gene therapy, it's prudent for us to use what's considered the gold standard preclinical model, which are non-human primates. So, again, acknowledging the prevalence of Parkinson's in late middle-aged individuals, we have undertaken studies in aged male and female Parkinsonian monkeys, and what we've shown is that decreasing the expression of the CaV 1.3 with our shRNA can provide again near complete protection against levodopa-induced dyskinesias over five and a half months of treatment. And this is when we've treated and given this gene therapy to monkeys that have had long standing severe Parkinsonian disability and all we've been able to look at so far is preventing dyskinesias. We want to look to see if we can reverse it, but the other thing that we've seen in both the rats and the monkeys is that we can prevent the dyskinesias without altering the anti-Parkinsonian motor benefit of levodopa. So that's very important, and there's a little bit of a hint that if you can maintain those dendritic spines and maintain the normal circuitry, it appears that we can also have enhanced motor benefit—and this is saying this cautiously optimistic—enhanced motor benefit of levodopa in these animal models.

Dan Keller 17:17 How long is the treatment with shRNA? Is it a one shot deal? Does it go on for a while? RNA is notably a rather sensitive labile molecule that may disappear pretty quickly, or does that not affect the shRNAs?

Dr. Kathy Steece-Collier 17:34 No, using a viral vector to encode production of the shRNA, it stays around for as long as we've looked. So it stays around, it continuously is producing the shRNA, and continuously the transgene expression is maintained for a very long time, so it appears to be able to be there continuously.

Dan Keller 17:57 You're not actually giving shRNA, you're giving a viral vector, which produces it over a long time. Is that right?

Dr. Kathy Steece-Collier 18:05 That is correct. We're using a viral vector that's linked to our shRNA. The vector allows us to get the shRNA into the cells of interest, right?

Dan Keller 18:16 Is there anything important to add or interesting that we've missed?

Dr. Kathy Steece-Collier 18:21 You know, it might be interesting to talk about the upsides and downsides of a gene therapy approach versus using a drug. I mean, drugs are always an optimal approach in that a person could take a pill; surgery can have unwanted consequences. But for this particular target, systemic administration of a drug is not effective. So one of the drawbacks to using systemic administration of a drug, taking a pill, is that it not only affects the brain region that we're interested in, but it also affects or has access to the whole brain and to the rest of the body. So CaV 1.3 calcium channels are involved in lots of other important processes in the brain, like learning and memory, and so trying to silence those CaV channels with a drug in areas outside of the striatum are not necessarily something that we're trying to do.

The other thing is that there is currently no 1.3-specific drug. People are working on developing one, but the drugs that are available for CaV 1.3 silencing are actually designed to optimally interact with a different calcium channel, CaV 1.2 calcium channels. CaV 1.2 calcium channels are very prominent in the cardiovascular system, and so the drug that we used as proof-of-principle investigations was isradipine, and that is a drug, DynaCirc, that's used for blood pressure. So if you want to get the drug concentration of isradipine high enough to be able to get effectively into the brain and silence those CaV channels, because it's not specifically designed for those CaV channels, the dose would be contraindicated in patients, because it would have off-target side effects in the cardiovascular system.

So, using a gene therapy approach, we can directly inject our viral vector containing the shRNA into the brain region of interest and selectively decrease CaV only in the cells we're interested in. And this gene therapy approach is very potent, and it's continuous. So if you take a drug, you have these increases and decreases in the amount of the drug in your body, depending on, you know, when you take the pill. So using gene therapy is a surgical approach, but in the realm of neurosurgical therapies, it's a relatively simple approach. Like we had talked about earlier, it's a one-time injection of the viral vector to deliver the shRNA. Neurosurgical approaches are done all the time, many of them much more aggressive—I'll say, for example, taking a biopsy, removing a tumor. Brain surgery is used for Parkinson's disease in the form of deep brain stimulation that involves putting hardware into the brain. So neurosurgical approaches are routinely used, and again, this is a relatively simple neurosurgical approach, just a single injection into the brain.

Dan Keller 21:30 It sounds like a very promising approach to overcome one of the most troubling side effects. So, thank you for explaining it all.

Dr. Kathy Steece-Collier 21:39 You are very welcome. I think the data that is coming out of the research is quite promising, and I am very hopeful that it might help patients with Parkinson's disease.

Dan Keller 21:58 To learn more about the gene silencing approach to preventing and treating dyskinesia, search our website at parkinson.org for gene silencing. There you can find an article titled Understanding Gene Silencing and its role in Parkinson's and dyskinesia. In it, Dr. Steece-Collier describes how she became interested in this line of research, what it involves, and how the international research grant from the Parkinson's Foundation facilitated her work. For an even more in-depth explanation of calcium channels' roles in normal neuronal function and in dyskinesia, see the article titled Levodopa-induced dyskinesias reversed in gene therapy study on the same page. It also describes her work and the therapeutic implications of it.

While the shRNA work appears promising as a long term solution to levodopa-induced dyskinesia, it's still in the animal experimentation stage, but for information on dyskinesia and what can be done for it today, search our website using the word dyskinesia. You can also find a link to our past podcast called Understanding Bradykinesia and dyskinesia.

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At the Parkinson's Foundation, our mission is to help every person diagnosed with Parkinson's live the best possible life today. To that end, we'll be bringing you a new episode in this podcast series every other week. Till next time, for more information and resources, visit parkinson.org or call our toll-free helpline at 1-800-4PD-INFO, that's 1-800-473-4636. Thank you for listening.

Kathy Steece-Collier, PhD is a Professor in the Department of Translational Neuroscience in the College of Human Medicine at Michigan State University. Over the past 20+ years, she has maintained her dedication to the development of improved therapeutics for individuals with PD with particular emphasis in understanding how, when the brain attempts to ‘fix itself’, aberrant remodeling of brain cells and their circuits results in the development of side-effects and/or lack of response to DA replacement therapies including levodopa medication and DA neuron grafting. By understanding how and why the brain changes in response to PD, her team hopes to find ways to prevent or repair the changes and improve therapeutic responses in individuals with this disease. Dr. Steece-Collier’s current research is focused on using a gene therapy approach that allows precise genetic silencing of a particular population of calcium channels, called CaV1.3 channels, to prevent aberrant ‘remodeling’ and reduce the often debilitating side-effect known as levodopa-induced dyskinesias in parkinsonian rats.