Discovering New ALS Therapeutic Avenues with Evangelos Kiskinis, PhD

“We're at a critical time where we've got this coming together of incredible resources, incredible technologies, and the coming together of the community to make meaningful therapeutics for these ALS patients who have been helpless.”  — Evangelos Kiskinis, PhD 

• Associate Professor of Neurology (Neuromuscular Disease) and Neuroscience 

While the underlying causes of genetic forms of ALS may be distinct, Kiskinis and his team have identified similar processes that disrupt the function of the motor nerves in ALS patients. Gained through the use of induced pluripotent stem cell technologies (iPSC), these findings may lead to novel therapeutics for ALS patients.    

• ​​ALS is a highly aggressive neurodegenerative condition that leads to complete muscular paralysis and eventual death. 
• Only about 10 percent of people diagnosed with ALS develop the disease through genetic factors, and this is due to roughly 30 genes that cause ALS when mutated.  
• Kiskinis’ lab is trying to understand whether the molecular causes driving the dysfunction of nerve cells in ALS patients are the same or different between distinct genetic subtypes of ALS. 
• Rather than traditional animal modeling, investigators are instead using induced pluripotent stem cell technologies (iPSC) to replicate nerve cells that degenerate in ALS patients.  
• The famous Ice Bucket Challenge funded the discovery of the NEK1 gene that causes 3% of all ALS cases. This led to further discoveries of how distinct genetic subtypes of ALS can converge in targeting the same molecular pathway in a nerve cell. 
• In a study published in Science Advances, Kiskinis and his team looked at the repeat expansion mutation in the C9ORF72 gene, the largest genetic cause of ALS. This mutation results in toxic dipeptide proteins that investigators remedied with so-called “RNA bait.” 
• In a study in Cell Reports, investigators used iPSC-derived spinal motor neuron cells to model a rare genetic subtype of ALS caused by mutations in the SOD1 gene.  
• All these findings could potentially have implications in the study of other neurodegenerative diseases, especially frontotemporal dementia, which shares both clinical and genetic overlap with ALS. 
• Studies show that while the genetic cause in each of these cases is different, there's always some sort of convergence, either directly, where two mutated genes are interacting with each other, or indirectly, where two distinct mutated genes are affecting the same cellular pathway.  

Additional Reading 

• Explore Kiskinis’ lab  
• Read the most recent publications from his lab  
• Discover other Northwestern Medicine ALS research at The Les Turner ALS Center

Recorded on December 12, 2023.

Target Audience

Academic/Research, Multiple specialties

Learning Objectives

At the conclusion of this activity, participants will be able to:

1. Identify the research interests and initiatives of Feinberg faculty.
2. Discuss new updates in clinical and translational research.

Accreditation Statement

The Northwestern University Feinberg School of Medicine is accredited by the Accreditation Council for Continuing Medical Education (ACCME) to provide continuing medical education for physicians.

Credit Designation Statement

The Northwestern University Feinberg School of Medicine designates this Enduring Material for a maximum of 0.50 AMA PRA Category 1 Credit(s)™. Physicians should claim only the credit commensurate with the extent of their participation in the activity.

American Board of Surgery Continuous Certification Program

Successful completion of this CME activity enables the learner to earn credit toward the CME requirement(s) of the American Board of Surgery’s Continuous Certification program. It is the CME activity provider's responsibility to submit learner completion information to ACCME for the purpose of granting ABS credit.

All the relevant financial relationships for these individuals have been mitigated.

Disclosure Statement

Evangelos Kiskinis, PhD, has nothing to disclose. Course director, Robert Rosa, MD, has nothing to disclose. Planning committee member, Erin Spain, has nothing to disclose. FSM’s CME Leadership, Review Committee, and Staff have no relevant financial relationships with ineligible companies to disclose.

[00:00:00] Erin Spain, MS: This is Breakthroughs, a podcast from Northwestern University Feinberg School of Medicine. I'm Erin Spain, host of the show. New Northwestern Medicine studies are deepening our understanding of ALS. Or amyotrophic lateral sclerosis, a progressive neurodegenerative disease that attacks motor neurons and the brain and spinal cord. This research from the lab of Evangelos Kiskinis could lead to new avenues for the development of targeted therapies. Dr. Kiskinis joins me today to discuss his work. He is an associate professor of neurology in the division of neuromuscular disease and of neuroscience at Feinberg. Welcome to the show. 

[00:00:53] Evangelos Kiskinis, PhD: Very happy to be here. Thank you for having me. 

[00:00:55] Erin Spain, MS: Well, let's talk about ALS. More than 32,000 Americans are currently living with this deadly condition, which is very difficult to diagnose, manage, and treat. Describe this condition to me and how you're studying it in your lab. 

[00:01:11] Evangelos Kiskinis, PhD: So ALS is really devastating neurodegenerative condition. The patients unfortunately exhibit a progressive inability to control their muscles that leads to eventual complete paralysis, which becomes fatal. Now, what's characteristic about this disease is, while all of this is happening in the majority of patients, your consciousness, your brain is completely unaffected. So it's also been described as locked in syndrome. Unfortunately, the current expectation of survival from diagnosis to eventual death is between two to five years. Not much time, so it's very aggressive, it's vastly progressing. The clinical presentation of ALS is caused by the underlying dysfunction and eventual degeneration of a very particular type of nerve cell in the human body that's called the motor neuron. And these motor neurons are the nerve cells that essentially connect our brains to our muscles and thus allow us to move, to walk, to speak, to swallow, to breathe, and execute all autonomic functions. For reasons that we do not quite understand, people that get diagnosed with this disease, these particular nerve cells progressively degenerate. 

[00:02:22] Erin Spain, MS: And tell me about the work in your lab to study this disease. We should mention most people have a sporadic form of this disease, but there is also a genetic form. Tell me about that and specifically how you're approaching this condition in your lab. 

[00:02:36] Evangelos Kiskinis, PhD: The overwhelming majority of ALS patients are characterized as suffering from sporadic disease. All that means is that there's no family history. That represents about 90 percent of cases. About 10 percent of cases run in families. There's multiple family members in every generation that get this disease. What we know is that the familial cases are caused by very strong highly penetrant genetic causes. So these are mutations that are found in particular genes that basically will guarantee that somebody will get that disease. That's what penetrance means. And sporadic disease, the short answer is we don't know exactly what causes it, but what we think is that it is likely a combination of genetic predisposition-- so people have maybe many mutations in different genes that interact with each other-- as well as environmental factors. We don't really understand what drives disease in sporadic cases. Now I will say that one of the reasons we do not know a lot about ALS is because historically it's been a very difficult disease to study. The nerve cells, the motor nerves that are affected in this disease and are driving it, are within the central nervous system. That means in our brains and our spinal cords, so they're really hard to access. And the second issue is that, as we've said, the sporadic disease is really hard to model. Traditionally neuroscientists have relied on animal models. But unfortunately, because in sporadic disease, it's probably a combination of genes or genes interacting with environmental factors, that's been hard to model. It's not amenable to traditional approaches. So, what my lab does, what, what we've proposed to do a few years ago at this point was to use an alternative approach, which is based on induced pluripotent stem cell technologies (iPSC). Induced pluripotent stem cells are embryonic stem cells that we can create from any given individual. This is a fantastic achievement that won the Nobel Prize a few years ago by a very famous scientist called Shina Yamanaka, who figured out that you could take any sort of somatic cell from an individual, a somatic cell could be a skin cell, a blood cell, and using a combination of molecular factors, you can turn those somatic cells into cells that behave just like embryonic stem cells. Really fascinating, incredible discovery. And embryonic stem cells have two unique characteristics: A, they can generate any cell type in the human body because at some point every one of us was just a bunch of those human embryonic stem cells, and eventually they differentiate and then they turn to different organs and tissues. And B, under the right conditions, they can divide indefinite. And that means that once we have them, we have them forever. We can make loads and loads of them, and we can coax them into making distinct cell types that represent the human body, like nerve cells, like the nerve cells that degenerate in ALS patients, the motor neurons, by applying to them a combination of either small molecules or molecular factors. This is really powerful because it allows us to make the motor neurons from a patient that has ALS, and we can study their motor neurons in the lab while this patient is still around. 

[00:06:01] Erin Spain, MS: So you've been using this technology to model ALS for about 15 years, but since coming to Northwestern, about eight years ago, you've really been laser focused on two overall objectives. Tell me about that. 

[00:06:15] Evangelos Kiskinis, PhD: Ultimately, we're trying to understand whether the molecular causes that drive the dysfunction of these nerve cells are the same or different between these distinct genetic subtypes of ALS. That's the primary goal. And the secondary goal is, once we define the molecular causes in these rare genetic subtypes of ALS, we can now make iPSCs or induced pluripotent stem cells from also sporadic cases. And then we can ask whether the mechanisms that drive the degeneration in these rare genetic subtypes are also relevant in sporadic disease. And as we discussed earlier, this is the first time that we can really model and go after sporadic disease using these technologies. 

[00:06:57] Erin Spain, MS: And you've made some discoveries recently. You've actually published three papers since August 2023. And I want to talk about some of those papers and what you've uncovered. So let's start with the first in Science Advances that was published in August 2023. Tell me about your discovery there. 

[00:07:14] Evangelos Kiskinis, PhD: That first paper that we published late in the summer describes our efforts to understand the causes of neuronal dysfunction and eventual degeneration in one of the newest discovered genetic causes of ALS. So this is one of the newer genes that was discovered a few years ago. And actually, what's fascinating about this gene, the gene is called NEK1, N-E-K-1. The genetic discovery was largely funded by money that was raised during the Ice Bucket Challenge. The Ice Bucket Challenge is this amazing cultural phenomenon that started by these two kids that said, well, we got to raise awareness around ALS. in the early, I guess, times of social media, they had this brilliant idea of getting a bucket of ice cold water and throwing it on somebody. The idea behind this is let's make an effort to sort of freeze ALS. Let's like figure out a way to make meaningful therapeutic discoveries for these diseases. So people would donate, people would challenge each other and it became a social media phenomenon. It raised a phenomenal amount of funds worldwide that were used to support research around ALS. One of the major genetic discoveries that came out of that effort is the discovery of this gene called NEK1 as a gene that can cause a disease. Now what's fascinating about this gene is that it is responsible potentially for as much as 3 percent of all ALS, which I know sounds like a small proportion, but when it comes to ALS, this is probably the third biggest genetic cause of the disease. So we got excited about that discovery, and to begin to address this question of how does this, how do mutations of this gene cause ALS, we made patient specific induced pluripotent stem cells from people that had mutations in this gene. We made nerve cells and we asked, using a combination of approaches, why do these nerve cells become dysfunctional? And what we essentially uncovered was that NEK1 is a protein that's known as a kinase. And what kinases do is they phosphorylate other target proteins to modulate the function of these target proteins. So what phosphorylation means is a modification, is a chemical modification on a protein cells utilize to change or modulate the function of that particular protein. So we discovered that this NEK1 targets a number of other proteins in human nerve cells. And most of these proteins, to our surprise, were involved in two fundamental cellular pathways in nerve cells. One of those pathways is known as nucleocytoplasmic transport. And all that that means is it's the pathway that regulates how proteins and RNAs go in and out of the nucleus. That's a very well safeguarded process, and there's particular proteins that transport things in and out of the nucleus. What we discovered was that NEK1 phosphorylates or can phosphorylate a number of these proteins. And this was an exciting discovery because that particular pathway of nuclear import had recently been highlighted as a pathway that becomes dysfunctional in other genetic subtypes of ALS. So this provides an example of how two distinct genetic subtypes of ALS meet or converge at targeting the same molecular pathway in a nerve cell. And that's the pathway of nuclear import. Now, what we think is happening is that nuclear import becomes a little bit less efficient. So fewer things that need to go into the nucleus eventually make it there. And as a result of that, the cells become dysfunctional. So that was one of the pathways that we uncovered. And the other one, which is just as exciting, is we found the NEK1 targets for phosphorylation proteins known as microtubules, or I should say tubulins, which assemble into microtubules. And what microtubules are, they're basically the structural, the building blocks of the cytoskeleton. So the structure of most cell types, including the nerve cell. And what we uncovered is that the lack of effective NEK1 activity leads to disruption of this microtubule network, which again is very exciting because disruptions of the microtubule network have been previously highlighted as another pathway that becomes dysfunctional in ALS, including in sporadic disease. 

[00:11:44] Erin Spain, MS: And in this study, you were able to introduce anti-cancer drugs. Tell me about that and how that fits into the discovery. 

[00:11:53] Evangelos Kiskinis, PhD: Anti-cancer drugs, this is a particular class of anti-cancer drugs that target this particular pathway, the microtubule pathway, because if cancer cells, which divide all the time, cannot properly control the microtubules, they stop dividing, they degenerate. So it turns out that in ALS neurons, this pathway is disrupted but in the opposite way. So it turns out that in ALS, nerve cells from ALS patients, the microtubules are less stable. And we thought, well, if we apply these anti-cancer drugs that stabilize microtubules, that could be a good thing in a nerve cell which doesn't divide, doesn't replicate, so it doesn't need to disassemble and reassemble its microtubules. And indeed, when we applied these drugs, we're able to stabilize microtubules and then patient nerve cells did a lot better. Their function improved. Obviously, if you applied these drugs into a cell that was dividing, you would have the opposite effect because it would not allow it to divide. And that's a good thing in cancer where you want to stop this continuous cell division. We're excited about this. I think it's a proof of principle approach. Obviously, turning this into a therapeutic would have many clinical challenges that we would need to overcome. But what we're doing right now is we're focusing on alternative ways to target the microtubules, perhaps using other classes of drugs. 

[00:13:21] Erin Spain, MS: A few months later, you published again in Science Advances. And this was looking at a particular gene: the C9ORF72 gene. Now this is the largest genetic cause of ALS. This was a study that could possibly impact even more people. So tell me, what was the leading question behind this study? 

[00:13:39] Evangelos Kiskinis, PhD: So C9ORF72 was discovered a few years ago. It was a phenomenal genetic breakthrough in ALS because as you mentioned, it's the largest genetic cause of the disease. So it's responsible for as much as 40 percent of all familial ALS, and it's also responsible for a good proportion of sporadic disease, about 5, maybe 7 percent of sporadic disease caused by these mutations. Now, it's unique relative to other ALS causal genes, because instead of a single mutation in the DNA, this is what we call a repeat expansion. So the way to think about this is a short part of the DNA, in this case, six bases, just get expanded in people that have ALS. So instead of having 10 copies of these, people that have ALS have hundreds or maybe thousands of copies of these. These are known as repeat expansion mutations and they can cause a number of other neurodegenerative diseases. So, what we knew about this mutation at the time when we started this study was that from this repeat expansion, a mutation in the DNA, we've got these irregular short proteins, we call them dipeptides, that are formed. And these are exclusively found in patients that have this mutation. We knew that these dipeptides can be toxic because when we express these peptides in nerve cells, they basically kill them. So we sought out to address how these dipeptides can become toxic. And using a combination of computational approaches as well as empirical, experimental methods, we determined that one of the mechanisms by which these dipeptide proteins cause toxicity in nerve cells is because they interact irregularly with RNA molecules. Now, RNA molecules are these messenger molecules. And what they do is they allow DNA to turn into protein. So DNA is found in the nucleus, turns into RNA, and RNA makes the proteins, which is what cells utilize to function. Now, these dipeptide proteins basically bind to a lot of RNA molecules. And using a technique known as CLIP-seq, we're able to determine the precise identities of the RNA molecules that these toxic peptides bind. And it turns out that these RNA molecules belong to a specific subclass of RNA molecules known as ribosomal RNAs. Once we discovered that, we thought, maybe we can use this knowledge to design a molecule that would block this toxicity. 

[00:16:09] Erin Spain, MS: You actually created something that you call RNA bait in this study. And I want you to tell me about that and how you used that. 

[00:16:16] Evangelos Kiskinis, PhD: So this is something that, again, we're very excited about, and it's inspired by other similar discoveries around neurodegenerative diseases. But the simple idea is if we know what a toxic protein binds to, maybe we can create something that looks like a thing that the toxic protein binds to in the cell and coax the toxic protein to bind to the thing that we put in the cell. We call this the bait molecule. Because now we had uncovered that this toxic C9ORF72 peptides bind to RNA molecules of ribosomal identities, we designed a molecule that looked like ribosomal RNA, but it had chemical modifications that allowed it to survive in cells for longer. And we asked, well, if we now load nerve cells from patients that have these mutations with these RNA baits, would it block the toxicity of the dipeptides? And again, to our surprise, we found that indeed this bait was able to coax these proteins away from the things that it shouldn't bind. And by this fashion, we made them less toxic. Initially we did this in simplistic cellular models, but eventually we're able to show that this bait molecules can be therapeutically meaningful in the context of human nerve cells that are derived from ALS patients. So we made nerve cells that have these mutations from patients that were recruited to ALS clinics. And when we treated these nerve cells with our bait molecules, we allowed them to survive for a lot longer in our cell culture. We then subsequently tested the ability of these bait molecules to block the toxicity in animal models of the disease. And again, we found that they were able to block this toxicity.  

[00:18:03] Erin Spain, MS: Finally, I want to dive into a third study. You recently published in Cell Reports where you were able to use patient derived ipsc spinal motor neuron cells to develop a model. Tell me more about this discovery. 

[00:18:19] Evangelos Kiskinis, PhD: In this particular study, we focused on yet another, rare genetic subtype of ALS caused by mutations in a gene called SOD1. As we previously discussed, SOD1 was the first gene that was ever discovered in ALS. A lot of people have been working on this gene for, you know, 25 years. Now we actually, for the first time, we have a rational therapeutic approach for patients that have SOD1 mutations. And that therapeutic approach is based on the discovery, the idea that the mutations in SOD1 cause a disease, but what we refer to as gain of function effects. So the protein, because it has that mutation, it's basically doing things that it shouldn't do in nerve cells. It's becoming toxic. And the therapeutic approach that people are using is these molecules known as antisense oligonucleotides that target this mutant SOD1 and degrade it. Now, while we're very excited by the fact that we have a drug that targets a mutation and people that are treated with this drug seem to be doing better with it, we still don't quite understand how this mutant SOD1 protein is toxic. How does it kill nerve cells? And to address that question, we made motor neurons, nerve cells from patients that have these SOD1 mutations, and we asked whether other proteins are affected in their stability and function within the patient's nerve cells. And what we uncovered, which was previously unknown, was that in these SOD1 ALS cases, one of the proteins that becomes unstable is a protein known as VCP, which itself causes another subtype of rare genetic ALS. So again, we've got another example where two distinct genetic subtypes of ALS converge mechanistically in ways that we have not previously appreciated. So in all three studies that we've discussed today, the message that's coming out is that while the genetic cause in each one of the three cases is different, there's always some sort of convergence, either direct: two genes that are mutated, interacting with each other; or indirect: two distinct genes that are mutated, affecting the same cellular pathway. And going back to how we started this discussion, this is suggesting to us that although the underlying cause of ALS in each one of these three cases is distinct, we're talking really about similar processes that disrupt the function of the motor nerves in ALS patients. That is important because, again, that will help us design, and we already have designed therapeutic approaches that could be applied to one or the other. 

[00:21:10] Erin Spain, MS: And I know we're talking about ALS today, but could these approaches also help other neurodegenerative diseases? 

[00:21:17] Evangelos Kiskinis, PhD: When it comes to our particular discoveries, they have potential applications for other subtypes, for other closely related neurodegenerative diseases to ALS. The first that comes to mind is frontotemporal dementia, which is a type of dementia that shares both clinical and genetic overlap with ALS. So in other words, genes that cause ALS can also cause this type of dementia. And people that have ALS can have this type of dementia and vice versa. A prominent example of this is this C9ORF72 mutation that is not only the leading genetic cause of ALS, but also the leading genetic cause of frontotemporal dementia. And in a separate project in my lab, we're trying to understand how mutation in the same gene can cause dementia in one patient, ALS in another patient, or ALS and dementia in a third patient. And again, the way we're doing that is we're making iPSC models, induced pluripotent stem cell models, personalized models from classes of patients that belong to either ALS or FTD or ALS FTD. 

[00:22:28] Erin Spain, MS: If you could look ahead 10 years or 15 years in the future, what would you like to see happening in your lab? Where would you like this to progress? 

[00:22:37] Evangelos Kiskinis, PhD: I just came back from an international ALS meeting. It's called the International Motor Neuron Disease Meeting, which was in Basel, Switzerland. And I witnessed what I think is a momentous event in the fight against ALS because for the first time we were presented with a new drug. Again, it's an antisense oligonucleotide drug that targets an exceptionally rare type of ALS. It usually affects teenagers. And it's exceptionally aggressive. And at this conference, the team of clinicians and scientists invited to the ceremony an actual ALS patient that had this disease, who was able to walk up and down the stage. And what's incredibly powerful is before this teenage girl started the treatment, she was bedridden. But once the team infused the patient with this particular therapeutic, they were not only able to halt the disease progression, but make the patient feel better. It was a very powerful moment. You know, there was a standing ovation for both the team of scientists and the patient. And when you ask me what I want to see over the next 10 years is more events like that. We're at a critical time point where we've got this coming together of incredible resources, incredible technologies, the coming together of the community to make meaningful therapeutics for these patients that have been really helpless. 

[00:23:59] Erin Spain, MS: I'm sure for a lot of people listening that gives them a lot of hope too for folks who have loved ones or who are interested in the science progressing. So thank you so much for sharing that story and talking me through these recent discoveries, and I hope that we can have you back on in the future to see where things have progressed. 

[00:24:15] Evangelos Kiskinis, PhD: Well, thank you so much for having me. It was a real pleasure chatting with you. I'd love to come back again.  

[00:24:29] Erin Spain, MS: Thanks for listening and be sure to subscribe to this show on Apple Podcasts or wherever you listen to podcasts and rate and review us. Also for medical professionals, this episode of Breakthroughs is available for CME credit. Go to our website, feinberg.northwestern.edu and search CME.