Dr. Brian Kaspar Discusses a New Method for Studying ALS, with Potential to Test Therapeutics in Individual Patients: April 2014

Guest: Brian Kaspar, PhD, is a principal investigator in the Center for Gene Therapy and the first recipient of the Grant Morrow, III, MD, Endowed Chair in Pediatric Research in The Research Institute at Nationwide Children’s Hospital. He is also an associate professor in the departments of Pediatrics and Neuroscience at The Ohio State University College of Medicine, and a recognized national expert in the discovery of new therapies for spinal muscular atrophy and amyotrophic lateral sclerosis.



Kevin: Welcome to this month in muscular dystrophy. I'm Kevin Flanigan from the Center for Gene Therapy at Nationwide Children's Hospital in Columbus, Ohio. Each month on this podcast we invite authors of recent publications to discuss how their work improves our understanding of inherited neuromuscular diseases and what their work might mean for the treatment of these diseases. Today it's my great pleasure to welcome back as our guest, Dr. Brian Kaspar who's associate professor of pediatrics and neuroscience at the Ohio State University and who holds the Grant Morrow, III Endowed Chair in Pediatric Research here at Nationwide Children's Hospital. Brian welcome.

Dr. Kaspar: Thank you Kevin, it's good to be here.

Kevin: I'll remind our listeners that there's a link to the people we're discussing today on our website at Nationwide Children's Hospital. We're discussing today your recent paper entitled "Direct conversion of patient fibroblasts demonstrates non-cell autonomous toxicity of astrocytes to motor neurons in familial and sporadic ALS." That's a bit of a mouthful so maybe we'll start talking to our listener's first about amyotrophic lateral sclerosis or ALS, what is it?


Dr. Kaspar: ALS is the most common adult motor neuron disease that leads to muscle weakness, muscle wasting, and ultimately death. It's a motor neuron disease where motor neuron fail and ultimately patient succumb to likely respiratory failure.

Kevin: So the sensory neurons are sphered as different from peripheral neuropathies in that regard.

Dr. Kaspar: That is correct; it's a motor neuron disease.

Kevin: And I know there are both sporadic and familial versions of these. You've studied both I believe.

Dr. Kaspar: Yes, indeed there are familial forms of the disease where there is a genetic link towards the disease. And one of the most commonly studied mutations is within the gene superoxide dismutase one.


Basically anywhere where one has a mutation within the SOD 1 gene, there's motor neuron disease will happen at some point in that individual's life. Now there's a recent familial form of the disease called C9 open reading frame 72, C9orf72 mutations. This is a protein found in many regions of the brain and disease mutations were discovered in October of 2011 by Rosa Rademakers at Mayo Clinic and Bryan Traynor at the National Institutes of Health. Now there's an expansion of repeats, so it's a hexanucleotide repeat and the C9 open reading frame 72, typically in normal patients would have about 30 repeats.


But in patients with ALS and front temporal dementia you can find hundreds of these repeats, and there's a lot of work now going on in the C9orf72 mutations because it's certainly is expanding upon the percentage of familial and even some sporadic cases of the disease. Still however sporadic disease accounts for the largest group of patients, and what we mean by sporadic is the aspect that we don't have a genetic cause of the disease and we don't know why the patients develop ALS or the motor neuron disease.

Kevin: So the familial version, it's around five to ten percent of cases, is that about right?

Dr. Kaspar: That's correct.

Kevin: And then those families we mentioned with the genetic defect where it's passed form mother or father to a child, that has a gene that you can approach for studying but the sporadic groups still remains quite unknown, they're remaining 90 or 95 percent.


Dr. Kaspar: That's correct. Geneticist are working very difficult these days, Kevin. As you know to try to identify additional gene mutations that can account for these motor neuron disease. I'd further like to add that despite many years of research and clinical developments ALS has a few drugs if any at all, that's actually one only one food, FDA approved drug, Riluzole, that is given to ALS patients. It has a modest effect of increase in survival, approximately three months in patients. So this is a very serious in most cases a deadly disease within two to four years after diagnosis. And the field is desperate, and patients are desperate for new drugs and new therapies.


Kevin: And I guess it's such a tough nut to crack as you said that new tools to study these new approaches are really important and I think that's where your paper comes in a way isn't it?

Dr. Kaspar: That's exactly right.

Kevin: What do we know about astrocytes and ALS like your title describes astrocytes, what do we know about them?

Dr. Kaspar: Decade ago some seminal studies really coming out of the laboratory of Don Cleveland at University of California at San Diego and the Ludwig Institute came up with this concept that ALS might be a non-cell autonomous disease.

Kevin: Can you explain what that means.

Dr. Kaspar: So we know it's a motor neuron disease and what Don's group did was to demonstrate that the onset of the disease, at least in a mouse, really derived from the motor neuron. But then disease progression was due to other cell types such as astrocytes, such as microglia, and such as oligodendrocytes.


So really astrocytes as one of the cell types involved in the disease is actually driving the disease progression and that's actually almost more important than the motor neuron itself because patients show up into the neurologist office as you know after the fact that the disease has already started. So basically the cells that are driving disease progression, like an astrocytes as it's been shown are really cell types that could potentially slow down the disease if we can have understanding or new drugs to treat these cells.

Kevin: For our listeners' maybe we can just go back for one second just to point out neurons, probably everyone knows what they do. They are involved in conducting nervous impulses for honestly speaking. What do astrocytes normally do in the central nervous system? What do oligodendrocytes normally do for example in nervous system?

Dr. Kaspar: So astrocyte has been taught of within the field of neuroscience as glue of the brain.


In fact the Greek meaning of astrocytes is actually glue. And this is taught to basically keep the brain held together, but I think the past several years, the neuroscientists have become much more appreciative of the diverse functions of astrocytes in communicating with neurons on having aspects to support neurons but also to remodel the neurons that's based upon different experiences that one has in really helping the process of development and maintenance of the central nervous system. I think it's just becoming an exciting time to really start unravelling the diverse capacity of these astrocytes.


Kevin: So your group I know has done this and you've doe previous studies where you've derived astrocytes from ALS patients. Can you tell us how you did that and what you're able to show with them?

Dr. Kaspar: The previous studies and the rodent models by a number of groups, Stan Appel's group, our own group, Don Cleveland's group had shown that astrocytes were involved from miles study. So this is a miles over expressed in the mutant SOD1. I think one of the questions that remain in the field, were these other cell types other than motor neurons really involved in the human disease and our laboratory as well as many others sought out to really test the ability for whether an astrocyte or other cell type might be involved in ALS. In 2011 our group published manuscripts where we took the approach to derive astrocytes. We took a very unique approach to deriving astrocytes back in this manuscript.


What we did was to harvest neuro stem cells from the spinal cord of either non-ALS patients or ALS patients, and only way that one can derive neuro stem cells from tissue is post mortem. So that is patients would die with the disease or it was a motor cycle crash or other event where we could harvest tissue and rapidly isolate these neuro stem cells from the post mortem autopsy material. And there we had now cells growing from those patients where we could instruct into neurons, astrocytes, and even oligodendrocytes. And we were interested in looking at astrocytes derived from these patients and when we study these astrocytes and did what we call a co-culture model that is taking astrocytes and mixing them with motor neurons.


Whenever motor neurons were exposed to ALS astrocytes, they shrunk their X on and neurite extensions, their cell body shrunk, and they ultimately die.

Kevin: So in the culture dish or test tube you would take normal neurons and put your ALS astrocytes on top than to do this test?

Dr. Kaspar: Correct. For the first time patients specific astrocytes growing from a person, unfortunately that was from a post mortem person and so we couldn't necessarily say that we could start screening for a drug or a therapy for that individual.

Kevin: Well that brings us to your current study really in which you began instead with fibroblast which I know can be obtained from a skin biopsy, grown out of skin biopsy. And in the study you've converted them or re-program them, what is re-programming cells mean?


Dr Kaspar: So this is work that really built of off Yamanaka's seminal discoveries where one could reprogram a fibroblast to induce pluripotent stem cell. This has really revolutionize science on the ability to take cells such as a skin cell and now push it into a manner where one can create any cell type of the human body.

Kevin: That's what pluripotent means then correct?

Dr. Kaspar: We did a similar approach but we didn't go all the way back to the induced pluripotent stem cell. We went back to an induced neural stem cell. So we didn't go all the way towards an embryonic like stem cell, we just went into an intermediate state in this recent work where we could now easily produce from a skin cell in a relatively quick manner, we're talking three weeks the ability to generate the patient's specific astrocytes in a tissue culture dish.


Kevin: That's extra ordinary and the first time it's been done in this fashion is that right?

Dr. Kaspar: This was the first time where we had modelled a live ALS patient where we could derive their skin cells and produce their astrocytes for studying potential therapies od to understand the mechanism behind the disease.

Kevin: So you took fibroblast from patients with sporadic ALS and familial ALS and did this to them. And when you put them in your co-culture model what happened? What did you find?

Dr. Kaspar: Very similar to what we found in the post mortem derived astrocytes, anytime a motor neuron was exposed to an ALS patient's astrocytes, those motor neurons would shrink and they would die in the tissue culture.


Whenever they were exposed to a non-ALS patient, the motor neurons were fine. So with 100 percent prediction we could in even blinded fashion, we could pick out the ALS patients in the tissue culture dish based upon motor neuron survival. I think this open the door, we worked with clinicians from around the world. Richard Smith in California, John Ravits in California, Pam Shaw in the UK, Ed Sheffield as well as Stephen Kolb at OSU where they were deriving skin cells from patients who have been diagnosed with ALS. And opening up the door to say we can now study your cells, your astrocytes in a tissue culture dish and start understanding your disease. And I think that offered a lot of hope towards patients within the ALS clinics to really feel that they were helping to advance the understanding of the disease, and that we could hopefully understand their disease better.


Kevin: So how is this work different from your last paper with post mortem cells, and why is this exciting to you?

Dr. Kaspar: That's a really good question Kevin because we were very excited on the aspect of our post mortem derived astrocytes. But others within the field started asking very valid questions and that is we derive neural stem cells from an inflamed hypoxic environment of what the ALS spinal cord is in an ALS patient. So we were taking the cells that we wanted to study to derive astrocytes from a very hostile environment and individuals had questioned within the field and their valid questions where those stems cells that we had isolated damaged.


And where we really truly study ALS in a tissue cultured dish, or where we study damaged neural stem cells due to an ALS environment. In this new work allowed us to now go outside of the central nervous system. A skin cell that could be isolated and those skin cells could then be programed into an astrocytes. And we had identical results, so we do believe that we were studying ALS in our first studies. But I think it also opens up the door of what we're doing today on deriving fibroblast. With the simple procedure that takes about less than half an hour, one can isolate fibroblast from a living patient today where we could follow the patient form first diagnose at different stages of the disease.


And we can offer to those patients that we can start studying them as they're living with the disease today, so I think that offers a great deal of hope. It also speeds up the process and reduces the cost for us to model these diseases. The post mortem derivation was costly, secondly it took a very long time to establish these cells. With the patient's fibroblast we can isolate hundreds of patients of fibroblast and within several weeks we can convert these cells into astrocytes, that's exactly what we did in this recent paper, we could expand upon the number of mutations that we had. We have the ability now to look at sporadic patients where we don't know the gene causing but we also had patients with the OSD1 mutation and then further we expand it into C9 open reading frame 72 expanded hexanucleotide repeats and we could study those patients in a dish.


And I think it's exiting from a biological stand point that astrocytes in all of these mutations and the sporadic diseases demonstrated toxicity towards motor neurons. That's not necessarily saying that it's the same mechanism but it certainly saying that we're seeing the same molecular, pathological hallmarks to the disease. And that's likely suggesting that there are common pathways that these astrocytes are using to kill a motor neuron and certainly is opening up now the door for us to be studying additional mutations within patients as well as expanding this to really determine from us cell biology perspective with this as say the similarities and differences between these patients.


Kevin: Well Brian what's next for your group?

Dr. Kaspar: It's quite exciting Kevin on the aspect that we have many exciting projects that are going on within the laboratory surrounding ALS in our understanding to this disease. I think what we're doing based upon this paper, we've expanded this induced astrocytes and performing this co-culture screens now in a model that we are ramping up the number of samples that we can work with at a time and this is called high-throughput screening. So we are just now starting high-throughput screening of drug compounds to be able to look at whether patients will respond to any potential therapies or drugs that might be targeting the astrocytes and or motor neuron.


So we're very excited about having this as say up and running and the tools and machinery to be able to do it. I think that there's also a lot of excitement on the aspect where we're starting to understand the biological mechanisms of how these astrocytes are going wrong in the disease and the better understanding that we have, the better we will to be able to develop therapies.

Kevin: Thank you very much for taking the time to come share your work with us today.

Dr. Kaspar: It's a pleasure, thank you Kevin.

Kevin: This podcast is brought to you by Nationwide Children's Hospital. You can find out more about the muscular dystrophy program and ongoing clinical trials at Nationwide Children's at our website nationwidechildrens.org/muscular-dystrophy-podcast. You'll also find a link to the published abstract to the study we discuss today and a link to Dr. Kaspar's laboratory page as well. Thank you very much for joining us.



Well. Thank you very much for joining us.