Research funded by The ALS Association recently identified a potential therapeutic target in the accumulation of a protein called CHMP7 that is connected to TDP-43 mislocalization and development of ALS.

Connecting ALS recently talked to Dr. Jeffrey Rothstein, professor of neurology and neuroscience and the founding director of the Robert Packard Center for ALS Research at Johns Hopkins University School of Medicine, and Dr. Alyssa Coyne, a postdoctoral fellow at Johns Hopkins about their research.

This transcript has been edited for brevity and clarity. You can listen to the entire conversation HERE.

Can you walk us through your findings and what it means for ALS research going forward?
I'll start off by introducing TDP 43 as it relates to ALS. TDP-43 is an RNA binding protein that regulates different aspects of RNA metabolism, regulates protein expression in the cell, ultimately. Previously in ALS and ultimately other neurodegenerative diseases like Alzheimer's, frontal temporal dementia, this normally nuclear protein is found present in the cytoplasm and disaggregates. And you can actually see that in end stage post-mortem tissues in ALS, Alzheimer's disease and frontotemporal dementia.

While that was identified about 15 years ago, no one really understands why that happens or how that happens. My work as a post-doc in the lab is really focused on identifying defects in the nuclear pore complex, which is sort of like the control center of the cell. And it forms a gate between the nucleus and the cytoplasm to regulate the compartmentalization of different proteins and MRNAs between the nuclear and cytoplasmic compartments of the cell.

TDP 43 is one of these proteins that can actually shuttle through this complex. We have hypothesized for a while now that perhaps an injury to the nuclear pore complex or control center could perhaps contribute to the mislocalization and dysfunction of TDP 43 that is seen in ALS-related neurodegenerative diseases.

CHMP7 is a protein that is involved in multiple cellular pathways, but it's predominantly known for its role in nuclear remodeling; and recent work from our collaborator, Patrick Lusk at Yale, has identified a role for CHMP7 and ESCRT-III complexes -- its protein partners -- in remodeling nuclear membranes and surveilling the nuclear pore complex to maintain homeostasis, to ultimately maintain trafficking and compartmentalization between the nucleus and the cytoplasm.

In ALS, this protein, CHMP7, actually accumulates in the nucleus and can initiate an injury to the nuclear pore complex structure, and this can ultimately be the loss of TDP 43 function within the nucleus and mislocalization of the TDP 43 protein itself from the nucleus to the cytoplasm.

Our previous research identified how the nuclear pore was defective. It was missing pieces, we'll call it. I like to use the term -- probably not properly -- as the pores being disassembled, and our most recent study explains the mechanism, how it becomes disassembled in ALS and then downstream of that, how that then affects TDP 43.

We want to emphasize, this is a pathology. It's come to all sporadic patients that is both the nuclear pore defect, but previously the defect and TDP-43, this loss of nuclear TDP-43.

How do these findings point to a potential therapeutic target?
ASO's, or antisense oligonucleotides, are elements of the nucleotides, the elements that the building blocks of either RNA or DNA, and they're complimentary,that is, they're matching partners. And when you add them to a cell artificially, as you can do with an ASO therapy, they cause a degradation of the endogenous RNA in the cell, and that's a way of essentially eliminating a toxic species in the cell. So, it's the earliest starting point in many diseases where there's defects in the nucleotide sequence. The ASO essentially eliminates that very first starting point. To use an old cliche, it’s like a domino.

This has actually been done for a while in ALS. It started with the SOD1 antisense trials, which are still now well underway, because exciting results have been reported in the last year. Quite honestly, it began there first, but sometime later that same kind of therapy, a different version of this drug, was used in spinal muscular atrophy. And it is what I consider the penicillin of the 21st century. It essentially stops the disease in those children, and they regain normal health. This has become a very powerful therapy that we can use.

And finally, the most important part, we know therapies take a long time to develop. It often takes 10 years to build a new molecule therapy. ASOs much faster. This research that Alyssa and I just published, we're already working with the drug companies to develop that ASO therapy. It will be, of course, their therapy to use, but it moves easily half the time a normal drug goes to development to a patient.

What are the next steps in the development and in the research process?
In the past, many therapies were first developed in a model, a rat or a mouse, for example. This gene product is very different than mice versus humans. And this is why the platform that Alyssa used, induced pluripotent cells, essentially a human neuron from our patients is the ideal platform to discover this drug in and to use this drug.

And so, we began without a mouse model, if you will, we went right to the human cells. We tend to refer to this as like the equivalent of a human biopsy from our patients.

We've worked this out with the help of Ionis, the pharmaceutical company that has been a real strong collaborator in this project. And so, we've worked at a molecule that works very good in the laboratory. They then as well as any commercial partner, whether they develop it for a drug or some other commercial partner have to then optimize it and we'll need to still do some testing in animals just to make sure it's relatively safe in animals, and of course we all know that ALS is a terrible disease and safety to some patients becomes a minor player when you have such a terrible disease, but they need to know before they'll ever bring it to patients that you can give it to a mouse and suddenly the analysis and dropped dead in two hours.

So, there are simple things that we need to know first and that's in their hands. We'll help them out a little bit, but it really ultimately goes to their hands. But in my past experience, an ASO moves within a few years to patients. Now, I can't tell you there'll be a few years for this molecule, but in my past experience, it's been more like a decade. This is going faster. If in fact it is a good molecule, that's where the next set of experiments are.

How much earlier are we talking about in terms of being able to think about earlier interventions in terms of earlier treatments? Is that something that we can conceptualize?
We believe that the nuclear accumulation and mislocalization of CHMP7 from the cytoplasm to the nucleus might be perhaps one of the earliest initiating events pathogenically in ALS.

We also know in a subset of ALS, such as C9 ALS for instance, that the initiating domino there is actually a genetic mutation in the gene, but we think we've landed on a very early event, at least in cultured IPS neurons.

Translating that to patients is a little harder. We don't really know. There have been very few studies of patients where brain tissue is examined before they actually had ALS --accidents, things like that. From the limited data that's available, it looks like that a very early event is this mislocalization followed by an event that many other labs have studied, which is this aggregation of TDP-43 outside the nuclease in what is known as the cytoplasm. Studies of real human neurons teach us that, that clearance is really likely the first event.

Now, can we translate that to the time we actually intervene in the clinic? I don't think we're ready to do, and no one is ready to do that yet. So, all we know is the earlier we get in ALS, the more hope we have that we will be impactful.

I've run clinical trials since 1995 and I can tell you when we looked very late in the disease, we haven't done so well in terms of drugs.

What's important to point out herethat we didn't mention yet in terms of translating this therapeutically is, when I did those ASO studies in these cultured neurons, I actually treated these neurons at a time point where the pathogenic cascade had already occurred.

These neurons already had nuclear pore injury. They already had TDP-43 dysfunction. They already had nuclear accumulation of CHMP7 initiating this all. And when I treat at that time point and now reduce CHMP7 protein levels, I can reverse the injuries that had already occurred.

So, the point being, it's not past the point of no return. We can still repair an injury once it's occurred, at least in this human cell line, which we hope recapitulates what goes on in patients.

What are the next steps in this particular line of research?
My future directions in terms of looking at CHMP7 research is we have been talking about identifying that first domino and identifying the initiating event. And so we know that nuclear accumulation of CHMP7 can initiate this pathogenic cascade, but this is a protein that's normally found in the cytoplasm and not the nucleus.

My open questions that I'm interested in pursuing, or one of them moving forward is, what actually initiates bringing CHMP7 into the nucleus and keeping CHMP7 and the nucleus to, quote unquote, disassemble the nuclear pore complex and initiate this pathogenic cascade, especially in sporadic ALS, where there's no known genetic mutation.

We have a few hypotheses that I'm working on testing and I'm interested in looking further into, in the future, just sort of identify this initiating event that initiates CHMP7 initiating nuclear pore, complex injury.

And one of these might be actually genetic variants in nuclear pore complex proteins or nuclear envelope proteins themselves that may act as a damage signal recruit this surveillance and homeostasis protein at pathway. That's a particularly exciting idea that we have in the group.

We all have normally acting proteins, but slight variations. If you look at all of us, we have slight differences in our face, eyes. And this surveillance pathway may pick up those slight differences over time, leading to the injury, as opposed to a strong genetic mutation like C9 mutation. So that's at least one way to explore this in sporadic ALS.

The other very important point, and we've seen this a lot now with things like COVID research, this fundamental research program, aside from giving us a fundamental understanding of cell biology, does begin to lead to new candidate drugs. When we started this years ago we were hoping to fix the nuclear pore. We didn't know this pathway would be the direction. It was a sequential series of basic research that led to this point. Continuing along those lines almost certainly will lead to those new opportunities.

As we continue to understand what initiates this whole cascade, we'll continue to refine and find new therapeutic opportunities for intervention, perhaps even earlier on.