2022 Annual ALS ONE Research Symposium: Dr. Kevin Eggan/Stathmin2 Update on biology and therapy.

Fantastic, coming through. Okay, how does everything look? Looks great. Thank you. I have the same throat bug that Bob does, so I'm going to do my best to muddle through this with occasional sips of iced coffee here from California. As Bob said, we have been in the process of winding down my research operation at Harvard over the last couple of years while I've been making the transition to BioMarin, where we're looking carefully at the ALS landscape and asking how we can get involved in bringing new therapeutic ideas forward. I really enjoyed what I've heard so far today, and I'm looking forward to providing you all an update on work we've been doing collaboratively with Bob's group and Matthias Forstner's group in Amsterdam. That has led to a couple of different therapeutics efforts to rescue stathmin-2 expression in the context of ALS models and now increasingly in the clinic. The story of how we got into working on stathmin-2 really comes directly from the original observations that TDP-43 pathology is present in the vast majority of ALS patients. This discovery has led to a cottage industry of groups trying to understand the many different roles that TDP-43 plays in neurobiology, specifically in cell biology more broadly. Those studies show that TDP-43 is an active regulator of many stages of RNA metabolism, ranging from regulating transcription to processivity to RNA metabolism at the level of splicing, or even how RNAs may be regulated at the translational level upon initiation of stress. It's become apparent that this is quite an important protein with respect to how cells integrate environmental information and respond. In particular, we became interested in the idea that a reason we may have failed to find changes in RNA metabolism in animal models that were predictive of changes in RNA metabolism in ALS patients themselves might have something to do with the evolution of RNA metabolism between those species. While TDP-43 might be doing the same types of things to RNAs in animals and in humans, the highly divergent nature of DNA sequence space between animals and mice might mean that the places we were looking specifically might mostly not be conserved. An easy thing to think about is the role in pre-mRNA splicing: if you look at the regulation of splicing between human and rodents, this is largely a divergent process, with dramatic reassessment over evolutionary time of where splicing should happen at a nucleotide level. This would mean that if you looked at TDP-43-dependent splicing changes in mice, even if those were occurring in humans, TDP-43 might be acting at very different places, so that could be part of why we had been striking out. With that in mind, we began to wonder whether even less mature motor neurons in culture from human sources, which make human transcripts and have a human regulatory environment, might be better predictors of the changes in RNA metabolism that we might see in patients. We set out to carry out a fairly simple experiment: take human embryonic stem cell lines that had been targeted to carry a motor neuron reporter gene, differentiate them into spinal motor neurons, expose them to either control siRNAs or siRNAs that knock down TDP-43, use flow cytometry to purify those motor neurons to homogeneity, and then examine changes in transcript abundance. Not surprisingly, given the pervasive role that TDP-43 plays in regulating RNA metabolism, we saw many changes. In the upper left-hand corner of this RNA sequencing experiment, predictably the thing that was most reliably changed was TDP-43 since we had knocked it down. While all of these changes are potentially fascinating with respect to understanding the changes in gene expression or RNA metabolism that may be happening in ALS, you can see in the upper left-hand corner, tracking very closely with the change in TDP-43, is a transcript encoding the protein stathmin-2. Because of this very close association between the amount of TDP-43 alteration and the amount of stathmin-2, and because of its function which seemed reminiscent of the sort of thing that is altered in ALS, we began focusing first on trying to understand the underlying mechanisms that connected TDP-43 alterations to stathmin-2, and indeed to test and play out this idea that these changes identified in human neurons when TDP-43 is altered would be more predictive of those in people. Stathmin-2 is a protein selectively expressed in neurons. Histopathological and in situ hybridization studies in the spinal cord suggest that it's very highly expressed in alpha motor neurons, the ones most sensitive to degeneration in ALS. It's encoded by a very abundant transcript which, as I'll show you, is subject to a mechanism regulated by TDP-43 in a variety of contexts. Interestingly, with respect to the last talk, it's a member of a family of proteins involved in dynamic regulation of the microtubule cytoskeleton. It localizes to both the Golgi apparatus and the growth cone, where it has an important role in regulating the microtubule skeleton. Early experiments looking at the stathmin family of proteins indicated that stathmin-2 played an important role in axonal outgrowth. To summarize a great deal of molecular biology work done in both my group and in Don Cleveland's lab, both groups identified that stathmin-2 is subject to regulation by TDP-43 through a mechanism in which TDP-43 directly interacts with five prime regulatory units within the gene of stathmin-2. In the presence of stathmin-2, transcription is processive and splicing omits an early cryptic exon, allowing processivity of the transcript and production of a full-length transcript and stathmin-2 protein. But when TDP-43 levels wane, there is premature transcriptional termination and splicing into a cryptic exon in the five prime region of the gene in an early intron. This may or may not result in the production of a 17-amino acid peptide, although that's not yet been empirically detected to my knowledge. You can see this phenomenon clearly when you look in RNA sequencing data, not just in all the models we've examined thus far, but also in patients. On the left, RNA sequencing reads from the spinal cord of control individuals who passed away in ways unrelated to ALS and were pathologically shown to be free of TDP-43 pathology. In contrast, on the right, the individuals all have TDP-43 pathology, and you can see that many of the transcripts splice into this premature cryptic exon, reducing the total amount of transcript being produced and splicing into downstream coding regions of the gene. A beautiful paper in which TDP-43-negative nuclei were flow-sorted and then RNA-sequenced demonstrates that in that fraction, this process is almost complete. The heterogeneity you see at a tissue level is probably the result of some cells having TDP-43 pathology and others not. Just as we were winding down the lab, Leslie Nash, a former postdoc now at the Canadian health authority, sought to design sensitive and quantitative qPCR methods for measuring these changes in RNA metabolism in different biofluids, particularly from exosomes. On the left-hand side, relative to healthy controls or individuals with hereditary paraplegia, levels found in either familial ALS or sporadic ALS are much higher with respect to their content of cryptic exon in the RNA. I really want to thank the ALS community, in particular Merit Cudkowicz and Niels, for access to these samples. On the right-hand side, you can also see that in these same biofluids derived from blood, there's a reduction in the amount of coding transcript we can detect in the circulation. This is highly encouraging because this would be not only a potential measure in patients of TDP-43 dysfunction that could be used for evaluating the success of any TDP-43-modifying therapy, but also an important potential biomarker for target engagement for any stathmin-2-specific intervention. Curalis, a company I was involved in founding, has been taking this forward and attempting to turn it into a qualified assay for use in clinical studies. Now I want to tackle a related question: now that we have begun to find targets of TDP-43 that seem to be changing in patients, is there a credible role for their misregulation in motor neuron degeneration or the motor neuropathy that we see unfolding in those patients that might suggest that correcting expression of those genes could be beneficial? We began this journey by knocking out the stathmin-2 protein in the mouse. Iruna Guerras, while she was in the lab, successfully produced cohorts of animals that completely lacked stathmin-2 protein expression. As she aged those animals, she found that they were subject to an age-dependent decline in their motor system. I'll show you a couple of aspects of this quickly. Here, whole-mount immunostaining of the neuromuscular junctions in these and control animals shows that while you can see the postsynaptic apparatus stained with bungarotoxin, after knocking out stathmin-2, the number of those that also have a presynaptic neuronal partner is decreased. In the controls, more of these pretzel-shaped neuromuscular junctions are yellow, but in the bottom panels, many of them lack their innervating motor neuron. This is severe enough and prolonged enough that you also begin to see another clear hallmark of motor neuron degeneration: postsynaptic neuromuscular junctions, if they lack synaptic input for long enough, begin to degrade. You can see this by a loss of complexity and simplification of those denervated neuromuscular junctions. This is very quantitatively apparent in this mouse model over time, as you can see both in the pictures on the left and in the quantification in the bottom right-hand corner. This is sustained enough that we also begin to see evidence of muscle injury and a muscle regenerative response. I apologize for some of the histopathology and processing. What you're looking for in these images is less the difference between the red and white space, which is an artifact, but instead where are the black speckles in these muscle fibers. In the controls, both early and late, those muscle nuclei are present near the periphery of those muscle cells, as one would expect from a well-differentiated and stable muscle. In contrast, on the right-hand side, early in the lives of these animals we see this peripheral localization of the nuclei, but later, after neuromuscular generation has occurred and we see evidence for degradation of the neuromuscular junction, those nuclei are more likely to be found in the center of those muscle fibers. This is a classic observation when muscle injury and regeneration are occurring and might be expected as a result of muscle denervation. So we see multiple lines of evidence to support changes in the neuromuscular axis. When we look at the behavior of these animals by either rotarod or hanging wire, we see a very significant decline in their performance in both assays. We've looked at this in several different cohorts of animals, both an initial cohort where we had made many different alleles by knocking out the gene with CRISPR and then looked directly at those animals, and then in the bottom panels, independent cohorts where we had bred out a specific allele that we knew was a loss-of-function allele and recapitulated these studies. So it's very clear that loss of stathmin-2 can have very severe effects on the motor system. We do not see those animals being subject to premature lethality; they are born at normal Mendelian frequency and seem to live a normal lifespan, albeit with greatly reduced motor neuron functionality. We don't know why this doesn't progress to fulminant ALS. The story may be that other targets of TDP-43 are important mediators of the complete picture of motor neuron disease, but I think this does underscore that the loss of even a single TDP-43 target could be contributing quite substantially to motor neuropathy in patients. With this in mind, and with the thought that rescuing stathmin-2 would be important not only for validating that the phenotypes we had seen in these models were dependent on stathmin-2, but also to create a model for studying human transcript regulation in vivo, we set out to produce BAC transgenic models carrying a humanized BAC transgene and to cross those into the stathmin-2 mutant background to ask whether they could rescue the phenotypes I just described. Leslie Nash was able to identify such a BAC and produce founder animals that demonstrated expression of all portions of the stathmin-2 transcript. One of the early questions we wondered was: if we induce TDP-43 pathology in a mouse, would we see alteration of the regulation of the human transcript from this BAC? This was something we had never seen for the mouse stathmin-2 transcript, explaining why it hadn't been identified before as a potential regulator of axonal function. To do that, she crossed the early BAC transgenics to the TAR4-4 animals that overexpress TDP-43, resulting in TDP-43 pathology. On the left, in this model where TDP-43 inclusions form, you also see nuclear clearance of TDP-43. Consistent with that and similar to our observation of knockdown of TDP-43 in human neurons, you can see a dramatic induction of the cryptic splicing event from this human BAC, suggesting that TDP-43 could regulate these sequences in stathmin-2 had the human sequences been there. Introducing them subjects those sequences to regulation by TDP-43. We found that when we crossed these human BAC transgenic animals to the loss-of-function animals, we could rescue stathmin-2 expression within those knockout animals and restore expression of human stathmin-2 from that human BAC to levels at or above those seen in wild-type animals. When we did that, we were able to rescue neuromuscular phenotypes within those animals, both at the NMJ level as well as at the level of motor behavior. This gives us a great deal of confidence that the deficits we're seeing in the knockout animals are a result of loss of stathmin-2, but also indicates that human stathmin-2 can complement the mouse deficiency and can be properly regulated in that context, offering an opportunity for exploring pharmacological impacts on stathmin-2 activities. I want to close with one last study and a couple of parting thoughts around what the molecular mechanisms might be that contribute to motor neuropathy when stathmin-2 levels wane. I think there's not an accidental alignment between these two talks because what we have found is that when stathmin-2 levels wane, there's a very substantial effect on microtubule stability. The importance of skeletal elements, as we heard about in the last talk, are important mediators of spatial regulation of autophagy within neuronal cell types. Here, in human motor neurons, if we eliminate stathmin-2, this dramatically affects the ratio of free to polymerized tubulin within these neurons. We've replicated this experiment with further controls. What's quite striking is that lacking stathmin-2 in human motor neurons is rather similar to hitting them with a substantial dose of nocodazole, a fairly striking finding given the effects of that small molecule on microtubule stability. We've followed this from human neurons into the loss-of-function animals. Interestingly, we don't seem to find that eliminating stathmin-2 has a big effect on the total amount of tubulin within those neurons, and it did not have a significant effect on the total free to polymerized tubulin when we looked at tubulin extracted from the total spinal cord. This wasn't necessarily surprising; we were surprised to see even such a strong trend toward effect because, remember, when we looked at the localization of stathmin-2 in the spinal cord, we find it most specifically expressed in alpha motor neurons and largely unexpressed in other cell types of the spinal cord, where presumably a lot of the free tubulin is coming from. In contrast, if we look at beta-3 tubulin, the neuronal-specific form of tubulin only expressed in neurons in the spinal cord, we see an absolutely huge effect on the ratio of free to polymerized microtubules, suggesting that stathmin-2 plays a very important role in regulating microtubule dynamics within the neuronal compartment, probably most specifically in alpha motor neurons. This is a very credible mechanistic explanation for the motor neuropathy we observe in these animals. In closing, I think something that the ALS community is going to continue to hear more about: we've worked closely with Bob and John Watts at UMass Medical School to identify oligonucleotides that bind to regions surrounding this cryptic exon. After TDP-43 knockdown, we've been able to show that these restore normal expression of stathmin-2. Bob has been overseeing an initial clinical study exploring dose escalation in an initial patient. That lead oligo, which has dramatic effects on stathmin-2 in culture, has been shown to be very tolerable up to what we would anticipate from these culture experiments to be an efficacious dose. Bob, as I understand it, is going to be working with Curalis to understand whether it also leads to restoration of stathmin-2 expression and quelling of the presence of the cryptic exon. Just in closing, we've been working hard to make sure we get these animal resources into Jackson Labs. We've also been able to distribute them to Bob and John's lab at UMass for use. We've now published most of this work in Neuron this year. I'd like to thank Bob for this opportunity to speak today on this work. We've managed to shutter the Harvard lab now, and both myself and my family are ensconced here in Marin County at BioMarin. I'm really looking forward to future interactions with the ALS community as what I hope will be a future of deep engagement. Bob, you probably didn't know this, but the CEO of BioMarin was the regulatory lead at Sanofi-Aventis for Riluzole and was central for getting Riluzole across the line all those years ago, and nobody thought it was possible. He has been a big believer ever since and was one of the big reasons I decided to come and join BioMarin.

Yes, so interesting. There had been a question early on in the community because some early experiments at JAX had suggested that there might be non-Mendelian inheritance in young animals and that animals might be being lost. We actually found that there's probably something else going on: stathmin-2 heterozygous and homozygous moms make them terrible mothers, and as a result we were losing a lot of pups. As we blew up our cohorts more and more, we found that we were able to overcome that with further breeding and a larger colony. I think some of those findings might have just been incidental findings with small Ns. But that question about whether there was an early phenotype actually led us to carry out an early time course to see when innervation begins. The youngest animals, as they wire the neuromuscular system, seem to have an early normally wired system. Then as the animals grow and become full-sized, this problem becomes apparent. We don't know exactly why it becomes apparent in these young adult animals. We have several hypotheses that we haven't had time to follow up that I hope the field will. One is that the demands on the motor neuron for a stable pool of properly regulated microtubules are not as great until the neurons become long. Another hypothesis is that early in development, stathmin proteins show a broader distribution of expression in the brain, so another intriguing idea is that other family members may cover for the expression of stathmin-2 until aspects of motor neuron specialization become more apparent. That is to say, early in motor neurons, stathmin-1 is also expressed, which covers for stathmin-2, and it's not until later that this is resolved. We don't know for sure; I think both of those are worth testing, and there are probably other ideas that could be true as well. With respect to the human phenotypes, yes, there are quite strong phenotypes. If you eliminate stathmin-2 and culture those human neurons for a long period of time, in addition to changes in microtubule dynamics, one also sees a dramatic reduction in the complexity of those neurons. They have fewer branches and a much simpler structure, which is consistent in our view with some changes in functionality in the growth cone as the cells grow and mature.

Yeah, we had just begun to look at this, Bob, as the lab began to wind down. It's such an interesting intersection, and we had not looked at it at all in the mice. We had begun to look at it in a different context, actually in the human motor neuron systems, because we did carry out experiments where we confirmed that knocking out SARM1 in human motor neurons does lead to axonal preservation after axotomy. So those experiments did confirm that that biology is indeed conserved in humans. Interestingly, we identified a chemical interaction between SARM1 and stathmin-2, which was never followed up on and I think would be quite an interesting thing to do. It makes sense given the SARM1 phenotype that there would be ultimately some tie-in to microtubule regulation, but validation of that interaction through independent means beyond the initial mass spec experiments where the pull-down found the putative interaction, and functional follow-up, are all wanting. But I think it's fascinating.