The International Symposium on ALS/MND is the largest gathering of researchers studying amyotrophic lateral sclerosis (ALS), otherwise known as motor neuron disease (MND), in the world. The conference, organized by the UK’s Motor Neurone Disease Association (MNDA) in partnership with the International Alliance of ALS/MND Associations, welcomes scientists, academics, and drug development researchers from around the world to share and discuss the latest research in ALS. The 2024 symposium took place in Montreal on December 6-8.
This year, five researchers from the ALS Therapy Development Institute (ALS TDI) took part in the symposium’s prestigious poster session, sharing their latest research with colleagues from around the world. The posters shared included:
- Kaly Mueller, Senior Associate Scientist, on her work with Profilin1 mouse models of ALS.
- Therese Dane, Senior Associate Scientist, on the potential role of C9orf72 mutations in TDP-43 function in some cases of ALS.
- Thomas (TJ) Krzystek, D., Scientist II, on research into C9orf72 mutations utilizing motor neurons derived from induced Pluripotent Stem Cells (iPSC).
- Anushka Bhargava, Ph.D., Scientist II, on research into the TDP-43 protein’s role in ALS conducted with iPSC-derived motor neurons.
- Val Tassinari, Associate Scientist III, on ALS TDI’s work to advance C9orf72 zebrafish models of ALS.
For the first time in ALS TDI’s history, scientist Kaly Mueller was honored with the prestigious Biomedical Poster Prize at the symposium. This highly competitive award recognizes outstanding poster presentations and is given to only three of the more than 450 submissions at the event. Mueller’s achievement marks a significant milestone for ALS TDI’s research team.
“Winning the biomedical poster prize at this year’s International Symposium on ALS/MND was a huge honor,” Kaly says, “as I never expected to be initially shortlisted for the prize let alone win (Fernando can attest to me saying “I’m not going to win” several times during the trip). When my name was announced, it felt amazing to have the work we have done to validate the Profilin1 mouse model recognized, and even better to represent ALS TDI, on such a large stage.”
For more in-depth summaries of these posters from the researchers themselves, read on:
Kaly Mueller
Key Takeaway: This work establishes the Profilin1 (PFN1) mouse as a valuable ALS animal model that complements the widely used SOD1G93A mouse, addressing the need for diverse preclinical models to better represent the heterogeneity of ALS.
Poster Summary: ALS is a heterogeneous disease, highlighting the need for additional animal models to complement the widely used SOD1G93A mouse and better translate preclinical findings to a broader patient population. Previous research suggested that the Profilin1 G118V mouse could be a potential model for testing therapies, and this work aimed to comprehensively characterize it and identify disease-relevant readouts for evaluating therapeutic efficacy.
Our poster summarizes several years of effort to validate the PFN1 model and our findings indicate that the PFN1 mouse exhibits a progressive ALS-like phenotype, including significant reductions in body weight, grip strength, and CMAP amplitude, along with hindlimb paralysis and decreased survival.
We were the first to longitudinally measure plasma neurofilament light chains (Nf-L), a known ALS biomarker, in these animals. We demonstrated that rising Nf-L levels correlated with disease onset and survival. Additionally, in collaboration with researchers at Brown University we revealed significant neuromuscular junction (NMJ) denervation in the hindlimbs. We are continuing the characterization of this model by performing histological evaluations.
Preliminary histological findings show a decrease in lumbar spinal cord neurons and increased neuroinflammation in this region. In summary, this work establishes the PFN1 mouse as a robust model that recapitulates key features of ALS and supports its use in preclinical pharmacology studies alongside the SOD1G93A mouse.
Therese Dane
Key Takeaway: This study highlights how C9orf72 mutations may disrupt the processing of genetic instructions in motor neurons and identifies a potential treatment approach that could help protect these cells by addressing those disruptions.
TDP-43 loss-of-function (LoF) pathology has emerged as a potential central hallmark for up to 97% of ALS patients. Here, we attempted to better understand the contribution of the C9orf72 hexanucleotide repeat expansion (HRE) in triggering TDP-43 LoF pathology, by examining improperly spliced TDP-43 regulated mRNA transcripts in aged iPSC-derived motor neurons. We found that day 49 motor neurons with the C9orf72-HRE had a significant elevation in transcripts containing cryptic exons (CEs), including stathmin-2 and UNC13A.
These improperly spliced transcripts were present in higher levels in the nucleus compared to the cytoplasm. We next asked if pharmacological modulation of protein arginine methyltransferases (PRMTs) alters the levels of these CE-transcripts, as they have been shown to modulate nuclear export of mRNA, and we previously found that they improve MN survival in response to toxic dipeptide repeat proteins. Interestingly, we found that treatment with a type-I PRMT inhibitor, MS023, significantly reduced the cytoplasmic levels of CE-transcripts, while increasing the levels in the nucleus. This suggests that MS023 is slowing the export of improperly spliced mRNA from the nucleus. Future work will focus on determining if the slowed nuclear mRNA export contributes to the beneficial effect on C9orf72-HRE neurons.
TJ Krzystek, Ph.D.
Key Takeaway: This study demonstrates how mutations in the C9orf72 gene may impair the movement of lysosomes—key cellular components responsible for waste management—within nerve cells and identifies potential drug candidates that could restore this essential function.
Our study investigates lysosomal transport dynamics in spinal motor neurons (MNs) derived from human induced pluripotent stem cells (hiPSCs) with C9orf72 hexanucleotide repeat expansions (HREs) and haploinsufficiency. Using a custom-built analysis workflow, we observed impaired lysosomal transport in axons of C9orf72-ALS and C9orf72-FTD patient lines, as evidenced by decreased lysosome velocity, long-distance track counts, and transport density compared to controls. Evidence from a haploinsufficiency model of C9orf72 suggests that both HRE mutations and C9orf72 loss-of-function could underscore these observed transport defects. Importantly, screening of rescue compounds revealed significant improvements in lysosomal transport metrics for specific candidates, demonstrating their potential therapeutic value.
This research suggests a critical role of C9orf72 in maintaining lysosomal dynamics and its disruption in neurodegenerative diseases. The findings provide a framework for therapeutic screening, emphasizing lysosomal dynamics as a measurable target for drug development. The study represents a significant step toward understanding the molecular mechanisms underlying C9orf72-ALS and FTD and identifying actionable interventions to restore neuronal function.
Anushka Bhargava, Ph.D.
Key Takeaway: This study developed a model of motor neuron cells missing a key protein, TDP-43, to mimic its loss of function in ALS and revealed how this disrupts crucial cellular functions, providing a powerful tool for understanding ALS and testing potential treatments.
A protein called the TAR DNA-binding protein 43 (TDP-43) plays a significant role in ALS, a disease that causes nerve cells to break down. While only a small number of people with ALS have changes (mutations) in the gene that makes TDP-43, most ALS patients (97%) show problems with how the TDP-43 protein behaves. In ALS, TDP-43 moves out of the cell's control center (nucleus) into the outer part of the cell (cytoplasm), where it forms clumps. These clumps damage nerve cells and may lead to their deterioration. In healthy cells, TDP-43 helps process the instructions that the gene encodes (RNA), which the cells use to make proteins. One of these many proteins is stathmin-2 (STMN2) which is crucial for nerve outgrowth and maintenance. When TDP-43 is not working properly in the nucleus, errors occur in the RNA processing for many proteins, including STMN2, leading to less of the normal functional protein being made. This adds to the nerve damage in ALS. To study how TDP-43 problems contribute to ALS, scientists currently use two main methods: models with rare genetic mutations seen in some familial ALS cases or methods that artificially stress cells to mimic disease. However, these approaches have significant drawbacks. TDP-43 mutations are rare (less than 0.5% of ALS cases), and stress-based models may create effects unrelated to ALS. Also, these models only show TDP-43 abnormalities in only a small number of cells. Therefore, to better understand how TDP-43 specifically contributes to ALS, researchers need more accurate models that mimic the disease more effectively.
In this study, we created a model to better understand the effects of completely losing TDP-43. To do this, we used human-induced pluripotent stem cells (iPSCs), which are lab-grown cells that can develop into almost any cell type in the body. We attempted to remove the TDP-43 protein from these cells and turn them into spinal motor neurons (the primary nerve cells affected in ALS). We then studied how the loss of TDP-43 affected their survival, ability to grow nerve fibers, transport of cell components, and processing of RNAs. To achieve our objective, we used the CRISPR/Cas9 technology, a tool for editing genes, to "knock out" (or completely lower the levels of) the TDP-43 protein in iPSCs. This process was carefully verified using various techniques, such as DNA sequencing, RNA analysis, and protein detection. Once the TDP-43 was successfully removed, we turned the stem cells into healthy and functional motor neurons in the lab. We then analyzed these neurons to see how losing TDP-43 impacted them. Loss of TDP-43 showed a decrease in stathmin-2 (STMN2) and an increase in a faulty version of STMN2 RNA, which matches what is seen in ALS with TDP-43 abnormalities. The motor neurons also showed a defect in transport of internal cellular organelles, which is crucial for a healthy cell to function, and a defect in nerve outgrowth.
This study demonstrates that we successfully created a cell model that lacks TDP-43 but can still grow into motor neurons. These neurons provide a valuable tool for studying how losing TDP-43 affects nerve cells and for testing new treatments aimed at reversing these changes, not just for ALS but for other diseases involving TDP-43 as well.
Valerie Tassinari
Key Takeaway: This study aimed to develop a zebrafish model that mimics some aspects of C9orf72 pathology using synthetic C9orf72 toxic repeat proteins, providing insights into their potential for use in testing experimental ALS therapies.
C9orf72 mutations, the most common cause of familial ALS, are postulated to cause production of toxic dipeptide repeat proteins (DPRs); ALS TDI has previously demonstrated toxicity of DPRs in cell-based assays1. More diverse disease models are needed to improve translatability of drugs into humans; to this end, we worked on developing a zebrafish toxic DPR challenge assay that attempts to recapitulate C9orf72 DPR toxicity in wild-type zebrafish. Synthetic DPRs (poly(PR) and poly(GR)) were applied to larval zebrafish in a medium-throughput format. Changes in survival and behavior were tracked, and a potentially protective compound was tested to characterize the effect of the synthetic DPRs. We found that exogenous PR(15) dose-dependently reduces survival in 4dpf wild-type zebrafish while GR(15) does not affect survival. We also found that exogenous GR(15) and PR(15) localize differently in 5dpf zebrafish. In behavior studies, preliminary results suggest that exogenous PR(15) does not reduce swimming distance in 6dpf zebrafish. Finally, pretreatment with a PRMT inhibitor, a compound that showed rescue in the cell-based assays, showed variable results. In conclusion, our findings suggest that exogenous PR(15) can induce dose-dependent toxicity in larval zebrafish. However, more characterization work still needs to be done before this model can be used for drug screening.
- Gill AL, Wang MZ, Levine B, Premasiri A, Vieira FG. Primary Neurons and Differentiated NSC-34 Cells Are More Susceptible to Arginine-Rich ALS Dipeptide Repeat Protein-Associated Toxicity than Non-Differentiated NSC-34 and CHO Cells. Int J Mol Sci. 2019 Dec 11;20(24):6238. doi: 10.3390/ijms20246238. PMID: 31835664; PMCID: PMC6941034.
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