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Current Methods In ALS Research

Published: March 3, 2023


Amyotrophic lateral sclerosis (ALS) is a devastating neurodegenerative disease that affects roughly 1 in 400 people in their lifetime. The disease initially presents as upper and lower motor neuron impairment and eventually progresses to paralysis and death as a result of respiratory failure within 2–5 years after symptom onset1. ALS can be hereditary, with over 30 different genetic mutations but only 4 gene variants (C9orf72, FUS, SOD1, TARDBP) accounting for about 55% of familial ALS. The majority of ALS cases, approximately 90%, represent sporadic ALS, for which the leading causes are still not fully understood2. There is an urgent need to unravel the mechanisms of ALS by using the appropriate tools and model organisms. In this methods collection, we provide an overview of the recent research progress in terms of mimicking this disease and, hopefully, ultimately finding treatment options. For example, the application of induced pluripotent stem cells (iPSCs) that can be differentiated into motor neurons or astrocytes offers a humanized model system3,4,5. Additionally, in this methods collection, animal models are presented, such as Drosophila to study glucose uptake and the neuromuscular junction (NMJ) in vivo6,7, mice to study cortical neurons8, and C. elegans or zebrafish to investigate motor impairments9,10 and post-mortem patient tissue11.

Zebrafish larvae are transparent, and their motor neurons are directly visible, making them a perfect tool for non-invasive in vivo studies. Asakawa et al. show the phase transition of optogenetically expressed TDP-43 in single spinal motor neurons9. After irradiation, the cytoplasmic relocation of TDP-43 can be observed and analyzed. The aggregation of cytoplasmic TDP-43 is a hallmark of degenerating motor neurons in ALS. This method allows for the functional study and analysis of ALS-associated proteins in a subcellular, temporal manner.

Employing super-resolution structured illumination microscopy (SIM), Coyne and Rothstein detail a protocol that isolates the nuclei and describe how to investigate nucleoporin complexes11. Nucleoporin complexes consist of multiple copies of about 30 different nucleoporin proteins (Nups). Nucleocytoplasmic transport (NCT) impairment and Nup alterations have been shown to be early hallmarks of many neurodegenerative diseases, including ALS. By extracting the nuclei, it is possible to investigate the individual Nup proteins within the NPC and nucleoplasm in 3D. Interestingly, this can be applied to not only iPSC-derived cells but also to post-mortem tissue.

Currey and Liachko describe two assays to discriminate between mild, moderate, and severe motor impairment in C. elegans models of ALS10. In the radial locomotion assay, crawling on a surface is measured, making this an easy and cost-effective assay. In their second method, the swimming assay, thrashing movements can be measured using a computer-based tracking method. The authors use this to study TDP-43 and tau.

Hayes et al. also describe a method to study NCT8. They apply a permeabilization method to neuronal cultures. Using primary mouse cortical neurons, they describe a method that maintains the nuclear membrane integrity by using hypotonic lysis combined with a bovine serum albumin cushion. By doing so, nuclear import still functions in an energy-dependent manner, thus providing a high-content microscopy and analysis platform. This platform will have broad applicability in the future for studying passive and active nuclear transport in primary neurons.

The quick assessment of how manipulation, disease-related proteins, or RNA impact synaptic processes and whether therapeutic drugs can restore these functions is essential for ALS research. Using iPSC-derived motor neurons as well as primary neurons from mice, Krishnamurthy et al. present a protocol that enables the real-time monitoring of presynaptic calcium influx dynamics and synaptic vesicle membrane fusion3. The authors demonstrate that C9orf72-(GA)50 transfection impairs synaptic transmission, highlighting the suitability of these methods for detecting mutation-based differences in synaptic function.

Altered glucose uptake is one of the pathobiological characteristics of ALS. In this Drosophila model, Loganathan et al. describe a FRET-based method to measure intracellular changes in glucose uptake in specific cells6. Using a genetically encoded glucose FRET sensor, they validate their method with TDP-43 expression neurons, which display higher glucose uptake. In the TDP-43G298S mutant line, increased glucose uptake is only detectable upon glucose stimulation. This method provides an important tool for studying glycolysis not only in ALS but also generally in relation to motor neuron regeneration.

Dissection techniques preserving the NMJ architecture are of the utmost importance for studying changes in the motor neurons along the Drosophila leg over time. Stilwell and Agudelo utilize a technique that allows the characterization of the NMJ for identifying motor neuron arbors using immunocytochemistry7. Interestingly, the adult neurons are present throughout the lifetime of a fly, which is approximately 90 days. Comparing a SOD1H71Y mutation to the wild-type, the authors demonstrate different markers for age-dependent bouton swelling, protein aggregates, and enlarged mitochondria.

The innovation of mimicking an NMJ using a co-culture system meets the urgent need to study the dissociation between motor neurons and myotubes. In terms of this method, Stoklund Dittlau et al. describe how to cultivate human iPSC-derived motor neurons and human primary mesoangioblast-derived myotubes to form functionally active NMJs4. The authors show their functionality by the activation of motor neurons with potassium chloride and calcium influx in Fluo-4-labeled myotubes thereafter, which was abolished by the administration of NMJ blockers.

Recently, co-culture systems have gained increasing attention. Studying not only one but multiple cell types in a dish has the benefit of mimicking physiological conditions better than methods using monocultured cells. ALS-related pathobiology, such as astrocyte-mediated toxicity and neuronal hyper-excitability, can be studied using this approach. In the video by Taga et al., the generation of cortical neurons and astrocytes in a co-culture combined with a multi-electrode array (MEA) setup is shown for monitoring electrophysiology5. The functional activity can be monitored over time, allowing flexibility in cellular composition as well as different culture conditions. This additionally provides a platform to test the therapeutic potential of drugs and their influence on functional activity.

Currently, there are only three FDA-approved treatments for ALS, all with limited application potential. To find more promising treatments, future research must understand the pathobiology better by employing multiple model systems and approaches. Without a doubt, human iPSC-derived models will provide an interesting platform to investigate the underlying molecular mechanisms. This, combined with model systems such as zebrafish, C. elegans, Drosophila, or rodents, will lead to progress in the field. Furthermore, future epidemiologic research will hopefully provide more insights into how environmental factors play a role in the development of ALS12. With the expanding datasets and bioinformatics developing at high speed, it will become easier to unravel the common denominators of neurodegenerative diseases in the future. This will lead to new avenues for therapy or even prevention.


The authors have nothing to disclose.


We thank all the authors for their contributions to this collection and our colleagues for the progress in the field. We also would like to thank the Fund for Scientific Research Flanders (FWO-Vlaanderen). Y.E.K. is an FWO PhD-fellow SB (#1S50320N). We would also like to acknowledge VIB, KU Leuven (C1 and “Opening the Future” Fund), the “Fund for Scientific Research Flanders” (FWO-Vlaanderen), the Thierry Latran Foundation, the “Association Belge contre les Maladies neuro-Musculaires – aide à la recherché ASBL” (ABMM), the Muscular Dystrophy Association (MDA), the ALS Liga België (A Cure for ALS), Target ALS, and the ALS Association (ALSA).


  1. Martin, S., Al Khleifat, A., Al-Chalabi, A. What causes amyotrophic lateral sclerosis. F1000Res. 6, 317 (2017).
  2. Talbott, E. O., Malek, A. M., Lacomis, D. The epidemiology of amyotrophic lateral sclerosis. Handbook of Clinical Neurology. 138, 225-238 (2016).
  3. Krishnamurthy, K., Trotti, D., Pasinelli, P., Jensen, B. Real-time fluorescent measurements of synaptic functions in models of amyotrophic lateral sclerosis. Journal of Visualized Experiments. (173), e62813 (2021).
  4. Stoklund Dittlau, K., et al. Generation of human motor units with functional neuromuscular junctions in microfluidic devices. Journal of Visualized Experiments. (175), e62959 (2021).
  5. Taga, A., et al. Establishment of an electrophysiological platform for modeling ALS with regionally-specific human pluripotent stem cell-derived astrocytes and neurons. Journal of Visualized Experiments. (174), e62726 (2021).
  6. Loganathan, S., Ball, H. E., Manzo, E., Zarnescu, D. C. Measuring glucose uptake in Drosophila. models of TDP-43 proteinopathy. Journal of Visualized Experiments. (174), e62936 (2021).
  7. Stilwell, G., Agudelo, A. Dissection and immunohistochemistry of the Drosophila. adult leg to detect changes at the neuromuscular junction for an identified motor neuron. Journal of Visualized Experiments. (180), e62844 (2022).
  8. Hayes, L. R., Duan, L., Vidensky, S., Kalab, P. Nuclear transport assays in permeabilized mouse cortical neurons. Journal of Visualized Experiments. (173), e62710 (2021).
  9. Asakawa, K., Handa, H., Kawakami, K. Optogenetic phase transition of TDP-43 in spinal motor neurons of zebrafish larvae. Journal of Visualized Experiments. (180), e62932 (2022).
  10. Currey, H. N., Liachko, N. F. Evaluation of motor impairment in C. elegans. models of amyotrophic lateral sclerosis. Journal of Visualized Experiments. (175), e62699 (2021).
  11. Coyne, A. N., Rothstein, J. D. Nuclei isolation and super-resolution structured illumination microscopy for examining nucleoporin alterations in human neurodegeneration. Journal of Visualized Experiments. (175), e62789 (2021).
  12. Al-Chalabi, A., Hardiman, O. The epidemiology of ALS: A conspiracy of genes, environment and time. Nature Reviews Neurology. 9 (11), 617-628 (2013).

Cite this Article

Klingl, Y. E., Da Cruz, S., Van Den Bosch, L. Current Methods In ALS Research. J. Vis. Exp. (193), e65016, (2023).More

Klingl, Y. E., Da Cruz, S., Van Den Bosch, L. Current Methods In ALS Research. J. Vis. Exp. (193), e65016, (2023).

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