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Quantitative Analysis of Axonal Degeneration and TDP-43 Aggregation in Compartmentalized Human iPSC-Derived Motor Neuron-Myotube Co-cultures.

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Paper

Loaded from Europe PMC by your browser, not stored by OSCR: doi.org · Europe PMC

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The authors' code

Python · 88 lines · 2.6 KB · MIT

  1. from ij import IJ
  2. from ij.io import DirectoryChooser
  3. from ij.plugin import ChannelSplitter
  4. from ij.gui import GenericDialog
  5. import os
  6. import random
  8. # Ask user which channel is NFH
  9. gd = GenericDialog("Select channel to open")
  10. gd.addNumericField("Which channel would you like to open? (enter channel number)", 1, 0)
  11. gd.showDialog()
  12. if gd.wasCanceled():
  13. IJ.error("User canceled dialog")
  14. exit()
  15. nfhChannel = int(gd.getNextNumber())
  17. # Ask user to select a folder
  18. dc = DirectoryChooser("Select a folder with images")
  19. inputDir = dc.getDirectory()
  20. if inputDir is None:
  21. IJ.error("No folder selected")
  22. exit()
  24. # Supported image extensions (case insensitive)
  25. extensions = [".ims", ".tif", ".tiff", ".czi", ".lif", ".dcm", ".ome.tif", ".nd2", ".svs"]
  27. # Get list of supported files in folder
  28. matching_paths = [f for f in os.listdir(inputDir) if any(f.lower().endswith(ext) for ext in extensions)]
  30. if len(matching_paths) == 0:
  31. IJ.error("No supported image files found in the folder.")
  32. exit()
  34. # Ask how many images to open — with input validation loop
  35. while True:
  36. n_input = IJ.getString("How many random images to open? (max: {})".format(len(matching_paths)), "1")
  37. if n_input is None:
  38. IJ.showMessage("Cancelled", "Operation cancelled by the user.")
  39. exit()
  40. try:
  41. n_to_open = int(n_input)
  42. if n_to_open <= 0 or n_to_open > len(matching_paths):
  43. raise ValueError
  44. break
  45. except:
  46. IJ.showMessage("Error", "Please enter a positive number up to {}.".format(len(matching_paths)))
  48. # Randomly sample the images
  49. selected_files = random.sample(matching_paths, n_to_open)
  51. for fileName in selected_files:
  52. fullPath = os.path.join(inputDir, fileName)
  53. #IJ.log("Processing: " + fileName)
  55. # Open image with Bio-Formats
  56. IJ.run("Bio-Formats Importer", "open=[" + fullPath + "] autoscale color_mode=Composite view=Hyperstack stack_order=XYCZT quiet")
  58. # Get current image
  59. imp = IJ.getImage()
  61. # Split channels
  62. channels = ChannelSplitter.split(imp)
  64. # Close original composite
  65. imp.close()
  67. # Validate NFH channel number
  68. if nfhChannel < 1 or nfhChannel > len(channels):
  69. IJ.log("Invalid channel number for image: " + fileName)
  70. for chImg in channels:
  71. chImg.close()
  72. continue
  74. # Close all channels except NFH channel
  75. for i, chImg in enumerate(channels):
  76. if i != nfhChannel - 1:
  77. chImg.close()
  79. # Get NFH channel image
  80. nfh_img = channels[nfhChannel - 1]
  82. # Convert to 8-bit
  83. #IJ.run(nfh_img, "8-bit", "")
  85. # Show NFH channel image
  86. nfh_img.show()
  88. #IJ.log("Processing completed.")

Choose_threshold.py at commit 237b34e, under MIT · at the source

Overview

Authors: Anand Ganapathy Subramaniam1, Lucas Keniger de Andrade Gensas1,2, Tal Gradus-Pery1, Eran Perlson1,2
  1. Gray Faculty of Medical & Health Sciences, Department of Neuroscience and Brain Disorders, Tel Aviv University, Tel Aviv, Israel
  2. Sagol School of Neuroscience, Tel Aviv University, Tel Aviv, Israel
Institutions: Tel Aviv University (Israel)
Journal: Bio-protocol, volume 16, issue 17, article e5801
Dates: received 22 March 2026; accepted 28 July 2026; published online 5 September 2026
Type: Methods article · Language: English
License: CC BY-NC
Identifiers: DOI 10.21769/bioprotoc.5801 · PMID 42729071 · PMCID PMC13561694 · OpenAlex W7196951841
Open access: diamond, a free copy (OpenAlex)
Status: code verified
Categories: human (organism), other condition (population), cellular / molecular (subfield)
Methods: Statistics, Machine learning
Keywords: Human iPSC-derived motor neuron–myotube co-culture, Axonal degeneration, TDP-43 aggregation, Microfluidic co-culture, Automated image analysis, Amyotrophic lateral sclerosis
Journal subjects: Biology, Clinical Protocols
Topic: Amyotrophic Lateral Sclerosis Research (Neurology, Medicine), according to OpenAlex
Funding: Muscular Dystrophy Association; Target ALS; Israel Science Foundation (ISF)
Citations: not cited yet (Europe PMC); 35 references in the paper

Abstract

Amyotrophic lateral sclerosis (ALS) is characterized by early and spatially restricted pathology in motor axons, including distal degeneration and accumulation of aggregation-prone proteins such as TDP-43. However, a major limitation in the field has been the lack of approaches that enable robust, quantitative, and compartment-specific analysis of these early axonal events, particularly in human-relevant systems. Here, we describe an integrated experimental and analytical framework that enables quantitative dissection of axonal degeneration and protein aggregation, specifically within distal motor axons. By combining compartmentalized human co-cultures with a dedicated image analysis strategy, this approach enables selective and quantitative analysis of pathological processes specifically within axons, independent of surrounding tissues such as muscle and other cellular compartments. This framework captures both structural degeneration and protein aggregation dynamics at subcellular resolution, enabling spatially resolved quantitative analysis of disease-relevant changes along axons. Importantly, the analytical framework is not limited to TDP-43 but is broadly applicable to diverse aggregation-prone proteins, thereby providing a generalizable platform to study axonal pathology across neurodegenerative diseases. Together, this work provides a scalable approach for investigating axonal pathology as an early and measurable feature of neurodegeneration, with potential applications in mechanistic studies and therapeutic targeting in ALS and related disorders.

Key features

• Compartmentalized human induced pluripotent stem cell (iPSC)-derived motor neuron–myotube co-cultures for modeling distal axonal pathology.

• Microfluidic separation of somatic and distal axonal compartments enabling spatial perturbation and analysis.

• Quantitative imaging of neurofilament heavy chain (NFH)-associated axonal degeneration and pTDP-43 accumulation.

• Semi-automated workflow for a reproducible, scalable, and modular pipeline for image quantification.

Reproduced under the paper's license (CC BY-NC), from the paper cited above.

Repository

Its files are read in the Code ↔ Paper reader above.

PerlsonLab/Bioprotocol

License: MIT
State: the link answers, verified on 26 September 2026
Evidence: files inventoried
Commit: 237b34ede8472c9d4f025aec6e010884ffc568cd, 9 September 2026
Languages: Python (2)
Size: 11 files, 2 scripts
Software Heritage: not archived
Found in: the text, “Software and datasets”
Holds: README, license file
Not found: CITATION.cff, environment file, tests, continuous integration, documentation
Tools: ImageJ / Fiji (2 files)
Availability: 1 check, the latest on 26 September 2026: the link answers
  • 26 September 2026: the link answers
4 files
At the source: github.com/PerlsonLab/Bioprotocol

Tracing map

Proposed by the machine: these links were found in the paper and verified at the source, without human review. The map will receive a Zenodo DOI once one of the paper's authors has validated it with their ORCID.

What the map holds:

  • 1 repository of the authors' code, each at its verified commit, with its license and how the link was found in the paper;
  • 2 scripts, each with its path and the digest of its content;
  • no match between paragraphs and code yet;
  • neither the text of the paper nor the code itself.

Its JSON (tracing-map.json) is deposited on Zenodo with its DOI once the map is validated.

Data

No dataset and no data link were found in the paper.

Versions

The history of this record: each version stored by the harvester or made by a correction of its authors or of the maintainers of its code, and what changed in its facts. The texts of the paper (its abstract, its availability statements) are not part of it; versions that changed only those are not listed.

Version 3, 28 September 2026

  • Funding: added Muscular Dystrophy Association; Target ALS; Israel Science Foundation

Version 1, 27 September 2026: the first record

Recorded: type, language, journal, volume, issue, pages, dates, 4 authors, 6 keywords, 35 references.

Cite

This paper

Subramaniam, A. G., de Andrade Gensas, L. K., Gradus-Pery, T., & Perlson, E. (2026). Quantitative Analysis of Axonal Degeneration and TDP-43 Aggregation in Compartmentalized Human iPSC-Derived Motor Neuron-Myotube Co-cultures. Bio-protocol, 16(17), e5801. https://doi.org/10.21769/bioprotoc.5801

BibTeX

@article{subramaniam2026quantitative,
author = {Subramaniam, Anand Ganapathy and de Andrade Gensas, Lucas Keniger and Gradus-Pery, Tal and Perlson, Eran},
title = {{Quantitative Analysis of Axonal Degeneration and TDP-43 Aggregation in Compartmentalized Human iPSC-Derived Motor Neuron-Myotube Co-cultures}},
journal = {Bio-protocol},
year = {2026},
month = sep,
volume = {16},
number = {17},
pages = {e5801},
publisher = {Bio-protocol, LLC},
issn = {2331-8325},
doi = {10.21769/bioprotoc.5801},
url = {https://doi.org/10.21769/bioprotoc.5801},
pmid = {42729071},
pmcid = {PMC13561694}
}

RIS

TY - JOUR
AU - Subramaniam, Anand Ganapathy
AU - de Andrade Gensas, Lucas Keniger
AU - Gradus-Pery, Tal
AU - Perlson, Eran
TI - Quantitative Analysis of Axonal Degeneration and TDP-43 Aggregation in Compartmentalized Human iPSC-Derived Motor Neuron-Myotube Co-cultures
T2 - Bio-protocol
J2 - Bio Protoc
PY - 2026
DA - 2026/09/05
VL - 16
IS - 17
SP - e5801
SN - 2331-8325
PB - Bio-protocol, LLC
DO - 10.21769/bioprotoc.5801
UR - https://doi.org/10.21769/bioprotoc.5801
LA - en
ER -

CSL-JSON

{
"id": "10.21769/bioprotoc.5801",
"type": "article-journal",
"title": "Quantitative Analysis of Axonal Degeneration and TDP-43 Aggregation in Compartmentalized Human iPSC-Derived Motor Neuron-Myotube Co-cultures",
"container-title": "Bio-protocol",
"author": [
{
"family": "Subramaniam",
"given": "Anand Ganapathy"
},
{
"family": "de Andrade Gensas",
"given": "Lucas Keniger"
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{
"family": "Gradus-Pery",
"given": "Tal"
},
{
"family": "Perlson",
"given": "Eran"
}
],
"container-title-short": "Bio Protoc",
"volume": "16",
"issue": "17",
"page": "e5801",
"DOI": "10.21769/bioprotoc.5801",
"PMID": "42729071",
"PMCID": "PMC13561694",
"ISSN": "2331-8325",
"publisher": "Bio-protocol, LLC",
"URL": "https://doi.org/10.21769/bioprotoc.5801",
"language": "en",
"issued": {
"date-parts": [
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2026,
9,
5
]
]
}
}

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