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

Authors

Anand Ganapathy Subramaniam, Lucas Keniger de Andrade Gensas, Tal Gradus-Pery, Eran Perlson

Published in

Bio-protocol. Volume 16. Issue 17. Pages e5801. Sep 05, 2026. Epub Sep 05, 2026.

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.

PMID:
42729071
Bibliographic data and abstract were imported from PubMed on 14 Oct 2026.

Read full publication at:
Please sign in to see all details.

Stats

1-terrible, 9-excellent. How would you rate this publication? Sign in in to submit your rating.

Post a comment

You need to be signed in to post comments. You can sign in here.

Comments

There are no comments yet.