TY  - JOUR
TI  - Quantitative Analysis of Axonal Degeneration and TDP-43 Aggregation in Compartmentalized Human iPSC-Derived Motor Neuron–Myotube Co-cultures
AU  - Subramaniam, Anand Ganapathy
AU  - de Andrade Gensas, Lucas Keniger
AU  - Gradus-Pery, Tal 
AU  - Perlson, Eran 
VL  - 16
IS  - 17
PY  - 2026
DA  - 2026/09/05
SP  - e5801
C1  - Bio-protocol 2026;16:e5801
DO  - 10.21769/BioProtoc.5801
UR  - https://doi.org/10.21769/BioProtoc.5801
AB  - 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.
KW  - Human iPSC-derived motor neuron–myotube co-culture
KW  - Axonal degeneration
KW  - TDP-43 aggregation
KW  - Microfluidic co-culture
KW  - Automated image analysis
KW  - Amyotrophic lateral sclerosis
JF  - Bio-protocol
SN  - 2331-8325
PB  - Bio-protocol LLC.
BIO101  - False