Method Profile
A semi-automated image pipeline distinguishes axonal fragmentation from protein puncta quantified inside intact distal axons.
Supply region-specific structural and molecular endpoints for mechanistic or intervention studies in a human ALS-related coculture.
Human iPSC-derived motor neuron–myotube coculture with cell bodies and distal axon/muscle regions separated by microfluidic grooves.
Fiji/ImageJ preprocessing and recorded channel thresholds precede CellProfiler object analysis; quantify NFH structural classes and pTDP-43 restricted to intact NFH-positive regions.
Model and markers
ALS-related TDP-43 M337V and isogenic cocultures; NFH axon marker and phosphorylated TDP-43 molecular signal.
Analysis resources
Fiji/ImageJ preprocessing, CellProfiler pipelines, macros and overlay review; source provides a public analysis repository.
Keep structural degeneration and molecular signal denominators distinct. The molecular endpoint excludes degenerated fragments and is not total compartmental aggregate burden.
Source protocol illustrates the analysis with disease/isogenic comparisons adapted from the 2025 validation study; post-validation image datasets were blinded and independently analyzed.
Figure 4: 28 and 18 imaging fields from 3 independently grown neuromuscular cocultures.
TDP-43 M337V versus isogenic control coculture, measured within the defined distal compartment.
The M337V/isogenic-control benchmark reports differences in pTDP-43 area within intact NFH-positive axons and in the NFH degeneration index (unpaired, two-tailed t-tests; P = 0.0001). Puncta-count density is an additional pipeline endpoint beyond the earlier area-based analysis.
A human motor-neuron–myotube protocol combines compartmentalized culture with Fiji and CellProfiler analysis. It measures axonal structure separately from pTDP-43 puncta restricted to intact axons, preserving the spatial meaning of each endpoint.
