Introduction:

Rationale: Modeling early synaptic dysfunction in ALS is critical for identifying therapies that preserve NMJ integrity and delay motor impairment.

Study focus: NMJ pathology was examined in a mouse model overexpressing human TDP-43 with a defective nuclear localization signal.

Objective: To determine how progressive ALS-like pathology affects neuromuscular innervation

 

Methods:

Mouse model: Transgenic rNLS8 mice expressing Dox-regulated human TDP-43ΔNLS were used to model inducible ALS-like pathology.

Disease induction: Mice on Biospective’s Low Dox protocol were used to induce gradual disease progression and provide an extended therapeutic window (≥8 weeks) .

Experimental groups: 28 mice total (N=14 Low Dox; N=14 tTA-matched controls); NMJ pathology was assessed 8 weeks post-induction.

Tissue processing & staining: Gastrocnemius muscles were collected after perfusion and co-stained with α-bungarotoxin (postsynaptic AChRs), SV2A (presynaptic terminals), and βIII-tubulin (motor axons).

Analysis: NMJ morphology and innervation were quantified using Biospective’s PERMITS™ platform, including presynaptic branching complexity, innervation status, and total axonal projections

 

Results:

A: Postsynaptic receptors (α-bungarotoxin, green), presynaptic terminals and axons (SV2A/βIII-tubulin, red), their overlay, and automated NMJ segmentation classifying innervation status.

B: Low Dox mice exhibit a significant reduction in innervation, presynaptic branching complexity, and total axonal projections. (Mean ± SEM; t-test)

C: Low Dox mice exhibit higher mean latency and lower max amplitude in Compound Muscle Action Potential (CMAP). (Mean ± SEM; t-test)

D: A strong correlation exists between CMAP amplitude and NMJ innervation

Sample size estimation for various NMJ metrics between Control (tTA) and Low Dox rNLS8. mice

 

Conclusion:

An 8-week Low Dox regimen in rNLS8 mice captures key features of ALS progression, including synaptic denervation, reduced presynaptic complexity, and impaired axonal connectivity. It provides a robust, translationally relevant platform for preclinical evaluation of therapeutics aimed at preserving NMJ integrity, muscle strength, and delaying motor decline.