Biomaterials: ALS mutations in human skeletal muscle and motor neurons have different effects on the integrity and function of neuromuscular junctions

Amyotrophic lateral sclerosis (ALS) is a debilitating adult-onset disease in which patients usually die
within 2-5 years of diagnosis.

On August 19, 2022, the James J.

Results of the study

1.

The previous work of the researchers has characterized the function of two SOD1 lines (SOD1L144P and SOD1E100G) and determined that they exhibit reduced myogenicity, myocyclic morphological atrophy, impaired mitochondrial function, and decreased
muscle fiber contractility in response to electrical stimulation 。 Next, the researchers co-cultured hSKMWT-hMNWT, hSKMSOD1-hMNWT, hSKMWT-hMNSOD1 and hSKMSOD1-hMNSOD1, including co-cultures paired with a mutant tissue and healthy tissue (i.

The results showed that the two cell types interacted closely, indicating NMJ formation (Figure 1), and functional NMJ tests on days 14 and 17 of co-culture verified the formation
of NMJ.

Figure 1: Immunocytochemical staining of nicotine acetylcholine receptors (nAChR) is green, indicating that skeletal muscle and synapsin (neuronal axonal markers) are red, showing the formation
of NMJs in the chamber.

2.

Next, the researchers quantified the number
of NMJs in hSKMSOD1-hMNWT, hSKMWT-hMNSOD1, and hSKMSOD1-hMNSOD1 on days 14 and 17 of co-culture.

Figure 2: The average NMJ number of hSKMWT-hMNSOD1, hSKMSOD1-hMNWT, and hSKMSOD1-hMNSOD1 co-cultures for all genetic groups were assessed
at day 14.

NMJ fidelity of SOD1 mutant cultures

NMJ fidelity is a measure of the reliability of hMN-hSKM transport across synapses to assess how SOD1 mutations in hMN or hSKM affect the functional integrity of NMJ in response to in
vivo stimuli.

It is also interesting to note that WThSKMs co-cultured with SOD1E100GhMNs typically exhibit a significant decrease in shrinkage fidelity (Figures 3A, 4A), while those cultured with SOD1L144PhMNs show no signs of shrinkage fidelity defects, suggesting some feed-forward deterioration signals from hMNs to hSKM, as well as phenotypic variation
between different mutations.

Figure 3: Analyze NMJ fidelity
on day 14 containing mutant SOD1 motor neurons and/or SKM with corresponding WT conditions.

Figure 4: Analysis of NMJ fidelity
of the NMJ system containing mutant SOD1 motor neurons and/or SKMs with corresponding WT conditions.

4.

The stability of NMJ formed due to the reduced stability of NMJ reported in ALS clinical pathology, which the investigators measured to examine the ability
of various NMJ models to maintain functional synapses after strenuous activity.

Figure 5: The average stability of NMJ was quantified when the two SOD mutant lines hSKMWT-hMNWT, hSKMWT-hMNSOD1, hSKMSOD1-hMNWT and hSKMSOD1-hMNSOD1 were quantified
.

Figure 6: hSKMWT-hMNWT, hSKMWT-hMNSOD1, hSKMSOD1-hMNWT and hSKMSOD1-hMNSOD1 NMJ fatigue index quantification
on the 14th day.

In summary, the work of James J.
Hickman's team confirmed that it has nothing to do with motor neurons, and that inherent morphological and functional defects in ALS hSKM also affect the integrity and function
of NMJ.
In addition, this work shows how symptom severity is increased in the presence of ALS hSKM
.
In this case, ALS hSKM is not the result of neuronal dysfunction, but may be an important factor in the pathogenesis of ALS
.

Therefore, since improving the condition of ALS hSKM may help reduce the severity of symptoms, ALS hSKM should be taken into account when developing treatments
.
Finally, it is important to note that LAS is a heterogeneous disease with many related gene mutations
.
While this work focuses on mutations in the SOD1 gene, the physiological platform described in this work can be used to explore other fALS-related mutations as well as sALS
.

References

Badu-Mensah A, Guo X, Nimbalkar S, Cai Y, Hickman JJ.
ALS mutations in both human skeletal muscle and motoneurons differentially affects neuromuscular junction integrity and function [published online ahead of print, 2022 Aug 19].
Biomaterials.
2022; 289:121752.
doi:10.
1016/j.
biomaterials.
2022.
121752

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