Reduced muscle strength in ether lipid-deficient mice is accompanied by altered development and function of the neuromuscular junction

Impaired acetylcholine receptor (AChR) clustering in vitro in Gnpat KO myotubes. Clustering of AChRs was induced by stimulation with neural agrin in myotubes derived from immortalized myoblasts from WT and Gnpat KO mice. AChRs were stained with Alexa Fluor 594-conjugated α-BTX and representative images of clusters (indicated by arrows) are shown in (a) for both genotypes; scale bar: 10 um. Quantifications of cluster number.

Dorninger F, Herbst R, Kravic B, Camurdanoglu BZ, Macinkovic I, Zeitler G, Forss-Petter S, Strack S, Khan MM, Waterham HR, Rudolf R, Hashemolhosseini S, and Berger J. (2017) Reduced muscle strength in ether lipid-deficient mice is accompanied by altered development and function of the neuromuscular junction.

In the nervous system, neurotransmitters are released into the synaptic space through a process called vesicular fusion. A synapse is the space between the axon terminal of a neuron and another neuron, gland, or muscle. When this connection is between a neuron and a muscle it is called a neuromuscular junction (NMJ). Acetylcholine is released into the NMJ, where it reversibly binds to ionotropic acetylcholine receptors (AChRs) along the muscle fiber. Post-synaptic folds, rich in AChRs, are unique to the NMJ. Plasmalogens, phospholipids containing a vinyl-ether bond at the sn-1 position of the glycerol backbone, give structure to membranes and are important in vesicular fusion. Dorninger et al examined the role of plasmalogens on the development and function of synapses through analyzing NMJs. These are the easiest type of synapse to study due to their larger size. This analysis was completed using the glyceronephosphate O-acyltransferase (Gnpat) knockout (KO) mouse model, which lack the first enzyme in the plasmalogen biosynthetic pathway, making them unable to synthesize plasmalogens. The role of plasmalogens was studied through behavioural assays, examining the function of the NMJ, and analyzing acetylcholine receptor (AChR) clustering.

When the Gnpat KO and control mice were tested in a balance beam assay to study motor performance, Gnpat KO mice showed severe difficulties, with their limbs slipping below the bar more frequently than the wild-type control mice. The Gnpat KO mice also showed reduced muscle strength. However, the two groups did not differ in scores during the inverted screen test, a method for evaluating grip strength. This was unsurprising, as humans with rhizomelic chondrodysplasia punctata (RCDP), caused by genetic mutations in the plasmalogen biosynthetic pathway, do not have issues with grip strength. The differences in motor performance were suggested to be due to alterations in acetylcholine signaling.

The diaphragm muscle of both embryonic wild-type and Gnpat KO mice was used to evaluate NMJ innervation and morphology. It was observed that Gnpat KO mice had widespread branching of the phrenic nerve and an enlarged endplate zone, causing the area covered by nerves to be increased compared to the wild-type mice. In addition, Gnpat KO mice had acetylcholine receptor (AChR) clusters on their muscle fibers covering a greater area than the wild-type diaphragms. This up-regulation of receptors is likely the body’s response to improper vesicular fusion hindering acetylcholine signaling. When this occurs, the receptors are upregulated to try and increase the amount of acetylcholine that can be bound by having more receptors present. To compare mature AChR clustering, hind limb muscles from both mouse lines were analyzed. It was found that Gnpat KO muscles had less area covered by clusters, and a reduced number of clusters compared to the wild-type muscles but no difference in the density of AChRs.

As morphological differences were present, the study also evaluated the functionality of the muscles by comparing their miniature endplate potentials (mEPPs), the depolarization caused by the release of a single vesicle into the synapse. Although the mEPP amplitude was increased in the Gnpat KO mice, the frequency was decreased, indicating a reduction in vesicle fusion. The quantal content, a measure to determine the number of vesicles released when stimulation occurs, was also evaluated, and found to be significantly reduced (~25% less) in the lipid deficient mice.

This NMJ model allows for clear examination of the role that plasmalogens have in vesicular fusion at the synapse and indicates the detrimental effects that arise in deficient neurons. Dorninger et al showed that vesicle number was reduced in the Gnpat KO model and when this occurs within the synapses between neurons, many signals would not be properly transmitted, demonstrated by reduced motor performance and strength in the plasmalogen deficient mice. These changes would be expected to functionally alter mechanisms involved in development, cognition, movement, and environmental response. These results indicate the significance that a plasmalogen deficiency can have on vesicular fusion and synaptic morphology and the downstream effects that these would have on neuronal signaling and observable behavior. Further work directly analyzing synaptic connections between neurons is necessary to confirm the role of plasmalogens in a deficient model and would provide information of how these neuronal alterations result in the clinical manifestations of human disorders caused by plasmalogen deficiency, such as RCDP.