Oxysterol-dependent pathway of regulation of synaptic transmission in the neuromuscular junction of mice
Authors: Zakyrjanova G. F. , Tsentsevitsky A. N. , Giniatullin A. R. , Kuznetsova E. A. , Petrov A. M. 
Published: May 2024

Cholesterol plays a crucial role in maintaining the stiffness, flexibility, and porosity of the bilipid layer. Moreover, cholesterol has a strong attraction to several membrane proteins, allowing it to modify their function, thereby influencing intracellular processes. Through enzymatic and oxidative reactions, cholesterol produces different types of oxysterols. One of the oxysterols synthesized is 25-hydroxycholesterol (25HC), formed with participation from the cholesterol-25-hydroxylase enzyme predominantly in macrophages, dendritic cells, and microglia.
25HC effectively regulates cholesterol homeostasis within individual cells, ensuring that cholesterol concentration remains at or below nanomolar levels. However, similar to the paracrine agent 25HC, macrophages and microglia produce it at significantly higher concentrations during inflammatory reactions.
Extensive research has focused on the role of 25HC in immune response during inflammation. 25HC appears to exert a variety of effects on the immune response. It promotes the secretion of inflammatory cytokines and chemokines such as IL-1 beta, IL-6, IL-8, CCL5, and macrophage colony-stimulating factor, and inhibits inflammation by blocking inflammasome activity. Increased production of 25HC via Toll-like receptor 4 activation reduces B-cell proliferation. 25HC has the ability to prevent viral penetration by integrating into the membrane and modifying its properties.
Macrophages play a significant role in both adaptive and innate immunity and are found in large numbers in skeletal muscle, suggesting the possible functional importance of 25HC in the interaction between the immune system and skeletal muscles. We discovered a concentration-dependent effect of 25HC on the neuromuscular synapse of mice: high concentrations (1–10 µM) enhance, while low concentrations (0.01–0.1 µM) inhibit the process of neuromuscular transmission [1].
25HC is a ligand for LX-receptors, which we observed to be expressed in the synaptic region of motor neuron axons. The high concentration of 25HC modulates synaptic transmission through an LX-receptor-dependent pathway. Furthermore, 25HC activates LX-receptors that are likely associated with estrogen receptors α, leading to the activation of the Gi-protein/βγ-dimer of G-protein/phospholipase C/Ca2+ protein kinase C signaling pathway. The potentiating effect of 25HC, which activates the LX-receptor/estrogen receptor α complex, depends on lipid rafts. This is because both receptors are localized in lipid microdomains, and the destruction of lipid rafts precludes the stimulating effects of 25HC. In addition, the study identified the contribution of reactive oxygen species (ROS) in the 25HC-dependent enhancement of synaptic transmission. Indeed, treatment with 25HC (1 µM) leads to an elevation in both ROS production in the synaptic region and hydrogen peroxide concentration in the extracellular environment. This phenomenon is dependent on an increase in intracellular calcium ions concentration. It is noteworthy that ROS, in this instance, act as signaling molecules since the level of lipid peroxidation is not impacted by 25НС.
Expression of 25HC increases in various neurodegenerative conditions. The concentration of 25HC escalates in amyotrophic lateral sclerosis, marked by progressive muscle atrophy resulting in death. High concentrations of 25HC above 5–30 µM can reduce the survival rate and trigger apoptosis of motor neurons. Nonetheless, low concentrations, 1 µM or lower, have the opposite effect, boosting the neurons’ survival rate.
The pivotal role of lipid rafts in ALS pathogenesis is evident. For instance, a decrement in caveolin-1 level in ALS brings about lipid raft disruption, thus accelerating disease progression. Moreover, in SOD1G93A ALS model mice during the pre-onset phase, alterations in membrane properties were observed, including lipid raft destabilization, lipid bilayer ordering, and heightened membrane fluidity [2]. One possible causal factor for this phenomenon could be muscle ceramide increase, which causes raft destabilization during motor unloading [3–5]. In fact, we observed elevated ceramide levels in the membrane of ALS model mice during the pre-onset stage. An increase in extracellular choline levels was detected in ALS, likely the result of heightened non-quantum secretion of the neurotransmitter, potentially due to lipid rafts disruption. Unregulated elevation of acetylcholine in ALS is a significant contributor to motor dysfunction and age-related morphological changes in the neuromuscular junction. An example of such changes would be the disruption of clustering of nicotine acetylcholine receptors in the postsynaptic membrane. Furthermore, disruption of lipid rafts in amyotrophic lateral sclerosis (ALS) was associated with elevated levels of hydroperoxides in muscle homogenates and lipid peroxidation in ALS model mice. Consequently, it can be inferred that the membrane properties are altered in ALS.
Furthermore, it was observed that 25HC can prevent the initial alterations of membrane properties by promoting the stabilization of lipid rafts in neuromuscular synapses in pre-onset ALS mouse models. Additionally, 25HC averted the accumulation of ceramide in the neuromuscular synapse. We observed that 25HC can inhibit synaptic alterations at the neuromuscular synapse in ALS. These alterations include elevated lipid peroxidation, increased extracellular choline levels, and disrupted clustering of nicotinic acetylcholine receptors [2].
Thus, 25HC has a multidirectional impact on neuromuscular transmission, hindering the recruitment of synaptic vesicles at low concentrations and enhancing the mobilization of vesicles at higher concentrations. Furthermore, 25HC exhibited a favorable impact on ALS, as it prevented the early manifestation of differences in the properties of the neuromuscular synapse in the ALS model. 25HC has the ability to relieve synaptic anomalies including increased membrane fluidity, ceramide accumulation, decreased membrane ordering, and lipid peroxidation. Furthermore, it decreases the heightened level of extracellular choline, which could potentially lead to neuromuscular synapse fragmentation in ALS.