A Novel Paradigm for Mechanistic Studies of Myalgic Ecephalomyelitis/Chronic Fatigue Syndrome 

Introduction: Myalgic encephalomyelitis/chronic fatigue syndrome (ME/CFS) is a debilitating illness characterized by extreme fatigue lasting for over six months. It is often developed following a viral or bacterial infection, does not improve with rest, and disproportionately affects women. Effective treatment options for ME/CFS are extremely limited and progress towards new treatments is hindered by a lack of suitable animal models for mechanistic and drug discovery studies. Although exact cause(s) of ME/CFS is (are) yet to be discovered, recent studies have shed light on potential mechanisms and the cascade of events that could lead to its development, which includes metabolic and autonomic dysfunctions, and brain abnormalities. Interestingly, broad-spectrum metabolomics performed on plasma taken from ME/CFS patients has revealed that metabolic features of this condition resemble those found in hibernating animals2.
The overall objective of this work is to investigate the hypothesis that ME/CFS is a result of perpetual activation of highly conserved brain networks, which in hibernating animals induce a hibernation state. Prior work has demonstrated that a hibernation-like state can be pharmacologically induced in Xenopus tadpoles, where swimming, sensory responses and metabolism are significantly suppressed. In this study we performed bulk RNA-sequencing (RNA-seq) experiments using tissue from tadpoles undergoing pharmacologically- induced hibernation to investigate the validity this model in mimicking molecular changes that are observed in human ME/CFS patients.
Materials & Methods: A Compound inducing hibernation-like state and a vehicle control were administered separately to two groups of Xenopus tadpoles for 1.5 hours, during which time their swimming behaviors were monitored. Treated tadpoles’ swimming rate was reduced to less than 20% of control by the end of the treatment. Following treatment, tadpoles were euthanized in 20X Tricaine, tissues were snap frozen in liquid nitrogen, and stored at -80C. RNA was extracted and mRNA-seq with polyA enrichment was performed by Novogene (N=3-4 samples/group). To validate the Xenopus hibernation model against clinical ME/CFS, a meta-analysis was conducted using publicly available human datasets: a set of publicly available bulk RNA-seq data1. The RNA-seq dataset was specific to post-infection ME/CFS and included samples from peripheral blood mononuclear cells (PBMCs) and muscle tissue. ClusterProfiler5 was used to perform gene set enrichment analysis (GSEA) and pathway over-representation analysis for each dataset to identify statistically significant genetic pathways within three gene ontology sets: cellular component, biological function, and metabolic function.
Results & Discussion: Comparison of the significantly activated and/or suppressed pathways in the Xenopus dataset with those in the human dataset, broken down by PBMC and muscle tissue samples, revealed areas of overlap as well as divergence. Characterization of pathways across Xenopus, male and female datasets included suppressed signaling receptor activity. Signaling receptor activity involves initiating a change in cell activity through transmitting a signal through the cell. However, this pathway is an umbrella for a plethora of immune cell activity such as cytokine, chemokine and receptor ligand activity. Such suppression of chemokine and cytokine activity were specifically observed in Xenopus through pathway enrichment. Consistent with this finding, the GSEA analysis of male patients’ muscle tissue revealed suppressed response to cytokines and regulation of immune response. The common suppression of signaling receptor activity warrants further analysis as it is suggestive of diminished immune response, a biological phenomenon established within the previous literature as a key component of the physiological characteristics of ME/CFS. Additionally, increased activation of the small nuclear ribonucleoprotein complex (snRNP complex) was observed in the male and female muscle tissue datasets. The small nuclear ribonucleoprotein complexes (snRNP) play a role in the spliceosome, splicing nuclear mRNAs and subsequently affecting the structure of nuclei, organelles, etc. The increased activation of the snRNP complex suggests possible changes in gene regulation that could impact multiple cellular functions. While the activity in the snRNP complex was exclusively within the human datasets, it is possibly indicative of a change due to prolonged effects of ME/CFS that should be considered for successive Xenopus models.
Conclusions: Past research has discussed transcriptional and metabolic perturbations within ME/CFS cohorts, the patterns being most unanimous in expression of inflammatory pathways. Overall, the Xenopus hibernation model shows some similarities to ME/CFS patient data, but further characterization of this model is required to make definitive conclusions on the reliability of the animal model. After the identification of a suitable model, future work will use a gene network-based drug prediction algorithm to identify existing drugs that may be able to reverse the disease state.