Drosophila model for muscular pain

Olga's is fundamentally interested in how nerve cells remain healthy and functional throughout the live and during ageing. Olga uses in-vitro cell cultures, fruit flies and mice to understand how cells cope with metabolic stress challenges, like deprivation of growth factors and loss of ability to remove toxic debris. Most intriguing questions are how the nerve cells try to adapt to sustained stress and how this changes their function and communication towards other cells in a possibly adverse way. Such adjustments are believed to go wrong in chronic pain conditions resulting in maladaptation. The nerve cells, which receive information from other organs, become over-sensitive and show a stronger tendency to respond to stimuli, which normally would not trigger a healthy system. During her fellowship Olga will use the fruit fly Drosophila melanogaster to study how defects in the muscle provoke maladaptation in the nerve cells. She will concentrate on specific factors which respond to metabolic stress and then change the activity of the nerve cells. The very specific abilities of those factors will be exploited to make pain visible in the fruit fly. The success of this study will give novel insights on maladaptive communication and misinterpretation of pain signals and provide a new model to substitute mouse studies in search for novel pain genes and therapeutics. Drosophila as a traditionally powerful genetic model offers an excellent platform to understand basic mechanisms and brings an extraordinary advantage compared to mice through a shorter lifespan. The uptake of the fruit fly as a model for chronic pain in the wider research community and industry would facilitate and accelerate the efforts to develop therapeutic approaches, ideally, aiming at re-establishment of the balance in the nerve cells, provoking them to re-learn to not interpret mild stimuli as pain.

Maladaptive homeostatic plasticity, as a consequence of non-cell autonomous insults, is one of the central aspects in chronic pain studies, where changes in excitability in the nociceptive system, such as sensory neurons, occur following a damaging insult elsewhere. Rbfox splicing factor family regulates genetic output of muscles and neurons by mediating alternative splicing and mRNA degradation. The ability of Rbfox family to respond to upstream activity changes and subsequently to regulate excitability and activity provide intriguing mechanisms for the adaptation of functional circuits to local environments resembling prerequisite characteristics for homeostatic plasticity. Preliminary data and literature suggest that Rbfox splicing may be involved in sensory neuron sensitization. The proposed study aims to establish Drosophila models to understand the role of Rbfox in sensorimotor circuit homeostasis, with particular focus on balance between the muscle and sensory neuron function. These Drosophila models, which allow monitoring Rbfox activity, will be tested for their potential to serve as a novel, so far unrepresented, sensor tool to study non-cell autonomous sensory neuron sensitization in chronic conditions affecting the skeletal muscle. The substantial functional consequences, like changes in mRNA splicing, translation and degradation as well as miRNA metabolism, upon altered Rbfox activity would provide complementary advantages to tools like calcium sensors. The power of the model will be demonstrated by subsequent analysis of genes already known to participate in mediation of chronic pain as well as known Rbfox targets, linked to sensory neuron sensitization. The relevance to inflammatory response and ability of the model to replace moderate severity limit protocols, such CFA-injection in the muscle of mice for studies of chronic inflammatory pain mechanisms, will be examined.

The proposed study will address the relevance of Rbfox mediated splicing for the sensorimotor circuit homeostasis, initially focusing muscle to neuron communication. Members of Rbfox splicing factor family have been shown to orchestrate genetic output of muscle and neuronal genes and participate in adaptation of neuronal circuits to homeostatic changes in the system. The outcomes of this study may provide original evidence that Rbfox factors are involved in regulating the activity balance between skeletal muscle and sensory neurons. The approach will generate novel paradigms and tools in Drosophila to study functional changes in sensory neurons upon muscle degeneration. Drosophila was extremely useful to identify genes required for acute nociception. Most approaches aimed to disrupt genes cell-intrinsically in the sensory neurons. Few studies addressed sensory neuron sensitization upon UV inflicted tissue damage in Drosophila larvae. The advantage of the proposed model is the ability to target genes in two separate systems, muscle and sensory neuron, simultaneously in the adult fly. The aims is to create Drosophila lines, which would allow a user to test a gene in question in one simple cross to a reporter line and use simple read-out, such as nociceptive response and flourescent Rbfox-sensor quantification. The success of the proposed study will expand and encourage the use of Drosophila as powerful genetic tool, to analyse and monitor non-cell autonomous sensory neuron sensitization as a model of chronic pain. Rbfox factors not only mediate alternative splicing and non-sense mediated decay on mRNA transcripts, but also have been linked translational inhibition and miRNA metabolism. Emerging role of miRNAs and splicing in pain perception advocate for usefulness of the proposed model for future mechanistic studies.
Most common mammalian models of muscle pain involve injecting an irritating compound like carrageenan, complete freund's adjuvant (CFA), or formalin into the muscle, which triggers a robust inflammatory response. In the host institution at least four groups utilize such models (McMahon, Denk, McNaughton, Malcangio). In 2016 number of publications which used: carrageenan was 69 in mice and 71 in rats; CFA was 34 in mice and 59 in rats; and formalin 156 in mice and 113 in rats. CFA-injections evoke a severe prolonged chronic inflammatory response that might occur in arthritis. Since mutation in one of the Rbfox family members is linked to inflammatory muscle pain and hand-osteoarthritis, this study aims to establish a complimentary model for chronic pain studies and to test whether the proposed Rbfox-based Drosophila models would mimic and replace the CFA-induced pain models.
Notably, companies, like Charles River and others (21 listed in UK alone), advertise CFA-induced models for analgesic compound testing. In particular in neurodegesneration research Drosophila finds strong attraction as a powerful genetic tool for mechanistic studies. With respect to proportion of CFA-induction in transgenic mice/total CFA-induction in mice, the analysis of the publication output suggests with 8/34 in 2016 (26%) and in 7/13 in 2017(50 %), the popularity continues. Such experiments typically utilize about 9 animals in control and mutant group each. Thus, if Drosophila was used instead, potentially at least about 150 animals less per year would be exposed to this chronic pain procedure classified at a moderate severity limit, which involves severe side effects , like chronic local inflammation, skin ulcerations, lymph node structural changes, systemic granulomas and chronic wasting disease.
Examples, where researches with main expertise in mammalian models, such as the applicant, partially move to Drosophila models, may lift some of the barriers within the pain research community. Communication of success in the immediate and widespread community will facilitate the transition of more mammalian work that can be done in Drosophila