microRNA-mediated Regulating of Axonal Structural Integrity and Nerve Injury Response

The function of the nervous system relies on the ability of neurons to signal to target cells, represented by other neuronal cells, muscles, or gland cells. Information is transmitted to receiving cells via protoplasmic protrusions called nerve fibers or axons. Axons are remarkable, long cellular projections, which in different species can extend from a few micrometers, to ~1 meter in the human sciatic nerve, and presumably over 30 meters in blue whales. All animals face the same formidable task of maintaining the structural integrity of axonal projection throughout the entire life span of the organism. Deciphering the molecular mechanisms underlying this process represents a challenging but fascinating scientific endeavor, with deep implications in the treatment of human neurological disorders. Indeed, axonal loss is one of the primary causes of disability in a rage of pathological conditions including traumatic injuries to brain (TBI) spinal cord (SCI) and peripheral nervous system, inflammatory neuropathies, inflammatory demyelinating conditions (such as multiple sclerosis), and certain neurodegenerative disorders. A notable feature of many human diseases is a pronounced alteration in the levels of numerous proteins. However, the upstream regulatory mechanisms controlling the aberrant production of these proteins remain unclear. For many years, it was thought that RNAs only function as structural scaffolds and messengers transferring genetic information from DNA to proteins. This view was drastically changed over a decade ago, following the seminal discovery of a class of regulatory RNAs called microRNAs. microRNAs are fascinating non-coding RNA molecules which are never translated into a polypeptide chain (protein), and are comprised of only 21 to 23 letters of the genetic alphabet. Strikingly, these tiny pieces of RNA appear to function as molecular rheostats of gene expression programs (protein production) in almost every living organism. Surprisingly, microRNAs have been shown to be dysregulated in many neurological disorders as well as traumatic axonal injuries. Still, it remains unclear whether microRNAs are central to the molecular mechanism controlling axonal structural integrity in intact and injury challenged neurons. Here, we propose to use the genetically tractable model organism Drosophila melanogaster (fruit fly) to shed light on the role of microRNAs in this process. The extensive conservation of molecular and cellular mechanisms governing nervous system development and function makes Drosophila an ideal experimental platform for attaining this goal. For these studies we will employ a multi-pronged strategy combining highly versatile transgenic technologies for microRNA loss and gain of function, state of the art transcriptional profiling, high-throughput microRNA target identification, and cutting-edge genome editing technologies for elucidating gene regulatory networks controlled by candidate microRNAs. While preventing clearance of severed axons in traumatic nerve injury appears to be detrimental for functional recovery, the knowledge acquired in this study may engender new targets for developing therapeutic strategies for other neuropathological conditions where axons are not severed but at risk of undergoing degeneration.

The architecture of the nervous system is dependent on sophisticated connections established by axons and dendrites. The ability of neurons to maintain the integrity of long axonal projections throughout the life span of an organism is a fascinating biological phenomenon. Elucidating the molecular mechanisms controlling this process under physiological conditions and pathological stress remains a fundamental scientific question. miRNAs are ~22 nucleotide non-coding RNAs that emerged as essential post-transcriptional regulators of gene expression programs in development and disease. Interestingly, miRNAs are abundantly expressed in the CNS, and have been implicated in modulating neurogenesis, axonogenesis, as well as synaptic morphogenesis and plasticity. Directly relevant to this proposal, both acute and prolonged alterations in the levels of several miRNAs following traumatic never injury have been reported, suggesting they may play important roles in neuronal injury response. We propose to explore the role of miRNAs in controlling axonal structural integrity and the response to traumatic nerve injury, using a multi-pronged and multi-disciplinary experimental strategy in the genetically tractable organism Drosophila melanogaster. Acute changes in miRNA homeostasis following traumatic nerve injury will be detected using state of the art miRNA profiling tools. High-throughput transgenic technologies will be used to discover miRNAs functionally required for axonal structural integrity and nerve injury response. Deciphering the regulatory pathways controlled by candidate miRNAs will be attained using cutting edge technologies for genome-wide miRNA target identification (Ago-HITS-CLIP). Finally, recently pioneered genome editing tools (TALEN/CRISPR) and epistasis experiments will be used to refine and validate direct effector genes. These studies promise to engender new insights into the molecular mechanisms underlying axonal structural integrity and pathophysiology.

The overarching goal of this research program is to understand the role of miRNAs in the molecular machinery regulating axonal structural integrity and nerve injury response. We anticipate that the outcome of the research proposed here may impact both basic academic and medical sciences.

Academic impact. The impact of this study on academic research is carefully detailed in the "Academic beneficiaries" section of this application. The information acquired over the course of this research project will engender new insights into the basic mechanisms maintaining the physiology and integrity of axonal projections during animal development and adult life. Furthermore, although focused on the nervous system, the interdisciplinary nature of this project and the technological platforms we propose to develop in Aims 3 and 4 will have broader consequences and will benefit microRNA research outside the neuroscience field.

Medical impact.
The fundamental knowledge emerging from this study may also uncover potential therapeutic targets (or strategies) for situations whereby axons have not been completely transected but are at risk of undergoing degeneration. These include inflammatory neuropathies, inflammatory demyelinating conditions (such as multiple sclerosis), and certain neurodegenerative disorders. Because microRNAs are smaller, less antigenic and display a stereotypical mechanism of action compared to protein-coding genes, the field of microRNA-based therapeutics is rapidly gaining momentum. Both microRNA replacement therapies and systemic microRNA inhibition have been successful in animal models of human disease, and a therapeutic strategy for Hepatitis C (Miravirsen) has now reached Phase II clinical trials at Santaris Pharma A/S. We hope that our findings will eventually benefit patients and we will make every effort to disseminate our findings to lay audiences and healthcare professionals.

NC3R and policymakers in the UK and abroad.
Because both microRNA identity and the molecular pathways governing nervous system development and function display a high degree of conservation from flies to humans, it is plausible that modifiers discovered in the fly model system, will have a similar impact in vertebrates. This will eliminate the need of exploratory screens in higher organisms and drastically reduce the time and effort required for these studies. A more targeted candidate approach will also reduce the number of protected animals used for studying pathological conditions where research is currently being pursued primarily in vertebrates, but Drosophila models already exist. These include traumatic brain injury, neurodegenerative diseases such as Alzheimer's and Parkinson's disease, and perhaps other conditions not directly related to the nervous system, such as wound healing and innate immune response. Therefore, in long term the studies proposed here could result in a significant reduction in the number of animals used for microRNA research in nervous system development and disease.