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Imaging neuroinflammatory axonal injury at nanoscale resolution

Subject Area Molecular Biology and Physiology of Neurons and Glial Cells
Molecular and Cellular Neurology and Neuropathology
Term from 2016 to 2025
Project identifier Deutsche Forschungsgemeinschaft (DFG) - Project number 317530061
 
Final Report Year 2024

Final Report Abstract

Maintaining the integrity of axons, the long projections of nerve cells, is essential for the proper functioning of the nervous system. Axons transmit electrical signals and transport cargo between nerve cell bodies and synapses. Due to their unique roles, axons can cover remarkable distances and are highly susceptible to injury. In neuroinflammatory diseases like multiple sclerosis, axons are particularly vulnerable, suffering damage from infiltrating immune cells. My previous research identified a novel form of neuroinflammatory axon loss—focal axonal degeneration (FAD). FAD is a sequential process triggered by neuroinflammatory oxidative stress. It is characterized by focal axonal swellings that may persist, progress to degeneration, or even spontaneously recover. Despite this discovery, many questions remain open, such as what determines axonal recovery, the events at the earliest injury sites, and the mediators of changes in axonal shape. In this project, we took an interdisciplinary approach to explore axonal biology, integrating cellular, molecular, chemical biology, and advanced microscopy techniques. Beyond conventional methods, we utilized super-resolution microscopy to gain nanoscale insights into neuronal organization. However, super-resolution microscopy presents challenges, particularly the need for new labelling techniques. Therefore, one key focus of this project was the development of minimally invasive fluorescent labelling tags for neuronal studies. Our approach relies on cutting-edge protein engineering tools, specifically the selective incorporation of unnatural amino acids (UAAs) and bioorthogonal click chemistry. Incorporating UAAs through genetic code expansion offers a unique opportunity to introduce new properties and functional groups into biological systems, such as fluorescent dyes for microscopy and capture tags for proteomics. However, this emerging protein engineering technology has mainly been used in conventional cell lines and at the proof-of-principle level. To fully realize its potential for fundamental and translational research, it was necessary to establish genetic code expansion-based click labelling of proteins in complex biological systems, such as primary neurons. We took on this challenge and, for the first time, established this protein labelling for advanced microscopy studies in primary neurons. In addition to imaging studies focused on cytoskeletal proteins and ion channels in healthy and injured neurons, another focus of this project was to apply this technology to investigate proteome dynamics during injury. To uncover unbiased changes in neuronal proteomes, we adapted UAA incorporation technology for cellspecific targeting, allowing us to study different cell types and proteome subsets. Ultimately, this approach will enable the identification of time-specific and cell-specific changes in proteomes, facilitating the discovery of novel mediators and signalling pathways involved in axonal injury.

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