Molecular regulation of axonal membrane resealing

Emmanouilidis, Ioanna (2026): Molecular regulation of axonal membrane resealing. Dissertation, LMU München: Graduate School of Systemic Neurosciences (GSN)

Abstract

Axonal degeneration is a critical determinant of neurological disability in central nervous system diseases or trauma. Previous work from our laboratory indicates that a key predictor of axonal survival upon mechanical or neuroinflammatory injury is rapid calcium influx through damaged axonal membranes. Sustained elevation of intra-axonal calcium subsequently triggers overt axonal degeneration. This, however, can be averted if calcium homeostasis is restored, likely via axolemma resealing. Therefore, the mechanisms by which axons can repair their membranes are the focus of this thesis. Despite its significance, membrane resealing in mammalian axons in vivo remains poorly understood, as most membrane repair research has been conducted primarily in cell culture systems and invertebrate model organisms. The present work aimed to optimize and apply experimental tools to investigate the mechanisms of axonal membrane repair in the mouse spinal cord. To this end, we established a two-photon in vivo imaging approach to the dorsal column, which enables the assessment of intra-axonal calcium levels and axonal fate, degeneration vs. recovery, over several hours following mild spinal cord contusion. We combined this with neonatal intraventricular viral injections to generate general and neuron-specific CRISPR/Cas9 knock-outs in marked axonal subsets. As proof of principle, we demonstrated the successful and extensive gene editing of loci of interest, known to regulate axonal membrane organization and repair, across various Cas9-expressing transgenic mouse lines. Our approach additionally yielded an effective reduction in protein levels of Caspr1 and demonstrated the efficient coupling of calcium imaging with CRISPR manipulations in vivo. With this experimental framework established, we conducted a small-scale screen of the effects of depleting molecules previously implicated in membrane repair. To compile the list of candidates, we created a single-nuclei transcriptomic dataset of dorsal root ganglia neurons to identify plasma membrane repair mediators from multiple pathways, expressed in sensory axons. We assessed the impact of knocking out Alix, Synaptotagmin 7 and Vps4b on axonal calcium handling and axonal fate following spinal cord contusion injury but could not observe any significant effect. In summary, despite not identifying key regulators of axonal membrane resealing in the spinal cord in vivo, we demonstrated the advantages and versatility of our methodological platform. Our combined application of two-photon calcium imaging and CRISPR/Cas9-mediated gene editing enables dynamic monitoring of axons bearing targeted manipulations within the intact central nervous system. Our current approach opens a unique window into the largely unknown, but highly disease-relevant biology of axonal membrane maintenance, providing a powerful toolset for future investigations.

• Item Type: Theses (Dissertation, LMU Munich)
• Keywords: axonal degeneration, axonal membrane resealing, spinal cord injury, CRISPR/Cas9 gene editing, in vivo imaging
• Subjects: 500 Natural sciences and mathematics; 500 Natural sciences and mathematics > 570 Life sciences
• Faculties: Graduate School of Systemic Neurosciences (GSN)
• Language: English
• Date of oral examination: 7. May 2026
• 1. Referee: Misgeld, Thomas
• Signature of the printed copy: 0001/UMC 32043