Effekte einer pharmakologischen Inhibition der ULK1-Kinase auf die akute axonalen Degeneration in vivo
Effects of pharmacological inhibition of the ULK1 kinase on acute axonal degeneration in vivo
von Jonas Sundermeyer
Datum der mündl. Prüfung:2022-09-06
Erschienen:2022-09-05
Betreuer:PD Dr. Jan C. Koch
Gutachter:Prof. Dr. Christine Stadelmann-Nessler
Gutachter:Prof. Dr. Ralf Dressel
Dateien
Name:Dissertation.eDISS.ohneLebenslauf.pdf
Size:26.5Mb
Format:PDF
Zusammenfassung
Englisch
Background: Axonal degeneration is a key and early pathological feature in neurodegenerative and traumatic disorders of the central nervous system. A focal lesion to an axon is followed by a rapid disintegration within several hours, named acute axonal degeneration (AAD). During AAD, the accumulation of autophagic proteins including Unc-51 like autophagy activating kinase 1 (ULK1) has been demonstrated, but its role is incompletely understood. Objective: The aim of this study was to investigate the effects of therapeutic ULK1 inhibition by the small-molecule ULK-1 inhibitor SBI-0206965 on acute axonal degeneration in vivo in a translational approach. Methods: To evaluate AAD in vivo, rat optic nerve crush and live imaging were performed. Intravitreal injections of AAV.EGFP were administered 14 days before nerve crush to label the axons. SBI-0206965 (5 µM or 50 µM) was injected intravitreally 2.5 h prior to nerve crush. Z-stack images were taken in the area of 400–500 µm proximal to the crush site 5–360 min after crush, and AAD was quantified by axonal integrity ratio and axonal bulb analysis. Quantification of LC3- and p62-puncta in the optic nerve were performed with immunohistochemistry and confocal microscopy 400 µm proximal and distal to the crush lesion. Results: Treatment with 50 µM SBI-0206965 resulted in a significant reduction of axonal fragmentation as measured by the axonal integrity ratio compared to control starting 4 h after crush. In contrast, the concentration of 5 μM SBI-0206965 showed no reduction in axonal integrity loss. Axonal bulb structures remained essentially unaffected in size and number within five hours postlesion. Treatment with 50 μM SBI-0206965 significantly lowered the number of distal LC3 puncta and was shown to increase the number of p62 puncta, indicating the inhibition of autophagosome formation by SBI. Conclusion: In a translational approach, we tested whether application of the ULK1 inhibitor SBI-0206965 affects AAD after rat optic nerve crush in vivo. Taken together, our data demonstrate that SBI could inhibit autophagy and attenuate AAD after lesion in vivo. Thus, ULK1 could represent a novel therapeutic target in traumatic and degenerative diseases of the central nerve system, but further research efforts are urgently needed.
Keywords: Autophagy; Acute axonal degeneration; ULK1