The Golgi apparatus drives microtubule nucleation and subsequent axon regeneration in injured vertebrate peripheral neurons
Alice E Mortimer, MBChB, MRes1,2, Raman M Das, PhD1 and Adam J Reid, MBChB, FRCS(Plast), PhD1,2, (1)The University of Manchester, Manchester, Greater Manchester, United Kingdom, (2)Department of Plastic Surgery and Burns, Manchester, Greater Manchester, United Kingdom
Introduction: Axon regeneration following injury of peripheral neurons is characterised by large-scale remodelling of the microtubule cytoskeletal network. However, the fundamental mechanisms directing de-novo microtubule nucleation following peripheral nerve injury (PNI) remain unknown. Evidence from embryonic drosophila supports a role for the Golgi apparatus (GA) as a microtubule organising centre in neurons and given that mature neurons lack a centrosome, we hypothesised that the GA functions as the primary MTOC during adult, vertebrate peripheral neuron regeneration, and that this process relies on the key microtubule nucleating proteins gamma-tubulin and AKAP9.
Materials and methods: We used a combination of high-resolution live-imaging and super-resolution fixed-imaging techniques in an in-vitro adult rat model of PNI, as well as donor-derived human peripheral neurons to study the spatial-temporal dynamics of microtubule nucleation, microtubule/GA interactions and axon regeneration. Furthermore, we performed disruption of GA architecture and gamma-tubulin-driven microtubule nucleation using the pharmacological inhibitors Brefeldin A and Gatastatin G2, respectively. We additionally performed CRISPR knock-out of AKAP9 in order to determine whether its function at the GA is required for microtubule nucleation and subsequent axon regeneration.
Results: We have observed that striking changes in GA conformation are accompanied by the emergence of ordered microtubule architecture in regenerating peripheral neurons, and that these conformational changes coincide with the onset of axon regeneration at 24-hours post-injury. This is accompanied by dynamic recruitment of the essential microtubule nucleating proteins gamma-tubulin and AKAP9 to the GA and de-novo microtubule nucleation from the GA, which we have successfully visualised using our high-resolution live-imaging assay [Figure 1; GA (Galt7NeonGreen) and growing microtubules (EB3mScarlet)]. Furthermore, disruption of GA organisation and/or gamma-tubulin function results in the cessation of GA-mediated microtubule nucleation and prevention of axon regeneration. This phenotype is also recapitulated with AKAP9 knock-out, suggesting that the recruitment of gamma-tubulin and AKAP9 to a structurally intact GA is required for microtubule nucleation and subsequent axon regeneration following PNI.
Conclusions: These findings considerably enhance our understanding of the cellular mechanisms leading to re-establishment of neuronal architecture following injury and identify the GA as a target for therapeutic strategies aimed at improving regeneration following PNI.
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