Meccanismi di crescita assonale nei neuroni cerebellari del purkinje: ruolo della kinasi c-Jun N-terminale

Purkinje cells neurons are an interesting model to investigate the cellular and molecular mechanisms of axon regeneration and plasticity, for their peculiarity of weak cell body response to axotomy, their strong resistance to injury and virtually absolute inability to regenerate severed neurites, also in presence of favourable environmental conditions. The development of Purkinje cells axons is accomplished by an intrinsic growth program whose execution is influenced by the cerebellar environment. Neurons plated in vitro at distinct developmental ages grow axons according to different patterns. Elongation of the main axon stem prevails in embryonic Purkinje axons, while postnatal neurites switch to arbour growth within a few days in vitro. Such patterns well correspond to distinct developmental stages, including a phase of elongation of the axon stem to reach the target territory, and the subsequent extension of the terminal arborization. Purkinje cells also grow thin processes along the stem, necessary for the formation of the infra and supra ganglionic plexus. This dynamic process of sprouting is finalized to the establishment of the correct pattern of Purkinje cells connections. We defined these processes as collaterals that sprout with an orthogonal angle from the axon stem and are thinner than the parent neurite. We found that high level of phosphorylation of the c-Jun N-terminal kinase (JNK) specifically characterize Purkinje cells main axon in vitro, while collaterals of the plexus and the terminal arbour are poorly labelled. To test the hypothesis that JNK cascade regulates the morphogenesis of the axon stem, we cultured mouse Purkinje cells, dissociated at embryonic and postnatal ages, and inhibited the JNK pathway by treatment with the cell permeable peptide D-JNK-1. Morphometric analysis of collaterals revealed that the peptide enhances the sprouting of collaterals along the stem and seems to modify their shape. Concomitantly, we observed a tendency to decrease the lengths of the stem and of the tree . Interestingly, the embryonic population was more affected by the treatment, in line with its capacity to develop more collaterals at this developmental time. A possible mechanistic explanation is that JNK acts on the cytoskeleton dynamics, reducing the rearrangement of its components. In summary, this study suggests the involvement of JNK in the morphogenesis of Purkinje cells terminal plexus and consequently in the correct establishment of Purkinje cells networks.