• axonal regeneration
• molecular mechanisms
• stretch growth

Axonal growth is a phenomenon of great interest for the community of neuroscientists. However, the knowledge regarding axon outgrowth mechanisms is still incomplete and requires further studies. It’s now known that mechanical forces play a crucial role in neuronal growth and development. To study the effects of extremely low tension on these mechanisms, a new protocol based on nanotechnologies was developed in the laboratory where this study was carried out. Specifically, magnetic nanoparticles (MNPs) made up by iron oxide were exploited to label the cells and an external magnetic field was applied to generate a dragging force, thus enabling the stretching of the neurons. Previous work performed by my team on hippocampal neurons, showed mechanical tension leads to an increase in axon length, mass addition and cytoskeletal remodeling. My thesis project consisted in exploring the still lacking knowledge about the local molecular mechanisms which take place at the axonal level when this stretching protocol is applied. To do so, I performed my experiments seeding neurons obtained from P0 mice hippocampi in microfluidic devices. This support allows to separate the somato-dendritic compartment from the axonal one, making it easier to study mechanisms taking place inside the axons. First, I performed analyses of the axonal transcriptome of stretched and unstreteched axons, to see which were the main players affected by the application of forces. To further investigate the involvement of local phenomena at axonal level, the study of translationally active ribosomes was carried out. I also investigated the possible functional interaction of these structures with late endosomes, whose involvement in the translation processes at the axonal level was recently demonstrated. Moreover, as previous electrophysiological studies revealed a higher synaptic maturation rate in stretched neurons, we decided to further investigate this aspect through histochemical labeling of synaptic markers. Neuronal maturation was also investigated through immunohistochemistry of Synapsin 1 (Syn1), a key regulator of synaptic vesicles dynamics.
Considering that in previous studies, our tension was found to increase axon length and neuronal maturation and considering that MNPs and magnetic fields are already used as tool in regenerative medicine, we decided to investigate the possibility to exploit our methodology in a model of axonal regeneration. To do this, human grade MNPs were used. I tested their possible toxicity, performed elongation studies, and investigated their capability to stimulate re-growth after axonal injury in the microfluidic devices.
In general, understanding the mechanisms that underlie axon growth following the introduction of a tension could have a strong impact in the field. First, it could allow to deeply understand mechanisms that take place during the development of the central nervous system. Last, but not least, it could allow to develop new therapeutic methods to promote axonal regeneration.