• Neuronal remodelling
• Neurons
• Mechanical force

Title: Investigating the effects of mechanical stimuli on neuronal remodelling in primary neurons.
Mechanotransduction, a mechanism by which cells convert mechanical stimuli into biochemical responses, plays a pivotal role in each stage of neuronal development regulating a plethora of molecular processes. This phenomenon is known as “stretch-growth”. As a matter of fact, neurons are mechanosensitive cells able to response at physical forces through complex intracellular and extracellular interactions. Recently, my team developed a method in which it is possible to apply mechanical stimuli, by generating extremely low forces lower than 10 pN. Magnetic nanoparticles (MNPs), combined with an external, static, magnetic field, are used to apply forces to mice hippocampal neurons. Interestingly, they found these extremely low forces promoted axonal elongation and sprouting. They also observed an alteration of intracellular calcium dynamics and the modulation of cytoskeletal dynamics, axonal transport, local translation, mainly at the axon level.
In my thesis project I focused on the understanding of molecular mechanisms globally activated. Specifically, I evaluated gene expression at the level of the soma, starting from data collected by my team. Next to this, I performed gene ontology (GO) analysis to highlight pathways mis-regulated between stretched and unstretched neurons. Starting from these data, I focused on cytoskeletal and metabolism pathways. Regarding, cytoskeletal components, I evaluated a microtubule-related protein at the axonal level. In particular, end binding protein 3 (EB3) that associates to plus-end of microtubules (MTs) was evaluated. The localization of EB3 in stimulated and unstimulated axons was investigated to understand the MT dynamics following mechanical force application. Being EB3 strictly related to MT assembly, the distribution of the protein was evaluated also following the administration of a drug that inhibits MT polymerization (i.e., Nocodazole). To further investigate MT dynamics, the abundance of the microtubule-associated protein tau was evaluated. Talking about metabolism dynamics, mitochondria distribution, abundance and functionality was evaluated. In addition, I focused on the effects of globally activated mechanical forces from transcriptomic data; in particular, the goal is to evaluate what happens at the nuclear level following the application of a mechanical stimulus and how these forces are transduced by evaluating cytoskeleton-nucleus interactions.
Furthermore, to establish if stretch-growth induces its effect in a model-dependent manner, I evaluated the role of tension in dorsal root ganglion neurons (DRGs). To do this, P3 mice pups were used. These cells require nerve growth factor (NGF) for survival. This factor is known to stimulate axonal outgrowth and development. Previous studies of the research team highlighted that neuron stimulated with neurotrophins do not respond to stretch-growth. For this, I tested several approaches to reduce the interference from NGF chemical signaling.
In conclusion, findings raised from my project could open interesting scenarios in the field. In particular, understanding mechanisms behind stretch-growth could enhance the knowledge about what happens during the development of the central nervous system. Next, they could be the starting point for the purpose of a novel strategies for the regeneration of sick or damaged neurons.

Candidate: Pietro Folino, CdLM Biologia Molecolare e Cellulare