Extremely Low Forces Induce Extreme Axon Growth

Abstract

Stretch-growth has been defined as a process that extends axons via the application of mechanical forces. In the present article, we used a protocol based on magnetic nanoparticles (NPs) for labeling the entire axon tract of hippocampal neurons, and an external magnetic field gradient to generate a dragging force. We found that the application of forces below 10 pN induces growth at a rate of 0.66 ± 0.02 µm h⁻¹ pN⁻¹. Calcium imaging confirmed the strong increase in elongation rate, in comparison with the condition of tip-growth. Enhanced growth in stretched axons was also accompanied by endoplasmic reticulum (ER) accumulation and, accordingly, it was blocked by an inhibition of translation. Stretch-growth was also found to stimulate axonal branching, glutamatergic synaptic transmission, and neuronal excitability. Moreover, stretched axons showed increased microtubule (MT) density and MT assembly was key to sustaining stretch-growth, suggesting a possible role of tensile forces in MT translocation/assembly. Additionally, our data showed that stretched axons do not respond to BDNF signaling, suggesting interference between the two pathways. As these extremely low mechanical forces are physiologically relevant, stretch-growth could be an important endogenous mechanism of axon growth, with a potential for designing novel strategies for axonal regrowth. <b>SIGNIFICANCE STATEMENT</b> Axon growth involves motion, and motion is driven by forces. The growth cone (GC) itself can generate very low intracellular forces by inducing a drastic cytoskeleton remodeling, in response to signaling molecules. Here, we investigated the key role of intracellular force as an endogenous regulator of axon outgrowth, which it has been neglected for decades because of the lack of methodologies to investigate the topic. Our results indicate a critical role of force in promoting axon growth by facilitating microtubule (MT) polymerization.