Neuron Replating, a Powerful and Versatile Approach to Study Early Aspects of Neuron Differentiation.

Neuron differentiation includes formation and outgrowth of neurites that differentiate into axons or dendrites. Directed neurite outgrowth is controlled by growth cones that protrude and retract actin-rich structures to sense environmental cues. These cues control local actin filament dynamics, steer growth cones toward attractants and away from repellents, and navigate neurites through the developing brain. Rodent hippocampal neurons are widely used to study the mechanisms underlying neuron differentiation. Genetic manipulation of isolated neurons including gene inactivation or reporter gene expression can be achieved by classical transfections methods, but these methods are restricted to neurons cultured for several days, after neurite formation or outgrowth. Instead, electroporation allows gene manipulation before seeding. However, reporter gene expression usually takes up to 24 h, and time course of gene inactivation depends on the half live of the targeted mRNA and gene product. Hence, these methods do not allow to study early aspects of neuron differentiation. In the present study, we provide a detailed protocol in which we combined electroporation-based gene manipulation of mouse hippocampal neurons before initial seeding with a replating step after 2 d in vitro (DIV) that resets neurons into an undifferentiated stage. By categorizing neurons according to their differentiation stage, thorough morphometric analyses, live imaging of actin dynamics in growth cones as well as guidance cue-mediated growth cone morphologic changes, we demonstrate that differentiation and function of replated neurons did not differ from non-replated neurons. In summary, we provide a protocol that allows to thoroughly characterize differentiation of mouse primary hippocampal neurons.

Mutual functional dependence of cyclase-associated protein 1 (CAP1) and cofilin1 in neuronal actin dynamics and growth cone function.

Neuron connectivity depends on growth cones that navigate axons through the developing brain. Growth cones protrude and retract actin-rich structures to sense guidance cues. These cues control local actin dynamics and steer growth cones towards attractants and away from repellents, thereby directing axon outgrowth. Hence, actin binding proteins (ABPs) moved into the focus as critical regulators of neuron connectivity. We found cyclase-associated protein 1 (CAP1), an ABP with unknown brain function, abundant in growth cones. Super-resolution microscopy and live cell imaging combined with pharmacological approaches on hippocampal neurons from gene-targeted mice revealed a crucial role for CAP1 in actin dynamics that is critical for growth cone morphology and function. Growth cone defects in CAP1 knockout (KO) neurons compromised neuron differentiation and was associated with impaired neuron connectivity in CAP1-KO brains. Mechanistically, by rescue experiments in double KO neurons lacking CAP1 and the key actin regulator cofilin1, we demonstrated that CAP1 was essential for cofilin1 function in growth cone actin dynamics and morphology and vice versa. Together, we identified CAP1 as a novel actin regulator in growth cones that was relevant for neuron connectivity, and we demonstrated functional interdependence of CAP1 and cofilin1 in neuronal actin dynamics and growth cone function.