Tubulin post-translational modifications in developing dog primary neurons obtained with methods according to the 3Rs principles

Microtubules play a crucial role during neuronal morphogenesis regulating many functions. In the study of these
phenomena in vitro cellular models have been employed, mainly resorting to housed experimental animals.
Among alternative models in neurobiological study, recently dog caught particular attention. In fact, the complexity
of the canine brain, the life long span and the neurodegenerative pathologies render the dog a species
more close to humans than rodents. Lately, growing interest in the limitation of the use of experimental animals,
has stimulated the search for alternative experimental protocols. Starting from fetal dog brain, obtained by
alternative way of sampling, we set neuronal primary cultures. Through immunofluorescence, we examined the
presence and the cellular distribution of tubulin post-translational modifications as tyrosinated and acetylated α-
tubulin, as markers of dynamic and stable microtubule respectively. In addition, we evaluated the pattern of two
associated proteins which may slide on these two tubulin modifications, i.e. CLIP-170 and Kinesin-1. A clear
positivity for tyrosinated and acetylated α-tubulin, was found. As far as the motor proteins are concerned, we
detected a prevalence of CLIP-170 compared to kinesin-1 with a better overlapping between tyrosinated α-
tubulin and CLIP-170. Our findings highlighted some original data about the role of the microtubular network
during early phases of canine neuronal morphogenesis. In addition, the experimental protocol underlined the
utility of this alternative model that allows to bypass both the scarcity of commercial canine neuronal cell lines
and the need to resort to experimental dogs, respecting the 3Rs principles (reduction, refinement, and replacement).