Abstract
The development of morphological neuronal polarity starts by the formation and elongation of an axon. At the same time the axon initial segment (AIS) is generated and creates a diffusion barrier which differentiate axon and somatodendritic compartment. Different structural and functional proteins that contribute to the generation of neuronal action potential are concentrated at the axon initial segment. While axonal elongation is controlled by signalling pathways that regulate cytoskeleton through microtubule associated proteins and tubulin modifications, the microtubule cytoskeleton under the AIS is mostly unknown. Thus, understanding which proteins modify tubulin, where in the neuron and at which developmental stage is crucial to understanding how morphological and functional neuronal polarity is achieved. In this study performed in mice and using a well established model of murine cultured hippocampal neurons, we report that the tubulin deacetylase HDAC6 is localized at the distal region of the axon, and its inhibition with TSA or tubacin slows down axonal growth. Suppression of HDAC6 expression with HDAC6 shRNAs or expression of a non-active mutant of HDAC6 also reduces axonal length. Furthermore, HDAC6 inhibition or suppression avoids the concentration of ankyrinG and sodium channels at the axon initial segment (AIS). Moreover, treatment of mouse cultured hippocampal neurons with detergents to eliminate the soluble pool of microtubules identified a pool of detergent resistant acetylated microtubules at the AIS, not present at the rest of the axon. Inhibition or suppression of HDAC6 increases acetylation all along the axon and disrupts the specificity of AIS cytoskeleton, modifying the axonal distal gradient localization of KIF5C to a somatodendritic and axonal localization. In conclusion, our results reveal a new role of HDAC6 tubulin deacetylase as a regulator of microtubule characteristics in the axon distal region where axonal elongation takes place, and allowing the development of acetylated microtubules microdomains where HDAC6 is not concentrated, such as the axon initial segment.
Highlights
The formation of the axon is the first step for a neuron to adopt its morphological and functional polarization
To assess whether tubulin deacetylases play a role in axonal growth, cultured hippocampal neurons were treated with trichostatin A (TSA), which inhibit HDACs but not sirtuins
From our data we can conclude that activity is necessary for axonal growth, we can not exclude that in neurons the increased acetylation due to HDAC6 inhibition can play an important role in neuronal development
Summary
The formation of the axon is the first step for a neuron to adopt its morphological and functional polarization. The axon initial segment (AIS) develops from the very beginning of axon formation [15], generates action potentials and filters axonal transport [16,17,18] How this specialized domain is generated is poorly understood, treatment of neurons with taxol, which increases tubulin acetylation, can disrupt axonal polarized traffic [14]. Together with the enrichment of acetylated tubulin at the axon, the axon cytoskeleton needs dynamic microtubules for its elongation and the specification of axonal domains and polarized cargo transport. This change in microtubules requires the participation of enzymes that reverse tubulin modifications, such as acetylation. HDAC6 interacts with and control the phosphorylation of Tau [30], and it co-immunoprecipitates with the microtubule end-tracking protein EB1 [25]
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