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Effects of ?TAT1 and HDAC5 on axonal regeneration in adult neurons.

ABSTRACT: The role of posttranslational modifications in axonal injury and regeneration has been widely studied but there has been little consensus over the mechanism by which each modification affects adult axonal growth. Acetylation is known to play an important role in a variety of neuronal functions and its homeostasis is controlled by two enzyme families: the Histone Deacetylases (HDACs) and Histone Acetyl Transferases (HATs). Recent studies show that HDAC5 deacetylates microtubules in the axonal cytoplasm as part of an injury-induced regeneration response, but little is known about how acetylation of microtubules plays a role. Alpha-tubulin acetyl transferase (?TAT1) is a microtubule specific acetyl transferase that binds to microtubules and directly affects microtubule stability in cells. We hypothesize that increasing tubulin acetylation may play an important role in increasing the rate of axonal growth. In this study, we infected cultured adult DRG neurons with ?TAT1 and ?TAT1-D157N, a catalytically inactive mutant, and HDAC5, using lentiviruses. We found that ?TAT1 significantly increases tubulin acetylation in 293T cells and DRG neurons but ?TAT1-D157N does not. Furthermore, in neurons infected with ?TAT1, a significant increase in acetylated tubulin was detected towards the distal portion of the axon but this increase was not detected in neurons infected with ?TAT1-D157N. However, we found a significant increase in axon lengths of DRG neurons after ?TAT1 and ?TAT1-D157N infection, but no effect on axon lengths after infection with HDAC5. Our results suggest that while ?TAT1 may play a role in axon growth in vitro, the increase is not directly due to acetylation of axonal microtubules. Our results also show that HDAC5 overexpression in the axonal cytoplasm does not play a crucial role in axonal regeneration of cultured DRG neurons. We expressed these genes in DRG neurons in adult rats and performed a sciatic nerve crush. We found that axons did not regenerate any better when infected with any of the constructs compared with control animals. Thus, while ?TAT1 may be important for axonal growth in vitro, neither ?TAT1 nor HDAC5 had an effect in vivo on the regeneration of sciatic nerves.

SUBMITTER: Lin S

PROVIDER: S-EPMC5432171 | biostudies-literature | 2017

REPOSITORIES: biostudies-literature

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Effects of αTAT1 and HDAC5 on axonal regeneration in adult neurons.

Lin Shen S Sterling Noelle A NA Junker Ian P IP Helm Courtney T CT Smith George M GM

PloS one 20170515 5

The role of posttranslational modifications in axonal injury and regeneration has been widely studied but there has been little consensus over the mechanism by which each modification affects adult axonal growth. Acetylation is known to play an important role in a variety of neuronal functions and its homeostasis is controlled by two enzyme families: the Histone Deacetylases (HDACs) and Histone Acetyl Transferases (HATs). Recent studies show that HDAC5 deacetylates microtubules in the axonal cyt ...[more]

PMID: 28505206

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Project description:Axonal regeneration is an essential condition to re-establish functional neuronal connections in the injured adult central nervous system (CNS), but efficient regrowth of severed axons has proven to be very difficult to achieve. Although significant progress has been made in identifying the intrinsic and extrinsic mechanisms involved, many aspects remain unresolved. Axonal development in embryonic CNS (hippocampus) requires the obligate activation of the insulin-like growth factor 1 receptor (IGF-1R). Based on known similarities between axonal growth in fetal compared to mature CNS, we decided to examine the expression of the IGF-1R, using an antibody to the βgc subunit or a polyclonal anti-peptide antibody directed to the IGF-R (C20), in an in vitro model of adult CNS axonal regeneration, namely retinal ganglion cells (RGC) derived from adult rat retinas. Expression of both βgc and the β subunit recognized by C20 antibody were low in freshly isolated adult RGC, but increased significantly after 4 days in vitro. As in embryonic axons, βgc was localised to distal regions and leading growth cones in RGC. IGF-1R-βgc co-localised with activated p85 involved in the phosphatidylinositol-3 kinase (PI3K) signaling pathway, upon stimulation with IGF-1. Blocking experiments using either an antibody which neutralises IGF-1R activation, shRNA designed against the IGF-1R sequence, or the PI3K pathway inhibitor LY294002, all significantly reduced axon regeneration from adult RGC in vitro (∼40% RGC possessed axons in controls vs 2-8% in the different blocking studies). Finally, co-transfection of RGC with shRNA to silence IGF-1R together with a vector containing a constitutively active form of downstream PI3K (p110), fully restored axonal outgrowth in vitro. Hence these data demonstrate that axonal regeneration in adult CNS neurons requires re-expression and activation of IGF-1R, and targeting this system may offer new therapeutic approaches to enhancing axonal regeneration following trauma.

Dataset Information

Effects of ?TAT1 and HDAC5 on axonal regeneration in adult neurons.

Publications

Effects of αTAT1 and HDAC5 on axonal regeneration in adult neurons.

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