Affiliation:
1. Department of Anatomy, Neuroscience Laboratory for Cognitive and Developmental Disorders, Medical College of Jinan University, Guangzhou 510630, China
Abstract
Aims:
Our work aims to revealing the underlying microtubule mechanism of neurites
outgrowth during neuronal development and also proposes a feasible intervention pathway for reconstructing
neural network connections after nerve injury.
Background:
Microtubule polymerization and severing form the basis for neurite outgrowth and
branch formation. However, the mechanisms that underlie the dynamic instability of microtubules
are unclear. Here, we showed that neurite outgrowth mediated by collapsing response mediator protein
2 (CRMP2) can be enhanced by spastin, which had an effect on the severing of microtubule cytoskeleton.
Objective:
To explore whether neurite outgrowth was mediated by coordination of CRMP2 and
spastin.
Methods:
Hippocampal neurons were cultured in vitro in 24-well culture plates for 4 days before
being used to perform the transfection. Calcium phosphate was used to transfect the CRMP2 and
spastin constructs and their control into the neurons. An interaction between CRMP2 and spastin
was examined by using pull down, CoIP and immunofluorescence colocalization assays. And immunostaining
was also performed to determine the morphology of neurites.
Results:
We first demonstrated that CRMP2 interacted with spastin to promote neurite outgrowth
and branch formation. Then our results identified that CRMP2 interacted with the microtubule-
binding domain of spastin via its C-terminus, and deleting these binding sites inhibited neurite
outgrowth and branch formation. In addition, we confirmed one phosphorylation site at S210
of spastin in hippocampal neurons. Spastin phosphorylation at S210 failed to alter the binding
affinity of CRMP2 but inhibited its binding to microtubules. Further study showed that phosphorylation
spastin at S210 inhibited the neurite outgrowth induced by CRMP2 and spastin interaction
through downregulation of microtubule-severing activity.
Conclusion:
Taken together, our data demonstrated that both CRMP2 and spastin interaction and
the microtubule-severing activity of spastin were required for neurite outgrowth and branch formation.
Funder
Guangzhou Institute of Pediatrics/ Guangzhou Women and Children’s Medical Center
Natural Science Foundation of Guangdong Province, China
National Natural Science Foundation of China
Publisher
Bentham Science Publishers Ltd.
Subject
Pharmacology,General Neuroscience
Cited by
9 articles.
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