Adaptor Protein 2 (AP-2) complex is essential for functional axogenesis in hippocampal neurons

Jae Won Kyung,In Ha Cho,Michael B Hoppa,Timothy A Ryan,Sung Hyun Kim,Sukmook Lee,Woo Keun Song

doi:10.1038/srep41620

Abstract

The complexity and diversity of a neural network requires regulated elongation and branching of axons, as well as the formation of synapses between neurons. In the present study we explore the role of AP-2, a key endocytic adaptor protein complex, in the development of rat hippocampal neurons. We found that the loss of AP-2 during the early stage of development resulted in impaired axon extension and failed maturation of the axon initial segment (AIS). Normally the AIS performs two tasks in concert, stabilizing neural polarity and generating action potentials. In AP-2 silenced axons polarity is established, however there is a failure to establish action potential firing. Consequently, this impairs activity-driven Ca2+ influx and exocytosis at nerve terminals. In contrast, removal of AP-2 from older neurons does not impair axonal growth or signaling and synaptic function. Our data reveal that AP-2 has important roles in functional axogenesis by proper extension of axon as well as the formation of AIS during the early step of neurodevelopment.

Highlights

Clathrin-mediated endocytosis has been identified as an important mechanism for enriching Na+ channels in epithelial tissue[21,22]
The efficiency of the knockdown in individual neurons was determined by retrospective immunocytochemistry against the AP-2 αsubunit as a proxy for the AP-2 complex as a whole, which showed that shRNA treatment targeting the μ2 subunit resulted in >90% loss of the AP-2 complex (Fig. 1a–c inset and supplementary Fig. 1)
Proper polarization was confirmed by sorting of endogenous microtubule associated protein 2 (MAP2), which was properly confined to the dendrites of both control and AP-2KD neurons

Summary

Introduction

Clathrin-mediated endocytosis has been identified as an important mechanism for enriching Na+ channels in epithelial tissue (a classic polarized cell)[21,22]. Endocytosis was identified as a critical process for modulating ligand-gated channels at the postsynaptic density of dendritic spines[23]. Taken together, these observations warrant a closer study of endocytic proteins in neuronal axogenesis and signaling. Removal of AP-2 once the axon has formed does not alter signal propagation and synaptic transmission Together, these data demonstrate a critical window during which AP-2 is needed in the process of establishing axogenesis including axonal branching and establishing the repertoire of ion channels critical for signal propagation to synapses

Methods

Results

Conclusion