Formin Activity and mDia1 Contribute to Maintain Axon Initial Segment Composition and Structure

Wei Zhang,Norah Boumedine-Guignon,Michaël Russier,Beatriz Achón,Juan José Garrido,Diana Retana,Dominique Debanne,María Ciorraga,Pablo Mendez

doi:10.1007/s12035-021-02531-6

Abstract

The axon initial segment (AIS) is essential for maintaining neuronal polarity, modulating protein transport into the axon, and action potential generation. These functions are supported by a distinctive actin and microtubule cytoskeleton that controls axonal trafficking and maintains a high density of voltage-gated ion channels linked by scaffold proteins to the AIS cytoskeleton. However, our knowledge of the mechanisms and proteins involved in AIS cytoskeleton regulation to maintain or modulate AIS structure is limited. In this context, formins play a significant role in the modulation of actin and microtubules. We show that pharmacological inhibition of formins modifies AIS actin and microtubule characteristics in cultured hippocampal neurons, reducing F-actin density and decreasing microtubule acetylation. Moreover, formin inhibition diminishes sodium channels, ankyrinG and βIV-spectrin AIS density, and AIS length, in cultured neurons and brain slices, accompanied by decreased neuronal excitability. We show that genetic downregulation of the mDia1 formin by interference RNAs also decreases AIS protein density and shortens AIS length. The ankyrinG decrease and AIS shortening observed in pharmacologically inhibited neurons and neuron-expressing mDia1 shRNAs were impaired by HDAC6 downregulation or EB1-GFP expression, known to increase microtubule acetylation or stability. However, actin stabilization only partially prevented AIS shortening without affecting AIS protein density loss. These results suggest that mDia1 maintain AIS composition and length contributing to the stability of AIS microtubules.

Highlights

The axon initial segment (AIS) serves as a barrier that controls membrane protein diffusion and cytoplasmic transport towards the axon, maintaining neuronal polarity [1,2,3,4,5]
Treatment with SMIFH2 (15 μM) in 14 DIV-cultured hippocampal neurons led to a significant ankyrinG decrease after 3 h of application (−25%, Fig. 1A, B) that was detected after 30 min (−15%, Fig. 1C)
To discard that SMIFH2 treatment was generating an indirect effect on neurons through formin inhibition in astrocytes, we applied SMIFH2 to neurons in the absence of astrocytes (Fig. S2A) and detected again an ankyrinG reduction (−28.80 ± 1.89%) compared to DMSO-treated neurons

Summary

Introduction

The axon initial segment (AIS) serves as a barrier that controls membrane protein diffusion and cytoplasmic transport towards the axon, maintaining neuronal polarity [1,2,3,4,5]. Peking University, Beijing, China 3 UNIS, INSERM, UMR 1072, Aix-Marseille Université, 13015 Marseille, France 4 Alzheimer’s Disease and Other Degenerative Dementias, Centro de Investigación Biomédica en Red de Enfermedades Neurodegenerativas (CIBERNED), Madrid, Spain of voltage-gated ion channels that allow action potential (AP) generation [6]. This high density of voltage-gated ion channels is achieved by a not completely understood protein complex formed by scaffold proteins, such as ankyrinG or βIV-spectrin, and a specialized actin and microtubule cytoskeleton [7]. AnkyrinG can bind to microtubules through EB1/3 proteins [13]

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Results

Conclusion