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

Area of Science:

• Neuroscience
• Cell Biology
• Molecular Biology

Background:

• The axon initial segment (AIS) is crucial for neuronal function, relying on a specialized actin and microtubule cytoskeleton.
• Existing knowledge on AIS cytoskeleton regulation and its impact on neuronal polarity, protein transport, and action potential generation is limited.
• Formins are key regulators of actin and microtubule dynamics, suggesting a potential role in AIS structure maintenance.

Purpose of the Study:

• To investigate the role of formins in regulating the actin and microtubule cytoskeleton at the AIS.
• To determine how formin inhibition affects AIS structure, protein composition, and neuronal excitability.
• To elucidate the molecular mechanisms by which formins contribute to AIS stability.

Main Methods:

• Pharmacological inhibition of formins in cultured hippocampal neurons and brain slices.
• Genetic downregulation of the mDia1 formin using interference RNAs (shRNAs).
• Assessment of F-actin density, microtubule acetylation, AIS protein (sodium channels, ankyrinG, βIV-spectrin) density, and AIS length.
• Measurement of neuronal excitability.
• Manipulation of microtubule acetylation and stability using HDAC6 downregulation and EB1-GFP expression.

Main Results:

• Formin inhibition reduced F-actin density and microtubule acetylation at the AIS.
• Pharmacological and genetic formin inhibition decreased AIS length and the density of key AIS proteins, including sodium channels, ankyrinG, and βIV-spectrin.
• Formin inhibition led to diminished neuronal excitability.
• Downregulation of HDAC6 or EB1-GFP expression partially rescued AIS shortening and ankyrinG decrease, suggesting a role for microtubule stability.
• Actin stabilization only partially prevented AIS shortening and did not affect AIS protein density loss.

Conclusions:

• The formin mDia1 plays a critical role in maintaining AIS composition and length.
• Formins contribute to AIS stability, at least in part, by regulating microtubule acetylation and stability.
• These findings reveal a novel mechanism for AIS regulation with implications for neuronal function and excitability.