Remodeling of the Actin/Spectrin Membrane-associated Periodic Skeleton, Growth Cone Collapse and F-Actin Decrease during Axonal Degeneration

Nicolas Unsain,Federico M Barabas,Alfredo O Cáceres,Luciano A Masullo,Catalina Von Bilderling,Sami Jalil,Philip A Barker,Gaby F Martinez,Mariano Bisbal,Martin D Bordenave,Aaron D Johnstone,Fernando D Stefani

doi:10.1038/s41598-018-21232-0

Abstract

Axonal degeneration occurs in the developing nervous system for the appropriate establishment of mature circuits, and is also a hallmark of diverse neurodegenerative diseases. Despite recent interest in the field, little is known about the changes (and possible role) of the cytoskeleton during axonal degeneration. We studied the actin cytoskeleton in an in vitro model of developmental pruning induced by trophic factor withdrawal (TFW). We found that F-actin decrease and growth cone collapse (GCC) occur early after TFW; however, treatments that prevent axonal fragmentation failed to prevent GCC, suggesting independent pathways. Using super-resolution (STED) microscopy we found that the axonal actin/spectrin membrane-associated periodic skeleton (MPS) abundance and organization drop shortly after deprivation, remaining low until fragmentation. Fragmented axons lack MPS (while maintaining microtubules) and acute pharmacological treatments that stabilize actin filaments prevent MPS loss and protect from axonal fragmentation, suggesting that MPS destruction is required for axon fragmentation to proceed.

Highlights

Axonal degeneration occurs in the developing nervous system for the appropriate establishment of mature circuits, and is a hallmark of diverse neurodegenerative diseases
We evaluated if changing media could induce transient growth cone collapse (GCC): intact cultures were compared with cultures whose media was taken out and added back, and found no effect in GCC (Fig. 1d, third bar)
Time-lapse microscopy of axonal tips shows that GCC start almost immediately after trophic factor withdrawal (TFW), with growth cone area reaching its minimum within 30 minutes (Fig. 1e,f) and was accompanied by increased filopodia extensions and retractions close to axonal tips (Fig. 1e arrows), which ceased by 1 hour of nerve growth factor (NGF) deprivation

Summary

Introduction

Axonal degeneration occurs in the developing nervous system for the appropriate establishment of mature circuits, and is a hallmark of diverse neurodegenerative diseases. Knowledge on how these cascades impair the structure and function of an axonal segment is still rudimentary Actin cytoskeleton regulators such as ROCK2, cofilin[3,4] or spectraplakin[5] have been reported to have a role in diverse modes of axonal degeneration. Enzymes related to actin dynamics were found to be highly regulated in injured CNS axons as shown by unbiased proteomics of soluble axoplasm[6] and drugs that promote F-actin disassembly activate pro-degenerative cascades[7]. This evidence suggests that the actin cytoskeleton is modified during degeneration with a negative impact on axonal stability. Scale bar: 1 μm. (f) Plot of growth cone areas of different axonal tips (#1, #2 and #3 shown in e) at different time points before and after TFW. (g,h)

Methods

Results

Conclusion