Cytoskeletal assembly in axonal outgrowth and regeneration analyzed on the nanoscale

Abstract

The axonal cytoskeleton is organized in a highly periodic structure, the membrane-associated periodic skeleton (MPS), which is essential to maintain the structure and function of the axon. Here, we use stimulated emission depletion microscopy of primary rat cortical neurons in microfluidic chambers to analyze the temporal and spatial sequence of MPS formation at the distal end of growing axons and during regeneration after axotomy. We demonstrate that the MPS does not extend continuously into the growing axon but develops from patches of periodic βII-spectrin arrangements that grow and coalesce into a continuous scaffold. We estimate that the underlying sequence of assembly, elongation, and subsequent coalescence of periodic βII-spectrin patches takes around 15 h. Strikingly, we find that development of the MPS occurs faster in regenerating axons after axotomy and note marked differences in the morphology of the growth cone and adjacent axonal regions between regenerating and unlesioned axons. Moreover, we find that inhibition of the spectrin-cleaving enzyme calpain accelerates MPS formation in regenerating axons and increases the number of regenerating axons after axotomy. Taken together, we provide here a detailed nanoscale analysis of MPS development in growing axons.

Pathology of Initial Axon Segments in Chronic Inflammatory Demyelinating Polyradiculoneuropathy and Related Disorders

“…Another important structure in the compartmentalization of the neuron is the submembrane cytoskeleton called the membrane-associated periodic skeleton (MPS) [ 22 ]. It consists of long filaments of actin organized in the braid pattern, forming ring-like structures connected with alpha- and beta-spectrin and other associated molecules [ 22 , 23 ]. MPS is present in the central and peripheral nervous system and plays a role in the mechanical resistance of axons [ 24 , 25 ].…”

“…MPS is present in the central and peripheral nervous system and plays a role in the mechanical resistance of axons [ 24 , 25 ]. membrane filtering at the AIS [ 21 ], maintenance of axonal microtubules [ 26 ] and regeneration of axons [ 23 ].…”

“…Degeneration manifests by the loss of MPS, and independently of this process, the activation of caspase apoptotic pathways [ 76 ]. The mechanisms by which the MPS reorganizes itself in the outgrowing or regenerating axon are not fully understood, although some evidence for the accumulation of spectrin in the growth cone and the accelerated formation of spectrin periodicity has already been gathered [ 23 ].…”

A fluid-walled microfluidic platform for human neuron microcircuits and directed axotomy

“…16,18 In this study, we exploit the advantages of fluid walls to establish a proof-of-concept model that recreates appropriate arrangements of human CNs and MSNs. We then go on to develop a method for targeted and localised axotomy of CNs, and compare the effects of pro-regenerative conditions on axonal regrowth; [18][19][20] brain-derived neurotrophic factor (BDNF) and postsynaptic MSNs both have positive effects. In combination, these approaches provide a method for screening drugs promoting developmental outgrowth of axons and regeneration of damaged ones.…”

A fluid-walled microfluidic platform for human neuron microcircuits and directed axotomy

“…In this study, we exploit the advantages of fluid walls to establish a proof-of-concept model that recreates appropriate arrangements of human CNs and MSNs. We then go on to develop a method for targeted and localised axotomy of CNs, and compare the effects of pro-regenerative conditions on axonal regrowth [19][20][21] ; brain-derived neurotrophic factor (BDNF) and postsynaptic MSNs both have positive effects. In combination, these approaches provide a method for screening drugs promoting developmental outgrowth of axons and regeneration of damaged ones.…”