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Soluble axoplasm enriched from injured CNS axons reveals the early modulation of the actin cytoskeleton

Soluble axoplasm enriched from injured CNS axons reveals the early modulation of the actin cytoskeleton
Soluble axoplasm enriched from injured CNS axons reveals the early modulation of the actin cytoskeleton
Axon injury and degeneration is a common consequence of diverse neurological conditions including multiple sclerosis, traumatic brain injury and spinal cord injury. The molecular events underlying axon degeneration are poorly understood. We have developed a novel method to enrich for axoplasm from rodent optic nerve and characterised the early events in Wallerian degeneration using an unbiased proteomics screen. Our detergent-free method draws axoplasm into a dehydrated hydrogel of the polymer poly(2-hydroxyethyl methacrylate), which is then recovered using centrifugation. This technique is able to recover axonal proteins and significantly deplete glial contamination as confirmed by immunoblotting. We have used iTRAQ to compare axoplasm-enriched samples from naïve vs injured optic nerves, which has revealed a pronounced modulation of proteins associated with the actin cytoskeleton. To confirm the modulation of the actin cytoskeleton in injured axons we focused on the RhoA pathway. Western blotting revealed an augmentation of RhoA and phosphorylated cofilin in axoplasm-enriched samples from injured optic nerve. To investigate the localisation of these components of the RhoA pathway in injured axons we transected axons of primary hippocampal neurons in vitro. We observed an early modulation of filamentous actin with a concomitant redistribution of phosphorylated cofilin in injured axons. At later time-points, RhoA is found to accumulate in axonal swellings and also colocalises with filamentous actin. The actin cytoskeleton is a known sensor of cell viability across multiple eukaryotes, and our results suggest a similar role for the actin cytoskeleton following axon injury. In agreement with other reports, our data also highlights the role of the RhoA pathway in axon degeneration. These findings highlight a previously unexplored area of axon biology, which may open novel avenues to prevent axon degeneration. Our method for isolating CNS axoplasm also represents a new tool to study axon biology.
1932-6203
e47552-[8pp]
Garland, Patrick
1d24a0cc-81f2-4ef1-82bd-77d2510e59d6
Broom, Lucy J.
a377b567-0943-45cf-b40a-8803d365cd7d
Quraishe, Shmma
cfc3aed4-f120-41aa-9127-0fc26c657ad2
Dalton, P.D.
ad77b93a-1348-445e-927d-6ac0c2c103fb
Skipp, Paul
1ba7dcf6-9fe7-4b5c-a9d0-e32ed7f42aa5
Newman, T.A.
322290cb-2e9c-445d-a047-00b1bea39a25
Perry, V.H.
8f29d36a-8e1f-4082-8700-09483bbaeae4
Garland, Patrick
1d24a0cc-81f2-4ef1-82bd-77d2510e59d6
Broom, Lucy J.
a377b567-0943-45cf-b40a-8803d365cd7d
Quraishe, Shmma
cfc3aed4-f120-41aa-9127-0fc26c657ad2
Dalton, P.D.
ad77b93a-1348-445e-927d-6ac0c2c103fb
Skipp, Paul
1ba7dcf6-9fe7-4b5c-a9d0-e32ed7f42aa5
Newman, T.A.
322290cb-2e9c-445d-a047-00b1bea39a25
Perry, V.H.
8f29d36a-8e1f-4082-8700-09483bbaeae4

Garland, Patrick, Broom, Lucy J., Quraishe, Shmma, Dalton, P.D., Skipp, Paul, Newman, T.A. and Perry, V.H. (2012) Soluble axoplasm enriched from injured CNS axons reveals the early modulation of the actin cytoskeleton. PLoS ONE, 7 (10), e47552-[8pp]. (doi:10.1371/journal.pone.0047552). (PMID:23115653)

Record type: Article

Abstract

Axon injury and degeneration is a common consequence of diverse neurological conditions including multiple sclerosis, traumatic brain injury and spinal cord injury. The molecular events underlying axon degeneration are poorly understood. We have developed a novel method to enrich for axoplasm from rodent optic nerve and characterised the early events in Wallerian degeneration using an unbiased proteomics screen. Our detergent-free method draws axoplasm into a dehydrated hydrogel of the polymer poly(2-hydroxyethyl methacrylate), which is then recovered using centrifugation. This technique is able to recover axonal proteins and significantly deplete glial contamination as confirmed by immunoblotting. We have used iTRAQ to compare axoplasm-enriched samples from naïve vs injured optic nerves, which has revealed a pronounced modulation of proteins associated with the actin cytoskeleton. To confirm the modulation of the actin cytoskeleton in injured axons we focused on the RhoA pathway. Western blotting revealed an augmentation of RhoA and phosphorylated cofilin in axoplasm-enriched samples from injured optic nerve. To investigate the localisation of these components of the RhoA pathway in injured axons we transected axons of primary hippocampal neurons in vitro. We observed an early modulation of filamentous actin with a concomitant redistribution of phosphorylated cofilin in injured axons. At later time-points, RhoA is found to accumulate in axonal swellings and also colocalises with filamentous actin. The actin cytoskeleton is a known sensor of cell viability across multiple eukaryotes, and our results suggest a similar role for the actin cytoskeleton following axon injury. In agreement with other reports, our data also highlights the role of the RhoA pathway in axon degeneration. These findings highlight a previously unexplored area of axon biology, which may open novel avenues to prevent axon degeneration. Our method for isolating CNS axoplasm also represents a new tool to study axon biology.

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Published date: 24 October 2012
Organisations: Faculty of Medicine, Centre for Biological Sciences

Identifiers

Local EPrints ID: 344626
URI: http://eprints.soton.ac.uk/id/eprint/344626
ISSN: 1932-6203
PURE UUID: 75d381aa-f5a9-46f3-8f92-f6a95c0ba40a
ORCID for Paul Skipp: ORCID iD orcid.org/0000-0002-2995-2959
ORCID for T.A. Newman: ORCID iD orcid.org/0000-0002-3727-9258

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Date deposited: 02 Nov 2012 10:03
Last modified: 18 Feb 2021 16:46

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