Investigation into the molecular mechanisms of axonal degeneration and characterisation of focal adhesion kinase in response to CNS injury

Rankine, Emma Louise (2006) Investigation into the molecular mechanisms of axonal degeneration and characterisation of focal adhesion kinase in response to CNS injury. University of Southampton, Doctoral Thesis.

Record type: Thesis (Doctoral)

Abstract

We raised polyclonal antibodies against components of sciatic nerves undergoing Wallerian degeneration. The antibodies have been screened against a range of naïve and injured tissues by immunohistochemistry and Western blotting. A proteomics approach has then been employed to deduce which antigen(s) the antibodies recognise. Immuno histochemistry on injured CNS tissues, using these novel antibodies, revealed staining of damaged axons from as early as 6 hours, which intensifies through to 7 days in the spinal cord and 28 days in the brain, the longest timepoints studied. Western blot analysis indicates a number of bands that are either upregulated or down regulated in response to injury in CNS tissue when compared to the appropriate naïve tissue. The bands identified by Western blot were subsequently analysed by matrix-assisted laser desorption/ionisation time-of-flight mass spectrometry (MALDI-TOF).

We identified changes in three proteins following CNS injury. Firstly, albumin appears to be taken up into damaged axons following injury prior to resealing. Secondly, kid-1, a transcription factor, was identified; Western blot and immunocytochemical analysis revealed no change in expression over the time course of injury investigated. And finally, focal adhesion kinase (FAK), a protein tyrosine kinase, was also identified.

Changes in FAK expression following injury were studied by Western blot and immunocytochemistry. Western blot analysis revealed a decrease in FAK expression following brain and spinal cord injury. Double immunofluorescence revealed that de novo FAK expression was present in oligodendrocytes as early as 6 hrs post injury. This is possibly the earliest event occurring in the oligodendrocyte in response to Wallerian degeneration of their associated axons. At later times FAK expression was detected, to a lesser extent, in astrocytes and possibly microglial cells.

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