Subcellular labelling of myelin protein (PLP and DM20) in the central nervous system

The Plp1 gene encodes the two proteins DM20 and PLP. Ultrastructural techniques will provide the best resolution to establish the sub-cellular localisation of the two myelin protein isoforms encoded by the Plp1 gene in mice and thus provide insight into the functions for the two isoforms. The gene is expressed primarily by oligodendroglia of the central nervous system (CNS), with some expression (primarily of DM20) in the Schwann cells of the peripheral nervous system. In the CNS, the DM20 isoform is expressed initially prior to myelin initiation and expression maintained throughout life. The function of DM20 is unknown. The expression of the PLP isoform appears to be related to myelination and have a structural role in compact myelin. The objective was to establish an immunolocalisation technique which would allow the hypothesis to be tested that DM20 is expressed in non-compacted regions of the myelinating oligodendrocyte, whilst PLP expression is confined to the compact myelin sheath. In order to differentially label PLP and DM20 at the paranodal region of myelinated axons, two different immunological techniques suitable for subcellular localisation were investigated. The photo-oxidation technique was applied to both cerebellar and cervical spinal cord vibratome sections. A brominated eosin conjugated secondary antibody was created and used to label a primary antibody against PLP and DM20. The photo-oxidation technique, a pre-embedding immunostaining, generated a very fine reaction product that was excellent at light microscopic resolution but only visible at electron microscopic resolution in sections that were not contrast stained. The immunogold technique was applied to sections from ventral columns in cervical spinal cord which has a dense population of medium to large myelinated axons. Assessments were made using anti-PLP antibodies in mature animals (with PLP at its peak of production and the myelin compacted and well established) and in young animals (with PLP in its beginning of production and less compacted). The immunogold technique, a post-embedding immuno-staining, produced a well-defined electron dense particle that was easily visualised in the EM. The technique proved insufficient to differentiate two different PLP antibodies, one labelling both isoforms (i.e. PLP and DM20) and the other specifically labelling only PLP. A substantially higher level of labelling was achieved, uniquely using the freeze substitution method of tissue preservation; however quantitative analysis could not be carried out because preservation was inconsistent both within the same tissue and from sample to sample.

Level dc:type.qualificationlevel

MSc(R)

Grantor dc:publisher.institution

University of Glasgow

Year dc:date.issued

2009

Author dc:creator

• McCulloch, Maj-Lis Christina

• QH301 Biology
• QR Microbiology

Language dc:language

en