Toward understanding the structure and plasticity of the human olfactory bulb in the normal and Parkinson's disease brain

Abstract

Hyposmia, or the diminished capacity to smell, is a robust precursor to the motor symptoms of Parkinson’s disease (bradykinesia, rigidity, a resting tremor, and a shuffling gait), preceding these by 4-10 years. In pursuit of what causes hyposmia in the virtually unstudied human olfactory bulb, a computerised 3D model of the olfactory bulb was created using a combination of automated, fluorescent immunohistochemistry and 3D reconstruction methods that provided quantitative parameters and a unique method for visualisation of the constituents of the olfactory bulb and its functional units (olfactory glomeruli). Antibodies to V-GLUT2 (vesicular glutamate transporter type 2) and NCAM (neural cell adhesion molecule) enabled the identification of olfactory glomeruli. The number of glomeruli, as counted from the 3D model, was highly variable amongst four normal cases (2, 436 – 9, 999 glomeruli) and not significantly different from three Parkinson’s disease bulbs (1, 840 – 8, 341 glomeruli). Total glomerular and bulb volume was also measured but no significant differences were observed. Nonetheless, the experimental and analytical approach employed in these studies represents a unique and powerful method for visualisation and objective quantification. The second major aspect to this thesis was an experimental approach to understanding the control mechanism of polysialic acidneural cell adhesion molecule (PSA-NCAM), a post-translational modification that aids cellular migration and dendritic foraging. The putative role of sialidase IV (NEU4) in the downregulation of PSA-NCAM was examined; the removal of polysialic acid being a critical step for correct positioning of neuroblasts upon migrating into the olfactory bulb. To do this, NEU4 was expressed in the TE671 rhabdomyosarcoma cell line, and its effects on PSA-NCAM expression were measured. No significant changes in overall PSA-NCAM levels were seen in cells expressing either the short or long isoform of NEU4. This was seen under conventional culture conditions and also when cells were cultured in an extracellular matrix. In order to detect more subtle changes that may be occurring at the cell surface, the same experiments were performed and analysed using immunocytochemistry and high content analysis. Depending on the mode of analysis small changes were detected suggesting that NEU4 may be involved in reducing the amount of PSA-NCAM present at the cell surface. However, owing to the experimental design, I was not able to definitively implicate or exclude NEU4 from involvement in the down-regulation of PSA-NCAM. Collectively, the findings of this thesis are of relevance when elucidating the mechanisms of olfactory bulb wiring and organisation, and how neural progenitors serve to maintain this brain region over a lifetime.