Graduate Program

Biological Sciences

Degree Name

Master of Science (MS)

Semester of Degree Completion

2006

Thesis Director

Britto Nathan

Thesis Committee Member

Charles Costa

Thesis Committee Member

Gary Fritz

Abstract

Previous studies have shown that apolipoprotein E (apoE), a lipid transporting protein, is involved in the growth and regeneration of injured neurons. apoE is a key molecule involved in lipid transport system associated with compensatory sprouting and synaptic remodeling after neuronal injury. Also, transport of cholesterol and phospholipids in brain is regulated by the coordinated expression of apoE and its main Low Density Lipoprotein (LDL) receptors during the different phases of the neuronal reinnervation processes. The discovery of apolipoprotein E4 genotype, a major risk factor for Alzheimer's disease (AD), is suggestive of involvement of dysfunctional apoE lipid transport system in the progression of AD. We used mouse olfactory system, olfactory epithelium (OE) and olfactory bulb (OB), as a model to study the role of apoE in the neuronal regeneration, maturation and synaptogenesis. Olfactory epithelium provides "window" on the neuronal development because it is a site for continual neurogenesis. Although the underlying mechanism(s) is incompletely understood, previous studies have shown that olfactory neurons reinnervate following lesioning. Recent studies including studies in our lab have shown that apoE is expressed at high concentrations in the olfactory nerve as well as in the olfactory bulb and that the apoE levels in these regions increase substantially following lesioning of the source of the olfactory nerves, the olfactory epithelium. However, the mechanism(s) underlying the contribution of apoE to the neuronal regeneration, maturation, and synaptogenesis are not clearly understood. We therefore hypothesize that apoE is essential for regeneration, maturation and synaptogenesis of mice olfactory epithelial neurons.

In this study, we have advanced a series of experiments to understand the role of apoE in regeneration, maturation and synaptogenesis of mice olfactory epithelial neurons. Aim 1 was used to test the hypothesis that apoE is involved in neuronal regeneration and maturation following olfactory epithelial lesioning. This hypothesis was tested by using immunoblotting technique to demonstrate time dependent change in levels of expression of apoE and Olfactory Marker Protein (OMP) at several stages of post OE lesioning induced by nasal irrigation of Triton X-100. The result revealed that apoE expression levels initially down-regulated at day 7 post-lesioning and reached to basal level at day 21 and significantly exceeded basal level at days 42 and 56. Whereas, OMP expression levels were down-regulated for 21 days post lesioning followed by a significant up-regulation at day 42 post lesioning in WT mice as compared to apoE KO mice. These collectively suggest that apoE is involved in neuronal regeneration and maturation of olfactory epithelium following olfactory epithelium lesioning.

of Aim 2 was used to test the hypothesis that apoE promotes synaptogenesis Olfactory Receptor Neuron (ORN) with OB neurons post OE lesioning. In this study, immunoblotting technique was used to examine the role of apoE on the rate of synaptic recovery following OE lesioning. The result revealed that Synaptophysin (Syn) levels declined sharply between 3 and 7 days post lesioning in both WT and KO mice. Following this precipitous decline, OB Syn levels steadily increased to about 80% of the normal levels by 56 days post lesion in WT mice. In contrast to WT mice, Syn density did not increase significantly in KO mice and was significantly less than that of WT mice on day 56. Since the rate of Syn recovery was delayed in apoE KO mice, suggesting apoE facilitates synaptogenesis of olfactory nerve post injury.

Aim 3 was used to test the hypothesis that apoE is involved in regeneration and maturation of ORN neurons following removal of the synaptic targets of ORN by bilateral olfactory bulb ablation. This was assessed by studying the level of expression of apoE and OMP in OE following day post bulbectomy. Olfactory bulbectomy (OBX) resulted in a significant increase in the expression of apoE level 3 days post bulbectomy, which further increased by 7 days and remained at high levels thereafter. Whereas, olfactory bulbectomy resulted in a significant decrease in the expression of OMP level for 14 days post bulbectomy and it increased slightly at day 21. This collectively suggests that apoE is involved in regeneration and maturation of OE neurons following removal of synaptic targets of ORN by OВХ.

Taken together, these results indicate that apoE is involved in regeneration and maturation as well as in the synaptic formation of ORN.

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