Degree Name

Master of Science (MS)

Semester of Degree Completion

2000

Thesis Director

Britto P. Nathan

Abstract

Previous studies have shown that apoE is upregulated in injured nerves, and that it may participate in nerve regeneration by recycling lipids from the degenerating myelin sheath to axonal growth cones. However, these studies fail to differentiate apoE function in degeneration and regeneration, because of temporal juxtaposition of degeneration and regeneration in these models. In this study, we characterized the apoE expression during olfactory nerve regeneration in mice, which occurs over an extended seven week period. Olfactory nerves were lesioned in 2-3-month-old mice by intransal irrigation of Triton X-100. Following lesioning the olfactory bulbs were collected at 0, 3, 7, 21, 42, and 56 days post-injury, and apoE levels in the bulbs were determined by immunoblot analysis. ApoE level peaked at 3 days-post lesion, reaching a concentration twice which is found in the normal olfactory bulb. The apoE level stayed elevated by approximately 1.5 times the normal level at 7 through 21 days after injury, and thereafter gradually returned to normal by 56 days. These data suggest that apoE functions in the central nervous system to promote efficient repair of neural structures following injury.

An extension of this observation is the hypothesis that if apoE is important in nerve repair, then nerve regrowth should be either incomplete or delayed in the absence of apoE. I tested this postulate by comparing olfactory nerve repair in apoE gene deficient/apoE gene knockout (apoE KO) with control mice. Olfactory nerve recovery was assessed by immunoblotting of GAP 43, a marker for juvenile olfactory neurons. Immunoblot analysis revealed that GAP 43 levels in control animals increased by approximately two times the normal level at 3 days post-lesion, and returned back to baseline at 7 days after injury. At 21 days post-lesion the GAP 43 level increased dramatically reaching a concentration 3 times which is found in the normal olfactory bulb. The GAP 43 level stayed elevated until 42 days, and thereafter gradually returned to normal by 56 days. Even though the GAP 43 time course in apoE KO mice followed a pattern similar to that observed in control animals, the major increase in GAP 43 was observed on 42 days post lesion. This is a two weeks delay from that observed in control animals, suggesting that the generation of new olfactory neurons is delayed by two weeks in apoE KO mice.

To confirm the GAP 43 data and examine the effects of apoE deficiency in the maturation of olfactory neurons, I examined the expression of the olfactory marker protein (OMP), a marker for adult olfactory neurons. Immunoblot analysis revealed that OMP levels in the bulb of control animals gradually declined following nerve lesioning, decreasing to approximately 50% of the normal level at 7 days post lesion. Thereafter OMP levels sharply increased to about 80% of the normal level at 21 days post lesion, and then gradually increased to normal by 56 days. In apoE KO animals the OMP time course was different than that of control animals. First, OMP levels continued to decrease in apoE KO animals until 21 days post-lesion decreasing to approximately 40% of normal OMP levels. Thereafter OMP levels increased slowly, reaching only 50% of normal level on 42 days post-lesion. The OMP levels gradually returned to normal after 56 days following injury in apoE KO animals, similar to that observed in control mice. Taken together the OMP data demonstrate that maturation of olfactory neurons is delayed by about two weeks in apoE KO mice as compared to control animals, and that it is consistent with the results from our GAP 43 studies.

The results from this study demonstrate that apoE has a tremendous impact on neurite outgrowth, and consequently has a significant effect on neuronal plasticity. The apoE is upregulated during periods predominated by degeneration of the olfactory nerve and at the early stages of regeneration. We hypothesize that apoE is upregulated to facilitate efficient repair of neural structures following injury. The association of apoE phenotype with dementing illnesses, like Alzheimer's disease, may represent a diminished ability to support a lifetime of nerve regeneration.

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