Degree Name

Master of Science (MS)

Semester of Degree Completion

2003

Thesis Director

Britto P. Nathan

Abstract

The apolipoprotein E4 (apoE4) genotype is a major risk factor for Alzheimer's disease (AD). Previous studies have shown that apoE4 is less effective than apoE3 in supporting neurite outgrowth in adult mice cortical cultures. However the mechanism underlying this phenomenon is not known. To understand this mechanism and the broader role of apoE isoforms on neuronal biology I examined the rate of apoE isoforms internalization in neurons, effects of apoE isoforms on lipid uptake by neurons, effects of apoE isoforms on neuronal environment - microglia and on neuroregeneration, or neurogenesis.

I examined the binding and internalization of recombinant human apoE3 and apoE4 in cortical cultures derived from adult apoE knockout mice. The cultures were incubated with apoE3 or apoE4 over various time periods at temperatures between 4°C and 37°C before immunocytochemistry for apoE was performed. The data revealed that cell-associated apoE3 uptake was significantly greater (≅2.5 times) than apoE4 regardless of the incubation time; the percent of total immunoreactivity in the cell body compared to dendritic immunoreactivity declined in the apoE3 group but did not change in the apoE4 group primarily because of increased total apoE per neuron.

To determine the lipoprotein receptors that mediate the differential uptake of apoE3 and apoE4 in neurons, I blocked the low-density lipoprotein (LDL) receptor related protein (LRP) and cell surface heparan sulfate proteoglycans (HSPG). The data revealed that inhibition of LRP decreased both apoE3 and apoE4 uptake. Blocking HSPG totally inhibited uptake to undetectable levels. The data suggests that HSPG has a pivotal role in apoE internalization.

I predicted that apoE3 would deliver more lipids to neurons than apoE4 because the rate of uptake of apoE3 was faster than apoE4 in neurons. To test this hypothesis, I examined fluorescently labeled dodecanoic acid accumulation in adult cortical neurons. Cells cultured with apoE3 internalized approximately twice as much vesicular accumulations of fluorescence as those with apoE4. ApoE4 roughly doubled the amount of lipid internalized compared to the absence of apoE. Therefore, although apoE4 was better than no apoE, it was less effective than apoE3 in providing lipids.

Previous studies demonstrated that apoE regulates microglial activation and inducible nitric oxide synthase (iNOS) in embryonic and postnatal mouse microglial cultures. I examined and compared the effects of apoE3 and apoE4 on microglial proliferation, morphology and activation in adult mouse cortical (AMC) and adult mouse microglial (AMM) cultures. Microglial proliferation and morphology for each apoE treatment in AMC and AMM cultures were assessed by immunocytochemistry; levels of iNOS expression were assessed by immuno-blotting. The results from this study demonstrated that apoE3 and apoE4 differentially regulate microglial proliferation and activation and iNOS production in a neuron-microglia independent manner and apoE4 induces microglial proliferation and activation, when compared to apoE3.

Recent findings concerning the regenerative potential of the adult brain showed that adult neurons have the ability to divide, a process known as neurogenesis. I examined the effects of apoE3 and apoE4 on neurogenesis in AMC culture. Neuronal proliferation in apoE3 or apoE4 treated cells was analyzed with MTT and BrdU cell proliferation kits, followed by ELISA and immunocytochemistry. The results show that the fraction of neurons in the DNA synthesis stage to total number of neurons in apoE3 treatment was significantly higher than in apoE4 and apoE KO treatment groups. These data suggests that apoE4 is less efficient in promoting neurogenesis. This study also revealed new phenomenon of BrdU-positive satellite stain outside the BrdU-positive nucleus in approximately 50% of dividing neurons. The significance of the satellite BrdU-positive stain found in adult neurons in both apoE isoform treatment groups is yet to be determined.

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