Date of Award
1-11-2022
Degree Name
Doctor of Philosophy (PhD)
Degree Type
Dissertation
Abstract
Alzheimer disease (AD) is characterized by two main pathological hallmarks, extracellular amyloid plaques primarily comprised of the amyloid-β (Aβ) peptide, and intracellular neurofibrillary tangles mainly comprised of the tau protein. Although the pathologies underlying AD were first described over one hundred years ago, researchers today are still trying to understand how the development of Aβ pathology is regulated in the hopes of developing more effective treatments. Advances in genetics over the last several decades have allowed for several genetic risk factors to be identified that increase or decrease ones likelihood for developing (AD). Of these genetic risk factors, the strongest one for developing late onset AD is apolipoprotein E (APOE). The ε2 allele of APOE (APOE2) is considered to be protective relative to the more common ε3 allele (APOE3), while the ε4 allele (APOE4) confers the strongest increased risk. For those who carry one copy of APOE4 there is an approximate 3-fold increase in risk while those with two copies of APOE4 have a 12-fold increase in risk. APOE4 has been shown to influence the development of Aβ pathology in several ways, including the ability to indirectly increase Aβ levels by competing for clearance mechanisms with Aβ and to increase fibrillization of Aβ through direct interactions. These influences of APOE4 on Aβ, as well as more recent findings that APOE4 can influence tau mediated neurodegeneration, likely underlie why APOE4 carriers also have an earlier age of onset of AD. Many studies, including work done in our lab, have shown potential interventions for improving AD pathology could come from targeting apoE. In particular, reducing the levels of apoE in mice has been shown to significantly reduce the overall level of Aβ plaque burden. While the ability to improve overall Aβ pathology by manipulating apoE levels holds promise as a potential therapeutic intervention, few studies have looked closely at how cells respond to, and are impacted by, changes in apoE levels in the presence of developing Aβ pathology, especially at an individual per plaque basis. To begin to address these questions, we investigated how a complete loss of apoE impacts Aβ plaque formation, and the subsequent microgliosis and neuritic dystrophy, in two mouse models of amyloidosis. Here, I report on how the loss of apoE resulted in the formation of fewer amyloid plaques and how the fibrillar plaques that did form had an altered morphology. These morphologically altered plaques also had a reduction in microgliosis as well as an increase in neuritic dystrophy. With these findings, we hypothesized that the phenotypes we were seeing were due not only to the loss of apoE from astrocytes but were also due in part to the loss of microglial apoE. However, little is known about the characteristics of microglial apoE and raises the question as to whether differences exist between microglial apoE and astrocytic apoE. The physical characteristics of apoE-containing lipoprotein particles have been previously investigated in an attempt to better understand how structural differences between APOE2, APOE3, and APOE4 may be exerting their impact on AD pathology. However, these studies focused primarily on the apoE particles that are produced by astrocytes with minimal characterization of the apoE lipoprotein particles that are produced by microglia. We hypothesized that the apoE-containing lipoprotein particles that are produced by microglia differ from the particles that are produced by astrocytes. We investigated these potential differences by using primary cultures enriched for either microglia or astrocytes that expressed either APOE2, APOE3, or APOE4. Serum-free media samples from these cultures were analyzed using gel?electrophoresis and run under non-denaturing conditions to determine the size of the secreted apoE particles. I found that microglia produced a far greater amount of smaller apoE particles (~8nm) compared to astrocytes, which produced more of the larger apoE particles (~10-17nm). Furthermore, by changing the cell culture conditions and shifting the state of the microglia, I found that the level, and to some extent the size, of the apoE particles could also be shifted. These drastic changes in microglial apoE levels provide evidence that microglia apoE expression is highly dynamic and can be altered depending on the activation state of the microglia. These findings provide insight into how microglia may regulate their apoE expression and associated lipid components depending on their environment and whether they encounter pathological conditions. After finding that astrocytes produce larger apoE particles than microglia, we wanted to further explore how a loss of astrocytic apoE might influence the development of Aβ pathology. To do so, we utilized new APOE knock-in (APOE-KI) mouse models that our lab recently developed. These mice express APOE2, APOE3, or APOE4 under the endogenous mouse APOE promoter and contain loxP sites within the APOE gene that allow for a cell-specific removal of APOE using Cre-recombinase. By crossing the APOE-KI mice with Aldh1l1-Cre/ERT2 mice, we were able to selectively remove apoE from astrocytes and examine the impact on Aβ pathology in the APPPS1-21 mouse model. I found that, similar to a global removal of apoE, the overall fibrillar Aβ plaque load was significantly reduced after the loss of astrocytic apoE. The overall levels of GFAP and dystrophic neurites in the cortex were also reduced. The fibrillar plaques that did form with a loss of astrocytic apoE still had apoE present in them, however, the intensity of the plaques was greatly reduced. Interestingly, microglia were less activated and the neuritic dystrophy was increased around plaques. These findings indicate that apoE produced by astrocytes is playing a significant role in regulating overall Aβ pathology and is also influencing how microglia are activated in the presence of Aβ plaques. While further work is needed to address how a reduction in microglial apoE may influence Aβ pathology, I have demonstrated that reducing astrocytic apoE may provide therapeutic opportunities to improve outcomes in AD
Language
English (en)
Chair and Committee
David Holtzman
Recommended Citation
Mahan, Thomas, "Cell-type Specific Expression of Apolipoprotein E by Astrocytes and Microglia: Implications for the Development of Amyloid-β Pathology in Alzheimer Disease" (2022). Arts & Sciences Electronic Theses and Dissertations. 3252.
https://openscholarship.wustl.edu/art_sci_etds/3252