<p>Microglia are a population of specialized,
tissue-resident immune cells that make up around 10% of total cells in our
brain. They actively prune neuronal synapses, engulf cellular debris, and
misfolded protein aggregates such as the Alzheimer’s Disease (AD)-associated amyloid-beta
(Aβ) by the process of phagocytosis. During AD, microglia are unable to
phagocytose Aβ, perhaps due to the several disease-associated changes affecting
their normal function. Functional molecules such as lipids and metabolites also
influence microglial behavior but have primarily remained uncharacterized to
date. The overarching question of this work is, <i>How do microglia become
dysfunctional in chronic inflammation</i>? To this end, we developed new
chemical tools to better understand and investigate the microglial response to
Aβ <i>in vitro</i> and <i>in vivo</i>. Specifically, we introduce three new
tools. (1) Recombinant human Aβ was developed via a rapid, refined, and robust
method for expressing, purifying, and characterizing the protein. (2) A
pH-sensitive fluorophore conjugate of Aβ (called Aβ<sup>pH</sup>) was developed
to identify and separate Aβ-specific phagocytic and non-phagocytic glial cells <i>ex
vivo</i> and <i>in vivo</i>. (3) New lysosomal, mitochondrial, and nuclei-targeting
pH-activable fluorescent probes (called LysoShine, MitoShine, and NucShine,
respectively) to visualize subcellular organelles in live microglia. Next, we asked,
<i>What changes occur to the global lipid and metabolite profiles of microglia in
the presence of Aβ in vitro and in vivo</i>? We screened 1500 lipids comprising
10 lipid classes and 700 metabolites in microglia exposed to Aβ. We found significant
changes in specific lipid classes with acute and prolonged Aβ exposure. We also
identified a lipid-related protein that was differentially regulated due to Aβ <i>in
vivo</i>. This new lipid reprogramming mechanism “turned on” in the presence of
cellular stress was also present in microglia in the brains of the 5xFAD mouse
model, suggesting a generic response to inflammation and toxicity. It is well
known that activated microglia induce reactive astrocytes during inflammation. Therefore,
we asked, <i>What changes in proteins, lipids, and metabolites occur in astrocytes
due to their reactive state? </i>We provide a comprehensive characterization of
reactive astrocytes comprising 3660 proteins, 1500 lipids, and 700 metabolites.
These microglia and astrocytes datasets will be available to the scientific community
as a web application. We propose a final model wherein the molecules secreted
by reactive astrocytes may also induce lipid-related changes to the microglial
cell state in inflammation. In conclusion, this thesis highlights chemical
neuroimmunology as the new frontier of neuroscience propelled by the
development of new chemical tools and techniques to characterize glial cell
states and function in neurodegeneration.</p>
| Identifer | oai:union.ndltd.org:purdue.edu/oai:figshare.com:article/14515074 |
| Date | 29 April 2021 |
| Creators | Priya Prakash (10725291) |
| Source Sets | Purdue University |
| Detected Language | English |
| Type | Text, Thesis |
| Rights | CC BY 4.0 |
| Relation | https://figshare.com/articles/thesis/Characterizing_Microglial_Response_to_Amyloid_From_New_Tools_to_New_Molecules/14515074 |
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