Neurodegenerative Diseases

Using the Caenorhabditis elegans as a model we have employed forward genetic screens to uncover several novel genetic contributors to dopamine (DA) signaling(1). Follow-up characterization of some of these novel contributors have been detailed in published work from our lab(2), while follow-up studies on other pathways are still underway. Moreover, using the powerful Million Mutation Project library, we have uncovered an important link between primary cilium formation and the regulation of the DA transporter dat-1(3). The focus of the body of work detailed in this manuscript is on a glial expressed gene, swip-10, uncovered from our original genetic screen(1, 4, 5). Unlike the other pathways uncovered from our genetic screening, swip-10 does not affect DA signaling via DAT-1 regulation, instead, loss of swip-10 produces excess DA signaling in a glutamate-signaling-dependent manner to cause swimming-induced paralysis (Swip)(4) as well as premature DA neuron degeneration(5). Specifically, the primary aim here was to uncover the molecular pathway by which swip-10 supports these phenotypes.

Cholesterol is a pivotal component of mammalian cell membranes and homeostasis. Due to its high concentration and heterogenous distribution in the brain, cholesterol is tightly regulated and its dyshomeostasis is frequently implicated in several neurodegenerative diseases. This dissertation reports the design, synthesis, and application of ten cholesterol naphthalimide probes (CND) to study cholesterol trafficking in live cells. The CND series was rationally designed by incorporating several of the structural features of endogenous cholesterol onto the naphthalimide (ND) scaffold conjugated via an ester bond. The modularity of the ND scaffold enabled all analogs to have the aliphatic tail of cholesterol, which is lacking in the most ubiquitously utilized probe, BODIPY-Cholesterol, a.k.a. Top-Fluor® Cholesterol (TFC). The CNDs were demonstrated to be optimal probes for cholesterol due to the ability to fluorescence exclusively in hydrophobic/membrane environments and exhibit low fluorescence in hydrophilic/aqueous environments. By incorporating a protonatable piperazine group on the C4 position of the ND scaffold, CND2 – CND4 possessed pH sensing capabilities, which were demonstrated to monitor intracellular vesicle turnover in neurons. The potential of the CNDs to bind to the lysosomal sterol transport protein NPC2 was investigated by molecular docking and molecular dynamics (MD) simulations. The docking pose with the CND’s aliphatic tail positioned inside the hydrophobic binding pocket was essential for mimicking endogenous cholesterol’s interactions and stabilizing the NPC2-ligand complex. Fluorescence confocal microscopy demonstrated a structure-dependent and cell-dependent intracellular distribution of the CND series in live cells.

Alzheimer’s disease (AD) is a common neurodegenerative disorder. The most recognized disease pathology is the Amyloid-β (Aβ) cascade hypothesis which states that the accumulation of Aβ plaques might be the cause of AD. In the AD brain, Aβ plaques stockpile a variety of molecular components including metals, lipids, nucleic acids, carbohydrates, and peptides, indicating Aβ aggregation might be influenced by these modulators. In this dissertation, we investigated the effects of Zn2+ and carnosine, phospholipids, and β-hairpins on Aβ aggregation to dissect their mechanistic roles in the amyloidogenesis of Aβ. We first systematically studied the kinetic impact of Zn2+ on the aggregation of Aβ40 and Aβ40-M. Our results show that the presence of Zn2+ transforms the Aβ40 aggregation kinetics from a single sigmoidal to a biphasic process, while the aggregation of Aβ40-M is significantly suppressed by Zn2+. We also found that a nature dipeptide, carnosine, remarkably decreases the activity of Zn2+ on modulating Aβ aggregation, although it has a weak direct effect on the peptide aggregation kinetics. Second, we investigated the activities of Aβ40 and Aβ42 in inducing membrane damage and the effects of lipid membranes on the aggregation of these peptides using liposome models containing mitochondrial-specific phospholipid–cardiolipin (CL).

Protein aggregation, oligomer and fibril formation is one of the dominant
characteristics in the pathogenesis of a number of neurodegenerative diseases, such as
Alzheimer’s disease (AD). Inhibition of toxic oligomer and fibril formation is one of
the approaches to find potential drug candidates for AD. Additionally, early diagnosis
of these amyloid species can provide mechanistic understanding of protein aggregation
and thus can pave the way for preventing the onset of AD. The aim of this dissertation
was 1) to explore the effects of charged cholesterol derivatives on the aggregation
kinetic behavior of Amyloid-β40 (Aβ40), 2) to probe Aβ40 oligomer and amyloid
formation in vitro using gold nanoparticles (AuNPs), and 3) to monitor the kinetic
effect of various natural product molecules on Aβ40 aggregation in vitro. In the first
chapter, a general introduction about AD as an amyloidogenic disease, amyloid cascade
hypothesis, and the manipulation of Aβ peptides aggregation kinetics using different
approaches was presented. In the second chapter, we studied the effects of oppositely charged cholesterol derivatives on the aggregation kinetics of Aβ. In the third chapter,
we developed a gold nanoparticles (AuNPs) assay to probe Aβ40 oligomers and
amyloid formation. In chapter IV, we monitored the effects of various small molecules
on the aggregation kinetics of Aβ40. In chapter V, we discussed the methods and
experimental details.