Understanding the cell biology of neurodegenerative risk factors using yeast and human neural cells (ongoing):
Various genome-wide association studies have identified a single nucleotide polymorphisms (SNPs) that correlate with increased neurodegnerative disease risk and more severe progression. The functional effects of many of these SNPs remains unknown. My postdoctoral work has taken an unconventional approach (pioneered in the Lindquist lab) to investigating the role of Alzheimer's disease (AD) risk SNPs; I have used a model organism, baker’s yeast (the same yeast used to make bread) to generate hypotheses on how Alzehimer's disease risk factors disrupt cell biology. This unusual approach relies on the observation that at a basic level, the inner machinery of human cells and yeast cells are very similar, and therefore, coding SNPs in these risk genes disrupt the same processes in both cell types. Most importantly, the tools currently available to genetically study yeast are much easier to work with than those available for studying human cells, especially complex human neural cell types. Previous work from the Lindquist lab and other groups has demonstrated that yeast screens can serve as great tools for hypothesis generation and mechanistic investigation of neurodegeneration-associated proteinopathies. However, yeast do not fully capture the complexity present in the diversified cell types of the human brain. Therefore, all hypotheses generated in yeast need to be tested in these complex cell types.
To accomplish this objective, I am working at the Tsai lab (MIT) to use induced pluripotent stem cell (iPSC)-derived neural cell types. Using genome editing technologies, isogenic sets of patient-derived iPSCs can be generated (that only differ in at the disease-associated risk SNP). We then use cell biology assays to test whether and how pathways perturbed by AD risk SNPs in yeast are affected in various iPSC-derived neural cell types (neurons, astrocytes, microglia).
Using single-molecule spectroscopy to probe protein aggregation (graduate work):
The aberrant aggregation of proteins into small toxic oligomers is a hallmark of many neurodegnerative diseases. Although they have been identified as the toxic species, they have been incredibly difficult to characterize due to their hetereogeneity in size and structure. My graduate work focused on accurately characterizing these oligomers and using these well-characterized preparations to understand the molecular origins of their cytotoxic effects. I applied a single-molecule fluorescence technique pioneered in the Klenerman lab and in collaboration with the Dobson Lab in Cambridge called two-color coincidence microscopy that allows the tracking of complexes to characterize the aggregation kinetics and themodynamics of oligomers of the amyloid-beta peptide (centrally involved in Alzheimer's disease pathogenesis). Using these well-characterized preparations of oligomeric species, I then used total internal reflection microscopy (TIRF), cell biology assays, and electrophysiological assays to investigate the mechanism of oligomer-induced cytotoxicity using human neuroblastoma cells, mouse hippocampal cultures, and hippocampal slices. I also investigated the effects of the extracellular chaperone (and AD risk factor) clusterin on all of the aforementioned assays. From these investigations, I identified that clusterin binds strongly to amyloid-beta oligomers and thereby inhibits the oligomers' cytotoxic effects. Much of this work would not have been possible without the unwavering support of collaborators at the Wilson lab (Wollongong), Wood lab (UCL), Cho lab (Bristol), and the St. George-Hyslop lab (Cambridge).