Mutations in the gene glucosidase, beta acid 1 (GBA) are not only the strongest genetic risk factor for Parkinson’s Disease (PD), but also accelerate the progression of PD. We hypothesize that GBA mutations accelerate disease progression by promoting propagation of Lewy pathology from cell to cell via dysregulated extracellular vesicles (EVs). To investigate this, we developed a Drosophila model of GBA deficiency (GBA^del) manifesting neurodegeneration and accelerated protein aggregation. We also developed a human neuronal model by generating human induced pluripotent stem cells (iPSCs) from an individual with PD heterozygous for a null GBA mutation (GBA^IVS PD). Neurons were differentiated from GBA^IVS PD iPSCs, isogenic GBA^WT PD iPSCs, and iPSCs from an age- and sex-matched healthy control. I performed immunocytochemistry and western blots to evaluate protein aggregation within neurons. Additionally, I isolated neuronal EVs by size exclusion chromatography and analyzed them using a ZetaView nanoparticle analyzer. We previously found the expression of wildtype GBA in muscles of GBA^del mutant flies rescued levels of protein aggregation in the brain. This non-cell autonomous rescue was accompanied by normalization of alterations observed in EVs from GBA^del flies. Similar to our fly model, I found human GBA^IVS PD neurons and EVs have increased ubiquitinated proteins when compared to GBA^WT PD or healthy control neurons and EVs collected from these neurons. Our results suggest that GBA deficiency mediates PD pathogenesis by accelerating propagation of pathogenic protein aggregation through the alteration of EV protein cargo. We are now further investigating how GBA influences endolysosomal trafficking and EV biogenesis and I will now test whether GBA^IVS PD EVs can propagate protein aggregation faster in recipient neurons than control EVs. Understanding mechanisms regulating the spread of protein aggregates could reveal novel therapeutic targets to slow the rate of progression of neurodegeneration.

Hypertrophic cardiomyopathy is a disease affecting millions of people worldwide, characterized by thickened heart tissue, which makes pumping difficult for the heart. Since the restructuring of the heart is influenced by remodeling at the cellular level, the overall goal of the project is to investigate the mechanics of how cells sense tension in their environment and its relationship to regulating cell remodeling. The direction in which sarcomeres, the basic contractile unit in the heart, are added influences the shape of the cells and consequently the shape of the overall heart. To study this relationship, it is necessary to visualize the sarcomeres for correlating acquired tension data from FRET sensors. By attaching a fluorophore to a sarcomeric protein such as alpha-actinin, it is possible to visualize sarcomeres using fluorescent microscopy. For this project, I developed a blue fluorescent protein (BFP)-tagged alpha-actinin plasmid through molecular cloning techniques. I used restriction enzymes and PCR to isolate and amplify the genes of interest from a different plasmid, then used Gibson Assembly to insert the genes into a plasmid containing ampicillin resistance to construct the final BPF-tagged alpha-actinin plasmid. Preliminary results showed successful expression of the transiently transfected BFP construct in cardiomyocytes. The next steps are to optimize the transfection for higher efficiency, adapt an existing data analysis pipeline for analyzing sarcomere dynamics, and develop a set of parameters for efficient image acquisition. Many existing therapies for hypertrophic cardiomyopathy only address symptoms but do not solve the underlying issue of systolic dysfunction. Rather than taking a genetic or biochemical approach, which can be difficult to develop, this research project focuses on the mechanical interactions in the heart and studying the contractile forces may yield more insight into this disease and build the informational foundation for developing future therapies to prevent or treat hypertrophic cardiomyopathy.

The kinetochore is a protein complex responsible for transmitting force between microtubules and the centromeric region of DNA on chromosomes. The OA and Mif2 protein subunits of the kinetochore make direct attachments to the chromosome. They both bind to another subunit called MIND, which forms a bridge to the microtubule-binding elements of the kinetochore. Preliminary research shows that OA and Mif2 exhibit different binding preferences for the MIND complex. OA binds constitutively, regardless of whether MIND is in an open or closed conformation, whereas Mif2 strongly prefers the open conformation. However, the binding affinities for both OA and Mif2 for MIND have not yet been measured. This study developed the experimental methods for performing immunoprecipitation assays with OA, Mif2, and MIND. By performing phosphomimetic and truncation mutations to promote the MIND open conformation, the binding affinities of OA and Mif2 will be quantified. It is important to quantify the subunit binding affinities to gain a deeper understanding of kinetochore assembly and force transmission between microtubules and chromosomes. Ultimately, this research has applications in cellular division and cancer research.

During mitosis, the kinetochore plays a central role in ensuring the proper segregation of chromosomes in the parent cell. It does so by forming attachments to spindle microtubules, which facilitate the equal distribution of chromosomes to daughter cells. In budding yeast, the Dam1 and Ndc80 complexes are essential protein complexes that bind the kinetochore to spindle microtubules. The Ndc80 complex functions as the direct contact between the kinetochore and the dynamic microtubule tip and it is required for the Dam1 complex to associate with kinetochores. The Dam1 complex strengthens the kinetochore-microtubule attachment. In the presence of microtubules, Dam1complex oligomerizes into a sliding ring. This self assembly has been observed to occur with nanomolar concentrations of the complex in the presence of microtubules but in the absence of microtubules, appreciable oligomerization occurs at concentrations of the complex in the micromolar range. Dimers of the complex appear to predominate in high salt concentrations (500 mM NaCl) in comparison to monomers. This is thought to be due to electrostatic interactions between the monomers. When yeast histones were swapped for human histones, several mutations occurred in the Dam1 complex, and one mutation in the Ndc80 complex, that rescued the yeast cells from defects in mitosis. Preliminary characterization of the mutant Dam1 complexes lead to the hypothesis that the mutations that allow the yeast cells to adapt to the humanized histones changed the monomer-dimer equilibrium for the Dam1 complex. To measure the affinity of Dam1 complex monomers for each other, I will purify the protein complex and use size-exclusion chromatography and western blotting to quantify the relative abundance of the monomer and dimer at different concentrations of the complex. This will contribute to a greater understanding of mitosis and in turn cancer because it focuses on the dynamics that control proper chromosome segregation in cells.