Parkinson’s disease (PD), the second most common neurodegenerative disorder, is characterized by Lewy bodies, pathogenic protein aggregates that include alpha-synuclein oligomers. The missense mutation p.G192R in the RAB39B gene was recently found to cause X-linked dominant PD. Loss of function mutations in RAB39B are associated with X-linked intellectual disability and autism spectrum disorder. RAB39B is a member of the human Rab GTPase family which plays a role in early autophagosome formation and is implicated in intracellular vesicular trafficking. This project investigates how defects in endolysosomal trafficking caused by the p.G192R mutation in RAB39B gene leads to parkinsonism and neurodegeneration. Because RAB39B is highly conserved, we developed a Drosophila model as human RAB39B and Drosophila RAB39 share 75% similarity in amino acid sequence, including 100% identity at p.G192 and flanking amino acids. Using CRISPR/Cas9 genome editing, we created a RAB39G196R Drosophila model that we are currently characterizing for possible neurodegenerative phenotypes. We are examining locomotor deficits and lifespan in RAB39G196R mutant flies compared to isogenic controls, as well as protein aggregation by Western blot. Complementary to the Drosophila model, we developed a human neuronal model by generating induced pluripotent stem cells (iPSCs) from peripheral blood mononuclear cells (PBMC) of an affected male and similar age unaffected male family member kindred with X-linked PD due to the p.G192R mutation. We are investigating endolysosomal trafficking defects in neurons differentiated from iPSCs using antibodies specific for early and late endosomes and lysosomes. We are also examining whether insoluble ubiquitinated protein aggregates and oligomerizes alpha-synuclein are present in RAB39BG192R neurons compared to control neurons. Understanding mechanisms underlying the pathogenesis of X-linked Parkinson’s disease will elucidate the development of PD and potential novel therapeutic targets.

Parkinson's Disease (PD) is a progressive neurodegenerative disease characterized by slowness or stiffness of movement and cognitive impairment. PD is characterized neuropathologically by Lewy Body (LB) aggregates that include lipids, proteins and oligomerized alpha-synuclein. Mutations in the gene glucosidase, beta acid 1 (GBA), are not only the most common genetic risk factor for PD but also accelerate the progression of the disease. We hypothesize that mutations in GBA may mediate faster spread of pathogenic protein aggregation from neuron to neuron. Our previous work has implicated GBA in extracellular vesicle (EV) regulation, suggesting a non-cell autonomous mechanism for GBA accelerating propagation of LBs. To test this hypothesis, we are first exploring how GBA influences EV biogenesis in neurons and astrocytes by examining endolysosomal trafficking in GBA mutated neurons and astrocytes, as well as controls, differentiated from human induced pluripotent stem cells (iPSCs). Our initial results indicate that neurons heterozygous for a GBA null mutation have impaired endolysosomal trafficking with enlarged early endosomes and lysosomes, while astrocytes heterozygous for GBA null do not have impaired early trafficking. These results suggest that GBA mutations differently affect different cell types in the brain and improve our understanding of how GBA influences the spread of LB pathology. I image the iPSC derived neurons and astrocytes using a confocal microscope, for endolysosomal markers and distributions. The goal of this work is to identify novel therapeutic targets for slowing PD progression. 

Dilated cardiomyopathy (DCM) is a leading cause of heart failure around the world. Inherited mutations cause the left ventricle of the heart to enlarge, thinning the heart muscle wall and decreasing the overall function of the heart. In my research project, I will determine if disrupting fibroblast function by knocking out a key developmental signaling factor, p38, can improve, or even reverse, DCM disease characteristics. Specific Aim 1 will be to determine the effects of p38 knockout-induced fibroblast dysfunction on cardiomyocyte function and structural remodeling in late-stage DCM. The rationale is that myocytes in DCM have poor contraction and structurally remodel to longer, thinner morphologies, which occurs in our DCM mouse model around 4 months of age. I expect to see less of these characteristics with the p38 knockout. Specific Aim 2 will assess cardiac fibroblast proliferation and fibrosis in response to disabling cardiac fibroblast function late into the DCM disease process. The rationale is that studying and observing the dynamics of the fibroblast population is critical when understanding the effects of fibroblasts and the p38 knockout model on DCM. In previous studies, the Davis lab identified that cardiac fibroblasts maladaptively respond to inherited DCM mutations in cardiac myocytes, worsening the whole heart. I expect to see less fibroblast proliferation in the p38 model. P38 is essential for fibroblast signaling pathways and functionality, so by knocking it out I will be able to test if fibroblasts are a viable therapeutic target for patients with DCM.

The paradigm for manganese (Mn) cycling in the marine environment has shifted over the past two decades to include not only the +IV and +II oxidation states. It is now recognized that dissolved Mn(III) can also exist when stabilized by organic ligands. Mn is critical for sustaining life and influences the cycling of many other bioactive elements. Because of this, further research is needed for understanding how Mn cycles in the environment, both between physical and chemical phases. My project aimed to look at how Mn cycles between dissolved and particulate phases and its three environmentally relevant oxidation states along the salinity gradient of the Mississippi River delta and what role organic ligands play in mediating Mn transformations. I hypothesized that the salinity gradient would influence the availability of organic ligands, which would promote the oxidation of dissolved Mn(II) and particulate Mn oxides (MnOx), making the cycling of Mn during estuarine mixing more complex than previously understood. To test this, I collected water from the Mississippi River and the Gulf of Mexico to conduct a mixing experiment to model the salinity gradient. UV-Vis spectrophotometry was used to analyze particulate and dissolved Mn speciation as well as the characteristics of the organic matter present. Inductively coupled plasma-mass spectrometry was used in analyzing dissolved Mn and Mn flocculants. Preliminary results show increases of dissolved Mn during mixing, and potential loss of particulate MnOx. Combined, these suggest redox cycling of Mn during estuarine mixing impacts its solubility and ultimately transport to the Gulf of Mexico. This region experiences heavy nutrient loading that leads to seasonal hypoxia. Understanding the cycling and solubility of Mn is imperative because it has broader implications for redox processes and element cycling in the Northern Gulf of Mexico, especially during hypoxic events.

Benzalkonium Chlorides (BACs) are widely used as an antimicrobial disinfectant in a variety of food and consumer goods processing. Exposure to BACs has increased significantly due to the COVID-19 pandemic. BACs have been reported in common foods like fruits, milk, and other dairy products, raising concerns about the impact of BACs on human health via oral exposure. Recent work in our lab has reported that BACs are metabolized by cytochrome P450 (CYPs) 4Fs and 2D6 in the liver. However, there is a gap in knowledge regarding how BACs and BAC metabolites are distributed throughout the body, post-oral exposure. We hypothesize that insight into BAC disposition and distribution following an oral exposure route could lead to valuable knowledge of BAC accumulation and subsequent toxicity. In this study, we exposed male and female C57BL/6 mice to deuterated C12- and C16-BACs at 120 μg/g/day for one week via a gel food diet. We harvested liver, lung, heart, spleen, and intestinal section tissues at the end of the study, as well as fecal samples at two time points, and a singular urine time point. Through a targeted BAC and BAC metabolite quantitation analysis using liquid chromatography-mass spectrometry, we found omega-oxidation of the alkyl chain to carboxylic acid followed by beta-oxidation to be a major route of metabolism. Additionally, we found that the liver and big intestine had a higher metabolizing capacity than other tissues and the C16 BACs were preferentially metabolized compared to the C12 BACs. This work provided a deeper look into the disposition and metabolism of BACs and revealed organs that are susceptible to BAC exposure for future studies

During mitosis, the kinetochore plays a central role in ensuring the proper segregation of chromosomes by connecting them to spindle microtubules, which facilitate the equal distribution of chromosomes to daughter cells. In budding yeast, the Dam1 complex is an essential protein complex that binds the kinetochore to spindle microtubules. The Dam1 complex strengthens the kinetochore-microtubule attachment by self-assembling into a sliding ring around microtubules. This self-assembly occurs at nanomolar concentrations of the complex in the presence of microtubules, but in their absence, appreciable oligomerization occurs at concentrations in the micromolar range. Dimers of the complex predominate in high salt concentrations (500 mM NaCl). This is thought to be due to hydrophobic interactions between the monomers. Yeast strains relying on human histones in place of their yeast histones grow slowly. This slow-growth phenotype is rescued by several different mutations in the Dam1 complex. Preliminary characterization of the mutant Dam1 complexes led 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 purified the wild-type and mutant protein complexes and used size-exclusion chromatography and mass photometry to determine the different quaternary structures that arise at different concentrations of the complex. I found that each mutation that enhances growth of yeast strains with humanized histones decreased the affinity of the Dam1 complex monomers for each other. The results of this investigation yielded a greater understanding of the requirements for accurate chromosome segregation.

Almost every form of cardiac disease is characterized by fibrosis, or the accumulation of collagen, an extracellular matrix (ECM) protein, secreted by the cardiac fibroblast. The buildup of fibrosis is a major clinical burden, as it contributes to diastolic dysfunction, or the heart’s inability to relax, and arrythmias, or an irregular heartbeat. In previous studies, the Davis lab has found that in chronic injury, the heart likely undergoes minor offenses along with periods of rest which accrue over a lifetime. Even when exposed to repeat injury stimuli, the heart is able to recover, and the cardiac fibroblasts can transcriptionally regress. Yet, what remains unclear is when the heart experiences repetitive stress, which is common with hypertension, will these once-activated cardiac fibroblasts have a more aggressive response? And if so, are the activation cues stored in the primed external environment, or are they intrinsic to the cell? To address this, we developed a fibroblast isolation and injection protocol that will ultimately allow us to isolate discrete populations of fibroblasts and study them in hearts void of injury. Our results found that fibroblasts from donor hearts that were subjected to a myocardial infarction injury were detectable at 4 and 14 days post cardiac injection but had little proliferation. However, there was an increase in host fibroblasts recruited to the graft site, many of which were proliferating, and fibrosis was found within these same regions. These results demonstrate that cardiac fibroblasts from the same strain can be isolated and adoptively transferred to other hearts, without exogenous ECM. We can apply this baseline protocol to further examine fibroblast memory in vivo in a model of intermittent hypertension.

The hallmark neuropathological finding of Parkinson’s Disease (PD) is the presence of intraneuronal protein aggregates, consisting of aggregated proteins and misfolded forms of alpha-synuclein. These intraneuronal protein aggregates, known as Lewy bodies, are implicated in many neurodegenerative diseases. Lewy pathology spread in a PD brain correlates with clinical disease progression. Glucosidase, beta, acid (GBA) gene mutations, the strongest genetic risk factor for PD, is also associated with accelerated disease progression and altered extracellular vesicles (EVs). EVs play a crucial role in intercellular communication and delivery of bioactive cargos throughout the central nervous system (CNS). I use a human neuronal cell culture model derived from induced pluripotent stem cells (iPSCs) to examine how GBA mutations alter EV composition, and investigate whether EVs truly act as a vehicle for the seeding of Lewy pathology in other cells, potentially accelerating the propagation of Lewy pathology throughout the CNS. To isolate and purify EVs from the conditioned media of neurons, I use centrifugation and size exclusion chromatography. I visualize and quantify the EV’s size and concentration using a ZetaView nanoparticle analyzer. I perform Western Blot Analysis for candidate cargo proteins within EVs, including alpha-synuclein, ubiquitinated proteins, and EV intrinsic proteins (CD-63 & CD-81). I isolate EVs from the media of GBA PD or WT control neurons expressing alpha-synuclein-GFP fusion protein and apply these EVs to GBA PD or WT neurons. I anticipate that EVs secreted by GBA versus control neurons will contain increased alpha-synuclein protein levels and that increased cell death, endolysosomal trafficking defects, and aggregation of endogenous alpha-synuclein will be associated with the uptake of GBA EVs by recipient neurons. This work will provide evidence supporting the role of GBA in influencing Lewy pathology propagation via EVs, which could elucidate a novel therapeutic mechanism that could be targeted to slow the progression of neurodegeneration.