Department of Biomedical Science

Amyloid beta (Aβ), a byproduct of amyloid precursor protein, is constantly cleared from the CNS. Aβ kinetics are visualized using 18F-florbetapir PET imaging, typically analyzed through 2D coregistration with MRI or CT followed by visual evaluation. Aβ was thought to coexist in the white matter of both Alzheimer’s disease (AD; Aβ+) patients and cognitively unimpaired (CU; Aβ-) individuals. However, this coexistence is likely a misperception of 2D imaging. In this study, data science techniques were used to evaluate PET images, transforming Aβ imaging into topographical pixel arrays for 3D reconstruction. Canal-like networks in the brain, skull, and neck were discovered to be part of the non-CNS fluid (NCF) compartment, which quarantines Aβ. In CU/Aβ- subjects, Aβ is transported to peripheral lymphatics. In AD/Aβ+ subjects, Aβ becomes congested in the NCF, diffusing into CNS interstitial fluid, leading to progression and neurodegeneration.

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.

There has been substantial progress in cancer research that has markedly enhanced patient outcomes. However, chemotherapy resistance persists and often leads to multidrug resistance, rendering cancer cells unresponsive to multiple chemotherapy drugs, presenting a significant challenge in the effective treatment of the disease. Dysregulation in gene expression patterns caused by abnormalities in epigenetic mechanisms have been identified as contributing factors to the development and progression of cancer. Epigenetic research offers potential to discover drugs that target specific epigenetic modifications to regulate gene expression patterns in the context of chemotherapy resistance. I hypothesize that histone modifications on histone H3 and histone H4 contribute to doxorubicin resistance. The data presented here provides an initial screening of the mutant monoallelic histone yeast strains to identify post-translationally modifiable amino acids in H3 and H4 that could contribute to doxorubicin resistance. The possible targets of histone modifications were then repeated in triplicate to obtain statistical significance. Finally, Western blot techniques were used to identify the modification occurring on the histone H3 and histone H4 amino acid sites that were previously identified to be statistically significant.

Cardiovascular disease is a broad term that encompasses a variety of disorders in which the heart and its associated blood vessels lose the capacity to deliver blood efficiently and effectively throughout the body. Cardiac endothelial cells play a vital role in maintaining the homeostatic balance of cardiac physiology. Research into c-Myc, a master regulator involved in the transcription of a large set of genes that regulate inflammation, has been the focus of new therapeutics aimed at treating or lessening the deleterious effects of cardiovascular disease. This project serves to explore how endothelial loss of c-Myc impacts cardiac function under normal and stress conditions, using ultrasound echocardiography image analysis to determine the key differences between all models.

The striatum, a region of the brain responsible for motor control and reward processing, plays a critical role in various neurological disorders, including Parkinson's disease, Huntington's disease, and addiction. Gnal encodes the heterotrimeric G-protein stimulatory alpha subunit, Gαolf. Gαolf is highly expressed in the striatum, a brain region that is highly relevant to psychosis and psychostimulant drug action. The Gγ7 protein is also enriched in the striatum, where we have previously shown that Gγ7 protein is required at the posttranscriptional level for the hierarchical assembly of the striatal-specific Gαo lfβ2γ7 heterotrimer, which represents the rate-limiting step for cAMP production in striatal D1R and D2R-expressing neurons in the D1 dopamine and A2a adenosine pathways.
Multiple transcripts with variable 3’ UTRs are produced from the Gng7 gene. Previous studies have shown that genes with these characteristics are post-transcriptionally regulated and can be subcellularly localized. Thus, we hypothesized that the γ7 transcripts with variable 3’UTRs act as signaling organizers that regulate the abundance and/or subcellular localization required for preferential assembly and specialized signaling by Golf heterotrimer in the brain. Our findings showed that striatal-enriched γ7 transcripts are post-transcriptionally regulated by virtue of regulatory elements outside of the coding region that bind to its long 3’UTR. These regulatory elements are responsible for translational repression of the γ7 protein. The different length 3’UTRs of the γ7 transcripts 1 and 3 allow for subcellar localization in the nuclei and the neuropil respectively.

Alzheimer’s disease (AD) is one of the most common neurodegenerative diseases affecting an estimated 20 million worldwide. The primary pathology of AD is the progressive loss of basal forebrain cholinergic neurons, which is responsible for the cognitive decline experienced by AD patients. The mechanisms underlying this selective vulnerability have not been fully elucidated. Furthermore, oxidative stress is a key factor behind the pathology of AD leading to this neuronal loss. The current literature suggests that there are limited in-vitro models available to accurately simulate the hallmark symptoms of Alzheimer's disease (AD). The SH-SY5Y cell line has been used extensively to study neuronal stress responses but the undifferentiated cell type has been predominantly used. Undifferentiated SH-SY5Y versus differentiated SH-SY5Y have been shown to have different interaction, expression and localization with AD hallmark, amyloid-b -42. This project sought to use differentiated cholinergic cells from the line SH-SY5Y to further isolate and elucidate, in-vitro, the mechanisms behind the oxidative stress response, a key stressor in the pathology of AD. Building upon previous studies, a protocol to differentiate SH-SY5Y cells with retinoic acid (RA) and neurotrophin (BDNF) to mature neurons of the cholinergic phenotype was optimized and implemented. The results showed successful differentiation into the cholinergic phenotype as evidenced via immunofluorescence imaging of choline acetyl transferase (ChAT) expression and mature neurite morphology. To simulate oxidative stress, we exposed both undifferentiated and differentiated SH-SY5Y cells to hypoxic conditions. Results indicated a stress response to mild hypoxic conditions with higher sensitivity in cholinergic differentiated SH-SY5Y. Understanding these hallmark mechanisms behind oxidative stress is crucial to developing mechanism-based therapeutics for AD.

Kleine-Levin Syndrome (KLS) is an extremely rare neurological disorder characterized by episodes of uncontrollable hypersomnia and various cognitive and behavioral abnormalities. There is neither a definitive etiology nor definite treatment modalities. Immunological studies for this condition are extremely limited, and this present study aims to investigate a potential autoimmune mechanism that underlies KLS. To achieve this, western blot and dot-blot assays analyzed the immunoreactivity of patients and control sera towards various brain tissue areas. Western blot did not show immunoreactivity with IgG-depleted brain tissue lysate. However, dot-blot assays revealed a significantly greater level of immunoreactivity with KLS patient sera towards the dorsolateral prefrontal cortex, hypothalamus, and parieto-temporal areas compared to KLS-negative sera. These areas have previously been shown to be hypo-perfused in KLS patients. Future studies are necessary to identify the specific antibodies that may be autoreactive in KLS patients.

Epigenetic dysregulation has been implicated in oncogenesis, with post-translational histone modifications being linked to cancer progression. WSTF/BAZ1B forms chromatin-remodeling complexes with other proteins and lowers cancer survival outcomes. Treatment resistance causes >90 % of all cancer deaths. In particular, cancers develop tolerance to cisplatin-induced genotoxicity. It is hypothesized that the BAZ1B bromodomain, PHD finger, and DDT domain recognize epigenetic modifications, contributing to cisplatin resistance in cancers. To test this, the domains were expressed in Rosetta 2 BL21(DE3) and Rosetta 2 BL21(DE3) PLysS Escherichia coli strains. Soluble proteins were extracted, purified, and then analyzed using pulldown assays and modified histone peptide arrays. The DDT and PHD finger domains were found to bind to specific histone modifications with the DDT domain also displayed DNA-binding properties. Some of the identified histone modifications have known roles/correlations in normal and cancer cells, implicating BAZ1B as an agent in oncogenesis, treatment resistance, and as a therapeutic target.

During eye lens development the lens receives oxygen from a network of capillaries that comprise of the tunica vasculosa lentis and the anterior pupillary membrane. In development there is regression of this capillaries with the vitreous and aqueous humor, which is the lens only source of oxygen, leaving the lens in low oxygen state. The lens contains a decreasing oxygen gradient from the surface to the core that parallels the differentiation of immature surface epithelial cells into mature core transparent fiber cells. These properties of the lens suggest a potential role for hypoxia and the master regulator of the hypoxic response, hypoxia-inducible transcription factor 1 alpha (HIF1a), in the regulation of genes required for lens fiber cell differentiation, structure, and transparency. Previous studies by our lab discovered the HIF1a-dependent gene expression patterns of lens genes by utilizing a Multiomics approach that integrated analysis from CUT&RUN, RNA-seq, and ATACseq. Additionally, our lab also established a hypoxia and HIF1a-dependent mechanism for the non-nuclear organelle degradation process required to form mature transparent fiber cells.

Lens differentiation begins with epithelial cells that undergo the process of cellular differentiation and remodelling into fiber cells (Bassnet et al., 2011; Menko 2002; Wride, 2011) that then will undergo terminal remodelling processes to eliminate their cellular organelles to achieve mature lens structure and transparency. We sought to determine if Serine 81, within the minimal essential region (MER) of the BNIP3L protein, is required for organelle elimination. Previous studies have shown that levels of phosphorylated P38 MAPK and ERK ½ peaked in the same region as phosphorylated S81 BNIP3L levels, the equatorial epithelium, where organelle degradation is initiated. The use of specific inhibitors of P38 MAPK (SB203580) or ERK ½ (U0126 or PD99089) and P38 MAPK activator Ansiomycin will be used to determine if P38 MAPK or ERK ½ phosphorylates BNIP3L at S81 to induce mitophagy of mitochondria, endoplasmic reticulum, and Golgi apparatus.