Alzheimer’s Disease (AD) and Traumatic Brain Injuries (TBI) are global societal problems affecting millions of people and costing billions of dollars per year.1,6 Hallmarks of AD include memory loss, cognitive decline, depression, and confusion due to unchecked inflammation in the brain caused by the overproduction of pro-inflammatory cytokines by the immune system.1,9,11 TBI occurs when a sudden trauma damages brain cells, which activate the immune response potentially leading to chronic inflammation and a multitude of symptoms affecting cognitive, somatic, and emotional processes.3,4,12 There is currently no cure for AD, nor is there an effective treatment for chronic inflammation caused by TBI. P2D Bioscience® has manufactured a series of drugs successfully targeting inflammation in a 3XTgAD mouse model.21 To understand the cellular mechanism of the novel drugs, we used SDS-PAGE electrophoresis and Western Blot analysis to investigate protein levels within the NFB pathway, which modulates inflammation. We monitored the inhibitor of NFb, IB, to determine whether the drugs were blocking the phosphorylation and degradation of IkBa and subsequently blocking the pro-inflammatory effects of activated NFB. We show that the drug is blocking the degradation of IB, and therefore the pro-inflammatory genes associated with the NFB pathway are not being transcribed. Increasing our understanding of the cellular mechanism of action is imperative for the progression of drug development because it can be used to evaluate potential side effects.

Alzheimer’s Disease (AD) is a neurodegenerative disease that primarily affects elderly populations. AD engenders memory loss and cognitive decline, and its prevalence is rapidly growing. It is estimated that 14 million Americans will have AD by the year 2050. Therefore, it is imperative for researchers to examine the underlying biological mechanisms responsible for AD. Previous research has demonstrated that chronic inflammation is linked to the hallmark AD pathology, amyloid beta (Aβ). Aβ is a protein that disrupts neuronal communication and increases the production of effector proteins called pro-inflammatory cytokines. Microglia function like immune cells in the brain, and when they are activated by inflammatory triggers, such as Aβ, they secrete pro-inflammatory cytokines. Although cytokine release is initially a healthy response, excess cytokine production is harmful to the brain and exacerbates AD pathologies. Prior research has demonstrated that pro-inflammatory cytokines are upregulated in the serum of AD patients. Therefore, cytokines are a crucial target for AD therapeutics.

The current experiment will examine the temporal inflammatory response of microglial cells following lipopolysaccharide (LPS) insult. LPS is a component of common bacteria and can induce inflammation in microglial cells. We will treat cells with several different concentrations of LPS and assess cytokine production at several different timepoints. To do this, we will collect cell supernatant (secretions) and measure multiple cytokines using an ultrasensitive electrochemiluminscent assay. Data collected from these experiments will be used in many future studies of potential therapeutics and dietary supplements. In fact, data from these experiments will be used by current and future departmental honors students to determine the optimal treatments and times for their experiments. This project is incredibly relevant because AD is currently the 6th leading cause of death in the United States. Data collected will help us pinpoint proper testing procedures for therapeutic compounds that are developed.

Cells use diverse mechanisms to prevent DNA damage and tumor formation. Two tumor suppressors employed in this effort are the focus of our study: breast cancer type 1 susceptibility protein (BRCA1) and BRCA-1-associated RING Domain protein 1 (BARD1). These two proteins form a complex that suppresses the generation of estrogen-derived free radicals. Inherited mutations in BRCA1 or BARD1 are associated with an increased risk of developing breast or ovarian cancer in humans. The model organism Caenorhabditis elegans possesses the orthologs BRC-1 and BRD-1 which can be readily mutated, offering an attractive model to study biochemical functions. However, it is unknown if BRC-1/BRD-1 also regulates the transcription of estrogen metabolism (cyp) genes to control the production of free radicals as noted for the human homologs. Utilizing gene expression analysis and estrogen exposure assays, this study demonstrates that BRC-1/BRD-1 has a conserved function of regulating cyp genes in C. elegans. However, our data also shows that BRC-1 and BRD-1 do not necessarily protect DNA from free radical damage upon estrogen exposure, despite its proven inhibition of cyp genes expression. Further investigation is required to determine the function of these cyp gene homologs in C. elegans. Our findings of this additional conserved function of the BRCA1/BARD1 homologs in C. elegans further validate its use as a model organism to better understand the myriad ways BRCA1/BARD1 protects the genome.

BRCA1 and p53 have been proven to interact in tumor suppressor pathways for hereditary breast and ovarian cancer. Finding the physical binding location associated with this interplay is important in assessing cancer risk and determining molecular details of the interaction. This project aimed to identify the protein binding region of p53 with the intrinsically disordered region of BRCA1. We cloned select regions of human BRCA1 and p53 protein into E. coli bacteria, then harvested and purified the proteins. A pull-down assay was performed to test binding affinity between a segment of p53 and two different length BRCA1 constructs. The assay showed that neither the construct that contained BRCA1 amino acids between 772-1126 nor the construct with amino acids between 896-1190 interacted with p53. This indicates that these amino acids alone are not sufficient for binding of p53 and BRCA1. Our results could also indicate that a third-party binding mediator is required in vivo. This information expands upon our knowledge of the p53 and BRCA1 binding interaction and can be used in a clinical setting to evaluate risk associated with mutations in the experimental regions.

Alzheimer’s Disease (AD) is a progressive neurodegenerative disease associated with old age and marked by deficits in memory and learning skills. AD pathology is characterized by amyloid-beta (AB) accumulation, which leads to plaque formation and ultimately neuronal death. Additionally, AB activates microglial cells, which function as an immune cell in the brain. Microglial cells secrete proteins that induce inflammation, known as pro-inflammatory cytokines. The chronic activation of pro-inflammatory cytokines engenders neuroinflammation and oxidative stress, which then further exacerbates AD pathologies. This project aims to study the effectiveness of cannabidiol (CBD) as a potential treatment for AD, due to its known anti-inflammatory properties. We will measure the inflammatory response of cultured BV2 immortalized mouse microglial cells following lipopolysaccharide (LPS) treatment. We will then include a CBD treatment to study its therapeutic capabilities in reducing inflammation. We hypothesize that treatment with CBD will decrease the pro-inflammatory cytokines TNF-alpha and IL-6 induced by LPS stimulation. We will perform enzyme-linked immunosorbent assays (ELISAs) to detect and quantify the cytokine levels.The overall goal of the research is to demonstrate the capacity of CBD to minimize the immunological mechanisms that drive AD pathologies. Our research will contribute to the understanding of the link between the immune system and central nervous system in AD development. AD is the sixth leading cause of death in America, but the availability of therapies is limited. CBD represents a natural and possible effective therapy for those suffering from Alzheimer’s disease, and our research will contribute to determining its efficacy.