Glioblastoma multiforme (GBM) is the most common and highly malignant brain cancer diagnosed in adults. Due to its high invasiveness and difficulty of surgical removal, there is a need for novel therapeutics to treat this disease. Recent evidence suggests that cannabidiol (CBD), a non-psychoactive compound from the Cannabis plant, possesses antitumor properties in a number of cancers. However, the mechanism by which CBD acts to decrease tumor viability and proliferation is unclear. To better understand the antitumor properties of CBD, we tested this compound against standard of care chemotherapy drugs BCNU, Temozolomide, and Cisplatin in human GBM cell lines T98G, U87MG, and U251. Using the water soluble tetra-zolium salt (WST-1) reagent and bromodeoxyuridine (BrDU) assay in vitro, we assessed the dose response of CBD on GBM cell viability and proliferation, respectively. Using the same assays, we also investigated the effects of CBD in combination with standard of care chemotherapy. We found that CBD dose-dependently (IC50 = 3.1µM) suppressed viability and proliferation in all GBM cell lines tested, and that this response was more potent than standard of care chemotherapy. In addition, CBD exhibited additive effects when combined with standard of care chemotherapy in vitro. Implications for this research include development of novel cannabinoid-based cancer therapeutics.

One of the leading causes of depression is stress, and yet not enough is known about the exact mechanism by which stress alters depression. Of the four opioid receptor systems (mu, delta, kappa, and opioid receptor like-1), the kappa opioid receptor (KOR) is what my research focuses on. KOR is activated by its endogenous ligand, dynorphin. This is responsible for promoting analgesia, and depression-like phenotypes such as swim-stress immobility. The mammalian target of rapamycin (mTOR) is a protein kinase involved in regulation of functions such as cell growth, and metabolism. When dysregulated, it may lead to obesity, depression or cancer. Both mTOR and KOR have been shown to independently regulate stress-induced depression. However, there are no studies which tie these two ideas together. One possible link could be the stimulation of p38 MAPK kinase. Stimulation of KOR causes G-protein coupled receptor kinase (GRK)-dependent activation of the p38. Additionally, it is known that p38 causes the inhibition of TSC2, promoting phosphorylation of mTOR. Based on our results, activation of KOR with U50,488 causes an increase in phosphorylation in mTOR in vitro. However, this is also present in negative control cells not expressing KOR, which could indicate that U50,488 also has an off-target effect on a non-KOR mediated pathway. A similar treatment on mice decreases phosphorylation of mTOR after 28 hours in the brain, indicating that after a stressful event, mTOR activation is transient and acute. Furthermore, decreased mTOR phosphorylation after 28 hours in the brain was found to be blocked in GRK3 knockout mice. This implies that decreased mTOR phosphorylation after 28 hours with U50,488, requires the GRK gene. Further experiments, including behavioral testing, will be needed to investigate a deeper relationship between KOR and mTOR. With further research, these studies may lead to developing novel anti-depressants, revolutionizing depression treatment.

The serotonin receptor subtype 5-HT6, a G protein-coupled receptor (GPCR), is a target of pharmacological agents that has been shown to have promising therapies to enhance cognitive function. Unfortunately, the mechanism by which drugs that activate 5-HT6 receptor signaling activity has on neuronal function is not well understood. Activation of 5-HT6 receptor activity induces a response in both canonical (ACIII) and non-canonical signaling pathways (Cdk5). Interestingly, endogenous 5-HT6 receptors are found solely on the neuronal primary cilia, a small organelle projecting from the cell body like an antenna. Primary cilia dysfunction has been correlated with several brain disorders, such as Meckel Syndrome and Joubert Syndrome. This study examines the role of the 5-HT6 receptors in the primary cilia in order to gain a better understanding of the receptor’s importance in neuronal morphology and cognitive processes. Using primary neuronal cultures from 5-HT6 receptor knockout mice, we rescued expression of 5-HT6 receptors using transgenic expression of wildtype receptors and mutant receptors deficient in a specific signaling pathway (Gs-DEAD-5-HT6 and Fyn/Cdk5-DEAD-5HT6). After the 5-HT6 receptor expression was restored, immunohistochemistry was used to determine the effect of rescuing 5-HT6 receptor expression on cilia morphology. Expression of a fluorescently-tagged dendritic arbor (Map2B-RFP) was used to measure the effect 5-HT6 receptor expression has on dendritic arborization. Preliminary results show that rescue of 5-HT6 receptor expression enhances dendritic outgrowth, and that this increase is dependent on Fyn-signaling pathway. Cilia length was only mildly affected by rescue of 5-HT6 receptor expression, and the 5-HT6 mutants only mildly affected cilia length.

Voltage-gated calcium channels are vital to electrical signaling in the human body, and their defects are associated with various neurological and cardiovascular disorders. Finding the 3-D molecular structure of these channels allows us to understand the physical basis for their function of selectively allowing calcium ions through the cell membrane. The Catterall lab uses X-ray crystallography, a technique in which a protein is grown into crystals and then diffracted with X-rays, to study the structure of Ca_vAb, a voltage-gated calcium channel from the bacterium Arcobacter butzleri. However, crystals of Ca_vAb are currently weak and give structures with relatively low resolution. This makes it difficult to see details in the protein’s structure and limits these structures' usefulness. To this end, I have introduced a zinc-binding site into Ca_vAb that increases the strength of the protein-protein interactions that cause it to crystallize. I anticipate that crystals of this altered form of Ca_vAb, when grown in the presence of zinc ions, will incorporate zinc into the introduced binding site, strengthening the crystal. Current results show some evidence for the inclusion of this zinc-binding site in crystals of Ca_vAb. The crystals show X-ray absorption and electron density within the binding site that indicate the presence of zinc. While further studies are needed to completely elucidate the effect of this zinc-binding site, current results are promising. In the future, we hope to take advantage of the anticipated crystal quality from this zinc-binding site for further structural studies on Ca_vAb. For instance, greater resolution will allow our lab to fully determine the mechanism of action by which calcium channel blocker drugs bind to a voltage-gated calcium channel. Furthermore, similar introduced metal-binding sites could be applied to the crystallization of other proteins that have so far evaded structure determination by crystallography.

A neuropeptide called corticotropin-releasing factor (CRF) is shown to be necessary in fear behavior, but it is currently unknown whether upregulated CRF expression in the central amygdala is sufficient for enhancing fear behavior. An increase in CRF expression in a controlled manner will specifically test CRF's ability to enhance fear learning. To achieve this, a Cas9-CIBN complex will be directed towards CRF promoter by small-guiding RNAs where it will recruit the Cry-Vp64 complex. When these two complexes come together, they form a light-inducible transcriptional effector (LITE) that can recruit the transcriptional machinery to the CRF promoter and up-regulate CRF expression in the presence of light. LITE machinery will also provide a method of improved spatial and temporal control to the upregulation compared to currently used methods of upregulation. However, in order to accurately analyze this uniquely controlled method, certain comparable controls are required. This project will serve as the comparison to temporally controlled CRF expression by creating a constitutive CRF overexpression vector. This will upregulate CRF without temporal control and cannot be switched “off”. This continuous effect may elicit compensatory changes to the system over time and reveal important differences between constitutive and temporally controlled CRF expression. The three objectives of this project are to 1) clone the CRF gene into a DNA vector called adeno-associated virus vector (AAV vector), 2) package the AAV-CRF vector into virus, and 3) inject the packaged virus into central amygdala for behavioral analysis. The first objective has been achieved using a standard cloning protocol. The second and third objectives are still in the preliminary phase.

Protein quality control degradation systems allow the cell to maintain normal function by eliminating anomalous proteins. The attachment of the small protein ubiquitin to lysine residues in other proteins is important for their proteasome-mediated degradation. Ubiquitin is attached to the lysines of proteins by the action of ubiquitin ligases. However, if ubiquitin ligases spuriously add ubiquitin to themselves, they could also be destroyed by the proteasome. Therefore, it is important that ubiquitin ligases have the means of preventing autoubiquitination. One particular ubiquitin ligase (San1) is involved in the degradation of misfolded proteins in the nucleus of yeast cells. We previously found that San1 prevents autoubiquitination from occurring by not having lysine residues in the N-terminal and C-terminal region of the protein. However when lysine residues are introduced to the N- and C-terminal regions of San1, it undergoes autoubiquitination, both within itself (in cis) and by other San1 ligases (in trans). The introduction of lysine residues also causes degradation of San1 by an additional protein. The premise of my experiments is to identify the novel form of degradation as well as the protein(s) involved. Retained stability of San1 within cells with blocked proteasomes indicates that the mechanism is proteasome dependent. Additionally, degradation of San1 in cells containing temperature dependent ubiquitin indicates that the mechanism of degradation is not ubiquitin dependent. Ubiquitin-like modifiers are now being knocked down to determine whether the novel mechanism may utilize them instead. The potential for uncovering a novel protein quality control mechanism relates to many neurodegenerative diseases such as Alzheimer's and Lou Gehrig's which all correlate strongly with high levels of misfolded protein aggregates.

As a consequence of unhealthy lifestyles, the prevalence of hypertensive heart disease is rising both in the United States and globally. Hypertension often results from the chronic activation of adrenergic receptors found in the walls of blood vessels. These receptors are normally activated in times of extreme stress by the sympathetic nervous system. However, chronic activation leads to elevated blood pressure which puts extra stress on the heart and can lead to complications that kill millions of people annually. Drugs such as beta-blockers treat this disease by blocking the endogenous ligand, epinephrine, from binding to the receptor, which relaxes the blood vessels and lowers blood pressure. However, this class of drugs ignores another important receptor found in vessel walls, the alpha-1D adrenergic receptor (ADRA1D). ADRA1D is known to contribute to hypertension and benign prostatic hypertrophy in humans, but its mechanism of action is poorly studied. The Hague Lab conducts pharmacological studies on ADRA1D to elucidate how this receptor confers disease. My project focuses on understanding how the receptor localizes to the surface of the cell where it can be activated. Recently, our lab conducted proteomic screenings to identify intracellular binding partners. The results showed that ADRA1D binds to two unique proteins, a signaling protein, alpha-syntrophin (SNTA1), and a cellular polarization protein, Scrbble (SCRIB). This interaction was later attributed to a conserved binding region known as a PDZ domain that interacts with the C-terminus of ADRA1D. SNTA1 has one of these domains while SCRIB has four. My project attempts to quantify the strength of these interactions to give a better understanding of ADRA1D’s intracellular landscape. By better understanding how this receptor locates to the surface and how it signals, drugs can be created to disrupt these processes and better treat patients with hypertensive heart disease.