Substance abuse leads to alterations in cognition that affects processes such as impulse control and valuation. Decreased impulse control and aberrant valuation are responsible for continued drug seeking and are thought to be escalated by external stress stimuli. Stress leads to release of an endogenous opioid neuropeptide called dynorphin which binds to the Kappa Opioid Receptor (KOR). Upon KOR binding, dynorphin induces a protein signaling cascade that also promotes drug seeking behavior. In this study, we investigated the dynorphin/KOR system in the medial prefrontal cortex (mPFC) due to its critical role in cognition. We examined the properties of dynorphin release in the mPFC of C57BL/6 mice in response to different external stressors to determine if this nucleus is a potential therapeutic target for stress-induced drug seeking behaviors. Using a pharmacological approach, we first showed that systemic administration of U50,488, a KOR agonist, leads to KOR activation in the mPFC. U50,488 administration also disrupted cognition by impairing performance in a working memory behavioral task. We next tested whether different stress modalities stimulated mPFC dynorphin release and disrupted cognitive performance. Surprisingly, repeated forced swim stress did not cause dynorphin release in the mPFC and did not disrupt cognitive performance although it did activate dynorphin release in the Dorsal Raphe nucleus, as expected. In contrast, different stressors, including repeated foot shock and precipitated morphine withdrawal did effectively lead to KOR activation in the mPFC. This indicates that dynorphin release in the mPFC is dependent on the type of behavioral stress. Future experiments will utilize an in-vivo dynorphin sensor, kLight, to detect dynorphin release in real-time in response to these stressors. Exploration of the differences in dynorphin/KOR system functioning in response to different stress modalities is important for establishing how this system may be targeted to alleviate stress-induced drug seeking behaviors.

Dopamine (DA) neurons found in the ventral tegmental area (VTA) are associated with reward feedback, and dysfunction in DA circuitry is associated with disorders such as Parkinson’s, schizophrenia, bipolar, and addiction to drugs. To adequately treat these diseases, we must have a more complete understanding of how dopamine contributes to emotional processes. This research project addresses this issue by investigating neuropeptide regulation of dopamine neurons in the VTA. The bed nucleus of the stria terminalis (BNST) is a brain region that expresses many neuropeptide genes and sends strong projections to the VTA. We utilized Cre driver lines to isolate neurons that produce the peptides Neurotensin, Neurokinin B, and Corticotropin Releasing Factor. We injected a virus into the BNST that induces the expression of a light activated ion channel and allows us to stimulate axon terminals in the VTA. I then conducted behavioral experiments to assess the effects of activating these peptidergic inputs. Dopamine-dependent behaviors relating to pleasure, reward, and anxiety were measured through the behavioral tests of Real Time Place Preference, operant conditioning, and Open Field respectively. Most likely due to low expression, my behavioral analyses did not yield statistically significant results. Moving forward, it may be necessary to increase viral titer for wider expression. In the future, I intend to use CRISPR/Cas9 technology to isolate neuropeptide function from fast neurotransmitter release in these circuits. This research, by producing findings that help explain how neuropeptides modulate DA neurons, has the potential to generate advances for the understanding and treatment of dopamine-related psychiatric disorders.

When all goes according to plan, newly synthesized proteins within cells fold down an energetic funnel into a functional, minimal energy configuration. If a protein does not fold properly, it is both energetically unfavorable and nonfunctional, often with hydrophobic parts exposed to the aqueous environment. This creates the potential for misfolded proteins to form insoluble aggregates, which are toxic to cells. These aggregates crowd the cellular environment and impair cellular functions, which on a single cell scale, leads to cell death, and on a larger organism scale, leads to diseases like Alzheimer's, Parkinson's, and Huntington's. To deal with this problem, cells have protein quality control (PQC) systems. PQC is composed of two classes of enzymes: chaperones that help proteins to fold properly and ubiquitin-protein ligases that tag misfolded proteins with ubiquitin, leading to degradation in the proteasome. Previous studies concluded that chaperones are required for protein degradation. In this study, we investigated yeast ubiquitin-protein ligase San1, which often requires chaperones, but can recognize substrates independently. The ubiquitin-protein ligase San1 recognizes patches of hydrophobicity on misfolded proteins and is able to accurately tag them for destruction in the proteasome. Primarily through the use of cycloheximide-chase degradation assays and fluorescent microscopy, I worked within our team and found that Hsp70 chaperone dependence is variable along a spectrum of independent to dependent. By studying the interactions of the folding and degradation enzymes, our lab is gaining new insights into the coordination of PQC pathways

Liver cancer is the world's second most deadly malignancy with a five-year survival rate of just 18%. Hepatocellular Carcinoma (HCC) accounts for most of the cases and its incidence is projected to rise to one million deaths per year by 2030. Unfortunately, HCC is extremely difficult to treat because several molecular pathways that promote drug resistance are upregulated in tumors, the most important of these being the epithelial-to-mesenchymal transition (EMT). Under physiological conditions the EMT regulates embryonic development, wound healing and tissue repair. However, cancer cells can hijack EMT signaling pathways to acquire a metastatic and drug resistant phenotype. Therefore, cell signaling enzymes that promote the cancer cell EMT are an attractive target for pharmacological intervention. Recently, we discovered that 71 protein kinases are highly enriched in mesenchymal HCC cells. To determine if these signaling enzymes are bona fide drivers of the EMT and drug resistance, we generated kinase RNAi knockout cell lines, quantified differences in EMT marker mRNA expression by qPCR, determined EMT pathway activation using quantitative proteomics, and tested differences in drug sensitivity. Here we demonstrate that inhibition of several of the 71 candidate EMT kinases reverses the phenotypic transition and sensitizes drug resistant HCC cells to chemotherapy. We conclude that these kinases could present attractive targets for the development of novel drugs that block cancer metastasis and overcome therapy resistance.

Current estimates by the centers for disease control (CDC) suggests that roughly 1 in 59 children in the US are affected by Autism Spectrum disorder (ASD). Despite its high prevalence, our current understanding on the biological etiology of ASD is limited. However, an ASD associated gene, TAOK2, which encodes a serine/threonine protein kinase, has been found to mediate neurodevelopmental disorders like ASD. Mutations within TAOK2 are associated with atypical neural connectivity in different brain regions, abnormal synapse formation, reduced cortical layering, and increases in brain size. Though TAOK2 plays a crucial role in neuronal development through phosphorylation of its substrate proteins, catalyzing or inhibiting their activity to regulate cellular processes, substrates of TAOK2 have not been fully elucidated and several candidate substrates have yet to be explored. Through my research, I explore TAOK2's interaction with putative substrate HDAC6, a histone deacetylase enzyme that functions to mediate a variety of cellular processes such as protein degradation, transcription, and the ability to regulate α-tubulin to mediate microtubule dependent cell motility. HDAC6 has been identified as a putative substrate of TAOK2 through mass spectrometry. Here, I present my findings regarding TAOK2's regulation of HDAC6 deacetylase activity and the potential it has for expanding our understanding of the molecular mechanisms underlying ASD.

The dorsomedial striatum (DMS) is a brain region that functions in mediating goal-directed action but the cellular, molecular, and circuit-level substrates controlling this remain unknown. Interestingly, the kappa opioid receptor (KOR) and its ligand dynorphin (dyn) are present in about half the cells in the DMS. KOR is a G protein-coupled receptor known to have dysphoric effects through dyn. Given the effects of KOR in the DMS are relatively unknown, we investigated the role of the dyn/KOR system on goal-directed action. We hypothesized that dyn-KOR signaling in the DMS constrained goal-directed actions and motivation. To investigate, we used transgenic mouse strains engineered to express loxp sites on either side of either pre-dynorphin or KOR (pDyn or KOR cKO) genes. We performed viral intracranial microinjections in the DMS of either an anterograde AAV expressing Cre recombinase (AAV-Cre) to remove pDyn in DMS cells or an AAVretro-Cre to delete KOR in neurons projecting to the DMS. We assessed goal-directed action in these mice via instrumental conditioning at varying contingencies, as well as using a wildtype control strain. Furthermore, we utilized progressive ratio testing where the amount of nose pokes required for a reward, a sucrose pellet, increased exponentially, as a measure of motivation. A greater number of nose pokes made indicated higher levels of motivation. Next, reversal training was done where the nose poke port that is active between the two was switched to determine whether the difference in nose pokes made was due to a learning issue or a true difference in goal-directed activity. Our studies aimed to address the involvement of dyn-KOR modulation of goal-directed behavior in the DMS.

People suffering from stressful events are likely to experience negative affective states. Previous studies have shown that stress induces the release of neuropeptides including dynorphin, which acts presynaptically on kappa opioid receptors (KOR) and may act to inhibit neurotransmitter release in the limbic brain areas. In this project, we investigated the interaction between KOR and dopaminergic systems in the ventral shell of the Nucleus Accumbens (NAc) to determine how dopamine release is altered during in vivo dynorphin activation of KORs. To monitor and manipulate dopamine and KOR dynamics, we injected genetically encoded fluorescent indicators of dopamine (dLight) and KOR (kLight) respectively and implanted optical fibers in the NAc ventral shell in transgenic mice brains. In collaboration with Lin Tian at UC Davis, we also characterized the in vivo fidelity and accuracy of a newly developed kLight—version 1.2a. To light-stimulate dynorphin neurons in vivo, we injected a red-shifted channelrhodopsin, Chrimson that acts as a light-gated ion channel to depolarize dynorphin neurons. We recorded dopamine/KOR activities during behavioral experiments including Pavlovian conditioning using sucrose pellets. We hypothesized that upon dynorphin neuron activation, there will be a decrease in the amount of dopamine release in the NAc, an increase in KOR light activity and fewer motivated behaviors observed in mice to obtain sucrose pellets. Here we will present our findings measuring dopamine and kappa opioid function in the NAc. These results could have implications for neuropsychiatric diseases including depression and addiction.

In order for cells to generate copies of themselves they must undergo a highly complex process called mitosis. During mitosis, many enzymes called protein kinases work together to ensure both daughter cells inherit the correct number of chromosomes when the cell divides. Polo-like kinase 1 (Plk1) is a protein kinase that regulates several events during mitosis including centrosome maturation, spindle assembly, sister chromatid cohesion, and cytokinesis. Recently, the A-kinase anchoring protein Gravin (AKAP12) has been implicated in regulating Plk1 function at mitotic centrosomes. Specifically, loss of Gravin has been linked to defective protein signaling at centrosomes, chromosome misalignment, and increased incidence of micronuclei (small nuclei, an aberration often seen in cancer). However, while previous studies used shRNA-mediated knockdown to reduce Gravin levels in cells, it remains unclear how complete loss of this scaffold in human cells influences mitotic signaling events. To test this, our lab generated Gravin knockout U2OS (osteosarcoma) cells using CRISPR/Cas9 genome editing. First, I employed a combination of immunohistochemical staining and quantitative imaging tools to assess how Gravin loss affected chromosome alignment, micronuclei formation, and gamma tubulin accumulation at centrosomes. I found that loss of Gravin in U2OS and HeLa cells caused misaligned chromosomes and micronuclei. Additional experiments I conducted revealed that Gravin-depleted U2OS, HeLa, and MEF cells presented aberrant gamma tubulin accumulation at mitotic spindle poles. Next, a local drug-targeting approach was used to specifically inhibit Plk1 activity at mitotic spindle poles in U2OS cells. I determined that localized inhibition of Plk1 produced similar mitotic defects as observed in cells lacking Gravin. Collectively, these findings suggest that Gravin is required for coordinating proper Plk1 signaling at centrosomes during mitosis while the loss of this scaffold protein leads to mitotic defects. Future work will uncover downstream substrates of Gravin-anchored Plk1 that becomes dysregulated in cells lacking Gravin.

The mitochondria are important organelles that regulate various processes such as oxidative phosphorylation, fatty acid oxidation and calcium homeostasis. They communicate with the rest of the cell to coordinate these functions. Mitochondrial communication is mediated by calcium signaling in which calcium ions pass through a calcium uniporter in the mitochondrial inner membrane. This signaling is altered when the cell undergoes stress and the mitochondria initiates the unfolded protein response (UPR^mito). We are interested in understanding the regulation of the mitochondrial calcium uniporter during UPR^mito. We previously have shown that calcium uptake becomes inhibited when UPR^mito is induced in HeLa cells. It is not known how the unfolded proteins initiate the UPR^mito gene expression pathway. However, we hypothesize that the calcium uniporter is involved in this process. To further characterize the behavior of the uniporter, we induced UPR^mito in another cell line, A549 cells, using different pharmacological agents. After the treatment, I analyzed and performed calcium uptake assays to determine the activity of the mitochondrial calcium uniporter. The results revealed that, similar to HeLa cells, UPR^mito inhibits the uniporter in A549 cells. We hypothesize that uniporter inhibition affects gene expression during UPRmito, especially in disease states where chronic UPR^mito is observed, such as Alzheimer’s and Parkinson’s diseases. To test this hypothesis, we want to develop a more physiologically relevant system for UPR^mito induction, the focus of my project is to induce chronic UPR^mito using low dose, long term drug treatment to be able to mimic diseases conditions better and to determine the effects of chronic UPR^mito on mitochondrial calcium uptake. This study has the potential to implicate the uniporter as a clinical drug target for treatment of diseases with chronic UPR^mito involvement.

Post-traumatic stress disorder (PTSD) is a widespread disorder that diminishes the quality of life for millions of adults annually. Symptoms of PTSD include flashbacks and unwanted memories in those who have suffered a traumatic experience. Research labs across the world are working diligently on understanding the interactions between brain regions affected by PTSD, with much of its effects still unclear. Our lab is researching the brain’s neural interactions during traumatic events, such as in PTSD. We are studying the connection between two specific brain regions in a mouse model: locus coeruleus (LC) and dentate gyrus (DG). Both regions are crucial for understanding the neural interactions of PTSD as they are associated with many of its symptoms. The LC is responsible for modulating anxiety and arousal, while the DG is responsible for memory formation and recall. The LC does this by releasing norepinephrine (NE) during a state of anxiety or arousal. We are altering the activity of the LC via optogenetics and identifying its effect on the formation of new memories. We measure the formation of memories by analyzing the behavior of the mice after the activation of the brain region. We also track the release of NE by with fluorescent imaging of the brain after analysis of the behavior. Under a fluorescent microscope, we are able to identify the parts of the brain norepinephrine travelled to and its effect on the behavior of the mice. Data collected thus far suggests that the norepinephrine released from the LC to the DG is associated with the inhibition of memory formation and impaired recall. These findings will help us better understand the underlying neural mechanisms that cause PTSD and other neurological disorders involving memory.

Adolescence alcohol use and opioid addiction in adults are systemic issues afflicting the world, and thus, it is important to elucidate the long-term individual and relational consequences of both substance abuse disorders. The purpose of this experiment was to examine the long-term consequences of voluntary adolescent alcohol use on morphine tolerance, fentanyl self-administration, and the effects of previous opioid exposure on fentanyl self-administration in adulthood. Using a preclinical model to examine this hypothesis, adolescent rats had access to alcohol in gelatin form for twenty days, after which a three week withdrawal period occured. Morphine was then administered intraperitoneally for five days and morphine tolerance was measured by a tail-flick test for those five days. Finally, fentanyl self-administration occured in an operant chamber and self-administration will be measure by the amount of fentanyl consumed. My expected results were that adolescent alcohol use will increase morphine tolerance as evidenced by decreased tail-flick time, and fentanyl self-administration will also be increased. Additionally, I expect that previous opioid exposure will increase fentanyl self-administration. Future research should examine the neurobiological mechanisms by which adolescent alcohol use increases morphine tolerance and fentanyl self-administration and how previous opioid exposure increases fentanyl self-administration since these biological mechanism are not well understood.