Fibrolamellar carcinoma (FLC) is a life-threatening variant of liver cancer affecting adolescents and young adults. FLC tumors do not respond to chemotherapy, thus, surgery remains the mainstay of therapy. FLC is characterized by a fusion event resulting in a novel chimeric protein that joins the N-terminal domain of DNAJ with the catalytic subunit of protein kinase A (PKAc). However, the underlying mechanism by which the FLC chimeric kinase drives FLC tumor growth remains unknown. Using CRISPR/Cas9 technology, the Scott Lab (UW) has re-created the human FLC fusion protein in a well-characterized and non-transformed hepatocyte cell line. The resulting cells retain hepatocytic differentiation while over-expressing DNAJ-PKAc compared with wild-type PKAc, akin to the human tumors. Using these model cell lines and a computational approach called, Kinome Regularization (KiR), I plan to uncover molecular mechanisms through which chimeric PKAc transforms hepatocytes in FLC. The concept of KiR is built around the fact that most kinase inhibitors are not specific but instead, affect a range of targets. By exploiting the property of polypharmacology in a set of well-characterized kinase inhibitors, we can make use of elastic net regression to compute and deconvolute the kinases that are responsible for a quantitative trait. To do so, we have identified a set of 30 broadly specific inhibitors that give us >85% coverage of the kinases studied. We can then test the effect of each of these 30 inhibitors on cell growth and use the results to identify the kinase important for cell growth. The KiR-based model will also be used to identify kinase inhibitors which can specifically reduce growth of FLC cells. Overall, these studies have a strong translational potential; a new therapeutic option for FLC could give unprecedented hope to the patients who currently face this disease with limited alternatives.

Stress-related disorders impact over 18% of the United States population. Further definition of the mechanisms behind stress responses in the brain is necessary for understanding and treating stress related disorders, such as anxiety and depression. Specifically, the lateral habenula (LHb) is a brain region known to modulate stress and anxiety responses, but the ways in which it modulates these responses is relatively unknown. In order to investigate potential mechanisms for this modulation, we examined differential gene expression in the pathways from the LHb to three brain regions: the ventral tegmental area (VTA), the dorsal raphe nucleus (DRN) and rostromedial tegmental nucleus (RMTg). Rats were stressed using a forced swim protocol and tissue from the LHb was homogenized in order to extract RNA using RiboTag. Using a combination of transgenic mice and a RiboTag viral vector we were able to extract highly specific RNA from complext brain tissue. We used a portion of our collected RNA to perform RNAseq, a molecular tool used to identify differential gene expression in certain pathways. Using the results from our RNAseq analysis, we used extracted RNA to perform quantitative PCR to verify the pathway specific genes found in the LHb. By determining the pathway specific patterns of differential gene expression, we propose a molecular mechanism for neuronal response to stress in the LHb.

The ability of an organism to dissociate environmental cues for either safe or threatening situations is key for survival. Generalized fear is an adaptation in which behavioral responses for threatening stimuli are produced to non-threatening cues. In mice, discriminative or generalized fear responses are modeled using a fear conditioning paradigm of two shock intensities, 0.3 mA or 0.5 mA. Mice trained at 0.3 mA intensities could discriminate between safe and threatening cues, yet mice trained at a 0.5 mA intensities displayed generalized fear behavioral responses. Previously, we had identified that dopaminergic neural activity was critical for the ability to discriminate between cues when shocked at 0.3 mA. Dopamine neurons express Kv7.3 potassium channel subunits, which modulate neural activity. I hypothesized that Kv7.3 subunits on dopamine neurons would be critical to threat discrimination and mice that had undergone mutagenesis of Kv7.3 would show generalized fear discrimination. My approach used transgenic mice expressing Cre-recombinase and viral-mediated gene delivery of cre-inducible CRISPR-Cas9 plasmids targeted for the specific mutagenesis of Kv7.3. Mice underwent a three day paradigm known as fear conditioning. Baseline freezing behavior was assessed by playing two tones, A and B. This was followed by fear conditioning trials where at the end of tone A, mice received a shock of either 0.3 mA or 0.5 mA (CS+) and at the end of tone B, mice received no shock (CS-). On the third day, mice underwent a probe trial to assess final ability to discriminate between threatening and non-threatening cues. These findings could provide insights into the mechanisms underlying neurological disorders and serve as a guide for future pharmacological interventions.

Rats produce ultrasonic vocalizations (USVs) in a range from 20-kHz to 95-kHz that vary in frequency and shape across social and motivational contexts and can correspond to the affective state of the animal. To assess these USVs accurately and efficiently, our lab created DeepSqueak, a novel machine learning software package that expedites the detection and analysis of rat USVs by using neural networks to differentiate them from noise. DeepSqueak also allows for automatic and unbiased classification of USVs into discrete categories using call parameters such as shape, frequency and duration. Prior to this unbiased categorization method, identified 14 subjective categories in 50-kHz rat vocalizations that could be manually identified by a trained experimenter. These categories have received some limited study, but the excessive labor and time needed for manual classification restricted broad adoption. We aim to use neural networks to quickly and automatically classify USVs into these categories to promote broad adoption and better our understanding of the relationship between USVs and behavior. The process of training our neural network to differentiate between vocalizations was approached in two ways. Audio files were converted to sonograms through DeepSqueak and manually labeled. Thousands of these labeled calls were then inputted as training data for the neural network. This method allowed the network to learn using a large set of labeled vocalization data. The second method is based around the manual selection of an optimal call for each subtype using DeepSqueak's "call clusters" function; the neural network was then trained around how closely vocalizations matched the optimal calls. We now plan to compare DeepSqueak's automated calls and clustering to manual scoring in order to develop the best possible system that reliably categorizes USVs, thus allowing for more specific analyses of USV categories and behavior.

Chronic stress induces the release of neuropeptides including dynorphin, which activates kappa opioid receptors (KOR) to encode the dysphoric components of the stress response. Dynorphin/KOR actions on dopamine neurons have been shown to underlie aversive learning, and it is hypothesized that potentiation of cocaine reward following stress is likely to occur through similar neural mechanisms. In this study, we investigate the neural substrates underlying stress-mediated enhancement of cocaine reward. First, we verified the reliability of a KOR (KT2) antibody. Immunostaining specificity was verified using KOR-Cre mice which express Cre-recombinase in KOR-containing neurons. By combining cell-specific targeting using Cre-dependent viral expression of fluorophores and immunohistochemistry staining, we confirmed the specificity of anti-KT2 to KOR containing neurons. Using this antibody and an anti-tyrosine hydroxylase antibody, we examined the co-localization of KOR and dopamine in the ventral tegmental area (VTA). Immunohistochemical analyses showed that KORs were expressed in a majority of dopamine neurons in the medial and lateral VTA. Furthermore, we investigated dynorphin-KOR circuitry in the mouse brain. Dynorphin-containing neurons projecting from the prefrontal cortex (PFC) and dorsal raphe nucleus (DRN) to the VTA were identified by injection of a retrograde virus (CAV2-DIO-ZsGreen) into the VTA of prodynorphin-Cre mice. In a separate cohort, an excitatory opsin (Channelrhodopsin2) was injected into the DRN of pDyn-Cre mice with an optic fiber implant. The DRN region was then optically stimulated and resulting KOR phosphorylation was measured in the VTA thereby verifying dynorphin-releasing projections from the DRN to the VTA. This projection was further investigated by examining the effect of DRN dynorphin on stress-induced potentiation of cocaine conditioned place preference (CPP). We found that deletion of dynorphin from the DRN, but not PFC, blocked stress induced enhancement of cocaine CPP. In conclusion, this experiment demonstrates a functional dynorphin/KOR circuit from the DRN to the VTA that mediates stress-induced increases in drug reward.

Mitosis is an essential cellular process in which a cell divides to produce two genetically identical daughter cells. When this process becomes dysregulated cells divide uncontrollably leading to diseases such as cancer. Polo-like kinase 1 (Plk1) is a key enzyme that is necessary for coordinating numerous events during mitosis. When Plk1 becomes dysregulated or mislocalized, mitotic spindle assembly, protein organization, and mitotic timing impairments may occur. One of the many subcellular locations where Plk1 carries out essential mitotic functions is the kinetochore. The kinetochore is the interface between the chromosomes and the mitotic spindle and is critical for ensuring proper DNA to microtubule attachments early on in mitosis. Historically, the small-molecule inhibitor drug BI2536 has been used to inhibit the activity of Plk1 in order to study its role in regulating various mitotic processes. However, traditional inhibitor drugs turn off entire protein kinase populations, inhibiting the activity of Plk1 all throughout the cell, not just at the kinetochore. This can lead to unwanted side effects and limits our understanding of Plk1’s role at specific subcellular locations. To improve the specificity of BI2536 drug delivery, we utilized SNAP-tag, a tool in which a self-labeling enzyme can irreversibly react with substrates linked to a chloropyrimidine (CLP) functional group. By genetically manipulating human bone cancer (U2OS) cells, we expressed a kinetochore localizing SNAP. We also generated a BI2536 conjugated to a CLP group. By treating our genetically modified cells with CLP-BI2536, we can target Plk1 inhibiting drug to the kinetochore to study Plk1’s role at this specific location. Using super-resolution structured illumination microscopy (SIM), we demonstrate that we can effectively target fluorescently labeled CLP substrates to kinetochores in our cell line. In future work, we will target our CLP-BI2536 drugs to the kinetochore and investigate how local Plk1 inhibition affects mitotic timing.

Epithelial-mesenchymal transition (EMT) refers to a biologic process that allows a polarized epithelial cell, which normally functions in the basement membrane of a cell, to undergo biochemical changes that makes it express as a mesenchymal cell phenotype. This mesenchymal phenotype allows the cell to have enhanced migratory capacity, invasiveness, elevated resistance to apoptosis, and increased production of extracellular matrix (ECM) elements. The process of EMT is considered completed once the underlying basement membrane breaks down, and the mesenchymal cell becomes migratory. Another component that proves EMT is the loss of e-cadherin. E-cadherin refers to cell-to-cell adhesion and the degradation of e-cadherin levels are a hallmark of EMT happening. There are three distinct types of EMTs; I will be focusing on type II EMT. Type II EMTs are associated with inflammation/wound repair but usually stops once inflammation subsides. However, in the context of organ fibrosis, type II EMTs can continue to over-respond to a persisting inflammation and can lead to organ death. In my experiment, I hypothesize that in HEK 293 human cells, SNAP -Δ1-91 alpha-1D adrenergic receptors undergo type II EMT. SNAP -Δ1-91 alpha-1D adrenergic receptors are a truncation of the extracellular portion of the receptor. Certain receptors undergo this truncation to increase its expression. It is shown that in SNAP – Full Length alpha-1D adrenergic receptors  (wild type receptors) do not undergo EMT. I will be able to observe the process of type II EMT through imaging the breakdown of the cell membrane in SNAP- Δ1-91 alpha-1D adrenergic receptors and the measuring of e-cadherin levels. The purpose of this research would be to potentially influence future therapeutic interventions that target wild type receptors to induce would repair. 

The process of gene expression known as transcription is an essential component in all eukaryotic cellular development. TBP Associated Factor 1 (Taf1) is the largest subunit within the the ubiquitously expressed transcription factor complex TFIID and plays an integral role in regulating gene transcription. In neuronal cells, an isoform of Taf1, dubbed N-Taf1, is expressed and is thought to be a key regulator of neuronal development. When under-expressed, N-Taf1 has been hypothesized to be associated with the neurodegenerative disease X-linked Dystonia Parkinsonism, which causes individuals to experience tremors, impaired movement, and uncontrolled rigidity. The goal of this study is to expand our knowledge of the functional role of N-Taf1 in neuron development and how impairment of this function may lead to neurodegenerative disease. To accomplish this, we investigate the expression of N-Taf1 and proteins that signify neuronal differentiation within various cell lines using western blot protocol and fluorescently active antibodies. Once an ideal cell line to study N-Taf1 function is identified, we selectively alter the expression of N-Taf1 using CRISP-Cas9 and compare the expression levels of our proteins of interest when N-Taf1 is under-expressed or unaltered. By analyzing this data, we can begin to draw connections between N-Taf1 expression levels and the process of neuronal development. By understanding this relationship, we hope to move closer to a treatment for X-linked Dystonia Parkinsonism and any other neurological impairments associated with N-Taf1.

2D cell models have traditionally been used in labs to test the effects of new drugs on certain cell types due to the ease and convenience of use. While 2D methods are great, they often simplify the cell-to-cell interactions and may not accurately represent cell systems in humans. 3D methods show the complex cell communication systems and better simulate actual organ systems. Research comparing these two methods can inform scientists on the benefits of 3D models which can help efficiency in creating new drugs. Our lab looked into various 3D models to determine their effectiveness and reliability and looked into the differences in perceived cell mechanics and functionality between 2D and 3D methods. We tried Corning Matrigel and Corning 3D Spheroid microplates for 3D cell modeling using HEK293 cells, which are human embryonic kidney cells that were grown in lab. They are known for being easy to grow and transfect. We used SNAP-Gels, which are protein assays that show the protein levels in the cells, to ensure that the protein levels were similar between the 2D and 3D systems. We then did florescent imaging to determine cell localization and EPIC dynamic mass redistribution (DMR) to determine cell functionality. We found Matrigel to have inconsistent results, so we focused on using the spheroid microplates. Based on our initial results, we saw increased functionality and expression levels for full-length protein cells compared to cells with a truncated N-terminal protein in the 3D method. This increase in functionality and expression levels was not seen in the 2D method. Our results show that 3D modeling methods can be reliable, and do show results that differ from 2D models. This is important for future studies that require cell modeling because 3D models can provide a more accurate and reliable modeling system to create novel therapeutics.

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 can become toxic to cells. These aggregates can crowd the cellular environment and impair cellular functions, which on a single cell scale can lead to cell death and on a larger organism scale, cause diseases like Alzheimer’s, Parkinson’s, and Huntington’s. To deal with this problem, cells have evolved protein quality control (PQC) systems that comprise two classes of action: chaperones that help proteins fold properly and ubiquitin-protein ligases that tag misfolded proteins with ubiquitin for destruction by the proteasome. Previous studies concluded that chaperones are required protein degradation. In our study, we find that Hsp70 chaperone dependence for protein degradation is variable along a spectrum of independent to dependent. My work specifically examined the function of yeast ubiquitin-protein ligase San1 by comparing degradation of various substrates between strains with or without San1 function, and with or without chaperone activity. By performing degradation whereby protein synthesis was halted and the stability of the synthesized pool of substrate was monitored by Western analyses, we were able to see the degree of substrate degradation by each strain over time. Degradation through San1 has been shown to require chaperones, but San1 also is known to recognize substrates independently without chaperones. From our work, San1 recognizes patches of hydrophobicity on misfolded proteins; a feature that is also recognized by chaperones. By studying the interactions of the folding and degradation enzymes, we are gaining a new understanding of how PQC pathways collaborate and coordinate to achieve optimal protection for the cell.
 

 Diabetes is the 7th leading cause of death in America and occurs when blood glucose levels are too high. The body regulates blood glucose levels with a hormone called insulin, which is made and secreted by pancreatic β-cells. Type I diabetes occurs when these β-cells cannot produce insulin. Type II diabetes occurs when the body does not respond to insulin efficiently. Our goal is to study how the protein phosphatase PP2B (also known as Calcineurin) impacts insulin release in pancreatic β-cells. Previous work in cells and genetically modified mice has found that PP2B signaling regulates insulin release, but the mechanism remains unknown. We are taking advantage of CRISPR/Cas9 gene-editing to replace a PP2B-anchoring protein with a modified version that allows biochemical labeling of proximal proteins. When combined with mass spectrometry analysis, this approach enables identification of PP2B substrates in β-cells. Future work will test these candidates for their impact on insulin production. Our long-term goals are to uncover how insulin is regulated and to find new targets for diabetes treatment.

The mammalian brain is composed of a wide variety of neurons, all of which signal through structurally and biochemically heterogeneous synapses. Understanding the proteomic makeup of these synapses is crucial for understanding brain function. However, biochemical isolation and characterization has proven difficult due to the morphological complexity of neurons. MS-based proteomics could be a useful tool for studying such diversity, but spatial information is lost because the analysis is performed post-lysis. In this study we proposed to overcome this limitation by using proximity biotin labeling of genetically specified neuronal subpopulations. I thus constructed APEX2-GFP fusion constructs with post-synaptic proteins which could be expressed in vivo using an adeno-associated virus (AAV). Four post-synaptic proteins were chosen: LRRTM1 and LRRTM2 to localize excitatory synapses, and NGLN2 and SLITRK3 to localize inhibitory synapses. A pAAV-CAG (AAV plasmid with CAG promoter) backbone was digested using NotI and AscI, and an APEX2-GFP fragment with NotI and AscI ends was generated via Phusion PCR. Following NotI/AscI double digests, the pAAV-CAG backbone and APEX2-GFP fragment were ligated together in order to make a pAAV-CAG-APEX2-GFP vector. The pAAV-CAG-APEX2-GFP vector was opened with a NotI digest, and gene fragments of the four post-synaptic proteins with Gibson overlaps were then generated via Phusion PCR. Gibson assembly was then used to assemble the pAAV-CAG-APEX2-GFP vector with the gene fragments, and pAAV-CAG-gene-APEX2-GFP vectors were generated for LRRTM1 and LRRTM2. The vectors were then expressed in HEK cells and monitored for GFP expression and biotinylation activity. The pAAV-CAG-APEX2-GFP vector showed bioluminescence and biotinylation activity, but the vectors with gene inserts did not. Although we successfully cloned two of the target constructs, current investigations are still under way in order to optimize them.

G-protein coupled receptors (GPCRs) - characterized by seven transmembrane alpha helical domains - are the largest family of membrane proteins, constituting ~1% of the human genome. The α1D-adrenergic receptor (A1DAR) is a GPCR that regulates function of the cardiovascular, urinary, and central nervous systems. Dysfunction of this receptor can lead to various diseases including schizophrenia, benign prostate hypertrophy, hypertension, and PTSD. Prazosin, a non-specific α1-antagonist is the first line treatment for PTSD, however, chronic use has deleterious side effects including orthostatic hypotension and potentially fatal reflex tachycardia due to interactions with off-target related receptors. Thus, understanding how A1DARs are regulated will allow for the development of targeted therapeutics. To this end, the Hague Lab has previously discovered that A1DAR undergoes an endogenous cleavage of its extracellular N-terminal domain, affecting its membrane localization and response to agonist stimulation. Located within the N-terminal domain of A1DAR are two glycosylation sites at amino acids 65 and 82. Currently, how glycosylation of these sites regulates the cleavage event remains unknown. To characterize this phenomena, I used molecular cloning to mutate the glycosylation sites of A1DAR in the pSNAP vector for expression in Human Embryonic Kidney 293 (HEK293) cells. Near Infrared PAGE analysis revealed that glycosylation of both amino acids is required for cleavage and proper expression of A1DAR. Sucrose density gradient and dynamic mass redistribution further showed that glycosylation controls function and trafficking of A1DAR to the membrane. These results allow for the development of targeted medications specific to the N-terminal glycosylation sites of A1DAR, further reducing the potential side effects experienced by patients.

G protein-coupled receptors (GPCRs) contain seven transmembrane domains and are the largest family of cell surface receptors, making up ~1% of the human genome. GPCRs can interact with a variety of ligands, such as odors, pheromones, hormones, and neurotransmitters. At least 30 human GPCRs contain a C-terminal Type-I PDZ ligand that allows for interactions with adapter proteins which can regulate receptor trafficking, stability, and signaling. The Hague Lab has previously found that the α1D-adrenergic receptor, which contains a C-terminal Type-I PDZ ligand, also undergoes an endogenous N-terminal cleavage event, which enhances receptor function and may play a role in which PDZ domain containing proteins interact with this receptor. We propose that this unique observation of the α1D-adrenergic receptor may be prototypical of a new class of GPCRs which contain a Type-I PDZ ligand and undergo an N-terminal cleavage. CysLT2, MAS1, and NPFFR2 are understudied GPCRs and potential members of this family with distinct PDZ ligands, though it remains unknown if their extracellular N-terminal domains regulates receptor function. Thus, I have cloned wildtype and N-terminal truncation mutants of these three GPCRs into the pSNAP vector to create fusion proteins with N-terminal SNAP tags. These constructs were transfected into HEK293 cells and subjected to near infrared PAGE analysis to elucidate the presence of N-terminal processing. Furthermore, dynamic mass redistribution revealed how the N-termini modulate receptor signaling. The combination of biochemical and pharmacological techniques allows me to determine if these receptors belong to this new subfamily of GPCRs. These results increase our understanding of how GPCRs are regulated within the cell, which can lead to the development of more efficient drugs.

Dravet syndrome (DS) is a form of intractable epilepsy that manifests itself in infancy. It is comorbid with behaviors resembling autism, motor deficits, and premature death. Human infants with DS also experience both febrile and non-febrile seizures. Therefore, research into the mechanistic pathway of DS in mice may shed light on the alleviation of DS symptoms detrimental to the human quality of life, as well as help to identify avenues for gene therapy and further research. Symptoms of DS have been shown to recruit certain immunologic entities such as microglia, a type of glial cell. Glial cells, or non-neuronal brain cells, have traditionally been thought to merely play the supportive role of providing nutrients to neurons. However, recent studies have shown that glia play a more active role in stabilizing information processing, brain function, and maintaining brain homeostasis. Following microglial recruitment, another type of glia, astroglia, are activated. Astroglia form the blood-brain barrier and collaborate with microglia to produce neuroinflammation through gliosis. Gliosis has been shown to interfere with normal neural function, which is postulated to contribute to Dravet phenotypes. Thus, DS symptoms may produce either hypertrophic astroglia or increased distribution of astroglia, both of which interfere with the ability of inhibitory interneurons to adequately regulate neural excitation as a result of induced neuroinflammation. Investigation into the link between glia and Dravet syndrome is the core of this research and has revealed sex differences in the distribution and size of astroglia. In particular, immunohistochemical staining of brain tissue on Dravet females have shown greater astroglial concentrations in the DG relative to their male counterparts, when coupled with behavioral analysis and EEG readings. This difference has implications in Dravet syndrome research and treatment.