Parkinson’s disease (PD) is a disorder of the nervous system that causes problems with movement (motor symptoms) as well as other problems such as mood disorders, cognitive changes, sleep disorders, constipation, pain, and other non-motor symptoms. The severity of PD symptoms worsens over time as the disease progresses, and while there are treatments for the motor and some non-motor symptoms there is no known cure for PD. Thus there is a high demand for therapies to slow the progressive neurodegeneration observed in PD. Two clinical trials at the University of Kentucky College of Medicine (NCT02369003, NCT01833364) are currently underway that aim to develop a disease-modifying therapy that slows the progression of PD. These clinical trials are evaluating the safety and feasibility of an autologous peripheral nerve graft to the substantia nigra in combination with Deep Brain Stimulation (DBS) for the treatment of PD. By grafting peripheral nerve tissue to the Substantia Nigra, the researchers aim to introduce peripheral nerve tissue, which is capable of functional regeneration after injury, to the degenerating Substantia Nigra of patients with PD. The central hypothesis of these clinical trials is that the grafted tissue will slow degeneration of the target brain region through neural repair actions of Schwann cells as well as other pro-regenerative features of the peripheral nerve tissue. This dissertation details analysis of the peripheral nerve tissue used in the above clinical trials with respect to tissue composition and gene expression, both of injury-naive human peripheral nerve as well as the post-conditioning injury nerve tissue used in the grafting procedure. RNA-seq analysis of sural nerve tissue pre and post-conditioning show significant changes in gene expression corresponding with transdifferentiation of Schwann cells from a myelinating to a repair phenotype, release of growth factors, activation of macrophages and other immune cells, and an increase in anti-apoptotic and neuroprotective gene transcripts. These results reveal in vivo gene expression changes involved in the human peripheral nerve injury repair process, which has relevance beyond this clinical trial to the fields of Schwann cell biology and peripheral nerve repair. To assess the neurobiology of the graft post-implantation we developed an animal model of the grafting procedure, termed Neuro-Avatars, which feature human graft tissue implanted into athymic nude rats. Survival and infiltration of human graft cells into the host brain were shown using immunohistochemistry of Human Nuclear Antigen. Surgical methods and outcomes from the ongoing development of this animal model are reported. To connect the results of these laboratory studies to the clinical trial we compared the severity of motor symptoms before surgery to one year post-surgery in patients who received the analyzed graft tissue. Motor symptom severity was assessed using the Unified Parkinson’s Disease Rating Scale Part III. Finally, the implications and future directions of this research is discussed. In summary, this dissertation advances the translational science cycle by using clinical trial findings and samples to answer basic science questions that will in turn guide future clinical trial design.

Glioblastoma multiforme (GBM) is a grade IV astrocytoma and is the most aggressive malignant primary brain tumor in adults. Without treatment, patients are expected to survive an average of three months. Conversely, current treatment regimens only extend survival to 12-14 months. Characteristically, GBM tumors are highly proliferative, invasive and stop responding to treatments relatively fast due to therapy resistance. Interestingly, GBM also exhibits high metabolic activity but manages to maintain a low level of reactive oxygen species (ROS). These ROS neutralization capabilities are sustained by system Xc–, a sodium-independent, electro neutral transporter that is found in the plasma membrane of GBM cells. System Xc– is composed of a regulatory heavy subunit (4F2hc) linked to a 12 transmembrane domain catalytic light chain subunit (xCT) that mediates the uptake of L-cystine into the cell, and L-glutamate out of the cell, at a 1:1 ratio. Imported cystine is quickly reduced to L- cysteine, the rate limiting substrate in glutathione (GSH) synthesis. Glutathione is a major antioxidant in the central nervous system that is responsible for maintaining intracellular redox homeostasis by neutralizing ROS by direct and indirect methods. The function of chemo and radiation therapy is to generate significant levels of ROS that tigger the cell to undergo apoptosis. High intracellular GSH levels in cancer cells are associated with drug resistance and detoxification of alkylating agents such as temozolomide (TMZ). Therefore, system Xc– represents a potential target to reduce glioma cell survival and reduce tumor progression. Sulfasalazine is an FDA approved drug in the treatment of arthritis and Crohne’s disease and has been shown to inhibit system Xc–. In vitro SASP studies demonstrated a strong antitumor potential in preclinical mouse models of malignant glioma. However, two clinical trials using sulfasalazine with standard chemo and radiation therapy to treat GBM patients were terminated due to off-target effects. Both results showed high toxicity and no change in the overall survival of patients. These studies demonstrate the need for a more effective inhibitor of system Xc–. To further elucidate the role of system Xc– in GBM survival, stable xCT knock-down and over-expressing U251 glioma cells were generated. These lines were characterized for survival, proliferation, apoptosis and resistance to oxidative and genotoxic insult. As expected xCT-knockdown cells exhibited lower GSH levels, increased intracellular ROS and markers for apoptosis after oxidative and genotoxic insult. The xCT-over-expressing cells displayed higher levels of GSH, increased resistance to hydrogen peroxide and various chemotherapy drugs including TMZ. An interesting unforeseen result of xCT over-expression in glioma cells was an increase in the metabolic activity as a result of increased mitochondria. Using xCT-modified glioma lines stably, we demonstrate for the first time that system XC– over-expression not only promotes survival under oxidative stress but may also decreases sensitivity to chemotherapy treatment and increase metabolic properties. Therefore, therapeutic manipulation of this transporter either alone or in combination with other treatments may improve clinical outcome in patients diagnosed with GBM.

Introduction: Current medical treatment for individuals diagnosed with schizophrenia (SCHZ) primarily relies on the inhibition of the dopamine D2 receptor that has been shown to be supersensitive in these patients. Treatment occurs through the use of antipsychotic medication which leads to a number of debilitating dose-dependent side effects, such as weight gain, agranulocytosis, and seizures. Patients diagnosed with SCHZ have also been shown to have increased inflammation in their central nervous system (CNS), particularly within specific brain regions such as the prefrontal cortex and hippocampus. This is in large part due to the interaction between a pro-inflammatory cytokine called tumor necrosis factor-alpha (TNFa) and microglia, which are resident CNS defense cells. TNFa is a cell-signaling protein, regulates a variety of immune cells, and is involved in the acute phase reaction of inflammation. Upon activation by TNFa secretion, microglial cells switch from being anti-inflammatory (M2) to pro-inflammatory (M1), thereby resulting in neuroinflammation as well as synaptic loss and neuronal death. In this project, we hypothesized oral administration through the diet of a novel TNFa modulator (PD2024) developed by P2D Biosciences, Inc. (Cincinnati, OH) would significantly reduce microglia activation in rats neonatally treated with Polyinosinic:polycytidylic acid (poly I:C). Methods and Results: To test our hypothesis, four groups (Neonatal Poly I:C/TNFa, Neonatal Poly I:C/Control, Neonatal Saline/TNFa, and Neonatal Saline/Control) were intraperitoneally injected with either poly I:C or saline during postnatal days (P)5-7. Poly I:C is an immunostimulant that mimics neonatal infection in humans, which also has been found to be a factor for the development of SCHZ later in life. Between days (P)30-(P)60, the Neonatal Poly I:C/TNFa and Neonatal Saline/TNFa groups were orally administered PD2024 through the diet. After (P)60, brain tissue was evaluated by immunohistochemistry (IHC) and confocal microscopy. Immunohistochemistry was used to label microglial cells in the prefrontal cortex and hippocampus with a green fluorescent dye attached to Iba1, a protein that specifically binds to these cells. Upon completion of IHC, tissue was evaluated using a confocal microscope and then analyzed with NIH ImageJ software. Analysis parameters included cell count, sampled cell body fluorescence, and overall image fluorescence. The results obtained showed a significant decrease in microglia activation for the Poly I:C/TNFa group when compared to the Poly I:C/Control group, as well as similarities in activation levels with the Saline/Control group. These results were demonstrated in both sampled cell body fluorescence and overall image fluorescence measurements. Conclusion: This data supports the hypothesis that PD2024 is successful in reducing microglia activation through the modulation of TNFa. Therefore, treatment with a TNFa modulator such as PD2024 alongside of current antipsychotic medication could mediate neuroinflammation and reduce the dose-dependent side effects. This approach could be a promising therapeutic treatment option for those diagnosed with schizophrenia, as well as potentially for other neurocognitive and behavioral disorders.

Breast cancer metastasizes to the brain in 15-30% of all breast cancer cases, and metastasis is the predominant cause of breast cancer-related deaths. Patients with HER2-enriched and triple-negative breast cancers (TNBCs) are more likely to develop brain metastases. While targeted therapies exist for HER2-enriched breast cancers, there are no effective treatments for TNBCs. Thus, a greater understanding of how these cancers spread to the brain is critical. In order to spread to the brain, disseminated breast cancer cells must overcome 2 major steps—crossing the blood-brain barrier (BBB) and survival and successful colonization of the distinctive and mostly cellular brain environment. Here, we report a novel role for breast cancer cell surface receptor, Syndecan-1 (Sdc1), a heparan sulfate proteoglycan, in promoting breast cancer cell transmigration across the BBB. We found that when we silenced Sdc1 expression in a highly metastatic TNBC cell line, MDA-MB-231, these cells exhibited reduced migration across an in vitro BBB model system. Further, in an in vivo experimental model of metastasis, mice injected with MDA-MB-231 Sdc1 KD (knock-down) cells developed less brain metastases than mice injected with control non-silencing (NS1) cells. Conversely, we found that overexpression of Sdc1 in a metastatic triple-negative mouse mammary carcinoma cell line, 4T1, led to an increase in brain metastases compared to empty vector control-treated mice. We predicted that a secreted factor(s) facilitated BBB disruption that allowed for Sdc1-mediated BBB transmigration, and found that silencing Sdc1 led to decreases in the production and/or release of various cytokines and chemokines implicated in BBB permeability and transmigration. In addition to supporting BBB transmigration, through an in vitro tissue section adhesion assay, we found that Sdc1 also facilitates adhesion of breast cancer cells to the brain, and not to the liver or lungs, revealing specificity for the brain. Further, we report that Sdc1 is expressed in 81% of breast cancer patient brain metastases in our tissue microarray study and that patients with TNBC and high Sdc1 expression have shorter disease-free survival based on a study performed using data from The Cancer Genome Atlas. Taken together, we predict that breast cancer cell Sdc1-regulated cytokines and chemokines promote BBB permeability and/or support transmigration to facilitate breast cancer metastasis to the brain. We also provide evidence for breast cancer-secreted extracellular vesicles, namely exosomes, in supporting the formation of a pro-metastatic brain environment. We compared exosomes derived from the metastatic 4T1 mouse mammary carcinoma cell line to a non-metastatic counterpart, the 67NR cell line, to assess their microRNA and protein composition and their effect(s) on recipient astrocytes, known mediators of brain metastasis. We found that there are inherent differences in both the microRNA and protein cargo from the metastatic 4T1 cells compared to the non-metastatic 67NR cells, whereby the metastatic 4T1 cells contained various tumor-promoting microRNAs and proteins, and also contained 4.5-fold more protein than the non-metastatic 67NR cells. Mouse astrocytes treated with the metastatic 4T1 exosomes exhibited a shift towards a pro-metastatic phenotype, characterized by upregulation of pro-inflammatory genes, and genes associated with astrocyte reactivity and cancer, whereby 67NR exosome-treated astrocytes exhibited a response profile that overlapped with untreated controls. Overall, these findings reveal an important role for exosomes in driving changes in the brain microenvironment to create a site conducive for cancer growth. Together, both studies help to elucidate how breast cancer cells can invade and colonize the unique brain environment.

Synthetic cathinones and related agents represent an international drug abuse problem, and at the same time an important class of clinically useful compounds. Structure-activity relationship studies are needed to elucidate molecular features underlying the pharmacology of these agents. Illicit methcathinone (i.e., MCAT), the prototype of the synthetic cathinone class, exists as a racemic mixture. Though the differences in potency and target selectivity between the positional and optical isomers of synthetic cathinones and related agents have been demonstrated to have important implications for abuse and therapeutic potential, the two MCAT isomers have never been directly compared at their molecular targets: the monoamine transporters (MATs). Additionally, previous studies have found that the carbonyl oxygen atom can be replaced with a methoxy group, but this results in two chiral centers (i.e., four possible optical isomers for synthesis and evaluation). Here, the individual isomers of MCAT, their racemate, and achiral MCAT analogs were prepared where necessary, and examined in vitro and in silico at the MATs. All agents were active as substrates, with a rank order of potency suggesting that α-position chirality, in either configuration, is favored but not required, with the S(-) configuration slightly preferred. Either chiral center removal approach resulted in a reduction in potency, suggesting both favorable interactions with the α-methyl, and limited bulk tolerance. To further investigate this possibility, docking studies were conducted using homology models of the MATs. Common binding modes were identified that were similar to the binding mode of S(+)amphetamine co-crystallized at drosophila DAT. Taken together, these studies supported our conclusions, as steric hindrance was observed in the α-methyl region of the proposed binding site for the R(+)MCAT isomer. Inclusion of the original synthetic cathinones among Schedule I controlled substances has driven the clandestine development of a second generation of agents, resulting in an array of new synthetic cathinones diverse in structure and effect.Pyrrolidinophenones are a major constituent of second-generation bath salts. Little is known about their structure-activity relationships. Here, we have synthesized and examined a series of aryl-substituted pyrrolidinophenone analogs, as well as an achiral pyrrolidinophenone analog, utilizing novel synthetic chemistry and an innovative cell-based epifluorescence Ca2+ imaging technique. Herein, we evaluated the neurochemical properties of these novel compounds at the dopamine transporter (DAT), considered to exert a major role in actions of drugs of abuse. For future structure-activity relationship studies, additional analogs of synthetic cathinone-related agents were produced using novel synthetic approaches, including analogs and isomers of known amphetamine drugs of abuse. Finally, though much has been learned about the role of the dopamine and serotonin transporters in the mechanisms of action of synthetic cathinones, the role of the norepinephrine transporter is poorly understood. Homology models of the human norephinephrine transporter were built and docking studies conducted to inform the study of MAT ligand selectivity, activity, and binding. In conclusion, these studies represent progress towards the establishment of comprehensive structure-activity relationships for synthetic cathinones and related agents. Particular emphasis was placed on the SAR of the phenylalkylamine α-carbon in the synthetic cathinone context, and the role of the norepinephrine transporter in their activity.

In humans and other organisms, monoaminergic systems are crucial in neuronal function and behavior. The monoamine transporters (MATs), which can be found on the presynaptic plasma membrane of neurons in the central nervous system (CNS), are crucial in the regulation of neurotransmitter concentration in the synaptic cleft. As the duration and concentration of neurotransmitters in the cleft affect further downstream signaling responses, these proteins are important targets for both understanding neuronal physiology and compounds of interest. Multiple theories exist proponing the contribution of MATs to a variety of mental and neurological disorders, including depression. This theory establishes that depression is caused by imbalances in monoamine neurotransmitters. Compounds such as Fluoxetine (FLX) are classified as selective serotonin reuptake inhibitors (SSRIs), these drugs selectively block the reuptake of neurotransmitters at the serotonin transporter (SERT). Since differences in MAT selectivity of inhibitory compounds are influential to selecting efficacious antidepressant treatments, we utilized a unique fluorescent analysis technique to explore three therapeutic compounds of interest (in-vitro) which contain structural similarity to FLX. Our results confirm the selectivity of FLX at SERT, and classify the novel compounds studied into different potential categories of reuptake inhibitors. We hope these compounds will be studied further to elucidate their potentially therapeutic roles and mitigation of undesired side effects seen in other medications.

Psychedelics are a subset of hallucinogenic drugs that exert their characteristic effects through agonist activity at the serotonin receptor 2A (5-HT2A). In this study, I aimed to characterize the modulatory role of the metabotropic glutamate subtype 2 receptor (mGluR2) in the 5-HT2A-specific rodent model of hallucinogenic action, head-twitch response (HTR). Secondly, I aimed to explore if 5-HT2A agonist-induced deficits in prepulse inhibition (PPI) of the startle response, an additional model of hallucinogenic action, could be produced in mice. Though 5-HT2A agonist-induced PPI deficits, which represent interruptions in normal sensorimotor gating, have been described in both rats and humans, attempts to translate this behavior to mice are rare. In contrast to prior gene knockout studies suggesting the mGluR2 is necessary for 5-HT2A agonist-induced HTR, mGluR2 knockout (Grm2-/-) mice still displayed HTR upon administration of the psychedelic 2,5-dimethoxy-4-iodoamphetamine (DOI). Additionally, DOI and lysergic acid diethylamide (LSD) produced unexpected improvements in PPI in male 126S6/Sv wild-type mice, depending on the experimental protocol used and the origin of the animals. Sex differences were observed as DOI-induced improvements in PPI were present in female 129S6/Sv mice of the same origin and tested with the same protocol as their male counterparts; this effect in females was absent in 5-HT2A knockout (Htr2a-/-) mice. The results of this study shed light on issues with replicability and reproducibility in science, the importance of highlighting the origin and background of animal subjects, and potential sex differences in hallucinogenic drug action.

I proposed the study of two distinct aspects of Ten-Eleven Translocation 2 (TET2) protein for understanding specific functions in different body systems. In Part I, I characterized the molecular mechanisms of Tet2 in the hematological system. As the second member of Ten-Eleven Translocation protein family, TET2 is frequently mutated in leukemic patients. Previous studies have shown that the TET2 mutations frequently occur in 20% myelodysplastic syndrome/myeloproliferative neoplasm (MDS/MPN), 10% T-cell lymphoma leukemia and 2% B-cell lymphoma leukemia. Genetic mouse models also display distinct phenotypes of various types of hematological malignancies. I performed 5-hydroxymethylcytosine (5hmC) chromatin immunoprecipitation sequencing (ChIP-Seq) and RNA sequencing (RNA-Seq) of hematopoietic stem/progenitor cells to determine whether the deletion of Tet2 can affect the abundance of 5hmC at myeloid, T-cell and B-cell specific gene transcription start sites, which ultimately result in various hematological malignancies. Subsequent Exome sequencing (Exome-Seq) showed that disease-specific genes are mutated in different types of tumors, which suggests that TET2 may protect the genome from being mutated. The direct interaction between TET2 and Mutator S Homolog 6 (MSH6) protein suggests TET2 is involved in DNA mismatch repair. Finally, in vivo mismatch repair studies show that the loss of Tet2 causes a mutator phenotype. Taken together, my data indicate that TET2 binds to MSH6 to protect genome integrity. In Part II, I intended to better understand the role of Tet2 in the nervous system. 5-hydroxymethylcytosine regulates epigenetic modification during neurodevelopment and aging. Thus, Tet2 may play a critical role in regulating adult neurogenesis. To examine the physiological significance of Tet2 in the nervous system, I first showed that the deletion of Tet2 reduces the 5hmC levels in neural stem cells. Mice lacking Tet2 show abnormal hippocampal neurogenesis along with 5hmC alternations at different gene promoters and corresponding gene expression downregulation. Through the luciferase reporter assay, two neural factors Neurogenic differentiation 1 (NeuroD1) and Glial fibrillary acidic protein (Gfap) were down-regulated in Tet2 knockout cells. My results suggest that Tet2 regulates neural stem/progenitor cell proliferation and differentiation in adult brain.

Mutant mRNA and protein both contribute to the clinical manifestation of many repeat-associated neurodegenerative and neuromuscular disorders. The presence of nuclear RNA clusters is a feature shared amongst these diseases, such as C9ORF72/ALS and myotonic dystrophy 1/2 (DM1/2); however, this pathological hallmark has not been conclusively demonstrated in Huntington’s disease (HD) in vivo. Investigations into HD – caused by a CAG repeat expansion in exon 1 of the huntingtin (HTT) gene – have largely focused on toxic protein gain-of-function as a disease-causing feature, with fewer studies investigating the role of mutant HTT mRNA in pathology or pathogenesis. Here we report that in two HD mouse models, YAC128 and BACHD-97Q-ΔN17, mutant HTT mRNA is preferentially retained in the nucleus in vivo. Furthermore, we observed the early, widespread formation of large mutant HTT mRNA clusters (approximately 0.6 to 5 µm3 in size) present in over 50-75% of striatal and cortical neurons. Affected cells were limited to one cluster at most. Endogenous wild-type mouse Htt or human HTT mRNA containing 31 or fewer repeats did not form clusters. Additionally, the aberrantly spliced N-terminal exon 1-intron 1 RNA fragment, HTT1a, also formed clusters that fully co-localized with the mutant HTT mRNA clusters. These results suggest that multiple repeat-containing transcripts can coalesce to form a single cluster in a given cell. Treating YAC128 mice with antisense oligonucleotides efficiently silenced individual HTT mRNA foci but had limited impact on clusters. Our findings identify mutant HTT mRNA clustering as an early, robust molecular signature of HD, further supporting HD as a repeat expansion disease with suspected mRNA involvement.

Nicotine dependence is responsible for perpetuating the adverse health effects due to tobacco use, the leading cause of preventable death worldwide. Nicotine is an agonist for nicotinic acetylcholine receptors, which are enriched in the mesocorticolimbic and habenulo-interpeduncular circuitries, underlying nicotine reward and withdrawal, respectively. Drugs of abuse, including nicotine, induce stable neuroadaptations, requiring protein synthesis through regulation of transcription factors, epigenetic mechanisms, and non-coding RNAs. It also been shown that miRNAs in brain are regulated by nicotine and that miRNA dysregulation contributes to brain dysfunction, including drug addiction. While much is known about the neurocircuitry responsible for the behaviors associated with nicotine reward or withdrawal, the underlying molecular mechanisms of how these changes in behavior are induced are less clear. Using miRNA-/mRNA-Seq, we demonstrate that there are widespread changes in both miRNA and mRNA expression in brain regions comprising the mesocorticolimbic circuit after chronic nicotine treatment, and the habenulo-interpeduncular circuit during acute nicotine withdrawal. Conserved, differentially expressed miRNAs were predicted to target inversely regulated mRNAs. We determined that expression of miR-106b-5p is up-regulated and Profilin 2 (Pfn2), an actin-binding protein enriched in the brain, is down-regulated in the interpeduncular nucleus (IPN) during acute nicotine withdrawal. Further we show that miR-106b-5p represses Pfn2 expression. We demonstrate that knockdown of Pfn2 in the IPN is sufficient to induce anxiety, a symptom of withdrawal. This novel role of Pfn2 in nicotine withdrawal-associated anxiety is a prime example of this dataset’s utility, allowing for the identification of a multitude of miRNAs/mRNA which may participate in the molecular mechanisms underlying the neuroadaptations of nicotine dependence.