Methylphenidate (MPH) is the most commonly prescribed drug to treat Attention Deficit Hyperactivity Disorder (ADHD). ADHD is now considered a life-long disorder; therefore, patients take MPH from adolescence into adulthood, highlighting the need for research studying chronic MPH use. MPH increases dopamine and norepinephrine within the synaptic cleft; therefore, chronic use of MPH may lead to changes within important dopaminergic pathways. One pathway, the mesolimbic pathway, includes the hippocampus, an area where adult neurogenesis occurs. We investigated the effects of chronic low and high doses of MPH on neurogenesis and examined levels of a few key proteins linked to cell proliferation in the hippocampus. Low dose MPH appears to increase cell proliferation and cell survival in the hippocampus, and these effects are accompanied by increases in vascular endothelial growth factor (VEGF), the receptor for brain-derived neurotrophic factor (TrkB), and beta-catenin. While high dose MPH may initially increase neuronal proliferation, newly-generated neurons are unable to survive long-term, and decreases in VEGF, TrkB, and beta-catenin are observed with chronic high dose MPH. Another major dopaminergic pathway is the nigrostriatal pathway, which is involved in motor control and degenerates with Parkinson’s disease. Chronic use of MPH appears to sensitize dopaminergic neurons within this pathway to the Parkinsonian toxin 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP), but the cause of this sensitization is unknown. The autooxidation of excess dopamine forms dopamine-quinones that lead to free radical production, but the antioxidant, glutathione, can protect neurons. However, we showed that chronic MPH increases dopamine-quinone formation and causes a subsequent glutathione depletion within the striatum. Therefore, oxidative stress may sensitize dopamine neurons to MPTP. We also assessed the vulnerability of dopaminergic neurons in the nigrostriatal pathway to MPTP after chronic MPH in females. Interestingly, proestrus (high estrogen) females were more sensitive to MPTP than anestrus (low estrogen) females. Similar to males, chronic MPH caused a depletion in glutathione that was further decreased following MPTP exposure. However, chronic MPH did not significantly alter dopaminergic neuronal numbers or quinone formation in females. These studies highlight some of the potential effects of chronic MPH use.

Despite 35 years of clinical trials, there has been little improvement in one-year survival rates with any chemotherapeutic regimen for the treatment of metastatic melanoma due to resistance to all known agents. Regardless of advances in detection and prevention, diagnosis of metastatic disease remains a death sentence. Resistance mechanisms, including aberrant kinase signaling and drug transport pumps, indicate a need for identification of other therapeutic targets that impinge upon multiple signaling pathways. The Abl family of non-receptor tyrosine kinases (c-Abl, Arg) has been indicted as a causative force in leukemia for more than three decades; however, their role in solid tumors has only recently been described. We first demonstrated that activated Abl family kinases promote breast cancer development and progression, and recently identified them to be novel therapeutic targets in metastatic melanoma cells by demonstrating that they promote proliferation, survival, invasion, and metastasis. We now present evidence that inhibitors of Abl family kinases abrogate resistance to a number of commonly used chemotherapeutics (i.e., 5-fluorouracil, cisplatin, paclitaxel, camptothecin) in a panel of breast cancer cells. We proceed to show that inhibitors of Abl family kinases, likewise, sensitize both breast cancer and melanoma cells to doxorubicin by blocking cell proliferation and dramatically inducing apoptosis. These findings were extended to advanced multi-drug resistant melanoma cells, in which we show for the first time that c- Abl promotes expression of the drug transporter, ABCB1, during acquired resistance, and drugs that inhibit c-Abl/Arg prevent ABCB1 expression and function. Moreover, c-Abl/Arg also promote acquired chemoresistance independent of ABCB1 by modulating multiple survival pathways. We demonstrate that c-Abl/Arg promote chemoresistance by upregulating STAT3, preventing doxorubicin-mediated conversion of NF-κB into a transcriptional repressor, activating an HSP27/p38/Akt survival pathway, and modulating ERK signaling. Therefore, c-Abl/Arg promote chemoresistance in highly resistant melanoma cells by impinging on drug transporter and cell survival pathways. Taken together, these data indicate that c-Abl/Arg inhibitors are likely to reverse acquired resistance in metastatic melanomas harboring activated c-Abl/Arg, and thus, may be effective in a combination regimen.

Migraine is one of the most common neurological disorders. The pathological conditions that initiate and sensitize afferent pain signaling are poorly understood. The goal of this study is to identify the ion channels and signaling proteins underlying activation and sensitization of meningeal nociceptors.In trigeminal neurons retrogradely labeled from the cranial meninges, approximately 80% responded to a pH 6.0 application with a rapidly activating and desensitizing ASIC-like current. Pharmacological experiments and kinetics analysis demonstrated that dural afferent pH-sensitive currents were mediated via activation of ASIC3. In addition, applications of decreased pH solutions were able to excite these neurons and generate action potentials. In awake animals, application of decreased pH solutions to the dura produced dose-dependent facial and hindpaw allodynia, which was also mediated through activation of ASIC3. Accumulating evidence indicates that meningeal inflammation induced sensitization of dural afferents contributes to migraine headache. We have demonstrated here that in the presence of mast cell mediators, dural afferents showed a decreased pH threshold and increased activity in response to pH stimuli both in vivo and in vitro. These data provide a cellular mechanism by which decreased pH in the meninges directly excites afferent pain-sensing neurons potentially contributing to migraine headache. It also indicates that inflammatory events within the meninges could sensitize afferent pain signaling and result in increased sensitivity of dural afferents.Intracranial Interleukin-6 (IL-6) levels have been shown to be elevated during migraine attacks, suggesting that this cytokine may facilitate pain signaling from the meninges. Here we reported that in awake animals, direct application of IL-6 to the dura produced dose-dependent facial and hindpaw allodynia via activation of the ERK signaling pathway. IL-6 application was also able to increase neuronal excitability in a manner consistent with phosphorylation of Nav1.7. These data provide a cellular mechanism by which IL-6 in the meninges causes sensitization of dural afferents therefore contributing to the pathogenesis of migraine.These findings are discussed in relation to how activation and sensitization of primary afferent neurons might initiate migraine pain signaling and how the research included in this dissertation relates to the development of new therapeutic strategies for migraine.

Glucocorticoids (GCs) are frequently prescribed pharmacological agents most notably for their immunosuppressant effects. Endogenous GCs mediate biological processes such as energy metabolism and tissue development. At the cellular level, GCs bind to the Glucocorticoid Receptor (GR), a cytosolic receptor that translocates to the nuclei upon ligand binding and alters gene transcription. Among a long list of genes activated by GCs is the Glucocorticoid Induced Leucine Zipper (GILZ). Although GC induced GILZ expression has been well established in lymphocytes, little is known whether cardiomyocytes respond to GCs by inducing GILZ. Unlike lymphocytes, in which GCs induce apoptosis and GILZ mediates GC induced apoptosis, cardiomyocytes respond to GCs by gaining resistance against apoptosis. We determined GILZ expression pattern in cardiomyocytes in vivo and in vitro. Our data demonstrate GILZ induction in cardiomyocytes both in vivo and in vitro by GCs and point to H9C2 cell line as a valid model for studying the biological function of GILZ in cardiomyocytes. I have also determined GILZ functions as GC induced cytoprotective protein against the known cardiac toxicant Doxorubicin. Finally I have determined GILZ stabilizes Bcl-xL pro-survival protein, providing a possible mechanism of cytoprotection in cardiomyocytes.

While there is currently active debate over possible therapeutic applications of marijuana and cannabis-based compounds, consistently their primary drawbacks have been the psychoactive properties, dependence, and abuse potential. Prolonged administration of ∆9-tetrahydrocannabinol (THC), the primary psychoactive constituent in marijuana, demonstrates both tolerance and physical withdrawal in both preclinical and clinical studies. Repeated THC administration also produces CB1 receptor adaptations in the form of reduced activation of receptors, along with a downregulation of membrane surface receptors, in many brain regions involved in THC-associated behaviors. The increased need for drug to maintain therapeutic effects, and a withdrawal syndrome following discontinuation of use, are common risk factors in drugs of abuse. Recently, compounds have been developed that prolong the availability of the major naturally occurring endogenous cannabinoids, anandamide (AEA) and 2-arachidonoylglycerol (2-AG), through inhibition of their catabolic breakdown by fatty acid amide hydrolase (FAAH) and monoacylglycerol lipase (MAGL), respectively. The overall objectives of this research are to elucidate the physiologic roles of these two endogenous ligands and to determine if either can produce beneficial therapeutic effects without negative cannabis-like CNS effects. Therefore, we tested the impact of acute and prolonged blockade of FAAH and MAGL on a variety of cannabinoid-mediated behaviors and on precipitated cannabinoid withdrawal. Despite that acute blockade of FAAH and MAGL produce similar efficacy in reducing nociceptive responses, and both can reduce THC-induced somatic withdrawal, sustained blockade of these enzymes leads to remarkably different adaptations in CB1 receptor functioning. Namely, prolonged elevations in brain 2-AG leads to marked antinociceptive tolerance, cross-tolerance to exogenous cannabinoid agonists, and physical dependence. In contrast, sustained elevations in brain anandamide continues to dampen pain responses without apparent signs of physical withdrawal, loss of CB1 receptor activation as measured by [35S]GTPγS, or receptor downregulation as measured by [3H]CP,55940. These results suggest that chronic 2-AG elicits greater compensatory changes in CB1 receptor functions than anandamide. With similar efficacy in most therapeutic endpoints tested, and evidence of reduced impact on long-term function of the endocannabinoid system, these results distinguish FAAH as a more promising therapeutic target to treat pain and other conditions than MAGL.

Previous studies from this and other laboratories have shown that the novel microtubule poison, JG-03-14, which binds to the colchicine binding site of tubulin, has the capacity to promote both autophagy and apoptosis in breast tumor cells, as well as interfering with endothelial cell function and potentially disrupting tumor vasculature. The current work was designed to investigate the interaction between JG-03-14 and cell culture models of colon carcinoma and melanoma, specifically HCT116 human colon carcinoma cells and B16F10 murine melanoma cells. In both cases, JG-03-14 promoted death in the bulk of the treated population. FACS analysis, DAPI and TUNEL staining indicated that only a small fraction of the cell population was undergoing apoptosis; furthermore, there was no evidence of mitotic catastrophe (micronuclei in bi-nucleated cells). Staining with acridine orange and monodansylcadaverine as well as electron microscopy demonstrated the formation of autophagic vesicles, consistent with the cells undergoing extensive autophagy. Cell cycle analysis indicated that cells had arrested in the G2/M stage, with evidence of a hyperdiploid population. Residual surviving cells appeared to be in a state of senescence; furthermore, the senescent cells failed to recover proliferative capacity, indicating that the cells were reproductively dead. Toxicity studies in cardiomyocytes with comparisons to combretastatin and taxol indicated that JG-03-14 was the less toxic of the microtubule poisons. In summary, our studies indicate that JG-03-14 induces autophagic and reproductive cell death in HCT116 colon carcinoma cells and B16F10 murine melanoma cells with limited toxicity to the normal cells that are generally susceptible to taxol and combretastatin. The possibility of alternative mode(s) of cell death (autophagy and irreversible senescence) induced by JG-03-14 makes it a potentially useful candidate as a chemotherapeutic drug that could be used to treat cancers resistant to apoptosis. The relative lack of toxicity of JG-03-14 provides additional support for its potential use in the treatment of malignancies.

Since its discovery, it was appreciated that the antifolate pemetrexed had multiple targets within folate metabolism. This laboratory was instrumental in showing that pemetrexed elicited its primary action as a thymidylate synthase inhibitor. Unusual for an antifolate, pemetrexed showed significant clinical activity against malignant pleural mesothelioma and non-small cell lung cancer. Accordingly, the FDA recently issued first-line approvals for pemetrexed in these diseases, leading us to question whether the effects of pemetrexed on other folate-dependent targets could explain this atypical clinical activity of the drug. Studies in this dissertation showed that in addition to thymidylate synthase inhibition, pemetrexed was also an inhibitor of aminoimidazolecarboxamide ribonucleotide formyltransferase (AICART), the second folate- dependent enzyme of de novo purine synthesis. Consequent of AICART inhibition, pemetrexed caused robust activation of a key energy-sensing regulatory enzyme of the PI3K-AKT signal transduction pathway, AMP-activated protein kinase (AMPK). AMPK activation resulted from xx accumulation of the AMP-mimetic, ZMP, behind the AICART block. Constituents of the PI3K- AKT cascade are frequently deregulated in human carcinomas, uncoupling nutrient supply from proliferative capacity. Therefore, interventions that reinstate control over aberrant signaling along this axis, such as AMPK activation, are of significant cancer therapeutic interest. The cellular consequences of AMPK activation in response to pemetrexed were assessed. In particular, effects on the downstream target of PI3K-AKT signaling, the mammalian target of rapamycin complex 1 (mTORC1), were studied. Unlike targeted mTORC1 inhibitors, such as rapamycin and its analogs, pemetrexed-mediated activation of AMPK also signaled to mTOR- independent controlling elements of protein and lipid synthesis, highlighting additional benefits of AMPK activating agents that extend beyond effects on mTOR signaling. We therefore propose that the unusual activity of pemetrexed in mesothelioma and non-small cell lung cancer is due in part to effects on signaling processes downstream of AMPK activation. These findings present a novel approach to AMPK activation secondary to an AICART block, define pemetrexed as a molecularly targeted agent, and ultimately extend the utility of antifolates beyond their traditional function.

CB1 receptors (CB1R) mediate the psychoactive and therapeutic effects of cannabinoids including ∆9-tetrahydrocannabinol (THC), the main psychoactive constituent in marijuana. However, therapeutic use is limited by side effects and tolerance and dependence with chronic administration. Tolerance to cannabinoid-mediated effects is associated with CB1R adaptations, including desensitization (receptor-G-protein uncoupling) and downregulation (receptor degradation). The objectives of this thesis are to investigate the regional-specificity in CB1R function and regulation. Previous studies have investigated CB1Rs in a subset of regions involved in cannabinoid effects, but an inclusive regional comparison of the relative efficacies of different classes of cannabinoids to activate G-proteins has not been conducted. A novel unbiased whole-brain analysis was developed based on Statistical Parametric Mapping (SPM) for 3D-reconstructed mouse brain images derived from agonist-stimulated [35S]GTPgS autoradiography, which has not been described before. SPM demonstrated regional differences in the relative efficacies of cannabinoid agonists methanandamide (M-AEA), CP55,940 (CP), and WIN55,212-2 (WIN) in mouse brains. To assess potential contribution of novel sites, CB1R knockout (KO) mice were used. SPM analysis revealed that WIN, but not CP or M-AEA, stimulated [35S]GTPgS binding in regions that partially overlapped with the expression of CB1Rs. We then examined the role of the regulatory protein Beta-arrestin-2 (βarr2) in CB1R adaptations to chronic THC treatment. Deletion of βarr2 reduced CB1R desensitization/downregulation in the cerebellum, caudal periaqueductal gray (PAG), and spinal cord. However in hippocampus, amygdala and rostral PAG, similar desensitization was present in both genotypes. Interestingly, enhanced desensitization was found in the hypothalamus and cortex in βarr2 KO animals. Intra-regional differences in the magnitude of desensitization were noted in the caudal hippocampus, where βarr2 KO animals exhibited greater desensitization compared to WT. Regional differences in βarr2-mediated CB1R adaptation were associated with differential effects on tolerance, where THC-mediated antinociception, but not catalepsy or hypothermia, was attenuated in βarr2 KO mice. Overall, studies using SPM revealed intra- and inter-regional specificity in the function and regulation of CB1Rs and underscores an advantage of using a whole-brain unbiased approach. Understanding the regulation of CB1R signaling within different anatomical contexts represents an important fundamental prerequisite in the therapeutic exploitation of the cannabinoid system.

The availability of murine genetic models is extremely advantageous to studying gastrointestinal function, but the benefits afforded by studying enteric neurons in mice has been hindered by their accessibility. Fetal (E13) and 2 day post-natal (P2) enteric neuron cell lines (IM-FEN and IM-PEN, respectively) were recently developed from the H-2Kb-tsA58 immortomouse. Our goal was to identify the electrophysiological properties of these cell lines and clarify their utility as a model of enteric neurons. IM-PEN cells stained positively for the neuron specific markers βIII-tubulin and PGP9.5 and were negative for the glial cell marker S100. Detection of mRNA for TRPA1, TRPV1, ClCa1, KCa3.1, NaV1.3 and NaV1.9 were present while CaV2.2 and TASK1 were very faint. No significant difference was observed in the passive membrane properties of IM-FEN and IM-PEN. The cells had depolarized resting membrane potentials -29.8 ± 0.9mV (n=30) and high input resistances ranging from 552 ± 104MΩ (IM-FEN, n=6) to 728 ± 128MΩ (IM-PEN, n=20). xiv In current clamp, hyperpolarizing current was given to obtain a holding potential of -60mV or -80mV, yet neither IM-FEN (n=6) nor the IM-PEN cells (n=20) were able to generate action potentials in response to depolarizing pulses. In whole cell voltage clamp depolarization induced an inward current which was identified as an L-type Ca2+ channel. Niflumic acid inhibited the outward current as well as the tail currents indicating a ClCa current supporting the mRNA data. A volume sensitive chloride channel was also identified that was DCPIB sensitive (n=7) and removed when chloride was replaced with gluconate (n=4), displaying characteristics of ICl,swell. As a result IM-PEN cells had a high chloride conductance resulting in a depolarized membrane potential, which is a characteristic of immature neurons. The transcription factor MASH1 has been found to be required for enteric neuron differentiation. Transfection of MASH1 after 4 and 8 days did not alter the electrophysiological characteristics of IM-PEN (n=6). We conclude that IM-PEN may represent immature enteric neurons and are a useful model to examine the effect of factors required for the development of enteric neurons.

Pain of various etiologies (e.g., visceral, inflammatory) can be a debilitating disorder that presents a problem of clinical relevance. While it is known that ∆9-tetrahydrocannabinol (THC) the primary psychoactive constituent found in marijuana produces analgesia in various rodent models of pain, its pharmacological properties are overshadowed by its psychomimetic effects. THC is the primary phytocannabinoid found in marijuana though other prevalent constituents such as the phytocannabinoids (e.g., cannabidiol (CBD), cannabinol (CBN), cannabichromene (CBC), tetrahydrocannabivarin (THCV)) may possess antinociceptive actions without the psychomimetic effects associated with THC. Indeed, these phytocannabinoids act upon the endocannabinoid system (ECS) that is comprised of the CB1 and CB2 cannabinoid receptors, endogenous ligands (anandamide (AEA), 2-arachidonoyolglycerol (2-AG)), and endocannabinoid biosynthetic and catabolic enzymes. We hypothesize that phytocannabinoids as well as endocannabinoid catabolic enzyme inhibitors reduce nociception preclinical models of pain. In the first series of studies, the antinociceptive effects of prevalent phytocannabinoids were evaluated in the acetic acid stretching test, a rodent visceral pain model. While CBN and THC both produced antinociceptive effects via a CB1 mechanism of action, CBC, and CBD had no effect on nociception. Conversely, THCV antagonized the antinociceptive effects of THC. These results suggest that various constituents of marijuana may interact in a complex manner to modulate pain. Since the THC and CBN displayed their effects via specific endogenous cannabinoid receptors, we investigated whether increasing endocannabinoids block nociceptive behavior. Blockade of the catabolic enzyme fatty acid amide hydrolase (FAAH) elevates AEA levels and elicits antinociceptive effects, without psychomimetic issues associated with THC. Similarly, blockade of another endocannabinoid catabolic enzyme monoacylglycerol lipase (MAGL) elevates (2-AG) and elicits antinociceptive effects. Therefore, we tested the hypothesis that FAAH and/or MAGL inhibition blocks nociception in the acetic acid abdominal stretching model, and the LPS-induced allodynia (i.e. painful response to a non-noxious stimuli) model of inflammation. Genetic deletion or pharmacological blockade of FAAH or pharmacological blockade of MAGL significantly reduced the total number of abdominal stretches in the visceral pain model. Additionally, blockade of both enzymes simultaneously produced an enhanced antinociceptive effect versus blocking the enzymes individually. These effects were mediated through CB1 receptors. However, in the LPS-induced allodynia model, FAAH inhibited anti-allodynic effects through a CB1 and CB2 receptor mechanismn. In both assays other potential targets of FAA substrates (i.e., mu-opioid, TRPV1, and PPAR-alpha receptors) did not play an apparent role in FAAH inhibited antinociceptive responses. Taken together, these results illustrate that targeting the endocannabinoid system via direct acting agonists such as the phytocannabinoids, or indirect methods (i.e. inhibiting degradative enzymes of the endogenous cannabinoids), represents a promising strategy to treat pain.