Recent studies have demonstrated that activation of the 5-HT4 receptors in the colonic mucosa can have healing and protective actions in experimental models of colitis. These actions include increased mucus secretion, increased epithelial proliferation, and enhanced epithelial migration. Since these studies involved chemically induced models of colitis, the current investigation was conducted to test whether a protective action of 5-HT4 receptor stimulation could be detected in Interleukin-10 knockout (IL-10 KO), which develop colitis spontaneously due to the absence of the anti-inflammatory cytokine, interleukin-10. Upon weaning, the IL-10 knockout mice were separated into two groups: an agonist group and a vehicle control group. The agonist group received 1 mg/kg tegaserod in a vehicle consisting of 0.9% saline each day by enema of dimethyl sulfoxide (DMSO) in saline each day, while the control group received daily enemas of vehicle over the course of 21 days. Several outcome measures were used to assess the effectiveness of the treatment. To evaluate the severity of colitis, disease activity index was monitored, and histologic damage was blindly scored. Administration of tegaserod by enema to the IL-10 KO mice had a significant protective effect on the treated mice. The disease activity index (DAI) of agonist treated mice was significantly better than that of vehicle treated mice over time (p<0.001; 2-way ANOVA). Mice treated with vehicle had a more significant decline in health over time versus the agonists, with more blood present in feces and a looser/diarrhea-like consistency in stool. The histological damage score (HDS) was also improved by 5-HT4 agonist treatment (p<0.05, t-test). Sections of vehicle treated colons showed significantly greater damage, including epithelial erosions, the presence of polymorphonuclear cells, and abnormal crypt architecture (cryptitis), than those treated with the 5-HT4 receptor agonist tegaserod. During the 21-day course of the current investigation, there was no difference in the survival data between the two groups. These data, when taken together, suggest that administration of the 5-HT4 receptor agonist tegaserod via enema to IL-10 KO mice has a greater healing and protective effect than seen in the IL-10 KO mice that received vehicle. We proposed to test the hypothesis that treatment of IL-10 knockout colitis with a 5-HT4 receptor agonist will attenuate the development of colitis and have healing and protective effects in the colons of the treated mice.

Polychlorinated biphenyls (PCBs) are industrial compounds whose ubiquitous environmental contamination has been known since the late 1960's. Incidents of human intoxication have been reported, most notably, the "Yusho" incident that affected over 1600 people in Japan in 1968. A broad spectrum of adverse effects are produced by PCBs, including some neurological symptoms. While examination of the toxic effects of PCBs has received much attention, there has been little work on the neurotoxicity of PCBs. The intent of the experiments presented in this thesis is to provide further information on the central neurotoxicity of PCBs in mice. A commercial mixture of PCBS (Aroclor 1254) was used in these experiments. The first set of experiments was initiated to evaluate the behavioral effects of orally administered Aroclor 1254 in mice. A dose of 500 mg/kg depressed spontaneous motor activity at times of 15 min to 3 hr after gavage, with the peak effect observed at 45 min. A dose-response curve generated at 45 min revealed statistically significant effects only at the highest dose, 500 mg/kg. Aroclor 1254 had no effect on the other behavioral measures evaluated - performance on the rotor rod and inverted screen and effect on pentylenetetrazol-induced convulsions. These results suggest that Aroclor 1254 may be exerting a CNS depressant effect as evidenced by a suppression of spontaneous activity in the absence of impaired motor coordination. In order to examine further the suggested CNS depressant effect, the interaction of Aroclor 1254 with a known CNS depressant, pentobarbital, was investigated. Pretreatment with a single dose of Aroclor 1254 (500 mg/kg) significantly enhanced pentobarbital-induced sleep time at pretreatment times of 0, 0.75, 2, 4 and 8 hr. A dose-response relationship was demonstrated with doses of 5 to 500 mg/kg of Aroclor‘ 1254 given at the pretreatment time of peak effect, 2 hr. By contrast, subchronic (14 day) administration of Aroclor 1254 (30 or 100 mg/kg) reduced pentobarbital induced sleep time in a dose-dependent fashion when pentobarbital was given 45 min after the last dose of Aroclor 1254 with a further reduction when given 24 hr after Aroclor 1254. Results of studies of the disposition of 14C-pentobarbital following Aroclor 1254-pretreatment suggested that acute Aroclor 1254-pretreatment inhibits pentobarbital metabolism while subchronic PCB-pretreatment enhances pentobarbital metabolism as evidenced by tissue levels of pentobarbital and metabolites. Therefore, it appears that the observed increase in pentobarbital-induced sleep time is not a result of altered CNS sensitivity or combined depressant effects of the two compounds, but rather is due to Aroclor 1254-induced alterations in the pharmacokinetics of pentobarbital. The next series of experiments was aimed at evaluating the effects of Aroclor 1254 on neurochemistry. Aroclor 1254 produced concentration-dependent alterations in the following parameters measured in vitro: inhibition of neurotransmitter and precursor uptake, enhancement of neurotransmitter release, enhancement of synaptosomal 45Ca++ uptake, stimulation of ATP-supported mitochondrial 45Ca++ uptake, inhibition of synaptosomal Na+ /K+ - and Mg++- ATPases, and inhibition of mitochondrial Mg++ -ATPases. Effective concentrations of Aroclor 1254 in these in vitro systems ranged from 10-6 to 10-4 M. Acute (up to 500 mg/kg) and subchronic (90 day) administration of Aroclor 1254 had no effect on' uptake of neurotransmitters or precursors by synaptosomes isolated from Aroclor 1254-treated mice. Nor was there any effect on ATPase activities in synaptosomes or mitochondria isolated from mice exposed to acute or subchronic (14 day) Aroclor 1254 regimens. This disparity between the results of in vitro and in vivo exposure to Aroclor 1254 prompted studies of the disposition of 14C-PCBs. A time course of radioactivity in plasma and brain following a 500 mg/kg dose of 14C-PCBS (a mixture of PCBs with 50% Cl by weight and uniformly labeled with 14C)compared favorably with that of the behavioral activity of Aroclor 1254. No differences in brain regional distribution of 14C-PCBs was observed. Most importantly, levels of radioactivity in synaptosomes and mitochondria of sub fractionated brains of mice that had been exposed to a behaviorally active dose of 14C-PCBS (500 mg/kg) were comparable to tissue levels in isolated synaptosomes and mitochondria that had been incubated with concentrations of 14C-PCBs (10-5 to 10-4M) that altered neurochemistry in vitro. Thus, effective concentrations of 14C-PCBS are apparently achieved in subcellular organelles of brain following oral administration of a behaviorally active dose. The lack of observable neurochemical effects in Aroclor 1254-treated mice may be due to compensatory mechanisms that maintain homeostasis in the intact brain. In summary, Aroclor 1254 has been shown to produce a depression of spontaneous motor activity in mice, changes in pentobarbital-induced sleep time (an effect that is probably a manifestation of changes in the pharmacokinetic behavior of pentobarbital rather than a direct effect of Aroclor 1254 on brain), and alterations in a number of neurochemical events in vitro. These results may help to expand the limited data base on the neurotoxicity of PCBs.

Studies were undertaken to characterize the immune status of mice bearing the Lewis lung carcinoma (LLC) and to maximize the cure rate of LLC and Madison l09 carcinoma (Ml09) bearing mice by employing the immunomodulator maleic vinyl ether (MVE) in combination with surgery or radiotherapy. The pattern of immunodeficiency in mice bearing the LLC appeared to be in contrast to most studies. LLC mice with a minimal tumor burden were found to have a diminished ability to phagocytize sheep erythorcytes (SRBC) or elicit an antibody response to the same antigen. Vascular clearance and phagocytic uptake of 51Cr-labeled SRBC into reticuloendothelial (RE) organs exhibited a triphasic response. Liver phagocytosis and vascular clearance were markedly suppressed 24 and 48 hours after tumor inoculation. The initial decrease in RE function was followed by a phase of increased activity which progressively decreased to control levels with increasing tumor burden. IgM antibody forming cells to SRBC were reduced to 18% of control values 7 days after inoculation and by day 27 no antibody forming cells could be detected in the LLC bearing mice. In contrast, delayed type hypersensitivity to SRBC remained intact until day 17 and no significant changes in inflammatory activity were noted. Surgical excision of the primary tumor burden on day l4 had no effect on survival time but partially restored the suppressed immune functions. Accordingly, immunotherapeutic regimens were designed to treat early in the disease state or after cytoreductive therapy in an effort to have immunocompetent animals with a minimal tumor burden. Yet, systemic MVE treatment by itself, or in combination with surgical excision or local radiotherapy of the primary tumor was ineffective in prolonging life span. Intravenous MVE treatment also failed to exhibit antitumor activity against the LLC when the metastatic tumor burden was markedly reduced by multiple cyclophosphamide injections. In contrast to systemic MVE treatment, an acute intralesional injection of MVE proved to be efficacious by increasing life span by 36%. Moreover, the combination of intralesional MVE with local radiation of the primary LLC tumor significantly prolonged survival time over the radiation controls. The M109 was shown to be relatively responsive to MVE treatment. Multiple injections of MVE in doses ranging from l0 to 50 mg/kg significantly prolonged the life span of the host and inhibited the growth of the primary tumor. The efficacy of MVE therapy appeared to be dependent on the distribution of the polymer. Weekly intralesional injections of MVE were most effective in prolonging life span early in the disease state, presumably by inhibiting the metastatic process. As the tumor metastasized to the lung only MVE introduced directly into the metastatic tumor bed by intrapleural or inhalation administration proved to be efficacious in prolonging life span. These data emphasize the importance of drug distribution in the treatment of neoplasia with nonspecific immunotherapy.

Chemotherapy-induced peripheral neuropathy (CIPN) is a side-effect of chemotherapy causing pain in the hands and feet. In particular, paclitaxel causes CIPN lasting for years without effective treatment. There is a strong need for analgesics to both treat and prevent CIPN. One system containing multiple targets to treat CIPN is the endogenous cannabinoid system. This system consists of cannabinoid type 1 (CB1) and type 2 (CB2) receptors primarily expressed on presynaptic neurons and cells of the immune system, respectively. Inhibition of monoacylglycerol lipase (MAGL), which hydrolyzes the endogenous cannabinoid 2- arachidonoylglycerol (2-AG), with JZL184 or MJN110 produces antinociceptive and anti- inflammatory effects in rodent pain models. In this dissertation, we test the hypothesis that MAGL inhibitors will both reverse and prevent mouse paclitaxel-induced mechanical allodynia. JZL184 and MJN110 reversed paclitaxel allodynia in dose-dependent manners with ED50 values (95% C.L.) of 8.4 (5.2-13.6) and 1.8 (1.0-3.3) mg/kg. Using genetic and pharmacologic approaches, we demonstrate that the anti-allodynic effects of both inhibitors require both cannabinoid receptors. As CIPN treatment could require repeated dosing, we demonstrate that repeated administration of 4 mg/kg JZL184 for six days produces anti-allodynic effects in contrast to tolerance development after repeated treatment with 40 mg/kg. We also show that MJN110 attenuates paclitaxel-induced inflammation in the spinal cord and dorsal root ganglia (DRG), namely monocyte chemoattractant protein-1 (MCP-1, CCL2) and phosphorylated p38 MAPK (phospho-p38) expression. Using the conditioned place preference (CPP) paradigm, we demonstrate that MJN110 produces a CPP in paclitaxel-treated, but not in control mice. As CIPN develops during chemotherapy, we also show that JZL184 does not interfere with the anti- proliferative and anti-apoptotic effects of paclitaxel in A549 or H460 lung cancer cell lines. Lastly, we show that co-administration of MAGL inhibitors with paclitaxel prevents the development of allodynia. Co-treatment with 5 mg/kg MJN110 or 40 mg/kg JZL184 prevents allodynia up to one or two week(s), respectively, after paclitaxel cessation. Treatment with 40 mg/kg JZL184 prevents allodynia in both CB1 (+/+) and (-/-) mice, suggesting that prevention is CB1-independent. Taken together, these results suggest that MAGL is a viable target for both the treatment and prevention of paclitaxel-induced allodynia in mice.

Several members of the schweinfurthin family are potent and selective inhibitors of cancer cell growth in the NCI 60-cell cancer screen. The schweinfurthins display unique activity in this screen which suggests that these compounds have a previously unexploited target. This activity encourages development of the schweinfurthins as anti-cancer agents; however, the scarcity of the natural products has hindered biological evaluation. For this reason, a program was initiated to synthesize the natural products and analogues thereof. These efforts have made 5 natural product schweinfurthins and over 60 analogues available for biological evaluation. Studies presented herein evaluate the biological activity, intracellular localization, and potential protein interactions of schweinfurthins analogues. To investigate the potent and differential effects of these compounds, a two-cell line screen was developed incorporating a sensitive cell (SF-295) and insensitive cell line (A549). The structure-function data obtained from this screen identified a schweinfurthin site amenable to modification. Based on this finding, fluorescent schweinfurthins were designed and synthesized. These compounds retain schweinfurthin-like potency and activity which is critical for their study. The intracellular localization of these schweinfurthins was investigated using fluorescence microscopy. These studies identified the lysosomes and peri-nuclear organelles as potential sites of schweinfurthin activity. Additionally, we describe the attachment of a fluorescent schweinfurthin to a solid support, which was utilized to identify protein-schweinfurthin interactions. This approach identified vimentin as a potential target of schweinfurthin activity.

Erythropoietin (Epo) acts through the erythropoietin receptor (EpoR) to influence survival, proliferation, and differentiation of erythroid progenitors. EpoR was recently found to be expressed in several cancers as well. The functional consequences of this expression are poorly understood but several findings suggest that EpoR contributes to the survival, migration, and drug resistance of cancer cells. The effects of Epo are mediated through activation of several signaling pathways including the Jak/Stat5 pathway and the Ras/Raf/MAPK pathway. The mevalonate pathway provides several products essential for cell function and survival. Cholesterol, ubiquinone, and dolichol are only few examples of such products. The isoprenoid intermediates, farnesyl pyrophosphate (FPP) and geranylgeranyl pyrophosphate (GGPP), are also generated from the mevalonate pathway and are essential for the process of isoprenylation, which is an essential post-translational modification of several cellular proteins. Many of the isoprenylated proteins are members of the Ras superfamily of small GTPases. Epo activates several members of this family including Ras, Rac and Rap1. The aim of this proposal is to understand the interaction between the mevalonate biosynthetic pathway and EpoR signal transduction and to investigate the mechanism(s) through which the isoprenoid intermediates affects EpoR signaling. We have shown that two major isoprenoid derivatives, GGPP and dolichol, are essential for EpoR signal transduction and surface expression. Furthermore, we identified new roles for EpoR in cancers of non-erythroid origin and characterized signaling pathways involved and means to manipulate them for the ultimate goal of finding new avenues for cancer treatment.

The goal of this dissertation research is to determine how post translational modifications of Mfn2, Drp1 and PP2A B'beta regulate protein function and stability. Mitofusin 2 (Mfn2) and Dynamin related protein 1 (Drp1) work in opposing manners to balance mitochondrial morphology and maintain organelle function. Loss of balanced fission and fusion results in mitochondrial dysfunction, a major contributor to the pathology of many neurodegenerative diseases and cancer. Regulation of fission is mediated through the reversible posttranslational modifications of Drp1, which has been shown to be (de)phosphorylated on multiple residues, (de)sumoylated, nitrosylated and is believed to be ubiquitinated. Yet there are currently no known post-translational modifications of Mfn2 that regulate fusion. The first two experimental chapters of this thesis focus on the regulation of PKA induced phosphorylation of mitochondrial fission and fusion proteins. In Chapter 2 I utilize a Mfn2 phospho Ser442 specific antibody to determine the phosphorylation state of both over-expressed and endogenous neuronal Mfn2. Using this technique I found that under the conditions studied, Mfn2 is not PKA phosphorylated on Ser442. To look at the requirement of Mfn2 Ser442 in promoting mitochondrial elongation and protecting hippocampal neurons from excitotoxic cell death, Hela cells and primary hippocampal neurons (PHN) were evaluated by immunofluorescence. Results from these studies suggest Mfn2's neuroprotective effects require Ser442, and are separate from its function to promote mitochondrial fusion, and independent of PKA phosphorylation. Chapter 3 utilizes biochemical and immunofluorescence techniques to determine whether the PKA induced phosphorylation of Drp1 on Ser656 and the nitrosylation of Drp1 on Cys663 are mutually exclusive events. While previous work demonstrated the requirement of Drp1 Cys663 in mediating nitric oxide (NO) induced mitochondrial fragmentation and cell death, results presented herein demonstrate Cys663 and Ser656 act independently to blunt, but not inhibit, mitochondrial fragmentation following NO treatment. Additional work demonstrated that blocking Drp1 nitrosylation with a conserved Cys663Val mutation enhanced the PKA mediated phosphorylation of Ser656 both biochemically and morphologically, while direct Drp1 nitrosylation had no effect. These results suggest Drp1 nitrosylation has no functional consequence, while Cys663 appears to be structurally important residue. The final chapter characterizes the formation of a novel E3 ubiquitin ligase complex, composed of KLHL15 and Cul3, that mediates the specific degradation of protein phosphatase 2A (PP2A) regulatory subunit B'beta. PP2A B'beta is expressed in neurons where it functions to regulate catecholamine synthesis through the dephosphorylation and inactivation of Tyrosine hydroxylase (TH). Here I establish the role of a novel kelch domain containing protein, KLHL15, which serves as an adaptor molecule, bridging the E3 ubiquitin ligase Cul3, though the N-terminal BTB domain, with the substrate B'beta through the C-terminal kelch domain. Additionally, the requirement of B'beta Tyr52 for its interaction with KLHL15 suggests MAPK phosphorylation of this residue might regulate the inclusion of PP2A B'beta in the complex, ultimately regulating downstream dopamine synthesis.

The Hippo signaling pathway, originally identified in Drosophila melanogaster and later shown to be conserved in mammals, is essential in regulating organ size and maintaining tissue homeostasis. It is now clear that functional inactivation of the Hippo pathway is common in variety of cancers and promotes their development and progression. This suggests re-activation of Hippo pathway activity may prove an effective anti-cancer therapy. Here, we describe two small molecule activators of the Hippo pathway that we have recently uncovered from a focused small-molecule inhibitor screen.

Biosynthesis of lipid mediators including the leukotrienes, lipoxins, eoxins, resolvins, and protectins entails the lipoxygenase (LOX) catalyzed synthesis of an allylic epoxide intermediate, designated as leukotriene A4 (LTA4) and LTA analogues, and heretofore considered too unstable for direct structural characterization. In this dissertation I developed methods involving biphasic reaction conditions for the LOX-catalyzed synthesis of LTA epoxides and their structural analysis by NMR. As proof of concept, human 15-LOX-1 was shown to convert 15S-hydroperoxyeicosatetraenoic acid (15S-HPETE) to the LTA analogue, 14,15-LTA4 (thus identifying eoxin A4). Using this methodology I then showed that recombinant Arabidopsis AtLOX1, an arachidonate 5-LOX, converts 5S-HPETE to the trans epoxide LTA4, and 5R-HPETE to the cis epoxide 5-epi-LTA4. The results are reconciled with a mechanism based on a dual role of the LOX non-heme iron in LTA epoxide biosynthesis. The same methodology was used to structurally characterize the LTA-related epoxides from omega-3 fatty acids, including identification of the proposed epoxide intermediate in protectin biosynthesis.

The serotonin transporter (SERT) mediates clearance of the neuromodulator serotonin (5-HT) following its release. The clinical efficacy of antidepressant drugs that block SERT activity indicates that serotonin transporter function is important for the maintenance of proper mood and affect. Biochemical studies demonstrate that SERTs form oligomeric protein complexes. The present study asks whether SERT function depends on interactions between subunits of the SERT oligomer under physiological conditions. Our data indicate that SERT function is intrinsically cooperative. Interactions between wild-type and mutant SERTs alter intrinsic properties of SERT function such as the Na+-dependent activation of 5-HT transport. Furthermore, the balance of 5-HT transport to 5-HT-induced current is sensitive to interactions between SERT and an as yet unidentified enabling factor. The model we present to explain our data postulates that a ternary complex composed of 5-HT, SERT, and the novel enabling factor dynamically regulates SERT function. Our findings have relevance to SERT function in the native context and transporter biology in general, and could lead to the development of novel therapies for diseases of serotonergic dysfunction.