In spontaneously hypertensive rats (SHR) the hypersensitivity of the renal vasculature to angiotensin II (Ang II), compared to Wistar-Kyoto rats (WKY), appears to be the determining factor in the development and progression of hypertension. Recent evidence indicates that the ERK cascade and NAD(P)H oxidase generation of superoxide are involved in smooth muscle contraction, and Phospholipase D (PLD) generation of phosphatidic acid is involved in activation of ERK and NAD(P)H. Importantly, Ang II-mediated PLD activity is greater in aortic smooth muscle from SHR compared with WKY; however, this signaling pathway has not been examined in the kidney vasculature. The purpose of these studies were to define Ang II-mediated signal transduction mechanism(s) involved in PLD regulation in WKY and SHR preglomerular smooth muscle cells (PGSMCs). The goals of this study were to determine: 1) whether Ang II-mediated PLD activity is greater in SHR; 2) the Ang II signaling pathway(s) responsible for regulating PLD activity, and whether they are altered in SHR; and 3) whether PLD-mediated generation of phosphatidic acid is involved in Ang II-induced activation of the ERK cascade. The data indicates that the mechanisms leading to activation of PLD are similar in WKY and SHR and PLD is required for Ang II activation of ERK; however, Ang II more potently activates PLD in SHR. Further analysis indicates that the AT2 receptor inhibits AT1 receptor/RhoA-dependent activation of PLD through a nitric oxide/cGMP-dependent phosphorylation of RhoA at serine 188, which promotes RhoGDI inhibition of RhoA. These experiments expose two key differences between WKY and SHR PGSMCs: 1) SHR have an increased AT1/AT2 receptor ratio; and 2) SHR are less sensitive to nitric oxide and cGMP. Therefore, the hypersensitivity of the SHR to Ang II may be due to an imbalance in Ang II receptors and/or impaired AT2 receptor-mediated signaling within the kidney vasculature.

Huntington's Disease (HD) belongs to the CAG repeat family of neurodegenerative diseases and is characterized by the presence of an expanded polyglutamine (polyQ) repeat in the huntingtin (htt) gene product. PolyQ-expanded htt accumulates within large aggregates in various subcellular compartments, but are more often localized within the nucleus. The sequestration of proteins essential to cell viability may be one mechanism that accounts for toxicity generated by polyQ-expanded proteins. Nuclear inclusions containing polyQ-expanded htt recruit the transcriptional cofactor, CREB-binding protein (CBP). PolyQ toxicity appears to involve alterations of gene transcription and reduced neuronal cell viability. In the HT22 hippocampal cell line, we found that toxicity within individual cells induced by polyQ-expanded htt was associated with the localization of the mutant htt within either nuclear or perinuclear aggregates. However, in addition to CBP recruitment, we found that CBP ubiquitylation and degradation can be selectively enhanced by polyQ-expanded htt. Thus, selected substrates may be directed to the ubiquitin/proteasome-dependent protein degradation pathway (UPP) in response to polyQ-expanded htt within the nucleus. While both the polyQ domain and the histone acetyltransferase domain (HAT) of CBP have been found to interact with polyQ-expanded htt, deletion of either domain does not affect its enhanced degradation in the presence of polyQ-expanded htt in HT22 cells. Thus, enhanced degradation of CBP in cells expressing polyQ-expanded htt may not involve a direct interaction between CBP and htt. It seems likely specific enzymes in the UPP may be activated by htt and selectively target proteins such as CBP for degradation. Since molecular chaperones are found in the aggregates containing polyQ-expanded proteins, misfolding of polyQ-expanded proteins may play a key role in polyglutamine disease pathogenesis. In a number of some studies, HDJ-2, a member of DnaJ family molecular chaperones, was found to reduce aggregation and toxicity induced by polyQ-expanded proteins. In contrast, we show that HDJ-2 is unable to rescue aggregate formation of polyQ-expanded htt in transfected HEK293 fibroblast cells, nor is it recruited into these aggregates in vivo in a HD transgenic mouse model. Thus, molecular chaperone effects on polyQ-expanded protein induced toxicity could be cell-type specific or influenced by the developmental state of the culturable cells. These factors must be considered in any attempts to use chaperones as potential therapeutic targets in polyglutamine diseases.

Deregulation of normal cell cycle control is essential for malignant transformation. The Cdc25A dual-specificity phosphatase promotes cell cycle progression by dephosphorylating and activating the cyclin-dependent kinases. Cdc25A has oncogenic and anti-apoptotic activity and is overexpressed in many human tumors. The mechanisms by which Cdc25A is overexpressed in human cancer are unknown. Cdc25A protein levels are downregulated by cell cycle checkpoints in response to genotoxic stress; cell cycle checkpoints are frequently compromised in tumor cells. In addition, under normal physiologic conditions, the half-life of Cdc25A protein is short. Alterations to physiologic Cdc25A regulatory mechanisms could be sufficient to result in oncogenic overexpression of this cell cycle regulatory protein. While Cdc25A downregulation in response to genotoxic stress occurs through defined signal transduction pathways, regulation of Cdc25A protein levels in non-stressed cells is poorly understood. The purpose of this thesis was to examine the physiological regulation of Cdc25A protein levels in human tumor cells. The goals of our studies were: 1) to investigate regulatory mechanisms of Cdc25A protein levels in non-stressed human tumor cells; 2) to understand how Cdk2 kinase activity regulates Cdc25A protein levels; and 3) to explore the mechanism by which Cdk2 kinase activity regulates Cdc25A protein turnover. The results of our studies revealed that Cdc25A protein half-life in non-stressed interphase cells is regulated, in part, by Cdk2 kinase activity, and that Cdk2 does not regulate Cdc25A turnover by affecting several known signal transduction pathways that control Cdc25A protein stability. Recent reports on the role of ubiquitin ligases in physiologic Cdc25A turnover have identified several phosphorylation sites that are necessary for efficient Cdc25A recruitment to ubiquitin ligases. The kinase(s) responsible for phosphorylating these serine residues remain to be identified, although Cdk2 could be one prime candidate. While initial reports of the interactions between Cdc25A and Cdk2 focused on an auto-amplification feedback loop that results in increased catalytic activity of both proteins, it now appears that Cdk2 also regulates Cdc25A stability and plays an important role in regulating Cdc25A protein levels during interphase progression.

The overall hypothesis of this thesis project was that bleomycin hydrolase (BLMH) has biologically specific and unique functions in the central nervous system (CNS). BLMH is a multifaceted papain superfamily cysteine protease that has importance in drug metabolism. The physiological functions of this protease are unknown as are factors regulating its expression. Immunohistochemical examination of B6.129Blmhtm1Geh/J null and control animals showed no gross abnormalities; however, marked global astrogliosis was observed in the null aged animals. To define the role of BLMH in the brain, the behavioral phenotype of hybrid [129S6-Blmhtm1Geh/J X B6.129 Blmhtm1Geh/J]F1 null and littermate controls was characterized using multiple behavioral paradigms. Deletion of Blmh was found to result in deficits among young animals in water maze probe trials. Retention of target platform location during the probe trials requires both learning and memory as well as sensory and locomoter skills. No overt sensory or motor deficits were noted in Blmh null F1 hybrids. The profile of BLMH expression and its regulation in the CNS was studied next. Inducible transcription of BLMH was evaluated in the context of a putative role in the processing of MHC I epitopes. BLMH was found to be differentially regulated in microglia and astrocytes. In microglia, Blmh protein was significantly induced by gamma interferon or tumor necrosis factor a, whereas in astrocytes, no change in protein expression was observed. Treatment of microglial derived cell lines with both gamma interferon and tumor necrosis factor a revealed synergistic effects between the cytokines. BLMH protein induction was accompanied by increased Blmh mRNA. These results suggest that cell specific regulation of BLMH is an important control mechanism for this protease. These data also provide further evidence for a targeted immune related biological function for BLMH. It is concluded that BLMH potentially has multiple unique and biologically important functions within the brain.

Mitochondria are essential for the function of all mammalian tissues, serving functions, such as ATP generation. Neurons are highly dependent on ATP production and consume more energy than other cells for their metabolism. Mitochondria are semi-autonomous organelles that contain their own DNA (mtDNA). Mutations and deletions in mtDNA lead to mitochondrial dysfunction that compromise neuronal viability. From the many approaches taken to investigate the role of mitochondria in neurodegeneration; however, few have focused on mtDNA dynamics. First, I investigated whether mtDNA replication impairment plays a role in neurotoxicity. For this purpose, I tested two neurotoxins, glutamate and rotenone, which induce neuronal damage by different mechanisms. Our results show that mitochondrial dysfunction induced by different neurotoxins does not correlate with effects on mtDNA replication. Glutamate, at excitotoxic concentrations, does not affect mtDNA replication while rotenone induces a time and concentration dependent decrease of mtDNA replication. Also, rotenone effect on mtDNA replication seems to be independent of its acute toxic effect. Several mechanisms have been proposed as responsible for rotenones toxicity, such as complex I inhibition and increased ROS production. Our experiments ruled out the implication of these two mechanisms in rotenone-induced mtDNA replication decrease. Mitochondrial nucleotides are key regulators of mtDNA replication. However, our experiments show that rotenone effect on mtDNA replication does not correlate with mitochondrial nucleotide imbalances. Therefore, our results suggest that rotenone-induced mtDNA replication decrease is mediated by a yet to be described mechanism. Mitochondrial function requires the coordination of all processes that take place at this organelle. I studied if a reduction in mtDNA replication could have an effect on mitochondrial membrane potential, movement and morphology. Experiments with rotenone treatments that reduce mtDNA replication have demonstrated that mtDNA replication decrease does not correlate with overall mitochondrial dysfunction at the time points used in this study. In summary, this dissertation provides a first attempt to study the dynamics of mtDNA upon neurotoxin exposure. I conclude that rotenone decreases mtDNA replication in the absence of overt toxicity. This effect could play an important role in its long term effects as neurons could accumulate mitochondria with decreased mtDNA content.

Earlier studies indicate that G₁ mediates enhanced renovascular responses to Ang II in SHR. The potentiation of Ang II by the G₁ pathway is blocked by pretreatment with pertussis toxin, an inhibitor of G₁. The G₁ pathway is also activated by receptors for PP-fold peptides; NPY, PYY, and PYY₃₋₃₆. Therefore, we hypothesize that in genetically predisposed models of hypertension PP-fold peptides augment renovascular responses to endogenous Ang II. Our study shows that LPNPY, an analogue of NPY selective for the Y₁ receptor, potentiates Ang II responses in SHR, but not WKY, kidneys in vitro. LPNPY'fs ability to potentiate Ang II renovascular responses is dependent on the Y₁ receptor and an intact G₁ pathway. The renal expression of Y₁ receptors is similar in SHR versus WKY. Our study also demonstrates that PYY₃₋₃₆, selective for the Y₂ receptor, potentiates renovascular responses to Ang II in SHR, but not WKY, in vitro. PYY₃₋₃₆ is dependent on an intact Y₂-G₁ pathway, and the Y₂ receptor is similarly expressed in the kidney of both strains. In comparing the PP-fold peptides, PYY is the most efficacious at potentiating Ang II-induced renovascular responses. Lower levels of these peptides have little effect on renal vasculature. Yet, these peptides are released with other G₁ coupled agonists, namely NE that acts on ∀₂-adrenoceptors. We observe a significant enhancement of Ang II-induced renal vasoconstriction with low level combinations of UK 14,304, an ∀₂-adrenoceptor agonist, and PYY/NPY. We demonstrate, in SHR, that nerve stimulation potentiates renal vasoconstrictive responses to Ang II. This interaction is dependent on an intact Y₁-G₁ pathway suggesting that NPY plays a predominate role in increasing renal vascular responses. PYY is a more potent agonist at augmenting renal vascular responses than is PYY₃₋₃₆. Blockade of the conversion of PYY to PYY₃₋₃₆ via a DPPIV inhibitor, P32/98, results in an increase in MABP in SHR. We also demonstrate that this effect is dependent on the Y₁ receptor pathway. This project demonstrates that PP-fold peptides may play a role in the etiology of genetic hypertension. This project is significant because it suggests a link between a high fat diet, sympathetic activation, and hypertension in a genetically susceptible animal.

The dissertation is based on the concept that pathogenesis of essential hypertension involves the kidney. In this regard, renal sodium ion transporters, responsible for sodium reabsorption and fluid balance, may be important candidates in hypertension. Many lines of evidence indicate that the sympathetic nervous system, via renal nerves, plays an important role in the pathogenesis of essential hypertension. The goals of the dissertation were to: 1) identify whether renal sodium ion transporter expression is altered in an animal model of essential hypertension, the Spontaneously Hypertensive Rat (SHR) and if so, its physiological significance; 2) determine the role of the sympathetic nervous system in regulation of renal sodium ion transporters and 3) elucidate the underlying molecular mechanism. Among the renal sodium transporters profiled in the SHR, the bumetanide-sensitive Na-K-2Cl cotransporter (BSC-1) of the thick ascending limb was found to be most elevated; suggesting that increase in BSC-1 abundance may contribute to altered tubular function in SHR. In support of this conclusion, our results demonstrate that the natriuretic response to furosemide is greater in SHR versus its normotensive counterpart the Wistar-Kyoto Rat (WKY), resulting in normalization of blood pressure. Additionally, progression from pre-hypertensive to hypertensive state in SHR is accompanied by an increase in steady state protein levels of BSC-1 and its distribution to plasma membrane. Thus our biochemical and pharmacological data are consistent with the hypothesis that BSC-1 is involved in the pathogenesis of hypertension in SHR. Activation of renal sympathetic efferent nerves releases norepinephrine and, if chronic, increases arterial pressure. We hypothesize that long-term exposure of kidney to norepinephrine increases expression of renal sodium transport systems. Our results indicate that chronic 14-day norepinephrine infusion increased abundance of BSC-1 along with an increase in mean arterial blood pressure; an effect that could explain altered sodium handling associated with an over-active renal sympathetic system. Finally, studies in an immortalized thick ascending limb cell line show that regulation of BSC-1 by norepinephrine involves post-transcriptional control mechanisms via the â-adrenoceptor-cAMP-PKA pathway, and involves in part MAP kinases and that the á-adrenoceptor negatively regulates BSC-1. Further elucidation of the mechanism would suggest new strategies to treat diseases associated with an over-active sympathetic nervous system such as essential hypertension.

The orphan nuclear receptors Pregnane X Receptor (PXR) and Constitutive Androstane Receptor (CAR) have been proposed to play an important role in the detoxification of xeno- and endobiotics by regulating the expression of detoxifying enzymes and transporters. We showed that the combined loss of PXR and CAR resulted in a significantly heightened sensitivity to bile acid toxicity in a sex-specific manner. The increased sensitivity in males was associated with genotype-specific suppression of bile acid transporters and loss of bile acid-mediated down regulation of small heterodimer partner, whereas the transporter suppression was modest or absent in the female DKO mice. The liver X receptors (LXRs), including the alpha and beta isoforms were identified as sterol sensors that regulate cholesterol and lipid homeostasis and macrophage functions. We found that activation of LXRÑ in transgenic mice or with LXR ligands confers a female-specific resistance to lithocholic acid (LCA)-induced hepatotoxicity and bile duct ligation (BDL)-induced cholestasis. In contrast, LXR alpha and beta double knockout mice (LXR DKO) exhibited heightened cholestatic sensitivity. The LCA and BDL resistance in transgenic mice was associated with an increased expression of bile acid detoxifying sulfotransferase 2A (SULT2A) and selected members of the bile acid transporters. We also showed that genetic or pharmacological activation of the orphan nuclear receptor liver X receptor (LXR) sensitized mice to cholesterol gallstone disease (CGD) induced by a high cholesterol lithogenic diet. LXR-promoted CGD was associated with increased expression of several canalicular transporters that efflux cholesterol and phospholipids, leading to higher biliary concentrations of cholesterol and phospholipids. The biliary bile salt concentration was reduced in these mice, resulting in increased cholesterol saturation index (CSI). Interestingly, the lithogenic effect of LXR was completely abolished in the low-density lipoprotein receptor (LDLR) null background or when the mice were treated with Ezetimibe, a cholesterol-lowering drug that blocks the intestinal dietary cholesterol absorption. We propose that LXRs have evolved to have dual function in maintaining cholesterol and bile acid homeostasis.

The overall hypothesis of this dissertation was that Cdc25B is an important regulator of the cellular response to DNA damage and defection from the normal response could promote tumorigenesis by enhancing genomic instability. Conventionally, DNA damage is generally thought to inhibit Cdc25 functionality to induce cell cycle arrest. However, recently a crucial role of Cdc25B in the cell cycle resumption after DNA damage was identified. To understand the precise regulation of Cdc25B following DNA damage, I examined the effect of mechanistically distinct DNA damaging agents on Cdc25B. Secondly, experiments were performed to elucidate how Cdc25B participates in the recovery from the checkpoints induced cell cycle arrest. Finally, the mechanism by which Cdc25B contributes to anti-BPDE induced tumorigenesis was investigated. The results of our studies revealed that Cdc25B was rapidly induced following DNA damage and levels of Cdc25B regulated the number of cells existing G2 into mitosis. Increased expression of Cdc25B did not affect the G2/M checkpoint engagement immediately following DNA damage; however, increased Cdc25B reduced the time required for cell cycle resumption. Using UV irradiation as the prototypic damaging agent, the increase in Cdc25B levels was found to be regulated by ATR/Chk1 via post-transcriptional mechanism, potentially by affecting Cdc25B protein stability. Furthermore, Cdc25B was found to be essential for anti-BPDE-induced neoplastic transformation. Additionally, Cdc25B facilitated resumption in the presence of DNA damage following anti-BPDE thus indicating that Cdc25B contributes to tumorigenesis by regulating premature recovery from checkpoints without completion of DNA repair. Finally, increased Cdc25B activated checkpoints in the absence of overt DNA damage suggesting that Cdc25B enables genomic instability by promoting selection of cells with deregulated checkpoint signaling. To conclude, studies presented in this dissertation identified a novel role of Cdc25B following DNA damage and elucidated the molecular mechanisms by which Cdc25B regulates anti-BPDE induced tumorigenesis.

The neuromuscular junction (NMJ) has a structure that is optimized to relay signals from nerve to muscle. As part of its organizational scheme, certain muscular proteins, like nicotinic acetylcholine receptors (AChRs), are clustered preferentially at the NMJ. Clustering of AChRs at the NMJ is essential for efficient neurotransmission. The major factor which strengthens and sustains the NMJ localization of AChRs is the motoneuron-derived glycoprotein agrin. Agrin acts via a receptor complex that includes the muscle-specific receptor tyrosine kinase (RTK) MuSK. Although MuSK has been well characterized, the signaling pathway by which it mediates agrin-induced clustering of AChRs remains elusive. Understanding this process will provide insights for the treatment of a variety of muscle weakness disorders, such as myasthenia gravis and muscular dystrophy. For instance, some forms of myasthenia gravis are caused by autoantibodies directed against MuSK. Future therapies could be designed to circumvent dysfunctional portions of the clustering cascade. In addition, studying this pathway may reveal mechanisms important for the formation and maintenance of synapses. A bacterial two-hybrid assay was used to screen a rat muscle cDNA library for binding partners of the cytoplasmic domain of mouse MuSK. The mammalian homologue of the Drosophila protein, tumorous imaginal discs, tid1, was identified as a specific MuSK binding protein. Interestingly, tid1 has recently been shown to bind to and to modulate the signaling of the ErbB2 and Trk families of RTKs. Biochemical assays confirmed that tid1 binds to MuSK. Tid1 was colocalized with AChR clusters in cultured myotubes and at rodent NMJs. Denervation dispersed tid1 and AChRs from the postsynaptic membrane of the NMJ. Overexpression of the N-terminal half of tid1 in myotubes induced aneural AChR clustering. Short hairpin RNA (shRNA)-mediated knockdown of tid1 inhibited spontaneous and agrin-induced AChR clustering in cultured myotubes and resulted in the disassembly of preformed NMJs in skeletal muscles of adult mice. Furthermore, the amplitudes of spontaneous miniature endplate potentials (MEPPs) and evoked endplate potentials (EPPs) were significantly reduced in muscles electroporated with tid1-targeted shRNA. These results implicate tid1 as a novel NMJ player and define a new class of molecules in the agrin/MuSK signaling cascade.