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.

Mutation of the mtATP6 gene, which encodes an essential subunit of the F0F1-ATP synthase (Complex V) in mitochondria, is known to cause a group of related encephalomyopathies. The ATP synthase acts as a hydrogen ion transporter that couples ion dissipation with ATP production. Diseases including NARP (neuropathy ataxia and retinitis pigmentosa) and MILS (maternally inherited Leighs syndrome) are caused by missense mutations in the ATP6 gene. Drosophila melanogaster, the common fruit fly, has a mitochondrial ATP6 missense mutation that models NARP/MILS diseases. Our aim is to develop a transgenic strategy where allotopic expression of a mitochondrial-targeted ATP6 mRNA may serve as a potential gene therapy for these devastating mitochondrial diseases. Mitochondria in metazoans are known to import nuclear encoded 5SrRNAs, which are thought to be essential for mitochondrial protein synthesis. We utilized a cluster of 100 individual 5S rRNA genes found at 56F region of the right arm of chromosome 2 in Drosophila melanogaster. Sequence comparisons revealed 17 groups of genomic variants and 14 processed rRNA counterparts. Identifying which, if any, of the known 5S rRNAs are competent for mitochondrial import was integral to our proposed gene therapy approach. A protocol was developed that utilizes gradient and percoll centrifugation steps to isolate highly purified mitochondria that lack detectable cytosolic contamination. RT-PCR and cloning were used to determine which 5S rRNAs were expressed and localized to the mitochondria. The cytoplasmic and mitochondrial derived clones and gDNA control clones support the assertion that, at least under normal in vivo conditions, ~ 60 % of the identified 5S rRNA genes are not expressed and are likely pseudogenes. One variant, 5S rRNA III, is predominantly expressed and localized to the mitochondria. Also, 8 novel and 3 possible 5S rRNA gene isoforms not currently categorized in sequence databases have been discovered. Clones capable of expressing chimeric rRNA::mRNAs in cells and in vivo were generated. These constructs could later be used to assess the ability of 5S rRNA to direct mitochondrial import of passenger mRNAs.

Human REV3L: Expression and Protein Interaction Studies Gregory N. Gan, Ph.D. University of Pittsburgh, 2007 REV3L is a specialized DNA polymerase essential for DNA damage-induced mutagenesis and for the ability of cells to tolerate DNA damage. Our understanding of REV3L biochemistry stems predominantly from studies done with the budding yeast homolog, Rev3. Yeast DNA polymerase zeta consists of two proteins, Rev3, the catalytic subunit, and Rev7, an accessory factor which enhances the activity of Rev3, in vitro. Yeast Rev1 acts as a scaffold by associating with Polymerase zeta and enhances its translesion bypass activity on a mismatch primer template. Because of the large size of the mammalian REV3L cDNA (10.6 kbp) and protein (353 kDa), work in this field has focused solely on functional genetic studies associated with disruption or knockdown of the gene. Loss of REV3L causes embryonic lethality in mice and leads to progressive chromosomal instability in Rev3L disrupted cell lines. In the developing mouse embryo, Rev3L transcript is found in all tissues. However, its expression pattern at the cellular level in the adult mouse has not been examined. Determining the protein interactions of REV3L will provide a better understanding of how the protein functions at the molecular level. In addition, elucidating how Rev3L is expressed and regulated in mammalian cells will indicate what role it may play in tissues of the adult organism and why it is essential for life. In order to study human REV3L biochemistry, this project focused on cloning, expressing, purifying and detecting full-length human REV3L protein. Human REV3L was hypothesized to interact with REV1 and/or REV7 based on knowledge about te yeast homologs. Furthermore, using a REV3L lacZ expression mouse model, the expression of REV3L in mouse organs containing proliferative cells was characterized. It was hypothesized that organs with highly proliferative tissue require REV3L. First, the results of immunoprecipitation studies demonstrated that full-length human REV3L interacts with REV1, but does not interact with REV7 in a DNA damage independent fashion. Preliminary analysis of deletion mutants indicates that the C-terminal domain of REV1 is required for the protein-protein interaction. Secondly, REV1 and REV3L are ubiquitinated in a DNA damage independent fashion and this covalent modification is not required for REV1-REV3L interaction. Finally, REV3L expression in mice is highest in testis, cardiac tissue and the smooth musculature of lung and intestines and low in lymphoid tissues. These sites of expression suggest that REV3L may be important for highly oxidative tissue compared to proliferative tissue. In summary, this dissertation provides insight on human REV3Ls protein-protein interaction with REV1 and REV7; their post-translational modification, and tissue-specific expression pattern in the adult mouse.

Alcohol (ethanol) has a prominent role in society and is one of the most frequently used and abused drugs. Despite the pervasive use and abuse of ethanol, the molecular mechanisms of ethanol action remain unclear. What is well known is that ethanol intoxication elicits a range of behavioral effects. These effects most likely occur through the direct action of ethanol on targets in the central nervous system. By studying behavioral effects, the role of individual targets can be determined. The function of &#x3B3;-amino butyric acid type A (GABAA) receptors is altered by ethanol, but due to multiple receptor subunits the exact role of individual GABAA receptor subunits in ethanol action is not known. This dissertation focused on the role of α1-containing GABAA receptors in ethanol action using gene knockin mice with ethanol insensitive α1 GABAA receptors. </br></br> In the second chapter, knockin mice were molecularly characterized and ethanol-induced behavioral effects were assessed. α1 was found to mediate acute tolerance to the motor ataxic effects of ethanol. In the third chapter, α1 involvement in ethanol induction of neuronal activity was assessed in discrete neuroanatomic regions using the immediate early gene c-fos. Specifically, c-fos immunohistochemistry was characterized after acute ethanol exposure, after chronic ethanol exposure, and finally during the ethanol withdrawal phase. α1 was found to be involved in ethanol-mediated effects in the dentate gyrus. </br></br> In the fourth chapter, α1 involvement in chronic tolerance to ethanol as well as physical dependence on ethanol was characterized. Results demonstrated that α1-GABAA-Rs play a role in the development of tolerance to chronic ethanol in motor ataxia. Intriguingly, α1 was implicated in dependence as assessed with ethanol withdrawal-related hyperexcitability. Knockin mice were more sensitive to ethanol's withdrawal-related hyperexcitability effects. In summary, this dissertation further supports α1 GABAA-Rs in the mechanism of ethanol action. By chiseling away at the various components of ethanol action we are beginning to elucidate the mechanism of ethanol action. Further elucidation of the mechanism of action of α1 GABAA-Rs in tolerance and dependence could deepen our understanding of the molecular mechanisms behind alcohol abuse and alcoholism. By understanding the molecular mechanisms of ethanol, alcohol abuse may be lessened and alcoholism could potentially be cured.

The gamma-aminobutyric acid (GABA) Type A receptor (GABAA-R) mediates the majority of rapid inhibition in the central nervous system and is the site of action for many clinically used drugs. GABAA-R mediated inhibition can occur via the conventional mechanism - the transient activation of synaptic receptors i.e. phasic inhibition, or via continuous activation of extrasynaptic, high affinity receptors by low concentrations of ambient GABA, leading to tonic inhibition. The GABAA-R alpha4 subunit is expressed at high levels in the dentate gyrus and thalamus and when partnered with the delta subunit, it is suspected to contribute to tonic inhibition. In vitro studies have found that GABAA-Rs containing alpha4 and delta are highly sensitive to ethanol and to competitive GABAA-R agonists such as gaboxadol and muscimol. In light of these findings, the central hypothesis tested in this thesis was that extrasynaptic GABAA-Rs mediate the depressant effects of these drugs. To provide a model for understanding the precise role of alpha4 containing GABAA-Rs in drug action, mice were engineered to lack the alpha4 subunit by targeted disruption of the Gabra4 gene. alpha4 Subunit knockout mice were viable and superficially indistinguishable from wild-type mice. In electrophysiological recordings, alpha4 knockout mice showed a lack of tonic inhibition in dentate granule cells and thalamic relay neurons. alpha4 knockout mice were also less sensitive to the behavioral effects of gaboxadol and muscimol. However, alpha4 knockout mice did not differ in ethanol-induced changes in anxiety, locomotion, ataxia, coordination, analgesia, or thermoregulation. These data demonstrate that tonic inhibition in dentate granule cells and thalamic relay neurons is mediated by extrasynaptic GABAA-Rs containing the alpha4 subunit and that gaboxadol and muscimol likely achieve their effects via the activation of this GABAA-R subtype. These data also suggest that GABAA-Rs containing the alpha4 subunit are not necessary for many acute behavioral responses to ethanol.