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.

Nonsteroidal anti-inflammatory drugs (NSAIDs) are effective in cancer prevention and have been shown to suppress the formation of colorectal tumors in both humans and rodents. The chemopreventive action of NSAIDs is believed to be mediated through induction of apoptosis in preneoplastic cells. However, the precise molecular mechanisms of NSAID-induced apoptosis remain unclear. Previous studies demonstrated that second mitochondria-derived activator of caspase (SMAC) plays an important role in executing NSAID-induced apoptosis in colon cancer cells. SMAC-knockout HCT116 colon cancer cells are resistant to NSAID-induced apoptosis, and are deficient in caspase activation and cytosolic release of cytochrome c and apoptosis inducing factor (AIF). In this study, we tested the hypothesis that SMAC regulates the release of cytochrome c and activation of caspase cascade through a feed-back amplification loop. We found that the N-terminal AVPI domain of SMAC is required for the proapoptotic activity of SMAC. Following NSAID treatment, SMAC promotes dissociation of caspase-3 from inhibitor of apoptosis proteins (IAPs), which in turn leads to mitochondrial dysfunction. We also studied the effects of pharmacological manipulation on NSAID-induced apoptosis by employing small-molecule compounds that functionally mimic the AVPI domain of SMAC. A synergistic action of NSAIDs and SMAC mimetics was observed in inducing a robust apoptotic response in several colon cancer cell lines, as well as in NSAID-resistant BAX-KO and SMAC-KO cell lines. SMAC mimetics appear to potentiate NSAID-induced apoptosis by stimulating the release of cytochrome c from mitochondria and activation of caspases. Together, these results suggest that SMAC may be useful as a target for the development of more effective chemopreventive agents.

Numerous reports correlate nitric oxide (NO) production with stalled S-phase progression, but the molecular mechanism(s) of cell cycle arrest remains elusive. Paradoxically numerous human tumors are exposed to vast quantities of nitric oxide and its reactive byproducts in situ, yet they continue to grow and proliferate. The dual-specificity phosphatase Cdc25A promotes cell cycle progression by dephosphorylating and activating cyclin-dependent kinases. Deregulation of Cdc25A is characteristic of human tumors, accelerates the cell cycle, and confers resistance to apoptosis, highlighting the importance of stringent Cdc25A control. Biochemical and structural analyses of Cdc25A indicate the potential for inhibition by S-nitrosation of the catalytic cysteine, providing a linkage between NO and cytostatic signaling. Thus, the overall hypothesis examined in this dissertation was that Cdc25A is a target and transducer of signaling by NO and NO-derived reactive species. The specific aims were to: 1) probe the susceptibility of Cdc25A to enzymatic regulation by NO-derived reactive species; 2) examine regulation of Cdc25A protein in nitrosatively challenged cells; and 3) determine whether Cdc25A activity was limiting for S-phase progression in nitrosatively-challenged tumor cells. My studies identified novel mechanisms controlling Cdc25A abundance and activity. S-Nitrosothiols rapidly S-nitrosated and inactivated Cdc25A in vitro, and Cdc25A activity was restored by reductants. Generation of nitrosative stress in cells either by iNOS-derived NO or the cell-permeable S-nitrosating agent S-nitrosocysteine ethyl ester (SNCEE) caused translational inhibition of Cdc25A via hyperphosphorylation and inhibition of the eukaryotic translational regulator eIF2á. Although iNOS-derived NO and SNCEE inhibited DNA synthesis coincident with Cdc25A loss, restoration of Cdc25A activity in nitrosatively-challenged cells did not alter DNA synthesis inhibition, distinguishing nitrosative inhibition of DNA synthesis from the canonical intra-S-phase checkpoint. SNCEE decoupled Cdc25A from ASK-1 and sensitized cells to chemotherapeutic-induced apoptosis, suggesting that Cdc25A suppression by nitrosative stress may lower the apoptotic threshold in nitrosatively-challenged cells by priming ASK-1 for activation. In summary, these studies describe novel regulation of Cdc25A translation and activity, and a model wherein selective inhibition of Cdc25A phosphatase-dependent and independent activities can occur under nitrosative stress, and implicate Cdc25A as a regulator of apoptotic threshold following nitrosative insult via priming of ASK-1.

There is an urgent need for novel effective drug regimens for the treatment of cancer. Current chemotherapy suffers from a slim therapeutic index, with significant toxicity from effective drug doses or tumor recurrence at low drug doses. Identifying synergistic interactions between drugs is a difficult process. To accelerate the discovery of potential drug combinations, I have developed a druggable genome siRNA synthetic lethal screen capable of rapidly identifying novel drug targets that would sensitize cancer cells to sublethal concentrations of microtubule destabilizing agents. I employed a high-throughput cell-based 16,560-siRNA screen to isolate a high-confidence list of genes that, when silenced, enhanced glioblastoma multiforme cancer cell chemosensitivity. Two gene products that were the major focus of my work were midline2 and the neurokinin receptor NK1R. Silencing of midline2, a PP2A-microtubule tether, sensitized cells to two microtubule destabilizing agents, vinblastine and disorazole C1, suggesting a mechanistic dependency of the phosphatidylinositol 3-kinase pathway on microtubule functionality. Combinations of phosphatidylinositol 3-kinase inhibitors with disorazole C1 and several vinca alkaloids confirmed this hypothesis. To verify microtubule destabilizing agent sensitization by NK1R silencing, I demonstrated a significant collaboration of neurokinin receptor NK1R antagonists with low concentrations of vinca alkaloids. These assay results and subsequent novel combination strategies demonstrate the tremendous ability of this synthetic lethal screen to predict potent collaborations between different classes of drugs, as well as identifying molecular constituents mediating those interactions.

Methyl-CpG binding protein 2 (MeCP2) is a protein associated with transcriptional repression of other proteins and its various mutants are found in Rett syndrome patients, which is a severe neurodevelopmental disease found in 1/15,000 females. It has specific binding affinity to methyl-CpG domain but also bind to architectural DNA structure. Here, its binding dynamics to Holliday junction structure was investigated by using single molecular detections; scanning confocal fluorescence microscope and wide-field evanescent field fluorescence microscope. Through these studies, we could distinguish the difference of transitions of the substrate with and without MeCP2. The effect of this MeCP2 binding to 4WJ will be discussed in terms of the structures and transition times.

Alzheimers disease (AD) is a neurodegenerative disorder that results from a loss of synaptic transmission and ultimately results in cell death. However, the mechanisms that induce neuronal cell death remain elusive. Amyloid plaques composed of amyloid fibrils (Ab) and neurofibrillary tangles (NFTs) composed of hyperphosphorylated tau (pTau) are the main pathogenic hallmarks of AD. Ab and NFT generation is influenced by reactive oxygen species and altered signaling pathways. Focal adhesion proteins assemble into intracellular complexes involved in integrin-mediated communication between the extracellular matrix (ECM) and the actin cytoskeleton, regulating many cell physiological processes. Interestingly, recent studies report that integrins bind to Ab fibrils, mediating Ab signal transmission from extracellular sites of Ab deposits into the cell and ultimately to the nucleus. Hydrogen peroxide-inducible-clone 5 (Hic-5) and paxillin are members of the group III LIM domain protein family that localize to both the nucleus and focal adhesions. Hic-5 and paxillin are expressed in numerous regions of the rat brain including cerebellum, striatum, prefrontal cortex, hippocampus, hypothalamus, thalamus, and spinal cord. While little is known about the specific roles of paxillin and Hic-5 in regulating focal adhesion signaling and gene expression within brain, non-genomic roles for both paxillin and Hic-5 in brain have been described. For example, in cultured neurons, paxillin is rapidly phosphorylated in the presence of fibrillar b-amyloid and colocalized with pTau, leading to altered focal adhesion turnover and loss of synaptic integrity. A functional role for Hic-5 in the brain was revealed by its ability to (1) decrease surface levels of dopamine transporter (DAT) in rat midbrain neuronal cultures and to (2) negatively affect dopamine uptake. A direct interaction between Hic-5 and DAT may be responsible for this effect. While these reports suggest biologic functions of Hic-5 and paxillin in brain, a detailed analysis of paxillin and Hic-5 expression and distribution in normal or AD brain has not been performed. Given the in vitro association between paxillin and b-amyloid-induced toxicity and Hic-5 response to oxidative stress, a blinded retrospective cross-sectional study of the human hippocampus for Hic-5 and paxillin was performed. The expression and subcellular distribution of Hic-5 and paxillin in AD and control hippocampus were determined by immunohistochemistry (IHC) from early and late-stage AD and age-matched control subjects. IHC was also used to examine the subcellular distribution of specific phosphorylated isoforms of paxillin. Laser scanning confocal microscopy (LSCM) was used to visualize or demonstrate colocalization of Hic-5, paxillin and phosphorylated isoforms of paxillin. Observations demonstrate changes in the subcellular distribution of Hic-5, paxillin and specific phosphorylated isoforms of paxillin within particular regions of the hippocampus in AD brain. Hic-5 and phosphorylated isoforms of paxillin colocalize with NFTs, while paxillin is predominantly found in reactive astrocytes (stellate-shaped) in the hippocampus of AD brains. Thus, important scaffolding proteins that link various intracellular signaling pathways to the ECM are modified and exhibit altered subcellular distribution in hippocampus during AD.