Peroxisome proliferator-activated receptor gamma (PPAR-gamma) belongs to the nuclear hormone receptor subfamily of transcription factors. PPARs are expressed in key target tissues such as liver, fat, and muscle and thus they play a major role in the regulation of energy balance. Because of PPAR-gamma's role in energy balance, signals originating from the gut (e.g., GIP), fat (e.g., leptin), muscle (e.g., myostatin), or bone (e.g., GILZ) can in turn modulate PPAR expression and/or function. Of the two PPAR-gamma isoforms, PPAR-gamma2 is the key regulator of adipogenesis and also plays a role in bone development. Activation of this receptor favors adipocyte differentiation of mesenchymal stem cells, while inhibition of PPAR-gamma2 expression shifts the commitment towards the osteoblastogenic pathway. Clinically, activation of this receptor by antidiabetic agents of the thiazolidinedione class results in lower bone mass and increased fracture rates. We propose that inhibition of PPAR-gamma2 expression in mesenchymal stem cells by use of some of the hormones/factors mentioned above may be a useful therapeutic strategy to favor bone formation.

Mutations in the LGI1 gene predispose to a hereditary epilepsy syndrome and is the first gene associated with this disease which does not encode an ion channel protein. In zebrafish, there are two paralogs of the LGI1 gene, lgi1a and lgi1b. Knockdown of lgi1a results in a seizure-like hyperactivity phenotype with associated developmental abnormalities characterized by cellular loss in the eyes and brain. We have now generated knockdown morphants for the lgi1b gene which also show developmental abnormalities but do not show a seizure-like behavior. Instead, the most striking phenotype involves significant enlargement of the ventricles (hydrocephalus). As shown for the lgi1a morphants, however, lgi1b morphants are also sensitized to PTZ-induced hyperactivity. The different phenotypes between the two lgi1 morphants support a subfunctionalization model for the two paralogs.

Eukaryotic cells begin to assemble discrete, nucleoplasmic repair foci within seconds after the onset of exposure to ionizing radiation. Real-time imaging of this assembly has the potential to further our understanding of the effects of medical and environmental radiation exposure. Here, we describe a microirradiation system for targeted delivery of ionizing radiation to individual cells without the need for specialized facilities. The system consists of a 25-micron diameter electroplated Nickel-63 electrode, enveloped in a glass capillary and mounted in a micromanipulator. Because of the low energy of the beta radiation and the minute total amount of isotope present on the tip, the device can be safely handled with minimum precautions. We demonstrate the use of this system for tracking assembly of individual repair foci in real time in live U2OS human osteosarcoma cells. Results indicate that there is a subset of foci that appear and disappear rapidly, before a plateau level is reached approximately 30 min post-exposure. This subset of foci would not have been evident without real-time observation. The development of a microirradiation system that is compatible with a standard biomedical laboratory expands the potential for real-time investigation of the biological effects of ionizing radiation.

Neuronal cell migration and cellular differentiation, major phases in the assembly process o f the mammalian neocortex, involve considerable organelle and cellular motility. While the cytoskeletal organization of migrating neurons is well documented, and the involvement of the cytoskeleton in modulating intracellular membrane transport events during neuronal cell differentiation is well appreciated, identification of selective cytoskeletal components underlying these processes is only beginning to emerge. Observations over the past two decades reveal that myosin motors are involved intimately in multiple actin-dependent membrane movements, including vesicular trafficking, organelle localization and organization, endocytosis, exocytosis, phagocytosis, lamellopodial extension, and the more classically defined functions such as cytokinesis, contractility, and cell motility or migration. Accordingly, our studies have been directed toward the identification and characterization of unconventional myosins that may participate in neuronal cell migration and/or differentiation events within the developing mammalian brain. Our analyses identified two myosin isoforms that contribute to a novel unconventional myosin class. We have cloned, sequenced, and designated these myosin isoforms as myr 8a and 8b (8th unconventional myosin from rat). Structurally, the head domain o f myr 8 contains a large N-terminal extension composed of multiple ankyrin repeats similar to myosin phosphatase. The motor domain is followed by a single putative light chain binding domain. The tail domain of myr 8a is comparatively shortwith a net positive charge, whereas the elongated tail domain of myr 8b bears an overall neutral charge and reveals several streches o f poly-proline residues. Phylogenetic analysis indicates that myr 8 is sufficiently divergent from known myosins as to comprise a new class of myosins. Northern analyses demonstrate that the m yr 8 myosins are expressed predominantly in the nervous system, and are detected principally at developmental timeperiods. Indirect-im munofluorescent studies reveal a pattern of im m unoreactivity within forming neuronal and astroglial cell processes located throughout the developing brain. Taken together these data suggest that this novel myosin may play a crucial role in membrane biogenetic events during neuronal and astroglial cell differentiation. Given the increasing identification of neurological dysfunctions that arise as a consequence of defective myosins, as well as from other cytoskeletal components, it is essential to unravel the selective roles in which this novel unconventional myosin may participate during neocortical development.

Severe vitamin E deficiency results in lethal myopathy in animal models. Membrane repair is an important myocyte response to plasma membrane disruption injury as when repair fails, myocytes die and muscular dystrophy ensues. Here we show that supplementation of cultured cells with α-tocopherol, the most common form of vitamin E, promotes plasma membrane repair. Conversely, in the absence of α-tocopherol supplementation, exposure of cultured cells to an oxidant challenge strikingly inhibits repair. Comparative measurements reveal that, to promote repair, an anti-oxidant must associate with membranes, as α-tocopherol does, or be capable of α-tocopherol regeneration. Finally, we show that myocytes in intact muscle cannot repair membranes when exposed to an oxidant challenge, but show enhanced repair when supplemented with vitamin E. Our work suggests a novel biological function for vitamin E in promoting myocyte plasma membrane repair. We propose that this function is essential for maintenance of skeletal muscle homeostasis.

Background: The pathology of diabetic neuropathy involves oxidative stress on pancreatic b-cells, and is related to decreased levels of Insulin-like growth factor 1 (IGF-1). Acylated steryl b-glucoside (PR-ASG) found in pre-germiated brown rice is a bioactive substance exhibiting properties that enhance activity of homocysteine-thiolactonase (HTase), reducing oxidative stress in diabetic neuropathy. The biological importance of PR-ASG in pancreatic b-cells remains unknown.

The N-methyl-D-aspartate receptor is the main coincidence detector in the brain. It is known to be necessary for many forms of learning and memory. Interestingly, the NR2 subunit composition of the NMDA receptor is modulated endogenously according to the location in the brain and the age of the animal, with the NR1 subunit being ubiquitously expressed. In the forebrain regions, including the cortex, hippocampus, striatum, and amygdala, the NR2A and NR2B subunits are the primary subunits expressed. While it is known that a high NR2B:NR2A ratio enhances memory and cognition, it is not directly known, the effects of a low NR2B:NR2A ratio. In this project, the effects of modulating the NR2A:NR2B ratio on multiple forms of learning and memory are explored by the use of a NR2A transgenic mouse. Our transgenic mice overexpress the NR2A subunit in the forebrain regions, driving the NR2A:NR2B ratio toward the expression of the NR2A. As the NR2B subunit is known to favor learning and memory; we also further explore the role of the N-terminal and membrane domains and the Cterminal domain in the observed enhancements. Our data indicate that a high NR2A:NR2B ratio constrains multiple forms of long-term memory in our transgenic animals. Additionally, we have observed additional forms of enhanced learning and memory in the NR2B transgenic mice that were not tested previously. We were able to show that the NR2B C-terminal tail, and thus the intracellular signaling cascades, is responsible for the enhancements seen in the NR2B animals. Using the NR2A and NR2B transgenic mice, we also investigated the long-held hypothesis that a low NR2B:NR2A ratio would be beneficial to hemorrhagic stroke recovery. Until now this hypothesis has only been investigated by the use of pharmaceuticals, whereby the NR2B subunit is antagonized. We found that while several physiological factors, including neurological deficit and survival rate were unchanged, lesion size and percent edema were significantly less in the NR2A transgenic mice than the NR2B transgenic mice. This demonstrates that a low NR2B:NR2A ratio may be beneficial for some aspects of hemorrhagic stroke recovery.

There are three distinct segments in this dissertation. First, I attempted to address the role of Schwann cells in mammalian neuromuscular junction (NMJ) development and function. Schwann cells at the NMJs do not form myelin sheaths and are known as terminal Schwann cells. Terminal Schwann cells are thought to be analogous to astrocytes in the central nervous system. Schwann cells (as described in details in the next section) provide trophic support to motor axons and modulate synaptic activity by sensing neurotransmitter release at the nerve terminal. However, the role of Schwann cells in synapse formation and maintenance remains unknown. Second, during NMJ formation, anterograde signals from nerve to muscle, and retrograde signals from muscle to nerve are critical for the establishment of a functional synapse. Research over the last three decades has contributed to our understanding of the role of the anterograde signaling at NMJ. However, identification of muscle-derived retrograde signals involved in motoneuron terminal differentiation remains scarce. Recent work from our laboratory suggests that genes that are transcriptionally regulated by p-catenin in muscles might play a crucial role in pre-synaptic differentiation at the NMJ.2 Third, Agrin-LRP4-MuSK signaling is critical for NMJ formation. At the NMJ, LRP4-mediated activation of MuSK by neural Agrin is required for post-synaptic differentiation. Mice that lack any one of the three genes fail to form NMJs and die at birth. Due to perinatal lethality of these null mice, less is known about how Agrin-LRP4-MuSK might regulate NMJ maintenance. Moreover, mutations in Agrin, LRP4, and MuSK have been reported in patients diagnosed with congenital myasthenic syndrome (CMS), and autoantibodies against MuSK and LRP4 have been detected in patients with myasthenia gravis (MG). However, the role of Agrin-LRP4-MuSK in the etiology of these neuromuscular disorders is not clear.

Given the involvement of Rabl la in each of these cellular processes and given the potential impact of Rabl la on human health and disease, we sought to further establish a role for Rabl la in plasma membrane recycling. Since other Rab proteins have numerous characterized interacting proteins and because the repetoire for Rabl la is currently limited to three identified interacting proteins, we hypothesized that other Rabl la binding partners exist as putative downstream effectors for the small GTPase. We therefore proposed the following three aims: Aim 1: Identify R ab lla interacting proteins. Aim 2: Determine the effect of interacting proteins on membrane recycling. Aim 3: Establish an organizational model of a putative Rabl la complex. The progression of studies herein provides insight into the dynamic and complex process of plasma membrane recycling. Yeast two hybrid screening of a parietal cell cDNA library utilizing dominant active Rabl laS20V as the bait identified Rabl 1-Family Interacting Protein 1 (Rabll-FIPl), a novel R ab lla interacting protein. EST database searches with the R abll-FIPl sequence identified three homologous proteins with high carboxyl-terminal identity. Chapter 1 introduces the new family of Rabl la interacting proteins and provides the initial characterization. Interestingly, these studies indicated an interaction between Rabll-Family Interacting Protein 2 (Rabll-FIP2) and myosin Vb tail. Chapter 2 further describes the Rabl l-FIP2/myosin Vb tail binding and provides functional data placing Rabll-FIP2 as an integral component of the plasma membrane recycling system. Finally, recent studies have indicated a recycling system dependence on different kinase activities. Through kinase inhibitor studies and immunofluorescence imaging, evidence presented in Chapter 3 suggests that R ab lla along with multiple Rabll-FIP proteins function as a complex beginning at the process of endocytosis with movement dependent on multiple phosphorylation events. The ultimate goal throughout these studies is to provide a clearer picture of Rabl la function in plasma membrane recycling so that one day a positive impact on human health can be achieved.

DNA damage caused by environmental mutagens or reactive metabolic byproducts induces DNA damage response (DDR), which regulates cell cycle transit, DNA repair and apoptosis. DDR involves the phosphorylation and activation of Ataxia Telangiectasia Mutated (ATM) and ATM and RAD3-related (ATR) proteins. ATR regulates the firing of the replication forks during S phase, and the repair of damaged replication forks to prevent premature onset of mitosis. ATR phosphorylates and activates CHK1 which phosphorylates and inactivates CDC25, thereby inhibiting CDK1 activation and cell cycle progression. In the present studies, we determined that treatment with an hsp90 inhibitor AUY922, without affecting the mRNA levels, dose-dependently depletes the protein levels of p-ATR (Ser 428), ATR and CHK1 in human breast and cervical cancer cells. Additionally, treatment with the pan-histone deacetylase inhibitor panobinostat (PS), which is known to induce hyperacetylation and inhibition of hsp90 function, also depleted ATR and CHK1 levels in cancer cells. Co-treatment with the proteasome inhibitor bortezomib (BZ) partially reversed AUY922- or PS-mediated depletion of ATR and CHK1 expression, indicating proteasome-mediated degradation of ATR and CHK1. Treatment with either AUY922 or PS markedly inhibited the binding of ATR with hsp90, induced polyubiquitylation of ATR, and decreased the half-life of both ATR and CHK1 proteins. Treatment with AUY922 also abrogated ionizing radiation (IR)-induced cell cycle arrest and increased the amount of DNA damage in the cancer cells following IR. Treatment with AUY922 also inhibited the recruitment of p-ATR, ATR and 53BP1 to the site of DNA damage. In addition, HDAC3 binds to and deacetylates hsp90 in the nucleus. Depletion of HDAC3 by either short hairpin RNA or genetic knockout induced hyperacetylation of nuclear hsp90, resulting in the inhibition of chaperone association of ATR with hsp90 and depletion of ATR. These findings demonstrate that 1) ATR is chaperoned by hsp90, 2) Inhibition of chaperone function of hsp90 results in proteasomal degradation of ATR and inhibition of DDR, 3) pan-HDAC inhibitors abrogate ATRCHK1 cell cycle checkpoint pathway by modulating chaperone activity of hsp90 and 4) HD AC3 plays a critical role in the regulation of DNA damage response by stabilizing the chaperone activity of nuclear hsp90.