Michael Forte - US grants

G proteins couple the receptors for a vast array of first messengers to a variety of second messenger systems which generate diverse biological effects. The study of these molecules in vivo has been limited to cell culture systems and the description of pathological conditions which alter the level or function of G proteins. A system does not currently exist ni which the role of these molecules in complex biological processes can be addressed in a systematic way. We intend to analyze the role of G proteins in these processes in the fruit fly, Drosophila. Preliminary studies have involved the characterization of the G proteins present in the adult fly CNS and the isolation of cDNAs from fly head libraries which code for proteins highly homologous to each of the main classes of G protein a subunit expressed in vertebrates. We will extend these studies by isolating and characterizing the genes coding for these cDNAs to determine their potential for the production of alternate transcripts. Antibodies will be generated to peptides specific for each of the G alpha subunit protein to provide us with tools to examine the distribution and expression of each protein during nervous system development. We will attempt to determine the functional homology between fly Gs alpha-like clones and the vertebrate equivalents by assessing the ability of fly cDNAs for this protein to complement the defect present in S49 cyc- cells. The maternal expression of several of the fly G protein alpha subunits will be manipulated to assess the role of these proteins in the early nervous system development. Using the genetic tools available in Drosophila, we will attempt to isolate mutations which abolish the expression of fly G alpha subunits. Such mutations will be studied in their own right and provide us with recipient strains in which to express G protein alpha subunit genes which have been altered in vitro buy site specific mutagenesis and other methods. Using the system we will establish, G proteins will be, for the first time, subjected to study by a number of combined approaches in vivo to determine the role they play in nervous system function and development.

biomedical equipment resource; biomedical equipment purchase;

The vast majority of eukaryotic cells contain many mitochondria. In this organelle, the oxidation of small molecules generates cellular ATP, thus providing most of the available energy for the cell. A unique feature of mitochondria and related intracellular organelles is that they are composed of two independent yet interacting membrane systems. Embedded in both of these membranes are a variety of channel and carrier molecules that are responsible for the monumental biochemical tasks necessitated by this structure; the transport of highly charged molecules into and out of the mitochondria. Many of the molecules responsible for these transport processes have been identified and characterized in recent months. Many of the recent advances in the field have been made by investigators applying disparate technologies and methodologies. This workshop on "Molecular Biology of Mitochondrial Transport System" will bring together scientists examining these systems by different approaches to begin to identify structural similarities and functionally important interactions. Although many of the individual players are known, the nature of potential functional interactions and conserved structural motifs have yet to be thoroughly explored. It is both important and timely to bring together a multidisciplinary group of investigators examining mitochondrial transport systems by different approaches but with the same overall goal.

DESCRIPTION: The objective of this proposal is to define how the activation of the G protein Gs alpha subunit (DGs) in Drosophila impacts on the development and function of a specific cell type using wing blistering as a paradigm. The investigator, using a sophisticated in vivo expression system based on GAL4 control elements, has noted that the expression of an activated mutant of DGs (DGs*) results in numerous and complex phenotypes. One of the simplest and most consistently observed is the formation of wing blisters by GAL4 lines that mediate expression in wing epithelium during late pupal periods. The blistering is proposed to occur from rupture of transalar connections through the failure of integrin-mediated adhesion processes during wing expansion. Of great interest, the DGs*-dependent blistering occurs even in the absence (null mutation) of protein kinase A, suggesting that DGs* utilizes either a cAMP-independent pathway or cAMP but not PKA. The first aim is to test the hypothesis that elevation of cAMP in wing epithelial cells is required for the generation of blistering. A system for monitoring intracellular cAMP levels based on expression of a beta-galactosidase reporter will be established, then cAMP will be modulated through overexpression of an adenylyl cyclase (rutabaga) or phosphodiesterase (dunce). The second aim will be to identify molecules which are components of the DGs*-activated pathway in wing epithelial cells. The investigator assumes that a subset of molecules mediating downstream effects exist at a critical level and will take advantage of the fact that most genes in Drosophila are dosage-compensated, in developing deficiency screens to identify genes which, when present in one copy, can suppress blister formation. The third aim is to test the idea that the function of the Gs pathway within wing epithelial cells is to regulate the adhesive properties of integrin molecules. Specific aspects of wing morphogenesis will be examined, as will the ability of genetic manipulations of genes encoding integrin proteins to enhance or suppress blister formation.

DESCRIPTION: The objective of this proposal is to define how the activation of the G protein Gs alpha subunit (DGs) in Drosophila impacts on the development and function of a specific cell type using wing blistering as a paradigm. The investigator, using a sophisticated in vivo expression system based on GAL4 control elements, has noted that the expression of an activated mutant of DGs (DGs*) results in numerous and complex phenotypes. One of the simplest and most consistently observed is the formation of wing blisters by GAL4 lines that mediate expression in wing epithelium during late pupal periods. The blistering is proposed to occur from rupture of transalar connections through the failure of integrin-mediated adhesion processes during wing expansion. Of great interest, the DGs*-dependent blistering occurs even in the absence (null mutation) of protein kinase A, suggesting that DGs* utilizes either a cAMP-independent pathway or cAMP but not PKA. The first aim is to test the hypothesis that elevation of cAMP in wing epithelial cells is required for the generation of blistering. A system for monitoring intracellular cAMP levels based on expression of a beta-galactosidase reporter will be established, then cAMP will be modulated through overexpression of an adenylyl cyclase (rutabaga) or phosphodiesterase (dunce). The second aim will be to identify molecules which are components of the DGs*-activated pathway in wing epithelial cells. The investigator assumes that a subset of molecules mediating downstream effects exist at a critical level and will take advantage of the fact that most genes in Drosophila are dosage-compensated, in developing deficiency screens to identify genes which, when present in one copy, can suppress blister formation. The third aim is to test the idea that the function of the Gs pathway within wing epithelial cells is to regulate the adhesive properties of integrin molecules. Specific aspects of wing morphogenesis will be examined, as will the ability of genetic manipulations of genes encoding integrin proteins to enhance or suppress blister formation.

DESCRIPTION (adapted from investigator's abstract): Programmed cell death (PCD or apoptosis) is an evolutionarily conserved mechanism essential for the development and maintenance of tissue and organismal homeostasis in multicellular organisms. All available evidence is consistent with the idea that one of the most biologically relevant classes of proteins regulating PCD processes at the effector phase common to all PCD processes is a family of proteins related to the bcl-2 gene product. The Principal Investigator intends to test current models of the action of this family of proteins which invoke regulation of the mitochondria permeability transition (PT) by bcl-2 family members, leading to modulation of early mitochondria events which characterize PCD pathways. The PT is inhibited by nanomolar concentrations of the immunosuppressant peptide cyclosporin A (CsA), as is PCD in a number of assay systems. A large body of data indicates that the only known target of CsA action in mitochondria is CyP-D, a mitochondria matrix cyclophilin whose enzymatic activity is inhibited by CsA and a variety of non-immunosuppressive analogs. The specific focus of this proposal is then to unambiguously resolve basic questions related to: 1) the mechanism of PT inhibition by CsA, 2) the influence of CyP-D on the PT and 3) the role of the PT in PCD and other pathological processes. Three aims are proposed. Aim 1 is to generate CyP-D knockout mice. The second aim is to characterize their phenotypes. Finally, the Principal Investigator will characterize the mice at the cellular level to determine the effects on mitochondrial function.

DESCRIPTION (appended verbatim from investigator's abstract): Programmed cell death (PCD) plays an indispensable role in the development and maintenance of homeostasis within all multicellular organisms. Thus, the defects in PCD pathways contribute to a variety of diseases including AIDS and oncogenesis. The evolutionarily conserved BCL2 family of proteins are thought to be central regulators, acting at or near a point in the PCD pathway that dictates whether or not cells are committed to die. Members of this family can roughly be divided into two groups; those that promote (BAX) and those that inhibit (BCLXL) apoptosis. Much recent evidence supports the view that critical life/death decisions are regulated by the interaction of BCL2 family members with mitochondria, yet a detailed understanding of many of the important underlying mechanisms and critical biochemical interactions is lacking. Our goal is to use the genetic and biochemical tools available in yeast to identify the mechanisms and interactions that underlie the function of BCL2 family members in caspase independent cell death pathways. All available evidence strongly indicates that BCL2 family members act directly upon highly conserved mitochondrial components in yeast that correspond exactly to their apoptotic substrates in mammalian cells. Our preliminary results are consistent with a model in which BCL2 proteins generate their effects through interaction with a conserved mitochondrial site. Although these interactions have been uncovered in yeast, our underlying general hypothesis is that they are reflective of a similar general relationship in mammalian cells. These results lead to the following specific aims: 1. Test the hypothesis that "BH3 only" proteins function by modifying the interaction of antiapoptotic members with a critical site in mitochondrial membranes. "BH3 only" members of the BCL2 family have all the characteristics expected of proteins which, when translocated to mitochondrial membranes in response to a death signal, function to restrict the ability of BCLXL to associate with critical sites. We propose to directly test this hypothesis. 2. Identify proteins forming the site recognized by BCL2 family members in mitochondria. It is our goal to use genetic approaches to specifically identify proteins forming the "receptor" for BCL2 family members in mitochondria, as well as proteins that function to promote BCLXL mediated cell survival. 3. Determine the mechanisms underlying the ability of BAX to mediate growth arrest in the absence of cell killing. A large body of evidence exists to indicate that cell cycle and cell death processes are interconnected, although the mechanisms and signals that integrate these two processes are completely unknown. Our preliminary studies have allowed us to genetically separate a BAX mediated cell killing process from a growth arrest process. We propose to use genetic strategies to identify the molecular components required for BAX mediated growth arrest, and subsequently, use these mutants to address the question of whether BAX generated cell killing requires growth arrest.

DESCRIPTION (adapted from investigator's abstract): Programmed cell death (PCD or apoptosis) is an evolutionarily conserved mechanism essential for the development and maintenance of tissue and organismal homeostasis in multicellular organisms. All available evidence is consistent with the idea that one of the most biologically relevant classes of proteins regulating PCD processes at the effector phase common to all PCD processes is a family of proteins related to the bcl-2 gene product. The Principal Investigator intends to test current models of the action of this family of proteins which invoke regulation of the mitochondria permeability transition (PT) by bcl-2 family members, leading to modulation of early mitochondria events which characterize PCD pathways. The PT is inhibited by nanomolar concentrations of the immunosuppressant peptide cyclosporin A (CsA), as is PCD in a number of assay systems. A large body of data indicates that the only known target of CsA action in mitochondria is CyP-D, a mitochondria matrix cyclophilin whose enzymatic activity is inhibited by CsA and a variety of non-immunosuppressive analogs. The specific focus of this proposal is then to unambiguously resolve basic questions related to: 1) the mechanism of PT inhibition by CsA, 2) the influence of CyP-D on the PT and 3) the role of the PT in PCD and other pathological processes. Three aims are proposed. Aim 1 is to generate CyP-D knockout mice. The second aim is to characterize their phenotypes. Finally, the Principal Investigator will characterize the mice at the cellular level to determine the effects on mitochondrial function.

DESCRIPTION (provided by applicant): Nervous system function depends on the construction of complex, ordered synaptic connections among neurons and targets during development. At the Drosophila larval neuromuscular junction (NMJ), and many other synapses, neural activity regulates cAMP levels through Ca2+ regulation of adenylyl cyclases (AC), leading to reductions in the levels of the homophilic cell adhesion molecules like FASII that act by restraining synaptic growth; thus down regulation of FASII through the activity-mediated increase in synaptic cAMP is necessary for structural expansion of the synapse. However, Ca+2 regulated ACs are important coincidence detectors, integrating increases in neuronal Ca+2 with the activation of transmembrane receptors coupled to the stimulation of ACs through the heterotrimeric G protein, Gs. To test the idea that receptor signaling through s plays a role in synaptic growth, we have taken advantage of the fact that all receptor-mediated pathways or activation of ACs require the a subunit of the Gs complex (Gsa). Consistent with a role for Gsa signaling, we have shown that the Gsa protein is concentrated in growing synaptic boutons and that mutations in the gene encoding Gsa inhibit neuronal arborization and bouton formation, leading to deficits in sensory-motor processes as assayed on both a behavioral and physiological level. Furthermore, restricted expression of Gsa indicates that Gsa pathways are likely involved in the reciprocal interactions between pre- and postsynaptic cells required for the growth and development of mature synapses. These and other preliminary results suggest that Gsa-dependent regulation of AC activity plays an important role during processes that underlie synaptic growth and plasticity. To further test this hypothesis, this proposal focuses on the following three specific aims:1. Comprehensive Assessment of the NMJ Phenotypes Generated by dgs Mutations.In order to investigate the formation of NMJ in hypomorphic dgs mutants at higher resolution, we will carefully quantify ultrastructural defects generated by these mutations at the E.M. level and use of electrophysiological approaches to determine if altered synaptic morphology is accompanied by altered synaptic transmission. In addition, to test our working hypothesis that hypomorphic dgs mutations result in attenuated, but not eliminated, signaling through Gsa, we will examine phenotypes generated by mutations in additional residues in the C terminus of Gsa and directly assess receptor-mediated signaling though individual mutant Gsa by biochemical approaches.2. Test Models of the Role of Gsa Signaling in NMJ Formation through Genetic Epistasis.The object of this aim is to test our revised model of NMJ formation by examining the functional relationship between processes defined by specific mutations through genetic epistasis. Our focus will be on mutations which have been used to develop existing models, since the utility of this approach has already been demonstrated (e.g., mutations affecting neuronal activity, cAMP, cell adhesion). We will use these studies to precisely define the role of receptor-dependent activation of adenylyl cyclase through Gsa within the context of each tissue (neurons and muscle) in the establishment and growth of synaptic connections at the larval NMJ.3. Identify Molecules that Participate in Pathways Activated By Gsa during NMJ Formation.A major advantage of the Drosophila NMJ is that powerful genetic approaches can be applied in the identification of molecules that participate in Gsa-regulation of synaptic plasticity. Thus, the goal of this aim is to identify participants in the Gsa-activated pathway operating specifically during NMJ formation through the use of the genetic interaction strategies possible in Drosophila.

DESCRIPTION (provided by applicant): Mitochondria play a pivotal role in cell survival and tissue development by virtue of their role in energy metabolism, regulation of cellular Ca 2+ homeostasis and apoptosis. Given this multifactorial role, Ca 2+ homeostasis, metabolism, and bioenergetics function as an integrated system since energy conservation is used to drive each process. Mitochondrial energy conservation (ATP production) requires the respiration-driven formation of a proton electrochemical potential difference (delta mu H) across the inner mitochondrial membrane (IMM), which is created by proton pumping by the respiratory complexes. Maintenance of the gradient demands a low permeability of the IMM to protons, charged species and solutes. Yet, mitochondria in vitro can easily undergo an IMM permeability increase to solutes with molecular masses of about 1,500 Da or lower. This permeability change, called the permeability transition (PT), is regulated by the opening of a membrane pore, the mitochondrial permeability transition pore (PTP). PTP opening in vitro has dramatic consequences on mitochondrial function (e.g., collapse of the delta mu H and depletion of pyridine nucleotides) and structure (release of cytochrome c) that lead to respiratory inhibition. This process has long been studied as a potential target for mitochondrial dysfunction in vivo and as a mediator of programmed cell death (PCD) through the release of cytochrome c and other intermembrane proteins active on the apoptotic machinery. However, despite detailed functional characterization over the last 30 years, the molecular components forming the PTP have been not been definitively established nor has the precise role of the PTP in vivo been defined. This proposal is based in the synergy possible through the combination of novel approaches available in our two laboratories. Our specific plans include the following aims: Aim 1: In screens for chemical inhibitors of the PTP, we have identified Ro 68-3400 in functional assays as a high affinity (nM) blocker of the PTP through covalent modification of isoform 1 of mammalian VDAC (VDAC1). Similar experiments have also demonstrated that yeast VDAC1 is specifically targeted by this compound. We plan to use our experience with both mammalian and yeast VDAC to pin-point the structural requirements for high affinity association of VDAC with this compound, examine other mammalian VDAC isoforms for their ability to be modified by Ro 68-3400 and test the sensitivity of mitochondria treated with this novel PTP blocker to proteins in the BCL-2 family. Aim 2: Traditionally, the PTP has been considered to be a dynamic multiprotein complex formed at inner/outer membrane contact sites through the interaction of the adenine nucleotide translocator (ANT) of the IMM, VDAC in the OMM and a matrix regulatory protein, mitochondrial cyclophilin D (CyP-D). However, evidence implicating the ANT in the PTP complex has not been supported by recent data. Therefore, in this aim we plan to take advantage of Ro 68-3400 as a specific tool to further define the core components forming the PTP, with a specific focus on the identification of the IMM partner for VDAC in the pore complex. Aim 3: Inhibition by cyclosporin A (CsA) and non-immunosuppressive analogs has become the standard diagnostic tool for the characterization of the PTP in isolated mitochondria, in living cells, and in vivo. The target of CsA in these studies, CyP-D, is the only component of the PTP whose role has been definitively established. The goal of this aim is to unambiguously resolve basic questions related to the influence of CyP-D on the PTP, the participation of the PTP in specific aspects of the apoptotic program, and its role in specific pathological processes of significance to human, disease through the use of mice in which the expression of CyP-D and MVDAC1 have been eliminated by "knock-out" strategies.

DESCRIPTION (provided by applicant): Activation of the mitochondrial permeability transition pore (PTP) clearly plays a key role in some of the most wide-spread and therapeutically challenging human diseases. Our studies have established that the PTP operates in two modes 1) transiently, whereby the PTP acts as a mitochondrial Ca2+ release channel or 2) persistently, which ultimately results in cell death and disease. Although well characterized on a functional level, we know remarkably little about the molecules that form the PTP or how it is regulated. We urgently need answers to basic questions concerning what proteins actually form the PTP channel and what modulates the opening of the PTP in vivo. As a result, our goal is to identify the molecules that contribute to the structure and regulation of the PTP in both normal and disease states. This information is critical if we are to be able to effectively identify and/or deign valuable therapeutics targeting the transition of the PTP from normal to pathological. Here, we will use biochemical and genetic tools to identify structural components of the PTP and how PTP activity can be dynamically regulated in vivo. The specific objectives of this application are based in the synergy possible through the unique combination of novel approaches available in the Forte and Bernardi laboratories. The specific objectives of this application are: Aim 1 - Test role of OMM proteins in the regulation of PTP activity: While the PTP is primarily an IMM event, it has long been appreciated that proteins in the OMM should prominently regulate PTP activity. We will initially focus on a specific OMM protein, Tspo, whose role in PTP regulation has been strongly suggested. Our studies here will allow us to gain a deeper understanding of how cytosolic elements can impact PTP activity. Aim 2 - Identify key structural components of the PTP: Despite our increasing appreciation of its fundamental role in normal and pathological cellular responses, the molecules that form the PTP have remained a mystery. Here, we will use information in available mitochondrial proteomes to identify proteins forming the pore of the PTP. It is our expectation that the identification of any single component forming the PTP will supply us with the missing hook, providing the opportunity to identify additional components. Aim 3 - Mitochondrial p66ShcA and ROS activation of the PTP: It is clear that pore open-closed transitions can be regulated at many levels and, by extension; misregulation of these upstream pathways can lead to persistent, pathological activation of the PTP. The goal of this aim is to investigate the hypothesis that ROS generated through the action of p66ShcA (p66) functions upstream of the activation of the PTP in conditions of excess oxidative stress. We anticipate on the completion of this aim to have clear understanding of the role of one novel upstream activator of PTP activity. These studies will set the stage for future interrogation aimed at extending our understanding of mitochondria and PTP activity in physiological and pathological settings. Clearly, these outcomes will be fundamental to developing novel therapeutic strategies specifically targeting the pore in the many disease processes in which the PTP has been clearly implicated.

DESCRIPTION (provided by applicant): Activation of the mitochondrial permeability transition pore (PTP) clearly plays a key role some of the most wide-spread and therapeutically challenging human diseases. Our studies have established that the PTP operates in two modes 1) transiently, whereby the PTP acts as a mitochondrial Ca2+ release channel or 2) persistently, which ultimately results in the cell death and disease. Although well characterized on a functional level, we have no small molecules that specifically target the PTP or the transition from transient to persistent - from normal to pathological. As a result, our goal in this applicatin is to use the resource available in the MLPCN network is to identify the probes that uniquely target the PTP. This information is critical if we are to be able to effectively identify and/or design valuable therapeutics targeting the transition of the PTP from normal to pathological. The specific objectives of this application are based in the synergy possible through the unique combination of novel approaches available in our three laboratories; Aim 1 - We will screen the available NIH SMR to identify small molecule probes able to inhibit PTP opening using a simple in vitro assay that has already been adapted to the 1536 plate format to allow screening in high throughput (HTS) formats. Aim 2 - Since the primary screen is designed to caste a wide net, secondary screens have been developed that can also be used in HTS formats to limit to our future studies to molecules that specifically target the PTP. Aim 3 - We will initiate studies on te mechanism of action of active compounds based on assays of mitochondrial function as assessed in an in situ, whole cell context. These tertiary screens will also serve as a mechanism to assess chemically modified active compounds in an attempt to improve their biological activity. These studies will set the stage for future interrogation aimed at extending our understanding of mitochondria and PTP activity in physiological and pathological settings. Clearly, these outcomes will be fundamental to developing novel therapeutic strategies specifically targeting the pore in the many disease processes in which the PTP has been clearly implicated.