Richard Mains - US grants

DESCRIPTION (provided by applicant): Proper control of the synthesis, storage and secretion of bioactive peptides is crucial to normal endocrine and neural function. Over the past several decades, we delineated the cell-type specific enzymatic steps leading from proopiomelanocortin (POMC) to its many product peptides. We selected one of these enzymes, peptidylglycine 1-amidating monooxygenase (PAM), as a focus of our studies because it is an integral membrane protein that can communicate information about the lumen of the regulated secretory pathway to cytosolic machinery and to the nucleus. Our focus on PAM led to the discovery of Kalirin, a GDP/GTP exchange factor (GEF) for small GTP binding proteins of the Rho family, that interacts with the cytosolic domain of PAM. Linkage studies associating Kalirin with coronary artery disease, the decrease in Kalirin expression associated with elevated iNOS in Alzheimer disease hippocampus, and the identification of Kalirin as one of the proteins essential for Ras-mediated epigenetic silencing of gene expression, motivate our studies of this complex, multidomain protein. By flanking an exon common to the major splice variants of Kalirin with loxp sites, Kalirin conditional knockout mice (KalCKO/CKO) and mice lacking the major isoforms of Kalirin (KalKO/KO) were created. While not yet well characterized, it is clear that normal storage and secretion of pituitary hormones require pituitary Kalirin. Using lentiviruses or mating with mice in which expression of Cre recombinase is driven by the POMC or growth hormone promoter, Kalirin expression in corticotropes or somatotropes will be eliminated. POMC and GH synthesis, processing and secretion will be evaluated in vivo and in cell culture. The hypothesis that G1q-mediated activation of the second GEF domain of Kalirin plays a key role in the ability of corticotropes to respond to specific secretagogues will be tested in vitro and in vivo. Endocytic trafficking of PAM will be evaluated to determine the role of Kalirin in recycling granule membrane proteins and PAM-mediated nuclear signaling. Proteomic analysis will be carried out on isolated immature granules, whose formation is regulated by Kalirin, and on isolated pituitary granules formed in the absence of Kalirin. Assays to detect activation of Rho GEFs and their effectors will be used to understand their physiological role in pituitary hormone secretion. Finally, we will use knowledge of its individual domains to test the hypothesis that Kalirin functions as a modular machine, coordinating multiple aspects of granule biogenesis and release. In particular, the physiological importance of the alternate N-termini of Kalirin, the ability of Kalirin to inhibit iNOS, and the ability of Kalirin to integrate signals from multiple pathways will be assessed in vivo and explored mechanistically in cell culture. PUBLIC HEALTH RELEVANCE: Precise control of the biosynthesis, storage and secretion of bioactive peptides requires the coordinate control of many different cellular processes. Genetically engineered mice were used to determine that Kalirin, a large protein with domains that allow it to interact with immature secretory granules, regulate the actin cytoskeleton, bind to lipid membranes and respond to multiple protein/protein interactions, plays an essential role in peptide hormone release. Mutations in the Kalirin gene or changes in Kalirin expression have been correlated with early-onset coronary artery disease, schizophrenia and Alzheimer Disease, making a better understanding of the functions of this complex protein relevant to human health.

DESCRIPTION (provided by applicant): Kalirin is a large, multidomain Rho GDP/GTP exchange factor (GEF) of the Dbl family found almost exclusively in the nervous system. Based on genetic studies in invertebrates and analysis of genes involved in X-linked mental retardation, Kalirin is expected to play a role in axonal pathfinding and synapse formation. Our studies in rat hippocampal, cortical and sympathetic neurons showed that Kalirin plays a role in axon initiation and outgrowth, dendritic growth, and spine formation and maintenance. Kalirin has two GEF domains with specificity for different Rho family GTPases and a potential kinase domain. In addition to these catalytic domains, it has interactor domains for a variety of proteins. Kalirin expression in the nucleus accumbens and striatum is stimulated by chronic administration of cocaine, with no change in cortical Kalirin levels; Kalirin is also increased following electroconvulsive shock stimulation. The exploratory studies proposed are based on our observations that antisense-mediated elimination of Kalirin expression in organotypic slices and dissociated hippocampal neurons leads first to a reduction in linear spine density followed by simplification of the dendritic tree. Aim 1 is to generate mutant mice that will facilitate studies of neuronal development and plasticity in multiple systems. In order to avoid embryonic or early postnatal lethality, and to allow generation of tissue-specific and developmentally regulated elimination of expression, two mouse lines will be created as floxed alleles. Both will be provided to all qualified investigators without restrictions. One allele will produce the Kalirin-7 null. Kalirin-7 is the most prevalent isoform in the adult brain, with a single Rho GEF domain, terminating with a unique PDZ binding motif. Kalirin-7 appears late in development and is localized to dendritic spines. The second allele will produce the total Kalidn knockout. Both will be bred with available tissue-specific and drug-inducible Cre recombinase mice. Aim 2 will examine the biochemical, histological, and developmental consequences of tissue-specific Kalirin conditional knockouts.

DESCRIPTION (provided by applicant): Rho GTPases play key roles in neuronal development and in the formation and function of dendritic spines. Mental retardation is associated with deficits in individual Rho proteins, in the guanine nucleotide exchange factors (GEFs) that activate Rho proteins and in downstream targets of activated Rho proteins. Deficits in expression of Kalirin, a large, dual Rho GEF protein, are associated with increased iNOS levels in Alzheimer's disease brain and with decreased spine density in post-mortem prefrontal cortex from schizophrenic patients. In addition, Kalirin interacts with DISC1 (a candidate schizophrenia gene) and HAP1 (a Huntingtin interactor). Single nucleotide polymorphisms predict a significant number of individuals heterozygous for Kalirin function, with only one expressed copy or a normal and a mutated copy. These observations make a compelling case for analyzing mice in which expression of Kalirin can be manipulated both during development and in the adult. Mammals also express Trio, a highly homologous, but non- redundant gene. The single Kalirin/Trio gene in Drosophila and C. elegans plays an essential role within and outside of the nervous system. Based on our studies in cultured neurons, Kalirin plays a central role in axon initiation and outgrowth and in dendritic growth. Over-expression of the major adult splice variant of Kalirin, Kalirin-7, increases the formation of dendritic spines in pyramidal neurons and in normally aspiny interneurons. Reductions in the expression of Kalirin-7 and Kalirin-7 (an N-terminally truncated variant generated from a different promoter) result in deficits in spine formation and maintenance. We generated mouse models in which expression of Kalirin-7 and Kalirin-7 can be varied. Mice lacking the single exon unique to Kalirin-7/ Kalirin-7 (Kal7KO) are born at half the expected frequency, but survive to adulthood and reproduce. Mice heterozygous for this exon (Kal7+/KO) have diminished levels of Kalirin-7 and Kalirin-7 and show deficits in synaptic transmission. At the ultrastructural level, Kal7KO mice have a reduced number of normal excitatory synapses, plus many aberrant synaptic profiles not seen in normal mice. When tested in the elevated zero maze, Kal7+/KO and Kal7KO mice show a graded decrease in anxiety-like behavior. Mice in which the Kal7 exon is surrounded by lox-p sites (Kal7CKO) allow tissue-specific, developmentally regulated elimination of Kalirin-7/ Kalirin-7. These mice will be assessed using behavioral tests, morphological assessment of pre- and post-synaptic elements, electrophysiological recordings of slices and biochemical analysis of subcellular fractions. Spine formation in hippocampal neurons prepared from Kal7KO mice can be rescued by expressing exogenous Kalirin-7, allowing detailed analysis of the role of Kalirin-7 and the isolated Sec14p, spectrin-like, DH and PH domains. The role of Kal7 in spine formation in response to proteins such as Shank3, GluR2 and Neuroligin-1 will be assessed. The ability of the six known human Kalirin-7 mutants to rescue spine formation and synaptic function will be assessed using the Kal7KO mice. PUBLIC HEALTH RELEVANCE: Excitatory synapses onto dendritic spines account for much of the communication that goes on between neurons. The number and shape of dendritic spines respond to developmental cues, environmental stimuli and hormonal changes. Changes in spine morphology play key roles in learning and memory and it is clear that many signaling pathways affect spine formation and function. Kalirin-7, an activator of small GTP binding proteins of the Rho family, is localized to dendritic spines and is one of a small number of factors known to be capable of increasing the number of dendritic spines. We plan to use the Kalirin-7 knockout mouse that we generated to elucidate the pathway(s) controlling spine formation and structure.

Judging by initial physiological responses, rapidity of acquisition of drug-seeking or self- administration behavior, and strength of tendency to relapse, women and female laboratory animals are far more sensitive to cocaine than men and male laboratory animals. While it is well known that women differ from men in many emotional and cognitive responses, our understanding of the molecular underpinnings of these differences is limited. We will use deep sequencing to provide insight into the mechanisms that contribute to the different responses to withdrawal from cocaine observed in females and males. We previously applied this approach to the nucleus accumbens of male mice withdrawing from experimenter administered cocaine and identified the Wnt/cadherin pathway and miR-8 family members as key players. Targeted analysis of specific pathways revealed cocaine-mediated changes in the expression of mRNAs encoding multiple components of the dopamine, glutamate, GABA, acetylcholine, neuropeptide and endocannabinoid signaling pathways in the nucleus accumbens. In Aim 1, four groups of mice will be examined: males, cycling females, ovariectomized females and estradiol replaced ovariectomized females. Mice injected with saline or cocaine for a week will be sacrificed after four weeks of withdrawal; nucleus accumbens, prefrontal cortex and ventral tegmental area will be harvested for preparation of RNA. Duplicate bar-coded libraries prepared from the nucleus accumbens of mice exhibiting locomotor sensitization will be sequenced simultaneously, with technical replicates. Bioinformatic analysis will be used to identify cocaine-responsive transcripts and pathways common to all groups (core cocaine response), unique to females and sensitive to estrogen. In Aim 2 we will select transcripts and pathways from the core cocaine response group and female sensitivity group for validation and further analysis. The data set will allow analysis of the effects of sex, estrogen and cocaine on alternative splicing and RNA editing. Bioinformatic analysis of estrogen responsive elements and transcription factor binding sites in the promoter regions of cocaine-responsive genes will be undertaken. With this high quality, validated data set, focused sequencing studies can be used to analyze the response of individual mice self-administering cocaine. This broad approach should allow identification of therapeutic targets unique to females or sensitive to estrogen.

? DESCRIPTION (provided by applicant): With purification of the first neuropeptide, vasopressin, it was clear that peptides play an essential role in our ability to respond to environmental changes. Production of bioactive peptides involves a carefully orchestrated set of enzymatic reactions, leading to their storage in secretory granules. Peptidylglycine ?-amidating monooxygenase (Pam), an integral membrane protein, is the only enzyme known to amidate peptides, a modification usually essential for receptor binding. PamKO mice are not viable and Pam+/- mice exhibit anxiety and thermoregulatory deficits, with older animals exhibiting impaired ability to handle a glucose load. While our analysis of Pam+/- mice revealed only a small decrease in amidated peptide levels, their copper homeostasis was altered. Using a neuroendocrine cell line engineered to allow control of PAM expression, we found that PAM alters cytoskeletal organization and regulated secretory pathway function. These observations led us to consider additional functions for PAM. A clue to these functions came when we found active integral membrane PAM in Chlamydomonas reinhardtii, a unicellular eukaryotic alga. Rat and Chlamydomonas PAM exhibit strikingly similar enzymatic properties and subcellular localizations, including their presence in cilia. Molecular oxygen, luminal copper and acidic pH are essential to PAM function. The P-type ATPase that pumps copper into the secretory pathway and the vacuolar ATPase (V-ATPase) that acidifies the luminal environment are evolutionarily ancient. We hypothesize that PAM interacts with both of these pumps. Using purified recombinant proteins, corticotrope tumor cells and Pam+/- mice, we will test the hypothesis that pH-dependent interactions between PAM and Atp7a enable direct transfer of copper from Atp7a to PAM. We will use Chlamydomonas to identify ancient interactions of PAM with the transporters needed to support its catalytic activity and to determine the role of PAM in cilia. Using pituitaries from Pam+/- mice and mouse embryo fibroblasts from PamKO mice, we will determine how interactions between PAM and the V-ATPase affect the regulated secretory pathway. Our hypothesis that PAM plays a role in coupling nutrient sensing to hormonal signaling networks, helping to adjust growth and development programs to an environment that is constantly changing, is novel. Knowledge of the role played by PAM in this coupling will identify sites for targeted pharmacological intervention. The recent association of mutations in hPAM with Type 2 diabetes and the endocrine deficits associated with ciliopathies and altered copper homeostasis indicate that there is much to be learned.