Daniel R. Storm - US grants

The long term objective of this proposal is to elucidate the mechanisms for regulation of brain adenylate cyclase by calmodulin (CaM) and the cAMP dependent protein kinase. Recent data from this laboratory using azido[125I]CaM and partially purified adenylate cyclase have implicated a 150,000 dalton polypeptide as the catalytic subunit of the CaM sensitive adenylate cyclase from bovine cerebral cortex. This polypeptide has been isolated in quantity by preparative SDS gel electrophoresis and shown to elicit polyclonal anti-cyclase antibodies with rabbits. We have also discovered that the catalytic subunit of protein kinase inhibits the activity of the CaM sensitive adenylate cyclase in the presence of ATP. It is our working hypothesis that direct phosphorylation of this adenylate cyclase may inhibit enzyme activity. The specific aims of this project include further purification of the CaM sensitive adenylate cyclase, preparation of monoclonal and polyclonal antibodies against specific polypeptides in the preparation, and elucidation of the quaternary structure of the enzyme complex. In addition, the significance of protein kinase inhibition of the enzyme will be evaluated.

The long term goal of this project is to characterize the structure and function of P-57, a novel calmodulin (CaM) binding protein from brain. We have recently purified P-57 to apparent homogeneity from bovine cerebral cortex membranes. In contrast to other CaM binding proteins, P-57 apparently has higher affinity for CaM in the absence of bound Ca2+ than in its presence and Ca2+ causes the release of CaM from P-57. It is hypothesized that P-57 may function to bind CaM at some local site within the cell and release CaM in response to increases in free Ca2+. The specific aims of this project include quantitation of binding between P-57 and CaM in the presence and absence of Ca2+, determining if P-57 interacts with other polypeptides in brain, examination of the tissue distribution of P-57, characterization of the physical properties of P-57, characterization of cAMP dependent protein kinase catalyzed phosphorylation of P-57, and determination of the influence of phosphorylation on the affinity of P-57 for CaM. Homogeneous P-57 in mg quantities, anti-P57 rabbit polyclonal antibodies, and fluorescent labeled CaM derivatives are currently available for the proposed experiments.

The long term goal of this proposal is to elucidate the quantitative relationships between fluctuations of free Ca2+ and cyclic nucleotides in heart muscle. The regulatory protein calmodulin (CaM) plays a key role in this system since it mediates Ca2+ stimulation of the Ca2+ sensitive phosphodiesterase and several other enzymes important for regulation of cardiac function. In this study, particular emphasis is placed on the energetics and chemistry for interactions between CaM and CaM regulated enzymes. Thermodynamic calculations for interactions between Ca2+, CaM, and CaM regulated enzymes predict that the Ca2+ concentration dependence for activation of each enzyme will vary in a manner tht can be quantitatively predicted from the free energy of coupling for binding of Ca2+ and the enzyme to CaM. It is also hypothesized that Ca2+ activation of CaM regulated enzymes will exhibit positive cooperativity to an extent which varies with the intrinsic energy coupling of the system, the concentration of the enzyme, and substrates. Specific aims of this proposal include quantitation of Ca2+ activation and Ca2+ binding to the CaM sensitive phosphodiesterase-CaM system and the myosin light chain kinase-CaM system and an examination of the effect of phosphorylation on energy coupling and Ca2+ sensitivity of the systems. Furthermore, we propose to quantitate binding of CaM to the CaM binding domain of myosin light chain kinase and related peptides in which the primary sequence has been systematically varied. These experiments will allow us to systematically define the chemistry for CaM-enzyme interactions and evaluate the importance of specific amino acid residues for the stability of CaM-enzyme complexes.

The long term goals of this project are to define the mechanism for calmodulin (CaM) regulation of Bordetella pertussis adenylate cyclase and to elucidate the mechanism for entry of the enzyme into animal cells. B. pertussis is the pathogen responsible for whooping cough and the organism releases several toxins into its growth media including a CaM sensitive adenylate cyclase. Recent evidence from our laboratory using a highly purified B. pertussis adenylate cyclase demonstrated that the enzyme preparation can cause significant increases in intracellular cAMP of animal cells. We hypothesize that the enzyme enters animal cells, is activated by CaM, and elevates intracellular cAMP levels. The specific objectives of this proposal include purification of the enzyme to homogeneity, elucidation of its subunit composition and quaternary structure and identification of its catalytic subunit and CaM binding subunits by affinity labeling with 8-azido[3H]ATP and azido[125I]CaM. Furthermore, we propose to demonstrate unambiguously that the enzyme enters animal cells and to define the mechanism for cellular entry. The availability of the highly purified toxin may aid in the development of a safer vaccine for whooping cough and the study should contribute to a better understanding of the molecular basis of B. pertussis pathology.

The long term goal of this project is to characterize the structure and function of P-57, an abundant, neurospecific calmodulin (CaM) binding protein that was discovered in this laboratory. We have purified this protein to homogeneity, determined a significant percentage of its primary structure and isolated a cDNA encoding for greater than 95% of its sequence. Furthermore, the CaM binding domain of P-57 has been identified as a nine amino acid sequence near the N-terminus. In contrast to other CaM binding proteins, P-57 has higher or equivalent affinity for CaM in the absence of Ca2+ compared to the presence of Ca2+ and it is phosphorylated by protein kinase C. Phosphorylated of P-57 by protein kinase C lowers its affinity for CaM. We hypothesize that P-57 may function to bind and localize CaM at specific sites within the cell, and that phosphorylation of P-57 and increases in free Ca2 occurring with cell stimulation may release CaM locally near its target enzymes. We propose to determine the complete amino sequence of bovine brain P-57 by conventional sequencing techniques. We will isolate a full length cDNA for P-57 and systematically modify the CaM binding domain by oligonucleotide directed mutagenesis to characterize structural elements that contribute to the unusual CaM binding properties of P-57 and construct a cDNA encoding for a P- 57 in which its CaM binding domain has been replaced by the CaM binding domain of myosin light chain kinase. Other specific aims include a search for proteins in brain that may interact with P-57 and screening of various single cell culture systems for P-57. We also propose to characterize phosphorylation of P-57 and identify the single phosphorylation site in the protein.

Neurogranin and neuromodulin (GAP-43) are neurospecific calmodulin (CaM) binding proteins that may play important roles for regulation of neuron growth by regulating the levels of free CaM within neurons. Although the overall sequence of the two proteins show low homology, neurogranin contains a 16 amino acid sequence which is almost identical to the CaM binding domain and protein kinase C phosphorylation site of neuromodulin. Neurogranin is dentritic specific and neuromodulin is concentrated in axonal growth cone membranes where it is transported by rapid axonal transport. Neuromodulin is one of only a limited number of proteins whose synthesis is specifically increased during axonal elongation. There is evidence that neuromodulin is attached to membranes by palmitylated cysteine residues near the N-terminus. We have discovered that protein kinase C (PKC) phosphorylation abolishes the binding of CaM to neuromodulin, and we propose that neuromodulin and neurogranin may function to localize CaM at specific sites in neurons and release free CaM in response to PKC phosphorylations. The general goals of this proposal are to test our hypotheses that phosphorylations of neuromodulin or neurogranin may control their interactions with CaM in vitro and in vivo. Specific objectives of this proposal are to quantitate neurogranin/ CaM interactions, determine if the levels of free CaM are regulated by PKC catalyzed phosphorylation of neuromodulin or neurogranin in vivo , determine if these proteins will inhibit CaM stimulation of CaM regulated enzymes, determine if neuromodulin interacts with components of the membrane cytoskeleton, and to develop an in vitro assay for palmitylation of neuromodulin.

DESCRIPTION: Ca2+/calmodulin-sensitive adenylyl cyclases (AC) are likely to be important for various types of synaptic plasticity. Transgenic mice deficient for type 1 adenylyl cyclase (I-AC) are deficient in certain types of spatial learning. Biochemical analysis of these mice indicate that the brain may contain at least one other Ca2+/calmodulin-sensitive AC. The long-term goals of this proposal are to determine if Ca2+/calmodulin-dependent ACs are required for LTP or spatial learning, determine which genes are in cultured primary neurons are regulated by cAMP, and determine if the Ca2+/calmodulin- sensitive ACs contribute to Ca2+ regulation of gene expression in cultured neurons. The sensitivity of IX-AC to CaM and Ca2+ will also be investigated. The specific aims of this project include the determination of the necessity of I-AC, III-AC and VIII-AC for specific forms of LTP or LTD and determining if the Ca2+/CaM sensitivity of I-AC is crucial for LTP and/or spatial learning. Furthermore, the investigator proposes to isolate the clone for IX-AC, determine if AC activity in neurons is regulated by membrane potential, determine what genes in cultured hippocampal neurons are regulated by cAMP, determine which forms of LTP activate CRE-mediated transcription, and determine if Ca2+/calmodulin-sensitive ACs contribute to regulation of gene expression in cultured neurons.

DESCRIPTION (from the applicant's abstract) The circadian organization of behavior determines how complex organisms respond to light/dark cues on a daily and seasonal basis. Disruption of the circadian rhythm in humans can lead to sleep disorders, mental fatigue, and depression, particularly with aging patients. The daily cycle of melatonin synthesis in the pineal is controlled by the circadian clock in the suprachiasmatic nucleus (SCN). Melatonin may stabilize the sleep cycle by maintaining steady-state entrainment of circadian pacemakers. Melatonin biosynthesis in the pineal and retina is regulated by cAMP which stimulates transcription of genes encoding enzymes important for circadian expression of melatonin. Nocturnal release of norepinephrine at the pineal increases cAMP by activation of beta- and alpha-1-adrenergic receptors. Calcium stimulated adenylyl cyclases (AC1 and AC8) may play a major role in regulating melatonin biosynthesis because of their unique regulatory properties. The applicants hypothesize that costimulation of AC1 by Ca++ and beta-adrenergic receptors may generate cAMP signals of sufficient strength and duration to regulate the transcription of specific genes important for melatonin synthesis. They hypothesize that the diurnal variation in expression of AC1 and its stimulation by nocturnal NE regulates the synthesis of melatonin. Furthermore, they hypothesize that cAMP may also play an important role in circadian time keeping in the SCN by regulating gene transcription through the cAMP response element (CRE). The overall goals of this project are to define the role of the Ca++-stimulated adenylyl cyclases and CRE-mediated transcription in the regulation of melatonin synthesis and in circadian rhythm. This proposal uses two unique tools developed in this laboratory; mutant mice lacking Ca++-sensitive adenylyl cyclases and a CRE-lacZ transgenic mouse strain that is used to monitor CRE-mediated transcription in vivo.

DESCRIPTION (provided by applicant): There is considerable interest in the cellular and molecular basis of memory formation. Studies of learning and memory are of fundamental importance for a better understanding of cognitive disorders in humans including Alzheimer's, autism, aging-related memory loss, and various types of mental retardation. It is the general hypothesis of this proposal that Ca2+ stimulation of the CREB/CRE (cAMP response element)-transcriptional pathway plays a pivotal role in long-lasting, long-term potentiation (L-LTP) and some forms of hippocampus-dependent long-term memory (LTM). Our long-term objectives are to define the mechanisms for Ca2+ stimulation of CRE-mediated transcription in hippocampal neurons and to understand why activation of this pathway is important for LTM and L-LTP. We hypothesize that Ca2+ activation of CRE-mediated transcription requires coactivation of the Erk/MAPK and camp signal transduction pathways. We propose that the critical cAMP signal increase originates from activation of calmodulin-stimulated adenylyl cyclases. We hypothesize that cAMP signaling is required for the nuclear translocation of Erk/MAPK and may also contribute to Ca2+ activation of Erk/MAPK. We also propose that proteolytic degradation of SCOP, a Ras inhibitor, may contribute to Ca2+ activation and sensitization of the Erk/MAPK signal transduction pathway. We hypothesize that long-lasting increases in CRE-mediated transcription, or transcriptional oscillations, in the hippocampus may be due to increased expression of gene products that function as positive-feedback regulators of the Erk/MAPK/CRE transcriptional pathway.

This proposal focuses on the role of the Ca2+ inhibited adenylyl cyclase (AC3) in the regulation of vascular smooth muscle (VSM) proliferation and smooth muscle contraction. cAMP is anti-proliferative for VSM and it antagonizes the actions of mitogens that stimulate proliferation through activation of the Erk/MAP kinase pathway. Furthermore, cAMP mediates relaxation of VSM caused by '-2-adrenergic agonists and counteracts vasoconstriction caused by a -1-adrenergic agonists. We hypothesize that Ca2+ inhibition of AC3 plays a major role in both processes by releaving the cAMP c check on proliferation and smooth muscle constriction. AC3 is strongly stimulated by Gs-coupled receptors including '-adrenergic receptors and it is inhibited by Ca2+. Ca2+ inhibition is mediated by CaM kinase II (CaMKII) which phosphorylates AC3 at Ser-1076. To explore the role of AC3 for VSM function we disrupted the AC3 gene in mice. The objectives of this proposal are to determine if Ca2+ inhibits the activity of AC3 in VSM by stimulating the phosphorylation of AC3 at Ser-1076, to determine if proliferation of cultured VSM cells from AC3 mutant mice is enhanced is enhanced compared to wild type mice, and to determine if proliferation of cultured VSM cells is reduced in CaMKII mutant mice. In addition, we propose to examine cardiodynamic parameters in AC3 mutant and CaMKII mutant mice. These studies should provide fundamental insight concerning the regulation of VSM functions by mitogens and adrenergic agents.

DESCRIPTION (provided by applicant): The circadian organization of behavior determines how complex organisms respond to light/dark cues encountered on a daily and seasonal basis. Circadian rhythms are generated and controlled by an endogenous clock in the suprachiasmatic nucleus (SCN) of the hypothalamus. Disruption of the circadian rhythm in humans can lead to sleep disorders, mental fatigue, and depression, particularly with aging patients. Retinal light signals mediated through the retinohypothalamic tract stimulate glutamate release in the SCN and maintain time-of-day congruence between the SCN and the external environment. Although the mechanism by which light entrains the circadian cycle in the SCN is not defined, it is apparently mediated through a transcriptional cycle involving several genes including CREB (cAMP response element binding protein) as well as the period and timeless genes. Light induces transcriptional changes of several genes in the SCN including the "clock" genes, period 1 (Per1) and period 2 (Per2). Per1 is upregulated after light pulses given during the subjective night and light-induced increases in Per1 expression correlate with phase shifts of the circadian rhythm. The objective of this proposal is to test the following proposal. We hypothesize that entrainment of the circadian cycle in the SCN is mediated, at least in part, by glutamate stimulation of the CREB/CRE transcriptional pathway and increased transcription of the Per1 gene through CREs in its promoter. We hypothesize that glutamate stimulation of the CREB/CRE transcriptional pathway and Per1 expression in SCN neurons is due to coactivation of the MAPK and cAMP signaling pathways by Ca2+. We propose that the cAMP signal required for this process arises from Ca2 + stimulation of the type 8 adenylyl cyclase (AC8), a Ca2+ stimulated adenylyl cyclase. This project uses several novel mouse strains developed in this lab, including a CRE/LacZ reporter strain and AC8 mutant mice.

DESCRIPTION (provided by applicant): Several human diseases including Alzheimer's and Rubinstein-Taybi syndrome are characterized by memory defects. The development of drugs to enhance the memory of compromised patients could have a major impact on the quality of life for these patients and others with memory defects. Evidence from several labs indicates that memory in mice may be enhanced using inhibitors of cyclic nucleotide phosphodiesterases (PDEs) to increase cAMP in the brain. Another promising drug target site to increase cAMP specifically in the brain is AC1, a neurospecific adenylyl cyclase that it is stimulated by activity-dependent calcium increases. To test this idea genetically, we made mice overexpressing AC1 in the hippocampus using the Ipha-CaM Kinase II promoter. These transgenic mice (AC1+) show enhanced LTP and memory for novel objects as well as a reduced rate of contextual memory extinction. Preliminary data indicate that the gain in memory may be due to enhanced signaling through the MAPK pathway. These data suggest the interesting possibility that AC1 may be a pharmacological "window of opportunity" to enhance memory formation. The major objective of this grant is to determine why AC1+ mice have superior memory. We hypothesize that AC1+ mice show memory enhancement because of the unique regulatory properties of AC1 which include its calcium sensitivity and synergistic activation by Gs-coupled receptors and calcium. We hypothesize that AC1+ mice show superior memory for novel objects because of more robust training- induced CRE-mediated transcription. This may be due to training-induced amplification MAPK activity, MAPK nuclear translocation, or postsynaptic depolarizations. We also propose that genetic enhancement of AC1 activity in the brain may overcome memory defects associated with Rubinstein-Taybi syndrome, a genetic disease due to a truncated form of the CREB binding protein (CBP).

DESCRIPTION (provided by applicant): Memory retrieval is a dynamic aspect of memory formation that either reinforces or alters stored memories through reconsolidation or extinction. Although several signaling pathways including the MAP kinase (MAPK) cascade are implicated in retrieval, the underlying signaling events are incompletely defined. The general objective of this grant is to identify signaling elements that contribute to retrieval of hippocampus-dependent, contextual memory. These studies may lead to the identification of specific drug target sites that can be exploited to modify memory retrieval. This proposal is based upon observations made by this lab that implicate PI3 kinase and MAPK in contextual memory retrieval. We discovered that PI3 kinase is activated in several areas of the hippocampus including areas CA1, CA3 and the dentate gyrus during retrieval of contextual memory. Furthermore, inhibition of PI3 kinase in area CA1 of the hippocampus in vivo reversibly blocked contextual memory retrieval. In addition, inhibitors of PI3 kinase blocked increases in MAPK activity associated with memory retrieval. This suggests that activation of PI3 kinase in the hippocampus is critical for memory retrieval and may be required for activation of MAPK during retrieval. Our data also indicate that cAMP-dependent protein kinase (PKA) activity is required for retrieval of contextual memory. There are a number of unanswered questions concerning the role of PI3 kinase in memory retrieval and the relationship between PI3 kinase signaling and other signaling events during retrieval. What is the mechanism for activation of PI3 kinase during retrieval of contextual memory? Are PI3 kinase and MAPK activated in the same neurons during contextual memory retrieval? Are the same neurons in the hippocampus activated during acquisition and retrieval of contextual memory? Is PKA activated in retrieval? Is Akt activity required for memory retrieval? Is activation of PI3 kinase in area CA3 or the dentate gyrus (DG) also required for contextual memory retrieval? PUBLIC HEALTH RELEVANCE This grant focuses on the molecular events underlying memory retrieval, a fundamental step in information processing. This study may identify drug target sites that can be used to modulate memory retrieval and be useful clinically. For example, drugs that impair memory retrieval may be used in the treatment of phobias.

DESCRIPTION (provided by applicant): Calcium activation of cAMP and MAP kinase (MAPK) signaling is required for consolidation of hippocampus- dependent memories and also plays a pivotal role in other forms of neuroplasticity including long-lasting, long- term potentiation (L-LTP). This project focuses on mechanisms by which Ca2+ regulates MAPK and adenylyl cyclase during memory formation and the role of MAPK oscillations for the persistence of hippocampus-dependent memory. This proposal is based upon several discoveries made by our lab during the last funding period including the finding that SCOP, a neurospecific protein, attenuates MAPK activity and CRE-mediated transcription in hippocampal neurons. Furthermore, we discovered that SCOP is proteolyzed by calpain, a calcium-stimulated protease, during memory formation. We found that over expression of SCOP in the hippocampus blocks consolidation of memory for novel objects, without affecting acquisition or short-term memory. We hypothesize that calcium-stimulated degradation of SCOP may play an important role in other forms of neuroplasticity including spatial memory and L-LTP. We also discovered that MAPK, PKA, and MSK-1, a CREB kinase, are coactivated in the same subset of neurons in the area CA1 of the hippocampus during memory formation and that these signaling events are not stimulated during training in mice lacking calcium-stimulated adenylyl cyclases (AC1 and AC8). Despite evidence supporting a role for cAMP, MAPK, and CREB-mediated transcription in memory consolidation, this hypothesis does not readily explain the duration of LTM which can persist well beyond the lifetime of gene products increased during memory formation. Therefore, we examined MAPK activity in the hippocampus over extended periods of time to determine if the pathway undergoes periodic reactivation. We discovered that cAMP, as well as MEK and MAPK activities undergo a circadian oscillation in the hippocampus. We hypothesize that the persistence of contextual fear memory may depend upon the circadian oscillation of this signaling pathway.