Edward Ziff - US grants

Adenovirus messenger RNA synthesis is potentially controlled at several levels: (i) Positive and negative regulation of the RNA initiation step; (ii) Transcription termination vs antitermination; (iii) Selection of 3' poly A site for mRNA; (iv) Differential splicing of nuclear precursors; (v) Changes in mRNA stablity. The major event in Ad-2 gene expression is the early-late switch which activates abundant mRNA synthesis from the major late transcription unit resulting in the expression of 10-20 late specific viral genes. The switch is made possible by a complex early period which prepares the cell for viral DNA replication and late gene expression. Despite great interest, little is known of the biochemical or molecular basis for the mechanisms which control Ad-2 gene expression. We therefore propose: 1) To identify specific RNA transcription and processing steps which are controlled during the early stage of infection, and the early-late transition. We shall introduce drug or mutant blocks during the early phase, and identify those steps which are coordinately inhibited, or permitted by the block. 2) To analyze in detail the mechanisms of activation of the late transcription unit, and the pathway of processing of late transcripts. 3) To analyze the structures of nuclear RNA-protein complexes which function in late RNA processing.

We propose to study the interaction of type C strains of adenovirus with proliferative and differentiation programs of the host cell. During its life cycle in natural hosts, adenovirus normally infects non-dividing cells. We propose to investigate the ability of the virus to induce in host cells proliferative functions which the cell normally expresses during active growth and which, according to our hypothesis, the virus diverts for the benefit of the viral life cycle. In so stimulating the infected cell, the virus may establish in its host a state normally occuring in rapidly dividing cells, or in cells with extended proliferative capacity, such as stem cells or progenitor cells. We shall also investigate whether the virus represses specialized differentiated cell functions which are expressed in terminally differentiated cells with limited proliferative capacity. The primary viral regulators of cellular proliferative functions are proposed to be the E1a proteins, which we suggest perform not only their well recognized role of regulating the transcription of viral early genes, but also act on cellular genes which are regulators of cell proliferation and differentiation.

Our laboratory has recently shown that induction of expression of the c-fos gene is a cellular response to a wide range of transmembrane signaling agents including growth factors, phorbol esters, and neurotransmitters. We proposed to study properties of the fos protein and its induction. Specifically we propose: 1. To determine the time course of synthesis of fos protein and the association of fos with a second cellular protein, p39 following growth factor induction in 3T3 cells. 2. To determine the molecular weight of the complex of fos protein with p39. From changes in this stoichiometry we will deduce the dynamics of fos-p39 complex formation. 3. To mutagenize the fos protein. We will determine the binding site on fos for p39 and compare this with sites of mutation which alter fos protein transforming ability, nuclear localization, post-translational modification and potential for autoregulation of expression. This will develop a functional topology for the fos protein. 4. To purify the p39 protein through isolation of its complex with fos with the objective of preparing reagents to study p39 expression and structure. 5. To assay the fos protein for a negative regulatory role acting at the fos promoter. 6. To assay other growth factor induced early response genes for fos transcription repressor activity using the antisense technique. 7. To identify proteins interacting with fos regulatory sequences using a novel DNA label transfer assay.

9318128 Smith The purpose of this proposal is to generate funds to upgrade our VAX 6000-410 computer to a DEC 7000-610, to increase our disk space by adding new SCSI disks, to purchase fiber-optic hub equipment allowing the new computer to be connected directly to the Medical Center's fiber-optic network, and to add two new X-terminals and a color printer for public use. The computer currently functions as a shared central resource within the Medical Center providing data bank storage and computing power for nearly all research scientists at the Medical Center whose work involves molecular biology. The current machine is overwhelmed by the rapidly increasing number of users, and the rapid growth of the molecular biology databases. Additionally, the resource is required to fill an expanding role as a file and compute server for a rapidly growing number of users who are choosing to purchase high power Macintoshes, IBM-PCs and workstations, in addition to serving a large group of peer reviewed and funded projects requiring basic computing facilities. The goal of our shared resource is to enhance current services and to provide new facilities, some of which are under development in the Medical Center. These resources consist of new databases, tools for the analysis of molecular structure, and selected image processing tools and resources. Our goal is to support research on biological structure at all levels from the molecular to the cellular. The PI and the CoPIs are currently--and will continue to be- -major users of the computer system. They share a commitment to a shared computing resource and are involved in projects that will enhance its value to the Medical Center and the broader international research community.

DESCRIPTION (provided by applicant): We will study the mechanism of trafficking of the GluR2 subunit of the AMPA receptor to synapses in cultured hippocampal neurons. Regulation of trafficking of AMPA receptors is thought to control AMPA receptor synaptic abundance and hence synaptic strength. We have identified three PDZ-containing proteins that associate with GluR2, ABP, GRIP and PICK1. These factors bind to the extreme carboxy terminal region of GluR2 and may serve as adaptors that link the receptor to the trafficking machinery or tether AMPA receptors at the synapse. We have also shown that the chaperone, N-ethylmaleimide sensitive fusion protein (NSF), binds specifically to GluR2. NSF may dissociate SNARE complexes associated with AMPA receptors and thereby "prime" vesicles containing GluR2 for transit to the synapse. Our work also suggests functional interactions between these two sets of proteins. We will determine the biochemical, molecular and cell biological consequences of mutating the GluR2 C terminus. We will express mutant GluR2 subunits and single pass chimeras bearing the GluR2 C terminus in hippocampal neurons from Sindbis virus vectors and measure a series of phenotypes during synapse formation and modification. This will divulge the contributions of different binding proteins to GluR2 trafficking and function. We will study subcellular structures formed by ABP and GRIP and analyze the contributions of ABP subdomains to ABP function. To assess NSF function in a possible priming of vesicles bearing GluR2, we will test for interaction of the NSF-GluR2 complex with the SNARE core complex. This will entail identifying proteins that bind to NSF while it is in contact with GluR2. We will also study the effects of mutation on activity dependent translocation of GluR2 subunits to synapses in cultured neurons. Together these studies will give a mechanistic view of the contributions of protein binding the GluR2 C terminal domain in AMPA receptor trafficking. These studies are pertinent to normal mechanisms of learning and to learning disabilities that occur during aging or as a result of neural degeneration or genetic mutation.

[unreadable] DESCRIPTION (provided by applicant): We will study the roles of PICK1 (Protein Interacting with C Kinase-1) and its regulator, NSF (N-ethylmaleimide Sensitive Factor) in AMPA receptor (AMPAR) trafficking. AMPAR are the major source of excitatory currents in the CNS and changes in AMPAR synaptic abundance regulate synaptic strength. PICK1 and NSF bind to the C-terminal, cytoplasmic domain of the AMPAR GluR2 subunit, and have opposing roles in controlling AMPAR synaptic levels. PICK1 binds to GluR2 via a PDZ domain and colocalizes with AMPAR in endosome-like vesicles in hippocampal pyramidal neurons. PICK1 expression reduces AMPAR synaptic membrane abundance and PICK is implicated in the mechanism of long term depression (LTD), all indicating a role for PICK1 in AMPAR endocytosis. NSF, together with the SNAP proteins, is known for dissociating SNARE complexes. In the case of AMPAR, the interaction of NSF with GluR2 is necessary for maintaining AMPAR currents and synaptic abundance. Our recent work has revealed a novel function for NSF. NSF binds together with the SNAPs to the PICK1-GluR2 complex and displaces PICK1 from GluR2. By displacing an endocytosis factor, NSF maintains AMPAR in the synapse. The Aims of this project are: 1) To determine the structural basis for assembly of the GluR2-PICK1-NSF-SNAP complex and its disassembly by NSF. We will define protein-protein contacts that stabilize the complex and determine whether the SNAPs transmit rotational torque from NSF to PICK1 during disassembly. We will determine the basis for the differential ability of alpha- and beta-SNAPs to disrupt PICK1-GluR2 complexes. 2) To determine the role of PICK1 in AMPAR trafficking. We will identify the starting and destination membranes and the roles of G proteins in the transport mechanism. 3) To determine regulation in vivo by physiologic stimuli, including the role of PICK1 in GluR2/GluR3 constitutive recycling. These studies will reveal basic mechanisms relevant to synapse regulation and memory formation.

DESCRIPTION (provided by applicant): Ca2+-permeable AMPA receptors are activated under less restrictive conditions than the NMDA receptor, are found in numerous neuron types, and may regulate pathways that are more generally controlled by NMDAR Ca2+ currents. We have shown that Ca2+-permeable AMPARs, like the NMDAR, can activate neuronal nitric oxide synthase (nNOS). In our first Aim we will determine if Ca2+-permeable AMPA receptors can activate successive regulatory phosphorylations of nNOS by Akt and CaMKII, previously shown by us (Rameau et al., 2007) to be induced by the NMDAR. We will study scaffolding structures and biochemical pathways Akt and CaMKII that contribute to nNOS control by Ca2+-permeable AMPARs. GluR2 when modified by RNA editing dominantly blocks the Ca2+ conductance of AMPAR channels. In contrast, unedited GluR2 is highly toxic through its Ca2+ permeability and constitutive trafficking (Mahajan and Ziff, 2007). Recently we have found that NMDAR activity can degrade the GluR2 pre mRNA editing enzyme, ADAR2. Our second Aim is to analyze the pathological, NMDAR-dependent proteolytic cleavage of ADAR2, the production of unedited GluR2 as RNA editing activity declines, and the mechanisms by which Ca2+-permeable AMPARs including the unedited GluR2 can kill neurons. To limit Ca2+-toxicity, cells have evolved mechanisms for restricting synaptic AMPAR Ca2+ currents in which Ca2+-impermeable AMPA receptors replace Ca2+-impermeable ones at the synapse. In preliminary studies, we have found that release of Ca2+ from ER stores cooperates with CaMKII to stimulate trafficking of GluR2 from the endoplasmic reticulum to the plasma membrane. In our third Aim, we will study the Ca2+-dependent trafficking of GluR2 from the ER, and distinguish if its control relies on the assembly of GluR2 into tetramers or release of GluR2 from ER retention. We will determine the domains of GluR2 that respond to Ca2+ and the roles of intracellular Ca2+ levels, CaMKII, PICK1 in the export mechanism, including the roles of PICK1-CaMKII complexes. These studies will provide a comprehensive analysis of physiologic and pathologic pathways controlled by Ca2+-permeable AMPARs and mechanisms for regulating Ca2+-permeable AMPAR function. PUBLIC HEALTH RELEVANCE: This grant is concerned with the physiologic functions of Ca2+ permeable AMPA receptors in brain regulation and synapse plasticity, and the consequences of formation of pathological Ca2+ permeable AMPA receptors, which have been implicated in Amyotrophic Lateral Sclerosis. Finally the grant will study pathways of trafficking of Ca2+-impermeable AMPAR receptors that block the Ca2+ permeable ones, and protect against neuron excitotoxic death.

DESCRIPTION (provided by applicant): AMPA receptors generate the major depolarizing currents that trigger the formation of action potentials. Alterations of the rates of insertion and removal of receptors at synapses can modify receptor synaptic abundance and control synaptic strength. AMPA receptor C-terminal domain phosphorylation is thought to be a major regulator of receptor interaction with proteins that control trafficking. Synapses convert ionic fluxes associated with synapse activity into biochemical signals that control kinases and CTD phosphorylation. Phosphorylation of the GluR1 subunit on serine 845, which lies near the middle of the GluR1 CTD, by PKA has been correlated with the level of GluR1 at extrasynaptic sites in the plasma membrane. We will use molecular and electrophysiological approaches to investigate a new mechanism of serine 845 phosphorylation by the cyclic GMP regulated kinase cGKII. cGKII is under the control of the NMDA receptor through NMDAR activation of nNOS, leading to the production of nitric oxide, activation of soluble guanylate cyclase and production of cGMP, which induces cGKII. The Aims of this project are: Aim 1. To analyze the roles of cGKII myristoylation, dimerization and activation in GluR1 trafficking and physiology. We will analyze how the structure of cGKII contributes to GluR1 regulation. Aim 2. To distinguish the mechanism of S845 phosphorylation in increasing GluR1 levels on the plasma membrane. We will determine the place within the cell that cGKII phosphorylates GluR1 and exerts its effect on GuR1 trafficking. We will attempt to distinguish whether cGKII regulates GluR1 exocytosis or if GluR1 delivered constitutively is stabilized on the plasma membrane by S845 phosphorylation. We will isolate factors that respond to S845 phosphorylation and control GluR1 surface levels. Aim 3. To study the cGKII regulated trafficking of GluR4 and GluR2L. GluR4 and GluR2L are AMPAR subunits that are structurally related to GluR1 in ways that suggest that they may also be controlled by cGKII. We will analyze the role of cGKII in the trafficking of these subunits to determine if the NMDAR-nNOS-cGKII pathway is more generally employed. This project will provide new information about mechanisms of activity dependent control of synapse function and may help to define the role of nitric oxide in these mechanisms. PUBLIC HEALTH RELEVANCE: Learning and memory depend upon changes in the strength of connections between neurons in the brain. This project will study a new pathway that can allow neurons to respond to their own activity and change the strength of their connections. This work is relevant to mechanisms of memory formation and to the origin of learning disabilities. It can provide the basis for devising new drugs to help maintain memory or lessen learning disabilities.

The goal of this proposal is to elucidate the mechanisms underlying calcium dysregulation and synapse loss in mouse models of Alzheimer's disease (AD). Many lines of evidence have shown that synapse loss occurs early in AD pathogenesis and is the best correlate of cognitive impairment in AD patients. Despite much effort, the mechanisms underlying synaptic loss in AD remain unclear. We have recently found that the dendrites of layer 2/3 pyramidal neurons exhibit abnormally long duration, high amplitude Ca2+ spikes in the APPPS1 mouse model of AD. The prolonged dendritic Ca2+ spikes are associated with the reduction in synaptic activity and size in this mouse model. By employing in vivo imaging, molecular and pharmacological approaches, we propose to determine whether the generation of abnormal dendritic Ca2+ spikes and their detrimental impact on synapse loss are a general phenomenon in mice carrying APP and PS1 mutations. Our preliminary studies show that NMDA receptor-dependent production of cyclic GMP and activation of the cyclic GMP regulated kinase II (cGKII) regulate the release of Ca2+ from endoplasmic reticulum to the cytosol. We will determine the important role of this cGKII-dependent signaling pathway in the generation of long-duration dendritic Ca2+ spikes in APP and PS1 mutant mice. To alleviate the generation of abnormal long-duration dendritic Ca2+ spikes and their detrimental consequences on synaptic plasticity, we will investigate the impact of reducing cGKII activity either by genetic or pharmacological manipulations in APP and PS1 mutant mice. The proposed experiments will reveal the mechanisms underlying the generation of abnormal dendritic Ca2+ spikes and their impact on synapse loss in AD. The proposed studies will also generate important new insights into the therapeutic treatment of AD aiming at reducing calcium dysregulation and synapse loss with cGKII inhibitors.