Jay M. Baraban - US grants

neurotransmitters; lithium;

The need to develop more effective strategies to combat neuronal degeneration has focused attention on defining the cellular-mechanisms underlying neuronal apoptosis. As Sindbis virus (SV) infection triggers neuronal apoptosis in vitro, this paradigm provides a useful model system for investigating this process. Previous studies from our laboratory reveal that oxidative stress can trigger the orchestrated events of neuronal apoptosis. These results, along with recent observations from other laboratories suggesting that the anti-apoptosis gene, bcl-2, may act as an antioxidant, cast oxidative stress induced death in a new light and suggest that if cellular defenses are inadequate to neutralize undesirable by-products of oxidative reactions, then the resultant unopposed oxidants may be a signal commonly utilized to initiate neuronal apoptosis. In preliminary studies, we have shown that: a) drugs with antioxidant capability, N-acetylcysteine (NAC) and pyrrolidinedithio-carbamate (PDTC), prevent SV-induced apoptosis in a neuroblastoma cell line, b) SV induces activation of the redox sensitive transcription factor NF-kB and this response is blocked by NAC and PDTC, and c) actinomycin-D, a selective inhibitor of host cell transcription, inhibits SV-induced apoptosis. Taken together, these results suggest that activation of a redox-sensitive host transcription factor pathway is required for SV-induced apoptosis. In this project, we propose to investigate the signalling pathways that lead to SV-induced host cell transcription and apoptosis. In particular, we intend: l) to define the spectrum of redox-sensitive transcription factors that are activated by SV infection, 2) to determine whether PDTC and NAC block SV-induced transcription factor activation by inhibiting SV-induced oxidative stress, 3) to determine whether expression of the proto-oncogene, bcl-2, a putative antioxidant, inhibits SV-induced activation of these transcription factors, and 4) to assess whether molecular manipulations of SV-activated transcription factor pathways that mimic PDTC/NAC's effects on them, also block viral- induced apoptosis.

neuropharmacology; transcription factor; gene expression; protein isoforms; cocaine; antipsychotic agents; phosphorylation; drug abuse; chemical binding; electrostimulus; nucleic acid sequence; haloperidol; gel mobility shift assay; nucleic acid probes; laboratory rat; molecular cloning;

As abnormalities in neuronal plasticity have been implicated in a wide variety of neurological and psychiatric disorders, one of the top priorities of neuroscience research is to decipher the molecular mechanisms that regulate this process. Recent studies have focused attention on the dynamic changes in neuronal morphology that appear to play an integral role in neuronal plasticity. Although the signaling pathways that mediate and regulate changes in neuronal morphology are poorly understood, recent studies indicate that the Rho family of GTPases play an important in this process. Accordingly, studies aimed at elucidating the regulation and function of these key signaling molecules in neurons are warranted. This R03 application seeks funds to conduct exploratory studies aimed at gaining clues to the function of Tech, a novel brain cDNA isolated by our laboratory. Preliminary studies indicate that the Tech transcript is enriched in cortex and hippocampus and is expressed in neurons. Analysis of the Tech cDNA sequence indicates that it is a member of the Rho GEF family of proteins that regulate the activity of Rho GTPases. Thus, elucidating the function of Tech is likely to provide new insights into the regulation and function of Rho signaling pathways in neurons, an important area of investigation. To explore the function of Tech, we plan to: 1) determine whether the Tech transcript encodes a functional Rho GEF as predicted by its cDNA sequence, 2) define the localization of Tech protein within neurons, and 3) carry out the initial phases of experiments aimed at identifying partner proteins that bind to its putative C-terminal PDZ ligand motif.

DESCRIPTION (provided by applicant): This revised R01 application focuses on pursuing our recent finding that dominant negative inhibitors of the Egr family of transcription regulatory factors represent a novel and highly effective means of suppressing activation of c-Jun. This observation emerged from our studies demonstrating that Egr inhibitor constructs block NGF induced neurite outgrowth in PC12 cells. In studies aimed at understanding how they exerted this effect, we have obtained compelling preliminary evidence that they do so by blocking the ability of NGF to induce phosphorylation and activation of c-Jun. Since c-Jun activation plays a critical role in several neuronal apoptosis paradigms, we examined whether Egr inhibitor constructs are effective in this context as well. To this end, we have found that Egr inhibitors are highly effective in protecting cerebellar granule cells from apoptotic cell death induced by potassium deprivation. Furthermore, we have obtained compelling evidence that they do so by blocking c-Jun activation. In preliminary studies aimed at deciphering the mechanism(s) mediating this effect, we have identified a novel effect of Egri, the prototype Egr family member, on c-Jun. We have found that Egri co-precipitates with c-Jun. This novel finding suggests that Egr proteins may control c-Jun activation via protein-protein interactions, rather than via its conventional mode of action, i.e. control of target gene expression by binding to its cognate DNA response element. Based on these findings, the overall goal of this proposal is to define how Egr family members control c-Jun activation and assess whether Egr inhibitors are also effective in blocking cell death in other neuronal apoptosis paradigms that are dependent on c-Jun activation.

Advances in understanding the molecular mechanisms mediating neuronal plasticity have provided important clues to the pathophysiology of drug abuse and addiction. Although substantial progress has been made in elucidating several key aspects of this process, e.g. the role of NMDA and AMPA receptors in mediating the induction and expression of activity-induced plasticity, respectively, critical features of this process are only partially understood. For example, it is now clear that enduring forms of synaptic plasticity require local translation of mRNAs that have been pre-positioned in the vicinity of synapses. However, we are only beginning to understand the mechanisms and molecules mediating mRNA trafficking and translation. In addition, recent studies have focused attention on the prevalence and importance of morphological changes associated with synaptic plasticity. However, how these changes occur and their relevance to functional plasticity are poorly defined. Accordingly, there is intense interest in understanding the molecular machinery that mediates and regulates these processes. To gain insights into these key features of synaptic plasticity, we plan to focus on two projects. In one, we will investigate the function and regulation of Tech, a RhoA GEF enriched in brain that has been implicated in regulating dendritic morphology. In the other, we will examine the role of the Translin/Trax RNA binding complex in mediating RNA trafficking in dendrites.

Recent studies have demonstrated that microRNA (miRNA) signaling pathways play a prominent role in regulating behavioral responses to cocaine. Therefore, studies aimed at understanding how the miRNA system operates are directly relevant to drug abuse research. Recent studies also indicate that the translin/trax RNAse complex plays a key role in miRNA processing. Furthermore, we have found, in preliminary studies, that translin knockout mice display reduced locomotor responses to cocaine. Accordingly, we plan to define: 1) the role of the translin/trax complex in regulating miRNA processing, and 2) its role in regulating responsiveness to cocaine. Accordingly, the specific aims of the proposed project are to: I. Identify direct miRNA targets of the translin/trax RNAse complex and determine its role in their processing. Although the translin/trax complex has been implicated in processing miRNAs, it is still unclear which specific miRNAs it targets directly. Accordingly, we plan to use a highly efficient UV-crosslinking procedure (PAR- CLIP) to identify RNAs that bind directly to the translin/trax complex in intact cells. II. Determine the impact of translin deletion on signaling pathways that regulate responsiveness to cocaine. As translin and trax are expressed in striatal neurons, a major site of cocaine action, we plan, in this set of studies, to conduct both candidate-based and screening approaches to identify alterations in striatal signaling pathways caused by translin deletion. III. Determine whether deletion of translin from striatal neurons mediates altered responsiveness to cocaine. Our initial studies demonstrating that translin deletion impairs the locomotor response to cocaine were performed in conventional ko mice. Accordingly, we plan, in this set of studies, to generate and use translin conditional ko mice to test the hypothesis that deletion of translin from D1R- and/or D2R-positive neurons mediates this behavioral phenotype. RELEVANCE (See instructions): To help develop improved approaches to prevent and treat drug abuse, a major goal of research in this field is to define the neurobiological changes that mediate the reinforcing properties of drugs of abuse. Recent studies have revealed that a newly identified class of RNA molecules, called microRNAs, play a key role in regulating behavioral responses to drugs of abuse. Accordingly, the goal of this proposal is to understand the role of the translin/trax comnlex in regulating microRNA nrocessina and behavioral responses to cocaine

Project Summary - Baraban Identification of more effective strategies to prevent and treat drug addiction is critically dependent on advances in deciphering the signaling pathways that mediate and regulate the behavioral effects of drugs of abuse. Recent studies indicate that the microRNA system plays a prominent role in this process. However, we are only beginning to understand its impact on neuronal signaling. In recent studies, we have found that the translin/trax RNase complex mediates degradation of a subset of microRNAs. Accordingly, to advance our understanding of the role of the microRNA system in dopamine signaling, we have checked whether translin KO mice display altered behavioral responses to cocaine. We have found that the ability of cocaine to increase locomotor activity is impaired in these mice. Furthermore, microdialysis studies revealed that cocaine's ability to elevate DA in the nucleus accumens is blunted in translin KO mice. In addition, fast scan cyclic voltammetry studies indicate that translin deletion blocks cocaine's ability to potentiate evoked release of DA without interfering with its ability to block the dopamine transporter. As recent studies have focused attention on the poorly understood ability of cocaine to potentiate evoked DA release as playing a key role in mediating its reinforcing properties, we plan to conduct studies aimed at understanding how translin deletion impairs this effect. In particular, we will assess whether this phenotype is: 1) due to loss of translin/trax RNase activity, and 2) due to loss of translin from DA neurons. Furthermore, we will use self-administration assays to assess whether translin deletion impairs the reinforcing properties of cocaine.