Lisa M. Monteggia - US grants

Ion channels are involved in the regulation of cellular excitability. A recently identified novel sodium (Na+) channel (NNC), highly enriched in the locus coeruleus (LC), may contribute to the intrinsic electrical properties of these neurons. Studies have demonstrated that alterations in LC neuronal activity, via activation of a sodium conductance that resembles NNC in many ways, occur following opiate administration. To better understand the role of NNC in vivo, studies will examine the regional profile of NNC mRNA and protein in brain. Biochemical studies will investigate the phosphorylation potential of NNC. In situ hybridization and Western blot analysis will determine whether morphine exposure induces alterations in NNC expression. To more directly assess the role of NNC in the LC, and to explore the possibility that this channel is a mediator in opiate dependence, I will begin work to create a line of NNC null ('knockout') mutant mice. Together, these integrated, multidisciplinary studies will advance my training as well as elucidate the role of NNC in regulating the excitability of LC neurons under normal conditions and after chronic opiate dependence.

DESCRIPTION (provided by applicant): Recent studies have suggested that brain-derived neurotrophic factor (BDNF) plays a role in depression and antidepressant-like behavioral effects. BDNF expression is decreased in the hippocampus, a brain region implicated in the pathophysiology of depression, by exposure to stress, a factor implicated in depression in some individuals. Conversely, multiple classes of antidepressants, as well as electrocpnvulsive therapy (ECT), increase BDNF expression in the hippocampus in a time course consistent with the therapeutic action of these drugs. BDNF, the most prevalent growth factor in the brain, can then exert alterations in neuronal plasticity through specific signaling pathways. However, a clear link between the role of endogenous BDNF and 'depression-like' behavior and in the behavioral responses to antidepressant drugs remains unclear. The main goal of this project is to investigate, 1) whether the loss of BDNF produces changes in 'depressive-like' behavior and antidepressant responses and, 2) whether chronic antidepressant treatment exerts effects on synaptic plasticity in a BDNF dependent manner. 1 major aim is to examine whether the loss of endogenous BDNF in the hippocampus of 3 complementary genetic mouse approaches produces a 'depressive' phenotype in animal models of depression. We also will examine whether these mice display attenuated behavioral responses to antidepressants. A second aim will focus on the role of BDNF in exerting downstream effects following antidepressant treatment. We have demonstrated an increase in the phosphorylation of the glutamate receptor subunit, N-methyl-D-aspartate 1 (NR1) on a protein kinase C (PKC) site following chronic antidepressant treatment. Previous data has shown that BDNF may regulate NR1 phosphorylation, which then potentiates NMDA receptor function. Changes in NMDA receptor function could mediate long-term consequences in synaptic plasticity. We will pursue the increase in NR1 phosphorylation by chronic antidepressant action to examine whether this upregulation is mediated via alterations in BDNF. Together, the proposed molecular, cellular, and behavioral studies promise to advance our understanding of the role of BDNF that chronic antidepressants induce in the hippocampus.

[unreadable] DESCRIPTION (provided by applicant): The major objective of this proposal is to delineate the role of MeCP2 in specific brain regions and at distinct developmental time points in mediating behavioral phenotypes observed in individuals patients afflicted with Rett Syndrome (RTT). We have recently developed a system in which genes in the mouse brain can be knocked out by targeted delivery of an adeno-associated virus (AAV) encoding Cre recombinase in a regional and temporal specific manner. We hypothesize that cessation of MeCP2 expression in specific brain regions (hippocampus, amygdala and motor cortex) will cause them to: 1) exhibit alterations in specific behaviors depending on the brain region targeted; and 2) provide a framework of the neural circuitry that is involved in mediating aspects of RTT. In these experiments, in addition to regional specificity, we will be able to deliver AAV- CRE at two distinct stages of development (1 month after birth or 4 months after birth.). One month after birth corresponds to a time before appearance of symptoms in mice. In this way, we will be able to see whether deletion of MeCP2 in a specific brain region after birth can recapitulate specific symptoms associated with the disease. Region specific deletion of MeCP2 four months after birth (a time point after emergence of symptoms) will enable us to find out if there is a developmental critical period for appearance of the symptoms. If symptoms still emerge after this late deletion, this would suggest that loss of MeCP2 at any developmental stage causes functional deficiencies indicating a role for MeCP2 in acute neuronal function in addition to a role in neuronal development. This information is important because it may provide a framework whereby particular pathways or specific brain regions can be targeted for the treatment of Rett Syndrome by taking advantage of their distinctive pharmacological profiles. Rett's syndrome (RTT) is a neurodevelopmental disorder that accounts for one of the leading causes of mental retardation and autistic behavior in females. In general, individuals affected with RTT experience normal development up to the age of 5-48 months at which time developmental problems occur. Most RTT defects are predominantly expressed in the CNS, including mental retardation, autism-like behavior, seizures, disturbances of sleep, problems with gait, and stereotypical hand movements. Recent work has demonstrated that RTT is an X-linked dominant disorder that in most instances (at least 76%) results from mutations in the Methyl-CpG-binding protein (MeCP2) gene that are predicted to result in loss of function of MeCP2. While these mutations have been identified in the majority of RTT cases, there is currently no direct link between loss of function of MeCP2 and the pathogenesis of RTT. To better understand the role of MeCP2 in mediating the behavioral phenotypes observed in RTT individuals, we propose to delete MeCP2 in specific regions of the brain and then examine these animals in a broad array of behavioral paradigms. We will also delete the MeCP2 gene in developing mice (before the appearance of a behavioral phenotype) and in adult mice (after the appearance of a behavioral phenotype) and then assess these animals in a broad array of behavioral models. This approach will allow a clearer interpretation of MeCP2's role in specific brain regions as well as in developmental time points in mediating behavioral phenotypes similar to those observed in RTT patients. The proposed studies should increase our understanding of the role of MeCP2 in mediating certain RTT associated behaviors as well as identify neural circuits that mediate these abnormalities. [unreadable] [unreadable] [unreadable]

[unreadable] DESCRIPTION (provided by applicant): The major objective of this proposal is to further delineate the role of MeCP2, the gene linked to Rett Syndrome (RTT), in excitatory neurotransmission through its function as a transcriptional repressor. We have recently shown that the loss of MeCP2 in neurons contributes to alterations in exicitatory, but not inhibitory, synaptic transmission. We also demonstrated that these deficits in excitatory transmission, a component of short term plasticity, appear due to MeCP2's role as a transcriptional repressor. Conversely we have recently found that overexpression of MeCP2 results in alterations in excitatory synaptic transmission and synaptic depression opposite to those obtained following the loss of MeCP2. This is important because if MeCP2 is a bona fide regulator of synaptic function then we would expect bidirectional changes in MeCP2 to result in reciprocal alterations in neurotransmission. The proposed studies will complement and extend our previous work by addressing three specific aims using molecular, cellular and electrophysiological methods. We will first, further characterize the loss of MeCP2 in short term plasticity to more fully discern its endogenous role in neuronal function. Second, we will characterize the overexpression of MeCP2 in short term plasticity to examine how alterations in MeCP2 expression effects synaptic transmission. This is important since recent studies have shown that duplication of the MeCP2 gene may underlie certain forms of mental retardation and progressive neurological symptoms. Lastly, we will elucidate the role of histone deacetylases (HDACs) 1 and 2, key repressors of gene repression that are part of a multiprotein complex with MeCP2 in regulating gene expression, on synaptic transmission. Our hypothesis is that alterations in MeCP2 expression contribute to effects on synaptic function. The studies proposed in this application will extend our original hypothesis by further characterizing how alterations in the expression of MeCP2 contributes to deficits in neurotransmission as well as how the HDAC components of the MeCP2 complex may be involved in these processes. This information is important because it will start to provide a framework in which to explore how synaptic alterations may underlie aspects of RTT. PUBLIC HEALTH RELEVANCE: The experiments proposed in this project represent a comprehensive effort to address the role of MeCP2, the gene linked to Rett Syndrome, in excitatory neurotransmission through its function as a transcriptional repressor. Currently, a thorough analysis of the role of MeCP2 in short-term synaptic plasticity in central synapses is lacking. In this project, we will examine via complementary approaches how the loss of MeCP2 or the overexpression of MeCP2 contributes to alterations in synaptic transmission. We will also elucidate the role of histone deacetylases (HDACs) 1 and 2, key repressors of gene repression that are part of a multiprotein complex with MeCP2 in regulating gene expression, on synaptic transmission. Information attained from these studies will provide new insight into the synaptic mechanisms that may be affected in Rett Syndrome as well as related disorders that involve alterations in MeCP2 expression. [unreadable] [unreadable]

DESCRIPTION (provided by applicant): Over the past ~5 years, we have examined the role of MeCP2 in neurotransmission as a transcriptional factor impacting gene expression. Our data has revealed that MeCP2 is a bona fide regulator of synaptic function with bidirectional changes in MeCP2 resulting in reciprocal alterations in neurotransmission. We have also shown alterations in MeCP2 expression in mice result in several behavioral phenotypes as well as deficits in specific measures of synaptic plasticity further implicating MeCP2 as a key mediator of synaptic processes. A rather surprising finding in the field of depression has been the demonstration that scopolamine, a muscarinic acetylcholine receptor antagonist, has rapid and long-lasting antidepressant responses in depressed individuals. We have started to investigate the mechanism of the antidepressant action of scopolamine, and have found it is dependent on MeCP2 expression as these effects are lost in Mecp2 knockout mice. Our findings also suggest that the antidepressant effects of scopolamine are dependent on MeCP2-dependent transcriptional mechanisms resulting in increased BDNF expression that is important for the behavioral effects. Our preliminary data further suggests that scopolamine triggers MeCP2 phosphorylation at Serine 421 (pMeCP2), which has been shown to regulate BDNF expression. In initial experiments, we find that scopolamine acts via blockade of the muscarinic M1 receptor to trigger pMeCP2. The objective of this grant is to explore the novel hypothesis that MeCP2- dependent transcriptional mechanisms underlie the fast acting antidepressant effects of scopolamine. We will use state of the art behavioral as well as cellular and biochemical approaches to examine our hypothesis. Collectively, these studies will contribute to a better understanding of mechanisms underlying fast-acting antidepressant responses as well as provide novel insight into MeCP2 regulation as a therapeutic target.

? DESCRIPTION (provided by applicant): In the previous funding period we investigated the mechanism of rapid antidepressant activity of ketamine, an ionotropic glutamatergic n-methyl-d-aspartate (NMDA) receptor antagonist. We demonstrated that Brain-derived neurotrophic factor (BDNF) is required for the fast acting antidepressant effects of ketamine as these effects are lost in forebrain specific BDNF knockout mice. We found that the antidepressant effects of ketamine require protein translation, but not transcription, resulting in increases in BDNF protein levels in the hippocampus that are important for the behavioral effect. Recent work has suggested a strong causal link between blockade of resting NMDA receptor activation and rapid increases in local dendritic protein translation. Blockade of NMDA receptor activation by spontaneous glutamate release has been shown to inactive eukaryotic elongation factor 2 kinase resulting in dephosphorylation of its only known substrate, eukaryotic elongation factor 2 (eEF2), thereby increasing protein translation of target transcripts. We showed that ketamine causes a decrease in phosphorylation of eEF2, which normally impedes translation in its phosphorylated state, suggesting translational de-repression of BDNF mRNA. Moreover, inhibitors of eEF2 kinase trigger a rapid antidepressant-like effect in mice and ketamine does not elicit an antidepressant effect in eEF2 kinase null mice. These data provide the basis for the novel hypothesis that ketamine, by blocking NMDA receptors at rest, inhibits the phosphorylation of eEF2 and augments subsequent expression of BDNF, critical determinants of ketamine-mediated antidepressant efficacy. The objective of this renewal is to delineate the role of BDNF-TrkB signaling in the hippocampus in ketamine-mediated antidepressant effects, as well as how eEF2 kinase acts as a transducer between NMDA receptor activity and BDNF regulation. Collectively, this information will provide novel information on the synaptic locus, as well as the key molecules, necessary for ketamine's rapid antidepressant effects.