Rodrigo Andrade - US grants

¿¿¿¿¿The¿neurotransmitter¿serotonin¿is¿thought¿to¿have¿broad¿functions¿in¿the¿central¿ nervous¿system,¿contributing¿to¿the¿biological¿basis¿underlying¿both¿behavior¿and¿ cognition.¿This¿neurotransmitter¿has¿also¿been¿implicated¿in¿mental¿disorders,¿ especially¿anxiety¿and¿depression,¿and¿several¿pharmacological¿agents¿used¿to¿treat¿ these¿diseases¿target¿serotonergic¿systems¿in¿the¿brain.¿¿ ¿¿¿¿¿Serotonin¿acts¿through¿multiple¿serotonin¿receptors¿to¿regulate¿neuronal¿activity¿and¿ work¿during¿the¿last¿decade¿has¿made¿extraordinary¿advances¿in¿our¿understanding¿of¿ the¿basic¿biology¿of¿these¿receptors.¿However¿our¿understanding¿of¿how¿these¿receptors¿ function¿within¿neurons,¿what¿they¿signal¿and¿how¿they¿signal¿in¿the¿central¿nervous¿ system,¿is¿still¿poorly¿understood.¿This¿is¿an¿important¿lacunae,¿since¿the¿biological¿ basis¿of¿mental¿disorders,¿as¿well¿as¿the¿development¿of¿better¿treatments,¿will¿be¿ grounded¿in¿the¿specific¿mechanisms¿used¿by¿these¿receptors.¿ ¿¿¿¿¿The¿long¿term¿goal¿of¿the¿PI's¿research¿program¿is¿to¿help¿develop¿a¿mechanistic¿ understanding,¿at¿the¿molecular¿level,¿of¿serotonin¿receptor¿function¿in¿central¿nervous¿ system¿neurons.¿Specifically¿in¿this¿application¿we¿propose¿to¿use¿a¿synergistic¿ combination¿of¿electrophysiological¿and¿molecular¿biological¿approaches¿to¿understand¿ the¿signaling¿mechanisms¿used¿by¿5 HT2A¿receptors¿in¿the¿cerebral¿cortex.¿¿ ¿¿¿The¿application¿proposes¿three¿Specific¿Aims.¿The¿first¿of¿these¿will¿elucidate¿the¿ proximal¿signaling¿mechanism¿used¿by¿5 HT2A¿receptors¿to¿regulate¿membrane¿ excitability¿in¿pyramidal¿cells¿of¿the¿prefrontal¿cortex.¿The¿second¿Aim¿will¿test¿the¿idea¿ that¿5 HT2A¿receptors¿inhibit¿the¿slow¿calcium activated¿potassium¿current¿present¿in¿ pyramidal¿cells¿by¿reducing¿the¿concentration¿of¿membrane¿PtdIns(4,5)2.¿The¿third¿ Specific¿Aim¿will¿determine¿how¿5 HT2A¿receptors¿depolarize¿and¿excite¿pyramidal¿ cells.¿¿ ¿¿¿The¿results¿of¿this¿project¿should¿contribute¿to¿our¿understanding¿of¿serotonergic¿ mechanisms¿in¿the¿brain¿and,¿as¿such,¿contribute¿to¿our¿understanding¿of¿mental¿ disorders¿while¿supporting¿the¿search¿for¿more¿effective¿treatments.¿ The¿neurotransmitter¿serotonin¿has¿been¿implicated¿in¿the¿genesis¿of¿anxiety¿and¿ depression¿and¿in¿the¿mechanism¿of¿action¿of¿several¿drugs¿effective¿in¿the¿treatment¿of¿ mental¿disorders.¿The¿research¿proposed¿in¿this¿application¿seeks¿to¿elucidate¿the¿ molecular¿mechanisms¿used¿by¿serotonin¿to¿regulate¿the¿function¿of¿brain¿cells¿and¿ brain¿circuits.¿The¿results¿of¿this¿project¿should¿help¿us¿better¿understand¿mental¿ disorders¿and¿contribute¿to¿the¿search¿of¿more¿effective¿treatments.¿

DESCRIPTION (adapted from the applicant's abstract): Cognitive impairment is a serious health concern in the United States. Epidemiological studies indicate a prevalence of over 1%, and this problem can be expected to worsen with the aging of the U.S. population. In spite of the seriousness of this problem, pharmacotherapeutic treatment for this condition remain extremely limited. Basic and clinical studies have long recognized the importance of cholinergic mechanisms for the maintenance of cognitive functioning. Indeed several lines of evidence support the idea that an AChergic deficit in cerebral cortex is a causative element in cognitive impairment. Thus, an understanding of AChergic mechanisms in cortex could substantially contribute to the search for effective pharmacotherapeutic treatments for this condition. The long term goal of this project is to elucidate the cellular and molecular mechanisms by which ACh regulates neuronal excitability and function in the cerebral cortex. To that effect, we have initially focused on responses mediated through mAChRs in PFC, an association area. During the past funding cycle, we have used electrophysiological techniques in vitro rat brain slices to identify and characterize a novel cation current that appears to be one of the principal targets for AChergic regulation in this region. In the present application we propose to extend these studies: (1) by examining the role of intracellular calcium (Ca2+) in the activation of this current; (2) determining the mAChR subtypes involved; (3) identifying the signaling mechanism by which this current is activated; and (4) determining whether this current is also activated by other neurotransmitters besides ACh. These studies should extend our understanding of the cellular and molecular mechanisms by which mAChRs regulate neuronal excitability in rat PFC. This knowledge should contribute to the search for novel and effective treatments for AChergic dysfunction in general, and cognitive impairment in particular.

Serotonin is a ubiquitous neurotransmitter in the mammalian brain. As such it has been implicated in the control of many behaviors and in the pathophysiology of mental diseases such as depression and obsessive compulsive disorders. It has long been recognized that serotonin acts through a variety of receptor subtypes to exert its effects in the brain. Thus specific serotonin receptor subtypes have long been regarded as promising targets for therapeutic intervention. Efforts to implement such interventions, however, are hampered by our limited understanding of the actions mediated by different serotonin receptor subtypes in the brain. This proposal seeks to address this issue by elucidating the mechanism of actions of two serotonin receptor subtypes that mediate excitatory responses to serotonin in the brain. The effects of serotonin in in vitro rat brain slices will be investigated using whole cell recording electrophysiological techniques. Specifically we will investigate the ability of serotonin to depolarize neurons of the anterior thalamus and prefrontal cortex. We hypothesize that these superficially similar responses involve two different receptors of the 5-HT7 and 5-H2A subtypes respectively. Furthermore we hypothesize that these responses will also be mediated though different cellular and ionic mechanisms. These studies should elucidate the mechanisms by which specific serotonin receptor subtypes regulate membrane excitability in the central nervous system. As such they should contribute to our understanding of the actions of serotonin in the brain. This knowledge should prove helpful in guiding the development of novel therapeutic strategies targeting serotonergic systems in the brain.

DESCRIPTION (provided by applicant): Serotonin is a ubiquitous neurotransmitter in the brain that has been linked to the pathophysiology of avariety of mental disorders. As such, there is great interest in understanding the cellular and molecular mechanisms by which serotonin regulates neuronal functionin the brain. Serotonin acts in the brain through 7 different families of serotonin receptors. Therefore, understanding the physiological function of different serotonin receptor subtypes is an important step in trying to understand how serotonin works in the brain. The long term goal of this project is to understand the physiological role of different serotonin receptor subtypes, and the cellular and molecular mechanisms that underlie these effects. During previous funding periods we have focused on several distinct classes of receptors including 5-HT1A, 5-HT4 and 5-HT7 receptors. In the present application we propose to expand these studies to receptors of the 5-HT2A subtype. These receptors are particularly interesting because they have been identified as the targets for classic hallucinogens, such as LSD, and may participate in the pathophysiology of schizophrenia and depression, For the present project we propose to combine electrophysiological and molecular biological approaches to understand how 5-HT2A receptors regulate neuronal function in the rat prefrontal cortex. In a first Specific Aim we propose to examine mechanistically how activation of 5-HT2A receptors increase glutamate synaptic transmission in this region. In the second Specific Aim we will test the idea that one important effect of serotonin receptors is to regulate synaptic plasticity in prefrontal cortex. Finally, in a third Specific Aim, we will test the hypothesis that scaffolding proteins, and specifically MUPP1, play an important role in organizing signaling by 5-HT2A receptor in the brain.Combined, these three Specific Aims will begin to fill some of the gaps in our understanding of what 5-HT2A receptors do, and how they do it, in the cerebral cortex. As such, these studies should contribute to our understanding of the biological basis underlying mental disorders. Equally important, we hope these studies will also contribute to the development on novel therapeuric approaches for the treatment of these disorders.

[unreadable] DESCRIPTION (provided by applicant): A strategy for the efficient, enantioselective synthesis of (+)-halichlorine, a marine natural product which inhibits VCAM-1 (vascular cell adhesion molecule-1) with an IC50 of 7 [unreadable]g/mL, is proposed. Due to its critical role in the regulation of inflammation, VCAM-1 has emerged as a potential target for drug discovery. Inhibitors of this protein could have a profound impact on the treatment of arteriosclerosis, asthma, and cancer. To date there only exists one total synthesis of halichlorine. The proposed plan outlines a novel synthetic route to halichlorine which can be modified to access precursors of pinnaic acid, a related alkaloid and potent inhibitor of cytosolic phospholipase A2. The plan utilizes cress-metathesis methodology for the construction of key olefinic bonds which heretofore has not been used in alkaloid synthesis. This represents a unique opportunity to study the scope of cross-metathesis in total synthesis. An intramolecular Kishi-Nozaki reaction is also proposed as an alternative macrocyclization step. Molecular modeling suggests the reaction will take place with a high degree of diastereoselectivity in favor of the desired stereoisomer. The synthesis will furnish sufficient quantities of halichlorine to assess its potential as a candidate for the treatment of the above conditions. [unreadable] [unreadable] [unreadable]

DESCRIPTION (provided by applicant): Serotonin is a ubiquitous neurotransmitter in the brain that has been linked to the pathophysiology of avariety of mental disorders. As such, there is great interest in understanding the cellular and molecular mechanisms by which serotonin regulates neuronal functionin the brain. Serotonin acts in the brain through 7 different families of serotonin receptors. Therefore, understanding the physiological function of different serotonin receptor subtypes is an important step in trying to understand how serotonin works in the brain. The long term goal of this project is to understand the physiological role of different serotonin receptor subtypes, and the cellular and molecular mechanisms that underlie these effects. During previous funding periods we have focused on several distinct classes of receptors including 5-HT1A, 5-HT4 and 5-HT7 receptors. In the present application we propose to expand these studies to receptors of the 5-HT2A subtype. These receptors are particularly interesting because they have been identified as the targets for classic hallucinogens, such as LSD, and may participate in the pathophysiology of schizophrenia and depression, For the present project we propose to combine electrophysiological and molecular biological approaches to understand how 5-HT2A receptors regulate neuronal function in the rat prefrontal cortex. In a first Specific Aim we propose to examine mechanistically how activation of 5-HT2A receptors increase glutamate synaptic transmission in this region. In the second Specific Aim we will test the idea that one important effect of serotonin receptors is to regulate synaptic plasticity in prefrontal cortex. Finally, in a third Specific Aim, we will test the hypothesis that scaffolding proteins, and specifically MUPP1, play an important role in organizing signaling by 5-HT2A receptor in the brain.Combined, these three Specific Aims will begin to fill some of the gaps in our understanding of what 5-HT2A receptors do, and how they do it, in the cerebral cortex. As such, these studies should contribute to our understanding of the biological basis underlying mental disorders. Equally important, we hope these studies will also contribute to the development on novel therapeuric approaches for the treatment of these disorders.

DESCRIPTION (provided by applicant): Serotonin is a ubiquitous neurotransmitter in the brain that has been linked to the pathophysiology of avariety of mental disorders. As such, there is great interest in understanding the cellular and molecular mechanisms by which serotonin regulates neuronal functionin the brain. Serotonin acts in the brain through 7 different families of serotonin receptors. Therefore, understanding the physiological function of different serotonin receptor subtypes is an important step in trying to understand how serotonin works in the brain. The long term goal of this project is to understand the physiological role of different serotonin receptor subtypes, and the cellular and molecular mechanisms that underlie these effects. During previous funding periods we have focused on several distinct classes of receptors including 5-HT1A, 5-HT4 and 5-HT7 receptors. In the present application we propose to expand these studies to receptors of the 5-HT2A subtype. These receptors are particularly interesting because they have been identified as the targets for classic hallucinogens, such as LSD, and may participate in the pathophysiology of schizophrenia and depression, For the present project we propose to combine electrophysiological and molecular biological approaches to understand how 5-HT2A receptors regulate neuronal function in the rat prefrontal cortex. In a first Specific Aim we propose to examine mechanistically how activation of 5-HT2A receptors increase glutamate synaptic transmission in this region. In the second Specific Aim we will test the idea that one important effect of serotonin receptors is to regulate synaptic plasticity in prefrontal cortex. Finally, in a third Specific Aim, we will test the hypothesis that scaffolding proteins, and specifically MUPP1, play an important role in organizing signaling by 5-HT2A receptor in the brain.Combined, these three Specific Aims will begin to fill some of the gaps in our understanding of what 5-HT2A receptors do, and how they do it, in the cerebral cortex. As such, these studies should contribute to our understanding of the biological basis underlying mental disorders. Equally important, we hope these studies will also contribute to the development on novel therapeuric approaches for the treatment of these disorders.

AWARD ABSTRACT

In this project funded by the Chemical Synthesis program of the Chemistry Division, Professor Rodrigo B. Andrade of Temple University (Department of Chemistry) is studying the development of new chemical reactions to quickly build complex nitrogen-containing molecules. An overarching goal is increasing the efficiency of making complex molecules having broad societal impact by minimizing the overall number of synthetic steps. An overwhelming number of such molecules are biologically active, and can be employed in molecular biology to understand biochemical pathways or directly applied in medicine to better understand disease on a molecular level. The chemical insight can then be applied in therapeutic regimes. To test the scope of these new methods, several complex molecules derived from nature are being prepared in the laboratory. During the development and application of these new methods, undergraduate and graduate students, including those from underrepresented groups, are being trained in the discipline of complex molecules synthesis.

This project systematically studies and applies a novel domino Michael/Mannich [4+2] annulation method to the synthesis of complex molecules. In Aim 1, the nucleophilic and electrophilic components of the method are being examined to determine the reaction scope. In Aim 2, the method is applied to the asymmetric total syntheses of the Aspidospermatan-type alkaloids (+)-epi-condyfoline, (+)-condyfoline, (+)-lagunamine, (+)-epi-lagunamine, (+)-condylocarpine, (+)-isocondylocarpine, and (+)-tubotaiwine. In Aim 3, the method is used to develop the asymmetric total syntheses of the bis-Aspidosperma alkaloids (-)-conophylline and (-)-conophyllidine.

Building on the foundation grounded in an interdisciplinary and multi-university collaborative research network and research program addressing health-related problems through basic and translational neuroscience, we will develop a comprehensive training and mentoring program that serves both faculty and students. There is a need on the Island to prepare competitive neuroscience researchers, and our program aims to fill this gap with Puerto Rican residents, who represent a considerable part of all authentic Hispanic Americans. Furthermore, due to the current economic situation on the Island, a significant number of our potential trainees come from disadvantaged backgrounds. Thus, creating on the Island a strong and efficient training and mentoring center in neuroscience has the potential to significantly promote workforce diversity in the U.S., which is clearly in accord with the goals of the SNRP. In the core of the program, we will have a group of productive senior faculty employed inside and outside UCC, who will provide mentoring to program participants and, along with others, will oversee training and career development activities. We will select the most promising junior faculty members and students as program participants, provide them with individual mentoring, and monitor their progress. We will work with participants to prepare individual career development plans. This will, include regular presentations both internally and at national and international meetings, placements for research practice experience in research intensive institutions, individual plans for grant proposal submission, and career guidance. Such plans will be worked out cooperatively by a program participant together with his/her mentoring committee and the Enrichment Core leadership. Besides individual mentoring, we will organize 1) expert workshops both on basic skills, such as grant writing and publication preparation, and cutting edge approaches in neuroscience, and 2) lectures, seminars, and career orientation sessions. Although these events, in part, will be targeted to train a small number of participants, they will also include sessions available to the broader neuroscience community as well as students, and thus will serve the neuroscience community of the Island. The details of our Enrichment Program are presented in the Enrichment Core section.

DESCRIPTION (provided by applicant): The cerebral cortex, including the median prefrontal cortex, receives a dense serotonergic innervation originating from the Dorsal and Median Raphe nuclei of the brainstem. It is now widely recognized that the prefrontal cortex plays an important role in the temporal organization of behavior and thus is an essential contributor to the pathophysiology of mental disorders including autism, anxiety and mood disorders and schizophrenia. Similarly, clinical and preclinical studies have also identified a strong serotonin component in the pathogenesis of these disorders and also in their pharmacological treatment. Thus there is a pressing need to understand how serotonin regulates the function of the prefrontal cortex. Yet, remarkably, our understanding of the cellular mechanisms by which serotonin regulates the activity of the prefrontal cortex remains frustratingly incomplete. Historically our ability to address serotonergic mechanisms in the prefrontal cortex has been hampered by the cellular complexity of the cerebral cortex and the lack of tools to address this complexity. Recent studies using molecular genetic approaches have begun to provide rich insights into the organization of the cerebral cortex and have generated powerful new tools for experimentally dealing with the different cell populations that make up the cerebral cortex. In this application we propose to take advantage of these developments to begin elucidating how serotonin regulates different genetically defined cell populations and hence regulates the neuronal networks that constitute the prefrontal cortex. The long term goal of this application is to contribute to a mechanistic understanding of mental disorders involving serotonin and the prefrontal cortex and the search for novel more efficacious therapeutic approaches.

DESCRIPTION (provided by applicant): Serotonin plays an important role in the pathophysiology and pharmacotherapeutic treatment of mood and anxiety disorders. Consequently there is an urgent need to understand the mechanisms that control serotonergic function in the brain. An important aspect of this task is understanding the factors that control the activity of serotonin secreting neurons. Previous studies have identified serotonin receptors of the 5-HT1A subtype as important regulators of the activity of serotonergic neurons. Specifically, these so called serotonin autoreceptors have been hypothesized to mediate autoinhibition in serotonergic nuclei and their downregulation has been hypothesized to mediate some of the effects of chronic antidepressant treatment on serotonergic function. Yet, surprisingly and in spite of considerable effort, we still know very little about the specific mechanisms supporting 5-HT1A autoreceptor mediated autoinhibition and how this process participates in the regulation of serotonergic cell activity. This important gap in our understanding reflects technical limitations in our ability to selectively control the activity of serotonergic neurons that have made it difficult to directly study autoreceptor mechanisms. In the current application we propose to use recent technical developments including optogenetics in genetically modified model mice to approach this problem. In preliminary results we show that this approach allows for the unambiguous recording of 5-HT1A receptor-mediated autoinhibitory currents in the Dorsal Raphe Nucleus, the principal serotonergic cell group innervating the forebrain. We propose to extend these findings in three directions, 1) to elucidate, at the level o their synaptic organization, how serotonergic neurons control their own activity through 5-HT1A autoreceptors, 2) to determine the conditions under which 5-HT1A autoreceptor-mediated autoinhibition becomes functional and 3) to investigate how chronic antidepressants modify 5-HT1A receptor-mediated autoinhibition and other cellular processes to modulate the activity of serotonergic neurons. By addressing these issues we hope to help develop a better understanding of the function and regulation of 5-HT1A autoreceptors in the brain and thus contribute to the development of more effective pharmacotherapeutic approaches for the treatment of mood and anxiety disorders.

The Chemical Synthesis Program of the Chemistry Division supports the project by Professor Rodrigo B. Andrade. Professor Andrade is a faculty member in the Department of Chemistry at Temple University. He is developing new chemical reactions to quickly build complex nitrogen-containing molecules. The overarching goal is to maximize the efficiency by which complex, three-dimensional molecules are prepared by using new reagents and reactions discovered in his laboratory. Such an approach reduces the time, energy, and cost associated with synthesizing complex molecules composed of carbon and nitrogen atoms. Many such molecules are biologically active, and so they can be employed as molecular probes to better understand biochemical pathways or as therapeutics to be used in medicine. Beyond the training of undergraduate and graduate students, including those from underrepresented groups, the broader impacts of this project touch the fields of organic chemistry (e.g., total synthesis, synthetic methodology), chemical biology, molecular biology, biochemistry, and medicine.

N-Sulfinyl metallodienamines (NSMDs) have emerged as powerful asymmetric synthons for chemical synthesis. The focus of this project is to determine both the mechanism and scope of reactions using acyclic NSMDs with a variety of electrophiles, which logically builds from previous efforts on arene-fused congeners. Specifically, in Aim 1 the researchers are studying the scope of the reactions of acyclic NSMDs with electrophilic olefins to afford cycloadducts through a Domino Michael/Mannich (formal Diels-Alder) process. In Aim 2, the research group is applying optimal reaction conditions determined from Aim 1 toward the step-efficient, asymmetric total syntheses of complex Iboga alkaloids (+)-catharanthine, (+)-coronaridine, and (+)-ibogamine. In Aim 3, the research students are systematically investigating the reactions of NSMDs with aldehydes (vinylogous aldol) and imines (vinylogous Mannich). In Aim 4, researchers are probing the reactions of NSMDs with electrophilic oxygen and nitrogen sources to access alpha-functionalized N-sulfinyl imines, which can be further modified to access chiral building blocks for organic synthesis. Computer modeling is used in all mechanistic analyses. Professor Andrade has demonstrated a strong commitment to the inclusion of undergraduates, particularly from underrepresented groups, in his research activities.

Neuronal co-transmission, the phenomenon whereby a neuron releases more than one neurotransmitter, is a generalized property of many synapses in the central and peripheral nervous system. Previous work has shown that serotonergic neurons can release both serotonin and glutamate and recent studies have shown that this co-transmission plays a significant role in the generation of behavioral output. However how the use of two neurotransmitters, serotonin and glutamate, uniquely shapes the regulation of postsynaptic targets remains poorly understood. Serotonergic neurons of the Median and Dorsal Raphe nuclei project to the hippocampus where they innervate pyramidal cells and interneurons. Surprisingly the synapses made by serotonergic neurons onto GABAergic interneurons of the SR and SLM appear to rely primarily on glutamate acting on ionotropic receptors, while those made onto pyramidal cells appear to rely primarily on serotonin acting on metabotropic 5-HT1A receptors. This suggests that co-transmission in hippocampus facilitates the differential regulation of distinct postsynaptic targets, a phenomenon that has previously been reported for co-transmission in invertebrates and in autonomic ganglia. In this application we propose to elucidate the mechanisms underlying this remarkable dichotomy. The experiments proposed in this application will be conducted using whole cell electrophysiological recordings in in vitro hippocampal brain slices. Slices will be prepared from SERT-Cre driver mice selectively expressing channelrhodopsin in serotonergic neurons. We and others have previously shown that serotonergic synapses onto GABAergic interneurons of the SR and SLM rely on GluA receptors to elicit robust EPSCs with only sparse evidence for serotonin co-transmission. In the first specific Aim we will examine the potential involvement of different mechanisms that can account for this phenomenon. Additionally we will also test the idea that expression of serotonergic co-transmission is stimulation frequency dependent. In contrast to the situation in interneurons, serotonergic synapses onto pyramidal cells rely primarily on serotonin to elicit 5-HT1A receptor mediated slow IPSCs. In the second Specific Aim we will use intersectional genetic strategies to separate synapses made by neurons co-releasing serotonin and glutamate and test the hypothesis that pyramidal cells are selectively innervated by neurons releasing only serotonin. We will also test the possibility that serotonergic neurons may release glutamate that acts on higher affinity NMDA receptors. The results of these experiments should clarify key mechanistic aspects of serotonin glutamate co-transmission in the hippocampus and contribute to a better understanding of the pathophysiology and therapeutics of diseases involving serotoninergic synapses.