Michael Ehlers - US grants

DESCRIPTION: (from applicant's abstract) Rapid communication between neurons is accomplished by the action of neurotransmitters on postsynaptic receptors. At excitatory synapses, these receptors belong to the family of ionotropic glutamate receptors. By regulating the activity and membrane density of glutamate-gated ion channels, neurons alter the strength of their synaptic inputs in response to developmental cues and sensory stimuli. Both inadequate and excessive activity of glutamate receptors has been linked to psychiatric disease and neurological deficit. Determining molecular features of glutamate receptor regulation is crucial for our understanding of normal excitatory synapse function, and should facilitate the design of rational strategies for the treatment of neurologic and psychiatric disease. The objective of this proposal is to understand how neurons assemble and regulate the postsynaptic molecular machinery present at excitatory synapses. In particular, the proposed research will determine molecular mechanisms which regulate the synaptic targeting and clustering of the N-methyl-D-aspartate (NMDA) class of ionotropic glutamate receptors. We have recently identified cytoplasmic domains of the NMDA receptor subunit NR1 important for receptor targeting and receptor inactivation. These regions of NR1 are thought to mediate their effects by interacting with cytoskeletal proteins and signaling molecules. To further elucidate the function of NMDA receptor cytoplasmic domains, we will first identify novel molecules which interact with NR1 subunits using both yeast two-hybrid screening and biochemical approaches. Second, the effects of these interactions on NMDA receptor channel activity and synaptic localization will be directly tested by electrophysiological and immunocytochemical methods. Third, NMDA receptor targeting motifs will be identified using fluorescent localization of tagged wild-type and mutant NR1 and NR2 subunit cytoplasmic domains expressed in cultured neurons. Finally, the effect of synaptic activity on the interaction of cellular proteins with native NMDA receptors will be assessed using covalent crosslinking techniques. Together, the proposed experiments will elucidate basic molecular mechanisms underlying synaptic transmission and synaptic plasticity.

DESCRIPTION (provided by applicant): Neurotransmission requires a precise number and arrangement of receptors and ion channels in the neuronal plasma membrane. Alterations in the localization or levels of these proteins in the membrane regulates synapse function, thereby strengthening or weakening synaptic connections in the brain. In all eukaryotic cells, the internalization, recycling, and degradation of membrane proteins is controlled by regulated trafficking through the endocytic pathway. Although previous studies have helped define this pathway in many nonneuronal cell types, the mechanisms underlying endocytic trafficking of postsynaptic receptors and the role of dendritic endosomal transport in synaptic transmission and plasticity remain unknown. To address these important questions, my laboratory has initiated a program of biochemical and cell biological studies to analyze the endocytic trafficking of AMPA-type glutamate receptors. AMPA receptors are the major mediators of fast excitatory transmission in the brain, and alteration of the number and function of AMPA receptors is a critical feature of synaptic plasticity. We have recently found that AMPA receptors are differentially sorted between recycling and degradative pathways following endocytosis. This sorting decision is in turn controlled by the relative activation of AMPA and NMDA-type glutamate receptors in the postsynaptic membrane. Taking advantage of these preliminary data and our ability to monitor and manipulate AMPA receptor trafficking in neurons, we propose to define the underlying molecular and cellular mechanisms of AMPA receptor endocytic trafficking and determine the functional consequences for synapse maturation and maintenance. This work will provide insight into fundamental mechanisms that underlie synapse formation and synaptic plasticity. Moreover, given the importance of excitatory synaptic transmission and AMPA receptor activation in the pathogenesis of numerous psychiatric and neurologic diseases, these studies hold promise for the development of novel therapeutic strategies.

DESCRIPTION (provided by applicant): A central goal of research in Alzheimer's disease (AD) is the identification and reversal of the earliest pathological changes in affected brain systems and neural circuits. Although numerous structural and biochemical changes have been documented in late-stage AD brains, the early microscopic events that initiate neuronal dysfunction provide potentially more attractive therapeutic targets. Among the initial targets of AD pathogenesis are neuronal synapses. In its earliest phases, AD is characterized by a remarkably pure impairment of memory that has been attributed to 'subpathological' alterations in excitatory synaptic transmission in the hippocampus. Recent studies strongly support the involvement of misprocessed amyloid precursor protein (APP) and A-beta deposition in the early synaptic and cognitive changes of AD. However, little is known about the molecular mechanisms by which exposure to A-beta affects synaptic plasticity, or potential compensatory mechanisms that could be used to counteract aberrant plasticity. In the proposed research, we will define the molecular targets for A-beta-induced synaptic dysfunction. A newly recognized mechanism for changing synaptic strength is the rapid removal of postsynaptic receptors via endocytosis. We have recently found that dendritic spines contain a zone of clathrin assembly and endocytosis adjacent to, but spatially segregated from, the postsynaptic density. Moreover, we have found that the protein machinery for postsynaptic endocytosis is functionally altered by aging and may be upregulated by exposure to A-beta. These findings present an opportunity to clarify in molecular detail the mechanisms by which A-beta influences excitatory transmission and synaptic plasticity. These studies will provide much-needed insight into the cell biological mechanisms that underlie AD-related changes in synaptic plasticity, and will identify molecular signaling pathways that may correct A-beta-induced changes in synaptic function. As such, the proposed research holds promise for the development of new therapeutic approaches for AD-associated memory loss and cognitive deficit.

DESCRIPTION (provided by applicant): Neural communication is modified throughout life by use-dependent changes in the number and function of glutamate receptors at excitatory synapses. Among the glutamate-gated ion channels, N-methyl-D-aspartate (NMDA) receptors play a central role in synapse formation, synaptic plasticity, neurological diseases, and psychiatric disorders including addiction. A principal mechanism controlling NMDA receptor signaling is accurate regulation of the number of NMDA receptors present at the synapse. Although most widely appreciated for AMPA-type glutamate receptors, dynamic regulation of the number of postsynaptic NMDA receptors is increasingly recognized as an integral feature of synapse maturation and synaptic plasticity. And yet, little is known about the molecular mechanisms for trafficking NMDA receptors to and from the synapse. To address these important questions, my laboratory has initiated a program of biochemical and cell biological studies to analyze the trafficking of NMDA receptors and the regulation of such trafficking by neuronal activity. We have recently found that export from the endoplasmic reticulum (ER) serves as an activity-dependent checkpoint for the synaptic delivery of NMDA receptors, Moreover, we have identified key molecular determinants within NMDA receptor subunits that control forward trafficking, and developed optical imaging approaches to visualize secretory trafficking in living dendrites. Taking advantage of these preliminary data, we propose to define the underlying cellular mechanisms that control NMDA receptor secretory trafficking, and to determine how activity directs NMDA receptor intracellular transport to effect synapse modification. This work will provide much-needed insight into fundamental mechanisms of synapse formation and plasticity. Moreover, because NMDA receptors participate in the pathogenesis of a wide range of neurologic disorders, psychiatric disease, and states of addiction, these studies hold promise for the development of novel therapeutic strategies.

DESCRIPTION (provided by applicant): Neurotransmission requires a precise number and arrangement of receptors, ion channels, and adhesion molecules at synapses. Alterations in the localization or levels of these proteins at the postsynaptic membrane regulates synapse function, thereby strengthening or weakening synaptic connections in the brain. In all eukaryotic cells, removal of membrane proteins of diverse types occurs by clathrin-mediated endocytosis. Although previous studies have helped define the endocytic machinery in nonneuronal cells and the presynaptic nerve terminal, the location and regulation of clathrin-mediated endocytosis within postsynaptic compartments and its functional role in synaptic signaling remain unknown. To address these important questions, my laboratory has initiated a program of biochemical and cell biological studies to analyze the endocytic machinery of dendritic spines - the primary postsynaptic compartment in the mammalian brain. We have recently found that dendritic spines contain a zone of clathrin assembly and endocytosis adjacent to, but spatially segregated from, the postsynaptic density. This endocytic zone forms and persists over long periods of time independent of synaptic activity, and serves to concentrate cargo destined for internalization. Taking advantage of these preliminary data and our ability to monitor and manipulate clathrin assembly and cargo uptake in neurons, we propose to define the underlying molecular and cellular mechanisms that form, maintain and regulate the endocytic zone of spines, and determine the functional consequences for spine maturation and synaptic transmission. This work will provide insight into fundamental mechanisms that underlie synapse formation and synaptic plasticity. Moreover, because clathrin-mediated endocytosis regulates neuronal responsiveness to a wide range of pathologic insults and therapeutic agents relevant to numerous neurologic and psychiatric diseases, these studies hold promise for the development of novel therapeutic strategies.

DESCRIPTION (provided by applicant): Mapping functional circuits is a major goal for both cellular and systems neuroscience. Current approaches for mapping neural circuits are limited by the lack of technologies for evoking cell-specific neural activity. Available methods of neural stimulation rely on either local application of undiscriminating fields of electrical currents, glutamate uncaging, or the presentation of artificial sensory stimuli. Although recent use of light- gated ion channels has provided optical control of neuronal activity on rapid time scales, such approaches are limited by the requirement for direct optical access to neuronal populations of interest, and are not currently suitable for activating large brain areas or disperse neuronal populations. A transformative technology for neuroscience would be non-invasive control over neural activity in genetically defined populations of neurons in the mammalian brain. Such a goal requires combining genetic sensitization of neuronal subsets with a means to manipulate their electrical activity remotely without surgery or intracranial implants. To create such a technology, my laboratory has initiated a program of in vivo chemical genetic and physiological studies to engineer a mouse model suitable for precise non-invasive manipulation of neural activity in genetically defined populations of neurons in vivo. We have developed a conditional mouse model that sensitizes genetically defined neurons to an artificial ligand (capsaicin) by cell type-specific expression of a heterologous receptor (TRPV1). We have found that application of capsaicin to neurons expressing TRPV1 induces strong inward currents, triggers robust firing of action potentials, and activates stereotyped behaviors. Taking advantage of these preliminary data, and the extensive pharmacological and biophysical characterization of TRPV1, we propose to extend and modify this model to enable peripheral administration of agonists for central activation of defined neuronal subsets. Moreover, because the large TRPV1 channel pore is permeable to small molecules, including the membrane-impermeant sodium channel blocker QX-314, we propose to test this novel mouse model to enable both activation and inhibition of neuronal activity. This work will allow for the development of a novel in vivo technology for chemical genetic regulation of neuronal activity that is (1) orthogonal to optical and optogenetic strategies, (2) based on the only current Cre/lox-based model for neuronal activation, (3) may allow for fully non-invasive CNS activation by drug injection, and (4) may enable targeted small molecule delivery to defined neuronal subsets. PUBLIC HEALTH RELEVANCE: The proposed research will develop a novel technology for non-invasive control over electrical activity in genetically defined populations of brain cells. Abnormal electrical activity in the brain contributes to epilepsy, memory decline, depression, autism, schizophrenia, and addiction. By developing a crucial new technology for targeted manipulation of brain cell activity and metabolism, the proposed research will define novel brain circuits and therapeutic strategies for treating these devastating neurological and psychiatric disorders, which currently have a profound negative impact on public health.