Sharon Swanger, PhD - US grants

DESCRIPTION (provided by applicant): Dendritic protein synthesis is essential for several forms of long-term synaptic plasticity that are thought to underlie learning and memory. However, the molecular mechanisms that regulate activity-induced dendritic translation remain unclear. One hypothesis is that activity may regulate mRNA binding proteins (RBP) that control the dendritic localization and translation of specific mRNAs. This proposal investigates the role of the RBP cytoplasmic polyadenylation element binding protein 1 (CPEB1) in dendritic mRNA targeting and local translation. The overall hypothesis is that CPEB1 associates with a multi-protein dendritic complex that regulates activity-induced transport and translation of known CPEB1 target mRNA CaMKIIa. The first aim of this proposal examines 1) dendritic localization of the multi-protein complex in CPEB1 null neurons compared to wild type and 2) the molecular mechanisms mediating activity regulation of the CPEB1 complex at synapses. The two proposed approaches for this aim are high-resolution quantitative immunofluorescence in cultured hippocampal neurons and biochemical analysis of synaptic fractions. The second aim addresses whether the CPEB1 complex regulates basal and activity-induced dendritic localization of CaMKIIa mRNA. CaMKIIa mRNA localization will be assayed in CPEB1 null neurons and following lentiviral-mediated knockdown of the CPEB1 complex proteins using in situ hybridization and live imaging of a tagged mRNA construct. The third aim evaluates whether the CPEB1 complex regulates dendritic CamKlla mRNA translation using live-cell imaging of a fluorescent mRNA reporter combined with lentiviral-mediated manipulation of the CPEB1 complex components. Consistent with the mission of NINDS to "pursue an understanding of the normal activities" of neurons, this proposal examines a potential mechanism by which activity might regulate the local synthesis of new proteins. This work contributes to the basic knowledge that founds the development of useful therapies for disorders of synaptic function. Lay summary: Deficits in learning and memory are symptoms of varied neurological conditions such as mental retardation, autism, traumatic brain injury and Alzheimer's disease. The proposed study investigates the regulation of experience-dependent modification of neuronal connections - the biological process that is thought to underlie learning and memory. Acquiring this basic knowledge is critical for understanding the pathology of these wide-spread disorders as well as the establishment of useful therapies.

DESCRIPTION (provided by applicant): The debilitating motor symptoms in Parkinson's disease are related to aberrant spike firing of neurons in the subthalamic nucleus (STN), a key structure within the basal ganglia motor circuit. Glutamatergic afferents to the STN critically regulate STN neuron firing frequency and pattern; however the functions of specific glutamate receptors in mediating synaptic transmission in the STN have not been well-studied. The N-methyl- D-aspartate (NMDA) family of ionotropic glutamate receptors are critical for excitatory neurotransmission throughout the brain. NMDA receptors are tetramers composed of two GluN1 subunits and two GluN2 subunits (GluN2A-2D), and the GluN2 subunits display different functional properties as well as expression patterns in the brain. The GluN2B and GluN2D subunits are expressed by STN neurons, however the lack of GluN2D-selective compounds has limited the study of this subunit in the brain. Newly developed allosteric modulators that are selective for GluN2C/D subunits will now allow investigation of the function of GluN2D in the STN as well as the potential of inhibition of GluN2D-containing NMDA receptors as a therapeutic strategy to correct the aberrant firing of STN neurons in Parkinson's disease. In this study, patch-clamp electrophysiology and optogenetics will be used to investigate the roles of GluN2B- and GluN2D-containing NMDA receptors at the different glutamatergic inputs to the STN. The specific aims of the proposed work are: 1) how NMDA receptor subtypes control excitatory synaptic transmission at anatomically distinct afferents to the STN, and 2) how specific NMDA receptors control tonic and activity-induced spike firing in STN neurons. These experiments will generate key data on the roles for NMDA receptors in controlling glutamatergic neurotransmission and spike firing in the STN.