J Julius Zhu, Ph.D. - US grants

Long-term synaptic depression (LTD) and depotentiation, the two forms of sustained synaptic depression after periods of repetitive synaptic activity, are extensively studied examples of vertebrate synaptic plasticity. The cellular and molecular mechanisms responsible for LTD and depotentiation will likely elucidate physiological and pathological phenomena of neural development, adaptation, learning and memory. There is now compelling evidence that repetitive synaptic activity leads to activation of NMDA- sensitive glutamate receptors (NMDA-Rs) and removal of postsynaptic AMPA-sensitive glutamate receptors (AMPA-Rs) from excitatory synapses during LTD and depotentiation. However, the biochemical pathways that link NMDA-R activity to AMPA-R trafficking are largely unknown. We have previously reported that small GTPase Rapl controls LTD via activation of p38MAPK. In a preliminary study, we observed that small GTPase Rap2 controls depotentiation via activation of JNK. Based on these findings, I proposed a new model that Rapl and Rap2 signal synaptic depression via two independent signaling pathways. We will test three hypotheses in this model with three aims, respectively, using an organotypic culture hippocampal slice preparation. This preparation allows us to manipulate synaptic activity and signaling molecules'activity using physiology, pharmacology and recombinant protein delivery methods. We will assay the effects of these manipulations by examining electrophysiologically tagged recombinant AMPA-R-mediated currents, measuring synaptic responses in GluRl and GluR2 knockout mice, as well as quantifying phosphorylated or active endogenous signaling molecules and glutamate receptors. Combining these approaches, we will determine whether: (Aim 1) Rapl-p38MAPK signals LTD whereas Rap2-JNK signals depotentiation;(Aim 2) different downstream signaling molecules relay Rapl-p38MAPK and Rap2-JNK pathways;and (Aim 3) different upstream signaling molecules control Rapl-p38MAPK and Rap2-JNK pathways. Because genetic defects in signaling molecules or enzymes controlling Rap signaling pathways lead to severe mental retardation, the findings from this study should also suggest additional molecular targets for novel genetic and pharmacological strategies that may efficaciously treat these insidious mental diseases.

[unreadable] DESCRIPTION (provided by applicant): How ascending sensory information is integrated in the neocortex has been extensively studied in the last several decades. This sustained interest in cortical sensory integration stems from the belief that sensory processing underlies key aspects of cognitive functions and dysfunctions, such as sensory perception, sensory discrimination, trauma and other cognitive dysfunctions. In sensory cortices, layer 4 constitutes the main target layer for specific thalamocortical afferents and is the layer in which cortical sensory processing begins. It is known that only approximately 5-15% of excitatory synapses onto layer 4 neurons are from thalamocortical (TC) fibers, whereas the majority of other excitatory synapses are from intracortical (IC) fibers. However, the relative importance of TC and IC connections to sensory processing is still in debate, due in part to the poor understanding of the cellular and molecular properties of these connections. Some studies have suggested that TC synapses are strong and reliable enough to convey sensory information into the cortex, while others have argued that recurrent IC synapses are indispensable in amplifying and dynamically regulating TC inputs, hi addition, how synaptic strengths of TC and IC connections are maintained and regulated is unclear. Combining electrophysiology, molecular biology and genetics techniques, we have recently found that synaptic responses at IC and TC synapses display different kinetics, which may represent a novel and important strategy for integrating and amplifying sensory inputs. Our preliminary data also suggest that the differences of kinetics at these synapses are due to synapse-specific delivery of distinct AMPA-sensitive glutamate receptors (-Rs). Based on these results, I hypothesize that postsynaptic regulation of transmission is synapse-specific in layer 4 cortical neurons. We will examine what controls the synaptic kinetics and efficacy of transmission at IC synapses. Moreover, we will determine what regulates synaptic kinetics and efficacy of transmission at TC synapses. Finally, we will investigate how IC and TC synapses maintain synaptic strength. Because different AMPA-Rs exhibit distinct properties in mediating synaptic transmission and in controlling synaptic efficacy, the results from this project should aid the understanding of sensory physiology of neocortex and cortical pathologies. [unreadable] [unreadable] [unreadable]

DESCRIPTION (provided by applicant): The multifaceted capability of neocortex emerges from its complicated cellular constituents, particularly the exceptionally diverse interneurons operating in an intricately organized circuit. Over the years many researchers have used primarily dual or triple whole-cell recordings to examine one or two, often ambiguously identified interneurons in incomplete circuits. As a consequence, most researchers have been overwhelmed by the differences between their results, much like the tale of three blind men and an elephant, and have come to believe that the cortex consists of at least a few dozen of distinct types of interneurons and these interneurons must thus form a sophisticated cortical inhibitory network. In this application, we plan to develop a stable simultaneous octuple whole-cell recording technology to directly record and compare multiple anatomically identified interneurons in complete cortical inhibitory circuits (to see a more complete picture of the elephant). Based on our preliminary data, we propose to test whether cortical interneurons may be classified into a handful of groups based on their axonal arborization, and whether these interneurons serve functionally different roles in column- (aim 1), laminar- (aim 2) and/or subcellular domain (aim 3)-specific circuits. Our central hypothesis is that the cortical interneuronal network is constituted by nine general types of interneurons and is organized following three elemental rules. The findings from this project will support these surprisingly simple cortical interneuron classification scheme and circuit organizational principles. Because altered interneuronal function is a common mechanism contributing to various neurological disorders, including autisms, epilepsy, depression, Huntington's disease, neurofibromatosis, schizophrenia, Tourette's syndrome and trauma, this project will also help to build the groundwork for future identification of specific interneuron type(s) and/or circuit(s) altered in each of these neurological diseases.

? DESCRIPTION (provided by applicant): T-type CaV3.2 calcium channels are widely expressed in various types of neurons and dysfunction of CaV3.2 channels has been strongly implicated in human childhood absence epilepsy (CAE). However, the role of these calcium channels in neurons remains unknown. More surprisingly, ~50% CAE patients did not respond to ethosuximide, a T-type Ca2+ channel antagonist and first-line drug used to treat CAE, and most of the nonresponsive patients carry gain-of-function Cav3.2 mutations. In this project, we plan to investigate the functional role of CaV3.2 channels in neuronal cells and the pathogenesis of ~20 CAE-linked human CaV3.2 channel mutations. In the preliminary investigation, we manipulated the activity of CaV3.2 channels genetically and pharmacologically, and monitored the effects with electrophysiological, two-photon imaging, electron microscopic and behavioral analyses. Our preliminary results consistently show that unlike other calcium channels, CaV3.2 channels function primarily to regulate NMDA-R-mediated transmission at synapses. Therefore, we hypothesize that CaV3.2 channels regulate synaptic NMDA transmission and that CAE- linked CaV3.2 channel mutations enhance susceptibility to absence seizures by potentiating glutamatergic transmission. Specifically, we will examine whether the activity of CaV3.2 channels enhances NMDA and AMPA responses in multiple different types of rat neurons in vitro and in vivo (Aim 1a). Moreover, we plan to study whether CaV3.2 channel activity-coupled synaptic calcium influx enhances NMDA responses that lead to the secondary potentiation of AMPA responses (Aim 1b). These results will define that the primary physiological function of neuronal CaV3.2 channels is to regulate glutamatergic synaptic transmission. In addition, we will examine how each of ~20 CAE- linked human CaV3.2 mutations may affect synaptic glutamatergic transmission (Aim 2a). Finally, we plan to investigate whether each of ~20 CAE-linked human CaV3.2 mutations may enhance the susceptibility to 2-4 Hz spike-and-wave discharges and absence-like seizures and if the seizures can be suppressed by glutamate receptor antagonists (Aim 2b). These results will shed new light on the mechanism and suggest new intervention for human CaV3.2 mutation-associated CAE.

? DESCRIPTION (provided by applicant): The previous investigation has revealed a pivotal role for cyclin-dependent kinase 5 (Cdk5) in synaptic plasticity, behavior and cognition, but also raised a fundamental question on how Cdk5 signaling regulates synaptic plasticity and behavior. To address this question, we examined Cdk5 signaling at hippocampal CA1 synapses in rat cultured slices and intact brains. We found that Cdk5, which is activated upon association with its neuron-specific regulatory subunit p35, depressed transmission using a homeostatic mechanism. Surprisingly, Cdk5 depressed transmission rapidly within 15?30 min. This result distinguishes Cdk5 from all known homeostatic transmission regulators (e.g., A? and Arc) that act in the time windows from hours to days. Moreover, we overexpressed p25, a cleavage product of p35 and more potent activator of Cdk5, in intact animals. Chronic overproduction of p25, seen in Alzheimer's patients, induced the concurrent reduction in synapse density and increase in synaptic size, the hallmark early synaptic pathology of Alzheimer's disease. This result designates p25 as the first molecule capable of inducing the characteristic synaptic pathology of the disease. Together, our preliminary data suggest a novel rapid transmission homeostasis at central synapses and a new mechanism for the early pathogenesis of Alzheimer's disease. Based on our preliminary findings, we propose to investigate how Cdk5 signaling regulates a novel rapid synaptic homeostasis at central synapses using a hippocampal cultured slice preparation (Aim 1). We expect that the investigation will define the synaptic role of Cdk5 signaling, and suggest a new molecule target (and strategy) for preventing the rapid status epilepticus. We also plan to extend the study into intact animals to examine how chronic overproduction of p25, seen in Alzheimer's patients, induces the characteristic early Alzheimer-like synaptic pathology and cognitive impairments (Aim 2). We expect that the examination will reveal a new mechanism for the pathogenesis of Alzheimer's disease, and establish an animal model for the pathogenesis. Finally, we will explore pharmacological and genetic manipulations that may reverse the synaptic pathology and cognitive impairments in the animal model (Aim 3). We expect that the exploration will develop alternative therapeutic options for Alzheimer's disease.

? DESCRIPTION (provided by applicant): Fragile X syndrome, the most common form of inherited mental impairment, is caused by the loss of function of the fragile X mental retardation protein (FMRP) encoded by gene Fmr1. Fragile X patients have the cognitive impairment that is particularly pronounced in attention-demanding learning. The impairment of the NMDA-sensitive glutamate receptor-dependent long-term potentiation, due to the selective impairment of small GTPase Ras signaling-mediated synaptic GluA1 trafficking in pyramidal neurons, is believed to be responsible for the deficit of attention-dependent learning in Fmr1 KO mice. However, how loss of function of FMRP leads to the impaired Ras signaling remains unclear. Recent studies showed that the interneuronal circuits mediated by parvalbumin- and somatostatin-expressing interneurons are altered in Fmr1 KO mice. The dysregulated inhibitions could account for the aberrant Ras signaling in pyramidal neurons of this animal model for fragile X syndrome. GABAergic progenitor cells derived from the embryonic medial and caudal ganglionic eminences (MGE and CGE) can repair defective interneuronal circuits. We recently transplanted MGE- and CGE-derived progenitor cells of wild type donor mice into the hippocampi of Fmr1 KO mice. We found that the transplantation enhanced synaptic plasticity in pyramidal neurons and rescued learning defects in Fmr1 KO mice, raising an intriguing possibility that MGE and CGE progenitors may rescue learning defects in Fmr1 KO mice via repairing the defective interneuronal circuits, Ras signaling and synaptic plasticity. Here, I propose to explore the cell therapy that improves learning in Fmr1 KO mice (aim 1) and examine how the cell therapy improves learning in Fmr1 KO mice (aim 2). The findings from this project should suggest alternative quick-to-clinic cell therapeutic options for treating fragile X patients.

PROJECT SUMMARY Acetylcholine (ACh) mediates cell-to-cell communication in the central and peripheral nervous systems, as well as non-neuronal systems. ACh released by neuronal and non-neuronal cells in these systems regulates complex brain functions, such as attention, perception, associative learning, and sleep/awake states, and various biological processes in other tissues and organs, including the heart, liver and pancreas. Dysregulation of cholinergic transmission is linked to a number of neurological diseases, including addiction, Alzheimer?s disease, epilepsy, schizophrenia, Parkinson?s disease and depression, as well as many other health problems, including cardiovascular diseases, obesity, diabetes, immune deficiency and cancer. Despite the significance of ACh in physiological and pathological conditions, the precise regulations and exact functional roles of cholinergic transmission in the majority of tissues and organs remain poorly understood, due primarily to the limitations of available tools for monitoring ACh. We recently initiated development of genetically-encoded G-protein-coupled receptor activation-based sensors for ACh (GACh) by coupling a circular permutated green fluorescent protein (cpGFP) with a muscarinic receptor. We are improving the sensors with large-scale site-directed mutagenesis and screening. Our preliminary data suggest that GACh sensors will have specificity, signal-to-noise ratio, kinetics and photostability suitable for real-time imaging of endogenous ACh signals. Here, I propose to complete the development and validation of GACh sensors following two specific aims: Aim 1 is to optimize and characterize GACh sensors. In our pilot work, we constructed a family of GACh sensors. We plan to use large-scale site-directed mutagenesis and screening to generate more GACh sensors with better performance (Aim 1a). Moreover, we will characterize the properties of GACh sensors in cultured cells and neurons (Aim 1b). We expect these experiments to optimize the specificity, signal-to-noise ratio, kinetics and photostability of GACh sensors. Aim 2 is to validate and utilize GACh sensors. In our preliminary study, we found that GACh sensors selectively detect exogenously applied ACh and endogenously released ACh. We will verify whether GACh sensors can be easily employed to detect ACh signals in various brain regions of both mice and rats (Aim 2a). Finally, we plan to explore the applications of GACh sensors in vitro and in vivo, and address a few fundamental questions about central cholinergic transmission (Aim 2b). We expect these experiments to testify the general applicability of GACh sensors in monitoring the dynamics of endogenous ACh signals and reveal some key features of cholinergic transmission.