Nien-Pei Tsai - US grants

Epilepsy affects 3 million people in the United States. Despite the development of current antiepileptic drugs to raise seizure threshold, one-third of epilepsy patients either respond poorly to the drugs or remain drug- resistant. Our research aims to facilitate the understanding neuronal excitability dysregulation in epilepsy with the intention to improve therapeutic outcome. We focus on ?-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid receptor (AMPA receptor; AMPAR), the most abundant receptor in the nervous system and one of the well- studied excitatory synaptic proteins. Elevated levels of AMPAR have been observed in epilepsy patients, and pharmacologically inhibiting AMPAR has been used in clinical practice for alleviating epilepsy. Despite all these facts, it remains unclear how the homeostasis of AMPAR mediates brain excitability and how dysregulated AMPAR contributes to epilepsy. We recently identified a novel ubiquitin E3 ligase for the GluA1 subunit of AMPAR, neural precursor cell expressed developmentally downregulated gene 4-like (Nedd4-2). Nedd4-2 is encoded by an epilepsy-associated gene, in which three missense mutations have been identified in patients with epilepsy. Our recent publication demonstrated that Nedd4-2 mediates neuronal and brain excitability in an AMPAR-dependent manner (Zhu et al., PLOS Genetics, 2017). However, it remains unknown (1) whether and how Nedd4-2 modulates AMPAR to affect excitatory synaptic transmission; and (2) how epilepsy-associated mutations affect the functions of Nedd4-2 in this regard. Aim 1 and Aim 2 are designed to answer these questions. Nedd4-2 is a target gene of, and transcriptionally repressed by, the tumor suppressor p53. Our work showed that inhibition of p53 reduces acute seizure susceptibility in mice in a Nedd4-2-dependent manner. This finding, together with our previous work, suggests p53-Nedd4-2 as a novel signaling axis to maintain brain excitability presumably through limiting AMPAR. Aim 3 will study the regulation of p53-Nedd4-2 signaling and AMPAR ubiquitination using a preclinical model of temporal lobe epilepsy in mice, and determine the roles of p53-Nedd4- 2 signaling in epileptogenesis in this model. Successfully accomplishing this project will improve the understanding of ion channel dysregulation in epilepsy and foster future development of therapies in treating epilepsy.

PROJECT SUMMARY/ABSTRACT Synapses mediate neurotransmission in our nervous systems. Although facilitation or stabilization of synaptic connections promotes signal transmission, synaptic elimination is vital to shaping the brain circuit during development, but is less understood. In autism spectrum disorders (ASDs), impaired synapse elimination has been observed and implicated to underlie parts of the cognitive deficits in ASD patients. It is well accepted that activation of gene transcription is required for synaptic elimination. However, the participating transcription factors and underlying molecular mechanisms remain elusive. Our recent work demonstrated that activity- dependent synapse elimination through myocyte enhanced factor 2 (MEF2) requires dephosphorylation of ubiquitin E3 ligase murine double minute-2 (Mdm2) (Tsai et al., 2017). Our latest data showed a reduction of ubiquitination of tumor suppressor p53, one of the Mdm2?s substrates, and an elevation of p53 activity upon MEF2 activation. This observation indicates a possible role of p53-mediated gene transcription in activity- dependent synapse elimination. Because we also observed elevated p53 ubiquitination in two ASD mouse models in which synapse number is known to be elevated, we propose to test a hypothesis that p53 mediates synapse elimination during development and upon neuronal activity stimulation, and that abnormally reduced p53 activity contributes to elevated synapse numbers in ASDs. In Aim 1 we will determine the role of p53 in synapse development. In Aim 2 we will explore the interplay between MEF2 and p53 in synapse elimination. In Aim 3 we will determine the contribution of elevated p53 ubiquitination in elevated synapse number in two ASD mouse models. Because of the extensive understanding of p53 within the field of cancer biology, we expect this research to quickly open a new avenue for the study of neurogenetics and synaptic biology. Furthermore, because many drugs related to p53 are clinically available, data from our research could allow basic researchers and clinicians to make rapid gains in the study of ASDs and other neurodevelopmental diseases.

PROJECT SUMMARY/ABSTRACT Epilepsy affects more than 3 million people in the United States. In addition to unprovoked seizures, cognitive decline and memory impairment are common comorbidities associated with epilepsy, but our knowledge in this area is limited. Our preliminary work suggests that an epilepsy associated ubiquitin E3 ligase Nedd4-2 mediates actin polymerization through promoting phosphorylation of an actin binding protein cofilin during the induction of long-term synaptic potentiation (LTP). LTP describes long?lasting increments of synaptic efficiency and is crucial for cognition and memory formation. In an effort to identify ubiquitination substrates of Nedd4-2 responsible for cofilin phosphorylation, we identified PAK3, an IDG-eligible understudied kinase, as a potential substrate of Nedd4-2 that contributes to cofilin phosphorylation during LTP. This pilot project is formulated to test our hypothesis that induction of LTP de-represses Nedd4-2 to stabilize PAK3 and subsequently induce cofilin phosphorylation and actin polymerization. In Aim 1, we will determine how PAK3 is ubiquitinated by Nedd4-2 and how this ubiquitination is disrupted by epilepsy associated mutations in Nedd4-2. We will also determine whether induction of LTP de-represses Nedd4-2 to allow PAK3 stabilization during LTP. In Aim 2, we will employ a recently developed optogenetic approach to rapidly stabilize PAK3 in Nedd4-2 conditional knockout (cKO) neurons during the induction of LTP, with the intention to restore cofilin phosphorylation and actin polymerization, and ultimately to restore LTP. We expect our project to further our knowledge of an IDG-eligible protein PAK3 in synaptic plasticity and help explain cognitive decline in epilepsy.

PROJECT SUMMARY/ABSTRACT Adaptation of living organisms to constantly changing environments depends on the plasticity of the nervous system. Neuronal plasticity often requires activity-dependent translation to rapidly supply selected proteins, for example, through activation of Group 1 metabotropic glutamate receptors (Gp1 mGluRs). Gp1 mGluRs, including mGluR1 and mGluR5, mediate translation-dependent synaptic plasticity, including long-term synaptic depression (LTD). Dysregulated Gp1 mGluR signaling is observed with various neurological and mental disorders, including Fragile X Syndrome (FXS) and autism spectrum disorders (ASDs). Although pharmacological correction of Gp1 mGluR activity reverses many of the phenotypes in animal models of those diseases, the molecular and cellular mechanisms underlying Gp1 mGluR-mediated synaptic plasticity have been elusive. Our published and preliminary data introduce the ubiquitin E3 ligase Murine double minute-2 (Mdm2) as a novel translational repressor and a ?switch? that permits Gp1 mGluR-induced protein translation (Liu et al., Hum Mol Genet., 2017). In our proposed research, we aim to characterize the role of Mdm2 in Gp1 mGluR- dependent synaptic plasticity (Aim 1) and determine the mechanism by which Mdm2 mediates activity-dependent protein translation (Aim 2). Our new data also show that Mdm2 is molecularly altered and unresponsive to Gp1 mGluR activation in the Fmr1 knockout (KO) mouse, the commonly used animal model for studying FXS (Tsai et al., Hum Mol Genet., 2017). In Aim 3 we will characterize the mechanism by which Fmr1 interconnects Gp1 mGluR signaling to permit translational activation through de-repressing Mdm2. Successful completion of this proposal will greatly facilitate the understanding of Gp1 mGluR-mediated synaptic plasticity through a novel mechanism of translational control. Building on the deep knowledge of Mdm2 in cancer biology, our research will also open a new avenue for the study of neurological disorders associated with abnormal Gp1 mGluR signaling.

A substantial amount of clinical reports have confirmed that patients with Alzheimer?s disease are at increased risk for developing seizures and/or epilepsy. This non-psychiatric comorbidity causes significant burden to the patients as well as the caregivers. However, our knowledge in this area is very limited. Understanding the mechanisms underlying Alzheimer?s disease-associated seizures may reveal novel risk factors and provide the opportunity to develop specific anti-epileptic therapies for Alzheimer?s disease patients. Our recent studies discovered that the activity of tumor suppressor p53 is positively correlated with neuronal excitability in vitro and seizure susceptibility in vivo. Because we and others have observed an up-regulation of p53 protein levels induced by amyloid beta (A?), we hypothesize that elevated p53 induced by A? contributes to elevated neuronal excitability and seizure susceptibility in Alzheimer?s disease. In Aim 1, we propose to determine the cellular mechanism by which p53 promotes neuronal excitability. In Aim 2, we propose to test whether pharmacologically or genetically inhibiting p53 is able to effectively reduce neuronal excitability in the presence of A? and seizure susceptibility in an Alzheimer?s disease mouse model. We expect that completion of our project will: (1) elucidate the mechanisms by which p53 promotes excitability; (2) uncover the key molecule (p53) that leads to elevated seizure susceptibility in Alzheimer?s disease; and (3) suggest novel therapeutic targets and potential therapies for Alzheimer?s disease-associated seizures and epilepsy.