Jenny Hsieh - US grants

? DESCRIPTION (provided by applicant): The adult hippocampus continuously generates new dentate granule neurons from neural stem cells. A number of factors that preferentially activate hippocampal circuits, such as exercise, enriched environment, and many pathological conditions, regulate new neuron development. Neurons transmit activity from cell to cell mainly through synapses. However, synapses of new neurons do not form until at least two weeks after birth. This suggests the presence of diffusible factors from active circuits to regulate new neuron development. This speculation has motivated us to examine signaling pathways reacting to circuit activity to regulate new neuron development. Sphingolipid signaling recently caught massive attention because of its role in mediating activity across cells in the immune system. In a pilot study, we tested the existence of this pathway in the dentate gyrus and found that sphingosine 1-phosphate receptor 1 (S1PR1) is enriched in new neurons. Moreover, both sphingosine kinase 1 and SPNS2 are enriched in existing but not new dentate granule cells. We therefore speculate that the SPNS2-S1PR1 pathway transmits the existing circuit activity to regulate the integration of newborn neurons. To test this hypothesis, we propose the following experiments: First, we will genetically perturb theSPNS2-S1PR1 pathway and test optogenetic activation of dentate granule cells-induced development of new dentate granule cells. Second, in the S1PR1 over-expressing new dentate granule cells, we will use a retroviral method to manipulate the Cdc42 and Akt pathways to examine their roles in mediating the S1PR1 regulation to new neuron development. We will perform the same genetic manipulation of the Cdc42 and Akt pathways in optogenetic-induced development of new dentate granule cells. Third, we will use pilocarpine-induced seizures as a model system to test the development of new neurons after genetically manipulating the SPNS2-S1PR1 pathway. Moreover, we will monitor epileptic activity after disrupting the SPNS2-S1PR1 pathway of new neurons. Our proposal aims to reveal the role of the SPNS2-S1PR1 pathway in propagating neural circuit activity to regulate new neuron development. This study will provide insights towards understanding how existing neural circuits dictate the development of new neurons. Our results may introduce a novel therapeutic target for the treatment of epilepsy.

? DESCRIPTION (provided by applicant): Acute seizures (or status epilepticus, SE) after a severe brain insult often leads to epilepsy and cognitive impairment. Aberrant hippocampal neurogenesis follows the insult but the role of adult-generated neurons in the development of chronic seizures or associated cognitive deficits remains to be determined. Recently, we found ablation of adult neurogenesis prior to acute seizures reduced chronic seizure frequency and normalized epilepsy-associated cognitive deficits. These data helped us formulate a clear objective for this grant proposal: to determine the effect of neurogenesis ablation after acute seizures in chronic seizure generation and epilepsy-associated memory function. Our central hypothesis is adult neurogenesis plays a key role in chronic seizure development and associated memory impairment, and our preliminary results suggest that targeting aberrant hippocampal neurogenesis may reduce recurrent seizures and restore cognitive function following a pro-epileptic brain insult. We will test this hypothesis in 3 specific aims: 1) To defie the therapeutic window of targeting adult neurogenesis to prevent epilepsy and associated cognitive deficits, 2) To evaluate the long-term effects of aberrant neurogenesis in preventing epilepsy, and 3) To identify molecules to target aberrant neurogenesis through studies on NeuroD. Aims 1 and 2 will utilize a Nestin-?-HSV-thymidine kinase transgenic mouse to genetically ablate newborn neurons. Aim 1 will also use an Ascl1-CreERT2; inducible DT-A model to ablate neurogenesis. Aim 3 will use a NeuroD conditional knockout mouse. In all 3 Aims, we will perform continuous video-EEG recording to measure chronic seizure frequency and duration. In Aims 1 and 2, novel location, novel object, and context-dependent fear conditioning tests will be performed to measure memory function. The conceptual framework and approach is innovative because we will apply state-of-the-art genetic and knockout mouse techniques to a mouse model of temporal lobe epilepsy and dissect underlying cellular- level mechanisms of SE-dependent neurogenesis. As our long-term goal is to understand the molecular mechanisms important for how aberrant neurogenesis drives chronic epilepsy, the proposed work is disease- relevant and highly significant. It will advance and expand our basic understanding of seizure activity- dependent molecular networks regulating neural stem cell proliferation, differentiation, survival and maturation of newborn neurons, which will advance our understanding of neurogenesis in both basal and pathological states. The proposed study is relevant to NIH's mission as it will allow us to gain fundamental insight regarding the fundamental underpinnings of epilepsy and associated comorbidities as well as acquiring knowledge towards new avenues for treating neurological and psychiatric disorders.

PROJECT SUMMARY/ABSTRACT Nearly 1% of the US population suffers from epilepsy (prevalence 5-8.4/1000), with a slightly higher prevalence in children. Despite this high frequency, the molecular and cellular basis for only a few types of epilepsy have been defined, while the basis for most remains unknown. Mutations in one gene, ARX, are of considerable interest as distinct mutations are associated with a spectrum of neurological disorders with epilepsy representing one of the few consistent features. ARX has 4 poly-alanine (pAla) tracts and expansions in the 1st or 2nd tract are consistently associated with epilepsy. pAla tract expansion mutations are a relatively newly described mutation type and are associated with a growing number of human developmental disorders, epilepsy being a component of several. How this mutation type results in human disorders and epilepsy in particular are not well understood. Our prior work has demonstrated that an expansion in the first pAla tract of ARX results in structural change in the protein and the resulting protein has differential effects on developing cortical interneuron- and projection neuron progenitor cells. In other studies, we have shown that the loss of Arx from each progenitor population accounts for specific components of the mouse and human phenotypes. In this multi-PI R01 proposal, building on our data from the past ten years, we seek to unite human stem cell models with mouse models to elucidate the pathobiology underlying ARX related epilepsy, and specifically the function of pAla tracts along with mutations in these tracts. Aim 1 will evaluate the cellular impact of ARX pAla mutations in patient-derived spheroids. Aim 2 will examine the role of ARX pAla mutations on cortical interneuron migration and network activity. Aim 3 will determine the effects of Arx pAla expansion mutations on brain development and function. This project will utilize human induced pluripotent stem cell (hiPSC) and spheroid models and complement these with mouse embryonic stem cell lines and behavioral and physiological assays in mice. Together, these studies are expected to provide a greater understanding of how pAla tracts function in normal and abnormal brain development, contribute to our understanding of the pathogenesis of epilepsy, and generate valuable resources and mouse models to test potential therapeutic strategies for developmental epilepsies.

PROJECT SUMMARY/ABSTRACT Epilepsy is frequently associated with Alzheimer's Disease (AD), but whether there are shared common mechanisms is largely unknown. Network hyperactivity due to altered functional connectivity of GABAergic interneurons is believed to underlie many cognitive disorders and a ?disease of interneurons? is the major hypothesis for epilepsy. However, little is known about the pathophysiology of interneurons particularly in patients with Apo4-associated AD and epilepsy. Understanding the role of ApoE4 in interneuron dysfunction requires direct investigation of interneuron properties in human neurons derived from patients with these mutations. Reprogramming patient somatic cells enables recapitulation of normal and pathological human tissue developmental properties in defined conditions and a new way to identify the cellular processes underlying complex human diseases, which can lead to mechanism-based drug discovery. Aim 1 will test the hypothesis that ApoE4 will cause degeneration of GABAergic neurons in 3D cortical spheroids which is associated with AD-related pathology by labeling spheroids with a Dlx1/2-GFP reporter to monitor interneuron behavior and correlating these cellular changes with AD-related pathology. Aim 2 will test the hypothesis that ApoE4-dependent degeneration will lead to hyperexcitability in 3D cortical spheroids by performing multi-electrode array recordings to measure baseline neural activity and after exposure with different anti-seizure drugs. Together, these studies are expected to provide a greater understanding of how ApoE4 functions in human cortical interneuron development and function at the network level, therefore contributing to the understanding of the pathophysiology of AD, which could help uncover new strategies to treat patients with AD and epilepsy.

PROJECT SUMMARY/ABSTRACT Neuropsychiatric disorders represent a leading cause of disability, affecting nearly 19% of the US population. Only 9% of neuropsychiatric drugs entering clinical trials reach the market, which is one of the lowest success rates across all therapeutic areas. Fundamental differences between the neurobiology of rodents and humans have been proposed to account for translational failures in development of effective therapeutic strategies to mitigate neurological or neurodegenerative diseases or disorders. Rodent behavioral assays are also variably effective in predicting clinically effective neuropsychiatric drugs. Nonhuman primates (NHPs) are recognized as a valuable, clinically relevant alternative to span the gap between rodents and humans in the development of therapies designed to advance brain health. Among NHPs, the common marmoset [Callithrix jacchus (cj)] affords a highly tractable option because of its small size, short lifespan, production of multiple offspring/year and accurate recapitulation of human neuroanatomy. However, the ultimate utility of the marmoset model remains in its infancy due to the paucity of efficient tools to facilitate studies requiring genetic modification, especially those needed to recapitulate complex aspects of brain health. To address this urgent need, we propose an innovative, more efficient approach to achieve gene editing and transgenesis in marmosets based on the novel use of highly manipulable induced pluripotent stem cells (iPSCs) that can be differentiated to form male germ cells that can ultimately be used to produce transgenic offspring carrying precisely edited alleles of genes relevant to brain health and disease. Specifically, we will combine 1) close proximity to one of two NIH-designated Marmoset Breeding Colonies, maintained at the Southwest National Primate Research Center, 2) experience with NHP pluripotent stem cells, iPSC derivation, and CRISPR/Cas9 editing, 3) a novel strategy to produce transplantable male germ cells from edited cjiPSCs, 4) documented expertise transplanting NHP germ cells into testes to produce sperm, 5) published experience in the use of cutting-edge single-cell genomics and multiparametric integrative epigenomics to assess normality of any cell type, and 6) leading expertise in brain health and disease in general and the neurogenetics of epilepsy in particular. In Aim 1, we will use CRISPR editing to generate mutant ARX alleles and reporter transgenes in cjiPSCs. In Aim 2, we will optimize derivation and transplantation of male cjiPSC-derived germ cells into recipient testes and grafts to foster development of transgenic sperm. In Aim 3, we will assess the impact of ARX mutations on marmoset cortical neuron development and migration. Together, these aims are designed to advance the utility of the marmoset model for brain research based on CRISPR/Cas9 editing of cjiPSCs, male germline-mediated transgenesis, development of cjiPSC-derived brain organoids, and specific knowledge of the neurological impact of ARX mutations.