Robert F. Hunt - US grants

DESCRIPTION (provided by applicant): Inhibitory interneurons perform important roles in regulating cortical network function, and their loss is associated with a number of neurological and psychiatric disorders. As such, transplantation of inhibitory neuronal progenitor cells might be a useful method to restore deficits in inhibition in the diseased brain. Our laboratory recently demonstrated that progenitor cells derived from the medial ganglionic eminence can migrate, differentiate into GABAergic interneurons, and suppress spontaneous seizures in a developmental epilepsy model. Despite these recent advances in transplantation strategies, the effect of inhibitory neural progenitor cell subtypes derived from other regions of the embryonic telencephalon on existing host circuitry remains largely unknown, especially after transplant into the adult brain. In this postdoctoral fellowship proposal, I will determine how GABA-progenitor cells derived from the caudal ganglionic eminence alter inhibitory circuitry after transplant into the adult dentate gyrus. Two specific aims are proposed, to determine: (1) progenitor cell migration and phenotypes of GABAergic subtypes generated after transplant into the adult hippocampus, and (2) how these new interneurons alter existing synaptic circuitry in the dentate gyrus. Electrophysiological results will be correlated with cellular, molecular, and anatomical characteristics. This study will provide detailed information about the synaptic connectivity of GABAergic progenitor cells after transplant into the adult brain. PUBLIC HEALTH RELEVANCE: A number of neurological disorders involve the loss of inhibitory interneurons, and a transplantation method to generate new cortical interneuron subtypes in affected brain regions might be a useful therapeutic approach. I will determine how embryonic inhibitory neural progenitor cells integrate into existing circuits after transplant into the adult hippocampus. By determining the synaptic connections formed by these cells, we will be able to develop methods for cortical interneuron replacement therapies to restore normal function in the human diseased brain.

Abstract Cortical interneurons represent a broad class of inhibitory neurons that are essential for controlling brain excitability and coordinating behavior. Disruption of inhibitory circuits has been implicated in a number of brain disorders, including epilepsy, intellectual disability, autism, schizophrenia, and head injury. Recently, advances in mouse and human stem cell research suggest that pluripotent cells can be used to generate enriched populations of cortical neurons and interneurons in vitro. However, few studies have systematically examined how exogenous inhibitory neurons derived from stem cell sources might be used as a cell-therapy to modify neural circuitry in vivo. The overall goal of this K99/R00 application is to determine how cortical interneurons derived from mouse and human induced pluripotent stem cells (iPS cells) functionally incorporate into the postnatal brain. The mentored phase of the award will be conducted at University of California, San Francisco under the guidance of Dr. Scott Baraban and the project will be continuted in my own laboratory after an independent faculty position is secured. In Specific Aims 1 and 2, I will use a promoter-based reporter construct to purify coritcal interneuron precursors generated from iPS cells and characterize their differentiation in vitro (Aim 1) and after transplantation (Aim 2) using a series of anatomical, molecular, and electrophysiological approaches. In Aim 3 (R00 phase), I will determine the connectivity patterns of iPS cell- derived interneurons grafted into the postnatal brain. Understanding how cortical interneurons generated from stem cell sources functionally incorporate into the recipient circuitry will provide new information about their functional plasticity and is a critical step toward translating these findings into new interneuron-based cell therapies. My long term goal is to build an independent dedicated to understanding mechanisms of neural circuit organization and to develop novel stem cell strategies for brain repair and regeneration, particularly for brain disorders associated with interneuron dysfunction. This research will require extensive training in stem cell biology, and UCSF is an outstanding institution to complete the mentored phase of this application, primarily due to the rich community of prominent neuroscientists and clinicians performing neural stem cell research and the pioneering role of UCSF in the stem cell field. In addition to Dr. Baraban's outstanding mentorship, I have assembled a team of internationally recognized scientists who will provide me with hands- on technical training, formal coursework, and career guidance during both phases of this proposal. Overall, these training experiences will be critical for me to successfully obtain an academic faculty position and establish my independent research program.

? DESCRIPTION (provided by applicant): Transplantation of neural progenitor cells has extraordinary potential for the treatment of many nervous system disorders. Several recent studies have shown that progenitor cells derived from the embryonic medial ganglionic eminence (MGE) retain a unique ability to migrate and differentiate into GABAergic interneurons following transplantation into the juvenile or adult rodent brain. We recently demonstrated that these cells are effective in suppressing spontaneous seizures in a mouse model of epilepsy. However, the therapeutic potential of this approach remains largely unexplored for traumatic brain injury, a disorder characterized by interneuron cell loss and a number of health problems linked to neural circuit hyperexcitability (e.g., epilepsy). Here, we propose studies to develop a novel MGE transplantation therapy specifically designed to replace interneurons lost after traumatic brain injury. MGE cells will be transplanted into a widely-used rodent model of closed-head injury at different stages following injury. Our approach involves in vitro patch-clamp recordings, immunofluorescence techniques and neural circuit mapping to evaluate the synaptic integration of MGE- grafted interneurons. A battery of behavioral assays and video-EEG monitoring will also be applied. Two specific aims are proposed: (i) evaluate the synaptic integration of MGE cells grafted into brain injured hippocampus, and (ii) assess the therapeutic potential of MGE cell grafts in a mouse model of traumatic brain injury. If successful, our results will provide new information about the functional plasticity of MGE progenitors in the injured adult brain and would establish relatively direct proof of concept for interneuron transplantation to treat traumatic brain injury, particularly for conditions where loss of inhibition is a major contributor.

ABSTRACT Stem cell-based platforms have transformed our understanding of many nervous system disorders, such as epilepsy. However, neurons develop in a highly complex environment of an intact brain, and functional maturation in vitro may not accurately reflect the developmental processes occurring in vivo. To accurately recapitulate the early developmental stages of synaptic and neuronal network dysfunction in these patients, direct access to patient neurons in an in vivo microenvironment is indispensable. Here, we propose an innovative approach to evaluate the in vivo development and function of neurons differentiated from human induced pluripotent stem cells. To accomplish this goal, we will develop a stem cell transplantation strategy to graft forebrain neurons into the developing brain. Using cell type-specific markers to fluorescently label subsets of neurons and a series of ex vivo and in vivo assays, we will record neuronal, synaptic and circuit-level properties of both the grafted and native-born neurons simultaneously for days to weeks. If successful, our work will provide an entirely new platform for studying epilepsy and other human nervous system disorders, one that would allow investigators to systematically and comprehensively evaluate how patient neurons become fully, and correctly, integrated into the developing brain circuitry and how they function in real time during behavior. It will also form the basis from which we can test emerging therapies on transplanted patient neurons.