Elizabeth Kirby - US grants

DESCRIPTION (provided by applicant): Stem cell therapies hold great promise for treatment of human disease, particularly for currently incurable neurodegenerative diseases. However, to effectively harness the healing potential of stem cells, it is necessary to understand how these cells interact with and respond to their environment. In transplants, stem cells may function as replacement cells that respond to local cues to help mend damaged tissue. They may also function as a source of protective cues, secreting in their undifferentiated state growth factors that facilitate local repair processes. This proposal focuses on this second, less well understood role of stem cells as neuromodulatory secretory cells. The goal of this proposal is to investigate the regulatory and neuroprotective role of adult neural stem/progenitor cell (NPC)-derived secretions in the hippocampus. Given the prominent degradation of the hippocampus in several neurodegenerative disorders such as temporal lobe epilepsy and Alzheimer's disease, this brain area provides an attractive target for stem cell therapy. The Wyss-Coray lab has previously shown that isolated adult hippocampal NPCs secrete a variety of growth factors in large quantities, most notably the highly neuroprotective vascular endothelial growth factor (VEGF). My goal is to investigate the hypothesis that NPC- derived VEGF regulates hippocampal function and provides neuroprotection from degenerative disease. In Aim 1, I will determine how NPC-derived VEGF regulates the proliferation and differentiation of other NPCs in vitro and in vivo using genetic knockdown techniques specific to NPCs. In Aim 2, I will use the in vivo knockdown models from Aim 1 to determine how NPC- derived VEGF regulates the local vascular environment and hippocampal behavior in adult mice. Finally, in Aim 3, I will test the neuroprotective qualities of NPC-derived VEGF from endogenous NPCs (and from NPC transplants) in an excitotoxic model of neural insult. These studies will help provide critical understanding of how NPCs may function as future therapies in vivo. This project combines basic neuroscience with translational research, requiring that I become proficient in a broad skill set. I will gain experience in in vitro NPC culture, shRNA, lox- cre genetics, stereotaxic injectios into the hippocampus and neurodegenerative models of disease. Through this project, it is my goal to acquire expertise in translational research designed to pursue treatment for neurodegenerative disease.

? DESCRIPTION (provided by applicant): Candidate: The candidate is a 3rd year postdoctoral fellow at Stanford University in the lab of Dr. Tony Wyss- Coray. The candidate's immediate goals are to gain independence and establish her own lab at an academic research-focused university or institute studying the role of undifferentiated neural stem or progenitor cells in hippocampal function. Her long-term goals are to build a prominent lab in the area of hippocampal plasticity and injury response at an academic research center. She aims to improve our understanding of how the hippocampus, an essential brain area for memory function, responds to the environment, either positively or negatively and thereby inform development of therapeutics for human brain health. She also plans to make high quality mentoring of young scientists a priority in her career. Environment: The proposed Mentored Phase research will be conducted in the lab of Dr. Tony Wyss-Coray at Stanford University. Co-mentors Dr. Theo Palmer and Dr. Tom Rando will also advise the candidate through quarterly individual meetings and yearly meetings of the candidate and the entire mentoring team. The Stanford Neurology department will hold yearly mandatory meetings where the candidate will present her research and career progress to senior faculty, along with other K99 awardees, and receive feedback and further guidance. Coursework at Stanford and Cold Spring Harbor Lab will provide formal instruction in lab management, grant writing and mentoring. A team of several consultants is also in place to help with technical training on several new procedures that the candidate will learn in the Mentored Phase, as well as study design using these new procedures. The transition to independence has defined milestones and will be tangibly supported by the primary mentor and co-mentors in the form of job opportunity referrals, practice with job talks, and advice on offers. The primary mentor will invest significant effort in the establishment of the candidate's lab by: 1) working with the neurology department chair to secure a tenure-track position for the candidate, 2) providing feedback on R01 preparation during the Mentored Phase and 3) promoting the candidate's new lab at national and international conferences. Research: The proposed experiments will investigate a novel functional role for undifferentiated neural stem and progenitor cells (NSPCs) in the adult brain after enhancement and injury. The proliferation of endogenous NSPCs in the adult hippocampus increases dramatically after both beneficial voluntary exercise and injurious seizures. Most previous studies have debated the function of the immature neurons that result from the differentiated products of these NSPCs. This proposal will determine the direct role of undifferentiated NSPCs in hippocampal function via secretion of growth factors after both seizures and exercise. A key motivation for this research is to inform future therapeutic targeting of NSPCs by determining how this important, unique cell population alters injury and plasticity responses in the adult hippocampus. This proposal will introduce functional implications for adult neurogenesis at a cellular age previously thought to be silent. Given the numerous environmental stimuli that impact NSPC proliferation, these findings could have wide-reaching implications for the role of NSPCs in hippocampal plasticity. The first aim of this project will characterize the growth factor response of NSPCs to physiological and pathological stimuli. Fluorescence-activated cell sorting techniques from acutely dissected murine adult hippocampus will be employed to isolate NSPCs after seizures or voluntary wheel running exercise. RNAseq will be used to contrast the profiles of NSPC transcriptional responses to pathological vs physiological stimuli. These findings will represent a rich dataset that will infor many future investigations of how NSPCs participate in regulating hippocampal function. To determine the direct role of NSPC-secreted growth factors, this proposal will use a novel mouse model for inducible, NSPC-specific knockdown of floxed growth factors, starting with an established model for inducible knockdown of the growth factor VEGF in NSPCs. For exercise, knockdown of a growth factor (like VEGF) in NSPCs will be induced in adulthood, followed by exposure to an unlocked running wheel. Behavioral and immunohistochemical markers of exercise-related enhancement of hippocampal function will then be quantified to determine whether loss of that growth factor from NSPCs prevents any benefits of exercise. For seizures, after growth factor knockdown, mice will be exposed to kainic acid either systemically or centrally to induce different levels of seizure severity. Behavioral and structural sequela will thn be quantified to determine whether loss of NSPC-growth factor is beneficial or detrimental to recovery from seizure. These studies will shed light on the function of undifferentiated neural progenitors, a largely uninvestigated cell population but one that may have important implications for design of therapies for disorders that differentially impact the hippocampus such as epilepsy.

Brain stem cells have great potential for treating neurological disorders, but this potential remains unrealized because of an incomplete understanding of how these cells maintain their immature status. In the brain of adult mammals, neural stem cells are found in a few, isolated places, including the hippocampus, where they continue to create new neurons that contribute to healthy memory function. Pilot data generated for this proposal found that adult hippocampal neural stem cells express a biochemical growth factor (vascular endothelial growth factor [VEGF]) that is essential for their long-term survival and maintenance. VEGF may act on these cells in several possible ways, and the proposed studies will determine which of these possible mechanisms VEGF uses to maintain brain stem cells. The results of this research will have a major impact on the design of stem cell-based therapies by revealing mechanisms that growth factors use to support stem cells and enhance their survival. In addition, this work will have an impact on the local community via a specialized program promoting high school stem cell education, support for a new Brain Bee outreach program, and general outreach that will educate the public about stem cell biology and its potential role in medicine.

The persistence of neural stem cells (NSCs) in the adult mammalian hippocampus is highly conserved across species, yet the molecular mechanisms that regulate maintenance of these NSCs are unresolved. Numerous studies show that local signals from neighboring cells help maintain NSCs, but less attention has been given to how NSCs self-regulate. Our preliminary data show that adult hippocampal NSCs and their progenitor progeny (NSPCs) self-maintain their own stemness via expression of vascular endothelial growth factor (VEGF). While soluble proteins are classically conceptualized as signaling by binding to receptors on the cell surface, they can also act through two alternative routes that are less-frequently studied: 1) via intracrine activation of receptors in endocytic vesicles, and 2) via transmission between cells in exosomes. Pilot data show that VEGF exists in all of these cellular compartments, but which ones are essential for VEGF maintenance of stemness is unknown. The proposed experiments will use in-vitro and in-vivo murine models to determine which of these methods of VEGF transmission (paracrine extracellular, intracrine and/or paracrine exosomal) are active in NSPCs and essential for self-maintenance of stemness. The proposed work will provide a major advance in understanding for the largely uninvestigated field of non-canonical pathways for adult NSPC self-maintenance via soluble signals.

This award reflects NSF's statutory mission and has been deemed worthy of support through evaluation using the Foundation's intellectual merit and broader impacts review criteria.

A unique neurogenic niche in the adult hippocampus hosts neural-lineage stem cells (NSCs) that generate new neurons in a wide range of adult mammals. This process of adult neurogenesis is essential for optimal hippocampal cognitive-emotional function and suggests an avenue for regenerating tissue in the adult brain. However, adult neurogenesis is sensitive to local niche signals, and depending on local signaling, it can fluctuate dramatically in quantity and net contribution to hippocampal function. Better understanding of the key regulatory components of stem cell-niche interactions is critically needed to advance efforts to support hippocampal function and repair. A major niche signal known to modulate adult neurogenesis in both healthy and diseased or injured states is the neurotransmitter glutamate. Excess glutamate stimulation is common in injuries and illnesses that differentially impact the hippocampus, including trauma, stroke, seizure, and neurodegeneration. Our objective in this application is to examine the mechanisms by which the excitatory neurotransmitter glutamate stimulates adult neurogenesis. Previous work on glutamatergic regulation of adult neurogenesis focuses on the role of glutamate receptor stimulation. Our preliminary data, in contrast, suggest an unexpected role for glutamate transporters from the excitatory amino acid transporter (EAAT) family in glutamate-induced stimulation of NSC proliferation. NSCs are widely known to express large quantities of EAATs yet their functional role has received little attention. The proposed experiments will investigate the central hypothesis that glutamate transport through EAAT1 promotes NSC activation and subsequent neurogenesis via cell depolarization. In Aim 1, we will use novel in vivo knockdown models to test the working hypothesis that NSC EAAT1 facilitates NSC proliferation and thereby stimulates adult neurogenesis. In Aim 2, we will use chemogenetic manipulation of NSC membrane potential and electrophysiology to test the working hypothesis that depolarization via EAAT1 drives NSC activation. These results of the proposed studies are expected to have a positive impact because they will introduce a novel molecular mechanism by which a major niche signal?glutamate?contributes to neurogenesis in the adult brain. The expected findings will have relevance both to fundamental understanding of hippocampal homeostasis and to design of therapeutic approaches that seek to capitalize on NSCs to support tissue repair.