Dong Sun - US grants

DESCRIPTION (provided by applicant): Major advances in Neurobiology have shown that the central nervous system can regenerate and repair itself in response to cell damage due to ischemia and trauma, yet these innate recovery mechanisms remain limited and poorly understood. Recently, endogenous cell proliferation has been recognized as a mechanism to replenish lost neurons following injury, and that this phenomenon is age-dependent being much more pronounced in juvenile and young animals than elderly. Traumatic brain injury (TBI) is the major cause of death and disability in persons under the age of 45. Over 5.3 million Americans currently live with disabilities due to TBI, ranging from cognitive impairment to vegatative state. Studies have shown that endogenous cells from the subventricular zone and the hippocampal subgranular zone constantly generate new neurons throughout life, and that this process is enhanced after injury. Moreover, we and others have shown that newly-generated hippocampal dentate granular neurons can integrate and form synaptic connections to the existing hippocampal circuitry. We have also shown that this injury-enhanced endogenous neurogenic response is associated with the expression levels of several trophic factors following TBI. We have further shown that administration of exogenous trophic factors can not only enhance neurogenesis but also greatly improve recovery of cognitive function in adult animals following injury. In this proposal, we will specifically investigate how the endogenous neurogenic capacity of the brain can be enhanced to repair damaged neuronal circuitry. We will first examine the extent to which the newly- generated neurons establish connections to their target and communicate with neighboring cells through synaptic connections;Secondly, to further explore the effect of trophic factors on neurogenesis and functional recovery, we plan to assess expression levels of a broad spectrum of growth factors in the hippocampus following TBI in juvenile, adult and aged animals using antibody array methods. Thirdly, we will then utilize this information to manipulate endogenous neurogenesis and test the extent to which cognitive recovery in the adult and elderly brain following TBI can be improved by administering trophic factors that promote neurogenesis. Collectively, these studies will provide the mechanistic underpinning for rational construction of clinical trials on trophic factor administatrion for severely head injured patients who have sustained hippocampal damage - one of the most common patterns of cognitive impairment in this common, and currently untreatable condition.

DESCRIPTION (provided by applicant): Using neural transplantation to replace lost cells in the brain and restore functions following TBI is a challenging but attractive strategy. To date, the few published studies investigating grafting of human neural cells have primarily utilized cells derived from fetal tissues or a human teratocarcinoma line (NT2 cell line). Despite some positive outcomes from these studies, however, cell source limitations, graft rejection and ethical controversies hinder their use in clinic. Recent studies show that the mature mammalian CNS harbors multipotent stem cells capable of differentiation into neurons and glia in various regions. These cells, when derived from the subventricular zone and the hippocampus, have been shown to be capable of generating new neurons that are able to integrate both anatomically and functionally into the existing neuronal circuitry and contribute to the maintenance of normal brain functions. These findings highlight the potential therapeutic value of adult derived neural stem and progenitor cells (NS/NPCs) in treating injured brain. Thus far, we and others have successfully isolated human NS/NPCs from neurosurgical resection tissues and established their long term culturing. Examination of these adult NS/NPCs as a potential candidate for neuronal cell replacement therapy, especially as a potential autologous cell source for TBI patients, is urgently needed. To date, very few studies have attempted to examine the behavior of adult human NS/NPCs in the injured mature CNS. In preliminary studies, we show that NS/NPCs derived from surgically removed tissues can survive in normal and injured rat brain after transplantation. However, it is not known whether these cells can survive for extended period in an injured environment and integrate into the host neuronal circuitry. We, therefore, plan to examine the long term fate of adult human NS/NPCs after transplantation into the injured brain. Thus, the overall focus of this proposal is aimed at exploring the long term survival and differentiation fate of transplanted adult human NS/NPCs in the injured rat brain, and to determine the extent to which these cells can integrate anatomically and functionally into the host neuronal circuitry and modify the functional recovery of the injured host. PUBLIC HEALTH RELEVANCE: To date, little information exists regarding the behavior, plasticity and fate of adult human neural stem cells after transplantation into the adult brain especially in an injured environment. This proposal is aimed at assessing the ability of survival, differentiation, anatomically and functionally integration of these cells into the adult host CNS following transplantation into normal and injured adult rat brain.

DESCRIPTION (provided by applicant): The long-term goal of this project is to develop patient-specific autologous cell source from induced pluripotent stem cells (iPSC) technology as a therapy for traumatic brain injury and other neurological diseases. Many current neural transplantation approaches have encountered problems associated with the source of donor cells. The ethnic controversies, limited cell availability, poor survival, lack of functional integration of grafted cells, the risk of tumor formation and immunological rejection in vivo are prominent issues need to be overcome before neural transplantation as a therapy to be used in clinic. It is essential, therefore, to identify optimal cell sources that are more conducive for cel replacement therapies. The emerging technology of human iPSCs generation, followed by directed differentiation into uniform populations of neural progenitor cells (hNPs) and neurons, holds great promise as an approach to reverse engineer human cells obtained from TBI patients. In this project, we are focusing our efforts on reprogramming adult human neural cells isolated from neurosurgical resection tissues. The specific hypothesis of this proposal is that functional iPSC-derived neural progenitor cells can be generated from neurosurgical resection tissues and utilized as cell replacement therapy for TBI. The hypothesis is based on our observations and research developments that (a) iPSCs can be generated from patients with specific neurodegenerative disorders, (b) directed differentiation of pluripotent stem cells can lead to uniform populations of hNPs, and (c) adult human stem-like cells can be isolated from neurosurgical resection tissues and successfully cultured as neurospheres and monolayers. To test the hypothesis, in this proposal, we will first generate iPSCs from adult human neural cells isolated from neurosurgical resection tissues and evaluate their pluripotent characteristics, directed differentiation capabilities in vitro. We will then assess the transplantation potential o these iPSC-derived hNPs in vivo in the brain in both intact immunodeficient animals and injured immunocompetent animals. The two proposed specific aims are: (1) Generate and characterize iPSCs from adult human neural cells isolated from neurosurgical resection tissues. (2) Evaluate the survival, differentiation and functionality of iPSC-derived NPs in the injured environment and assess the role of gender differences on cell survival. Results from the proposed study will have significant impact in the development of patient-specific hiPSCs as autologous cell replacement strategies for TBI and other neurological disorders.

Project Summary/Abstract Pediatric brain trauma is a significant debilitating health problem among children in the United States. To date, there is no effective treatment to structurally repair the injured brain and restore the lost neurological functions associated with brain trauma. Cell transplantation offers hope to treat the injured brain through direct neural replacement to replenish cells lost to injury and reconstruct the disrupted neuronal circuitry or/and by stimulating endogenous repair mechanisms. However, current approaches have encountered prominent issues such as ethical controversies of cell source, limited cell availability, poor graft survival, lack of functional integration of grafted cells, the risk of tumor formation and immunological rejection. Recent advances in tissue engineering and induced pluripotent stem cells (iPSCs) have instilled new hope to develop patient-specific autologous cell source to overcome the issues that neural transplantation has encountered. Utilizing iPSCs and our well-constructed injectable hydrogel system, the goal of this proposal is to develop a biomaterial- assisted cell transplantation strategy through modulating the host microenvironment to enhance brain structure remodeling and improve survival, neuronal differentiation and functional integration of grafted cells achieving repair and regeneration of the injured pediatric brain following traumatic injury. We have previously developed a series of injectable in-situ cross-linkable hydrogels to promote the reconstruction of a complete vascular network in the injured adult brain. We have also optimized our hydrogel system for supporting three dimensional growths of cells and as a delivery vehicle releasing bioactive reagents. In this proposal, we plan to utilize our hydrogel system to modify the host environment to promote long term survival, neuronal differentiation and functional integration of the transplanted iPSC-derived neural stem cells (iPSC-NPs) in the injured pediatric brain via reconstruction of a complete vasculature network and conditioning of focal tissue microenvironment at the site of injury. We will test the central hypothesis that a combination of iPSCs transplantation with targeted tissue bioengineering will achieve optimal brain tissue regeneration and enhance functional recovery following pediatric brain injury. The hypothesis will be tests in two Specific Aims, 1) Optimize the biomechanical and biochemical properties of our injectable hydrogel system to promote the growth of primary neurons, endothelial cells isolated from neonatal brain and iPSC-NPs in vitro, 2) Utilize injectable hydrogels optimized for neonatal brain cell growth to reconstruct local vasculature and deliver growth promoting molecules in combination with iPSC-NPs to promote tissue structural regeneration and functional recovery following pediatric brain injury. The results of this study will advance the understanding of the key microenvironmental requirements for successful neural transplantation therapy and will have translational significance not only for TBI but also for other neurological diseases.

It is now well established that the mature brain is capable of mounting a reparative response as neural stem cells (NSCs) within the neurogenic regions of the brain, the subventricular zone and the hippocampus, proliferate and generate functional neurons under homeostatic condition and following brain insults. However, the regenerative potential of NSCs is diminished with aging. Thus far, the regulatory mechanisms which drive activation of regenerative NSCs during neuropathological conditions particularly following TBI is largely unknown. Furthermore, it is also unclear whether there is an age-related difference in regulating regenerative NSC response following brain injury. Developmental studies have established that Notch signaling pathway is essential for NSC maintenance, proliferation and survival during CNS development. Recent studies have also shown that Notch signaling is a key player for NSCs in the adult brain under homeostatic condition. As the injured brain recapitulates many aspects as the developing brain, we speculate that Notch signaling is likely the key regulator responsible for the activation and function of regenerative NSCs following brain injury and aging. In our preliminary studies, we have found that following brain injury, the expression level of Notch pathway proteins is elevated in the neurogenic regions in young adult brain which is correlated to the increased NSC proliferation and neurogenesis. In contrast, in the aged brain, the neurogenic regions display diminished expression of Notch pathway in normal condition and following TBI which parallels to the decreased NSC response in the aging brain. From these observations, we hypothesize that Notch signaling is necessary for activation of regenerative NSCs in the injured brain and reduced Notch signaling during aging contributes to the decreased regenerative response of NSCs in the aged brain following injury. To test this hypothesis, in Aim 1 of this proposal, we will determine the importance of Notch signaling in regenerative NSC response and functional recovery after TBI. In Aim 2, we will assess the significance of Notch signaling on regenerative NSCs on cellular learning, memory and plasticity in the hippocampus and olfactory bulb following TBI. In Aim 3, we will examine how Notch pathway activation in neurogenic niches affects the function of regenerative NSCs in the injured aged brain. As the significance of endogenous repair mechanism through NSCs in the adult brain is increasingly recognized and has attracted increasing interests for development of NSC-based therapies, it is necessary to understand the fundamental principles governing NSC activation under regenerative conditions. The goal of this proposal is to examine the regulatory signaling pathway responsible for regenerative NSC activation and functioning following TBI and aging with the focus on the role of Notch. .

Neuroinflammation has been recognized as an essential player in the pathogenesis of Alzheimer's disease (AD), especially for the late-onset AD. This notion is supported by the facts that glial activation and elevated cytokines have been observed in AD animal models and patients. Furthermore, genome-wide associated studies have identified inflammatory genes in the innate immune system, such as CLU, CR1 and TREM2, as AD risk factors. Recently, the NLRP3 inflammasome, a multiprotein platform that tightly regulates the innate immune response, has been suggested to play critical roles in AD development. Activation of the NLRP3 inflammasome is responsible for the production of pro-inflammatory interleukin (IL)-1? and IL-18, ultimately leading to inflammatory responses. Given the important role of the NLRP3 inflammasome and IL-1? in AD, development of selective NLRP3 inflammasome inhibitors (NLRP3Is) as chemical probes with well- defined mode of action will not only enhance our current knowledge on the NLRP3 inflammasome in AD pathogenesis, but also provide translational promise to this disease. Recently, we developed a lead inhibitor that blocks the assembly and activation of the NLRP3 inflammasome, resulting in inhibition of IL-1? production both in vitro and in vivo. The central hypothesis of this proposal is that the NLRP3 inflammasome is involved in chronic inflammatory responses of AD, and pharmacological suppression with small molecule inhibitors that directly target the NLRP3 inflammasome platform will prevent or inhibit AD disease progression. In support of this hypothesis, our preliminary studies showed that the lead inhibitor engaged the NLRP3 inflammasome, reduced AD pathology, and improved cognitive functions in transgenic AD mouse models, thus providing proof-of-concept for developing NLRP3Is as in vivo probes. Furthermore, our preliminary structure activity relationship (SAR) studies confirmed that this chemical scaffold can be optimized to improve inhibitory potency and pharmacokinetic properties. The goal of this proposal is to understand the chemical space of this scaffold and to develop more potent analogs by comprehensive SAR studies as in vivo probes and three specific aims are proposed to achieve our objective in this application. In Aim 1, new analogs of this lead structure will be designed and synthesized to provide understanding of SAR for this scaffold that will guide the development of more potent inhibitors. In Aim 2, the designed new analogs will be evaluated in tiered biological systems for potency, selectivity, target engagement, and immunotoxicity. In aim 3, the top candidate inhibitor identified from Aim 2 will be tested to confirm the in vivo efficacy in a transgenic AD mouse model. The proposed research is highly significant because successful development of novel and selective NLRP3Is will not only provide effective pharmacological tools to precisely define the contribution of the NLRP3 inflammasome in disease pathogenesis, but also provide promising candidates for clinical studies, thus offering translational potential to achieve clinical benefits.