John A. Kessler - US grants

DESCRIPTION (Applicant's abstract reproduced verbatim): The proposed research is a continuation of our ongoing studies of the development of the peripheral nervous system. These studies will employ a combined in vitro/in vivo approach to define the role of bone morphogenetic protein 4 (BMP4) in regulating the survival and differentiation of peripheral sympathetic, dorsal root ganglion, and cranial sensory ganglion neurons. The first set of studies will examine the proapoptotic and anti-proliferative effects of BMP4 and the role of the factor in inducing neuronal dependence on growth factors for survival in vitro. They will further examine the role of the factor in the induction and regulation of trkC expression and the facilitation of neuronal responses to neurotrophin 3. To define the actions of BMP4 in vivo we have constructed transgenic animals in which a keratin 14 promoter (K14) or a neuron specific enolase (NSE) promoter drives expression of either BMP4 or of the BMP inhibitor, noggin. This approach will allow examination of the effects of both loss of BMP function and gain of function in the developing peripheral nervous system. The NSE animals will be particularly useful for examining ganglion development during the period of time when neuroblasts stop dividing, begin to extend processes to targets, and switch the neurotrophin receptors they express. The Kl4 animals will be particularly useful for examining the effects of BMP4 on neurons that are innervating a target (skin). Specifically these studies will define trk expression and distribution, neuron numbers, and neuronal phenotype in the peripheral nervous system of the transgenic animals. They will further determine whether there are abnormalities in peripheral nerve function in these animals. In a broader sense these studies seek to define the role of intercellular communication in development and function of the nervous system. It is hoped that these studies will indicate biochemical loci where therapeutic intervention in disease processes may lead to a return to normal neuronal function.

The proposed research is a continuation of ongoing studies of neuronal growth, development, and transmitter phenotypic expression. Using a combination of biochemical, immunocytochemical, and pharmacological techniques, we have defined factors governing embryogenesis in sympathetic and sensory neurons, the factors regulating peptide neurotransmitter phenotypic expression, and the role of intercellular communication in neuronal ontogeny. In particular, we have focused on defining the molecular mechanisms regulating development of the putative peptide neurotransmitters, substance P (SP) and somatostatin (SS). The present studies will examine neuron target interactions and the role of growth factors in mediating the effects of these interactions on neuronal phenotypic expression. More specifically, we hope to a) Examine the effects of a recently described peptide stimulating factor on SP and SS development in sensory, sympathetic, and striatal neurons in vivo, b) Define the effects of this growth factor on cholinergic development in striatal neurons in vivo and in culture, c) Define the role of SP and SS in regulating sympathetic, noradrenergic development, d) Examine interactions between the overlapping sensory, sympathetic, and parasympathetic innervations of the iris, e) Use the above information to understand processes governing neuronal phenotypic expression and interneuronal communication. It is hoped that these studies will elucidate mechanisms leading to abnormal neuronal development and indicate new therapeutic approaches to diseases of disordered neural development.

DESCRIPTION (adapted from the applicant's abstract): The proposed studies are a continuation of studies that have been ongoing for 15 years to explore the role of cytokines in regulating neural development. The proposed studies will focus on the role of the bone morphogenetic proteins (BMPs), particularly BMP4, in progenitor cell lineage commitment and brain development. Combined in vitro and in vivo approaches will assess BMP4 on progenitor cell survival, proliferation, and commitment to astrocytic and neuronal lineages. The first set of studies the differential effects of BMP on the genesis and fate of progenitor cells from the ventricular (VZ) and subventricular zones (SVZ) in vitro. These studies will include examining the role of epidermal growth factor receptor (EGF) signaling as modulating the effects of BMP, examining the effects of BMP on neuronal differentiation and survival. The second set of studies will define the actions of BMP4 in vivo using transgenic animals in which a glial fibrillary acidic acid promoter (GBMP) or a neuron specific enolase promoter (NSEBMP) drives overexpression of BMP4. These transgenic animals will allow examination of effects of BMP4 on the genesis and fate of the developing brain in vivo, including the regulation of neuronal differentiation. These studies will test the hypotheses that the BMPs promote astrocytic, but suppress oligodendroglial lineage commitment, in the developing brain, and that they regulate growth of dendrites and/or axons from specific populations of cortical, hippocampal, and cerebellar neurons.

cell bank /registry; tissue /cell culture; biomedical facility; growth media;

Description (Abstract reproduced verbatim): The overall goal of these studies is to define the role of gap junctions in neural progenitor cell survival, proliferation, and commitment to neuronal and glial lineages, and in the progressive development of neurons and glia. The basic hypotheses underlying these studies are that gap junctional communication coordinates proliferation and survival of subpopulations of progenitor cells and orchestrates early lineage commitment, and that functional uncoupling of progenitor species is subsequently necessary for exit from cell cycle and commitment to specific lineages. Thereafter re-expression of other connexins with different biophysical properties influences cell function. The development of progenitor cells can be manipulated in vitro by changing the cellular microenvironment to promote neurogenesis or gliogenesis. The proposed studies will examine the functional role of gap junctions in regulating development of these populations in vitro, and will define connexin expression in the developing brain in vivo. They will examine the relationship between the regulation of gap junctional communication and exit of progenitor cells from cell cycle and the role of connexins in the subsequent maturation of neuronal progeny. They will define mechanisms that determine which connexin is expressed by developing neurons and whether the expression of gap junction channels with different biophysical properties alters the phenotype of developing neurons. Particular focus will be placed on the study of connexin 36, a major neuronal gap junction protein in the brain. These studies should provide insight into mechanisms regulating neural development, lineage commitment, and phenotypic expression, and into the role of gap junctions in regulating these processes.

DESCRIPTION:(Adapted from the Applicant's Description) Neural tube defects represent a common, heterogeneous set of congenital malformations whose etiology is poorly understood. Although some environmental factors have been implicated, a significant genetic contribution is implied by the analysis of murmne lines with NTDs and the familial clustering in humans. Additionally, numerous syndromes and chromosomal anomalies are associated with NTDs. Both deletions and trisomies of chromosome 13 have been associated with neural tube defects raising the possibility that certain genes on chromosome 13 contribute to the etiology of NTDs when their dosage is altered. Nested sets of deletions of chromosome 1 3q, including one recently identified in the investigators clinic, implicate a fairly small region on chromosome 1 3q33-34. Although the exact genes required for producing an NTD in a 13q deletion are not known, several genes found in this region are important for early neural development in murine models, making them excellent candidates to test for involvement in human NTDs. The investigators propose to test the hypothesis that candidate genes in the chromosome 1 3g critical region are associated with NTDs in patients who lack chromosomal deletions. They will map the chromosome 1 3q critical region using patients with deletions and NTDs to narrow further the number of candidate genes. They will then choose genes to screen in detail based upon evidence of their involvement in early neural development or evidence of linkage disequilibrium. The candidates will be screened for mutations in a large set of individuals with NTDs using the resources of the NTD collaborative group. The investigators will test for linkage disequilibrium of all available candidate genes in the 1 3q critical region with the transmission disequilibrium test. These studies are likely to define mutations or polymorphisms associated with NTDs. The ability to identify genetic factors that place families at risk might help reduce the incidence of NTDs or make early intervention more feasible.

Embryonic stem (ES) cells are pluripotent cells derived from the inner cell mass of the blastocyst stage embryo that can differentiate in vivo and in vitro into all cell lineages of the adult animal. The ability of these cells to adopt.multiple fates makes ES cells prime candidates for use in stem cell therapies. The mechanisms that maintain sternness in human ES cells (hESCs) are poorly understood. The first part of the proposed studies will examine the signaling pathways responsible both for maintenance of sternness and for neural differentiation of hESCs. Successful use of hESCs in transplantation strategies will require an extracellular milieu appropriate for directing survival and differentiation of the cells. We have found that a self-assembling peptide amphiphile that can be safely injected directly into neural tissue promotes neuronal differentiation of neural stem cells and facilitates functional recovery after spinal cord injury. The second aspect of the proposed research will examine the effects of such nanoengineered materials on hESCs and will define the mechanisms underlying effects of the nanogels on hESC properties. The stem cell technology and nanotechnology that are developed as part of these studies will then be used to address the problem of spinal cord injury. The lack of regenerationfollowing injury to the adult spinal cord reflects a number of factors including the presence of molecules inhibitory to axon outgrowth, the paucity of molecules that foster regeneration, and the formation of a cavity and of a glia scar that can physically impede axonal outgrowth. This study will therefore adopt a multifactorial approach that addresses each of these issues. First, hESCs will be engineered to secrete blockers of the major, well-studied inhibitors, Nogo, MAG, and Omgp. Second, hESCs will be engineered to secrete neurotrophic factors that have been shown to facilitate motor and sensory tract regeneration. Third, the nanoengineered self-assembling gel will be used in conjunction with the hESCs to fill the cavity and to inhibit formation of the glia scar. The long term goal of these studies is to develop techniques for combining advances in stem cell biology and nanotechnology for the repair/ regeneration of the damaged central nervous system. The studies described in this proposal will only utilize NIH approved embryonic stem cell lines lines including WA01, UC06, and TE03.

[unreadable] Description (provided by applicant): [unreadable] [unreadable] This project will address some fundamental questions about the biology of human embryonic stem cells. Our overall, long-term goal is that the answers to these questions should help to hasten the development of novel stem-cell based strategies for treating human diseases. We will pursue the following aims. First, we will evaluate a range of antibodies directed against cytoplasmic intermediate filament (IF) proteins as markers to help identify specific classes of progenitor cells in cultures of human embryonic stem cells (hESC). We will use these antibodies to track the changing patterns of IF protein expression as cells progress to terminal differentiation. Second, we will use small interfering RNAs (siRNAs) to down regulate specific IF proteins to help determine the functional significance of the different IF protein expression profiles that emerge during development. Third, we will make a detailed study of the structure and function of nestin, a type IV IF protein widely-used as a neural progenitor cell marker, focussing on our recent finding that its C terminus interacts with the insulin degrading enzyme and may regulate its activity. The experiments will make use of an extensive collection of mono- and polyclonal antibodies raised against cytoskeletal IF proteins that we have developed and tested in our laboratory over the years. We will employ high resolution, confocal fluorescence microscopy to study fixed, immunostained cells. Time lapse studies will be made of living cells expressing various GFP-IF protein constructs and the dynamic properties of IF of varying protein composition will be studied using fluorescence recovery after photobleaching (FRAP). Public health relevance of this research: Human embryonic stem cells have the potential to give rise to all cell types of the human body, and thus have great utility for treating diseases such as Parkinsons' Disease and diabetes, as well as spinal cord injuries. The proposed research will advance our understanding of the cell biological events that take place as stem cells change into more highly differentiated cell types. This information is crucial for guiding cells down the right pathway so that they can eventually be used to treat patients. [unreadable] [unreadable]

DESCRIPTION (provided by applicant): Regenerative medicine is one of the great biomedical challenges of this century, requiring research at the interface of the physical and life sciences as well as engineering. Cell-based therapies for tissue and organ regeneration will involve not only stem cell biology but also the crafting of bioactive matrices to support tissue growth. This will require cutting- edge supramolecular chemistry, nanotechnology, and micro scale process engineering. In addition, understanding and regulating immune responses to regenerated tissues will be essential for long term survival and function of reconstituted organs. The most effective regenerative strategies will include not only cell-based therapies but also the development of man-machine interfaces, particularly in the case of the damaged nervous system. No single discipline is likely to be able to overcome all of the obstacles to developing techniques for repairing or regenerating damaged tissues and organs. It is therefore necessary to design and implement programs that fuse disparate disciplines into a unified effort to achieve the goals of regenerative medicine. The goal of this training program is to provide predoctoral and postdoctoral trainees with integrated interdisciplinary training in this area. The training faculty members represent 10 different departments/divisions in the Feinberg School of Medicine, 3 departments in the McCormick School of Engineering and Applied Sciences, 2 departments/divisions at Children's Medical Research Center, and 1 department at the Rehabilitation Institute of Chicago. Collectively the preceptors include 5MD PhDs, 12 PhDs, and 3 MDs. The faculty has been carefully constituted to be able to provide oversight in critical areas for the field including stem cell and developmental biology, materials science and nanomedicine, extracellular matrix, immune tolerance to tissues, and neural engineering and robotics. It is anticipated that trainees will be drawn from six unified graduate programs at Northwestern University. Predoctoral trainees selected for the program will fulfill the rigorous requirements for the granting of the PhD degree in their respective programs. Due to the interdisciplinary nature of this research area, the training program core curriculum will have additional unique and non-traditional aspects in which both predoctoral and postdoctoral trainees will participate. The overall theme involves the wedding of bioengineering and materials science with stem cell biology and animal modeling. The goal is to train a cadre of investigators with the requisite interdisciplinary skills necessary both to overcome the barriers to organ and tissue regeneration and to translate advances in the life and physical sciences into clinical medicine.

DESCRIPTION (provided by applicant): There is a critical need for clinician-scientists possessing the medical training and research experience to conduct basic, clinical and translational research on the mechanisms and treatment of neurological disorders. This Neuroscience Research Education Program at Northwestern (R25) is designed to foster the development of clinician neuroscientists in the Departments of Neurology at Northwestern University Feinberg School of Medicine (NUFSM) via research and educational experiences that will prepare these trainees to successfully compete for individual fellowships and mentored career development awards that would enhance their ability to become independent investigators. Specifically we propose to: 1. Recruit at least one neurology resident each year who demonstrates aptitude for and interest in research to prepare them to compete for K08 or K23 awards. 2. Create and implement a research training curriculum for the later years of neurology residency and the first year of a clinical neuroscience research fellowship using a mentoring team comprising a Primary Mentor and Collaborating faculty, and utilizing established programs and institutional resources at Northwestern. 3. Enhance existing research mentorship opportunities available at Northwestern to be available to the residency trainees within this R25 program. 4. Conduct ongoing evaluation of the R25 program. Northwestern has excellent institutional resources including NIH funded Clinical and Translational Science Institute, outstanding cadre of Neurology and Collaborating faculty with extensive track record of mentorship, strong pool of trainees interested in research career and rich culture of interdepartmental collaborations to assure success of the trainees in preparation for independent research careers. One trainee will be selected from the pool of neurology residents. Individual training plan will created for each trainee. Training team will consist of the Primary Mentor and collaborating faculty with direct supervision and oversight by the Program Directors. Trainees will be assured at least nine months of protected research time with 80% time dedicated to research during residency and 12 months dedicated to research during fellowship. Success of the program will be determined by the number of trainees who receive career development awards and ultimately continue with academic career. The quality of the program will be evaluated on the regular basis.

Summary and Relevance: The ability to reprogram somatic cells into pluripotent stem cells (induced pluripotent stem cells - iPSCs) has transformed both the study of basic human cellular neurobiology and the examination of the cellular basis of human diseases of the nervous system. However the creation, culturing and differentiation of iPSCs and their progeny are technically challenging and labor intensive which has inhibited use of iPSCs by many investigators. This core will provide the facilities and technical expertise to enable NINDS investigators to incorporate the technology into their research efforts.

? DESCRIPTION (provided by applicant): Mechanisms Underlying Heterotopic Ossification Heterotopic ossification (HO) is bona fide bone formation outside of the normal skeletal system. The precise mechanisms underlying HO are unknown, although numerous signaling pathways and cellular components, local and systemic, have been implicated. Our previous work demonstrated that dysregulation of the neuroinflammatory factor, substance P (SP), along with mast cells and macrophages, leads to elevated bone morphogenetic protein (BMP) signaling that triggers the pathological process. Glast and Tie2 expressing mesenchymal stem/progenitor cells (MSCs) respond to the inductive signals and form ectopic bone through endochondral ossification. Our central hypothesis is that unlike normal skeletogenesis, the process of HO is an injury/inflammation induced, abnormal local morphogenic phenomenon. Specifically, multiple disease specific contributing factors, including both cellular and extracellular molecular elements, co-ordinate with each other to form a unique hierarchical microenvironment, similar to endogenous stem cells niches, to induce and propagate HO. In the niche, MSCs recruited from local tissues are the cells of origin of HO, and specific local supportive cellular (e.g. local neres, mast cells) and molecular (e.g. SP) components are crucial to regulate the stereotyped self-renewal, proliferation, chondrogenic and osteogenic differentiation of these MSCs. Thus development of therapeutic approaches must consider the role of the local innervation and mast cells as well as local sources of cells of origin of HO. This requires an interdisciplinary approac to study the niche as a minimum functional unit, the goal of this proposal. Understanding the characteristics of the cells of origin of HO will be necessary for precise therapeutic targeting to prevent or limit the disorder. The first part of the proposed studies will examine the niche dwelling cells that directly participate in HO. The second part will study the niche supportive cells and key molecular components that initiate and propagate HO. A major focus will be to examine the therapeutic effectiveness in preventing or limiting HO by inhibiting factors, such as SP, that play a central role in the HO niche. Thus the overall goal is to develop a therapy for thi debilitating disorder for which there currently is no effective treatment.

? DESCRIPTION (provided by applicant): Aging often leads to a functional decline across multiple cognitive domains, but the physiologic and anatomic changes underlying these impairments are not fully understood. A number of changes in hippocampal structure and connectivity are associated with aging including a decline in neurogenesis in the subgranular zone of the dentate gyrus (DG) and decreased performance on hippocampus-dependent tasks. Levels of bone morphogenetic protein 4 (BMP4) in the mouse DG increase more than 10-fold between 8 and 52 weeks of age. A similar aging-related increase in BMP4 expression is found in the human DG. Conversely, levels of the BMP inhibitor, noggin, in the mouse DG decrease by about 70% during this time. This results in an extraordinary 30- fold aging-related increase in BMP signaling in the DG measured by levels of phosph-SMAD1/5/8. Reducing BMP signaling in aged mice by either intraventricular infusion or transgenic overexpression of noggin reverses aging-related changes in both neurogenesis and cognition. Conversely, transgenic overexpression or intraventricular infusion of BMP4 in aged mice prevents the beneficial effects of exercise on neurogenesis and cognitive performance. These findings lead to the hypothesis that changes in BMP signaling underlie the decreases in neurogenesis and in hippocampus-dependent behavior associated with aging. To test this hypothesis we will first investigate the cellular and behavioral effects of inducible cre-mediated ablation of BMPRII in the DG neural stem cells (NSCs) of aged mice. To identify BMP targets associated with aging related neural stem cell quiescence, we will perform genomic scale gene expression profiling of NSCs isolated from BMPRII intact and ablated aged mice. Finally, we will define changes in expression and cellular origin of BMP ligands, receptors, and inhibitors in the hippocampus of aging humans and examine correlations between BMP levels, neurogenesis, and age-associated cognitive decline in human. The goal of the studies is to identify specific molecular loci where therapeutic intervention in the aged nervous system may lead to a return to normal neurological function.

Project Summary Aging often leads to a functional decline across multiple cognitive domains, and there is a signficantly increased risk of depression and anxiety disorders in the aged. However, the physiologic and anatomic changes underlying these impairments are not fully understood. A number of changes in hippocampal structure and connectivity are associated with aging including a decline in neurogenesis in the subgranular zone of the dentate gyrus (DG) and decreased performance on hippocampus-dependent tasks. Levels of bone morphogenetic protein 4 (BMP4) in the mouse DG increase more than 10-fold between 8 and 52 weeks of age. A similar aging- related increase in BMP4 expression is found in the human DG. Conversely, levels of the BMP inhibitor, noggin, in the mouse DG decrease by about 70% during this time. This results in an extraordinary 30-fold aging-related increase in BMP signaling in the DG measured by levels of phosph-SMAD1/5/8. Reducing BMP signaling in aged mice by either intraventricular infusion or transgenic overexpression of noggin reverses aging-related changes in both neurogenesis and cognition, and it reduces depression-like behavior. Conversely, transgenic overexpression or intraventricular infusion of BMP4 in aged mice prevents the beneficial effects of exercise on neurogenesis and on cognitive and affective behavior. These findings lead to the hypothesis that changes in BMP signaling underlie the decreases in neurogenesis and in hippocampus- dependent behavior associated with aging. To test this hypothesis, we will first investigate the cellular and behavioral effects of inducible cre-mediated ablation of BMPRII in neural stem cells in the DG of aged mice. We then will examine the potential causal relationship between changes in neurogenesis and behavior. To begin to develop a potential therapeutic approach, we will use a new technology, spherical nucleic acid nanoparticle conjugates (SNAs), as an RNAi-based therapeutic approach to enable us to specifically target BMPR signaling in the brain to enhance adult neurogenesis. Finally, we will define changes in expression and cellular origin of BMP ligands, receptors, and inhibitors in the hippocampus of aging humans and examine correlations between BMP levels, neurogenesis, and age-associated cognitive decline in humans. The goal of the studies is to identify specific molecular loci where therapeutic intervention in the aged nervous system may lead to a return to normal neurological function.

Aging is the leading risk factor for developing sporadic Alzheimer?s disease (sAD) suggesting that the aging process is linked to the pathophysiology of the disease. The aging process is highly variable, and some aged individuals develop non-degenerative cognitive impairment while others are resilient. The biological basis of this heterogeneity is unknown. However, aging is associated with biochemical and morphological changes that may shed insight into the both the phenotypic heterogeneity in aging and the age-related susceptibility to AD. In particular, changes in hippocampal structure and connectivity are associated with aging. Among these changes is a decline in neurogenesis in the dentate gyrus (DG) with decreased performance on hippocampus- dependent cognitive tasks. BMP signaling in the brain increases dramatically with age, and we have been exploring the hypothesis that this underlies aging-related changes neurogenesis and cognition. BMP signaling and neurogenesis are also both altered significantly in AD. However, it is unclear whether changes in BMP signaling and in neurogenesis in AD are an exacerbation of the aging process or rather due to unrelated mechanisms. The goal of this proposed supplement is to use both the knowledge and the tools developed as part of our ongoing studies of the role of BMP signaling in the aging brain to define the role of this signaling pathway in the development of AD. Second to age, the human apolipoprotein E (hAPOE) genotype is the strongest known risk factor for sAD. Further, in aging mice, neuronal expression of the ?4 isoform results in neuron loss and impaired learning and memory, and APOE regulates hippocampal neurogenesis. The convergence of the effects of the aging related increase in BMP signaling and of APOE on cognition and on neurogenesis suggest possible interactions between the aging related increase in BMP signaling, APOE genotype, and the development of sAD. Specifically, we hypothesize that the aging related increase in BMP signaling in brain predisposes neurons to the effects of APOE4 and to development of AD, and that that it explains, at least in part, why aging is the greatest risk factor for sAD. We will use the iPSC lines we derived from sAD patients to explore interactions between APOE genotype and BMP signaling on tau phosphorylation, amyloid ß secretion, and neurite preservation, and on neuronal survival after increasing calcium influx in neurons derived from the iPSCs. We also will examine the role of BMP signaling in development of pathology and of behavioral changes in 5XFAD mice.

Traumatic Brain Injury (TBI) is a major health issue. After the primary injury, there is substantial secondary injury attributable to infiltrating immune cells, cytokine release, reactive oxygen species, excitotoxicity, and other mechanisms. Despite many preclinical and clinical trials designed to limit such secondary damage, no successful therapies have emerged. However, we have found that Immune-modifying nanoParticles (IMP) are a strong candidate for a clinically translatable acute pharmacologic intervention for TBI. IMP are highly negatively charged, 500 nm-diameter particles composed of the FDA-approved biodegradable biopolymer, carboxylated poly(lactic-co-glycolic) acid (PLGA-COOH). After intravenous (IV) administration, IMP bind to the macrophage receptor with collagenous structure (MARCO) on monocytes. Monocytes bound to IMP no longer travel to sites of inflammation, but instead are sequestered in the spleen. Because IMP specifically target the MARCO+ subset of monocytes, it is distinctly different from other approaches that non-specifically target all monocyte/macrophage lineage cells including microglia. IV treatment with IMP in two different TBI models profoundly reduced the number of immune cells infiltrating into the brain, mitigated the inflammatory status of the infiltrating cells, and reduced levels of an array of cytokines and chemokines. More importantly, IMP treatment resulted in attenuated edema, preservation of brain tissue, and significant preservation of both physiologic visual and motor function. The proposed studies will examine IMP-mediated changes in gene expression that alter the inflammatory status of infiltrating cells, limit gliosis, reduce edema, and promote neuronal survival. They also will examine effects of IMP on other cell types including microglia, progenitor cells, and other immune cells. Notably, IMP are made of an FDA-approved material that is stable at room temperature and could easily be given immediately IV after TBI in the field by EMTs or in the emergency room. Mechanistically the proposed studies will help to understand more clearly the effects of infiltrating hematogenous monocyte-derived macrophages after TBI. Significantly, they also will help to develop a potentially effective and practical therapy for human TBI.

Summary Major depressive disorder (MDD) is one of the leading causes of disability and lost productivity. Nearly half of all clinically depressed patients fail to respond to the first prescribed antidepressant, and about a third fail to respond to all medications. Development of new approaches will require better understand of the mechanisms underlying the disorder. This project has identified and is examining a signaling pathway not previously implicated in anxiety and depression-like behavior, bone morphogenetic protein (BMP) signaling. MDD is associated with reductions in volume of the hippocampus (HC) in humans and in neurogenesis in the HC in animal models of the disorder. Reduction of BMP signaling in the HC in mice is sufficient to produce antidepressant-like changes in behavior and to increase neurogenesis. Treatment with several different classes of antidepressant drugs reduces BMP signaling in the HC, and prevention of this reduction in BMP signaling blocks the effects of the drugs on both behavior and neurogenesis. Inhibition of BMP signaling in the HC also blocks the effects of unpredictable chronic mild stress on both depression- like behavior and neurogenesis. Thus BMP signaling in the hippocampus regulates both depression-like behavior. However, a causal link between the changes in neurogenesis and behavior has not been established. The proposed studies will determine whether there is a causal relationship between changes in neurogenesis, electrophysiological activity of newly generated neurons, and behavior after inhibition of BMP signaling in HC stem/progenitor cells. They also will define the role of BMP signaling in cellular and behavioral responses to stress, and test the hypothesis that that gene expression changes due to elevated BMP signaling contribute to the decrease in neurogenesis, increased proportion of quiescent neural stem cells, and behavioral changes associated with stress/depression.