Marion S. Buckwalter - US grants

DESCRIPTION (provided by applicant): Dr. Buckwalter is a stroke neurologist and neurointensivist with training in genetics and molecular biology. She is interested in how the aged brain's response to injury affects repair and functional recovery. Aging is associated with less effective recovery from brain injury. The reasons for this decline are unknown, but a reduced capacity of the aging brain to form new neurons may be in part responsible. The applicant has generated preliminary data that demonstrate a marked inhibition of adult hippocampal neurogenesis by transforming growth factor beta-1 (TGF-beta1), a cytokine that is increased by aging and injury. This data, and the known effect of TGF-beta on the cell cycle, led her to propose the hypothesis that TGF-beta1 acts directly on neural progenitor cells to inhibit hippocampal neurogenesis. Thus, too much TGF-beta1 in the aging brain, and especially in the injured and aged brain, may inhibit neurogenesis. She will determine how TGF-beta1 affects the cell cycle and number of neural progenitor cells, how this translates into fewer new neurons, and whether the reversal of this effect leads to cognitive improvement. At the conclusion of these studies, we will have a significantly better understanding of the mechanism by which TGF-beta1 inhibits hippocampal neurogenesis. This application also details a carefully thought out career development plan that includes rich interaction with other laboratories at Stanford, exposure to seminars and scientific meetings, supervision of students and technicians, and classes in neuroscience and immunology. Dr. Buckwalter has strong institutional support that will allow her to focus on these studies with minimal distractions. This K08 Mentored Clinical Scientist Career Development Award will facilitate Dr. Buckwalter's transition from a neurologist with training in genetics to a physician scientist who is fully competent to investigate the injury response in aging in her own laboratory through independent, investigator-initiated funding. This research is directly related to public health because millions of people in the US are living with disability due to traumatic brain injury or stroke. Our data shows that elevated brain TGF-beta 1, which is seen in all these diseases, inhibits our ability to grow new neurons from cells in our own brains. We hope that the experiments in this application will help us to someday design strategies to grow new brain cells to replace those lost to these devastating diseases.

[unreadable] DESCRIPTION (provided by applicant): Dr. Buckwalter is a stroke neurologist and neurointensivist with training in genetics and molecular biology. She is interested in how the aged brain's response to injury affects repair and functional recovery. Aging is associated with less effective recovery from brain injury. The reasons for this decline are unknown, but a reduced capacity of the aging brain to form new neurons may be in part responsible. The applicant has generated preliminary data that demonstrate a marked inhibition of adult hippocampal neurogenesis by transforming growth factor beta-1 (TGF-beta1), a cytokine that is increased by aging and injury. This data, and the known effect of TGF-beta on the cell cycle, led her to propose the hypothesis that TGF-beta1 acts directly on neural progenitor cells to inhibit hippocampal neurogenesis. Thus, too much TGF-beta1 in the aging brain, and especially in the injured and aged brain, may inhibit neurogenesis. She will determine how TGF-beta1 affects the cell cycle and number of neural progenitor cells, how this translates into fewer new neurons, and whether the reversal of this effect leads to cognitive improvement. At the conclusion of these studies, we will have a significantly better understanding of the mechanism by which TGF-beta1 inhibits hippocampal neurogenesis. This application also details a carefully thought out career development plan that includes rich interaction with other laboratories at Stanford, exposure to seminars and scientific meetings, supervision of students and technicians, and classes in neuroscience and immunology. Dr. Buckwalter has strong institutional support that will allow her to focus on these studies with minimal distractions. This K08 Mentored Clinical Scientist Career Development Award will facilitate Dr. Buckwalter's transition from a neurologist with training in genetics to a physician scientist who is fully competent to investigate the injury response in aging in her own laboratory through independent, investigator-initiated funding. This research is directly related to public health because millions of people in the US are living with disability due to traumatic brain injury or stroke. Our data shows that elevated brain TGF-beta 1, which is seen in all these diseases, inhibits our ability to grow new neurons from cells in our own brains. We hope that the experiments in this application will help us to someday design strategies to grow new brain cells to replace those lost to these devastating diseases. [unreadable] [unreadable] [unreadable]

DESCRIPTION (provided by applicant): Stroke is the leading cause of long-term disability in the United States. Although acute revascularization therapies can be used to abort or reduce stroke burden, there are currently no drugs that improve recovery after a stroke has happened. The inflammatory response is a promising target for such therapies as it occurs in the days and weeks after a stroke and can be both detrimental and beneficial. A major unanswered question is how the injured brain modulates immune responses, and if there are molecular pathways that can be utilized to exert beneficial or limit detrimental effects on functional recovery via modulating the overall immune response. Astrocytes are a key component of the brain's injury response - so-called "reactive astrocytes" are ubiquitous after brain injury. They are also increasingly recognized as key components of the brain's innate immune system. We propose to ask if Transforming Growth Factor Beta (TGF[unreadable]) signaling in astrocytes modulates inflammation after stroke because it is a master regulator of immune responses. TGF[unreadable] can resolve immune responses after injury and drive immune cell phenotypes towards less inflammatory states. Our preliminary experiments show that TGF[unreadable] signaling is increased in the brain after stroke, persists for weeks, and occurs in reactive astrocytes. To test if TGF[unreadable]'s function in reactive astrocytes mirror its role in other types of immune cells we constructed mice in which TGF[unreadable] signaling is decreased only in astrocytes. We have found that primary astrocytes from these mice exhibit a more "pro-inflammatory" phenotype after oxygen- glucose deprivation, and the mice themselves demonstrate increased inflammatory responses after stroke. Based on this data we hypothesize that after stroke, TGF[unreadable] signaling (1) occurs in reactive astrocytes, (2) limits the inflammatory response, and (3) improves functional recovery. We plan to test our hypothesis in three Specific Aims. In Aim 1 we will use reporter mice and immunohistochemistry to determine patterns of TGF[unreadable] signaling after stroke. We hypothesize that there are increased responses to TGF-[unreadable] for weeks after stroke, and that reactive astrocytes are responding to TGF[unreadable] after stroke. In Aim 2 we will test the function of astrocytic TGF[unreadable] signaling in the neuroinflammatory response to ischemia, using genetic and pharmacological approaches and in vivo and in vitro experiments to target TGF[unreadable] signaling in astrocytes. We hypothesize that astrocytic TGF[unreadable] signaling drives resolution of the immune response to stroke. In Aim 3 we will use a genetic mouse model to ask if stroke-induced astrocytic TGF[unreadable] signaling is beneficial or detrimental for functional recovery. We predict that astrocytic TGF[unreadable] signaling improves recovery from stroke. With the completion of the proposed experiments we will have defined the length and cell specificity of TGF[unreadable] responses after stroke. We will gain insight into how astrocytes influence the immune response to stroke, and into the functional diversity of reactive astrocytes. Our findings may lead to therapies that will target the brain's immune responses and benefit patients who present for medical care in the days after stroke. PUBLIC HEALTH RELEVANCE: Stroke is the third leading cause of death in the US, and a leading cause of disability, and there are currently no drugs that improve recovery after stroke. Neuroinflammation affects many processes important for recovery from stroke and modulating neuroinflammation is therefore likely to be a way we can improve recovery from stroke. In this application we propose to study the effects of a master regulator of neuroinflammation, transforming growth factor beta, to determine how its effects in astrocytes can be manipulated to increase successful recovery from stroke.

DESCRIPTION (provided by applicant): Cerebrovascular disease is a major risk factor for dementia. All-cause dementia, (vascular dementia, Alzheimer's disease, and mixed disorders) is diagnosed in up to 48% of stroke survivors, with about 20% incidence in the first year after stroke. Several stroke risk factors, including hypertension, high cholesterol, diabetes, and age, also independently increase the risk of dementia. But multiple studies demonstrate that even after controlling for risk factors, stroke alone still doubles the risk of new onset dementia. We propose to model how stroke increases the risk of developing dementia by developing a new mouse model where stroke causes delayed cognitive dysfunction. In our model mice undergo DH stroke as a result of distal middle cerebral artery occlusion followed by hypoxia. This confers a primarily cortical infarct. Mice display normal working memory 8 days after stroke but by 6-7 weeks later they develop deficits in working and spatial memory, accompanied by a prolonged inflammatory response. A part of this inflammatory response is the appearance of B cells in the stroke core, in follicle-like structures. Such structures in the CNS have recently been linked to several neurodegenerative diseases. We propose here to develop our model and test whether B cells are pathogenic in our model. In Aim 1 we will use a panel of cognitive tests at key timepoints to assess which areas of cognition are affected in this model, and also carefully phenotype the delayed inflammatory response to stroke. We hypothesize that pathogenic features of neuroinflammation will precede or coincide with cognitive impairment. In Aim 2 we will use a B cell-depleting antibody and B cell knockout mice to ask whether B cells are required for cognitive dysfunction to occur. This work has high potential to produce significant impact because there are currently no well-accepted models and little is known about the genesis of this common and debilitating disorder. At the conclusion of these experiments we will have characterized the cognitive deficit and inflammatory responses in our model and will know whether B cells are causative. This information will allow us to pursue critical future studies on the mechanisms that underlie post-stroke dementia, and in addition will provide a way to test potential therapies.

Project Summary. Stress strongly influences the immune system, particularly antigen-specific adaptive immune responses, via sympathetic outflow and activation of adrenergic receptors. However, although there are consistent findings that the sympathetic neurotransmitters epinephrine and norepinephrine regulate antigen presenting cells and T and B lymphocytes via adrenergic receptors, the results are context dependent?either activation or suppression of antigen-dependent responses is observed. Importantly, the cues that mediate these opposing effects are not well understood. We propose here to generate tools to understand whether spleen glia play an unrecognized role in regulating these context-dependent effects of sympathetic outflow on adaptive immunity. Sympathetic terminals carrying adrenergic neurotransmitters enter the spleen around the arterial blood supply and innervate the white pulp, where antigen-specific T and B lymphocyte responses are continually developing. So glia accompanying the sympathetic nerves are optimally positioned to influence stress effects on antigen-dependent adaptive immune responses. The barrier to testing this is, however, that spleen glia have not previously been described. We are therefore requesting R21 funding to describe their anatomy and translatome (translating mRNAs), and to determine if they change their expression of immune genes during the extreme physiological stress of a large stroke. At the conclusion of these experiments, we will have generated tools to study not only spleen glia, but also other peripheral glia, and also have established whether spleen glia are important participants in sympathetically-mediated effects on immune responses. These studies are appropriate for R21 funding as they are high-risk initial studies in a new scientific area, the study of the functions of spleen glia.