Todd A. Fiacco, Ph.D. - US grants

DESCRIPTION (provided by applicant): Numerous lines of evidence suggest that astrocytes actively participate in regulating neuronal excitability, but the role of astrocyte swelling in conrol of neuronal excitability has never been directly tested. Our long-term goal is to identify and understand astrocytic mechanisms controlling neuronal excitability. The objective in this particular application is to determine how specific manipulations of astrocyte swelling and swelling-evoked glutamate release lead to changes in neuronal excitability in situ and in vivo. The central hypothesis is that astrocyte swelling and glutamate release from astrocytic volume-regulated anion channels (VRAC) is both necessary and sufficient to elevate neuronal excitability in situ and in vivo. The rationale for the proposed research is that, identification o novel astrocytic pathways controlling neuronal excitability will provide new astrocytic drug targets for the treatment of neurological disorders and neurodegenerative disease. Guided by strong preliminary data, the central hypothesis will be tested by pursuing three specific aims: 1) Determine the extent to which astrocyte swelling-evoked glutamate release is necessary to increase neuronal excitability in situ; 2) Determine the extent to which astrocyte swelling-evoked glutamate release is sufficient to increase neuronal excitability in situ; and 3) Determine the contribution of astrocyte swelling to the control of neuronal excitability in vivo. Astrocyte swellng and glutamate release will be selectively manipulated using patch clamp and transgenic approaches, together with real-time imaging of astrocyte volume changes during recording of NMDA receptor activity in CA1 pyramidal neurons in acute hippocampal slices (Aims 1 and 2), and the effects of hypoosmolarity, hyperosmolarity and selective inhibitors on astrocytic volume changes and neuronal excitability will be assayed in vivo (Aim 3). Our approach is innovative, in our opinion, because it represents a significant departure from the status quo of assessing the role of astrocyte Ca2+-dependent gliotransmission in regulating neuronal excitability, and because techniques have been developed and proven feasible in our hands to selectively and specifically manipulate astrocyte volume changes and release of glutamate. The proposed re- search is significant, because once astrocytic mechanisms controlling neuronal excitability become clarified, novel astrocyte-directed therapies can be devised to prevent excessive levels of neuronal excitability while leaving basal levels of neuronal excitability and normal cognitive function intact. Such knowledge will also pro- vide new strategies to treat neurological disorders associated with cellular volume changes (including various forms of edema), while also fundamentally advancing our understanding of glial-neuronal interactions.

PROJECT SUMMARY Common to virtually all neurodegenerative diseases and brain disorders are changes in a glial cell type called an astrocyte, which become ?reactive?. Astrocytes ordinarily provide critical support for neurons and only turn into reactive astrocytes (RAs) following injury or development of disease. A longstanding issue which has remained unknown is whether RAs contribute to, or help alleviate, disease progres- sion. Our long-term goal is to increase understanding of the molecular and cellular underpinnings of diseases and disorders of the nervous system through increased understanding of the role of reactive astrocytes. The objective of this proposal is to deliver a new strategy to selectively alter (eliminate, in- crease, or decrease) the function of only RAs at any point in the progression of nervous system disor- ders. One such disorder for which new treatments are needed is epilepsy. Despite being the third most common neurological disorder in the U.S. after Alzheimer?s disease and stroke, epilepsy is among the least understood of the major chronic medical conditions. Two aims are proposed, with the goal of characterizing this new strategy and providing proof-of-principle for using the approach for the selective manipulation of RAs in disease: In Aim 1, we will first characterize a novel reactive astrocyte inducible Cre transgenic strategy to selectively manipulate RAs in brain disease. In Aim 2, we will then use the new approach to either selectively eliminate RAs or reprogram them back into non-reactive astrocytes at various stages during the development of epilepsy. Our work will provide new knowledge on the pro- tective vs. detrimental roles of reactive astrocytes in the development of epilepsy. The rationale for the proposed research is that new insight into the role of reactive cell types in disease is an important goal for the development of more efficacious treatments. We anticipate that this research will be transforma- tive, as we will introduce to the research community a powerful new strategy to investigate the role of reactive astrocytes in any disease or disorder of the nervous system.

PROJECT SUMMARY The proposed studies will dissect the role of Reactive Astrocytes (RAs) in Alzheimer?s Disease (AD). Common to virtually all neurodegenerative diseases and brain disorders are changes in glial cells called astrocytes, which become ?reactive?. Astrocytes ordinarily provide critical support for neurons and only turn into RAs following injury or development of disease. A longstanding issue which has re- mained unknown is whether RAs contribute to, or help alleviate, Alzheimer?s Disease progression. The long-term goal of our research is to increase understanding of the molecular and cellular underpinnings of AD and Alzheimer?s Disease Related Dementias through increased understanding of the role of re- active astrocytes. The objective of this proposal is to deliver a new strategy to selectively alter (elimi- nate, increase, or decrease) gene expression only in RAs at any point during the progression of AD. Despite being one of the most common neurological diseases, the mechanisms underlying synaptic loss and cognitive decline in AD are poorly understood. It is critical to gain new insights into AD mecha- nisms if new treatment targets are to be developed. Two aims are proposed, with the goal of character- izing our new strategy and providing proof-of-principle for using the approach to selectively target and manipulate RAs in AD: In Aim 1, a novel reactive astrocyte inducible Cre transgenic strategy to selec- tively manipulate RAs in AD will be characterized for specificity of expression in reactive cell types in the diseased brain. In Aim 2, the new strategy will be used to either selectively eliminate RAs or repro- gram them back into non-reactive astrocytes at various stages during the development of AD. The pro- posed work will provide new knowledge on the protective vs. detrimental roles of reactive astrocytes in the development and progression of AD. The rationale for the proposed research is that new knowledge on the role of reactive cell types in AD is an important goal for the development of more effi- cacious treatments. It is anticipated that this research will be transformative, as a powerful new strategy will be introduced to the research community to investigate the role of reactive astrocytes in Alzheimer?s Disease, and potentially any disease or disorder of the nervous system.

PROJECT SUMMARY While numerous transgenic tools and approaches exist to enable manipulation of gene expression in many cell types in the healthy brain, tools designed to target and study cells present only in the dis- eased or damaged brain are lacking. Common to virtually all neurodegenerative diseases, brain injuries and infections is a neuroinflammatory and immune response characterized by changes in astrocytes, which become ?reactive?. Astrocytes ordinarily provide critical support for neurons and only turn into reactive astrocytes (RAs) in brain disease and inflammation. A longstanding issue which has remained unknown is whether RAs contribute to or help alleviate disease progression. The objective of this appli- cation is to deliver a new combinatorial transgenic strategy and toolkit to specifically target RAs in dis- ease. This toolkit will enable researchers to selectively alter (eliminate, increase, or decrease) gene ex- pression only in RAs at any point in the progression of brain disease and inflammation. Brain infection by the parasite Toxoplasma gondii will serve as a model of brain inflammation stemming from infection. Three aims are proposed: In Aim 1, we will first characterize the Cre transgenic strategy to selectively manipulate gene expression only in RAs in brain disease. In Aim 2, we will then use the new approach to selectively ablate, prevent, or reprogram RAs back into non-reactive astrocytes at various stages during the acute and chronic inflammatory process. Our work will provide new information on the role of reactive astrocytes in the early vs. sustained stages of brain inflammation. In Aim 3, we will perform ?translatome? analysis to identify genes uniquely altered in reactive astrocytes during brain inflamma- tion for the first time, providing novel targets for future study. Our innovative approach will allow detec- tion of both inductions and reductions in gene expression with unprecedented signal-to-noise over ex- isting approaches. The rationale for the proposed research is that improving understanding of the cellu- lar and molecular mechanisms of brain disease will provide novel insights into the development of more effective treatments. We anticipate that this research will be transformative, as we will introduce to the research community a powerful new strategy to investigate the role of reactive cell types in any disease or disorder of the nervous system.