Michelle L. Olsen, Ph.D. - US grants

DESCRIPTION (provided by applicant): Traumatic brain injury (TBI) affects over 1.7 million Americans each year and is the leading cause of death and disability in young children in the United States. For children with TBI, current treatment options are largely extrapolated from studies on adults, despite significant differences between pediatric and adult brains. Therefore, this proposal aims to specifically study pediatric injury, using clinically-relevant models of childhood disease. Specifically, we will focus on the role of astrocytes in pediatric TBI, a highly understudied area of research. It is well established that astrocytes in the brain and spinal cord play a major role in both acute and long term response to injury. Astrocytes associated with injured tissue, termed reactive astrocytes, are characterized by profound changes in protein expression leading to changes in the fundamental properties of these cells. Yet, little is known about the genetic regulation of the astrocytic injury response. This proposal seeks to address this question by examining the regulation of two essential functions of astrocytes following injury: the ability to buffer extracellular K+ ions and to regulate extracellular glutamate concentrations. These two astrocytic functions are largely mediated via the inwardly-rectifying potassium channel, Kir4.1, and the astrocytic glutamate transporter, GLT-1. In the adult spinal cord and brain, dysregulated K+ and glutamate homeostasis in the extracellular space leads to neuronal hyperexcitability, changes in synaptic physiology, and plasticity. Furthermore, both proteins are developmentally regulated with the most significant increases in expression in humans and rodents during early postnatal development at the peak of glutamatergic synaptogenesis, establishing an important role for these two proteins in the immature brain. This developmental period also correlates with the age group highest at risk for TBI. Despite the importance of these two proteins in brain function, very little is known regarding their regulation during development or in response to injury. Using a highly clinical relevant model of TBI, this proposal aims to specifically ask the following questions: 1) Following pediatric injury, is there persistent decrease in Kir4.1 and GLT-1, leading to neuronal hyperexcitability? 2) Is loss of these proteins a direct result of epigenetic modulation of gene transcription? 3) Can manipulation of DNA methylation using FDA-approved drugs reverse the loss of Kir4.1 and GLT-1 in pediatric injury models? This proposal seeks to enhance our understanding of the role of astrocytes, the most abundant cells in the CNS, in the pathophysiology of pediatric traumatic brain injury and abnormal brain development following injury. Results from these experiments could lead to novel therapeutic strategies using FDA-approved drugs for the treatment of TBI in pediatric patients.

? DESCRIPTION (provided by applicant): Rett syndrome (RTT) is a neurodevelopmental disorder caused by loss-of-function mutations in the gene encoding methyl-CpG-binding protein 2 (MECP2). Symptoms of RTT include mental disability, autistic behavior, and seizures. In addition, severe respiratory dysfunction contributes significantly to poor quality of life and is associated with high mortality rate in this population. Evidence from RTT mouse models suggest that disordered breathing in RTT may result from disruption of central chemoreceptors (neurons that regulate breathing in response to CO2/H+), yet the cellular and molecular basis of MeCP2-dependent control of breathing remains largely unknown. MeCP2 is highly expressed throughout the nervous system and recent evidence shows that loss of MeCP2 from astrocytes contributes to symptoms of RTT including disordered breathing. Astrocytes in a brainstem region called the retrotrapezoid nucleus (RTN) are known to control breathing by sensing CO2/H+ by inhibition of inward rectifying K+ channels (Kir4.1) and releasing ATP to stimulate nearby chemosensitive neurons. Preliminary data presented here demonstrates Kir4.1 expression is significantly decreased in multiple brain regions in MeCP2 deficient mice, suggesting expression of this channel is regulated by MeCP2. Therefore, we hypothesize that MeCP2 is required for expression of Kir4.1 in RTN astrocytes and loss of MeCP2 from astrocytes disrupts RTN chemoreceptor function and contributes to disordered breathing in RTT. In this proposal, we use an established mouse model of RTT and the newly developed inducible astrocyte specific Kir4.1 knockouts in conjunction with molecular, genetics, slice electrophysiology, and whole-animal plethysmography to determine if MeCP2 and Kir4.1 in astrocytes are essential for control of breathing. The three Specific Aims of this project are: 1) determine whether MeCP2 is required for expression of Kir4.1 in RTN astrocytes; 2) determine if loss of MeCP2 affects excitability chemosensitive RTN neurons; 3) determine the essential roles of Kir4.1 in RTN astrocytes for control of breathing. By understanding contributions of MeCP2 and Kir4.1 in astrocytes to RTN physiology in vitro and in vivo, we will provide insight into the cellular and molecular basis of disordered breathing in RTT and in doing so create new avenues for treatment of life-threatening symptoms of this disease.

Astrocytes contribute to many facets of ?normal? central nervous system (CNS) physiology, including regulation of neurotransmitters and K+ ions concentration, synaptic development, and synapse stabilization. These functions are largely mediated at distal, fine, peripheral astrocyte processes (PAPs). It is at these processes that astrocytes communicate with their neighbors, regulate ion and neurotransmitter levels and contribute to synapse development and stabilization. Despite decades of research indicating astrocytes enwrap or contact excitatory and inhibitory synaptic elements, with increased coverage of mature synapses, there is little known regarding signals that recruit astrocyte PAPs to synaptic structures. RNA sequencing data we have generated (and confirmed using multiple public resources) indicate astrocytes express very high levels of the BDNF receptor, TrkB. Isoform specific identification demonstrates astrocytes predominately express the truncated form, TrkB.T1. In cortex, TrkB.T1 is found almost exclusively in astrocytes. Global and astrocyte specific genetic deletion of TrkB.T1 results in astrocytes with significantly reduced volume and branching complexities. Astrocytes lacking TrkB.T1 show dysregulated expression of both perisynaptic genes associated with mature astrocyte function and pro-synaptogenic genes. In vitro and in vivo we observed that TrkB.T1 KO astrocytes do not support normal excitatory synaptogenesis or function as assessed by evaluation of pre and post synaptic excitatory elements and neuronal mEPSC analysis. Preliminary in vitro data also indicate that TrkB.T1 KO astrocytes fail to enwrap glutamatergic synapses, a phenotype we readily observe in WT astrocytes. In the current proposal we test the hypothesis that BDNF signaling through the astrocytic TrkB.T1 receptor serves as a key signaling pathway in recruiting astrocyte perisynaptic processes to glutamatergic synapses thus facilitating actin mediated structural plasticity. In the current work we use ultrastructural imaging in WT and astrocyte specific TrkB.T1 KO mice to determine if BDNF/TrkB.T1 signaling in astrocytes is necessary for astrocyte structural plasticity and function at glutamatergic synapses (Aim 1). We evaluate the loss of astrocyte TrkB.T1 on neuronal synapse development and function (Aim 2) and we use a combination of in vitro and in vivo approaches to identify the key signaling mechanisms by which BDNF binding to astrocyte TrkB.T1 receptors engage downstream signaling mechanisms, providing a molecular mechanistic framework linking astrocyte BDNF/TrkB.T1 signaling to actin cytoskeletal reorganization, morphological refinement, process outgrowth and synapse enwrapment. These studies identify a completely novel signaling pathway in astrocyte structural plasticity and have the potential to significantly advance our understanding of astrocyte-synapse interactions. While disrupted BDNF/TrkB signaling is implicated in many CNS disorders the relevance of BDNF/astrocytic TrkB.T1 signaling has not been considered.