Stefanie Robel - US grants

Project Summary After years of assuming that neurological diseases are caused by direct damage to neurons, we now know that impaired astrocyte physiology and function precedes and is essential for the progression of many of these diseases. This revelation hints toward the reason why anti-epileptic drugs that exclusively target neurons do not prevent the development of epilepsy after traumatic brain injury (TBI), the largest group of acquired epilepsies. For more than a decade, data have accumulated showing that astrocytes become reactive and lose their homeostatic functions indispensable for normal neuronal operation in epilepsy patients and animal models. Yet, a direct causal link between astrocyte dysfunction and post-traumatic epilepsy (PTE) has not been established beyond the fact TBI triggers astrogliosis. This may be in part due to the complexity of TBI, which induces many pathobiological mechanisms in parallel. Astrogliosis has mostly been studied in focal TBI, where layers of different types of reactive astrocytes surround a site of primary brain damage. Yet, this injury type presents in isolation in less than 10% of TBI patients and induces additional mechanisms that could trigger seizures, limiting our ability to determine if a causal relationship between astrocyte dysfunction and the development of PTE exists. Current PTE models are induced by focal TBI, but the vast majority of human TBIs include diffuse or concussive injury induced by rapid acceleration/deceleration of the brain tissue. Even patients who incur a single mild diffuse TBI are at increased risk for the development of PTE. Therefore, a new PTE mouse model that recapitulated diffuse TBI without focal injury was developed. This new PTE model induced spontaneous seizures at higher incidence than previous PTE models but with only a subset of cellular and tissue level changes, markedly reducing complexity of the underlying pathobiology. Data obtained in this model point to a surprisingly different response of astrocytes to diffuse TBI, suggesting that early loss of astrocytes may contribute to the development PTE. Yet, the upstream molecular mechanism inducing astrocyte loss and the downstream physiological consequences on neurons and neighboring astrocytes must be identified to ultimately find targets for interrupting the progression of TBI to PTE. This proposal aims to determine the primary cause for astrocyte loss using modified Folch extraction and fractionation techniques to narrow down the list of candidates. It further tests the hypothesis that astrocyte loss causes neurons and close-by astrocytes to become dysfunctional, initiating the formation of a seizure focus. This hypothesis will be tested using a combination of imaging, electrophysiology and EEG recordings in PTE mice or after specific ablation of cellular players. Given that the incidence of TBI has increased over the last decade, PTE as a lifelong complication of TBI is not only debilitating for those afflicted, but represents an ever-rising social and economic burden in the US. This proposal will examine astrocyte loss as a root cause initiating epileptogenesis after TBI, and will provide a basis for developing interventions that prevent the progression of TBI toward PTE.

PROJECT SUMMARY After years of assuming that neurological diseases are caused by direct damage to neurons, we now know that impaired astrocyte physiology and function precedes and is essential for the progression of many of these diseases. This revelation hints toward the reason why anti-epileptic drugs that exclusively target neurons do not prevent the development of epilepsy after traumatic brain injury (TBI), the largest group of acquired epilepsies. For more than a decade, data have accumulated showing that astrocytes become reactive and lose their homeostatic functions indispensable for normal neuronal operation in epilepsy patients and animal models. Identifying and targeting astrocytic molecular mechanisms contributing to this dysfunction might provide the long-missing puzzle piece to interrupt the slow progression from TBI to post-traumatic epilepsy (PTE). Yet, astrocytic molecular mechanisms driving PTE have not been identified. This may be due to the complexity of TBI, which induces many pathobiological mechanisms in parallel, as well as the lack of unbiased approaches to identifying new targets. To address these problems, a new mouse model of post-traumatic epilepsy that recapitulates diffuse/concussive TBI without focal injury was developed reducing the number of pathobiological mechanisms that have to be taken into consideration. This model was used to identify specific molecular targets that might contribute to astrocyte dysfunction after diffuse TBI using unbiased RNA sequencing of the astrocytic transcriptome. This approach pointed to the downregulation of cell communication pathways, which was corroborated by the reduced spread of stimulated astrocytic calcium waves in vivo after diffuse TBI. Astrocytes are coupled by and communicate via gap junctions of which Cx43 is a major component. Importantly, altered astrocyte gap junction coupling has been reported in patients who suffer from temporal lobe epilepsy and in animal models of this disease. In addition to forming gap junctions, Cx43 is present in undocked hemichannels. These abnormally open under pathological conditions, releasing neurotoxic molecules such as ATP and glutamate. Our preliminary results confirm abnormal expression pattern of Cx43, a loss of gap junction plaque formation and relocalization in the cytoplasm following TBI. Cx43 localization and activity is regulated by several sites within the Cx43 carboxy-terminus (CT) that mediate protein?protein interaction and allow for post-translational modifications. This proposal uses two proprietary Cx43 mimetic peptides, aCT1 and JM2, which modulate Cx43 localization and function by increasing Cx43 gap junction plaque formation and blocking Cx43 hemichannel activity, respectively. The overall objective of this proposal is to determine in which way abnormal Cx43 following diffuse TBI affects astrocyte function and to develop new therapeutic strategies to prevent PTE using Cx43 mimetic peptides.