Diverse Functions of Astroglial Cells

Astrocytes perform many functions in the adult brain and even act as neural stem cells after brain injury (Buffo et al., 2008) or in regions where neurogenesis persists, e.g. in the subependymal zone of the lateral ventricle. The stem cell astrocytes possess an apicobasal polarity as they are coupled by adherens junctions to neighbouring ependymal cells and possess an apical membrane domain with CD133 and Par complex proteins and a basolateral membrane domain including contact of processes to the basement membrane (BM). This is notably different from parenchymal astrocytes that only have contacts to the BM under physiological conditions. The major underlying question is how differences between neural stem cells and 'normal' astrocytes are generated and how polarity mechanisms may be involved in generating this difference. Here, I set out to determine the role of BM contact and the Par complex for astrocyte function in the normal brain parenchyma as well as in the neurogenic niche. First, I examined the influence of BM-mediated signaling by conditional deletion of β1-integrin, one of the major BM receptors in the CNS. The use of specific Cre lines resulted in a loss of β1-integrin protein only at postnatal stages either in both glia and neurons or specifically in neurons. Strikingly, only the former resulted in reactive gliosis, with the hallmarks of reactive astrocytes comprising astrocyte hypertrophy and upregulation of the intermediate filaments GFAP and Vimentin as well as pericellular components, such Tenascin-C and the 473HD proteoglycan. This reaction to the loss of β1-integrin was further accompanied by non-cell autonomous activation of microglial cells. However, neither reactive astrocytes nor microglia divided, suggesting that the loss of β1-integrin-mediated signaling is not sufficient to elicit proliferation of these cells. Interestingly, this partial reactive gliosis appeared in the absence of cell death and blood-brain barrier disturbances. As these effects did not appear after neuron-specific deletion of β1-integrin, we conclude that β1-integrin-mediated signaling in astrocytes is required to promote their acquisition of a mature, non-reactive state. Interestingly, neural stem cell astrocytes in the SEZ were not affected in their proliferation and fate, suggesting that β1-integrins are not involved in the regulation of these stem cell properties. However, loss of β1-integrins interfered with the normal dedifferentiation of astrocytes into stem cells after brain injury. Next, I examined the role of Cdc42, a key activator of the Par complex, but also a mediator of β1-integrin signalling in adult stem cell astrocytes. Therefore, I genetically deleted this small RhoGTPase in astroglia at adult stages. In contrast to what has been observed during development, loss of Cdc42 had no influence on proliferation or fate of subependymal zone astrocytes. These effects on adult astroglia-like stem cells differ profoundly from effects on parenchymal astrocytes upon injury. Here, deletion of Cdc42 resulted in severe defects of astrocyte polarity as measured by centrosome reorientation and oriented process extension in the scratch assay in vitro. In vivo, astrocytes could still orient towards the injury site suggesting the existence of compensating signaling pathways. However, the increase of astrocyte numbers around the injury site was reduced. Impaired proliferation certainly contributes to this phenotype. Most importantly, loss of Cdc42 resulted in a significantly increased size of brain injury enlightening the importance of this pathway in the wound reaction towards brain injury. Conversely, no effects were seen by Cdc42 deletion in astrocytes in the absence of injury, suggesting that integrin-mediated signaling from the BM maintains the hallmarks of mature non-reactive astrocytes while Cdc42, most likely via activation of the Par complex, regulates polarity and dedifferentiation after injury. Taken together, this work elucidated for the first time specific signaling pathways regulating the role of astrocytes as stem cells during wound reaction of the injured brain

Conditional deletion of β1‐integrin in astroglia causes partial reactive gliosis

Astrocytes play many pivotal roles in the adult brain, including their reaction to injury. A hallmark of astrocytes is the contact of their endfeet with the basement membrane surrounding blood vessels, but still relatively little is known about the signaling mediated at the contact site. Here, we examine the role of β1‐integrin at this interface by its conditional deletion using different Cre lines. Thereby, the protein was reduced only at postnatal stages either in both glia and neurons or specifically only in neurons. Strikingly, only the former resulted in reactive gliosis, with the hallmarks of reactive astrocytes comprising astrocyte hypertrophy and up‐regulation of the intermediate filaments GFAP and vimentin as well as pericellular components, such as Tenascin‐C and the DSD‐1 proteoglycan. In addition, we also observed to a certain degree a non‐cell autonomous activation of microglial cells after conditional β1‐integrin deletion. However, these reactive astrocytes did not divide, suggesting that the loss of β1‐integrin‐mediated signaling is not sufficient to elicit proliferation of these cells as observed after brain injury. Interestingly, this partial reactive gliosis appeared in the absence of cell death and blood brain barrier disturbances. As these effects did not appear after neuron‐specific deletion of β1‐integrin, we conclude that β1‐integrin‐mediated signaling in astrocytes is required to promote their acquisition of a mature, nonreactive state. Alterations in β1‐integrin‐mediated signaling may hence be implicated in eliciting specific aspects of reactive gliosis after injury. © 2009 Wiley‐Liss, Inc

Applying Proteomics and Computational Approaches to Identify Novel Targets in Blast-Associated Post-Traumatic Epilepsy

Traumatic brain injury (TBI) can lead to post-traumatic epilepsy (PTE). Blast TBI (bTBI) found in Veterans presents with several complications, including cognitive and behavioral disturbances and PTE; however, the underlying mechanisms that drive the long-term sequelae are not well understood. Using an unbiased proteomics approach in a mouse model of repeated bTBI (rbTBI), this study addresses this gap in the knowledge. After rbTBI, mice were monitored using continuous, uninterrupted video-EEG for up to four months. Following this period, we collected cortex and hippocampus tissues from three groups of mice: those with post-traumatic epilepsy (PTE+), those without epilepsy (PTE−), and the control group (sham). Hundreds of differentially expressed proteins were identified in the cortex and hippocampus of PTE+ and PTE− relative to sham. Focusing on protein pathways unique to PTE+, pathways related to mitochondrial function, post-translational modifications, and transport were disrupted. Computational metabolic modeling using dysregulated protein expression predicted mitochondrial proton pump dysregulation, suggesting electron transport chain dysregulation in the epileptic tissue relative to PTE−. Finally, data mining enabled the identification of several novel and previously validated TBI and epilepsy biomarkers in our data set, many of which were found to already be targeted by drugs in various phases of clinical testing. These findings highlight novel proteins and protein pathways that may drive the chronic PTE sequelae following rbTBI