Stefanie Robel

The stem cell potential of glia: lessons from reactive gliosis.

Astrocyte-like cells, which act as stem cells in the adult brain, reside in a few restricted stem cell niches. However, following brain injury, glia outside these niches acquire or reactivate stem cell potential as part of reactive gliosis. Recent studies have begun to uncover the molecular pathways involved in this process. A comparison of molecular pathways activated after injury with those involved in the normal neural stem cell niches highlights strategies that could overcome the inhibition of neurogenesis outside the stem cell niche and instruct parenchymal glia towards a neurogenic fate. This new view on reactive glia therefore suggests a widespread endogenous source of cells with stem cell potential, which might potentially be harnessed for local repair strategies.

Glutamate release by primary brain tumors induces epileptic activity

Epileptic seizures are a common and poorly understood comorbidity for individuals with primary brain tumors. To investigate peritumoral seizure etiology, we implanted human-derived glioma cells into severe combined immunodeficient mice. Within 14–18 d, glioma-bearing mice developed spontaneous and recurring abnormal electroencephalogram events consistent with progressive epileptic activity. Acute brain slices from these mice showed marked glutamate release from the tumor mediated by the system xc− cystine-glutamate transporter (encoded by Slc7a11). Biophysical and optical recordings showed glutamatergic epileptiform hyperexcitability that spread into adjacent brain tissue. We inhibited glutamate release from the tumor and the ensuing hyperexcitability by sulfasalazine (SAS), a US Food and Drug Administration–approved drug that blocks system xc−. We found that acute administration of SAS at concentrations equivalent to those used to treat Crohns disease in humans reduced epileptic event frequency in tumor-bearing mice compared with untreated controls. SAS should be considered as an adjuvant treatment to ameliorate peritumoral seizures associated with glioma in humans.

A neurocentric perspective on glioma invasion.

Malignant gliomas are devastating tumours that frequently kill patients within 1 year of diagnosis. The major obstacle to a cure is diffuse invasion, which enables tumours to escape complete surgical resection and chemo- and radiation therapy. Gliomas use the same tortuous extracellular routes of migration that are travelled by immature neurons and stem cells, frequently using blood vessels as guides. They repurpose ion channels to dynamically adjust their cell volume to accommodate to narrow spaces and breach the blood–brain barrier through disruption of astrocytic endfeet, which envelop blood vessels. The unique biology of glioma invasion provides hitherto unexplored brain-specific therapeutic targets for this devastating disease.

Disruption of astrocyte–vascular coupling and the blood–brain barrier by invading glioma cells

Astrocytic endfeet cover the entire cerebral vasculature and serve as exchange sites for ions, metabolites, and energy substrates from the blood to the brain. They maintain endothelial tight junctions that form the blood-brain barrier (BBB) and release vasoactive molecules that regulate vascular tone. Malignant gliomas are highly invasive tumors that use the perivascular space for invasion and co-opt existing vessels as satellite tumors form. Here we use a clinically relevant mouse model of glioma and find that glioma cells, as they populate the perivascular space of pre-existing vessels, displace astrocytic endfeet from endothelial or vascular smooth muscle cells. This causes a focal breach in the BBB. Furthermore, astrocyte-mediated gliovascular coupling is lost, and glioma cells seize control over regulation of vascular tone through Ca2+-dependent release of K+. These findings have important clinical implications regarding blood flow in the tumor-associated brain and the ability to locally deliver chemotherapeutic drugs in disease.

Reactive Astrogliosis Causes the Development of Spontaneous Seizures

Epilepsy is one of the most common chronic neurologic diseases, yet approximately one-third of affected patients do not respond to anticonvulsive drugs that target neurons or neuronal circuits. Reactive astrocytes are commonly found in putative epileptic foci and have been hypothesized to be disease contributors because they lose essential homeostatic capabilities. However, since brain pathology induces astrocytes to become reactive, it is difficult to distinguish whether astrogliosis is a cause or a consequence of epileptogenesis. We now present a mouse model of genetically induced, widespread chronic astrogliosis after conditional deletion of β1-integrin (Itgβ1). In these mice, astrogliosis occurs in the absence of other pathologies and without BBB breach or significant inflammation. Electroencephalography with simultaneous video recording revealed that these mice develop spontaneous seizures during the first six postnatal weeks of life and brain slices show neuronal hyperexcitability. This was not observed in mice with neuronal-targeted β1-integrin deletion, supporting the hypothesis that astrogliosis is sufficient to induce epileptic seizures. Whole-cell patch-clamp recordings from astrocytes further suggest that the heightened excitability was associated with impaired astrocytic glutamate uptake. Moreover, the relative expression of the cation-chloride cotransporters (CCC) NKCC1 (Slc12a2) and KCC2 (Slc12a5), which are responsible for establishing the neuronal Cl− gradient that governs GABAergic inhibition were altered and the NKCC1 inhibitor bumetanide eliminated seizures in a subgroup of mice. These data suggest that a shift in the relative expression of neuronal NKCC1 and KCC2, similar to that observed in immature neurons during development, may contribute to astrogliosis-associated seizures.

Glia as drivers of abnormal neuronal activity

Reactive astrocytes have been proposed to become incompetent bystanders in epilepsy as a result of cellular changes rendering them unable to perform important housekeeping functions. Indeed, successful surgical treatment of mesiotemporal lobe epilepsy hinges on the removal of the glial scar. New research now extends the role of astrocytes, suggesting that they may drive the disease process by impairing the inhibitory action of neuronal GABA receptors. Here we discuss studies that include hyperexcitability resulting from impaired supply of astrocytic glutamine for neuronal GABA synthesis, and epilepsy resulting from genetically induced astrogliosis or malignant transformation, both of which render the inhibitory neurotransmitter GABA excitatory. In these examples, glial cells alter the expression or function of neuronal proteins involved in excitability. Although epilepsy has traditionally been thought of as a disease caused by changes in neuronal properties exclusively, these new findings challenge us to consider the contribution of glial cells as drivers of epileptogenesis in acquired epilepsies.

Conditional deletion of beta1-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.

SLC7A11 expression is associated with seizures and predicts poor survival in patients with malignant glioma

SLC7A11, the catalytic subunit of the cystine/glutamate antiporter, System xc− (SXC), is up-regulated in a subpopulation of patient gliomas, where it is responsible for excitotoxic glutamate release, accelerated tumor growth, and tumor-associated seizures. Seizing an opportunity to study glioma Gliomas are the most common type of malignant brain tumors, and they frequently cause seizures. A new study by Robert et al. uncovers some of the mechanisms involved in this process, showing how a specific cystine/glutamate transporter contributes to excitotoxic glutamate release, causing the death of surrounding cells and inducing seizures. The authors also showed that tumors expressing this transporter were more aggressive and grew more quickly, possibly because the destruction of surrounding normal cells allowed the tumors to expand more rapidly. These findings suggest that the expression of this cystine/glutamate transporter may be useful as a predictor of outcome and a potential therapeutic target in glioma. Glioma is the most common malignant primary brain tumor. Its rapid growth is aided by tumor-mediated glutamate release, creating peritumoral excitotoxic cell death and vacating space for tumor expansion. Glioma glutamate release may also be responsible for seizures, which complicate the clinical course for many patients and are often the presenting symptom. A hypothesized glutamate release pathway is the cystine/glutamate transporter System xc− (SXC), responsible for the cellular synthesis of glutathione (GSH). However, the relationship of SXC-mediated glutamate release, seizures, and tumor growth remains unclear. Probing expression of SLC7A11/xCT, the catalytic subunit of SXC, in patient and mouse-propagated tissues, we found that ~50% of patient tumors have elevated SLC7A11 expression. Compared with tumors lacking this transporter, in vivo propagated and intracranially implanted SLC7A11-expressing tumors grew faster, produced pronounced peritumoral glutamate excitotoxicity, induced seizures, and shortened overall survival. In agreement with animal data, increased SLC7A11 expression predicted shorter patient survival according to genomic data in the REMBRANDT (National Institutes of Health Repository for Molecular Brain Neoplasia Data) database. In a clinical pilot study, we used magnetic resonance spectroscopy to determine SXC-mediated glutamate release by measuring acute changes in glutamate after administration of the U.S. Food and Drug Administration–approved SXC inhibitor, sulfasalazine (SAS). In nine glioma patients with biopsy-confirmed SXC expression, we found that expression positively correlates with glutamate release, which is acutely inhibited with oral SAS. These data suggest that SXC is the major pathway for glutamate release from gliomas and that SLC7A11 expression predicts accelerated growth and tumor-associated seizures.

Genetic Deletion of Cdc42 Reveals a Crucial Role for Astrocyte Recruitment to the Injury Site In Vitro and In Vivo

It is generally suggested that astrocytes play important restorative functions after brain injury, yet little is known regarding their recruitment to sites of injury, despite numerous in vitro experiments investigating astrocyte polarity. Here, we genetically manipulated one of the proposed key signals, the small RhoGTPase Cdc42, selectively in mouse astrocytes in vitro and in vivo. We used an in vitro scratch assay as a minimal wounding model and found that astrocytes lacking Cdc42 (Cdc42Δ) were still able to form protrusions, although in a nonoriented way. Consequently, they failed to migrate in a directed manner toward the scratch. When animals were injured in vivo through a stab wound, Cdc42Δ astrocytes developed protrusions properly oriented toward the lesion, but the number of astrocytes recruited to the lesion site was significantly reduced. Surprisingly, however, lesions in Cdc42Δ animals, harboring fewer astrocytes contained significantly higher numbers of microglial cells than controls. These data suggest that impaired recruitment of astrocytes to sites of injury has a profound and unexpected effect on microglia recruitment.

GABAergic disinhibition and impaired KCC2 cotransporter activity underlie tumor-associated epilepsy

Seizures frequently accompany gliomas and often escalate to peritumoral epilepsy. Previous work revealed the importance of tumor‐derived excitatory glutamate (Glu) release mediated by the cystine‐glutamate transporter (SXC) in epileptogenesis. We now show a novel contribution of GABAergic disinhibition to disease pathophysiology. In a validated mouse glioma model, we found that peritumoral parvalbumin‐positive GABAergic inhibitory interneurons are significantly reduced, corresponding with deficits in spontaneous and evoked inhibitory neurotransmission. Most remaining peritumoral neurons exhibit elevated intracellular Cl− concentration ([Cl−]i) and consequently depolarizing, excitatory gamma‐aminobutyric acid (GABA) responses. In these neurons, the plasmalemmal expression of KCC2, which establishes the low [Cl−]i required for GABAAR‐mediated inhibition, is significantly decreased. Interestingly, reductions in inhibition are independent of Glu release, but the presence of both decreased inhibition and decreased SXC expression is required for epileptogenesis. We suggest GABAergic disinhibition renders peritumoral neuronal networks hyper‐excitable and susceptible to seizures triggered by excitatory stimuli, and propose KCC2 as a therapeutic target. GLIA 2015;63:23–36