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Perineuronal nets (PNNs) are extracellular matrix assemblies of highly negatively charged proteoglycans that wrap around fast-spiking parvalbumin (PV) expressing interneurons in the cerebral cortex. PNNs play important roles in neuronal plasticity and modulate biophysical properties of the enclosed interneurons. Various central nervous system diseases including schizophrenia, Alzheimer disease and epilepsy present with qualitative alteration in PNNs, however prior studies failed to quantitatively assess such changes at single PNN level and correlate them with functional changes in disease. We describe a method to quantify the structural integrity of PNNs using high magnification image analysis of Wisteria Floribunda Agglutinin (WFA)-labeled PNNs in combination with cell-type-specific marker such as PV and NeuN. A polyline intensity profile of WFA along the entire perimeter of cell shows alternate segments with and without WFA labeling, indicating the intact chondroitin sulfate proteoglycan (CSPG) and holes of PNN respectively. This line intensity profile defines CSPG peaks, where intact PNN is present, and CSPG valleys (holes) where the PNN is missing. The average number of peaks reflect the integrity of the lattice assembly of PNN. The average size of PNN holes can be readily computed using image analysis software. Furthermore, degradation of PNNs using a bacterial-derived enzyme, Chondroitinase ABC (ChABC), allows to experimentally manipulate PNNs in situ brain slices during which biophysical properties can be assessed by patch-clamp recordings. We describe optimized experimental parameters to degrade PNNs in brain slices before as well as during recordings to study the possible change in function in real time. Our protocols provide effective and appropriate methods to modulate and quantify the PNN's experimental manipulations.

Epilepsy is a neurological disorder afflicting ~65 million people worldwide. It is caused by aberrant synchronized firing of populations of neurons primarily due to imbalance between excitatory and inhibitory neurotransmission. Hence, the historical focus of epilepsy research has been neurocentric. However, the past two decades have enjoyed an explosion of research into the role of glia in supporting and modulating neuronal activity, providing compelling evidence of glial involvement in the pathophysiology of epilepsy. The mechanisms by which glia, particularly astrocytes and microglia, may contribute to epilepsy and consequently could be harnessed therapeutically are discussed in this Review.

A bio-inspired hydrogel for 3D bioprinting of soft free-standing neural tissues is presented. The novel filler-free bioinks were designed by combining natural polymers for extracellular matrix biomimicry with synthetic polymers to endow desirable rheological properties for 3D bioprinting. Crosslinking of thiolated Pluronic F-127 with dopamine-conjugated (DC) gelatin and DC hyaluronic acid through a thiol-catechol reaction resulted in thermally gelling bioinks with Herschel-Bulkley fluid rheological behavior. Microextrusion 3D bioprinting was used to fabricate free-standing cell-laden tissue constructs. The bioinks exhibited flattened parabolic velocity profiles with tunable low shear regions. Two pathways were investigated for curing the bioink: chelation and photocuring. The storage modulus of the cured bioinks ranged from 6.7 to 11.7 kPa. The iron (III) chelation chemistry produced crosslinked neural tissues of relatively lower storage modulus than the photocuring approach. In vitro cell viability studies using the 3D bioprinted neural tissues showed that the cured bioink was biocompatible based on minimal cytotoxic response observed over seven days in culture relative to control studies using alginate hydrogels. Rodent Schwann cell-, rodent neuronal cell-, and human glioma cell-laden tissue constructs were printed and cultured over seven days and exhibited comparable viability relative to alginate bioink controls. The ability to fabricate soft, free-standing 3D neural tissues with low modulus has implications in the biofabrication of microphysiological neural systems for disease modeling as well as neural tissues and innervated tissues for regenerative medicine.

3D bioprinting is an emerging biofabrication process for the production of adherent cell-based products, including engineered tissues and foods. While process innovations are rapidly occurring in the area of process monitoring, which can improve fundamental understanding of process-structure-property relations as well as product quality by closed-loop control techniques, in-line sensing of the bioink composition remains a challenge. Here, we report that hollow multifunctional fibers enable in-line impedimetric sensing of bioink composition and exhibit selectivity for real-time classification of cell type, viability, and state of differentiation during bioprinting. Continuous monitoring of the fiber impedance magnitude and phase angle response from 102 to 106 Hz during microextrusion 3D bioprinting enabled compositional and quality analysis of alginate bioinks that contained fibroblasts, neurons, or mouse embryonic stem cells (mESCs). Fiber impedimetric responses associated with the bioinks that contained differentiated mESCs were consistent with differentiation marker expression characterized by immunocytochemistry. 3D bioprinting through hollow multifunctional fiber impedimetric sensors enabled classification of stem cells as stable or randomly differentiated populations. This work reports an advance in monitoring 3D bioprinting processes in terms of in-line sensor-based bioink compositional analysis using fiber technology and provides a non-invasive sensing platform for achieving future quality-controlled bioprinted tissues and injectable stem-cell therapies.

Unprovoked recurrent seizures are a serious comorbidity affecting most patients who suffer from glioma, a primary brain tumor composed of malignant glial cells. Cellular mechanisms contributing to the development of recurrent spontaneous seizures include the release of the excitatory neurotransmitter glutamate from glioma into extracellular space. Under physiological conditions, astrocytes express two high affinity glutamate transporters, Glt-1 and Glast, which are responsible for the removal of excess extracellular glutamate. In the context of neurological disease or brain injury, astrocytes become reactive which can negatively affect neuronal function, causing hyperexcitability and/or death. Using electrophysiology, immunohistochemistry, fluorescent in situ hybridization, and Western blot analysis in different orthotopic xenograft and allograft models of human and mouse gliomas, we find that peritumoral astrocytes exhibit astrocyte scar formation characterized by proliferation, cellular hypertrophy, process elongation, and increased GFAP and pSTAT3. Overall, peritumoral reactive astrocytes show a significant reduction in glutamate and potassium uptake, as well as decreased glutamine synthetase activity. A subset of peritumoral astrocytes displayed a depolarized resting membrane potential, further contributing to reduced potassium and glutamate homeostasis. These changes may contribute to the propagation of peritumoral neuronal hyperexcitability and excitotoxic death. Funding was provided by the National Institutes of Health (NIH) RO1 NS036692 (HS), RO1 NS082851 (HS), RO1 NS052634 (HS), RO1 NS105807 (SR). SR was also supported by the Epilepsy Foundation and the American Brain Tumor Association (ABTA). Accepted version

Reduced cerebral blood flow impairs cognitive function and ultimately causes irreparable damage to brain tissue. The gliovascular unit, composed of neural and vascular cells, assures sufficient blood supply to active brain regions. Astrocytes, vascular smooth muscle cells, and pericytes are important players within the gliovascular unit modulating vessel diameters. While the importance of the gliovascular unit and the signals involved in regulating local blood flow to match neuronal activity is now well recognized, surprisingly little is known about this interface in disease. Alzheimer's disease is associated with reduced cerebral blood flow. Here, we studied how the gliovascular unit is affected in a mouse model of Alzheimer's disease, using a combination of ex vivo and in vivo imaging approaches. We specifically labelled vascular amyloid in living mice using the dye methoxy-XO4. We elicited vessel responses ex vivo using either pharmacological stimuli or cell-specific calcium uncaging in vascular smooth muscle cells or astrocytes. Multi-photon in vivo imaging through a cranial window allowed us to complement our ex vivo data in the presence of blood flow after label-free optical activation of vascular smooth muscle cells in the intact brain. We found that vascular amyloid deposits separated astrocyte end-feet from the endothelial vessel wall. High-resolution 3D images demonstrated that vascular amyloid developed in ring-like structures around the vessel circumference, essentially forming a rigid cast. Where vascular amyloid was present, stimulation of astrocytes or vascular smooth muscle cells via ex vivo Ca(2+) uncaging or in vivo optical activation produced only poor vascular responses. Strikingly, vessel segments that were unaffected by vascular amyloid responded to the same extent as vessels from age-matched control animals. We conclude that while astrocytes can still release vasoactive substances, vascular amyloid deposits render blood vessels rigid and reduce the dynamic range of affected vessel segments. These results demonstrate a mechanism that could account in part for the reduction in cerebral blood flow in patients with Alzheimer's disease.media-1vid110.1093/brain/awv327_video_abstractawv327_video_abstract.

Brain tumor patients commonly present with epileptic seizures. We show that tumor-associated seizures are the consequence of impaired GABAergic inhibition due to an overall loss of peritumoral fast spiking interneurons (FSNs) concomitant with a significantly reduced firing rate of those that remain. The reduced firing is due to the degradation of perineuronal nets (PNNs) that surround FSNs. We show that PNNs decrease specific membrane capacitance of FSNs permitting them to fire action potentials at supra-physiological frequencies. Tumor-released proteolytic enzymes degrade PNNs, resulting in increased membrane capacitance, reduced firing, and hence decreased GABA release. These studies uncovered a hitherto unknown role of PNNs as an electrostatic insulator that reduces specific membrane capacitance, functionally akin to myelin sheaths around axons, thereby permitting FSNs to exceed physiological firing rates. Disruption of PNNs may similarly account for excitation-inhibition imbalances in other forms of epilepsy and PNN protection through proteolytic inhibition may provide therapeutic benefits., Brain tumours are associated with epilepsy. Here the authors show, using a mouse model, that the degradation of perineuronal nets around fast spiking interneurons near the tumour contribute to seizures by increasing their membrane capacitance and firing.

Malignant gliomas are glial-derived, primary brain tumors that carry poor prognosis. Existing therapeutics are largely ineffective and dramatically affect quality of life. The standard of care details a taxing combination of surgical resection, radiation of the resection cavity, and temozolomide (TMZ) chemotherapy, with treatment extending life by only an average of months (Maher et al., 2001; Stupp et al., 2005). Despite scientific and technological advancement, surgery remains the most important treatment modality. Therapeutic obstacles include xenobiotic protection conveyed by the blood-brain barrier (Zhang et al., 2015), invasiveness and therapeutic resistance of tumor cell populations (Bao et al., 2006), and distinctive attributes of secondary glioma occurrence (Ohgaki and Kleihues, 2013). While these brain malignancies can be classified by grade or grouped by molecular subclass, each tumor presents itself as its own complication. Based on all of these obstacles, new therapeutic approaches are urgently needed. These will likely emerge from numerous exciting studies of glioma biology that are ongoing and reviewed here. These show unexpected roles for ion channels, amino-acid transporters, and connexin gap junctions in supporting the invasive growth of gliomas. These studies have identified a number of proteins that may be targeted for therapy in the future.

Translational challenges associated with reductionist modeling approaches, as well as ethical concerns and economic implications of small animal testing, drive the need for developing microphysiological neural systems for modeling human neurological diseases, disorders, and injuries. Here, we provide a comprehensive review of microphysiological brain and neural systems-on-a-chip (NSCs) for modeling higher order trajectories in the human nervous system. Societal, economic, and national security impacts of neurological diseases, disorders, and injuries are highlighted to identify critical NSC application spaces. Hierarchical design and manufacturing of NSCs are discussed with distinction for surface- and bulk-based systems. Three broad NSC classes are identified and reviewed: microfluidic NSCs, compartmentalized NSCs, and hydrogel NSCs. Emerging areas and future directions are highlighted, including the application of 3D printing to design and manufacturing of next-generation NSCs, the use of stem cells for constructing patient-specific NSCs, and the application of human NSCs to 'personalized neurology'. Technical hurdles and remaining challenges are discussed. This review identifies the state-of-the-art design methodologies, manufacturing approaches, and performance capabilities of NSCs. This work suggests NSCs appear poised to revolutionize the modeling of human neurological diseases, disorders, and injuries.

Huntington disease is a neurodegenerative disease caused by a single mutated gene on chromosome 4 that encodes for the huntingtin protein. Huntingtin is believed to function as a regulator of nervous system development and protein trafficking. The normal huntingtin gene contains a trinucleotide (CAG) sequence that encodes for glutamine and therefore adds a variable number of glutamines to the huntingtin protein. In the mutated gene this stretch is expanded. Once more than 39 CAG sequences are present, the resulting mutated protein impairs neuronal health and has a tendency to fold incorrectly and accumulate as inclusion bodies that also contain other proteins that regulate transcription, protein synthesis, folding, and degradation. The disease primarily affects cortical and striatal neurons involved in the control of voluntary movement, motor planning, and cognition. As these neurons die, patients develop abnormal, involuntary dance-like body movements called chorea. In addition, diffuse neuronal loss throughout the cortex and frontal lobe causes emotional and cognitive dysfunction. The disease is inherited in an autosomal dominant fashion. If 36–39 repeats are present, disease may or may not develop. If 40 or more repeats are present, all affected individuals develop disease. In those instances the likelihood of a child also developing the disease is at least 50% if one of the parents carries the mutation. The disease is universally fatal, causing death within 15–20 years of diagnosis, and no disease-modifying treatments currently exist. However, genetic testing allows families to determine the risk of having offspring affected by disease. Interestingly, within families with an affected parent or relative, the choice to be tested is often not pursued since no treatment exists and because of fear that this will upset the family dynamics.

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 GABAA R-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.

Redox homeostasis is especially important in the brain where high oxygen consumption produces an abundance of harmful oxidative by-products. Glutathione (GSH) is a tripeptide non-protein thiol. It is the central nervous system’s most abundant antioxidant, and the master controller of brain redox homeostasis. The glutamate transporters, System xc− (SXC) and the Excitatory Amino Acid Transporters (EAAT), play important, synergistic roles in the synthesis of GSH. In glial cells, SXC mediates the uptake of cystine, which after intracellular reduction to cysteine, reacts with glutamate during the rate-limiting step of GSH synthesis. EAAT3 mediates direct cysteine uptake for neuronal GSH synthesis. SXC and EAAT work in concert in glial cells to provide two intracellular substrates for GSH synthesis, cystine and glutamate. Their cyclical basal function also prevents a buildup of extracellular glutamate, which SXC releases extracellularly in exchange for cystine uptake. Maintaining extracellular glutamate homeostasis is critical to prevent neuronal toxicity, as well as glutamate-mediated SXC inhibition, of which each could lead to a depletion of intracellular GSH and loss of cellular redox control. Many neurological diseases show evidence of GSH dysfunction, and increased GSH has been widely associated with chemotherapy and radiotherapy resistance of gliomas. We present evidence suggesting that gliomas expressing elevated levels of SXC are more reliant on GSH for growth and survival. They have an increased inherent radiation resistance, yet inhibition of SXC can increase tumor sensitivity at low radiation doses. GSH depletion through SXC inhibition may be a viable mechanism to enhance current glioma treatment strategies and make tumors more sensitive to radiation and chemotherapy protocols.

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.

Glioblastoma multiforme are highly motile primary brain tumors. Diffuse tissue invasion hampers surgical resection leading to poor patient prognosis. Recent studies suggest that intracellular Ca(2+) acts as a master regulator for cell motility and engages a number of downstream signals including Ca(2+) -activated ion channels. Querying the REepository of Molecular BRAin Neoplasia DaTa (REMBRANDT), an annotated patient gene database maintained by the National Cancer Institute, we identified the intermediate conductance Ca(2+) -activated K(+) channels, KCa3.1, being overexpressed in 32% of glioma patients where protein expression significantly correlated with poor patient survival. To mechanistically link KCa3.1 expression to glioma invasion, we selected patient gliomas that, when propagated as xenolines in vivo, present with either high or low KCa3.1 expression. In addition, we generated U251 glioma cells that stably express an inducible knockdown shRNA to experimentally eliminate KCa3.1 expression. Subjecting these cells to a combination of in vitro and in situ invasion assays, we demonstrate that KCa3.1 expression significantly enhances glioma invasion and that either specific pharmacological inhibition with TRAM-34 or elimination of the channel impairs invasion. Importantly, after intracranial implantation into SCID mice, ablation of KCa3.1 with inducible shRNA resulted in a significant reduction in tumor invasion into surrounding brain in vivo. These results show that KCa3.1 confers an invasive phenotype that significantly worsens a patient's outlook, and suggests that KCa3.1 represents a viable therapeutic target to reduce glioma invasion.

Here, a 3D printed multiplexed competitive migration assay is reported for characterizing a chemotactic response in the presence of multiple spatially distributed chemoattractants. The utility of the assay is demonstrated by examining the chemotactic response of human glioblastoma cells to spatially opposing chemotactic gradients of epidermal growth factor (EGF) and bradykinin (BK). Competitive migration assays involving spatially opposing gradients of EGF and BK that are optimized in the absence of the second chemoattractant show that 46% more glioblastoma cells migrate toward EGF sources. The migration velocities of human glioblastoma cells toward EGF and BK sources are reduced by 7.6 ± 2.2% and 11.6 ± 6.3% relative to those found in the absence of the spatially opposing chemoattractant. This work provides new insight to the chemotactic response associated with glioblastoma-vasculature interactions and a versatile, user-friendly platform for characterizing the chemotactic response of cells in the presence of multiple spatially distributed chemoattractants.

Grade IV astrocytomas, or glioblastomas (GBMs), are the most common malignant primary brain tumor in adults. The median GBM patient survival of 12&ndash, 15 months has remained stagnant, in spite of treatment strategies, making GBMs a tremendous challenge clinically. This is at least in part due to the complex interaction of GBM cells with the brain microenvironment and their tendency to aggressively infiltrate normal brain tissue. GBMs frequently invade supratentorial brain regions that are richly innervated by neurotransmitter projections, most notably acetylcholine (ACh). Here, we asked whether ACh signaling influences the biology of GBMs. We examined the expression and function of known ACh receptors (AChRs) in large GBM datasets, as well as, human GBM cell lines and patient-derived xenograft lines. Using RNA-Seq data from the &ldquo, The Cancer Genome Atlas&rdquo, (TCGA), we confirmed the expression of AChRs and demonstrated the functionality of these receptors in GBM cells with time-lapse calcium imaging. AChR activation did not alter cell proliferation or migration, however, it significantly increased cell invasion through complex extracellular matrices. This was due to the enhanced activity of matrix metalloproteinase-9 (MMP-9) from GBM cells, which we found to be dependent on an intracellular calcium-dependent mechanism. Consistent with these findings, AChRs were significantly upregulated in regions of GBM infiltration in situ (Ivy Glioblastoma Atlas Project) and elevated expression of muscarinic AChR M3 correlated with reduced patient survival (TCGA). Data from the Repository for Molecular Brain Neoplasia Data (REMBRANDT) dataset also showed the co-expression of choline transporters, choline acetyltransferase, and vesicular acetylcholine transporters, suggesting that GBMs express all the proteins required for ACh synthesis and release. These findings identify ACh as a modulator of GBM behavior and posit that GBMs may utilize ACh as an autocrine signaling molecule.

Malignant gliomas are devastating tumors, frequently killing those diagnosed in little over a year. The profuse infiltration of glioma cells into healthy tissue surrounding the main tumor mass is one of the major obstacles limiting the improvement of patient survival. Migration along the abluminal side of blood vessels is one of the salient features of glioma cell invasion. Invading glioma cells are attracted to the vascular network, in part by the neuro-peptide bradykinin, where glioma cells actively modify the gliovascular interface and undergo volumetric alterations to navigate the confined space. Critical to these volume modifications is a proposed hydrodynamic model that involves the flux of ions in and out of the cell, followed by osmotically obligated water. Ion and water channels expressed by the glioma cell are essential in this model of invasion and make opportune therapeutic targets. Lastly, there is growing evidence that vascular-associated glioma cells are able to control the vascular tone, presumably to free up space for invasion and growth. The unique mechanisms that enable perivascular glioma invasion may offer critical targets for therapeutic intervention in this devastating disease. Indeed, a chloride channel-blocking peptide has already been successfully tested in human clinical trials.

An important event during apoptosis is regulated cell condensation known as apoptotic volume decrease (AVD). Ion channels have emerged as essential regulators of this process mediating the release of K+ and Cl−, which together with osmotically obliged water, results in the condensation of cell volume. Using a Grade IV human glioblastoma cell line, we examined the contribution of calcium-activated K+ channels (KCa channels) to AVD after the addition of either staurosporine (Stsp) or TNF-α-related apoptosis-inducing ligand (TRAIL) to activate the intrinsic or extrinsic pathway of apoptosis, respectively. We show that AVD can be inhibited in both pathways by high extracellular K+ or the removal of calcium. However, BAPTA-AM was only able to inhibit Stsp-initiated AVD, whereas TRAIL-induced AVD was unaffected. Specific KCa channel inhibitors revealed that Stsp-induced AVD was dependent on K+ efflux through intermediate-conductance calcium-activated potassium (IK) channels, while TRAIL-induced AVD was mediated by large-conductance calcium-activated potassium (BK) channels. Fura-2 imaging demonstrated that Stsp induced a rapid and modest rise in calcium that was sustained over the course of AVD, while TRAIL produced no detectable rise in global intracellular calcium. Inhibition of IK channels with clotrimazole or 1-[(2-chlorophenyl) diphenylmethyl]-1 H-pyrazole (TRAM-34) blocked downstream caspase-3 activation after Stsp addition, while paxilline, a specific BK channel inhibitor, had no effect. Treatment with ionomycin also induced an IK-dependent cell volume decrease. Together these results show that calcium is both necessary and sufficient to achieve volume decrease and that the two major pathways of apoptosis use unique calcium signaling to efflux K+ through different KCa channels.

SUMMARY Purpose: Patients with gliomas frequently present with seizures, but the factors associated with seizure development are still poorly understood. In this study, we assessed peritumoral synaptic network activity in a glioma animal model and tested the contribution of aberrant glutamate release from gliomas on glioma-associated epileptic network activity. Methods: In vitro brain slices were made from gliomaimplanted mice. Using extracellular field recordings, we analyzed peritumoral epileptiform activity induced by Mg 2+ -free medium in slices from tumor-bearing animals and sham-operated controls. We assessed the effect of sulfasalazine (SAS), a blocker of system x c and glutamate release, on spontaneous and evoked activity in tumorassociated slices. Key Findings: Tumor-associated cortical networks were hyperexcitable. The onset latency of Mg 2+ -free‐induced epileptiform activity was significantly shorter in tumorbearing slices, and the incidence of Mg 2+ -free‐induced ictal-like events was higher. Block of glutamate release from system x c decreased the response area of evoked activity and completely blocked Mg 2+ -free‐induced ictallike, but not interictal-like events. Significance: Control of seizures in patients with gliomas is an essential component of clinical management; therefore, understanding the origin of seizures is vital. This work provides evidence that peritumoral synaptic network activity is disrupted by tumor masses resulting in network excitability. We show that blocking glutamate release via system x c with SAS, a drug already approved by the U.S. Food and Drug Administration (FDA), can inhibit Mg 2+ -free‐induced ictal-like epileptiform events similar to other chemicals used to decrease seizure activity. We, therefore, suggest that further studies should consider SAS a promising agent to aid in the treatment of seizures associated with gliomas.

Recent research has identified an important role for a cystine--glutamate anti-porter (system Xc) in the biology of malignant brain tumors. This transporter is effectively inhibited by sulfasalazine, a drug widely used to treat a number of chronic inflammatory conditions such as Crohn’s disease. Preclinical data show that sulfasalazine is an effective inhibitor of tumor growth and tumor-associated seizures. These studies suggest that the cystine--glutamate anti-porter is a valuable drug target for which tumor-specific drugs can be generated. In the meantime, sulfasalazine may be considered as adjuvant treatment for malignant gliomas.

In animals, hypoxic preconditioning has been used as a form of neuroprotection. The exact mechanism involved in neuroprotective hypoxic preconditioning has not been described, yet could be valuable for possible neuroprotective strategies. The overexpression of the cystine-glutamate exchanger, system Xc−, has been demonstrated as being neuroprotective (Shih, Erb et al. 2006). Here, using immunohistochemistry, we demonstrate that C57BL/6 mice exposed to hypoxia showed an increase in system Xc− expression, with the highest level of intensity in the hippocampus. Western Blot analysis also showed an almost 2-fold increase in system Xc− protein in hypoxia-exposed versus control mice. The mRNA for the regulatory subunit of system Xc−, xCT, and the xCT/actin ratio were also increased under hypoxic conditions. Experiments using hypoxia-inducible factor (HIF-1α) siRNA showed a statistically significant decrease in HIF-1α and system Xc− expression. Under hypoxic conditions, system Xc− activity, as determined by cystine uptake, increased 2-fold. Importantly, hypoxic preconditioning was attenuated in neural stem cells by pharmacological inhibition of system Xc− activity with S4-carboxyphenylglycine. These data provide the first evidence of hypoxic regulation of the cystine glutamate exchanger system Xc−.

A hallmark of high-grade cancers is the ability of malignant cells to invade unaffected tissue and spread disease. This is particularly apparent in gliomas, the most common and lethal type of primary brain cancer affecting adults. Migrating cells encounter restricted spaces and appear able to adjust their shape to accommodate to narrow extracellular spaces. A growing body of work suggests that cell migration/invasion is facilitated by ion channels and transporters. The emerging concept is that K+and Cl−function as osmotically active ions, which cross the plasma membrane in concert with obligated water thereby adjusting a cell's shape and volume. In glioma cells Na+-K+-Cl−cotransporters (NKCC1) actively accumulate K+and Cl−, establishing a gradient for KCl efflux. Ca2+-activated K+channels and voltage-gated Cl−channels are largely responsible for effluxing KCl promoting hydrodynamic volume changes. In other cancers, different K+or even Na+channels may function in concert with a variety of Cl−channels to support similar volume changes. Channels involved in migration are frequently regulated by Ca2+signaling, most likely coupling extracellular stimuli to cell migration. Importantly, the inhibition of ion channels and transporters appears to be clinically relevant for the treatment of cancer. Recent preclinical data indicates that inhibition of NKCC1 with an FDA-approved drug decreases neoplastic migration. Additionally, ongoing clinical trials demonstrate that an inhibitor of chloride channels may be a therapy for the treatment of gliomas. Data reviewed here strongly indicate that ion channels are a promising target for the development of novel therapeutics to combat cancer.

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 x(c)(-) 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 x(c)(-). We found that acute administration of SAS at concentrations equivalent to those used to treat Crohn's 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.

The majority of malignant primary brain tumors are gliomas, derived from glial cells. Grade IV gliomas, Glioblastoma multiforme, are extremely invasive and the clinical prognosis for patients is dismal. Gliomas utilize a number of proteins and pathways to infiltrate the brain parenchyma including ion channels and calcium signaling pathways. In this study, we investigated the localization and functional relevance of transient receptor potential canonical (TRPC) channels in glioma migration. We show that gliomas are attracted in a chemotactic manner to epidermal growth factor (EGF). Stimulation with EGF results in TRPC1 channel localization to the leading edge of migrating D54MG glioma cells. Additionally, TRPC1 channels co-localize with the lipid raft proteins, caveolin-1 and β-cholera toxin, and biochemical assays show TRPC1 in the caveolar raft fraction of the membrane. Chemotaxis toward EGF was lost when TRPC channels were pharmacologically inhibited or by shRNA knockdown of TRPC1 channels, yet without affecting unstimulated cell motility. Moreover, lipid raft integrity was required for gliomas chemotaxis. Disruption of lipid rafts not only impaired chemotaxis but also impaired TRPC currents in whole cell recordings and decreased store-operated calcium entry as revealed by ratiomeric calcium imaging. These data indicated that TRPC1 channel association with lipid rafts is essential for glioma chemotaxis in response to stimuli, such as EGF.

Several important and previously unrecognized roles for the neurotransmitter glutamate in the biology of primary brain tumors have recently been elucidated. Glutamate is produced and released from glioma cells via the system x(c) (-) cystine glutamate transporter as a byproduct of glutathione synthesis. Glutamate appears to play a central role in the malignant phenotype of glioma via multiple mechanisms. By binding to peritumoral neuronal glutamate receptors, glutamate is responsible for seizure induction and similarly causes excitotoxicity, which aids the expansion of tumor cells into the space vacated by destroyed tissue. Glutamate also activates ionotropic and metabotropic glutamate receptors on glioma cells in a paracrine and autocrine manner. α-Amino-3-hydroxy-5-methyl-4-isoaxazolepropionate acid (AMPA) glutamate receptors lack the GluR2 subunit rendering them Ca(2+) permeable and capable of activating the AKT and MAPK pathways. Furthermore, these receptors are critical in aiding the invasion of glioma cells into normal brain. AMPA-Rs accumulate at focal adhesion sites where they may indirectly mediate interactions between the extracellular matrix and integrins. Glutamate receptor stimulation results in activation of focal adhesion kinase, which is critical to the regulation of growth factor and integrin-stimulated cell motility and invasion. The multitude of effects of glutamate on glioma biology supports the rationale for pharmacological targeting of glutamate receptors and transporters. Several ongoing and recently completed clinical trials are exploring the therapeutic potential of interrupting glutamate-mediated brain tumor growth.

Movement toward the source of a chemoattractant gradient is a basic cellular property in health and disease. Enhanced migration during metastasis involves deregulated growth factor signaling. Growth factor stimulation and cell migration converge both on the important second messenger Ca(2+). To date, the molecular identification of Ca(2+) entry pathways activated by growth factors during chemotaxis is still an open issue. We investigated the involvement of the nonselective Ca(2+) channel TRPC1 (transient receptor potential canonical 1) in FGF-2 guided chemotaxis by means of time-lapse video microscopy and by functional Ca(2+) measurements. To specifically address TRPC1 function in transformed MDCK cells we altered the expression levels by siRNA or overexpression. We report that TRPC1 channels are required for the orientation of transformed MDCK cells in FGF-2 gradients because TRPC1 knockdown or pharmacological blockade prevented chemotaxis. Stimulation with FGF-2 triggered an immediate Ca(2+) influx via TRPC1 channels that depended on phospholipase C and phosphatidylinositol 3-kinase signaling. Impeding this Ca(2+) influx abolished chemotaxis toward FGF-2. This functional connection correlated with clustering of FGF receptors and TRPC1 channels as was observed by immunolabeling. These findings show the important interplay between growth factor signaling and Ca(2+) influx in chemotaxis.

Glutathione (GSH) is an essential antioxidant responsible for the maintenance of intracellular redox homeostasis. As tumors outgrow their blood supply and become hypoxic, their redox homeostasis is challenged by the production of nitric oxide and reactive oxygen species (ROS). In gliomas, the sustained import of L-cystine via the L-cystine/L-glutamate exchanger, system x(c)(-), is rate-limiting for the synthesis of GSH. We show that hypoxia causes a significant increase in NO and ROS but without affecting glioma cell growth. This is explained by a concomitant increase in the utilization of GSH, which is accompanied by an increase in the cell-surface expression of xCT, the catalytic subunit of system x(c)(-), and L-cystine uptake. Growth was inhibited when GSH synthesis was blocked by buthionine sulfoximine (BSO), an inhibitor of the enzyme required for GSH synthesis, or when cells were deprived of L-cystine. These findings suggest that glioma cells show an increased requirement for GSH to maintain growth under hypoxic conditions. Therefore, approaches that limit GSH synthesis such as blocking system x(c)(-) may be considered as an adjuvant to radiation or chemotherapy.

Glial-derived tumors, gliomas, are highly invasive cancers that invade normal brain through the extracellular space. To navigate the tortuous extracellular spaces, cells undergo dynamic changes in cell volume, which entails water flux across the membrane through aquaporins (AQPs). Two members of this family, AQP1 and AQP4 are highly expressed in primary brain tumor biopsies and both have a consensus phosphorylation site for protein kinase C (PKC), which is a known regulator of glioma cell invasion. AQP4 colocalizes with PKC to the leading edge of invading processes and clustered with chloride channel (ClC2) and K(+)-Cl(-) cotransporter 1 (KCC1), believed to provide the pathways for Cl(-) and K(+) secretion to accomplish volume changes. Using D54MG glioma cells stably transfected with either AQP1 or AQP4, we show that PKC activity regulates water permeability through phosphorylation of AQP4. Activation of PKC with either phorbol 12-myristate 13-acetate or thrombin enhanced AQP4 phosphorylation, reduced water permeability and significantly decreased cell invasion. Conversely, inhibition of PKC activity with chelerythrine reduced AQP4 phosphorylation, enhanced water permeability and significantly enhanced tumor invasion. PKC regulation of AQP4 was lost after mutational inactivation of the consensus PKC phosphorylation site S180A. Interestingly, AQP1 expressing glioma cells, by contrast, were completely unaffected by changes in PKC activity. To demonstrate a role for AQPs in glioma invasion in vivo, cells selectively expressing AQP1, AQP4 or the mutated S180A-AQP4 were implanted intracranially into SCID mice. AQP4 expressing glioma cells showed significantly reduced invasion compared to AQP1 and S180 expressing tumors as determined by quantitative stereology, consistent with a differential role for AQP1 and AQP4 in this process.

Malignant gliomas metastasize throughout the brain by infiltrative cell migration into peritumoral areas. Invading cells undergo profound changes in cell shape and volume as they navigate extracellular spaces along blood vessels and white matter tracts. Volume changes are aided by the concerted release of osmotically active ions, most notably K+ and Cl−. Their efflux through ion channels along with obligated water causes rapid cell shrinkage. Suitable ionic gradients must be established and maintained through the activity of ion transport systems. Here, we show that the Sodium-Potassium-Chloride Cotransporter Isoform-1 (NKCC1) provides the major pathway for Cl− accumulation in glioma cells. NKCC1 localizes to the leading edge of invading processes, and pharmacologic inhibition using the loop diuretic bumetanide inhibits in vitro Transwell migration by 25% to 50%. Short hairpin RNA knockdowns of NKCC1 yielded a similar inhibition and a loss of bumetanide-sensitive cell volume regulation. A loss of NKCC1 function did not affect cell motility in two-dimensional assays lacking spatial constraints but manifested only when cells had to undergo volume changes during migration. Intracranial implantation of human gliomas into severe combined immunodeficient mice showed a marked reduction in cell invasion when NKCC1 function was disrupted genetically or by twice daily injection of the Food and Drug Administration–approved NKCC1 inhibitor Bumex. These data support the consideration of Bumex as adjuvant therapy for patients with high-grade gliomas. Cancer Res; 70(13); 5597–606. ©2010 AACR.

Despite decades of research, primary brain tumors, gliomas, lack effective treatment options and present a huge clinical challenge. Particularly, the most malignant subtype, Glioblastoma multiforme, proliferates extensively and cells often undergo incomplete cell divisions, resulting in multinucleated cells. We now present evidence that multinucleated glioma cells result from the functional loss of transient receptor potential canonical 1 (TRPC1) channels, plasma membrane proteins involved in agonist-induced calcium entry and reloading of intracellular Ca(2+) stores. Pharmacological inhibition or shRNA mediated suppression of TRPC1 causes loss of functional channels and store-operated calcium entry in D54MG glioma cells. This is associated with reduced cell proliferation and, frequently, with incomplete cell division. The resulting multinucleated cells are reminiscent of those found in patient biopsies. In a flank tumor model, tumor size was significantly decreased when TRPC1 expression was disrupted using a doxycycline inducible shRNA knockdown approach. These results suggest that TRPC1 channels play an important role in glioma cell division most likely by regulating calcium signaling during cytokinesis.

Glioblastoma multiforme is the most common and lethal primary brain cancer in adults. Tumor cells diffusely infiltrate the brain making focal surgical and radiation treatment challenging. The invasion of glioma cells into normal brain is facilitated by the activity of ion channels aiding dynamic regulation of cell volume. Recent studies have specifically implicated ClC-3, a voltage-gated chloride channel, in this process. However, the interaction between ClC-3 activity and cell movement is poorly understood. Here, we demonstrate that ClC-3 is highly expressed on the plasma membrane of human glioma cells where its activity is regulated through phosphorylation via Ca(2+)/calmodulin-dependent protein kinase II (CaMKII). Intracellular infusion of autoactivated CaMKII via patch pipette enhanced chloride currents 3-fold, and this regulation was inhibited by autocamtide-2 related inhibitory peptide, a CaMKII-specific inhibitor. CaMKII modulation of chloride currents was also lost upon stable small hairpin RNA knockdown of ClC-3 channels indicating a specific interaction of ClC-3 and CaMKII. In ClC-3-expressing cells, inhibition of CaMKII reduced glioma invasion to the same extent as direct inhibition of ClC-3. The importance of the molecular interaction of ClC-3 and CaMKII is further supported by our finding that CaMKII co-localizes and co-immunoprecipitates with ClC-3. ClC-3 and CaMKII also co-immunoprecipitate in tissue biopsies from patients diagnosed with grade IV glioblastoma. These tumor samples show 10-fold higher ClC-3 protein expression than nonmalignant brain. These data suggest that CaMKII is a molecular link translating intracellular calcium changes, which are intrinsically associated with glioma migration, to changes in ClC-3 conductance required for cell movement.

During brain development, progenitor cells migrate over long distances through narrow and tortuous extracellular spaces posing significant demands on the cell's ability to alter cell volume. This phenotype is recapitulated in primary brain tumors. We demonstrate here that volume changes occurring spontaneously in these cells are mediated by the flux of Cl− along with obligated water across the cell membrane. To do so, glioma cells accumulate Cl− to ∼100 mM, a concentration threefold greater than predicted by the Nernst equation. Shunting this gradient through the sustained opening of exogenously expressed GABA-gated Cl− channels caused a 33% decrease in cell volume and impaired the ability of cells to migrate in a spatially constrained environment. Further, dividing cells condense their cytoplasm prior to mitosis, a phenomenon which is associated with the release of intracellular Cl− as indicated by a 40-mM decrease in [Cl−]i. These findings provide a new framework for considering the role of intracellular Cl− in glioma cells. Here, Cl− serves as an important osmotically active regulator of cell volume being the energetic driving force for volume changes required by immature cells in cell migration and proliferation. This mechanism that was studied in CNS malignancies may be shared with other immature cells in the brain as well.

Astrocytes and oligodendrocytes are characterized by a very negative resting potential and a high resting permeability for K(+) ions. Early pharmacological and biophysical studies suggested that the resting potential is established by the activity of inwardly rectifying, Ba(2+) sensitive, weakly rectifying Kir channels. Molecular cloning has identified 16 Kir channels genes of which several mRNA transcripts and protein products have been identified in glial cells. However, genetic deletion and siRNA knock-down studies suggest that the resting conductance of astrocytes and oligodendrocytes is largely due to Kir4.1. Loss of Kir4.1 causes membrane depolarization, and a break-down of K(+) and glutamate homeostasis which results in seizures and wide-spread white matter pathology. Kir channels have also been shown to act as critical regulators of cell division whereby Kir function is correlated with an exit from the cell cycle. Conversely, loss of functional Kir channels is associated with re-entry of cells into the cell cycle and gliosis. A loss of functional Kir channels has been shown in a number of neurological diseases including temporal lobe epilepsy, amyotrophic lateral sclerosis, retinal degeneration and malignant gliomas. In the latter, expression of Kir4.1 is sufficient to arrest the aberrant growth of these glial derived tumor cells. Kir4.1 therefore represents a potential therapeutic target in a wide variety of neurological conditions.

(+/–)-1-Aminocyclopentane-trans-1,3-dicarboxylic acid (t-ACPD) is an equimolar mixture of two enantiomers: (1S,3R)-1-Aminocyclopentane-1,3-dicarboxylic acid (SR-ACPD) and 1R,3S-1-Aminocyclopentane-1,3-dicarboxylic acid (RS-ACPD). t-ACPD and SR-ACPD have been commonly used as agonists for metabotropic glutamate receptors (mGluR). Here we demonstrated that RS-ACPD, but not SR-ACPD, is transported into astrocytes with a Km of 6.51 ± 2.38 mm and Vmax of 22.8 ± 3.4 nmol/mg/min. This low-affinity transport is Na+-dependent and is competitively blocked by glutamate or other substrates for the glutamate transporter. RS-ACPD therefore is probably taken up by the glutamate transporter. Prolonged incubation with high levels of RS-ACPD (> 500 µm) induced significant swelling of astrocytes. At lower concentrations (100 µm), RS-ACPD reduced intracellular glutamate content ([Glu]i) by > 50% without obvious morphological changes. The reduction in [Glu]i was accompanied by an increase in [glutamine]i. The RS-ACPD's effect on [Glu]i required glutamine and high levels of phosphate, suggesting that RS-ACPD inhibited phosphate-activated glutaminase (PAG). These data suggest that astrocytic PAG is actively involved in determining the equilibrium between intracellular glutamate and glutamine. By reducing [Glu]i, RS-ACPD reduces the amount of glutamate available for release.

Over the past two decades it has become apparent that essentially all living cells express voltage-activated ion channels. While the role of ion channels for electrical signaling between excitable cells is well known, their function in non-excitable cells is somewhat enigmatic. Research on cancer cells suggests that certain ion channels, K+ channels in particular, may be involved in aberrant tumor growth and channel inhibitors often lead to growth arrest. An unsuspected role for K+ and Cl− channels has now been documented for primary brain tumors, glioma, where the concerted activity of these channels promotes cell invasion and the formation of brain metastasis. Specifically, Ca2+-activated K+ (BK) channels colocalize with ClC-3 Cl− channels to the invading processes of these tumor cells. Upon a rise in intracellular Ca2+, these channels activate and release K+ and Cl− ions together with obligated water causing a rapid shrinkage of the leading process. This in turn facilitates the invasion of the cell into the narrow and tortuous extracellular brain spaces. The NKCC1 cotransporter accumulates intracellular Cl− to unusually high concentrations, thereby establishing an outward directed gradient for Cl− ions. This allows glioma cells to utilize Cl− as an osmotically active anion during invasion. Importantly, the inhibition of Cl− channels retards cell volume changes, and, in turn, compromises tumor cell invasion. These findings have led to the clinical evaluation of a Cl− channel blocking peptide, chlorotoxin, in patients with malignant glioma. Data from this clinical trial shows remarkable tumor selectivity for chlorotoxin. The experimental therapeutic was well tolerated and is now evaluated in a multi-center phase II clinical trial. A similar role for Cl− and K+ channels is suspected in other metastatic cancers, and lessons learned from studies of gliomas may pave the way towards the development of novel therapeutics targeting ion channels.

Programmed cell death (apoptosis) is important in tissue maintenance. Hallmarks of apoptosis include caspase activation, DNA fragmentation and an overall reduction in cell volume. Whether this apoptotic volume decrease (AVD) is a mere response to initiators of apoptosis or whether it is functionally significant is not clear. In this study, we sought to answer this question using human malignant glioma cells as a model system. In vivo, high grade gliomas demonstrate an increased percentage of apoptotic cells as well as upregulation of death ligand receptors. By dynamically monitoring cell volume, we show that the induction of apoptosis, via activation of either the intrinsic or extrinsic pathways with staurosporine or TRAIL, respectively, resulted in a rapid AVD in D54-MG human glioma cells. This decrease in cell volume could be prevented by inhibiting the efflux of Cl– through channels. Such suppression of AVD also reduced the activation of caspases 3, 8 and 9 and suppressed DNA fragmentation. Importantly, experimental manipulations that reduce the cell volume to 70% of the original volume for periods of at least 3 hours were sufficient to initiate apoptosis even in the absence of death ligands. Hence, this data suggests that cell condensation is both necessary and sufficient for the induction of apoptosis.

Glioma cells prominently express a unique splice variant of a large conductance, calcium-activated potassium channel (BK channel). These channels transduce changes in intracellular calcium to changes of K+ conductance in the cells and have been implicated in growth control of normal and malignant cells. The Ca2+ increase that facilitates channel activation is thought to occur via activation of intracellular calcium release pathways or influx of calcium through Ca2+-permeable ion channels. We show here that BK channel activation involves the activation of inositol 1,4,5-triphosphate receptors (IP3R), which localize near BK channels in specialized membrane domains called lipid rafts. Disruption of lipid rafts with methyl-β-cyclodextrin disrupts the functional association of BK channel and calcium source resulting in a >50% reduction in K+ conductance mediated by BK channels. The reduction of BK current by lipid raft disruption was overcome by the global elevation of intracellular calcium through inclusion of 750 nm Ca2+ in the pipette solution, indicating that neither the calcium sensitivity of the channel nor their overall number was altered. Additionally, pretreatment of glioma cells with 2-aminoethoxydiphenyl borate to inhibit IP3Rs negated the effect of methyl-β-cyclodextrin, providing further support that IP3Rs are the calcium source for BK channels. Taken together, these data suggest a privileged association of BK channels in lipid raft domains and provide evidence for a novel coupling of these Ca2+-sensitive channels to their second messenger source.

Cell growth and osmotic volume regulation are undoubtedly linked to the progression of the cell cycle as with each division, a newly generated cell must compensate for loss of half of its volume to its sister cell. The extent to which size influences cell cycle decisions, however, is controversial in mammalian cells. Further, a mechanism by which cells can monitor and therefore regulate their size has not been fully elucidated. Despite an ongoing debate, there have been few studies which directly address the question in single cell real-time experiments. In this study we used fluorescent time-lapse imaging to quantitatively assess volume in individual spontaneously dividing cells throughout the cell cycle. Together with biophysical studies, these establish that the efflux of salt and water brings about a condensation of cytoplasmic volume as glioma cells progress through mitosis. As cells undergo this pre-mitotic condensation (PMC) they approach a preferred cell volume preceding each division. This is functionally linked to chromatin condensation, suggesting that PMC plays an integral role in mitosis.

The discovery of microtube structures that link tumour cells in some invasive brain tumours reveals how these cancers spread, and how they resist treatment.

Reduced cerebral blood flow impairs cognitive function and ultimately causes irreparable damage to brain tissue. The gliovascular unit, composed of neural and vascular cells, assures sufficient blood supply to active brain regions. Astrocytes, vascular smooth muscle cells, and pericytes are important players within the gliovascular unit modulating vessel diameters. While the importance of the gliovascular unit and the signals involved in regulating local blood flow to match neuronal activity is now well recognized, surprisingly little is known about this interface in disease. Alzheimer's disease is associated with reduced cerebral blood flow. Here, we studied how the gliovascular unit is affected in a mouse model of Alzheimer's disease, using a combination of ex vivo and in vivo imaging approaches. We specifically labelled vascular amyloid in living mice using the dye methoxy-XO4. We elicited vessel responses ex vivo using either pharmacological stimuli or cell-specific calcium uncaging in vascular smooth muscle cells or astrocytes. Multi-photon in vivo imaging through a cranial window allowed us to complement our ex vivo data in the presence of blood flow after label-free optical activation of vascular smooth muscle cells in the intact brain. We found that vascular amyloid deposits separated astrocyte end-feet from the endothelial vessel wall. High-resolution 3D images demonstrated that vascular amyloid developed in ring-like structures around the vessel circumference, essentially forming a rigid cast. Where vascular amyloid was present, stimulation of astrocytes or vascular smooth muscle cells via ex vivo Ca(2+) uncaging or in vivo optical activation produced only poor vascular responses. Strikingly, vessel segments that were unaffected by vascular amyloid responded to the same extent as vessels from age-matched control animals. We conclude that while astrocytes can still release vasoactive substances, vascular amyloid deposits render blood vessels rigid and reduce the dynamic range of affected vessel segments. These results demonstrate a mechanism that could account in part for the reduction in cerebral blood flow in patients with Alzheimer's disease.media-1vid110.1093/brain/awv327_video_abstractawv327_video_abstract.

Stem cells present in the adult brain are regulated by neuronal activity; malignant gliomas, which most likely originate from this population of cells, could also be regulated in this manner. A recent study by Venkatesh et al. published in Cell has identified Neuroligin-3 (NLGN3) as a mitogen promoting high-grade glioma growth.

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.

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.

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.

Amyotrophic lateral sclerosis is the most common neurodegenerative disorder in younger to middle-aged adults. The progressive loss of upper (cortical) and lower (midbrain and spinal) motor neurons involved in voluntary movements causes a gradual paralysis that eventually affects all muscle groups, including those required for breathing, while completely sparing sensory neurons. Only one FDA approved drug exist that marginally slows disease, and respiratory distress is ultimately the main cause of death. Approximately 10% of familial cases show mutations in one of several genes that cause disease, yet in the majority of cases the cause of disease remains a mystery. Affected genes include DNA -and-RNA binding proteins as well as the enzyme superoxide dismutase 1 ( SOD1 ). Mutations generally cause aberrant RAN metabolism, protein misfolding and aggregation. Thus far, no disease modifying treatments exist, and patient outlook is grim. Promising clinical studies that seek to replace dying neurons with stem cells, curb neurotoxicity by attenuating glutamate concentrations, and/or attempt to remove misfolded proteins are underway.

Most central nervous system (CNS) pathogens infect the nervous system nonspecifically, and disease is the result of the brain's immune response. This is exemplified by viral and bacterial meningitis, which cause extensive inflammation of the brain or its coverings. However, some pathogens show neural tropism and specifically infect certain neurons, as illustrated by motor neuron infections underlying poliomyelitis, neurosyphilis, or infection of sensory neurons by herpes simplex. Similarly, infestation of the brain by larvae (cysticerci) from intestinal tapeworms nonspecifically causes neurological disease in millions of people in the developing world, whereas rare paralytic amoeba actively invade the brain through the nasal canal and feast on brain tissue. By far the most important neurological disease caused by a pathogen is neuro–acquired immunodeficiency syndrome (AIDS), the development of cognitive decline and dementia in the late stages of AIDS due to brain infection with human immunodeficiency virus. Given the magnitude of the global AIDS endemic, these cases will soon rival more traditional age-related dementia. Neuroscientists are unlikely to contribute to the development of more effective treatments, a task that rests on microbiologists and immunologists who to develop vaccines and antivirals. However, several neurotoxins (Botox) and neurotropic viruses (herpes simplex virus 1) are providing tools to treat neurological disease through targeted delivery of genes or paralytic therapeutics.