Mitochondrial Superoxide Contributes to Hippocampal Synaptic Dysfunction and Memory Deficits in Angelman Syndrome Model Mice.

Angelman syndrome (AS) is a neurodevelopmental disorder associated with developmental delay, lack of speech, motor dysfunction, and epilepsy. In the majority of the patients, AS is caused by the deletion of small portions of maternal chromosome 15 harboring the UBE3A gene. This results in a lack of expression of the UBE3A gene because the paternal allele is genetically imprinted. The UBE3A gene encodes an enzyme termed ubiquitin ligase E3A (E6-AP) that targets proteins for degradation by the 26S proteasome. Because neurodegenerative disease and other neurodevelopmental disorders have been linked to oxidative stress, we asked whether mitochondrial reactive oxygen species (ROS) played a role in impaired synaptic plasticity and memory deficits exhibited by AS model mice. We discovered that AS mice have increased levels of superoxide in area CA1 of the hippocampus that is reduced by MitoQ 10-methanesuflonate (MitoQ), a mitochondria-specific antioxidant. In addition, we found that MitoQ rescued impairments in hippocampal synaptic plasticity and deficits in contextual fear memory exhibited by AS model mice. Our findings suggest that mitochondria-derived oxidative stress contributes to hippocampal pathophysiology in AS model mice and that targeting mitochondrial ROS pharmacologically could benefit individuals with AS. SIGNIFICANCE STATEMENT: Oxidative stress has been hypothesized to contribute to the pathophysiology of neurodevelopmental disorders, including autism spectrum disorders and Angelman syndrome (AS). Herein, we report that AS model mice exhibit elevated levels of mitochondria-derived reactive oxygen species in pyramidal neurons in hippocampal area CA1. Moreover, we demonstrate that the administration of MitoQ (MitoQ 10-methanesuflonate), a mitochondria-specific antioxidant, to AS model mice normalizes synaptic plasticity and restores memory. Finally, our findings suggest that antioxidants that target the mitochondria could be used therapeutically to ameliorate synaptic and cognitive deficits in individuals with AS.

A proinvasive role for the Ca(2+) -activated K(+) channel KCa3.1 in malignant glioma.

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.

Cl- and K+ channels and their role in primary brain tumour biology.

Profound cell volume changes occur in primary brain tumours as they proliferate, invade surrounding tissue or undergo apoptosis. These volume changes are regulated by the flux of Cl(-) and K(+) ions and concomitant movement of water across the membrane, making ion channels pivotal to tumour biology. We discuss which specific Cl(-) and K(+) channels are involved in defined aspects of glioma biology and how these channels are regulated. Cl(-) is accumulated to unusually high concentrations in gliomas by the activity of the NKCC1 transporter and serves as an osmolyte and energetic driving force for volume changes. Cell volume condensation is required as cells enter M phase of the cell cycle and this pre-mitotic condensation is caused by channel-mediated ion efflux. Similarly, Cl(-) and K(+) channels dynamically regulate volume in invading glioma cells allowing them to adjust to small extracellular brain spaces. Finally, cell condensation is a hallmark of apoptosis and requires the concerted activation of Cl(-) and Ca(2+)-activated K(+) channels. Given the frequency of mutation and high importance of ion channels in tumour biology, the opportunity exists to target them for treatment.

KCa3.1 modulates neuroblast migration along the rostral migratory stream (RMS) in vivo.

From the subventricular zone (SVZ), neuronal precursor cells (NPCs), called neuroblasts, migrate through the rostral migratory stream (RMS) to become interneurons in the olfactory bulb (OB). Ion channels regulate neuronal migration during development, yet their role in migration through the adult RMS is unknown. To address this question, we utilized Nestin-CreER(T2)/R26R-YFP mice to fluorescently label neuroblasts in the adult. Patch-clamp recordings from neuroblasts reveal K(+) currents that are sensitive to intracellular Ca(2+) levels and blocked by clotrimazole and TRAM-34, inhibitors of intermediate conductance Ca(2+)-activated K(+) (KCa3.1) channels. Immunolabeling and electrophysiology show KCa3.1 expression restricted to neuroblasts in the SVZ and RMS, but absent in OB neurons. Time-lapse confocal microscopy in situ showed inhibiting KCa3.1 prolonged the stationary phase of neuroblasts' saltatory migration, reducing migration speed by over 50%. Both migration and KCa3.1 currents could also be inhibited by blocking Ca(2+) influx via transient receptor potential (TRP) channels, which, together with positive immunostaining for transient receptor potential canonical 1 (TRPC1), suggest that TRP channels are an important Ca(2+) source modulating KCa3.1 activity. Finally, injecting TRAM-34 into Nestin-CreER(T2)/R26R-YFP mice significantly reduced the number of neuroblasts that reached the OB, suggesting an important role for KCa3.1 in vivo. These studies describe a previously unrecognized protein in migration of adult NPCs.

Bradykinin-induced chemotaxis of human gliomas requires the activation of KCa3.1 and ClC-3.

Previous reports demonstrate that cell migration in the nervous system is associated with stereotypic changes in intracellular calcium concentration ([Ca(2+)](i)), yet the target of these changes are essentially unknown. We examined chemotactic migration/invasion of human gliomas to study how [Ca(2+)](i) regulates cellular movement and to identify downstream targets. Gliomas are primary brain cancers that spread exclusively within the brain, frequently migrating along blood vessels to which they are chemotactically attracted by bradykinin. Using simultaneous fura-2 Ca(2+) imaging and amphotericin B perforated patch-clamp electrophysiology, we find that bradykinin raises [Ca(2+)](i) and induces a biphasic voltage response. This voltage response is mediated by the coordinated activation of Ca(2+)-dependent, TRAM-34-sensitive K(Ca)3.1 channels, and Ca(2+)-dependent, 4,4'-diisothiocyanato-stilbene-2,2'-disulfonic acid (DIDS)-sensitive and gluconate-sensitive Cl(-) channels. A significant portion of these Cl(-) currents can be attributed to Ca(2+)/calmodulin-dependent protein kinase II (CaMKII) activation of ClC-3, a voltage-gated Cl(-) channel/transporter, because pharmacological inhibition of CaMKII or shRNA-mediated knockdown of ClC-3 inhibited Ca(2+)-activated Cl(-) currents. Western blots show that K(Ca)3.1 and ClC-3 are expressed in tissue samples obtained from patients diagnosed with grade IV gliomas. Both K(Ca)3.1 and ClC-3 colocalize to the invading processes of glioma cells. Importantly, inhibition of either channel abrogates bradykinin-induced chemotaxis and reduces tumor expansion in mouse brain slices in situ. These channels should be further explored as future targets for anti-invasive drugs. Furthermore, these data elucidate a novel mechanism placing cation and anion channels downstream of ligand-mediated [Ca(2+)](i) increases, which likely play similar roles in other migratory cells in the nervous system.

Calcium entry via TRPC1 channels activates chloride currents in human glioma cells.

Malignant gliomas are highly invasive brain cancers that carry a dismal prognosis. Recent studies indicate that Cl(-) channels facilitate glioma cell invasion by promoting hydrodynamic cell shape and volume changes. Here we asked how Cl(-) channels are regulated in the context of migration. Using patch-clamp recordings we show Cl(-) currents are activated by physiological increases of [Ca(2+)]i to 65 and 180nM. Cl(-) currents appear to be mediated by ClC-3, a voltage-gated, CaMKII-regulated Cl(-) channel highly expressed by glioma cells. ClC-3 channels colocalized with TRPC1 on caveolar lipid rafts on glioma cell processes. Using perforated-patch electrophysiological recordings, we demonstrate that inducible knockdown of TRPC1 expression with shRNA significantly inhibited glioma Cl(-) currents in a Ca(2+)-dependent fashion, placing Cl(-) channels under the regulation of Ca(2+) entry via TRPC1. In chemotaxis assays epidermal growth factor (EGF)-induced invasion was inhibition by TRPC1 knockdown to the same extent as pharmacological block of Cl(-) channels. Thus endogenous glioma Cl(-) channels are regulated by TRPC1. Cl(-) channels could be an important downstream target of TRPC1 in many other cells types, coupling elevations in [Ca(2+)]i to the shape and volume changes associated with migrating cells.

Differential role of IK and BK potassium channels as mediators of intrinsic and extrinsic apoptotic cell death.

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 (K(Ca) 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 K(Ca) 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]-1H-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 K(Ca) channels.

The pan erbB inhibitor PD168393 enhances lysosomal dysfunction-induced apoptotic death in malignant peripheral nerve sheath tumor cells.

Malignant peripheral nerve sheath tumors (MPNSTs) are rapidly progressive Schwann cell neoplasms. The erbB family of membrane tyrosine kinases has been implicated in MPNST mitogenesis and invasion and, thus, is a potential therapeutic target. However, tyrosine kinase inhibitors (TKIs) used alone have limited tumoricidal activity. Manipulating the autophagy lysosomal pathway in cells treated with cytostatic agents can promote apoptotic cell death in some cases. The goal of this study was to establish a mechanistic basis for formulating drug combinations to effectively trigger death in MPNST cells. We assessed the effects of the pan erbB inhibitor PD168393 on MPNST cell survival, caspase activation, and autophagy. PD168393 induced a cytostatic but not a cytotoxic response in MPNST cells that was accompanied by suppression of Akt and mTOR activation and increased autophagic activity. The effects of autophagy modulation on MPNST survival were then assessed following the induction of chloroquine (CQ)-induced lysosomal stress. In CQ-treated cells, suppression of autophagy was accompanied by increased caspase activation. In contrast, increased autophagy induction by inhibition of mTOR did not trigger cytotoxicity, possibly because of Akt activation. We thus hypothesized that dual targeting of mTOR and Akt by PD168393 would significantly increase cytotoxicity in cells exposed to lysosomal stress. We found that PD168393 and CQ in combination significantly increased cytotoxicity. We conclude that combinatorial therapies with erbB inhibitors and agents inducing lysosomal dysfunction may be an effective means of treating MPNSTs.

Transient receptor potential canonical channels are essential for chemotactic migration of human malignant gliomas.

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 ß-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.