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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.

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 theUBE3Agene. This results in a lack of expression of theUBE3Agene because the paternal allele is genetically imprinted. TheUBE3Agene 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 STATEMENTOxidative 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.

Previous reports demonstrate that cell migration in the nervous system is associated with stereotypic changes in intracellular calcium concentration ([Ca2+]i), yet the target of these changes are essentially unknown. We examined chemotactic migration/invasion of human gliomas to study how [Ca2+]iregulates 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 Ca2+imaging and amphotericin B perforated patch-clamp electrophysiology, we find that bradykinin raises [Ca2+]iand induces a biphasic voltage response. This voltage response is mediated by the coordinated activation of Ca2+-dependent, TRAM-34-sensitive KCa3.1 channels, and Ca2+-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 Ca2+/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 Ca2+-activated Cl−currents. Western blots show that KCa3.1 and ClC-3 are expressed in tissue samples obtained from patients diagnosed with grade IV gliomas. Both KCa3.1 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 slicesin 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 [Ca2+]iincreases, which likely play similar roles in other migratory cells in the nervous system.

Electrical transduction of DNA hybridization events is a major challenge in genoelectronics. Here we report on a new strategy for amplifying electrical DNA sensing based on the use of microsphere tags loaded internally with a redox marker. The resulting “electroactive beads” are capable of carrying a huge number of the ferrocenecarboxaldehyde marker molecules and hence offer a remarkable amplification of single hybridization events. This allows chronopotentiometric detection of target DNA down to the 5.1 × 10-21 mol level (∼31 000 molecules) in connection to 20 min of hybridization and “release” of the marker in an organic medium. The dramatic signal amplification advantage is combined with a remarkable discrimination against a huge excess (107) of noncomplementary nucleic acids. Such electroactive beads hold great promise for multitarget detection (in connection to spheres loaded with different redox markers) and for enhancing the sensitivity of other bioassays.

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.