We report that KCC2 activity, monitored with wide-field fluorescence, is inhibited by intracellular Zn 2+ , a key component of neuronal injury. Zn 2+ -mediated KCC2 inhibition produced a depolarizing shift of GABA A reversal potentials in rat neurons. Moreover, oxygen-glucose deprivation attenuated KCC2 activity in a Zn 2+ -dependent manner. The link between Zn 2+ and KCC2 activity provides a novel target for neuroprotection and may be important in activity-dependent regulation of inhibitory synaptic transmission. KeywordsZinc; potassium-chloride co-transporter; ischemiaThe K + /Cl − co-transporter-2 (KCC2) is the major Cl − outward transporter in neurons, creating a Cl − equilibrium potential negative to the resting membrane voltage and rendering GABA inhibitory. However, following acute and chronic neuronal injury, KCC2 activity is decreased and GABA becomes excitatory 1,2,3 , a process that has been tightly linked to neurodegeneration 4 . Another important contributor to neuronal injury and death is an increase of cytosolic Zn 2+ concentrations ([Zn 2+ ] i ) 5 . Although both rise in [Zn 2+ ] i and decrease in KCC2 have been associated with neuronal injury, whether Zn 2+ itself can influence KCC2 activity is unknown.To test whether [Zn 2+ ] i modulates KCC2 activity we first monitored KCC2-mediated ion transport in HEK 293T cells expressing the co-transporter 6 . NH 4 + was used as a surrogate ion for K + and changes in intracellular pH were monitored using the fluorescent dye BCECF (see Supplemental methods online). KCC2-expressing cells showed a 3-fold faster NH 4 + -induced acidification rate than cells transfected with empty vector (Fig. 1a,b). Increasing [Zn 2+ ] i by a 2 min application of Zn 2+ with the Zn 2+ ionophore pyrithione (ZnPyr) was immediately followed by a decrease of KCC2-mediated acidification rate, with an IC 50 of ~50 µM Zn 2+ (Fig. 1a-c (Fig. 1b). Moreover, TPEN alone increased KCC2 activity (Fig. 1a,b (Fig. 1d). Increasing [Zn 2+ ] i with ZnPyr blocked the Cl − influx (Fig 1d,e), while TPEN enhanced it (Fig. 1d,e), similar to the results observed with NH 4 + -induced acidification.We then tested whether KCC2 inhibition by Zn 2+ is also observed in cortical neurons, which endogenously express the co-transporter. In BCECF-loaded immature neurons (7 days in vitro, DIV), expressing low levels of KCC2, NH 4 + had little effect on intracellular acidification, similar to empty vector-transfected HEK 293T cells (Fig. 1f). In contrast, in mature neurons (>25 DIV), which express high levels of KCC2, NH 4 + -induced acidification was pronounced. Importantly, in mature neurons ZnPyr also effectively attenuated KCC2 activity (Fig. 1f).The Zn 2+ -dependent changes in KCC2 activity should affect the Cl − gradient and thereby the reversal potential of GABA A receptor-mediated currents (E GABA ). We measured E GABA using the gramicidin perforated patch technique, which leaves intracellular Cl − undisturbed. A 2 min ZnPyr treatment produced a gradual positive shift in E GABA as, presumably, the...

Behavioral (nonphotic) stimuli can shift circadian rhythms by serotonin (5-HT) and/or neuropeptide Y (NPY) inputs to the suprachiasmatic nucleus (SCN) circadian clock. Based on the idea that behavioral phase resetting is modulated by endogenous changes in postsynaptic sensitivity to such transmitters, hamsters were exposed to constant light (LL; approximately 250 lx) for 1-3 days, which suppresses locomotor activity and eliminates the daily rhythm of SCN 5-HT release measured by microdialysis. Groups subjected to brief LL or maintained under a light/dark cycle (LD) received phase-resetting treatments with the 5-HT(1A,7) agonist (+/-)-2-dipropyl-amino-8-hydroxyl-1,2,3,4-tetrahydronapthalene (8-OH-DPAT) or sleep deprivation (SD). Animals were released to constant darkness at the start of the treatments. Phase advances to 8-OH-DPAT and SD during the day were 11 and 3 h for LL vs. 2 and 1 h for LD, respectively. Phase delays during the night were -12 and -5 h for LL vs. no responses for LD, respectively. Phase-transition curves for both LL treatments had slopes approximating 0, indicative of Type 0 phase resetting. For all treatments, the degree of locomotor suppression by LL was not correlated with the phase shift magnitude. Re-establishing locomotor activity by overnight food deprivation did not prevent potentiated shifting to SD. However, re-establishing peak extracellular 5-HT levels by intra-SCN 5-HT reverse microdialysis perfusion in LL did significantly reduce potentiated 8-OH-DPAT phase advances. Constant light also enhanced intra-SCN NPY-induced phase advances during the day (6 vs. 2 h for LD). These results suggest that LL promotes Type 0 phase resetting by supersensitizing 5-HT and/or NPY postsynaptic responses and possibly by attenuating the amplitude of the circadian pacemaker, thus enhancing circadian clock resetting nonspecifically.

Oxidative stress induced by glutathione depletion in the mouse HT22 neuroblastoma cell line and embryonic rat immature cortical neurons causes a delayed, sustained activation of extracellular signal-regulated kinase (ERK) 1/2, which is required for cell death. This sustained activation of ERK1/2 is mediated primarily by a selective inhibition of distinct ERK1/2-directed phosphatases either by enhanced degradation (i.e., for mitogenactivated protein kinase phosphatase-1) or as shown here by reductions in enzymatic activity (i.e., for protein phosphatase type 2A). The inhibition of ERK1/2 phosphatases in HT22 cells and immature neurons subjected to glutathione depletion results from oxidative stress because phosphatase activity is restored in cells treated with the antioxidant butylated hydroxyanisole. This leads to reduced ERK1/2 activation and neuroprotection. Furthermore, an increase in free intracellular zinc that accompanies glutathioneinduced oxidative stress in HT22 cells and immature neurons contributes to selective inhibition of ERK1/2 phosphatase activity and cell death. Finally, ERK1/2 also functions to maintain elevated levels of zinc. Thus, the elevation of intracellular zinc within neurons subjected to oxidative stress can trigger a robust positive feedback loop operating through activated ERK1/2 that rapidly sets into motion a zinc-dependent pathway of cell death.

Microglial cells are critical components of the injurious cascade in a large number of neurodegenerative diseases. However, the precise molecular mechanisms by which microglia mediate neuronal cell death have not been fully delineated. We report here that reactive species released from activated microglia induce the liberation of Zn(2+) from intracellular stores in cultured cortical neurons, with a subsequent enhancement in neuronal voltage-gated K(+) currents, two events that have been intimately linked to apoptosis. Both the intraneuronal Zn(2+) release and the K(+) current surge could be prevented by the NADPH oxidase inhibitor apocynin, the free radical scavenging mixture of superoxide dismutase and catalase, as well as by 5,10,15,20-tetrakis(4-sulfonatophenyl)porphyrinato iron(III) chloride. The enhancement of K(+) currents was prevented by neuronal overexpression of metallothionein III or by expression of a dominant negative (DN) vector for the upstream mitogen-activated protein kinase apoptosis signal regulating kinase-1 (ASK-1). Importantly, neurons overexpressing metallothionein-III or transfected with DN vectors for ASK-1 or Kv2.1-encoded K(+) channels were resistant to microglial-induced toxicity. These results establish a direct link between microglial-generated oxygen and nitrogen reactive products and neuronal cell death mediated by intracellular Zn(2+) release and a surge in K(+) currents.