Dong Ho Woo

GABA from reactive astrocytes impairs memory in mouse models of Alzheimer's disease

In Alzheimers disease (AD), memory impairment is the most prominent feature that afflicts patients and their families. Although reactive astrocytes have been observed around amyloid plaques since the disease was first described, their role in memory impairment has been poorly understood. Here, we show that reactive astrocytes aberrantly and abundantly produce the inhibitory gliotransmitter GABA by monoamine oxidase-B (Maob) and abnormally release GABA through the bestrophin 1 channel. In the dentate gyrus of mouse models of AD, the released GABA reduces spike probability of granule cells by acting on presynaptic GABA receptors. Suppressing GABA production or release from reactive astrocytes fully restores the impaired spike probability, synaptic plasticity, and learning and memory in the mice. In the postmortem brain of individuals with AD, astrocytic GABA and MAOB are significantly upregulated. We propose that selective inhibition of astrocytic GABA synthesis or release may serve as an effective therapeutic strategy for treating memory impairment in AD.

TREK-1 and Best1 Channels Mediate Fast and Slow Glutamate Release in Astrocytes upon GPCR Activation

Astrocytes release glutamate upon activation of various GPCRs to exert important roles in synaptic functions. However, the molecular mechanism of release has been controversial. Here, we report two kinetically distinct modes of nonvesicular, channel-mediated glutamate release. The fast mode requires activation of G(αi), dissociation of G(βγ), and subsequent opening of glutamate-permeable, two-pore domain potassium channel TREK-1 through direct interaction between G(βγ) and N terminus of TREK-1. The slow mode is Ca(2+) dependent and requires G(αq) activation and opening of glutamate-permeable, Ca(2+)-activated anion channel Best1. Ultrastructural analyses demonstrate that TREK-1 is preferentially localized at cell body and processes, whereas Best1 is mostly found in microdomains of astrocytes near synapses. Diffusion modeling predicts that the fast mode can target neuronal mGluR with peak glutamate concentration of 100 μM, whereas slow mode targets neuronal NMDA receptors at around 1 μM. Our results reveal two distinct sources of astrocytic glutamate that can differentially influence neighboring neurons.

Direct activation of Transient Receptor Potential Vanilloid 1(TRPV1) by Diacylglycerol (DAG)

The capsaicin receptor, known as transient receptor potential channel vanilloid subtype 1 (TRPV1), is activated by a wide range of noxious stimulants and putative ligands such as capsaicin, heat, pH, anandamide, and phosphorylation by protein kinase C (PKC). However, the identity of endogenous activators for TRPV1 under physiological condition is still debated. Here, we report that diacylglycerol (DAG) directly activates TRPV1 channel in a membrane-delimited manner in rat dorsal root ganglion (DRG) neurons. 1-oleoyl-2-acetyl-sn-glycerol (OAG), a membrane-permeable DAG analog, elicited intracellular Ca2+ transients, cationic currents and cobalt uptake that were blocked by TRPV1-selective antagonists, but not by inhibitors of PKC and DAG lipase in rat DRG neurons or HEK 293 cells heterologously expressing TRPV1. OAG induced responses were about one fifth of capsaicin induced signals, suggesting that OAG displays partial agonism. We also found that endogenously produced DAG can activate rat TRPV1 channels. Mutagenesis of rat TRPV1 revealed that DAG-binding site is at Y511, the same site for capsaicin binding, and PtdIns(4,5)P2binding site may not be critical for the activation of rat TRPV1 by DAG in heterologous system. We propose that DAG serves as an endogenous ligand for rat TRPV1, acting as an integrator of Gq/11-coupled receptors and receptor tyrosine kinases that are linked to phospholipase C.

Caffeine-Mediated Inhibition of Calcium Release Channel Inositol 1,4,5-Trisphosphate Receptor Subtype 3 Blocks Glioblastoma Invasion and Extends Survival

Calcium signaling is important in many signaling processes in cancer cell proliferation and motility including in deadly glioblastomas of the brain that aggressively invade neighboring tissue. We hypothesized that disturbing Ca(2+) signaling pathways might decrease the invasive behavior of giloblastoma, extending survival. Evaluating a panel of small-molecule modulators of Ca(2+) signaling, we identified caffeine as an inhibitor of glioblastoma cell motility. Caffeine, which is known to activate ryanodine receptors, paradoxically inhibits Ca(2+) increase by inositol 1,4,5-trisphospate receptor subtype 3 (IP(3)R3), the expression of which is increased in glioblastoma cells. Consequently, by inhibiting IP(3)R3-mediated Ca(2+) release, caffeine inhibited migration of glioblastoma cells in various in vitro assays. Consistent with these effects, caffeine greatly increased mean survival in a mouse xenograft model of glioblastoma. These findings suggest IP(3)R3 as a novel therapeutic target and identify caffeine as a possible adjunct therapy to slow invasive growth of glioblastoma.

Bestrophin-1 Encodes for the Ca2+-Activated Anion Channel in Hippocampal Astrocytes

In mammalian brain, neurons and astrocytes are reported to express various chloride and anion channels, but the evidence for functional expression of Ca2+-activated anion channel (CAAC) and its molecular identity have been lacking. Here we report electrophysiological evidence for the CAAC expression and its molecular identity by mouse Bestrophin 1 (mBest1) in astrocytes of the mouse brain. Using Ca2+ imaging and perforated-patch-clamp analysis, we demonstrate that astrocytes displayed an inward current at holding potential of −70 mV that was dependent on an increase in intracellular Ca2+ after Gαq-coupled receptor activation. This current was mediated mostly by anions and was sensitive to well known anion channel blockers such as niflumic acid, 5-nitro-2(3-phenylpropylamino)-benzoic acid, and flufenamic acid. To find the molecular identity of the anion channel responsible for the CAAC current, we analyzed the expression of candidate genes and found that the mRNA for mouse mBest1 is predominantly expressed in acutely dissociated or cultured astrocytes. Whole-cell patch-clamp analysis using HEK293T cells heterologously expressing full-length mBest1 showed a Ca2+-dependent current mediated by mBest1, with a complete impairment of the current by a putative pore mutation, W93C. Furthermore, mBest1-mediated CAAC from cultured astrocytes was significantly reduced by expression of mBest1-specific short hairpin RNA (shRNA), suggesting that the CAAC is mediated by a channel encoded by mBest1. Finally, hippocampal CA1 astrocytes in hippocampal slice also showed mBest1-mediated CAAC because it was inhibited by mBest1-specific shRNA. Collectively, these data provide molecular evidence that the mBest1 channel is responsible for CAAC function in astrocytes.

A disulphide-linked heterodimer of TWIK-1 and TREK-1 mediates passive conductance in astrocytes

TWIK-1 is a member of the two-pore domain K(+) (K2P) channel family that plays an essential part in the regulation of resting membrane potential and cellular excitability. The physiological role of TWIK-1 has remained enigmatic because functional expression of TWIK-1 channels is elusive. Here we report that native TWIK-1 forms a functional channel at the plasma membrane of astrocytes. A search for TWIK-1-binding proteins led to the identification of TREK-1, another member of the K2P family. The TWIK-1/TREK-1 heterodimeric channel is formed via a disulphide bridge between residue C69 in TWIK-1 and C93 in TREK-1. Gene silencing demonstrates that surface expression of TWIK-1 and TREK-1 are interdependent. TWIK-1/TREK-1 heterodimers mediate astrocytic passive conductance and cannabinoid-induced glutamate release from astrocytes. Our study sheds new light on the diversity of K2P channels.

Protease activated receptor 1-induced glutamate release in cultured astrocytes is mediated by Bestrophin-1 channel but not by vesicular exocytosis

BackgroundGlutamate is the major transmitter that mediates the principal form of excitatory synaptic transmission in the brain. It has been well established that glutamate is released via Ca2+-dependent exocytosis of glutamate-containing vesicles in neurons. However, whether astrocytes exocytose to release glutamate under physiological condition is still unclear.FindingsWe report a novel form of glutamate release in astrocytes via the recently characterized Ca2+-activated anion channel, Bestrophin-1 (Best1) by Ca2+ dependent mechanism through the channel pore. We demonstrate that upon activation of protease activated receptor 1 (PAR1), an increase in intracellular Ca2+ concentration leads to an opening of Best1 channels and subsequent release of glutamate in cultured astrocytes.ConclusionsThese results provide strong molecular evidence for potential astrocyte-neuron interaction via Best1-mediated glutamate release.

Optical properties of AlxGa1−xP (0⩽x⩽0.52) alloys

We report optical properties of AlxGa1−xP (0⩽x⩽0.52) alloys grown by gas source molecular-beam epitaxy on S-doped GaP(001) substrates. Room-temperature pseudodielectric function spectra from 1.5 to 6.0 eV were obtained by spectroscopic ellipsometry. By applying the parabolic-band critical point model to numerically calculated second energy derivatives of these spectra, we obtained accurate room-temperature values of the E1, E0′, E2, and E2′ critical point energies and their dependence on composition x.

Ca2+ Entry is Required for Mechanical Stimulation-induced ATP Release from Astrocyte

Astrocytes and neurons are inseparable partners in the brain. Neurotransmitters released from neurons activate corresponding G protein-coupled receptors (GPCR) expressed in astrocytes, resulting in release of gliotransmitters such as glutamate, D-serine, and ATP. These gliotransmitters in turn influence neuronal excitability and synaptic activities. Among these gliotransmitters, ATP regulates the level of network excitability and is critically involved in sleep homeostasis and astrocytic Ca2+ oscillations. ATP is known to be released from astrocytes by Ca2+-dependent manner. However, the precise source of Ca2+, whether it is Ca2+ entry from outside of cell or from the intracellular store, is still not clear yet. Here, we performed sniffer patch to detect ATP release from astrocyte by using various stimulation. We found that ATP was not released from astrocyte when Ca2+ was released from intracellular stores by activation of Gαq-coupled GPCR including PAR1, P2YR, and B2R. More importantly, mechanical stimulation (MS)-induced ATP release from astrocyte was eliminated when external Ca2+ was omitted. Our results suggest that Ca2+ entry, but not release from intracellular Ca2+ store, is critical for MS-induced ATP release from astrocyte.

THERMAL STABILITY OF SULFUR-TREATED INP INVESTIGATED BY PHOTOLUMINESCENCE

The effect of sulfur (S) treatments on InP is investigated by low‐temperature photoluminescence (PL) measurements. For both n‐ and p‐InP, the PL intensity is observed to increase about four times in magnitude if the scattering by the S overlayer is relatively small. Some PL bands are observed to disappear after S treatments and then reappear if the S‐treated surface is heat treated at 220 °C in a vacuum of 10−3 Torr. By observing their dependence on the excitation power density, the doping level of the samples, and measurement temperature, these PL bands are ascribed to the optical transitions via surface states. Our results thus indicate that the S‐treated InP surface may not be stable at a subsequent processing temperature of about 250 °C.