Jinu Lee

Metabotropic glutamate receptors: Phosphorylation and receptor signaling

Metabotropic glutamate receptors (mGluRs) play important roles in neurotransmission, neuronal development, synaptic plasticity, and neurological disorders. Recent studies have revealed a sophisticated interplay between mGluRs and protein kinases: activation of mGluRs regulates the activity of a number of kinases, and direct phosphorylation of mGluRs affects receptor signaling, trafficking, and desensitization. Here we review the emerging literature on mGluR phosphorylation, signaling, and synaptic function.

Calmodulin dynamically regulates the trafficking of the metabotropic glutamate receptor mGluR5

Metabotropic glutamate receptors (mGluRs) 1–8 are G protein-coupled receptors (GPCRs) that modulate excitatory neurotransmission, neurotransmitter release, and synaptic plasticity. PKC regulates many aspects of mGluR function, including protein–protein interactions, Ca2+ signaling, and receptor desensitization. However, the mechanisms by which PKC regulates mGluR function are poorly understood. We have now identified calmodulin (CaM) as a dynamic regulator of mGluR5 trafficking. We show that the major PKC phosphorylation site on the intracellular C terminus of mGluR5 is serine 901 (S901), and phosphorylation of this residue is up-regulated in response to both receptor and PKC activation. In addition, S901 phosphorylation inhibits mGluR5 binding to CaM, decreasing mGluR5 surface expression. Furthermore, blocking PKC phosphorylation of mGluR5 on S901 dramatically affects mGluR5 signaling by prolonging Ca2+ oscillations. Thus, our data demonstrate that mGluR5 activation triggers phosphorylation of S901, thereby directly linking PKC phosphorylation, CaM binding, receptor trafficking, and downstream signaling.

Zinc-induced NF-κB inhibition can be modulated by changes in the intracellular metallothionein level

Metallothionein (MT), a small metal-binding protein, is involved in the regulation of cellular metal homeostasis. Sequestration and the release of metals to and from MT plays an important role in the attenuation or amplification of signal transduction. Zinc has been suggested to be an important regulator of nuclear factor kappaB (NF-kappaB). In this study, the effect of MT expression on the zinc-induced inhibition of NF-kappaB activity was examined. In HeLa cells, pyrrolidine dithiocarbamate (PDTC), a zinc ionophore, and zinc itself inhibited NF-kappaB activity. When the cells were pretreated with MT-inducers, cadmium, or dexamethasone, PDTC did not inhibit NF-kappaB activity. We transfected HeLa cells with a DNA construct in which expression of MT-IIA is controlled by tet operator protein. Treatment of HeLa cells with doxycycline, a tetracycline analogue, induced the expression of MT-IIA, which attenuated the effect of PDTC on NF-kappaB activity. These results implicate MT in the zinc regulation of NF-kappaB and identify MT as one of the potential intracellular modulators of NF-kappaB activation.

In vivo high-throughput profiling of CRISPR–Cpf1 activity

CRISPR from Prevotella and Francisella 1 (Cpf1) is an effector endonuclease of the class 2 CRISPR–Cas (clustered regularly interspaced short palindromic repeats–CRISPR-associated proteins) gene editing system. We developed a method for evaluating Cpf1 activity, based on target sequence composition in mammalian cells, in a high-throughput manner. A library of >11,000 target sequence and guide RNA pairs was delivered into human cells using lentiviral vectors. Subsequent delivery of Cpf1 into this cell library induced insertions and deletions (indels) at the integrated synthetic target sequences, which allowed en masse evaluation of Cpf1 activity by using deep sequencing. With this approach, we determined protospacer-adjacent motif sequences of two Cpf1 nucleases, one from Acidaminococcus sp. BV3L6 (hereafter referred to as AsCpf1) and the other from Lachnospiraceae bacterium ND2006 (hereafter referred to as LbCpf1). We also defined target-sequence-dependent activity profiles of AsCpf1, which enabled the development of a web tool that predicts the indel frequencies for given target sequences (http://big.hanyang.ac.kr/cindel). Both the Cpf1 characterization profile and the in vivo high-throughput evaluation method will greatly facilitate Cpf1-based genome editing.

Thiol antioxidant reversal of pyrrolidine ditiocarbamate-induced reciprocal regulation of AP-1 and NF-κB

Abstract Pyrrolidine dithiocarbamate (PDTC) has been shown to have unique reciprocal activities in activating AP-1 and inhibiting NF-κB, two oxidative stresssensitive transcription factors. The opposing effects of PDTC on these two transcription factors have been attributed to its thiol antioxidant properties. In the present study, PDTC activation of AP-1, like its inhibition of NFκB, in bovine cerebral endothelial cells (BCECs) was zinc-dependent, consistent with the contention that PDTC acts as a zinc ionophore and the apparent reciprocal actions of PDTC are mediated by zinc. Unlike PDTC, other thiols and non-thiol antioxidants did not activate AP-1 on their own. Thiol, but not nonthiol, antioxidants reversed PDTC actions on AP-1 and NF-κB. PDTC reduced the intracellular glutathione content, and depletion of the cellular glutathione store by buthionine sulfoximine (BSO) further augmented PDTC actions on AP-1 and NF-κB. Nacetyl cysteine (NAC), a thiol antioxidant, reversed PDTC actions even after irreversible depletion of the cellular glutathione store by BSO. These findings together suggest that thiol antioxidant reversal of PDTC actions on AP-1 and NF-κB is independent of their established roles in scavenging oxygen free radicals or repleting the cellular glutathione content. The results in the present and earlier studies suggest that thiol antioxidants are likely to act as metal chelators that buffer zinc mediation of the reciprocal actions of PDTC on AP-1 and NF-κB.

Zinc stimulates tau S214 phosphorylation by the activation of Raf/mitogen-activated protein kinase-kinase/extracellular signal-regulated kinase pathway.

Hyperphosphorylated tau is a main component of neurofibrillary tangles, a pathological hallmark of Alzheimers disease (AD). There is evidence that various protein kinases are involved in tau hyperphosphorylation. However, little is known about AD-related stimuli that activates tau kinases. We investigated the role of zinc, a metal involved in AD pathology, in tau phosphorylation. Zinc increased the phosphorylation of serine 214 (S214) in tau protein in human wild-type tau1-441-expressing SH-SY5Y cells. The phosphorylation was inhibited by suppressing the Ras-Raf/mitogen-activated protein kinase kinase/extracellular signal-regulated kinase (ERK) pathway. Mutation of serine to alanine at residue 214 of tau reduced microtubule polymerization impairment by ERK phosphorylation. These data suggest that zinc induces S214 phosphorylation in tau through ERK activation and interferes with microtubule polymerization.

dTULP, the Drosophila melanogaster Homolog of Tubby, Regulates Transient Receptor Potential Channel Localization in Cilia

Mechanically gated ion channels convert sound into an electrical signal for the sense of hearing. In Drosophila melanogaster, several transient receptor potential (TRP) channels have been implicated to be involved in this process. TRPN (NompC) and TRPV (Inactive) channels are localized in the distal and proximal ciliary zones of auditory receptor neurons, respectively. This segregated ciliary localization suggests distinct roles in auditory transduction. However, the regulation of this localization is not fully understood. Here we show that the Drosophila Tubby homolog, King tubby (hereafter called dTULP) regulates ciliary localization of TRPs. dTULP-deficient flies show uncoordinated movement and complete loss of sound-evoked action potentials. Inactive and NompC are mislocalized in the cilia of auditory receptor neurons in the dTulp mutants, indicating that dTULP is required for proper cilia membrane protein localization. This is the first demonstration that dTULP regulates TRP channel localization in cilia, and suggests that dTULP is a protein that regulates ciliary neurosensory functions.

Heparin Inhibits NF-κB Activation and Increases Cell Death in Cerebral Endothelial Cells after Oxygen-Glucose Deprivation

Heparin is a classic anticoagulant that is commonly used in the treatment of acute ischemic stroke (AIS). Its use remains controversial, however, due to the risk of cerebral hemorrhagic transformation. In addition to anticoagulant effects, diverse effects on transcription factors can be caused by heparin. Among the transcription factors potentially affected is nuclear factor kappa B (NF-κB), a protein that is reportedly related to the survival of cerebral endothelial cells. We investigated the effect of heparin on NF-κB activation and cell death following oxygen-glucose deprivation (OGD), an experimental model of AIS. We subjected bEnd.3 cells from a murine cerebral microvascular endothelial cell line to OGD. We examined the effect of heparin on OGD-induced NF-κB activation and its mechanism of action, using electrophoretic mobility shift assays, reporter gene analysis, real-time RT-PCR, Western blot analysis, and confocal microscopy. We also measured the effect of heparin on OGD-induced cell death using an MTT assay. Heparin inhibited both tumor necrosis factor α- and OGD-induced NF-κB activation. Heparin was taken up by endocytosis and then entered the nucleus. Heparin did not affect the nuclear translocation of NF-κB, but instead inhibited the DNA binding of NF-κB in the nucleus. Cells were more susceptible to OGD-induced cell death after heparin treatment. Besides producing an anticoagulation effect, heparin also inhibits NF-κB activation, resulting in increased susceptibility to OGD-induced cell death. This effect may be responsible for hemorrhagic transformation in patients following heparin treatment for AIS.

Zinc Inhibits Amyloid β Production from Alzheimer's Amyloid Precursor Protein in SH-SY5Y Cells

Zinc released from excited glutamatergic neurons accelerates amyloid beta (Abeta) aggregation, underscoring the therapeutic potential of zinc chelation for the treatment of Alzheimers disease (AD). Zinc can also alter Abeta concentration by affecting its degradation. In order to elucidate the possible role of zinc influx in secretase-processed Abeta production, SH-SY5Y cells stably expressing amyloid precursor protein (APP) were treated with pyrrolidine dithiocarbamate (PDTC), a zinc ionophore, and the resultant changes in APP processing were examined. PDTC decreased Abeta40 and Abeta42 concentrations in culture media bathing APP-expressing SH-SY5Y cells. Measuring the levels of a series of C-terminal APP fragments generated by enzymatic cutting at different APP-cleavage sites showed that both beta- and alpha-cleavage of APP were inhibited by zinc influx. PDTC also interfered with the maturation of APP. PDTC, however, paradoxically increased the intracellular levels of Abeta40. These results indicate that inhibition of secretase-mediated APP cleavage accounts -at least in part- for zinc inhibition of Abeta secretion.

Gap junctions contribute to astrocytic resistance against zinc toxicity.

Astrocytic gap junctions have been implicated in the regulation of cell viability. High amounts of extracellular zinc, which is released during ischemia, seizure, and brain trauma, can be cytotoxic to astrocytes. We tested whether gap junction coupling between astrocytes plays an important role in modulating zinc toxicity in hippocampal astrocytes. Zinc induces cell death in a dose-dependent manner in primary cultured hippocampal astrocytes. Two gap junction inhibitors, 18β-glycyrrhetinic acid and arachidonic acid, had no effect on zinc-induced cell death in low-confluence culture, where physical separation prevents gap junctions from forming. However, these inhibitors can potentiate zinc toxicity in high-confluence astrocyte cultures. Zinc toxicity was substantially suppressed upon connexin 43 overexpression, whereas knockdown caused a significant enhancement of the toxicity in high-confluence cultures. These data suggest that gap junctions in hippocampal astrocytes provide a protective role against zinc toxicity.