Publications by authors named "Wen-Hai Chou"

24 Publications

  • Page 1 of 1

Lipocalin-2 mediates the rejection of neural transplants.

FASEB J 2021 Feb;35(2):e21317

Center for Neuropsychiatric Research, National Health Research Institutes, Miaoli, Taiwan.

Lipocalin-2 (LCN2) has been implicated in promoting apoptosis and neuroinflammation in neurological disorders; however, its role in neural transplantation remains unknown. In this study, we cultured and differentiated Lund human mesencephalic (LUHMES) cells into human dopaminergic-like neurons and found that LCN2 mRNA was progressively induced in mouse brain after the intrastriatal transplantation of human dopaminergic-like neurons. The induction of LCN2 protein was detected in a subset of astrocytes and neutrophils infiltrating the core of the engrafted sites, but not in neurons and microglia. LCN2-immunoreactive astrocytes within the engrafted sites expressed lower levels of A1 and A2 astrocytic markers. Recruitment of microglia, neutrophils, and monocytes after transplantation was attenuated in LCN2 deficiency mice. The expression of M2 microglial markers was significantly elevated and survival of engrafted neurons was markedly improved after transplantation in LCN2 deficiency mice. Brain type organic cation transporter (BOCT), the cell surface receptor for LCN2, was induced in dopaminergic-like neurons after differentiation, and treatment with recombinant LCN2 protein directly induced apoptosis in dopaminergic-like neurons in a dose-dependent manner. Our results, therefore, suggested that LCN2 is a neurotoxic factor for the engrafted neurons and a modulator of neuroinflammation. LCN2 inhibition may be useful in reducing rejection after neural transplantation.
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http://dx.doi.org/10.1096/fj.202001018RDOI Listing
February 2021

Drosophila R8 photoreceptor cell subtype specification requires hibris.

PLoS One 2020 14;15(10):e0240451. Epub 2020 Oct 14.

Department of Neurology, Department of Ophthalmology, Dell Medical School, University of Texas at Austin, Austin, Texas, United States of America.

Cell differentiation and cell fate determination in sensory systems are essential for stimulus discrimination and coding of environmental stimuli. Color vision is based on the differential color sensitivity of retinal photoreceptors, however the developmental programs that control photoreceptor cell differentiation and specify color sensitivity are poorly understood. In Drosophila melanogaster, there is evidence that the color sensitivity of different photoreceptors in the compound eye is regulated by inductive signals between cells, but the exact nature of these signals and how they are propagated remains unknown. We conducted a genetic screen to identify additional regulators of this process and identified a novel mutation in the hibris gene, which encodes an irre cell recognition module protein (IRM). These immunoglobulin super family cell adhesion molecules include human KIRREL and nephrin (NPHS1). hibris is expressed dynamically in the developing Drosophila melanogaster eye and loss-of-function mutations give rise to a diverse range of mutant phenotypes including disruption of the specification of R8 photoreceptor cell diversity. We demonstrate that hibris is required within the retina, and that hibris over-expression is sufficient to disrupt normal photoreceptor cell patterning. These findings suggest an additional layer of complexity in the signaling process that produces paired expression of opsin genes in adjacent R7 and R8 photoreceptor cells.
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http://journals.plos.org/plosone/article?id=10.1371/journal.pone.0240451PLOS
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7556441PMC
December 2020

Neutralization of Lipocalin-2 Diminishes Stroke-Reperfusion Injury.

Int J Mol Sci 2020 Aug 29;21(17). Epub 2020 Aug 29.

Department of Biological Sciences, School of Biomedical Sciences, Kent State University, Kent, OH 44242, USA.

Oxidative stress is a key contributor to the pathogenesis of stroke-reperfusion injury. Neuroinflammatory peptides released after ischemic stroke mediate reperfusion injury. Previous studies, including ours, have shown that lipocalin-2 (LCN2) is secreted in response to cerebral ischemia to promote reperfusion injury. Genetic deletion of LCN2 significantly reduces brain injury after stroke, suggesting that LCN2 is a mediator of reperfusion injury and a potential therapeutic target. Immunotherapy has the potential to harness neuroinflammatory responses and provides neuroprotection against stroke. Here we report that LCN2 was induced on the inner surface of cerebral endothelial cells, neutrophils, and astrocytes that gatekeep the blood-brain barrier (BBB) after stroke. LCN2 monoclonal antibody (mAb) specifically targeted LCN2 in vitro and in vivo, attenuating the induction of LCN2 and pro-inflammatory mediators (iNOS, IL-6, CCL2, and CCL9) after stroke. Administration of LCN2 mAb at 4 h after stroke significantly reduced neurological deficits, cerebral infarction, edema, BBB leakage, and infiltration of neutrophils. The binding epitope of LCN2 mAb was mapped to the β3 and β4 strands, which are responsible for maintaining the integrity of LCN2 cup-shaped structure. These data indicate that LCN2 can be pharmacologically targeted using a specific mAb to reduce reperfusion injury after stroke.
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http://dx.doi.org/10.3390/ijms21176253DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7503651PMC
August 2020

Protective effects of ischemic preconditioning against neuronal apoptosis and dendritic injury in the hippocampus are age-dependent.

J Neurochem 2020 11 15;155(4):430-447. Epub 2020 Jun 15.

Department of Neurology, Faculty of Medicine, School of Medicine, National Yang-Ming University, Taipei, Taiwan.

Ischemic preconditioning with non-lethal ischemia can be protective against lethal forebrain ischemia. We hypothesized that aging may aggravate ischemic susceptibility and reduce brain plasticity against preconditioning. Magnetic resonance diffusion tensor imaging (DTI) is a sensitive tool to detect brain integrity and white matter architecture. This study used DTI and histopathology to investigate the effect of aging on ischemic preconditioning. In this study, adult and middle-aged male Mongolian gerbils were subjected to non-lethal 5-min forebrain ischemia (ischemic preconditioning) or sham-operation, followed by 3 days of reperfusion, and then lethal 15-min forebrain ischemia. A 9.4-Tesla MR imaging system was used to study DTI indices, namely fractional anisotropy (FA), mean diffusivity (MD), and intervoxel coherence (IC) in the hippocampal CA1 and dentate gyrus (DG) areas. In situ expressions of microtubule-associated protein 2 (MAP2, dendritic marker protein) and apoptosis were also examined. The 5-min ischemia did not cause dendritic and neuronal injury and any significant change in DTI indices and MAP2 in adult and middle-aged gerbils. The 15-min ischemia-induced significant delayed neuronal apoptosis and early dendritic injury evidenced by DTI and MAP2 studies in both CA1 and DG areas with more severe injury in middle-aged gerbils than adult gerbils. Ischemic preconditioning could improve neuronal apoptosis in CA1 area and dendritic integrity in both CA1 and DG areas with better improvement in adult gerbils than middle-aged gerbils. This study thus suggests an age-dependent protective effect of ischemic preconditioning against both neuronal apoptosis and dendritic injury in hippocampus after forebrain ischemia.
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http://dx.doi.org/10.1111/jnc.15029DOI Listing
November 2020

Serum Level and Activity of Butylcholinesterase: A Biomarker for Post-Stroke Dementia.

J Clin Med 2019 Oct 24;8(11). Epub 2019 Oct 24.

Center for Neuropsychiatric Research, National Health Research Institutes, Miaoli County 35053, Taiwan.

Cholinergic neurotransmission regulates the immune response and inhibits cytokine release after stroke. The changes in the level/activity of blood cholinesterase (ChE) in patients with post-stroke dementia (PSD) are less known. This study aimed to examine post-stroke plasma acetylcholinesterase (AChE) and butylcholinesterase (BChE) and determine whether they are biomarkers for PSD. Thirty patients with PSD, 87 post-stroke patients without dementia (PSNoD), and 117 age- and gender-matched healthy controls were recruited. Missense genetic variants rs1799806 and rs1803274 were genotyped. The plasma AChE level did not differ between the PSD and PSNoD groups. However, BChE levels were significantly lower in the PSD than in the PSNoD group (3300.66 ± 515.35 vs 3855.74 ± 677.60 ng/mL, respectively; = 0.0033). The activities of total ChE, BChE, and AChE were all lower in the PSD group (19,563.33 ± 4366.03, 7650.17 ± 1912.29, 11,913.17 ± 2992.42 mU/mL, respectively) than in the PSNoD group (23,579.08 ± 5251.55, 9077.72 ± 1727.28, and 14,501.36 ± 4197.17 mU/mL, respectively). When further adjusting for age and sex, significance remained in BChE level and activity and in total ChE activity. rs1803274 was associated with reduced BChE activity, while rs1799806 did not influence AChE activity. The level and activity of BChE, but not of AChE, were decreased in PSD patients and may therefore aid in PSD diagnosis.
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http://dx.doi.org/10.3390/jcm8111778DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6912582PMC
October 2019

A Study of D-Amino Acid Oxidase in Blood as an Indicator of Post-stroke Dementia.

Front Neurol 2019 26;10:402. Epub 2019 Apr 26.

Center for Neuropsychiatric Research, National Health Research Institutes, Miaoli, Taiwan.

Stroke is an important risk factor for dementia. Epidemiological studies have indicated a high incidence of dementia in stroke patients. There is currently no effective biomarker for the diagnosis of post-stroke dementia (PSD). D-amino acid oxidase (DAO) is a flavin-dependent enzyme widely distributed in the central nervous system. DAO oxidizes D-amino acids, a process which generates neurotoxic hydrogen peroxide and leads to neurodegeneration. This study aimed to examine post-stroke plasma DAO levels as a biomarker for PSD. In total, 53 patients with PSD, 20 post-stroke patients without dementia (PSNoD), and 71 age- and gender-matched normal controls were recruited. Cognitive function was evaluated at more than 30 days post-stroke. Plasma DAO was measured using the enzyme-linked immunosorbent assay. White matter hyperintensity (WMH), a neuroimaging biomarker of cerebral small vessel diseases, was determined by magnetic resonance imaging. We found that plasma DAO levels were independently higher in PSD subjects than in PSNoD subjects or the controls and were correlated with the WMH load in stroke patients. Using an area under the curve (AUC)/receiver operating characteristic analysis, plasma DAO levels were significantly reliable for the diagnosis of PSD. The sensitivity and specificity of the optimal cut-off value of 321 ng/ml of plasma DAO for the diagnosis of PSD were 75 and 88.7%, respectively. In conclusion, our data support that plasma DAO levels were increased in PSD patients and correlated with brain WMH, independent of age, gender, hypertension, and renal function. Plasma DAO levels may therefore aid in PSD diagnosis.
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http://dx.doi.org/10.3389/fneur.2019.00402DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6497996PMC
April 2019

PKCε phosphorylation regulates the mitochondrial translocation of ATF2 in ischemia-induced neurodegeneration.

BMC Neurosci 2018 Nov 29;19(1):76. Epub 2018 Nov 29.

Department of Biological Sciences, School of Biomedical Sciences, Kent State University, Kent, OH, 44242, USA.

Background: Global cerebral ischemia triggers neurodegeneration in the hippocampal CA1 region, but the mechanism of neuronal death remains elusive. The epsilon isoform of protein kinase C (PKCε) has recently been identified as a master switch that controls the nucleocytoplasmic trafficking of ATF2 and the survival of melanoma cells. It is of interest to assess the role of PKCε-ATF2 signaling in neurodegeneration.

Results: Phosphorylation of ATF2 at Thr-52 was reduced in the hippocampus of PKCε null mice, suggesting that ATF2 is a phosphorylation substrate of PKCε. PKCε protein concentrations were significantly reduced 4, 24, 48 and 72 h after transient global cerebral ischemia, resulting in translocation of nuclear ATF2 to the mitochondria. Degenerating neurons staining positively with Fluoro-Jade C exhibited cytoplasmic ATF2.

Conclusions: Our results support the hypothesis that PKCε regulates phosphorylation and nuclear sequestration of ATF2 in hippocampal neurons during ischemia-induced neurodegeneration.
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http://dx.doi.org/10.1186/s12868-018-0479-zDOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6267029PMC
November 2018

Genetic inhibition of PKCε attenuates neurodegeneration after global cerebral ischemia in male mice.

J Neurosci Res 2019 04 29;97(4):444-455. Epub 2018 Nov 29.

Department of Biological Sciences, School of Biomedical Sciences, Kent State University, Kent, Ohio.

Global cerebral ischemia that accompanies cardiac arrest is a major cause of morbidity and mortality. Protein Kinase C epsilon (PKCε) is a member of the novel PKC subfamily and plays a vital role in ischemic preconditioning. Pharmacological activation of PKCε before cerebral ischemia confers neuroprotection. The role of endogenous PKCε after cerebral ischemia remains elusive. Here we used male PKCε-null mice to assess the effects of PKCε deficiency on neurodegeneration after transient global cerebral ischemia (tGCI). We found that the cerebral vasculature, blood flow, and the expression of other PKC isozymes were not altered in the PKCε-null mice. Spatial learning and memory was impaired after tGCI, but the impairment was attenuated in male PKCε-null mice as compared to male wild-type controls. A significant reduction in Fluoro-Jade C labeling and mitochondrial release of cytochrome C in the hippocampus was found in male PKCε-null mice after tGCI. Male PKCε-null mice expressed increased levels of PKCδ in the mitochondria, which may prevent the translocation of PKCδ from the cytosol to the mitochondria after tGCI. Our results demonstrate the neuroprotective effects of PKCε deficiency on neurodegeneration after tGCI, and suggest that reduced mitochondrial translocation of PKCδ may contribute to the neuroprotective action in male PKCε-null mice.
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http://dx.doi.org/10.1002/jnr.24362DOI Listing
April 2019

Lipocalin-2 in Stroke.

Neuro 2015 Aug 17;2(1):38-41. Epub 2015 Apr 17.

Department of Biological Sciences, School of Biomedical Sciences, Kent State University, Kent, OH 44224, USA.

Stroke is a leading cause of adult disability in the United States. However, limited number of molecularly targeted therapy exists for stroke. Recent studies have shown that Li-pocalin-2 (LCN2) is an acute phase protein mediating neuroinflammation after ischemic and hemorrhagic strokes. This review is an attempt to summarize some LCN2-related research findings and discuss its role in stroke.
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http://dx.doi.org/10.17140/NOJ-2-109DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6287915PMC
August 2015

Identification of lipocalin-2 as a PKCδ phosphorylation substrate in neutrophils.

J Biomed Sci 2015 Mar 20;22:21. Epub 2015 Mar 20.

Department of Biological Sciences, School of Biomedical Sciences, Kent State University, Kent, OH, 44224, USA.

Background: PKCδ expressed in neutrophils is implicated in promoting reperfusion injury after ischemic stroke. To understand the molecular and cellular actions of PKCδ, we employed a chemical-genetics approach to identify PKCδ substrates in neutrophils.

Results: We recently generated knock-in mice endogenously expressing analog-specific PKCδ (AS-PKCδ) that can utilize ATP analogs as phosphate donors. Using neutrophils isolated from the knock-in mice, we identified several PKCδ substrates, one of which was lipocalin-2 (LCN2), which is an iron-binding protein that can trigger apoptosis by reducing intracellular iron concentrations. We found that PKCδ phosphorylated LCN2 at T115 and this phosphorylation was reduced in Prkcd (-/-) mice. PKCδ colocalized with LCN2 in resting and stimulated neutrophils. LCN2 release from neutrophils after cerebral ischemia was reduced in PKCδ null mice.

Conclusions: These findings suggest that PKCδ phosphorylates LCN2 and mediates its release from neutrophils during ischemia-reperfusion injury.
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http://dx.doi.org/10.1186/s12929-015-0129-zDOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4396066PMC
March 2015

Lipocalin-2 released in response to cerebral ischaemia mediates reperfusion injury in mice.

J Cell Mol Med 2015 Jul 20;19(7):1637-45. Epub 2015 Feb 20.

Department of Biological Sciences and School of Biomedical Sciences, Kent State University, Kent, OH, USA.

Thrombolysis remains the only effective therapy to reverse acute ischaemic stroke. However, delayed treatment may cause serious complications including hemorrhagic transformation and reperfusion injury. The level of lipocalin-2 (LCN2) is elevated in the plasma of ischaemic stroke patients, but its role in stroke is unknown. Here, we show that LCN2 was acutely induced in mice after ischaemic stroke and is an important mediator of reperfusion injury. Increased levels of LCN2 were observed in mouse serum as early as 1 hr after transient middle cerebral artery occlusion (tMCAO), reaching peak levels at 23 hrs. LCN2 was also detected in neutrophils infiltrating into the ipsilateral hemisphere, as well as a subset of astrocytes after tMCAO, but not in neurons and microglia. Stroke injury, neurological deficits and infiltration of immune cells were markedly diminished in LCN2 null mice after tMCAO, but not after permanent MCAO (pMCAO). In vitro, recombinant LCN2 protein induced apoptosis in primary cultured neurons in a dose-dependent manner. Our results demonstrate that LCN2 is a neurotoxic factor secreted rapidly in response to cerebral ischaemia, suggesting its potential usage as an early stroke biomarker and a novel therapeutic target to reduce stroke-reperfusion injury.
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http://dx.doi.org/10.1111/jcmm.12538DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4511361PMC
July 2015

Generation and characterization of ATP analog-specific protein kinase Cδ.

J Biol Chem 2015 Jan 10;290(4):1936-51. Epub 2014 Dec 10.

From the Department of Biological Sciences, School of Biomedical Sciences, Kent State University, Kent, Ohio 44242, the Ernest Gallo Clinic and Research Center, Department of Neurology, University of California, San Francisco, Emeryville, California 94608, and

To better study the role of PKCδ in normal function and disease, we developed an ATP analog-specific (AS) PKCδ that is sensitive to specific kinase inhibitors and can be used to identify PKCδ substrates. AS PKCδ showed nearly 200 times higher affinity (Km) and 150 times higher efficiency (kcat/Km) than wild type (WT) PKCδ toward N(6)-(benzyl)-ATP. AS PKCδ was uniquely inhibited by 1-(tert-butyl)-3-(1-naphthyl)-1H-pyrazolo[3,4-d]pyrimidin-4-amine (1NA-PP1) and 1-(tert-butyl)-3-(2-methylbenzyl)-1H-pyrazolo[3,4-d]pyrimidin-4-amine (2MB-PP1) but not by other 4-amino-5-(4-methylphenyl)-7-(t-butyl)pyrazolo[3,4-d]pyrimidine (PP1) analogs tested, whereas WT PKCδ was insensitive to all PP1 analogs. To understand the mechanisms for specificity and affinity of these analogs, we created in silico WT and AS PKCδ homology models based on the crystal structure of PKCι. N(6)-(Benzyl)-ATP and ATP showed similar positioning within the purine binding pocket of AS PKCδ, whereas N(6)-(benzyl)-ATP was displaced from the pocket of WT PKCδ and was unable to interact with the glycine-rich loop that is required for phosphoryl transfer. The adenine rings of 1NA-PP1 and 2MB-PP1 matched the adenine ring of ATP when docked in AS PKCδ, and this interaction prevented the potential interaction of ATP with Lys-378, Glu-428, Leu-430, and Phe-633 residues. 1NA-PP1 failed to effectively dock within WT PKCδ. Other PP1 analogs failed to interact with either AS PKCδ or WT PKCδ. These results provide a structural basis for the ability of AS PKCδ to efficiently and specifically utilize N(6)-(benzyl)-ATP as a phosphate donor and for its selective inhibition by 1NA-PP1 and 2MB-PP1. Such homology modeling could prove useful in designing molecules to target PKCδ and other kinases to understand their function in cell signaling and to identify unique substrates.
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http://dx.doi.org/10.1074/jbc.M114.598698DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4303651PMC
January 2015

Rhodopsin 5- and Rhodopsin 6-mediated clock synchronization in Drosophila melanogaster is independent of retinal phospholipase C-β signaling.

J Biol Rhythms 2012 Feb;27(1):25-36

School of Biological and Chemical Sciences, Queen Mary, University of London, London, United Kingdom.

Circadian clocks of most organisms are synchronized with the 24-hour solar day by the changes of light and dark. In Drosophila, both the visual photoreceptors in the compound eyes as well as the blue-light photoreceptor Cryptochrome expressed within the brain clock neurons contribute to this clock synchronization. A specialized photoreceptive structure located between the retina and the optic lobes, the Hofbauer-Buchner (H-B) eyelet, projects to the clock neurons in the brain and also participates in light synchronization. The compound eye photoreceptors and the H-B eyelet contain Rhodopsin photopigments, which activate the canonical invertebrate phototransduction cascade after being excited by light. We show here that 2 of the photopigments present in these photoreceptors, Rhodopsin 5 (Rh5) and Rhodopsin 6 (Rh6), contribute to light synchronization in a mutant (norpA(P41) ) that disrupts canonical phototransduction due to the absence of Phospholipase C-β (PLC-β). We reveal that norpA(P41) is a true loss-of-function allele, resulting in a truncated PLC-β protein that lacks the catalytic domain. Light reception mediated by Rh5 and Rh6 must therefore utilize either a different (nonretinal) PLC-β enzyme or alternative signaling mechanisms, at least in terms of clock-relevant photoreception. This novel signaling mode may distinguish Rhodopsin-mediated irradiance detection from image-forming vision in Drosophila.
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http://dx.doi.org/10.1177/0748730411431673DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4405110PMC
February 2012

Mouse model of middle cerebral artery occlusion.

J Vis Exp 2011 Feb 13(48). Epub 2011 Feb 13.

Department of Neurology, Ernest Gallo Clinic and Research Center, University of California, San Francisco, USA.

Stroke is the most common fatal neurological disease in the United States. The majority of strokes (88%) result from blockage of blood vessels in the brain (ischemic stroke). Since most ischemic strokes (~80%) occur in the territory of middle cerebral artery (MCA), many animal stroke models that have been developed have focused on this artery. The intraluminal monofilament model of middle cerebral artery occlusion (MCAO) involves the insertion of a surgical filament into the external carotid artery and threading it forward into the internal carotid artery (ICA) until the tip occludes the origin of the MCA, resulting in a cessation of blood flow and subsequent brain infarction in the MCA territory. The technique can be used to model permanent or transient occlusion. If the suture is removed after a certain interval (30 min, 1 h, or 2 h), reperfusion is achieved (transient MCAO); if the filament is left in place (24 h) the procedure is suitable as a model of permanent MCAO. This technique does not require craniectomy, a neurosurgical procedure to remove a portion of skull, which may affect intracranial pressure and temperature. It has become the most frequently used method to mimic permanent and transient focal cerebral ischemia in rats and mice. To evaluate the extent of cerebral infarction, we stain brain slices with 2,3,5-triphenyltetrazolium chloride (TTC) to identify ischemic brain tissue. In this video, we demonstrate the MCAO method and the determination of infarct size by TTC staining.
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http://dx.doi.org/10.3791/2761DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3197421PMC
February 2011

GABAA receptor trafficking is regulated by protein kinase C(epsilon) and the N-ethylmaleimide-sensitive factor.

J Neurosci 2010 Oct;30(42):13955-65

Ernest Gallo Clinic and Research Center, Department of Neurology, University of California, San Francisco, Emeryville, California 94688, USA.

Disturbances in GABA(A) receptor trafficking contribute to several neurological and psychiatric disorders by altering inhibitory neurotransmission. Identifying mechanisms that regulate GABA(A) receptor trafficking could lead to better understanding of disease pathogenesis and treatment. Here, we show that protein kinase Cε (PKCε) regulates the N-ethylmaleimide-sensitive factor (NSF), an ATPase critical for membrane fusion events, and thereby promotes the trafficking of GABA(A) receptors. Activation of PKCε decreased cell surface expression of GABA(A) receptors and attenuated GABA(A) currents. Activated PKCε associated with NSF, phosphorylated NSF at serine 460 and threonine 461, and increased NSF ATPase activity, which was required for GABA(A) receptor downregulation. These findings identify new roles for NSF and PKCε in regulating synaptic inhibition through downregulation of GABA(A) receptors. Reducing NSF activity by inhibiting PKCε could help restore synaptic inhibition in disease states in which it is impaired.
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http://dx.doi.org/10.1523/JNEUROSCI.0270-10.2010DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC2994917PMC
October 2010

Disruption of photoreceptor cell patterning in the Drosophila Scutoid mutant.

Fly (Austin) 2009 Oct-Dec;3(4):253-62. Epub 2009 Oct 7.

Department of Cell and Developmental Biology, University of Colorado Denver, School of Medicine, Aurora, CO, USA.

Cell fate determination in many systems is based upon inductive events driven by cell-cell interactions. Inductive signaling regulates many aspects of Drosophila compound eye development. Accumulating evidence suggests that the color sensitivity of the R8 photoreceptor cell within an individual ommatidium is regulated by an inductive signal from the adjacent R7 photoreceptor cell. This signal is thought to control an induced versus default cell-fate switch that coordinates the visual pigment expression and color sensitivities of adjacent R7 and R8 photoreceptor cells. Here we describe a disruption in R7 and R8 cell patterning in Scutoid mutants that is due to inappropriate signals from Rh4-expressing R7 cells inducing Rh5 expression in adjacent R8 cells. This dominant phenotype results from the misexpression of the transcriptional repressor snail, which with the co-repressor C-terminal-Binding-Protein represses rhomboid expression in the developing eye. We show that loss of rhomboid suppresses the Scutoid phenotype. However in contrast to the loss of rhomboid alone, which entirely blocks the normal inductive signal from the R7 to the R8 photoreceptor cell, Scutoid rhomboid double mutants display normal Rh5 and Rh6 expression. Our detailed analysis of this unusual dominant gain-of-function neomorphic phenotype suggests that the induction of Rh5 expression in Scutoid mutants is partially rhomboid independent.
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http://www.ncbi.nlm.nih.gov/pmc/articles/PMC2836898PMC
http://dx.doi.org/10.4161/fly.10546DOI Listing
March 2010

rhomboid mediates specification of blue- and green-sensitive R8 photoreceptor cells in Drosophila.

J Neurosci 2009 Mar;29(9):2666-75

Department of Cell and Developmental Biology, University of Colorado Denver, School of Medicine, Aurora, Colorado 80045, USA.

Color vision is based on the differential color sensitivity of retinal photoreceptors, however the developmental programs that control photoreceptor cell differentiation and specify color sensitivity are poorly understood. In Drosophila there is growing evidence that the color sensitivity of the R8 cell within an individual ommatidium is regulated by an inductive signal from the adjacent R7 cell. We previously examined the retinal patterning defect in Scutoid mutants, which results from a disruption of rhomboid expression. Here we show that loss of rhomboid blocks the induction of Rh5 expression and misexpression of rhomboid leads to the inappropriate induction of Rh5. These effects are specific to rhomboid, because its paralogue roughoid is neither required nor sufficient for the induction of Rh5 expression. We show that rhomboid is required cell-autonomously within the R8 photoreceptor cells and nonautonomously elsewhere in the eye for Rh5 induction. Interestingly, we found that the Epidermal growth factor receptor is also required for Rh5 induction, and its activation is sufficient to rescue the loss of Rh5 induction in a rhomboid mutant. This suggests that rhomboid may function in R8 cells to activate Epidermal growth factor receptor signaling in R7 cells and promote their differentiation to a signaling competent state.
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http://dx.doi.org/10.1523/JNEUROSCI.5988-08.2009DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC2679528PMC
March 2009

Hypertensive encephalopathy and the blood-brain barrier: is deltaPKC a gatekeeper?

J Clin Invest 2008 Jan;118(1):17-20

Ernest Gallo Clinic and Research Center, Department of Neurology, UCSF, Emeryville, California 94608, USA.

Hypertensive encephalopathy is a life-threatening condition due to elevation of cerebral perfusion pressure beyond the limits of autoregulation. Breakdown of the blood-brain barrier (BBB) leads to cerebral edema and reduced blood flow. In this issue of the JCI, Mochly-Rosen and colleagues demonstrate a novel molecular strategy for preserving the BBB in a model of hypertension-induced encephalopathy (see the related article beginning on page 173). Using a rationally designed peptide inhibitor of deltaPKC, they stabilized the BBB and improved mortality in hypertensive rats. This study highlights the therapeutic potential of deltaPKC inhibitors in hypertensive encephalopathy and provides incentive to elucidate deltaPKC signaling pathways that mediate BBB dysfunction in other disease states.
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http://dx.doi.org/10.1172/JCI34516DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC2147677PMC
January 2008

Protein kinase C epsilon regulates gamma-aminobutyrate type A receptor sensitivity to ethanol and benzodiazepines through phosphorylation of gamma2 subunits.

J Biol Chem 2007 Nov 17;282(45):33052-63. Epub 2007 Sep 17.

Ernest Gallo Clinic and Research Center, Department of Neurology, University of California-San Francisco, 5858 Horton Street, Emeryville, CA 94608, USA.

Ethanol enhances gamma-aminobutyrate (GABA) signaling in the brain, but its actions are inconsistent at GABA(A) receptors, especially at low concentrations achieved during social drinking. We postulated that the epsilon isoform of protein kinase C (PKCepsilon) regulates the ethanol sensitivity of GABA(A) receptors, as mice lacking PKCepsilon show an increased behavioral response to ethanol. Here we developed an ATP analog-sensitive PKCepsilon mutant to selectively inhibit the catalytic activity of PKCepsilon. We used this mutant and PKCepsilon(-/-) mice to determine that PKCepsilon phosphorylates gamma2 subunits at serine 327 and that reduced phosphorylation of this site enhances the actions of ethanol and benzodiazepines at alpha1beta2gamma2 receptors, which is the most abundant GABA(A) receptor subtype in the brain. Our findings indicate that PKCepsilon phosphorylation of gamma2 regulates the response of GABA(A) receptors to specific allosteric modulators, and, in particular, PKCepsilon inhibition renders these receptors sensitive to low intoxicating concentrations of ethanol.
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http://dx.doi.org/10.1074/jbc.M707233200DOI Listing
November 2007

A semisynthetic epitope for kinase substrates.

Nat Methods 2007 Jun 7;4(6):511-6. Epub 2007 May 7.

Graduate Program in Chemistry and Chemical Biology, University of California, San Francisco, San Francisco, California 94143, USA.

The ubiquitous nature of protein phosphorylation makes it challenging to map kinase-substrate relationships, which is a necessary step toward defining signaling network architecture. To trace the activity of individual kinases, we developed a semisynthetic reaction scheme, which results in the affinity tagging of substrates of the kinase in question. First, a kinase, engineered to use a bio-orthogonal ATPgammaS analog, catalyzes thiophosphorylation of its direct substrates. Second, alkylation of thiophosphorylated serine, threonine or tyrosine residues creates an epitope for thiophosphate ester-specific antibodies. We demonstrated the generality of semisynthetic epitope construction with 13 diverse kinases: JNK1, p38alpha MAPK, Erk1, Erk2, Akt1, PKCdelta, PKCepsilon, Cdk1/cyclinB, CK1, Cdc5, GSK3beta, Src and Abl. Application of this approach, in cells isolated from a mouse that expressed endogenous levels of an analog-specific (AS) kinase (Erk2), allowed purification of a direct Erk2 substrate.
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http://dx.doi.org/10.1038/nmeth1048DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC2932705PMC
June 2007

Acute functional tolerance to ethanol mediated by protein kinase Cepsilon.

Neuropsychopharmacology 2007 Jan 15;32(1):127-36. Epub 2006 Mar 15.

Ernest Gallo Clinic and Research Center, Emeryville, CA 94608, USA.

A low level of response to ethanol is associated with increased risk of alcoholism. A major determinant of the level of response is the capacity to develop acute functional tolerance (AFT) to ethanol during a single drinking session. Mice lacking protein kinase C epsilon (PKCepsilon) show increased signs of ethanol intoxication and reduced ethanol self-administration. Here, we report that AFT to the motor-impairing effects of ethanol is reduced in PKCepsilon (-/-) mice when compared with wild-type littermates. In wild-type mice, in vivo ethanol exposure produced AFT that was accompanied by increased phosphorylation of PKCepsilon and resistance of GABA(A) receptors to ethanol. In contrast, in PKCepsilon (-/-) mice, GABA(A) receptor sensitivity to ethanol was unaltered by acute in vivo ethanol exposure. Both PKCepsilon (-/-) and PKCepsilon (+/+) mice developed robust chronic tolerance to ethanol, but the presence of chronic tolerance did not change ethanol preference drinking. These findings suggest that ethanol activates a PKCepsilon signaling pathway that contributes to GABA(A) receptor resistance to ethanol and to AFT. AFT can be genetically dissociated from chronic tolerance, which is not regulated by PKCepsilon and does not alter PKCepsilon modulation of ethanol preference.
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http://dx.doi.org/10.1038/sj.npp.1301059DOI Listing
January 2007

Protein kinase C isozymes in stroke.

Trends Cardiovasc Med 2005 Feb;15(2):47-51

Ernest Gallo Clinic and Research Center, University of California San Francisco, Emeryville, California 94608, USA.

Stroke is a devastating neurologic disease and a leading cause of death and disability worldwide. Thrombolytic agents have been used to re-establish circulation in thromboembolic stroke, but their utility is limited by hemorrhage and reperfusion injury. Studies with experimental stroke models, mouse genetics, and selective peptide inhibitors and activators have implicated protein kinase C (PKC) epsilon in ischemic preconditioning and PKCdelta and gamma in tissue injury. PKCdelta, resident both in neutrophils and in the brain, appears particularly essential for reperfusion injury, and recent work using PKCdelta-specific peptide inhibitors suggests that PKCdelta inhibitors could prove useful in attenuating reperfusion injury and improving outcome following thrombolysis.
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http://dx.doi.org/10.1016/j.tcm.2005.01.003DOI Listing
February 2005

Neutrophil protein kinase Cdelta as a mediator of stroke-reperfusion injury.

J Clin Invest 2004 Jul;114(1):49-56

Ernest Gallo Clinic and Research Center at the University of California San Francisco, Emeryville, California 94608, USA.

Thrombolysis is widely used to intervene in acute ischemic stroke, but reestablishment of circulation may paradoxically initiate a reperfusion injury. Here we describe studies with mice lacking protein kinase Cdelta (PKCdelta) showing that absence of this enzyme markedly reduces reperfusion injury following transient ischemia. This was associated with reduced infiltration of peripheral blood neutrophils into infarcted tissue and with impaired neutrophil adhesion, migration, respiratory burst, and degranulation in vitro. Total body irradiation followed by transplantation with bone marrow from PKCdelta-null mice donors reduced infarct size and improved neurological outcome in WT mice, whereas marrow transplantation from WT donors increased infarction and worsened neurological scores in PKCdelta-null mice. These results indicate an important role for neutrophil PKCdelta in reperfusion injury and strongly suggest that PKCdelta inhibitors could prove useful in the treatment of stroke.
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http://dx.doi.org/10.1172/JCI21655DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC437973PMC
July 2004

Heterologous expression of limulus rhodopsin.

J Biol Chem 2003 Oct 23;278(42):40493-502. Epub 2003 Jun 23.

Department of Ophthalmology, SUNY Upstate Medical University, Syracuse, New York 13210, USA.

Invertebrates such as Drosophila or Limulus assemble their visual pigment into the specialized rhabdomeric membranes of photoreceptors where phototransduction occurs. We have investigated the biosynthesis of rhodopsin from the Limulus lateral eye with three cell culture expression systems: mammalian COS1 cells, insect Sf9 cells, and amphibian Xenopus oocytes. We extracted and affinity-purified epitope-tagged Limulus rhodopsin expressed from a cDNA or cRNA from these systems. We found that all three culture systems could efficiently synthesize the opsin polypeptide in quantities comparable with that found for bovine opsin. However, none of the systems expressed a protein that stably bound 11-cis-retinal. The protein expressed in COS1 and Sf9 cells appeared to be misfolded, improperly localized, and proteolytically degraded. Similarly, Xenopus oocytes injected with Limulus opsin cRNA did not evoke light-sensitive currents after incubation with 11-cis-retinal. However, injecting Xenopus oocytes with mRNA from Limulus lateral eyes yielded light-dependent conductance changes after incubation with 11-cis-retinal. Also, expressing Limulus opsin cDNA in the R1-R6 photoreceptors of transgenic Drosophila yielded a visual pigment that bound retinal, had normal spectral properties, and coupled to the endogenous phototransduction cascade. These results indicate that Limulus opsin may require one or more photoreceptor-specific proteins for correct folding and/or chromophore binding. This may be a general property of invertebrate opsins and may underlie some of the functional differences between invertebrate and vertebrate visual pigments.
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http://dx.doi.org/10.1074/jbc.M304567200DOI Listing
October 2003