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1.  GSK3 INHIBITION PREVENTS LEARNING DEFICITS IN DIABETIC MICE 
Journal of neuroscience research  2013;91(4):506-514.
There is an increasing awareness that diabetes has an impact on the central nervous system, with reports of impaired learning, memory and mental flexibility being more common in diabetic subjects than in the general population. Insulin-deficient diabetic mice also display learning deficits associated with defective insulin-signaling in the brain and increased activity of GSK3. In the present study, AR-A014418, a GSK3β inhibitor and TX14(A), a neurotrophic factor with GSK3 inhibitory properties, were tested against the development of learning deficits in mice with insulin-deficient diabetes. Treatments were started at onset of diabetes and continued for 10 weeks.
Treatment with AR-A014418 or TX14(A) prevented the development of learning deficits, assessed by the Barnes maze, while only AR-A014418 prevented memory deficits, as assessed by the object recognition test. Diabetes-induced increased levels of amyloid beta protein and phosphorylated tau were not significantly affected by the treatments. However the diabetes-induced decrease in synaptophysin, a presynaptic protein marker of hippocampal plasticity, was partially prevented by both treatments. These results suggest a role for GSK3 and/or reduced neurotrophic support in the development of cognitive deficits in diabetic mice that are associated with synaptic damage.
doi:10.1002/jnr.23192
PMCID: PMC3570718  PMID: 23362012
Diabetes; Brain; GSK3; cognitive functions; synaptic plasticity
2.  O-Linked β-N-Acetylglucosaminylation in Mouse Embryonic Neural Precursor Cells 
Journal of neuroscience research  2009;87(16):3535-3545.
In neural stem cells (NSCs), glycoconjugates and carbohydrate antigens are known not only to serve as excellent cell surface biomarkers for cellular differentiation and development but also to play important functional roles in determining cell fate. O-linked β-N-acetylglucosamine (O-GlcNAc), which modifies nuclear and cytoplasmic proteins on the serine and threonine residues, is also expected to play an important regulatory role. It is not known, however, whether O-GlcNAc is expressed in NSCs or what the function of this expression is. In this study, we evaluated the patterns and possible functions O-GlcNAcylation in mouse embryonic neuroepithelial cells (NECs), which are known to be rich in NSCs. We confirmed the expression of O-GlcNAc transferase, O-GlcNAcase, and several O-GlcNAcylated proteins in NECs. Treatment of NECs with O-GlcNAcase inhibitors, PUGNAc and streptozotocin, induced robust accumulation of O-GlcNAc in NECs and reduction of number of NECs. In O-GlcNAcase inhibitor-treated NECs, the Rasmitogen-activated protein kinase pathway and the phosphoinositide 3-kinase-Akt pathway, important for proliferation and survival, respectively, were intact, but caspase-3, an executioner for cell death, was activated. These results suggest the possibility that O-GlcNAc is involved in cell death signaling in NECs. Furthermore, for NECs, we identified an O-GlcNAc-modified protein, Sp1 transcription factor. Our study is the first to evaluate expression and functions of O-GlcNAc in NECs.
doi:10.1002/jnr.22170
PMCID: PMC3956749  PMID: 19598243
development; carbohydrate; signal transduction
3.  Sulindac Sulfide Inhibits Sarcoendoplasmic Reticulum Ca2+ ATPase, Induces Endoplasmic Reticulum Stress Response, and Exerts Toxicity in Glioma Cells: Relevant Similarities To and Important Differences From Celecoxib 
Journal of neuroscience research  2012;91(3):393-406.
Malignant gliomas have low survival expectations regardless of current treatments. Nonsteroidal anti-inflammatory drugs (NSAIDs) prevent cell transformation and slow cancer cell growth by mechanisms independent of cyclooxygenase (COX) inhibition. Certain NSAIDs trigger the endoplasmic reticulum stress response (ERSR), as revealed by upregulation of molecular chaperones such as GRP78 and C/EBP homologous protein (CHOP). Although celecoxib (CELE) inhibits the sarcoendoplasmic reticulum Ca2+ ATPase (SERCA), an effect known to induce ERSR, sulindac sulfide (SS) has not been reported to affect SERCA. Here, we investigated these two drugs for their effects on Ca2+ homeostasis, ERSR, and glioma cell survival. Our findings indicate that SS is a reversible inhibitor of SERCA and that both SS and CELE bind SERCA at its cyclopiazonic acid binding site. Furthermore, CELE releases additional Ca2+ from the mitochondria. In glioma cells, both NSAIDS upregulate GRP78 and activate ER-associated caspase-4 and caspase-3. Although only CELE upregulates the expression of CHOP, it appears that CHOP induction could be associated with mitochondrial poisoning. In addition, CHOP induction appears to be uncorrelated with the gliotoxicity of these NSAIDS in our experiments. Our data suggest that activation of ERSR is primarily responsible for the gliotoxic effect of these NSAIDS. Because SS has good brain bioavailability, has lower COX-2 inhibition, and has no mitochondrial effects, it represents a more appealing molecular candidate than CELE to achieve gliotoxicity via activation of ERSR.
doi:10.1002/jnr.23169
PMCID: PMC3595008  PMID: 23280445
GRP78; gliotoxicity; Ca2+; NSAIDs
4.  Activation of Toll-Like Receptors Inhibits Herpes Simplex Virus-1 Infection of Human Neuronal Cells 
Journal of neuroscience research  2009;87(13):2916-2925.
Toll-like receptors (TLRs) play an essential role in initiating intracellular type I interferon (IFN)-mediated innate immunity against viral infections. We examined whether human neuronal cells (primary human neurons, NT2-N and CHP-212 cells) express TLRs and mount type I IFN-mediated innate immunity against herpes simplex virus-1 (HSV-1) infection. Human neuronal cells expressed TLR family members 1–10 and IFN-α/β. The activation of TLR3 or TLR8 by double-stranded RNA (poly-I:C) or single-stranded RNA (ssRNA) induced IFN-α/β expression. In addition, HSV-1 infection of human neuronal cells induced IFN-α expression. Investigation of the mechanisms showed that poly-I:C or ssRNA treatment enhanced the expression of several IFN regulatory factors. Importantly, the activation of TLR3 or TLR8 by poly-I:C or ssRNA prior to HSV-1 infection reduced the susceptibility of the neuronal cells to infection. These observations indicate that human neuronal cells possess intracellular TLR-mediated innate immune protection against HSV-1 infection.
doi:10.1002/jnr.22110
PMCID: PMC3935181  PMID: 19437550
human neuronal cells; Toll-like receptor; poly-I:C; ssRNA; herpes simplex virus-1; type I interferons
5.  UPREGULATION OF BRAIN-DERIVED NEUROTROPHIC FACTOR EXPRESSION IN NODOSE GANGLIA AND THE LOWER BRAINSTEM OF HYPERTENSIVE RATS 
Journal of neuroscience research  2012;91(2):220-229.
Hypertension leads to structural and functional changes at baroreceptor synapses in the medial nucleus tractus solitarius (NTS), but the underlying molecular mechanisms remain unknown. Our previous studies show that brain-derived neurotrophic factor (BDNF) is abundantly expressed by rat nodose ganglion (NG) neurons, including baroreceptor afferents and their central terminals in the medial NTS. We hypothesized that hypertension leads to upregulation of BDNF expression in NG neurons. To test this hypothesis, we used two mechanistically distinct models of hypertension: the spontaneously hypertensive rat (SHR) and the deoxycorticosterone acetate (DOCA)-salt rat. Young adult SHRs, whose blood pressure was significantly elevated compared to age-matched Wistar-Kyoto (WKY) control rats, exhibited dramatic upregulation of BDNF mRNA and protein in the NG. BDNF transcripts from exon 4, known to be regulated by activity, and exon 9 (protein-coding region) showed the largest increases. Electrical stimulation of dispersed NG neurons with patterns that mimic baroreceptor activity during blood pressure elevations led to increases in BDNF mRNA that were also mediated through promoter 4. The increase in BDNF content of the NG in vivo was associated with a significant increase in the percentage of BDNF-immunoreactive NG neurons. Moreover, upregulation of BDNF in cell bodies of NG neurons was accompanied by a significant increase in BDNF in the NTS region, the primary central target of NG afferents. A dramatic increase in BDNF in the NG was also detected in DOCA-salt hypertensive rats. Together, our study identifies BDNF as a candidate molecular mediator of activity-dependent changes at baroafferent synapses during hypertension.
doi:10.1002/jnr.23158
PMCID: PMC3927140  PMID: 23172808
Baroreceptor; Blood Pressure; DOCA; Nodose Ganglion; SHR
6.  Copper signaling in the mammalian nervous system: synaptic effects 
Copper (Cu) is an essential metal present at high levels in the CNS. Its role as a co-factor in mitochondrial ATP production and in other essential cuproenzymes is well defined. Menkes and Wilson’s diseases are severe neurodegenerative conditions that demonstrate the importance of Cu transport into the secretory pathway. Brain levels of Cu, which is almost entirely protein bound, exceed extracellular levels by more than a hundred-fold. Cu stored in the secretory pathway is released in a Ca2+-dependent manner and can transiently reach concentrations over 100 µM at synapses. The ability of low µM levels of Cu to bind to and modulate the function of γ-aminobutyric acid type A (GABAA) receptors, N-methyl-D-aspartate (NMDA) receptors and voltage-gated Ca2+ channels contributes to its effects on synaptic transmission. Cu also binds to amyloid precursor protein and prion protein; both proteins are found at synapses and brain Cu homeostasis is disrupted in mice lacking either protein. Especially intriguing is the ability of Cu to affect AMP-activated protein kinase (AMPK), a monitor of cellular energy status. Despite this, few investigators have examined the direct effects of Cu on synaptic transmission and plasticity. Although the variability of results demonstrates complex influences of Cu that are highly method-sensitive, these studies nevertheless strongly support important roles for endogenous Cu and new roles for Cu-binding proteins in synaptic function/plasticity and behavior. Further study of the many roles of Cu in nervous system function will reveal targets for intervention in other diseases in which Cu homeostasis is disrupted.
doi:10.1002/jnr.23143
PMCID: PMC3926505  PMID: 23115049
7.  N-Methyl-D-Aspartate Receptor-Mediated Axonal Injury in Adult Rat Corpus Callosum 
Journal of neuroscience research  2012;91(2):240-248.
Damage to white matter such as corpus callosum (CC) is a pathological characteristic in many brain disorders. Glutamate (Glut) excitotoxicity through AMPA receptors on oligodendrocyte (OL) was previously considered as a mechanism for white matter damage. Recent studies have shown that N-methyl-D-aspartate receptors (NMDARs) are expressed on myelin sheath of neonatal rat OL processes and that activation of these receptors mediated demyelization. Whether NMDARs are expressed in the adult CC and are involved in excitotoxic axonal injury remains to be determined. In this study, we demonstrate the presence of NMDARs in the adult rat CC and their distributions in myelinated nerve fibers and OL somata by means of immunocytochemical staining and Western blot. Incubation of the CC slices with Glut or NMDA induced axonal injury as revealed by analyzing amplitude of CC fiber compound action potentials (CAPs) and input–output response. Both Glut and NMDA decreased the CAP amplitude and input–output responses, suggesting an involvement of NMDARs in Glut- and NMDA-induced axonal injury. The involvement of NMDAR in Glut-induced axonal injury was further assayed by detection of β-amyloid precursor protein (β-APP) in the CC axonal fibers. Treatment of the CC slices with Glut resulted in β-APP accumulation in the CC fibers as detected by Western blot, reflecting an impairment of axonal transport function. This injurious effect of Glut on CC axonal transport was significantly blocked by MK801. Taken together, these results show that NMDARs are expressed in the adult CC and are involved in excitotoxic activity in adult CC slices in vitro.
doi:10.1002/jnr.23150
PMCID: PMC3546500  PMID: 23161705
NMDA receptor; excitotoxicity; myelinated fibers; oligodendrocyte; corpus callosum
8.  Selective activation of PKC epsilon in mitochondria is neuroprotective in vitro and reduces focal ischemic brain injury in mice 
Journal of neuroscience research  2013;91(6):799-807.
Activation of PKCε confers protection against neuronal ischemia/reperfusion. Since activation of PKCε leads to its translocation to multiple intracellular sites, a mitochondrial-selective PKCε activator was used to test the importance of mitochondrial activation to the neuroprotective effect of PKCε. PKCε can regulate key cytoprotective mitochondrial functions including electron transport chain activity, reactive oxygen species (ROS) generation, mitochondrial permeability transition, and detoxification of reactive aldehydes. We tested the ability of mitochondrial selective activation of PKCε to protect primary brain cell cultures or mice subjected to ischemic stroke. Pre-treatment with either general PKCε activator peptide, ψεRACK, or mitochondrial-selective PKCε activator, ψεHSP90, reduced cell death induced by simulated ischemia/reperfusion in neurons, astrocytes, and mixed neuronal cultures. The protective effects of both ψεRACK and ψεHSP90 were blocked by the PKCε antagonist, εV1–2, indicating protection requires PKCε interaction with its anchoring protein, εRACK. Further supporting a mitochondrial mechanism for PKCε, neuroprotection by ψεHSP90 was associated with a marked delay in mitochondrial membrane depolarization and significantly attenuated ROS generation during ischemia. Importantly, ψεHSP90 reduced infarct size and reduced neurological deficit in C57/BL6 mice subjected to middle cerebral artery occlusion and 24 hours of reperfusion. Thus selective activation of mitochondrial PKCε preserves mitochondrial function in vitro and improves outcome in vivo, suggesting potential therapeutic value clinically when brain ischemia is anticipated, including neurosurgery and cardiac surgery.
doi:10.1002/jnr.23186
PMCID: PMC3905808  PMID: 23426889
mitochondria; astrocytes; acute stroke; cell culture; animal models
9.  Vitamin E Suppression of Microglial Activation Is Neuroprotective 
Journal of neuroscience research  2001;66(2):163-170.
Neurotoxic microglial-neuronal interactions have been implicated in the pathogenesis of various neurodegenerative diseases such as Alzheimer’s disease, and vitamin E has been shown to have direct neuroprotective effects. To determine whether vitamin E also has indirect neuroprotective effects through suppression of microglial activation, we used a microglial-neuronal coculture. Lipopolysaccharide (LPS) treatment of a microglial cell line (N9) induced a time-dependent activation of both p38 mitogen-activated protein kinase (p38 MAPK) and nuclear factor-κB (NFκB), with consequent increases in interleukin-1α (IL-1α), tumor necrosis factor-α (TNF-α), and nitric oxide (NO) production. Differentiated neuronal cells (PC12 cells treated with nerve growth factor) exhibited marked loss of processes and decreased survival when cocultured with LPS-activated microglia. Preincubation of microglia with vitamin E diminished this neurotoxic effect, independently of direct effects of the antioxidant on the neuronal cells. Microglial NO production and the induction of IL-1α and TNFα expression also were attenuated by vitamin E. Such antiinflammatory effects of vitamin E were correlated with suppression of p38 MAPK and NFκB activation and were mimicked by an inhibition of either p38 MAPK (by SB203580) or NFκB (by decoy oligonucleotides). These results suggest that, in addition to the beneficial effects of providing direct antioxidant protection to neurons reported by others, vitamin E may provide neuroprotection in vivo through suppression of signaling events necessary for microglial activation.
PMCID: PMC3903400  PMID: 11592111
Alzheimer’s disease; interleukin-1; NFκB; nitric oxide; p38 mitogen-activated protein kinase; tumor necrosis factor; vitamin E
10.  Intracerebral Transplantation of Neural Stem Cells Combined with Trehalose Ingestion Alleviates Pathology in a Mouse Model of Huntington’s Disease 
The present investigation examined the neuroprotective benefits for combined trehalose administration with C17.2 neural stem cell transplantation in a transgenic mouse model of Huntington’s disease (HD), R6/2. C17.2 neural stem cells have the potential of differentiating into a neuronal phenotype in vitro and have been shown to be effective in the treatment of a variety of lysosomal lipid storage disorders in the nervous system. In this study, we transplanted these cells into the lateral ventricle of R6/2 transgenic mice in order to examine the efficacy of using these cells for correcting the accumulated polyglutamine storage materials in HD. To improve efficacy, animals were fed with a diet rich in trehalose, which has been shown to be beneficial to retard aggregate formation. The combined treatment strategy not only decreased ubiquitin-positive aggregation in striatum, alleviated polyglutamine aggregation formation, and reduced striatal volume, but also extended lifespan in the R6/2 animal model. Behavioral evaluation showed that the combination treatment improved motor dysfunction. Statistical analysis revealed that the combination treatment was more effective than treatment with trehalose alone based on the above biochemical and behavioral criteria. This study provides a strong a basis for further developing an effective therapeutic strategy for HD.
doi:10.1002/jnr.21817
PMCID: PMC3895944  PMID: 18683244
Huntington’s disease; neural stem cell; cell transplantation; polyQ; polyglutamine; trehalose
11.  Homozygous inactivation of the Lgi1 gene results in hypomyelination in the peripheral and central nervous system 
Journal of neuroscience research  2010;88(15):10.1002/jnr.22496.
Mutations in the LGI1 gene in humans predispose to the development of Autosomal Dominant Partial Epilepsy with Auditory Features (ADPEAF). Homozygous inactivation of the Lgi1 gene in mice results in an epilepsy phenotype characterized by clonic seizures within 2–3 weeks after birth. Before onset of seizures the 2–3 week-old null mutant mice show poor locomotor activity and neuromuscular strength. EM analysis of the sciatic nerve demonstrates impaired myelination of axons in the peripheral nervous system. Although heterozygous mutant mice do not show any locomotor phenotypes, they also demonstrate an intermediate level of hypomyelination compared with the wild type mice. Hypomyelination was also observed in the central nervous system which, although relatively mild, was still significantly different to the wild type mice. These data suggest a role for LGI1 in the myelination functions of Schwann cells and oligodendrocytes.
doi:10.1002/jnr.22496
PMCID: PMC3885985  PMID: 20857514
LGI1; hypomyelination; mutant null mouse; CNS; PNS
12.  B2 attenuates polyglutamine-expanded androgen receptor toxicity in cell and fly models of spinal and bulbar muscular atrophy 
Journal of neuroscience research  2010;88(10):10.1002/jnr.22389.
Expanded polyglutamine tracts cause neurodegeneration through a toxic gain of function mechanism. Generation of inclusions is a common feature of polyglutamine diseases and other protein misfolding disorders. Inclusion formation is likely to be a defensive response of the cell to the presence of unfolded protein. Recently, the compound B2 has been shown to increase inclusion formation and decrease toxicity of polyglutamine-expanded huntingtin in cultured cells. We explored the effect of B2 on spinal and bulbar muscular atrophy (SBMA). SBMA is caused by expansion of polyglutamine in the androgen receptor (AR) and is characterized by the loss of motor neurons in the brainstem and spinal cord. We found that B2 increases the deposition of mutant AR into nuclear inclusions without altering the ligand-induced aggregation, expression, or subcellular distribution of the mutant protein. The effect of B2 on inclusions was associated with a decrease in AR transactivation function. Importantly, we show that B2 reduces mutant AR toxicity in cell and fly models of SBMA, further supporting the idea that accumulation of polyglutamine-expanded protein into inclusions is protective. Our findings suggest B2 as a novel approach to therapy for SBMA.
doi:10.1002/jnr.22389
PMCID: PMC3881232  PMID: 20336775
polyglutamine disease; SBMA; androgen receptor; B2; inclusions
13.  Tissue Inhibitor of Metalloproteinase (TIMP)-1: The TIMPed Balance of Matrix Metalloproteinases in the Central Nervous System 
Journal of neuroscience research  2003;74(6):10.1002/jnr.10835.
Astrocytes are intimately involved in the mechanisms of neural injury and repair. They participate in a variety of homeostatic functions and elicit repair responses as balance mechanisms. Currently, there is a growing appreciation of a more active role of astrocytes in neuronal signaling and function. One key homeostatic mechanism of astrocytes in tissue repair is maintained through their production of tissue inhibitors of metalloproteinases (TIMPs). The family of TIMPs (1–4) plays a central regulatory role as inhibitors of matrix metalloproteinases (MMPs), enzymes involved in extracellular matrix maintenance and remodeling. Recently, TIMP-1, the inducible form, has been identified as a multifunctional molecule with divergent functions. It participates in wound healing and regeneration, cell morphology and survival, tumor metastasis, angiogenesis, and inflammatory responses. An imbalance of MMP/TIMP regulation has been implicated in several inflammatory diseases of the central nervous system (CNS). Here we review the conundrums of TIMP-1 regulation in CNS pathophysiology. We propose that astrocyte-TIMP-1 may play an important role in CNS homeostasis and disease. Astrocyte TIMP-1 expression is differentially regulated in inflammatory neurodegenerative diseases and may have significant therapeutic relevance.
doi:10.1002/jnr.10835
PMCID: PMC3857704  PMID: 14648584
astrocytes; neurodegenerative diseases; inflammation
14.  Comparison of Nerve Growth Factor Receptor Binding Models Using Heterodimeric Muteins 
Journal of neuroscience research  2012;90(12):2259-2271.
Nerve growth factor (NGF) is a homodimer that binds to two distinct receptor types, TrkA and p75, to support survival and differentiation of neurons. The high-affinity binding on the cell surface is believed to involve a heteroreceptor complex, but its exact nature is unclear. We developed a heterodimer (heteromutein) of two NGF muteins that can bind p75 and TrkA on opposite sides of the heterodimer, but not two TrkA receptors. Previously described muteins are Δ9/13 that is TrkA negative and 7-84-103 that is signal selective through TrkA. The heteromutein (Htm1) was used to study the heteroreceptor complex formation and function, in the putative absence of NGF-induced TrkA dimerization. Cellular binding assays indicated that Htm1 does not bind TrkA as efficiently as wild-type (wt) NGF but has better affinity than either homodimeric mutein. Htm1, 7-84-103, and Δ9/13 were each able to compete for cold-temperature, cold-chase stable binding on PC12 cells, indicating that binding to p75 was required for a portion of this high-affinity binding. Survival, neurite outgrowth, and MAPK signaling in PC12 cells also showed a reduced response for Htm1, compared with wtNGF, but was better than the parent muteins in the order wtNGF > Htm1 > 7-84-103 >> Δ9/13. Htm1 and 7-84-103 demonstrated similar levels of survival on cells expressing only TrkA. In the longstanding debate on the NGF receptor binding mechanism, our data support the ligand passing of NGF from p75 to TrkA involving a transient heteroreceptor complex of p75-NGF-TrkA.
doi:10.1002/jnr.23116
PMCID: PMC3674885  PMID: 22903500
TrkA-p75 heteroreceptor complex; high-affinity binding; ligand passing model
15.  GLUT-1 GLUCOSE TRANSPORTERS IN THE BLOOD-BRAIN BARRIER: DIFFERENTIAL PHOSPHORYLATION 
Journal of neuroscience research  2011;89(12):10.1002/jnr.22738.
Glucose is the primary metabolic fuel for the mammalian brain and a continuous supply is required to maintain normal CNS function. The transport of glucose across the blood-brain barrier (BBB) into the brain is mediated by the facilitative glucose transporter GLUT-1. Prior studies (Simpson et al. 2001) had revealed that the conformations of the GLUT-1 transporter were different in luminal (blood facing) and abluminal (brain facing) membranes of bovine cerebral endothelial cells, based on differential antibody recognition. In this study we have extended these observations and using a combination of 2D-PAGE/Western blotting and immunogold electron microscopy we determined that these different conformations are exhibited in vivo and arise from differential phosphorylation of GLUT-1 and not from alternative splicing or altered O- or N-linked glycosylation.
doi:10.1002/jnr.22738
PMCID: PMC3835747  PMID: 21910135
16.  Novel Role of TGF-β in Differential Astrocyte-TIMP-1 Regulation: Implications for HIV-1-Dementia and Neuroinflammation 
Journal of neuroscience research  2006;83(7):10.1002/jnr.20787.
Astrocyte production of tissue inhibitor of metalloproteinase (TIMP)-1 is important in central nervous system (CNS) homeostasis and inflammatory diseases such as HIV-1-associated dementia (HAD). TIMPs and matrix metalloproteinases (MMPs) regulate the remodeling of the extracellular matrix. An imbalance between TIMPs and MMPs is associated with many pathologic conditions. Our recently published studies uniquely demonstrate that HAD patients have reduced levels of TIMP-1 in the brain. Astrocyte-TIMP-1 expression is differentially regulated in acute and chronic inflammatory conditions. In this and the adjoining report (Gardner et al., 2006), we investigate the mechanisms that may be involved in differential TIMP-1 regulation. One mechanism for TIMP-1 downregulation is the production of anti-inflammatory molecules, which can activate signaling pathways during chronic inflammation. We investigated the contribution of transforming growth factor (TGF)-signaling in astrocyte-MMP/TIMP-1-astrocyte regulation. TGF-β1 and β2 levels were upregulated in HAD brain tissues. Co-stimulation of astrocytes with IL-1β and TGF-β mimicked the TIMP-1 downregulation observed with IL-1β chronic activation. Measurement of astrocyte-MMP protein levels showed that TGF-β combined with IL-1β increased MMP-2 and decreased proMMP-1 expression compared to IL-1β alone. We propose that one of the mechanisms involved in TIMP-1 downregulation may be through TGF-signaling in chronic immune activation. These studies show a novel extracellular regulatory loop in astrocyte-TIMP-1 regulation.
doi:10.1002/jnr.20787
PMCID: PMC3820372  PMID: 16496359
transforming growth factor; neurodegene-ration; chronic inflammation; HIV-1-associated dementia; extracellular matrix; astrocyte-activation
17.  Differential Alteration of Hippocampal Excitatory Synaptic Transmission by Cannabinoid Ligands 
Journal of neuroscience research  2009;87(3):10.1002/jnr.21889.
Cannabinoid compounds affect synaptic activity and plasticity in numerous brain areas by activating CB1 receptors (CB1). In hippocampus, varying results have been obtained on the extent and site of cannabinoid actions on excitatory transmission, ranging from no effect to complete obliteration of synaptic responses. Here we used the rat hippocampal slice preparation to study and compare the effect of various synthetic and endogenous CB1 ligands on excitatory synaptic transmission. The full CB1 agonist WIN55212-2 (WIN2) greatly decreased excitatory synaptic transmission by 62%. The effect of WIN-2 was concentration dependent (EC50 of 200 nM) and completely prevented by CB1 antagonists. The nondegradable partial CB1 agonist R1-methanandamide (mAEA) decreased transmission by 25% and the endocannabinoids 2-arachid-onylglycerol (2-AG) and anandamide (AEA) had no significant effect. The action of AEA was improved by inhibiting its degradation but not its transport. The effect of 2-AG was enhanced upon inhibition of COX-2 but remained unchanged with blockade of monoacylglycerol lipase (MAGL). The observed effects were prevented by CB1 antagonists regardless of the ligand used, and paired-pulse paradigms pointed to presynaptic mechanisms of cannabinoid action. Our results show that cannabinoid effects on neuronal activity differ widely according to the CB1 ligand used. We observed large differences between full (synthetic) and partial (endogenous) CB1 agonists in altering synaptic transmission, notably because of the involvement of active degradation mechanisms.
doi:10.1002/jnr.21889
PMCID: PMC3816768  PMID: 18816788
CB1; anandamide; 2-arachidonoylglycerol; degradation; hippocampus
18.  Protective role of µ opioid receptor activation in intestinal inflammation induced by mesenteric ischemia/reperfusion in mice 
Journal of neuroscience research  2012;90(11):2146-2153.
Intestinal ischemia is a clinical emergency with high morbidity and mortality. We investigated whether activation of µ opioid receptor (µOR) protects from the inflammation induced by intestinal ischemia and reperfusion (I/R) in mice. Ischemia was induced by occlusion of the superior mesenteric artery (45 min) and followed by reperfusion (5 hours). Sham Operated (SO) and normal (N) mice served as controls. Each group received subcutaneously: (1) saline solution; (2) the µOR selective agonist, [D-Ala2, N-Me-Phe4, Gly5-ol]-enkephalin (DAMGO) (0.01 mg.kg−1); (3) DAMGO and the selective µOR antagonist [H-D-Phe-Cys-Tyr-D-Trp-Arg-Thr-Pen-Thr-NH2] (CTAP) (0.1 mg.kg−1) or (4) CTAP alone. I/R induced intestinal inflammation as indicated by histological damage and the significant increase in myeloperoxidase (MPO) activity, index of tissue neutrophil accumulation. TNF-α and IL-10 mRNA levels were also increased in I/R mice compared to SO. DAMGO significantly reduced tissue damage, MPO activity and TNF-α mRNA levels in I/R and these effects were reversed by CTAP. By contrast, DAMGO did not modify IL-10 mRNA levels and gastrointestinal transit. DAMGO effects are receptor-mediated and are likely due to activation of peripheral µORs since it does not readily cross the blood brain barrier. These findings suggest that activation of peripheral µOR protects from the inflammatory response induced by I/R through a pathway involving the pro-inflammatory cytokine, TNF-α. Reduction of acute inflammation might prevent I/R complications, including motility impairment, which develop at a later stage of reperfusion and are likely due to inflammatory cell infiltrates.
doi:10.1002/jnr.23108
PMCID: PMC3439549  PMID: 22806643
intestinal injury; myeloperoxidase activity; cytokines; DAMGO
19.  Differential Effects of Distinct CNS Regions on Cell Migration and Axonal Extension of Neural Precursor Transplants 
Journal of neuroscience research  2012;90(11):2065-2073.
Transplantation of neural precursor cells (NPCs) is a promising therapeutic strategy in CNS injury. However, the adult CNS lacks instructive signals present during development and, depending on the region and type of transplant, may be inhibitory for neuron generation and axonal growth. We examined the effects of the white matter in different regions of the adult CNS on the properties of NPC transplants with respect to cell survival, differentiation, migration, and axonal growth. NPCs were prepared from day 13.5 embryonic spinal cord of transgenic rats that express the human placental alkaline phosphatase (AP) reporter. These NPCs were injected unilaterally into the cervical spinal cord white matter and into the corpus callosum of adult rats and were analyzed immunohistochemically 2 weeks later. NPCs survived in both regions and differentiated into astrocytes, oligodendrocytes, and neurons, with no apparent differences in survival or phenotypic composition. However, in the spinal cord white matter, graft-derived cells, identified as precursors and glial cells, migrated from the injection site rostrally and caudally, while in the corpus callosum, graft-derived cells did not migrate and remained at the injection site. Importantly, graft-derived neurons extended axons from the grafting site along the corpus callosum past the midline, entering into the contralateral side of the corpus callosum. These results demonstrate dramatic differences between white matter regions in the spinal cord and brain with respect to cell migration and axonal growth and underscore the importance of considering the effects of the local CNS environment in the design of effective transplantation strategies.
doi:10.1002/jnr.23099
PMCID: PMC3439565  PMID: 22740505
axonal growth; cell migration; corpus callosum; spinal cord; white matter
20.  A Novel Organotypic in vitro Slice Culture Model of Intraventricular Hemorrhage of Premature infants 
Journal of neuroscience research  2012;90(11):2173-2182.
Mechanisms of brain injury in intraventricular hemorrhage (IVH) of premature infants are elusive; and no therapeutic strategy exists to prevent the brain damage in these infants. Therefore, there is a need to develop an in vitro organotypic forebrain slice-culture model to advance mechanistic studies and therapeutic developments for this disorder. We cultured forebrain slices from E29 rabbit pups and treated the cultured slices (CS) with moderate (50 μl) or large (100 μl) amount of autologous blood to mimic moderate and severe IVH. Blood-induced damage to CS was evaluated for propidium-iodide staining, LDH levels, microglial density, neuronal degeneration, myelination, and gliosis over 2 weeks after the initiation of culture. CS were viable for at least 14 days in vitro (DIV). The application of blood induced significant neural cell degeneration. Degenerating cells were more abundant and LDH levels were elevated in a dose-dependent manner in CS treated with 50 or 100 μl of blood compared to untreated controls. Microglial density was higher in blood-treated CS compared to controls at both 7 and 14 days post-treatment. Myelination was reduced and gliosis enhanced in blood-treated CS. Selective application of blood fractions revealed that CS treated with plasma displayed more hypomyelination and gliosis compared to RBC-treated slices. This study developed and characterized a novel rabbit forebrain-slice culture model of IVH that exhibits neuropatholgical changes similar to human infants with IVH. Importantly, plasma appears to induce greater white matter damage than erythrocytes in IVH, indicating plasma a source of neurotoxic components.
doi:10.1002/jnr.23102
PMCID: PMC3439584  PMID: 22806625
Intraventricular hemorrhage; Slice culture; Gliosis; forebrain; Myelin; Microglia
21.  δ-Catenin/NPRAP: A New Member of the Glycogen Synthase Kinase-3β Signaling Complex That Promotes β-catenin Turnover in Neurons 
Journal of neuroscience research  2010;88(11):10.1002/jnr.22414.
Through a multiprotein complex, glycogen synthase kinase-3β (GSK-3β) phosphorylates and destabilizes β-catenin, an important signaling event for neuronal growth and proper synaptic function. δ-Catenin, or NPRAP (CTNND2), is a neural specific member of the β-catenin superfamily, and is also known to modulate neurite outgrowth and synaptic activity. In this study, we investigated the possibility that δ-catenin expression is also affected by GSK-3β signaling, and it participates in the molecular complex regulating β-catenin turnover in neurons. Immunofluorescent light microscopy revealed co-localization of δ-catenin with members of the molecular destruction complex: GSK-3β, β-catenin, and APC in rat primary neurons. GSK-3β formed a complex with δ-catenin, and its inhibition resulted in increased δ-catenin and β-catenin expression levels. LY294002 and amyloid peptide, known activators of GSK-3β signaling, reduced δ-catenin expression levels. Furthermore, δ-catenin immunoreactivity increased and protein turnover decreased when neurons were treated with proteasome inhibitors, suggesting that the stability of δ-catenin, like that of β-catenin, is regulated by proteasome-mediated degradation. Co-immunoprecipitation experiments showed that δ-catenin overexpression promoted GSK-3β and β-catenin interactions. Primary cortical neurons and PC12 cells expressing δ-catenin treated with proteasome inhibitors showed increased ubiquitinated β-catenin forms. Consistent with the hypothesis that δ-catenin promotes the interaction of the destruction complex molecules, cycloheximide treatment of cells overexpressing δ-catenin showed enhanced β-catenin turnover. These studies identify δ-catenin as a new member of the GSK-3β signaling pathway and further suggest that δ-catenin is potentially involved in facilitating the interaction, ubiquitination, and subsequent turnover of β-catenin in neuronal cells.
doi:10.1002/jnr.22414
PMCID: PMC3813950  PMID: 20623542
Glycogen synthase kinase-3β; δ-catenin/NPRAP; β-catenin; proteasome; ubiquitination
22.  Decreased NR1 Phosphorylation and Decreased NMDAR Function in Hibernating Arctic Ground Squirrels 
Journal of neuroscience research  2006;84(2):291-298.
Heterothermic mammals such as ground squirrels tolerate ischemia and N-methyl-D-aspartate (NMDA) better than homeothermic mammals such as rats both in vivo and in vitro, and this tolerance is enhanced in the hibernating state. However, the cellular mechanisms underlying this tolerance remain unclear. NMDA receptors (NMDAR) play a key role in excitotoxicity. The purpose of the current study was therefore to test the hypothesis that NMDAR are down-regulated in hibernating Arctic ground squirrels (hAGS; Spermophilus parryii). To address this hypothesis, we used Western blot analysis to investigate NMDAR phosphorylation, an activator of NMDAR function, and internalization in naïve hippocampal tissue from hAGS, interbout euthermic AGS (ibeAGS), and rats. Furthermore, we used fura-2 calcium imaging to examine NMDAR function in cultured hippocampal slices from hAGS, ibeAGS, and rats. We report that phosphorylation of the NMDAR1 (NR1) subunit is decreased in hippocampal tissue from hAGS and that the NMDAR component of Glu-induced increase in [Ca2+]i is decreased in hippocampal slices from hAGS. Moreover, the fraction of NR1 in the functional membrane pool in AGS is less than that in rats.
doi:10.1002/jnr.20893
PMCID: PMC3796386  PMID: 16676330
hibernation; hippocampal slices; excitotoxicity; stroke; fura-2
23.  Altered Global and Regional Brain Mean Diffusivity in Patients with Obstructive Sleep Apnea 
Journal of neuroscience research  2012;90(10):2043-2052.
Obstructive sleep apnea (OSA) is a common and progressive disorder accompanied by severe cardiovascular and neuropsychological sequelae, presumably induced by the brain injury resulting from the intermittent hypoxia and cardiovascular processes accompanying the syndrome. However, whether the predominant brain tissue pathology is acute or chronic in newly-diagnosed, untreated OSA subjects is unclear, an assessment essential to reveal pathological processes. Diffusion tensor imaging (DTI)-based mean diffusivity (MD) procedures can detect and differentiate acute from chronic pathology, and may be useful to reveal processes in the condition. We collected four DTI series from 23 newly-diagnosed, treatment-naïve OSA and 23 control subjects, using a 3.0-Tesla magnetic resonance imaging scanner. Mean diffusivity maps were calculated from each series, realigned, averaged, normalized to a common space, and smoothed. Global brain MD values of each subject were calculated using normalized MD maps and a global brain mask. Mean global brain MD values and smoothed MD maps were compared between groups using analysis of covariance (covariate: age). Mean global brain MD values were significantly reduced in OSA over controls (p=0.01). Multiple brain sites in OSA, including medullary, cerebellar, basal-ganglia, prefrontal and frontal, limbic, insular, cingulum bundle, external capsule, corpus callosum, temporal, occipital, and corona radiata regions showed reduced regional MD values over controls. The results suggest that global brain MD values are significantly reduced in OSA, with certain regional sites especially affected, presumably a consequence of axonal, glial, and other cell changes in those areas. The findings likely represent acute pathological processes in the newly-diagnosed OSA subjects.
doi:10.1002/jnr.23083
PMCID: PMC3418429  PMID: 22715089
Diffusion tensor imaging; Acute condition; Cytotoxic edema; Vasogenic edema; Sleep-disordered breathing
24.  Dnmt3a regulates both cell proliferation and differentiation of mouse neural stem cells 
Journal of neuroscience research  2012;90(10):1883-1891.
DNA methylation is known to regulate cell differentiation and neuronal function in vivo. Here we examined whether deficiency of a de novo DNA methyltransferase, Dnmt3a, affects in vitro differentiation of mouse embryonic stem cells (mESCs) to neuronal and glial cell lineages. Early passage neural stem cells (NSCs) derived from Dnmt3a-deficient ESCs exhibited a moderate phenotype in precocious glial differentiation compared to wild-type counterparts. However, successive passaging to passage six (P6), when wild-type NSCs become gliogenic, revealed a robust phenotype of precocious astrocyte and oligodendrocyte differentiation in Dnmt3a−/− NSCs, consistent with our previous findings in the more severely hypomethylated Dnmt1−/− NSCs. Mass-spectrometry analysis revealed total levels of methylcytosine in Dnmt3a−/− NSCs at P6 were globally hypomethylated. Moreover, Dnmt3a−/− NSC proliferation rate was significantly increased when compared to control from P6 on. Thus, our work revealed a novel role for Dnmt3a in regulating both the timing of neural cell differentiation and cell proliferation in the paradigm of mESC-derived-NSCs.
doi:10.1002/jnr.23077
PMCID: PMC3418436  PMID: 22714992
DNA methylation; Cell differentiation; Cell proliferation; Glial cells; Neural stem cells; p53
25.  Retinal Pigment Epithelial Cells Promote Spatial Reorganization and Differentiation of Retina Photoreceptors 
Journal of neuroscience research  2008;86(16):3503-3514.
Retina differentiation involves the acquisition of a precise layered arrangement, with RPE cells in the first layer in intimate contact with photoreceptors in the second layer. Here, we developed an in vitro coculture model, to test the hypothesis that RPE cells play a pivotal role in organizing the spatial structure of the retina. We cocultured rat retinal neurons with ARPE-19 epithelial cells under various experimental conditions. Strikingly, when seeded over RPE cells, photoreceptors attached to their apical surfaces and proceeded with their development, including the increased synthesis of rhodopsin. Conversely, when we seeded RPE cells over neurons, the RPE cells rapidly detached photoreceptors from their substrata and positioned themselves underneath, thus restoring the normal in vivo arrangement. Treatment with the metalloproteinase inhibitor TIMP-1 blocked this reorganization, suggesting the involvement of metalloproteinases in this process. Reorganization was highly selective for photoreceptors because 98% of photoreceptors but very few amacrine neurons were found to redistribute on top of RPE cells. Interestingly, RPE cells were much more efficient than other epithelial or nonepithelial cells in promoting this reorganization. RPE cells also promoted the growth of photoreceptor axons away from them. An additional factor that contributed to the distal arrangement of photoreceptor axons was the migration of photoreceptor cell bodies along their own neurites toward the RPE cells. Our results demonstrate that RPE and photoreceptor cells interact in vitro in very specific ways. They also show that in vitro studies may provide important insights into the process of pattern formation in the retina.
doi:10.1002/jnr.21813
PMCID: PMC3768016  PMID: 18709656
spatial reorganization; metalloproteinases; axonal outgrowth

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