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1.
Mov Disord ; 35(4): 640-649, 2020 04.
Article in English | MEDLINE | ID: mdl-31967361

ABSTRACT

BACKGROUND: NYX-458 is a N-methyl-d-aspartate receptor (NMDAR) modulator that enhances synaptic plasticity. Dopaminergic cell loss in Parkinson's disease (PD) leads to NMDAR dysregulation in the cortico-striato-pallidal-thalmo-cortical network and altered plasticity in brain regions important to cognitive function. We hypothesize that targeting the NMDAR may be an efficacious approach to treating cognitive impairment in PD. OBJECTIVES: NYX-458 was evaluated in 2 nonhuman primate models of PD. The first, a chronic low-dose 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP)-administration model, was used to assess the effects of NYX-458 on cognitive domains impacted early in PD including attention, working memory, executive function, and visuospatial learning. The second, a high-dose MPTP-administration model, was used to assess potential for NYX-458 induced change in motor symptoms. METHODS: NYX-458 was evaluated in the chronic low-dose MPTP model using the variable delayed response measure to assess attention and working memory and simple discrimination reversal to assess executive function. NYX-458 was also assessed in the high-dose MPTP model as a monotherapy and in combination with low-dose or high-dose levodopa to assess potential impact on motor symptoms. RESULTS: NYX-458 administration resulted in rapid and long-lasting improvement in cognitive function across the domains of attention, working memory, and executive function. Dose levels effective in improving cognitive performance had no effect on PD motor symptoms, the antiparkinsonian benefit of levodopa, or dyskinesia. CONCLUSIONS: NYX-458 provides benefit in specific domains known to be impaired in PD in a dopamine depletion model of PD-like cognitive impairment. These data support the continued evaluation of NYX-458 as a potential therapeutic for cognitive decline in PD. © 2020 International Parkinson and Movement Disorder Society.


Subject(s)
Parkinson Disease , 1-Methyl-4-phenyl-1,2,3,6-tetrahydropyridine , Animals , Antiparkinson Agents , Cognition , Disease Models, Animal , Levodopa/pharmacology , Parkinson Disease/complications , Parkinson Disease/drug therapy , Primates
2.
Curr Opin Neurobiol ; 60: 192-200, 2020 02.
Article in English | MEDLINE | ID: mdl-31958623

ABSTRACT

The use of ultrasonic vocalizations as an experimental tool for studying emotional states in rodents has led to an increased understanding of the basic science of affect as well as the development of novel diagnostics and therapeutics for the treatment of affective disorders. At the behavioral level, the rules that govern the generation of affective 'feeling' states are similar to those of the psychophysics of sensory perception. Emotions are elicited primarily in response to active social stimuli. A linear increase in affective response requires a logarithmic increase in stimulation and habituation of a given affective response allows for transition across the cycle of emotional/affective states (approach→consummatory phase→avoidance). At the neuronal level, the coordinated expression of affective responses in the medial prefrontal cortex is orchestrated by rhythmic activity, which is initiated and maintained by a variety of short-term and long-term synaptic plasticity processes. An objective measure of affective states may emerge from these psychophysical and neuronal properties of emotion. Enhancing synaptic plasticity with pharmacological agents that modulate NMDA receptor activity as well as IGFI receptor activity may have therapeutic potential for the treatment of affective disorders.


Subject(s)
Ultrasonics , Vocalization, Animal , Animals , Emotions , Neurobiology , Neuronal Plasticity , Rats
3.
Psychopharmacology (Berl) ; 236(12): 3687-3693, 2019 Dec.
Article in English | MEDLINE | ID: mdl-31392357

ABSTRACT

BACKGROUND: NYX-2925 is a novel N-methyl-D-aspartate receptor (NMDAR) modulator that has been shown to facilitate both NMDAR-dependent long-term potentiation (LTP) in vitro and learning and memory in vivo. OBJECTIVE: The present studies examine the effects of NYX-2925 on NMDAR-dependent auditory LTP (aLTP) in vivo. METHODS: NMDAR-dependent aLTP and NMDAR-dependent auditory mismatch negativity (MMN) was measured, as well as changes in resting-state qEEG power. RESULTS: NYX-2925 (1, 10 mg/kg PO) increased aLTP 1 h after auditory tetanus measured by the post- minus pre-tetanus difference waveform 140-180 ms post tone onset. NYX-2925 (0.1, 1 mg/kg PO) facilitated MMN measured by the difference waveform (i.e., deviant minus standard tones). NYX-2925 (0.1, 1, 10 mg/kg PO) also enhanced resting-state alpha qEEG power. Conversely, the NMDAR glutamate site antagonist CPP (10 mg/kg IP) reduces alpha power and MMN and produces an opposite effect as NYX-2925 on aLTP. CONCLUSIONS: Together, these data suggest that the activation of the NMDAR by NYX-2925 enhances synaptic plasticity in vivo, which may both reduce symptoms of neurological disorders and serve as a biomarker for drug effects. This is the first demonstration of a long-lasting (1-h post-tetanus) effect of NMDAR modulation on synaptic plasticity processes in vivo using a noninvasive technique in freely behaving animals.


Subject(s)
Electroencephalography/methods , Neuronal Plasticity/drug effects , Receptors, N-Methyl-D-Aspartate/physiology , Spiro Compounds/pharmacology , Translational Research, Biomedical/methods , Animals , Electroencephalography/drug effects , Excitatory Amino Acid Antagonists/pharmacology , Learning/drug effects , Learning/physiology , Long-Term Potentiation/drug effects , Long-Term Potentiation/physiology , Male , Memory/drug effects , Memory/physiology , Neuronal Plasticity/physiology , Rats , Rats, Sprague-Dawley , Receptors, N-Methyl-D-Aspartate/agonists
4.
Neuroscience ; 308: 202-11, 2015 Nov 12.
Article in English | MEDLINE | ID: mdl-26343295

ABSTRACT

Rapastinel (GLYX-13) is an N-methyl-d-aspartate receptor (NMDAR) modulator that has characteristics of a glycine site partial agonist. Rapastinel is a robust cognitive enhancer and facilitates hippocampal long-term potentiation (LTP) of synaptic transmission in slices. In human clinical trials, rapastinel has been shown to produce marked antidepressant properties that last for at least one week following a single dose. The long-lasting antidepressant effect of a single dose of rapastinel (3mg/kg IV) was assessed in rats using the Porsolt, open field and ultrasonic vocalization assays. Cognitive enhancement was examined using the Morris water maze, positive emotional learning, and contextual fear extinction tests. LTP was assessed in hippocampal slices. Dendritic spine morphology was measured in the dentate gyrus and the medial prefrontal cortex. Significant antidepressant-like or cognitive enhancing effects were observed that lasted for at least one week in each model. Rapastinel facilitated LTP 1day-2weeks but not 4weeks post-dosing. Biweekly dosing with rapastinel sustained this effect for at least 8weeks. A single dose of rapastinel increased the proportion of whole-cell NMDAR current contributed by NR2B-containing NMDARs in the hippocampus 1week post-dosing, that returned to baseline by 4weeks post-dosing. The NMDAR antagonist 3-(2-carboxypiperazin-4-yl)propyl-1-phosphonic acid (CPP) blocked the antidepressant-like effect of rapastinel 1week post dosing. A single injection of rapastinel also increased mature spine density in both brain regions 24h post-dosing. These data demonstrate that rapastinel produces its long-lasting antidepressant effects via triggering NMDAR-dependent processes that lead to increased sensitivity to LTP that persist for up to two weeks. These data also suggest that these processes led to the alterations in dendritic spine morphologies associated with the maintenance of long-term changes in synaptic plasticity associated with learning and memory.


Subject(s)
Antidepressive Agents/pharmacology , Depressive Disorder/drug therapy , Hippocampus/drug effects , Neuronal Plasticity/drug effects , Oligopeptides/pharmacology , Prefrontal Cortex/drug effects , Animals , Dendritic Spines/drug effects , Dendritic Spines/pathology , Dendritic Spines/physiology , Depressive Disorder/pathology , Depressive Disorder/physiopathology , Disease Models, Animal , Dose-Response Relationship, Drug , Hippocampus/pathology , Hippocampus/physiopathology , Male , Maze Learning/drug effects , Maze Learning/physiology , Membrane Potentials/drug effects , Membrane Potentials/physiology , Memory/drug effects , Memory/physiology , Neuronal Plasticity/physiology , Prefrontal Cortex/pathology , Prefrontal Cortex/physiopathology , Rats, Sprague-Dawley , Receptors, N-Methyl-D-Aspartate/metabolism , Time Factors , Tissue Culture Techniques
5.
Neuroscience ; 192: 515-23, 2011 Sep 29.
Article in English | MEDLINE | ID: mdl-21645591

ABSTRACT

In rats, hedonic ultrasonic vocalizations (USVs) is a validated model of positive affect and is best elicited by rough-and-tumble play. Here we report that modulation of GluN2B-containing NMDA receptors (NMDAR) in the medial prefrontal cortex (MPFC) is involved in positive emotional learning. Rough and tumble play increased both GluN1 and GluN2B NMDAR subunit mRNA and protein levels in the frontal cortex. GLYX-13, a GluN2B-preferring, NMDAR glycine-site partial agonist (1 mg/kg, i.v.) significantly increased positive emotional learning whereas the GluN2B receptor-specific antagonist, ifenprodil (10 mg/kg, i.p.), inhibited positive emotional learning. Animals selectively bred for low rates of hedonic USVs were returned to wild-type levels of positive emotional learning following GLYX-13 treatment. MPFC microinjections of GLYX-13 (0.1-10 µg/side) significantly increased rates of positive emotional learning. Thus GluN2B-containing NMDARs may be involved in positive emotional learning in the MPFC by similar mechanisms as spatial/temporal learning in the hippocampus.


Subject(s)
Emotions/physiology , Learning/physiology , Prefrontal Cortex/metabolism , Receptors, N-Methyl-D-Aspartate/metabolism , Animals , Blotting, Western , Male , Oligonucleotide Array Sequence Analysis , Oligopeptides/pharmacology , Protein Subunits/metabolism , Rats , Rats, Sprague-Dawley , Real-Time Polymerase Chain Reaction
6.
Cell Mol Neurobiol ; 31(6): 819-34, 2011 Aug.
Article in English | MEDLINE | ID: mdl-21424270

ABSTRACT

Repeated electrical stimulation results in development of seizures and a permanent increase in seizure susceptibility (kindling). The permanence of kindling suggests that chronic changes in gene expression are involved. Kindling at different sites produces specific effects on interictal behaviors such as spatial cognition and anxiety, suggesting that causal changes in gene expression might be restricted to the stimulated site. We employed focused microarray analysis to characterize changes in gene expression associated with amygdaloid and hippocampal kindling. Male Long-Evans rats received 1 s trains of electrical stimulation to either the amygdala or hippocampus once daily until five generalized seizures had been kindled. Yoked control rats carried electrodes but were not stimulated. Rats were euthanized 14 days after the last seizures, both amygdala and hippocampus dissected, and transcriptome profiles compared. Of the 1,200 rat brain-associated genes evaluated, 39 genes exhibited statistically significant expression differences between the kindled and non-kindled amygdala and 106 genes exhibited statistically significant differences between the kindled and non-kindled hippocampus. In the amygdala, subsequent ontological analyses using the GOMiner algorithm demonstrated significant enrichment in categories related to cytoskeletal reorganization and cation transport, as well as in gene families related to synaptic transmission and neurogenesis. In the hippocampus, significant enrichment in gene expression within categories related to cytoskeletal reorganization and cation transport was similarly observed. Furthermore, unique to the hippocampus, enrichment in transcription factor activity and GTPase-mediated signal transduction was identified. Overall, these data identify specific and unique neurochemical pathways chronically altered following kindling in the two sites, and provide a platform for defining the molecular basis for the differential behaviors observed in the interictal period.


Subject(s)
Gene Expression Regulation , Kindling, Neurologic/physiology , Limbic System/metabolism , Amygdala/metabolism , Animals , Glutamates/metabolism , Hippocampus/metabolism , Male , RNA, Messenger/genetics , RNA, Messenger/metabolism , Rats , Rats, Long-Evans , Receptors, N-Methyl-D-Aspartate/metabolism , Signal Transduction/genetics , Software , Transcriptome
7.
Neuroscience ; 168(3): 769-77, 2010 Jul 14.
Article in English | MEDLINE | ID: mdl-20350589

ABSTRACT

Positive emotional states have been shown to confer resilience to depression and anxiety in humans, but the molecular mechanisms underlying these effects have not yet been elucidated. In laboratory rats, positive emotional states can be measured by 50-kHz ultrasonic vocalizations (hedonic USVs), which are maximally elicited by juvenile rough-and-tumble play behavior. Using a focused microarray platform, insulin-like growth factor I (IGFI) extracellular signaling genes were found to be upregulated by hedonic rough-and-tumble play but not depressogenic social defeat. Administration of IGFI into the lateral ventricle increased rates of hedonic USVs in an IGFI receptor (IGFIR)-dependent manner. Lateral ventricle infusions of an siRNA specific to the IGFIR decreased rates of hedonic 50-kHz USVs. These results show that IGFI plays a functional role in the generation of positive affective states and that IGFI-dependent signaling is a potential therapeutic target for the treatment of depression and anxiety.


Subject(s)
Emotions , Insulin-Like Growth Factor I/physiology , Vocalization, Animal , Animals , Gene Knockdown Techniques , Injections, Intraventricular , Insulin-Like Growth Factor I/pharmacology , Microinjections , Oligonucleotide Array Sequence Analysis , RNA, Small Interfering/pharmacology , Rats , Rats, Inbred F344 , Rats, Long-Evans , Receptor, IGF Type 1/genetics , Reverse Transcriptase Polymerase Chain Reaction
8.
Dev Psychobiol ; 50(4): 322-31, 2008 May.
Article in English | MEDLINE | ID: mdl-18393285

ABSTRACT

In rats, the rates of 50 kHz ultrasonic vocalizations (USVs) can be used as a selective breeding phenotype and variations in this phenotype can be an indicator of affective states. The 50 kHz USV is elicited by rewarding stimuli (e.g., food, sexual behavior) and therefore can express a positive affective state. Conversely, the 22 kHz USV is elicited by aversive stimuli (e.g., presence of a predator, social defeat) indicating a negative affective state. In the present study, we tested the effect of selectively breeding for 50 kHz USVs on a variety of maternal social/emotional behaviors in young rat pups (PND 10-12). These measures consisted of an assessment of isolation calls and conditioned odor preference paradigm. Results indicate that animals selected for low levels of 50 kHz USVs show the greatest alterations in social behaviors compared to the control animals. The low line animals had an increase in isolation calls tested during place preference conditioning and a decrease in 50 kHz ultrasonic calls in all conditions. These same low line animals failed to show a typical preference for a maternally-associated odor during the place preference test. The different social behaviors of the high line animals did not consistently vary from those of the control group. These results have important implications for the study of genetic and epigenetic mechanisms underlying emotional states, and possibly contribute to the research underlying the emotional changes in developmental disorders such as autistic spectrum disorder by providing a novel animal model that displays communication deficits that are interdependent with significant social behavioral impairments.


Subject(s)
Selection, Genetic , Social Behavior , Ultrasonics , Vocalization, Animal/physiology , Affect , Animal Communication , Animals , Conditioning, Psychological , Female , Genotype , Locomotion , Male , Motivation , Odorants , Phenotype , Rats , Reward , Social Isolation
9.
Neuroscience ; 137(1): 37-49, 2006.
Article in English | MEDLINE | ID: mdl-16289586

ABSTRACT

Gene expression profiles in the cortex of adult Long-Evans rats as a function of a stressful social loss and victory in inter-male fighting encounters were examined. This social dominance and subordination model has been postulated to simulate early changes in the onset of depression in the losers. Microarrays were fabricated containing 45mer oligonucleotides spotted in quadruplicate and representing 1178 brain-associated genes. Dynamic range, discrimination power, accuracy and reproducibility were determined with standard mRNA "spiking" studies. Gene expression profiles in dominant and subordinate animals were compared using a "universal" reference design [Churchill GA (2002) Fundamentals of experimental design for cDNA microarrays. Nat Genet 32 (Suppl):490-495]. Data were analyzed by significance analysis of microarrays using rank scores [Tusher VG, Tibshirani R, Chu G (2001) Significance analysis of microarrays applied to the ionizing radiation response. Proc Natl Acad Sci USA 98:5116-5121; van de Wiel MA (2004) Significance analysis of microarrays using rank scores. Kwantitatieve Methoden 71:25-37]. Ontological analyses were then performed using the GOMiner algorithm [Zeeberg BR, Feng W, Wang G, Wang MD, Fojo AT, Sunshine M, Narasimhan S, Kane DW, Reinhold WC, Lababidi S, Bussey KJ, Riss J, Barrett JC, Weinstein JN (2003) GoMiner: a resource for biological interpretation of genomic and proteomic data. Genome Biol 4(4):R28]. And finally, genes of special interest were further studied using quantitative reverse transcriptase polymerase chain reaction. Twenty-two transcripts were statistically significantly differentially expressed in the neocortex between dominant and subordinate animals. Ontological analyses revealed that significant gene changes were clustered primarily into functional neurochemical pathways associated with protein biosynthesis and cytoskeletal dynamics. The most robust of these were the increased expression of interleukin-18, heat shock protein 27, beta3-tubulin, ribosome-associated membrane protein 4 in subordinate animals. Interleukin-18 has been found to be over-expressed in human depression and panic disorder as well as other physiological stress paradigms [Takeuchi M, Okura T, Mori T, Akita K, Ohta T, Ikeda M, Ikegami H, Kurimoto M (1999) Intracellular production of interleukin-18 in human epithelial-like cell lines is enhanced by hyperosmotic stress in vitro. Cell Tissue Res 297(3):467-473] and heat shock proteins have been shown to be involved in the pathogenesis of many neurodegenerative and psychiatric disorders [Iwamoto K, Kakiuchi C, Bundo M, Ikeda K, Kato T (2004) Molecular characterization of bipolar disorder by comparing gene expression profiles of postmortem brains of major mental disorders. Mol Psychiatry 9(4):406-416; Pongrac JL, Middleton FA, Peng L, Lewis DA, Levitt P, Mirnics K (2004) Heat shock protein 12A shows reduced expression in the prefrontal cortex of subjects with schizophrenia. Biol Psychiatry 56(12):943-950]. Thus, the gene expression changes that we have observed here are consistent with and extend the observations found in the clinical literature and link them to the animal model used here thereby reinforcing its use to better understand the genesis of depression and identify novel therapeutic targets for its treatment.


Subject(s)
Depression/etiology , Disease Models, Animal , Dominance-Subordination , Gene Expression , Neocortex/physiology , Animals , Gene Expression Profiling , Interleukin-18/genetics , Interleukin-18/metabolism , Intracellular Signaling Peptides and Proteins , Male , Membrane Proteins/genetics , Membrane Proteins/metabolism , Oligonucleotide Array Sequence Analysis , Protein Serine-Threonine Kinases/genetics , Protein Serine-Threonine Kinases/metabolism , Rats , Rats, Long-Evans , Reverse Transcriptase Polymerase Chain Reaction , Tubulin/genetics , Tubulin/metabolism
10.
J Neurochem ; 89(6): 1436-44, 2004 Jun.
Article in English | MEDLINE | ID: mdl-15189346

ABSTRACT

Human glioma cell line U-373 MG expresses CMP-NeuAc : Galbeta1,3GlcNAc alpha2,3-sialyltransferase [EC No. 2.4.99.6] (alpha2,3ST), UDP-GlcNAc : beta-d-mannoside beta1,6-N-acetylglucosaminyltransferase V [EC 2.4.1.155] (GnT-V) and UDP-GlcNAc3: beta-d-mannoside beta1,4-N-acetylglucosaminyltransferase III [EC 2.4.1.144] (GnT-III) but not CMP-NeuAc : Galbeta1,4GlcNAc alpha2,6-sialyltransferase [EC 2.4.99.1] (alpha2,6ST) under normal culture conditions. We have previously shown that transfection of the alpha2,6ST gene into U-373 cells replaced alpha2,3-linked sialic acids with alpha2,6 sialic acids, resulting in a marked inhibition of glioma cell invasivity and a significant reduction in adhesivity. We now show that U-373 cells, which are typically highly resistant to cell death induced by chemotherapeutic agents (< 10% death in 18 h), become more sensitive to apoptosis following overexpression of these four glycoprotein glycosyltransferases. U-373 cell viability showed a three-fold decrease (from 20 to 60% cell death) following treatment with staurosporine, C2-ceramide or etoposide, when either alpha2,6ST and GnT-V genes were stably overexpressed. Even glycosyltransferases typically raised in cancer cells, such as alpha2,3ST and GnT-III, were able to decrease viability two-fold (from 20 to 40% cell death) following stable overexpression. The increased susceptibility of glycosyltransferase-transfected U-373 cells to pro-apoptotic drugs was associated with increased ceramide levels in Rafts, increased caspase-3 activity and increased DNA fragmentation. In contrast, the same glycosyltransferase overexpression protected U-373 cells against a different class of apoptotic drugs, namely the phosphatidylinositol 3-kinase inhibitor LY294002. Thus altered surface protein glycosylation of a human glioblastoma cell line can lead to lowered resistance to chemotherapeutic agents.


Subject(s)
Glioma/enzymology , Glycoconjugates/biosynthesis , N-Acetylglucosaminyltransferases/genetics , Sialyltransferases/genetics , Sphingosine/analogs & derivatives , Antineoplastic Agents/pharmacology , Caspase 3 , Caspases/metabolism , Cell Death/genetics , Cell Line, Tumor , Cell Membrane/metabolism , Cell Survival/drug effects , Cell Survival/genetics , Chromones/pharmacology , Dose-Response Relationship, Drug , Drug Resistance, Neoplasm/genetics , Enzyme Inhibitors/pharmacology , Etoposide/pharmacology , Glioma/genetics , Glycosylation , Humans , Morpholines/pharmacology , N-Acetylglucosaminyltransferases/metabolism , Oligosaccharides/metabolism , Sialyltransferases/metabolism , Sphingosine/pharmacology , Staurosporine/pharmacology , Transfection , beta-D-Galactoside alpha 2-6-Sialyltransferase
11.
Curr Drug Targets ; 2(3): 331-45, 2001 Sep.
Article in English | MEDLINE | ID: mdl-11554557

ABSTRACT

According to a recent World Health Organization survey, there are over four hundred million people worldwide suffering from mental and neurological disorders; schizophrenia affects some forty-five million people, and unipolar major depression ranked fifth in major causes of disability and death. Clearly it is of the utmost importance to develop new, effective, and safe neuro-pharmaceuticals with this increasing "global burden of disease". To this end, we have developed a strategy of generating monoclonal antibodies that act as modulators of the cell-surface central nervous system receptor-ion channel complexes. In this review we will focus on the generation and characterization of a monoclonal antibody that acts as a partial agonist to the N-methyl-D-aspartate receptor. The creation of peptide mimetics, derived from this monoclonal antibody, that may be useful as cognitive enhancers and protect neurons hypoxic and ischemic insults caused by stroke, will also be discussed.


Subject(s)
Antibodies/chemistry , Receptors, N-Methyl-D-Aspartate/drug effects , Receptors, N-Methyl-D-Aspartate/immunology , Amino Acid Sequence , Animals , Antibodies/pharmacology , Antibodies, Monoclonal/chemistry , Antibodies, Monoclonal/pharmacology , Humans , Long-Term Potentiation/drug effects , Models, Molecular , Molecular Sequence Data , Rabbits , Receptors, N-Methyl-D-Aspartate/chemistry
12.
Cancer Res ; 61(18): 6822-9, 2001 Sep 15.
Article in English | MEDLINE | ID: mdl-11559557

ABSTRACT

Human gliomas express very high levels of cell-surface alpha2,3-linked terminal sialic acids on glycoproteins bearing N-linked oligosaccharides, most notably on alpha3beta1 integrin, which is the predominant integrin found in these tumors. Alpha2,6-linked terminal sialic acids, however, are not expressed. Two stable transfectants were made using a tumorigenic human glioma cell line, U-373 MG. Galbeta1,4GlcNAc alpha2,6-sialyltransferase (ST6Gal I) transfectants were made to replace the endogenous alpha2,3-linked sialic acids with alpha2,6-linked sialic acids. And Galbeta1,3(4)GlcNAc alpha2,3-sialyltransferase (ST3Gal III) transfectants were made to increase further the expression of cell-surface, N-glycan, alpha2,3-linked sialic acids. Although ST3Gal III transfection resulted in increased invasivity when compared with parental U-373 MG and vector-transfected control cells in vitro, ST6Gal I transfection abolished invasion in vitro and induced alterations in both cell morphology, cell-spreading, and adhesion-mediated protein tyrosine phosphorylation. Furthermore, the ST6Gal I transfectants produced no intracranial tumors in severe combined immunodeficient mice, whereas parental U-373 MG cells, the vector-transfected control cells, and ST3Gal III-transfected U-373 MG cells did. These results suggest that both the linkage and expression levels of the terminal sialic acids of alpha3beta1 integrin N-glycans play an important role in glioma cell-extracellular matrix interactions. Thus, manipulating ST6Gal I gene expression may have therapeutic potential for the treatment of malignant gliomas.


Subject(s)
Brain Neoplasms/metabolism , Brain Neoplasms/pathology , Glioma/metabolism , Glioma/pathology , Polysaccharides/metabolism , Animals , Brain Neoplasms/genetics , Extracellular Matrix/metabolism , Glioma/genetics , Humans , Integrin alpha3beta1 , Integrins/metabolism , Isoenzymes/genetics , Isoenzymes/metabolism , Polysaccharides/biosynthesis , Rats , Sialic Acids/metabolism , Sialyltransferases/genetics , Sialyltransferases/metabolism , Transfection , Tumor Cells, Cultured , beta-Galactoside alpha-2,3-Sialyltransferase
13.
Cancer Lett ; 156(2): 191-8, 2000 Aug 11.
Article in English | MEDLINE | ID: mdl-10880769

ABSTRACT

A two-step strategy was developed consisting of differential display reverse transcriptase polymerase chain reaction (DDRT-PCR) with cultured normal human fetal astrocytes and U-373MG glioma cells followed by reverse Northern analysis of normal brain and primary tumor tissues. hu-dek, alpha-NAC, ribosomal proteins L7a and L35a, and five novel genes were identified. Since none of these genes has been previously shown to be associated with malignant brain tumor formation, this approach may be useful to identify novel targets for the diagnosis and treatment of brain tumors.


Subject(s)
Brain Neoplasms/genetics , Drosophila Proteins , Glioblastoma/genetics , Glioma/genetics , Receptors, Eph Family , Reverse Transcriptase Polymerase Chain Reaction/methods , Astrocytes/physiology , Blotting, Northern , Brain/physiology , Gene Expression , Genetic Therapy/methods , Humans , Middle Aged , Molecular Chaperones , Receptor Protein-Tyrosine Kinases/biosynthesis , Receptor Protein-Tyrosine Kinases/genetics , Ribosomal Proteins/biosynthesis , Ribosomal Proteins/genetics , Trans-Activators/biosynthesis , Trans-Activators/genetics , Tumor Cells, Cultured
14.
Cancer Res ; 60(1): 134-42, 2000 Jan 01.
Article in English | MEDLINE | ID: mdl-10646865

ABSTRACT

The metastatic potential of tumor cells has been shown to be correlated with the expression of tri- and tetra-antennary beta1,6-N-acetylglucosamine (beta1,6-GlcNAc)-bearing N-glycans, which are recognized by Phaseolus vulgaris leukoagglutinating lectin (L-PHA). The expression of beta1,6-GlcNAc-bearing N-glycans also has been used as a marker of tumor progression in human breast and colon cancers. In this report, the role of N-glycan branching in regulating glioma migration and invasion was examined. The expression of beta1,6-GlcNAc-bearing N-glycans was found in human glioma specimens, whereas astrocytes from normal adult brain were negative. The expression of N-acetylglucosaminyltransferase V (GnT-V) mRNA, which is responsible for the biosynthesis of beta1,6-GlcNAc-bearing N-glycans, was high in glioma cell lines with robust ets-1 expression. To study the molecular mechanism of GnT-V expression in human glioma cells, an inducible ets-1 gene was stably transfected into SNB-19 cells using a tetracycline repressor system. GnT-V mRNA expression was increased by the induction of c-ets-1, suggesting that the Ets-1 transcription factor directly regulates the transcription of GnT-V. Stable transfection of GnT-V into human glioma U-373 MG cells resulted in changes in cell morphology and focal adhesions and a marked increase in glioma invasivity in vitro. L-PHA has little effect on cell migration. On the contrary, Phaseolus vulgaris erythroagglutinating lectin (E-PHA), which recognizes bisecting beta1,4-GlcNAc-bearing N-glycans, strongly inhibits cell migration (haptotaxis) on a fibronectin substrate in U-373 MG transfectants and other glioma cell lines tested. These results suggest that the increased beta1,6-GlcNAc-bearing N-glycan expression found in malignant gliomas is modulated by GnT-V through the Ets-1 transcription factor, and that the branching of complex type N-glycans plays a major role in glioma invasivity.


Subject(s)
Brain Neoplasms/metabolism , Glioma/metabolism , N-Acetylglucosaminyltransferases/metabolism , Neoplasm Proteins/metabolism , Polysaccharides/metabolism , Proto-Oncogene Proteins/metabolism , Transcription Factors/metabolism , Adult , Brain/metabolism , Brain Neoplasms/pathology , Cell Movement/drug effects , Glioma/pathology , Humans , Neoplasm Invasiveness , Phytohemagglutinins/pharmacology , Proto-Oncogene Protein c-ets-1 , Proto-Oncogene Proteins c-ets , RNA, Messenger/metabolism , Tumor Cells, Cultured
15.
Cancer Res ; 59(1): 234-40, 1999 Jan 01.
Article in English | MEDLINE | ID: mdl-9892212

ABSTRACT

Glycosphingolipids expressed in cancer cells have been implicated in the modulation of tumor cell growth through their interaction with transmembrane signaling molecules such as growth factor receptors. For glycosphingolipids to interact with growth factor receptors, the presence of sialic acid seems to be essential. Stable transfection of a gene encoding a soluble Mr 42,000 sialidase into a human epidermoid carcinoma cell line (A431) provided an approach by which the level of terminal lipid-bound sialic acid on the cell surface could be altered. In the sialidase-positive clones, the level of ganglioside GM3 was diminished, and little change was observed in protein sialylation. Sialidase-transfected cells grew faster than control cells. Sialidase expression did not modify the binding of epidermal growth factor (EGF) to its receptor but enhanced EGF receptor (EGFR) tyrosine autophosphorylation as compared to that of parental cells or cells transfected with the vector (pcDNA3) alone. Moreover, the phosphorylation of the EGFR, as well as other protein substrates, was observed at low EGF concentrations, suggesting an increase in the receptor kinase sensitivity. These data provided evidence that changes in ganglioside expression in cancer cells by appropriate gene transfection can dramatically affect EGFR kinase activity. Hence, the modulation of ganglioside expression may represent an approach to alter tumor cell growth.


Subject(s)
Carcinoma, Squamous Cell/metabolism , ErbB Receptors/metabolism , Gene Expression Regulation, Neoplastic , Neuraminidase/genetics , Carcinoma, Squamous Cell/genetics , ErbB Receptors/genetics , Gene Transfer Techniques , Humans , Signal Transduction/genetics , Tumor Cells, Cultured
16.
Virchows Arch ; 431(2): 139-47, 1997 Aug.
Article in English | MEDLINE | ID: mdl-9293896

ABSTRACT

Several studies have shown that immunoenzymatic staining of formalin-fixed, paraffin-embedded astrocytomas with keratin antibodies frequently yields positive labelling, but no biochemical evidence of keratin expression in astrocytomas has been reported. We have investigated the presence of keratin in astrocytoma and normal brain tissues both by immunofluorescence on frozen sections and by 1D and 2D immunoblotting using seven monoclonal antibodies that, collectively, recognize most keratin polypeptides. Four of these antibodies did not stain neural tissues by immunofluorescence and were also negative by immunoblotting. The remaining three keratin antibodies stained normal brain and/or a high proportion of astrocytomas. Two of these three antibodies only stained glial fibrillary acidic protein (GFAP)-positive cells, while the third only stained GFAP-negative cells. 1D and 2D immunoblotting analysis showed that positive immunofluorescence staining of normal brain and/or astrocytomas seen with these three keratin antibodies was due to cross-reactivity with non-keratin proteins, such as GFAP. These results demonstrate that, contrary to earlier suggestions, keratin polypeptides are not frequently expressed in astrocytomas. Our studies also emphasize that keratin antibodies should be used cautiously for the differential diagnosis of undifferentiated gliomas from tumours of non-glial origin.


Subject(s)
Astrocytoma/metabolism , Brain Neoplasms/metabolism , Brain/metabolism , Keratins/metabolism , Antibodies, Monoclonal/immunology , Astrocytoma/pathology , Brain Neoplasms/pathology , Cross Reactions/immunology , False Positive Reactions , Fluorescent Antibody Technique, Direct , Glial Fibrillary Acidic Protein/immunology , Glial Fibrillary Acidic Protein/metabolism , Humans , Immunoblotting , Keratins/immunology
17.
J Neurochem ; 68(6): 2566-76, 1997 Jun.
Article in English | MEDLINE | ID: mdl-9166754

ABSTRACT

Glycosyltransferase gene transfection into cell lines has been an approach used successfully to elucidate the functional role of cell surface glycoconjugates. We have transfected the rat CMP-NeuAc:Galbeta1,4GlcNAc alpha2,6-sialyltransferase (EC 2.4.99.1) gene into a human, tumorigenic, glioma cell line, U373 MG. This transfection led to a marked inhibition of invasivity, alterations in adhesivity to fibronectin and collagen matrices, and inappropriately sialylated alpha3beta1 integrin. Adhesion-mediated protein tyrosine phosphorylation was reduced in the transfectants despite increased expression of focal adhesion kinase, p125fak. Furthermore, the transfectants showed a distinct cell morphology, an increased number of focal adhesion sites, and different sensitivity to cytochalasin D treatment than control U373 MG cells. These results suggest that inappropriate sialylation of cell surface glycoconjugates, such as integrins, can change focal adhesion as well as adhesion-mediated signal transduction and block glioma cell invasivity in vitro.


Subject(s)
Neoplasm Invasiveness/physiopathology , Sialyltransferases/metabolism , Animals , Antigens, CD/genetics , Antigens, CD/metabolism , Cell Adhesion/physiology , Cell Adhesion Molecules/metabolism , Cloning, Molecular , Collagen/metabolism , Cytoskeleton/metabolism , Fibronectins/metabolism , Focal Adhesion Kinase 1 , Focal Adhesion Protein-Tyrosine Kinases , Glioma , Humans , Integrin alpha3beta1 , Integrins/metabolism , Phosphorylation , Protein-Tyrosine Kinases/metabolism , Rats , Receptor, Insulin/metabolism , Receptors, Laminin/metabolism , Sialic Acids/metabolism , Sialyltransferases/genetics , Signal Transduction/physiology , Transfection , Tumor Cells, Cultured/chemistry , Tumor Cells, Cultured/enzymology , Tyrosine/metabolism , beta-D-Galactoside alpha 2-6-Sialyltransferase
18.
Brain Res ; 755(1): 175-9, 1997 Apr 25.
Article in English | MEDLINE | ID: mdl-9163557

ABSTRACT

CMP-NeuAc: Galbeta1,3(4)GlcNAc alpha2,3-sialyltransferase (alpha2,3-ST) mRNA was expressed in human glioma specimens, human fetal astrocytes, and a panel of brain tumor cell lines. Maackia amurensis agglutinin staining revealed the presence of alpha2,3-linked sialic acids on glioma cell surfaces and extracellular matrices whereas normal human adult astrocytes were negative. Increased expression of alpha2,3-linked glycoprotein sialylation may play a role in glial tumorigenesis.


Subject(s)
Brain Neoplasms/metabolism , Glioma/metabolism , Glycoproteins/metabolism , RNA, Messenger/metabolism , Sialyltransferases/genetics , Adenocarcinoma/metabolism , Astrocytes/metabolism , Astrocytoma/metabolism , Brain Neoplasms/secondary , Carbohydrate Conformation , Glioblastoma/metabolism , Humans , Tumor Cells, Cultured , beta-Galactoside alpha-2,3-Sialyltransferase
19.
J Biol Chem ; 272(14): 9275-9, 1997 Apr 04.
Article in English | MEDLINE | ID: mdl-9083062

ABSTRACT

N-linked oligosaccharides appear to be important for the function of the epidermal growth factor (EGF) receptor. In a previous study (Rebbaa, A., Yamamoto, H., Moskal, J. R., and Bremer, E. G. (1996) J. Neurochem. 67, 2265-2272), we showed that binding of the erythroagglutinating phytohemagglutin lectin from Phaseolus vulgaris to the bisecting structures on the EGF receptor from U373 MG glioma cells blocked EGF binding and receptor autophosphorylation. In this study we examined the consequences of overexpression of the bisecting structure on the EGF receptor by gene transfection of U373 MG cells with the N-acetylglucosaminyltransferase III (GnT-III). This modification leads to a significant decrease in EGF binding and EGF receptor autophosphorylation. In addition, the cellular response to EGF was found to be altered. Proliferation of U373 MG cells in serum-free medium is inhibited by EGF. In contrast, proliferation of the GnT-III-transfected cells was stimulated by EGF. These data demonstrate that changes in EGF receptor glycosylation by GnT-III transfection reduces the number of the active receptors in U373 MG cells and that this change results in change in the cellular response to EGF.


Subject(s)
Acetylglucosamine/metabolism , ErbB Receptors/metabolism , Glioma/genetics , Oligosaccharides/metabolism , Transfection , Epidermal Growth Factor/metabolism , Glioma/metabolism , Glycosylation , Humans , N-Acetylglucosaminyltransferases/genetics , N-Acetylglucosaminyltransferases/metabolism , Phosphorylation , Phytohemagglutinins/metabolism , RNA, Messenger/metabolism , Tumor Cells, Cultured
20.
J Neurochem ; 67(6): 2265-72, 1996 Dec.
Article in English | MEDLINE | ID: mdl-8931457

ABSTRACT

Little is known about the role of the N-linked oligosaccharides in the function of the epidermal growth factor (EGF) receptor (EGF-R). In a human glioma cell line, U373 MG, EGF-Rs contain the bisecting N-linked oligosaccharide sequence recognized by erythroagglutinating phytohemagglutinin lectin from Phaseolus vulgaris (E-PHA). Incubation of E-PHA with cultured U373 MG cells results in inhibition of EGF binding to its receptor and consequently inhibition of EGF-induced autophosphorylation of the receptor. Consistent with the inhibitory effects on the EGF-R, phenotypic events that depend on EGF-R signaling, such as cell spreading and proliferation, were also found to be modified. The effect of this lectin seems to be specific because leukoagglutinating phytohemagglutinin lectin from P. vulgaris (L-PHA), an isolectin of E-PHA, had no effect on EGF-R activity or the biological functions of these cells even though L-PHA was able to bind to the EGF-R. These findings suggest the presence of an important bisecting N-linked oligosaccharide structure in close proximity to the EGF binding site on the receptor. Furthermore, these results suggest the possibility that E-PHA lectin binding may provide an additional approach to blocking EGF-dependent glioma cell growth.


Subject(s)
ErbB Receptors/agonists , ErbB Receptors/physiology , Phytohemagglutinins/metabolism , Signal Transduction/physiology , Cell Division/drug effects , Glioma , Humans , Isomerism , Oligosaccharides/analysis , Phosphorylation , Phytohemagglutinins/chemistry , Phytohemagglutinins/pharmacology , Signal Transduction/drug effects , Tumor Cells, Cultured/cytology , Tumor Cells, Cultured/drug effects , Tumor Cells, Cultured/physiology
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