Plasticity-related Gene 1 Research Articles

Mutations in plasticity-related-gene-1 (PRG-1) protein contribute to hippocampal seizure susceptibility and modify epileptic phenotype.

The Phospholipid Phosphatase Related 4 gene (PLPPR4, *607813) encodes the Plasticity-Related-Gene-1 (PRG-1) protein. This cerebral synaptic transmembrane-protein modulates cortical excitatory transmission on glutamatergic neurons. In mice, homozygous Prg-1 deficiency causes juvenile epilepsy. Its epileptogenic potential in humans was unknown. Thus, we screened 18 patients with infantile epileptic spasms syndrome (IESS) and 98 patients with benign familial neonatal/infantile seizures (BFNS/BFIS) for the presence of PLPPR4 variants. A girl with IESS had inherited a PLPPR4-mutation (c.896C > G, NM_014839; p.T299S) from her father and an SCN1A-mutation from her mother (c.1622A > G, NM_006920; p.N541S). The PLPPR4-mutation was located in the third extracellular lysophosphatidic acid-interacting domain and in-utero electroporation (IUE) of the Prg-1p.T300S construct into neurons of Prg-1 knockout embryos demonstrated its inability to rescue the electrophysiological knockout phenotype. Electrophysiology on the recombinant SCN1Ap.N541S channel revealed partial loss-of-function. Another PLPPR4 variant (c.1034C > G, NM_014839; p.R345T) that was shown to result in a loss-of-function aggravated a BFNS/BFIS phenotype and also failed to suppress glutamatergic neurotransmission after IUE. The aggravating effect of Plppr4-haploinsufficiency on epileptogenesis was further verified using the kainate-model of epilepsy: double heterozygous Plppr4-/+|Scn1awt|p.R1648H mice exhibited higher seizure susceptibility than either wild-type, Plppr4-/+, or Scn1awt|p.R1648H littermates. Our study shows that a heterozygous PLPPR4 loss-of-function mutation may have a modifying effect on BFNS/BFIS and on SCN1A-related epilepsy in mice and humans.

Synaptic activity-dependent changes in the hippocampal palmitoylome.

Dynamic protein S-palmitoylation is critical for neuronal function, development, and synaptic plasticity. Synaptic activity-dependent changes in palmitoylation have been reported for a small number of proteins. Here, we characterized the palmitoylome in the hippocampi of male mice before and after context-dependent fear conditioning. Of the 121 differentially palmitoylated proteins identified, just over half were synaptic proteins, whereas others were associated with metabolic functions, cytoskeletal organization, and signal transduction. The synapse-associated proteins generally exhibited increased palmitoylation after fear conditioning. In contrast, most of the proteins that exhibited decreased palmitoylation were associated with metabolic processes. Similar results were seen in cultured rat hippocampal neurons in response to chemically induced long-term potentiation. Furthermore, we found that the palmitoylation of one of the synaptic proteins, plasticity-related gene-1 (PRG-1), also known as lipid phosphate phosphatase-related protein type 4 (LPPR4), was important for synaptic activity-induced insertion of α-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid receptors (AMPARs) into the postsynaptic membrane. The findings identify proteins whose dynamic palmitoylation may regulate their role in synaptic plasticity, learning, and memory.

PRG-1 prevents neonatal stimuli-induced persistent hyperalgesia and memory dysfunction via NSF/Glu/GluR2 signaling

SummaryNeonatal repetitive noxious stimuli (RNS) has been shown to cause long-term harmful effects on nociceptive processing, learning, and memory which persist until adulthood. Plasticity-related gene 1 (PRG-1) regulates synaptic plasticity and functional reorganization in the brain during neuronal development. In this study, neonatal RNS rats were established by repetitive needle pricks to neonatal rats on all four feet to model repetitive pain exposure in infants. Neonatal RNS caused thermal hyperalgesia, mechanical allodynia, learning, and memory impairments which manifested in young rats and persisted until adulthood. Hippocampal PRG-1/N-ethylmaleimide sensitive fusion protein (NSF) interaction was determined to be responsible for the RNS-induced impairment via enhanced extracellular glutamate release and AMPAR GluR2 trafficking deficiency in a cell-autonomous manner. These pathways likely act synergistically to cause changes in dendritic spine density. Our findings suggest that PRG-1 prevents the RNS-induced hyperalgesia, learning, and memory impairment by regulating synaptic plasticity via NSF/Glu/GluR2 signaling.

PRG-1 relieves pain and depressive-like behaviors in rats of bone cancer pain by regulation of dendritic spine in hippocampus.

Rationale: Pain and depression, which tend to occur simultaneously and share some common neural circuits and neurotransmitters, are highly prevalent complication in patients with advanced cancer. Exploring the underlying mechanisms is the cornerstone to prevent the comorbidity of chronic pain and depression in cancer patients. Plasticity-related gene 1 (PRG-1) protein regulates synaptic plasticity and brain functional reorganization during neuronal development or after cerebral lesion. Purinergic P2X7 receptor has been proposed as a therapeutic target for various pain and neurological disorders like depression in rodents. In this study, we investigated the roles of PRG-1 in the hippocampus in the comorbidity of pain and depressive-like behaviors in rats with bone cancer pain (BCP).Methods: The bone cancer pain rat model was established by intra-tibial cell inoculation of SHZ-88 mammary gland carcinoma cells. The animal pain behaviors were assessed by measuring the thermal withdrawal latency values by using radiant heat stimulation and mechanical withdrawal threshold by using electronic von Frey anesthesiometer, and depressive-like behavior was assessed by sucrose preference test and forced swim test. Alterations in the expression levels of PRG-1 and P2X7 receptor in hippocampus were separately detected by using western blot, immunofluorescence and immunohistochemistry analysis. The effects of intra-hippocampal injection of FTY720 (a PRG-1/PP2A interaction activator), PRG-1 overexpression or intra-hippocampal injection of A438079 (a selective competitive P2X7 receptor antagonist) were also observed.Results: Carcinoma intra-tibia injection caused thermal hyperalgesia, mechanical allodynia and depressive-like behaviors in rats, and also induced the deactivation of neurons and dendritic spine structural anomalies in the hippocampus. Western blot, immunofluorescence and immunohistochemistry analysis showed an increased expression of PRG-1 and P2X7 receptor in the hippocampus of BCP rats. Intra-hippocampal injection of FTY720 or A438079 attenuated both pain and depressive-like behaviors. Furthermore, overexpression of PRG-1 in hippocampus has similar analgesic efficacy to FTY720. In addition, they rescued neuron deactivation and dendritic spine anomalies.Conclusion: The results suggest that both PRG-1 and P2X7 receptor in the hippocampus play important roles in the development of pain and depressive-like behaviors in bone cancer condition in rats by dendritic spine regulation via P2X7R/PRG-1/PP2A pathway.

Altered synaptic phospholipid signaling in PRG-1 deficient mice induces exploratory behavior and motor hyperactivity resembling psychiatric disorders

Plasticity related gene 1 (PRG-1) is a neuron specific membrane protein located at the postsynaptic density of glutamatergic synapses. PRG-1 modulates signaling pathways of phosphorylated lipid substrates such as lysophosphatidic acid (LPA). Deletion of PRG-1 increases presynaptic glutamate release probability leading to neuronal over-excitation. However, due to its cortical expression, PRG-1 deficiency leading to increased glutamatergic transmission is supposed to also affect motor pathways. We therefore analyzed the effects of PRG-1 function on exploratory and motor behavior using homozygous PRG-1 knockout (PRG-1−/−) mice and PRG-1/LPA2–receptor double knockout (PRG-1−/−/LPA2−/−) mice in two open field settings of different size and assessing motor behavior in the Rota Rod test. PRG-1−/− mice displayed significantly longer path lengths and higher running speed in both open field conditions. In addition, PRG-1−/− mice spent significantly longer time in the larger open field and displayed rearing and self-grooming behavior. Furthermore PRG-1−/− mice displayed stereotypical behavior resembling phenotypes of psychiatric disorders in the smaller sized open field arena. Altogether, this behavior is similar to the stereotypical behavior observed in animal models for psychiatric disease of autistic spectrum disorders which reflects a disrupted balance between glutamatergic and GABAergic synapses. These differences indicate an altered excitation/inhibition balance in neuronal circuits in PRG-1−/− mice as recently shown in the somatosensory cortex [38]. In contrast, PRG-1−/−/LPA2−/− did not show significant changes in behavior in the open field suggesting that these specific alterations were abolished when the LPA2-receptor was lacking. Our findings indicate that PRG-1 deficiency led to over-excitability caused by an altered LPA/LPA2-R signaling inducing a behavioral phenotype typically observed in animal models for psychiatric disorders.

Effects of physical exercise on seizure-induced cognitive deficits

Objective To explore the long-term effects of physical exercise on learning, memory and the expression of plasticity-related gene-1 (PRG-1) in the cerebral cortex of rats with penicillin-induced developmental seizures. Methods Twenty-four 21-day Sprague-Dawley rats were randomly divided into a control group (CONT1), an exercised control group (CONT2), a seizure group (EXP1) and a seizure plus exercises group (EXP2), each of 6 using a random number table. Penicillin was injected intraperitoneally to the rats in the EXP1 and EXP2 groups to induce seizures, while those in the CONT1 and CONT2 groups received saline injections. Morris water-maze tests were performed to evaluate spatial learning and memory capacity. The rats in the CONT2 and EXP2 groups were administered an aerobic exercise program 30 min per day for 6 consecutive days. The other groups were maintained on the treadmill for the same time but without exercising. Real-time polymerase chain reactions were used to quantify the expression of PRG-1 mRNA in the cerebral cortex. Results There was a decreasing trend in marginal searching and increasing taxis and linear searching in all four groups. Ridit analysis showed that in the water-maze tests on days 2 and 4 the average scores of the control groups were significantly higher than those of the EXP1 and EXP2 groups. However, significant increases in the average scores were observed in the maze tests of the EXP1 group after day 2 and with the EXP2 group from day 4 on. The average scores of the control group were significantly lower than those of the other 3 groups. In the first maze test, the average memory scores of the two seizure groups were significantly lower than those of the controls. In the second maze test, however, only the EXP1 group′s average score was significantly worse than those of the other groups. That of the EXP2 group had improved significantly, and was not significantly different from that of the CONT2 group. The expression of PRG-1 was much higher in the CONT2, EXP1 and EXP2 groups than in the CONT1 group. The average expression of PRG-1 in the EXP2 group was not significantly different from that in the EXP1 group. Conclusions Physical exercise can significantly relieve the cognitive deficits induced by long-term seizures, which may be associated with the regulation of PRG-1 expression in the cerebral cortex. Key words: Seizures; Exercise; Plasticity-related genes; Cognition

Plasticity-Related Gene 1 Affects Mouse Barrel Cortex Function via Strengthening of Glutamatergic Thalamocortical Transmission.

Plasticity-related gene-1 (PRG-1) is a brain-specific protein that modulates glutamatergic synaptic transmission. Here we investigated the functional role of PRG-1 in adolescent and adult mouse barrel cortex both in vitro and in vivo. Compared with wild-type (WT) animals, PRG-1-deficient (KO) mice showed specific behavioral deficits in tests assessing sensorimotor integration and whisker-based sensory discrimination as shown in the beam balance/walking test and sandpaper tactile discrimination test, respectively. At P25-31, spontaneous network activity in the barrel cortex in vivo was higher in KO mice compared with WT littermates, but not at P16-19. At P16-19, sensory evoked cortical responses in vivo elicited by single whisker stimulation were comparable in KO and WT mice. In contrast, at P25-31 evoked responses were smaller in amplitude and longer in duration in WT animals, whereas KO mice revealed no such developmental changes. In thalamocortical slices from KO mice, spontaneous activity was increased already at P16-19, and glutamatergic thalamocortical inputs to Layer 4 spiny stellate neurons were potentiated. We conclude that genetic ablation of PRG-1 modulates already at P16-19 spontaneous and evoked excitability of the barrel cortex, including enhancement of thalamocortical glutamatergic inputs to Layer 4, which distorts sensory processing in adulthood.

Molecular cause and functional impact of altered synaptic lipid signaling due to a prg-1 gene SNP.

Loss of plasticity-related gene 1 (PRG-1), which regulates synaptic phospholipid signaling, leads to hyperexcitability via increased glutamate release altering excitation/inhibition (E/I) balance in cortical networks. A recently reported SNP in prg-1 (R345T/mutPRG-1) affects ~5million European and US citizens in a monoallelic variant. Our studies show that this mutation leads to a loss-of-PRG-1 function at the synapse due to its inability to control lysophosphatidic acid (LPA) levels via a cellular uptake mechanism which appears to depend on proper glycosylation altered by thisSNP. PRG-1(+/-) mice, which are animal correlates of human PRG-1(+/mut) carriers, showed an altered cortical network function and stress-related behavioral changes indicating altered resilience against psychiatric disorders. These could be reversed by modulation of phospholipid signaling via pharmacological inhibition of the LPA-synthesizing molecule autotaxin. In line, EEG recordings in a human population-based cohort revealed an E/I balance shift in monoallelic mutPRG-1 carriers and an impaired sensory gating, which is regarded as an endophenotype of stress-related mental disorders. Intervention into bioactive lipid signaling is thus a promising strategy to interfere with glutamate-dependent symptoms in psychiatric diseases.

NT-3 protein levels are enhanced in the hippocampus of PRG1-deficient mice but remain unchanged in PRG1/LPA2 double mutants

The plasticity-related gene 1 (PRG1) modulates bioactive lipids at the postsynaptic density and is a novel player in neuronal plasticity and regulation of glutamatergic transmission at principal neurons. PRG1, a neuronal molecule, is highly expressed during development and regeneration processes at the postsynaptic density, modulates synaptic lysophosphatidic acid (LPA) levels and is related to epilepsy and brain injury. In the present study, we analyzed the interaction between the synaptic molecules PRG1 and LPA2R with other plasticity-related molecules the neurotrophins. The protein levels of NGF, BDNF and NT-3 were measured using ELISA in hippocampal tissue of homozygous (PRG−/−) and heterozygous (PRG+/−) PRG1 deficient mice and compared to their wild type (PRG+/+/WT) littermates. In the hippocampus, protein levels of NT-3 were significantly increased in PRG−/− mice (compared to WT-litters) while protein levels of NGF and BDNF were not affected. Since PRG1 deficiency leads to increased neuronal excitability and higher hippocampal network activity, which may well influence neurotrophin levels, we further assessed PRG1 deficient mice on an LPA2-receptor (LPA2R) deficient background, reported to normalize hippocampal over-excitability in PRG1−/− mice. However, on an LPA2R deficient background, protein levels of NT-3 in PRG1−/− mice (PRG1−/−/LPA2R−/−) were not significantly different when compared to WT animals. Since PRG1 deficient mice showed over-excitability in glutamatergic neurons. This was normalized by additional LPA2R deletion, and we conclude the increased NT3-levels were directly or indirectly attributable to increased hippocampal network activity, possibly exerting a protective effect against over-excitability.

Analysis of Lipid Experiments (ALEX): A Software Framework for Analysis of High-Resolution Shotgun Lipidomics Data

Global lipidomics analysis across large sample sizes produces high-content datasets that require dedicated software tools supporting lipid identification and quantification, efficient data management and lipidome visualization. Here we present a novel software-based platform for streamlined data processing, management and visualization of shotgun lipidomics data acquired using high-resolution Orbitrap mass spectrometry. The platform features the ALEX framework designed for automated identification and export of lipid species intensity directly from proprietary mass spectral data files, and an auxiliary workflow using database exploration tools for integration of sample information, computation of lipid abundance and lipidome visualization. A key feature of the platform is the organization of lipidomics data in ”database table format” which provides the user with an unsurpassed flexibility for rapid lipidome navigation using selected features within the dataset. To demonstrate the efficacy of the platform, we present a comparative neurolipidomics study of cerebellum, hippocampus and somatosensory barrel cortex (S1BF) from wild-type and knockout mice devoid of the putative lipid phosphate phosphatase PRG-1 (plasticity related gene-1). The presented framework is generic, extendable to processing and integration of other lipidomic data structures, can be interfaced with post-processing protocols supporting statistical testing and multivariate analysis, and can serve as an avenue for disseminating lipidomics data within the scientific community. The ALEX software is available at www.msLipidomics.info.

Plasticity-related gene-1 inhibits lysophosphatidic acid-induced vascular smooth muscle cell migration and proliferation and prevents neointima formation

Plasticity-related gene-1 (PRG-1) protects neuronal cells from lysophosphatidic acid (LPA) effects. In vascular smooth muscle cells (VSMCs), LPA was shown to induce phenotypic modulation in vitro and vascular remodeling in vivo. Thus we explored the role of PRG-1 in modulating VSMC response to LPA. PCR, Western blot, and immunofluorescence experiments showed that PRG-1 is expressed in rat and human vascular media. PRG-1 expression was strongly inhibited in proliferating compared with quiescent VSMCs both in vitro and in vivo (medial vs. neointimal VSMCs), suggesting that PRG-1 expression is dependent on the cell phenotype. In vitro, adenovirus-mediated overexpression of PRG-1 specifically inhibited LPA-induced rat VSMC proliferation and migration but not platelet-derived growth factor-induced proliferation. This effect was abolished by mutation of a conserved histidine in the lipid phosphate phosphatase family that is essential for interaction with lipid phosphates. In vivo, balloon-induced neointimal formation in rat carotid was significantly decreased in vessels infected with PRG-1 adenovirus compared with β-galactosidase adenovirus (-71%; P < 0.05). PRG-1 overexpression abolished the activation of the p42/p44 signaling pathway in LPA-stimulated rat VSMCs in culture and in balloon-injured rat carotids. Taken together, these findings provide the first evidence of a protective role of PRG-1 in the vascular media under pathophysiological conditions.

Long-term effects of bubble baths on seizure-induced neurobehavioral deficits in newborn rats and related gene expressions in the hippocampus

Objective To explore the long-term effects of bubble baths on seizure-induced neurobehavior deficits and the expression of apoptotic/autophagic marker B cell lymphoma/leukemia-2 ( Bcl-2 ),Beclin-1,and plasticity-related gene-1 ( PRG-1 ) in newborn rats. Methods Sixty rats aged 6 days were randomly divided into 4 groups:a control group (CON),a control hydrotherapy group (HCON),a recurrent-seizure group (RS) and a recurrent-seizure hydrotherapy group (HRS),with 15 in each group.Flurothyl was used to induce 30 min of seizures daily for 6 consecutive days in the RS and HRS groups.Rats in the CON and HCON groups were placed in the same container for equal duration without exposure to flurothyl.Rats in the HCON and HRS groups were given bubble baths for 28 consecutive days after the end of the last seizure.Neurobehavioral damage was observed using open field behavior at postnatal day 26 (P26) and Morris water maze performance at postnatal day 43 to 49 (P43-P49) and a single-blind method.PRG-1,Bcl-2 and Beclin-1 protein levels in the hippocampus were detected by Western blotting at postnatal day 50 (P50). Results ①The average open field test scores of the RS rats decreased significantly compared with those of the CON and HRS rats at P26.②In the Morris water maze test the average latencies of all rats decreased gradually from the 1st to 5th days (d1 to d5) after establishment of seizure model.The average escape latency was significantly longer for rats of the RS group than for CON group rats at the 4th and 5th days ( d4 and d5 ) after establishment of seizure model.The escape latency was significantly shorter for rats of the HRS group than for RS group rats at d4.③The level of Bcl-2 protein in the hippocampus was much lower in the RS group than in the HRS and control groups.In addition,the expression of PRG-1 in the RS group was significantly higher than in the CON group. Conclusions Recurrent prolonged seizures cause long-term neurobehavior deficits,which might be associated with the down-regulated expression of Bcl-2 and up-regulated expression of PRG-1 in the hippocampus.Bubble baths can improve the neurobehavioral sequelae from seizures,perhaps through up-regulation of hippocampal Bcl-2 expression. Key words: Seizures; Bubble baths; Neonates; B cell lymphoma/leukemia-2 gene; Plasticity-related gene-1

PRG-1 transcriptional regulation independent from Nex1/Math2-mediated activation.

Plasticity-related gene 1 (PRG-1) is a novel player in glutamatergic synaptic transmission, acting by interfering with lysophosphatidic acid (LPA)-dependent signaling pathways. In the central nervous system, PRG-1 expression is restricted to postsynaptic dendrites on glutamatergic neurons. In this study, we describe the promoter architecture of the PRG-1 gene using RNA ligase-mediated rapid amplification of cDNA ends (RLM-Race) and PCR analysis. We found that PRG-1 expression is under the control of a TATA-less promoter with multiple transcription start sites. We demonstrated also that 200-kb genomic environment of the PRG-1 gene is sufficient to mediate cell type-specific expression in a reporter mouse model. Characterization of the PRG-1 promoter resulted in the identification of a 450-bp sequence, mediating ≈40-fold enhancement of transcription in cultured primary neurons compared to controls, and which induced reporter expression in slice cultures in neurons. Recently, the regulation of PRG-1 by the basic helix-loop-helix transcription factor Nex1 (Math2, NeuroD6) was reported. However, our studies in Nex1-null-mice revealed that Nex1-deficiency induces no change in PRG-1 expression and localization. We detected an additional Nex1-independent regulation mechanism that increases PRG-1 expression and mediates neuron-specific expression in an organotypic environment.

Identification and characterization of PRG-1 as a neuronal calmodulin-binding protein

Intracellular Ca2+-dependent cellular responses are often mediated by the ubiquitous protein CaM (calmodulin), which, upon binding Ca2+, can interact with and alter the function of numerous proteins. In the present study, using a newly developed functional proteomic screen of rat brain extracts, we identified PRG-1 (plasticity-related gene-1) as a novel CaM target. A CaM-overlay and an immunoprecipitation assay revealed that PRG-1 is capable of binding the Ca2+/CaM complex in vitro and in transfected cells. Surface plasmon resonance and zero-length cross-linking showed that the C-terminal putative cytoplasmic domain (residues 466-766) of PRG-1 binds equimolar amounts of CaM in a Ca2+-dependent manner, with a relatively high affinity (a Kd value for Ca2+/CaM of 8 nM). Various PRG-1 mutants indicated that the Ca2+/CaM-binding region of PRG-1 is located between residues Ser554 and Gln588, and that Trp559 and Ile578 potentially anchor PRG-1 to CaM. This is supported by pronounced changes in the fluorescence emission spectrum of Trp559 in the PRG-1 peptide (residues 554-588) upon binding to Ca2+/CaM, showing the stoichiometrical binding of the PRG-1 peptide with Ca2+/CaM. Immunoblot analyses revealed that the PRG-1 protein is abundant in brain, but is weakly expressed in the testes. Immunohistochemical analysis revealed that PRG-1 is highly expressed in forebrain structures and in the cerebellar cortex. Furthermore, PRG-1 localizes at the postsynaptic compartment of excitatory synapses and dendritic shafts of hippocampal neurons, but is not present in presynaptic nerve terminals. The combined observations suggest that PRG-1 may be involved in postsynaptic functions regulated by intracellular Ca2+-signalling.

Upregulated expression of plasticity-related gene 1 protein in cerebral cortex in a rat model of recurrent neonatal seizures

Objective To explore the dynamic expression of a new phospholipid phosphatase, plasticity related gene 1 ( PRG-1 ) in cerebral cortex following recurrent neonatal seizures.Methods A seizure was induced by inhalant flurothyl daily in neonatal Sprague-Dawley rats from postnatal day 6 (P6).Rats were assigned into the recurrent-seizure group (seizures induced in six consecutive days) and the control group.At 3 h,12 h,48 h,14d after the last convulsion,PRG-1 protein level in cerebral cortex was detected by western blot method.Results At 3 h, 12 h,48 h, 14d after the last convulsion in control group, the expression of PRG-1 in cerebral cortex respectively was 1.363 ± 0.742,1.278 ± 0.687 ,0.763 ± 0.374,1.004 ± 0.113, in experimental group , the expression of PRG-1 in cerebral cortex respectively was 1.818 ± 1.093,1.562 ± 0.782,1.024 ± 0.510,1.378 ± 0.279.At 3 h, 12 h,48 h, the expression of PRG-1 in cerebral cortex was not significantly different between the experimental and control group( t = 0.843,0.668,1.011 ,all P ＞ 0.05 ).However, at 14 d, the level of PRG-1 protein in cerebral cortex of experimental group was significantly higher than that of control ( t = 3.041, P ＜ 0.05 ).Conclusion The up-regulated expression of PRG-1 in cerebral cortex may be associated with the recurrent neonatal seizure-induced brain damage. Key words: Plasticity-related gene 1; Recurrent seizures; Cerebral cortex; Developmental

Abstract 412: Isolation and analysis of metastasis-related genes in an oral cancer cells with autocrine stromal cell-derived factor −1/CXCR4 system

Abstract We have previously demonstrated that distant metastasis might require the autocrine stromal cell-derived factor-1 (SDF-1; CXCL12)/CXCR4 system in cases of oral squamous cell carcinoma (SCC). In this study, we examined the expression of metastasis-related genes in B88-SDF-1 cells, which have an autocrine SDF-1/CXCR4 system and distant metastatic potentials in vivo. Microarray analysis revealed that 418 genes were upregulated in B88-SDF-1 cells. To confirm the specificity of the microarray analysis, 3 highly upregulated genes, metabotropic glutamate receptor 5 (mGluR5), plasticity related gene 1 (PRG1), and unknown gene were reanalyzed by RT-PCR. These 3 genes were upregulated in B88-SDF-1 cells in comparison with those in mock cells, but AMD3100, a specific antagonist of CXCR4, abrogated the upregulation of 2 (mGluR5 and PRG1) out of 3 genes in B88-SDF-1 cells. Thus, we analyzed the expression of metabotropic glutamate receptor 5 (mGluR5), one of the genes that were upregulated in B88-SDF-1 cells. A specific mGluR5 antagonist, MPEP did not affect the growth of B88-SDF-1 cells, but did inhibit the enhanced-migration of B88-SDF-1 cells. When the B88-SDF-1 cells were transplanted into the vein, numerous metastatic foci were detected. Under the present conditions, MPEP significantly inhibited the lung metastasis of the B88-SDF-1 cells, ameliorated body weight loss, and improved the survival rate of tumor-bearing nude mice. These results suggested that mGluR5 might be one of a possible metastasis-related gene induced by an autocrine SDF-1/CXCR4 system in oral SCC. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 412.

Synaptic PRG-1 Modulates Excitatory Transmission via Lipid Phosphate-Mediated Signaling

Plasticity related gene-1 (PRG-1) is a brain-specific membrane protein related to lipid phosphate phosphatases, which acts in the hippocampus specifically at the excitatory synapse terminating on glutamatergic neurons. Deletion of prg-1 in mice leads to epileptic seizures and augmentation of EPSCs, but not IPSCs. In utero electroporation of PRG-1 into deficient animals revealed that PRG-1 modulates excitation at the synaptic junction. Mutation of the extracellular domain of PRG-1 crucial for its interaction with lysophosphatidic acid (LPA) abolished the ability to prevent hyperexcitability. As LPA application in vitro induced hyperexcitability in wild-type but not in LPA(2) receptor-deficient animals, and uptake of phospholipids is reduced in PRG-1-deficient neurons, we assessed PRG-1/LPA(2) receptor-deficient animals, and found that the pathophysiology observed in the PRG-1-deficient mice was fully reverted. Thus, we propose PRG-1 as an important player in the modulatory control of hippocampal excitability dependent on presynaptic LPA(2) receptor signaling.

I039 Role of PRG-1, a neuronal phospholipid phosphatase, in the function of vascular smooth muscle cells

Proliferation and migration of vascular smooth muscle cells (VSMCs) leading to neointima formation are a hallmark of development and progression of some cardiovascular diseases. LPA (lysophosphatidic acid) which has been shown to induces a dedifferenciation of VSCM, resulting in cell proliferation and migration in vitro, and vascular remodelling in vivo. The aim of our project is to understand the role of PRG-1 (Plasticity Related Gene-1), or LPPR4 (Lipid Phosphate Phosphatase Related protein 4), in the regulation of VSMCs proliferation and migration induced by LPA. This enzyme acts as en ecto-enzyme dephosphorylating LPA, thereby inhibiting its activity, and was found in previous studies to be down-regulated in the media of aging and hypertensive rats. We showed that PRG-1 is expressed in the rat and human aortic media and rat VSMCs by PCR, immunofluorescence and western blot. This expression was highly decreased in cultured VSMCs as compared with what was observed in the aortic media, and restored by cells starvation, suggesting that PRG-1 expression is dependent on the differentiated phenotype of these cells. We studied the response to LPA of VSMCs infected with an adenovirus containing the human sequence of PRG1 (PRG1-VSMCs) or with an adenovirus expressing LacZ (LacZ-VSMCs). In vitro, LPA-induced migration of PRG1-VSMCs (measured by scrapewound assays) and LPA-induced proliferation (measured by BrdU incorporation) were inhibited (by 60 % and 40 % respectively) as compared with LacZ-VSMCs. In vivo, experiments of left carotid injury by ballooning followed by gene transfer with adenovirus injection, showed a 70 % reduction of neointima formation when carotids were infected with PRG-1 adenovirus as compared with infection with LacZ adenovirus. Current investigations indicate that PRG-1 seems to block Erk1/2 signalling pathway activated by LPA. Our results demonstrate that PRG-1 is expressed in the VSMCs of the media ; and this expression is modulated in conditions of vascular remodelling and primary culture. PRG-1 overexpression inhibits VSMCs migration and proliferation in vitro and neotintima formation in vivo. Taken together, these findings suggest that, in the media, PRG-1 plays a protective role in pathophysiological conditions.

ZnT-1, ZnT-3, CaMK II, PRG-1 expressions in hippocampus following neonatal seizure-induced cognitive deficit in rats

Epilepsy in children is associated with a broad spectrum of cognitive deficits, which is associated with hippocampal mossy fiber sprouting. The underlying molecular mechanisms involved in mossy fiber sprouting in hippocampus following developmental seizures are not completely known. We studied the timing of cognitive dysfunction following neonatal seizures and the relation of this cognitive impairment to zinc transporter 1 (ZnT-1), 3 (ZnT-3), calcium/calmodulin-dependent protein kinase II (CaMK II), plasticity-related gene 1 (PRG-1) expression in hippocampus. A seizure was induced by inhalant flurothyl daily in neonatal Sprague-Dawley rats from postnatal day 6 (P6). Rats were assigned into the single-seizure group (SS), the recurrent-seizure group (RS, seizures induced in six consecutive days), and the control group. During P41–P46 and P85–P90, the rats were tested for spatial learning and memory abilities with automatic Morris water maze task. At P90, mossy fiber sprouting and gene expression in hippocampus were determined subsequently by Timm staining and RT-PCR methods. The escape latencies from the water maze were significantly longer in rats of RS group than those of the control and SS groups at d4 of the first maze test and at d3, d4 of the second maze test. As far as Spatial Probe Test was concerned, the frequency of passing through the platform quadrant was significantly decreased in RS group than that in control and SS groups in the entire two probe tests. In rats with recurrent seizures (RS group), there was an increased distribution of Timm granules in both the supragranular region of the dentate gyrus and the stratum pyramidale of CA3 subfield in RS group, while remaining barely visible in control and SS groups; the Timm scores in CA3 and dentate gyrus in the RS animals were significantly higher than that in the control and SS groups. RT-PCR densitometry analysis showed that the ratios of hippocampal ZnT-1 to β-actin of SS and RS group were decreased significantly compared with that of control group. Meanwhile, CaMK II to β-actin of RS group was markedly lower compared with those of SS and control groups. Our results suggest that the long-term adverse effects of recurrent neonatal seizures on cognition and mossy fiber sprouting may be associated with the down-regulated expression of ZnT-1 and CaMK II in hippocampus.

Improved outcome of facial nerve repair in rats is associated with enhanced regenerative response of motoneurons and augmented neocortical plasticity

Within a recent study on the vibrissae motor performance after facial nerve repair in strains of blind (SD/RCS) and sighted (SD) rats we found that, despite persisting myotopic disorganization in the facial nucleus, the blind animals fully restored vibrissal whisking. Here we searched for morphological substrates of better recovery in the regenerating motoneurons and in the cerebral motor cortex. Expression analyses of the neurite growth-related proteins f-actin, neuronal class III beta-tubulin and plasticity-related gene-1, and stereological estimates of growth cone densities revealed a more vigorous regenerative response in the proximal nerve stump of blind SD/RCS rats compared with SD animals at 5-7 days after buccal nerve transection. Using c-Fos immunoreactivity as a marker for neuronal activation, we found that the volume of the cortex acutely responding to nerve transection (facial muscles reactive volume, FMRV) in both hemispheres of intact sighted rats was twofold smaller than that measured in blind animals. One month after transection and suture of the right facial nerve (FFA) we found a twofold increase in the FMRV in both rat strains compared with intact animals. The FMRV in SD/RCS animals, but not in SD rats, returned to the values in intact rats 2 months after FFA. Our findings suggest that enhanced plasticity in the CNS and an augmented regenerative response of the injured motoneurons contribute to better functional recovery in blind rats.