Impaired P2X and P2Y receptor-mediated signaling in HPRT-deficient B103 neuroblastoma cells

Defect of hypoxanthine phosphoribosyl transferase (HPRT) causes Lesch-Nyhan disease (LND), but the link between HPRT deficiency and the self-injurious behavior of LND is unknown. In a previous study (Pinto et al., J. Neurochem. 72 (2005) 1579-1586) we reported on a decrease in nucleotidase activity in membranes of several HPRT⁻ cell lines and fibroblasts from LND patients. Since nucleotidases are involved in ATP-induced signal transduction, in the present study, we tested the hypothesis that P2X and P2Y receptor-mediated signal transduction is impaired in HPRT deficiency. As model we studied rat B103 neuroblastoma cells. Compared to control cells, in HPRT⁻ cells, NTP and NDP-induced Ca²⁺ influx across the membrane and Ca²⁺ mobilization from intracellular stores were impaired. Both P2X and P2Y receptors were involved in the responses. Quantitative real-time PCR revealed reduced expression of receptors P2X₃, P2X₅, P2Y₂, P2Y₄, P2Y₁₂, P2Y₁₃ and P2Y₁₄ in HPRT deficiency. Collectively, HPRT deficiency is associated with abnormal purinergic signaling, encompassing P2X and P2Y receptors and nucleotidases.     

The cortical stab injury induces beading of fibers expressing ecto-nucleoside triphosphate diphosphohydrolase 3

The ecto-nucleoside triphosphate diphosphohydrolase 3 (NTPDase3), an enzyme involved in degradation of extracellular adenosine triphosphate (ATP), is expressed on nerve fibers in different brain regions, including cortex. Here we studied the expression and role of this enzyme after unilateral cortical stab injury in rats. In cortical sections of control rats, NTPDase3 immunoreactivity was associated with two types of fibers: thin processes, occasionally with small mushroom-like protrusions and slightly thicker fibers with more pronounced and more frequent varicosities, whereas immunopositive neuronal perycaria were never observed. Although NTPDase3–positive thin processes and thicker fibers, by general appearance, size and shape, could be dendrites and axons, respectively, they were never immunopositive for microtubule associated protein-2 or neurofilament H subunit. Cortical stab injury induced rapid (within 4 hours) focal varicose swelling that evolved over time to prominent beading of NTPDase3–positive fibers. The NTPDase3–positive fibers in all experimental groups also abundantly express NTPDase1, ecto-5′-nucleotidase and P2X2 receptor channels. Because the brain injury causes a massive ATP release, it is reasonable to conclude that purinoreceptors and ectonucleotidases play an important role in the process of neuritic beading.     

Changes in Presenilin 1 gene methylation pattern in diet-induced B vitamin deficiency

We have previously shown that a nutritional model of B vitamin deficiency and homocysteine cycle alteration could lead to increased amyloid β deposition, due to PSEN1 and BACE over-expression and consequent increase in secretase activity. We hypothesize that nutritional factors causing homocysteine cycle alterations (i.e. hyperhomocysteinemia) could induce sequence-specific DNA hypomethylation and “aberrant” gene activation.Aim of present study was to analyze the methylation pattern of PSEN1 promoter in SK-N-BE neuroblastoma cells and TgCRND8 mice, in a B vitamin (folate, B12 and B6) deficiency paradigm. PSEN1 methylation status has been evaluated through bisulphite modification and genomic sequencing. We demonstrate that B vitamin deficiency induces hypomethylation of specific CpG moieties in the 5′-flanking region; S-adenosylmethionine has been supplemented as methyl donor to reverse this effect. PSEN1 promoter methylation status is correlated with gene expression. These findings pinpoint a direct relationship between B vitamin-dependent alteration of homocysteine cycle and DNA methylation and also indicate that PSEN1 promoter is regulated by methylation of specific CpG moieties.     

Indirubin-3′-monoxime inhibits β-amyloid-induced neurotoxicity in neuroblastoma SH-SY5Y cells

Increasing evidence suggests that the inappropriate activation of cyclin-dependent kinases (CDKs) could induce neuronal apoptosis in Alzheimer's disease (AD), which means that the pharmacological inhibitors of cell-cycle progression may effectively impede the development or progression of AD. Indirubin-3′-monoxime (IMX), a known effective inhibitor of CDKs, has been shown to have therapeutic effects on learning and memory deficits induced by beta-amyloid (Aβ) intracerebroventricular infusion in rats. In the present study, we investigated the neuroprotective effects of IMX on Aβ_25–35-induced neuronal apoptosis and its potential mechanisms in human neuroblastoma SH-SY5Y cells. Aβ_25–35-induced apoptosis, characterized by decreased cell viability, neuronal DNA condensation, and fragmentation, was associated with an increase in tau protein hyperphosphorylation. IMX, however, attenuated Aβ_25–35-induced cell death in a dose-dependent manner. Furthermore, expression of hyperphosphorylation tau protein was significantly decreased with IMX treatment. Our study suggests that IMX may usefully prevent or delay the neuronal loss of AD.     

The effect of intra-arterial papaverine on ECoG activity in the ketamine anesthetized rat

The opium alkaloid papaverine (PPV) causes vasodilatation of the cerebral arteries through direct action on smooth muscle that reduces the constriction of smooth muscle. Intra-arterial papaverine (IAP) has been used widely to increase the regional cerebral blood flow in order to reverse the cerebral vasospasm that occurs during endovascular procedures. IAP-induced seizures have been reported, although PPV has anticonvulsive effects. This study determined the effects of IAP on electrocorticography (ECoG) in the ketamine anesthetized rats. We used 24 Sprague–Dawley male rats weighing 200–250 g. The animals were divided randomly into four groups: three treatment groups (groups 1–3) and a control (group 4). Groups 1, 2, and 3 were given 1, 7, and 14 mg/kg IAP, respectively. The ECoG was compared across groups. Our results indicated that IAP did not cause seizures and that it decreased the frequency of ketamine-induced epileptiform activity in the 14 mg/kg group.     

Lesch-Nyhan syndrome: mRNA expression of HPRT in patients with enzyme proven deficiency of HPRT and normal HPRT coding region of the DNA

Inherited mutation of the purine salvage enzyme, hypoxanthine guanine phosphoribosyltransferase (HPRT) gives rise to Lesch-Nyhan syndrome (LNS) or Lesch-Nyhan variants (LNV). We report a case of two LNS affected members of a family with deficiency of activity of HPRT in intact cultured fibroblasts in whom mutation could not be found in the HPRT coding sequence but there was markedly decreased HPRT expression of mRNA.     

Classical swine fever virus NS5A protein interacts with 3'-untranslated region and regulates viral RNA synthesis

To investigate the function of classical swine fever virus (CSFV) NS5A protein, the experiments for viral RNA synthesis and viral replication were performed in the co-presence of NS5A and NS5B. Results showed that small concentrations of NS5A stimulated, large concentrations of NS5A inhibited, viral RNA synthesis and viral replication. Affinity chromatography experiments and UV-crosslinking assays revealed that CSFV NS5A and NS5B bound its cognate 3′UTR and that NS5A had higher affinity than NS5B protein in binding to 3′UTR. 200 ng of NS5A inhibited NS5B-3′UTR complex formation by about 95%. CSFV 3′UTR was found to contain two NS5A-binding sites, located in 3′UTRSL-1 (nt 161-231) and 3′UTRSL-2 (nt 90-160), respectively, a NS5B-binding site, also located in 3′UTRSL-1. The 3′UTRSL-1 is the common binding site for NS5A and NS5B. Furthermore, competitive electrophoretic mobility shift assays indicated that binding of CSFV NS5A to 3′UTRSL-1 is more efficiently than to 3′UTRSL-2. These results suggested that the different concentrations of NS5A, the different binding activities of NS5A and NS5B to 3′UTR and binding of NS5A to different regions of 3′UTR might contribute at least partially to modulation of CSFV replication.     

Phenylbutenoid dimers isolated from Zingiber purpureum exert neurotrophic effects on cultured neurons and enhance hippocampal neurogenesis in olfactory bulbectomized mice

Trans-3-(3′4′-dimethoxyphenyl)-4-[(E)-3″,4″-dimethoxystyryl]cyclohex-1-ene (Comp.1) and cis-3-(3′4′-dimethoxyphenyl)-4-[(E)-3″,4″-dimethoxystyryl]cyclohex-1-ene (Comp.2), phenylbutenoid dimers, have been isolated as neurotrophic molecules from an Indonesian medicinal plant, Zingiber purpureum. The aim of this study was to explore the neurotrophic effects of Comp.1 and Comp.2 in vitro and in vivo. Comp.1 (10–30 μM) or Comp.2 (30 μM) significantly induced neurite sprouting in PC12 cells. Comp.1 (0.03–3 μM) or Comp.2 (0.3–3 μM) significantly increased the neurite length and number of neurites in primary cultured rat cortical neurons. Comp.1 (30 μM) and Comp.2 (3–30 μM) also provided significant protection against cell death caused by deprivation of serum. The in vivo effects of both Comp.1 and Comp.2 were evaluated on hippocampal neurogenesis in olfactory bulbectomized (OBX) mice, an experimental depression and dementia animal model. Comp.1 (50 mg/kg p.o.), Comp.2 (50 mg/kg p.o.), or fluoxetine (10 mg/kg i.p.), an antidepressant, were administrated once a day on days 15–28 after OBX. Neurogenesis was assessed by analysis of cells expressing NeuN, a neuronal marker, and 5-bromo-2′-deoxyuridine (BrdU) uptake. Immunohistochemical analysis showed that the number of BrdU/NeuN double-labeled cells in the dentate gyrus was significantly decreased 30 days after OBX. Chronic treatment with Comp.1, Comp.2 or fluoxetine significantly increased the number of BrdU/NeuN double-labeled cells. These results indicate that Comp.1 and Comp.2 have neurotrophic effects, and have the potential for disease modification in depression and dementia.     

The large form of human 2′,5′-Oligoadenylate Synthetase (OAS3) exerts antiviral effect against Chikungunya virus

Chikungunya virus (CHIKV) becomes one of the most important mosquito-borne alphavirus in the medical field. CHIKV is highly sensitive to antiviral activity of Type-I interferons (IFN-α/β). Here, we investigated the role of IFN-induced 2′,5′-Oligoadenylate Synthetase (OAS) family in innate immunity to CHIKV. We established inducible human epithelial HeLa cell lines expressing either the large form of human OAS, OAS3, or the genetic variant OAS3-R844X which is predicted to lack about 20% of the OAS3 protein from the carboxy terminus. HeLa cells respond to ectopic OAS3 expression by efficiently inhibiting CHIKV growth. The characteristic of the antiviral effect was a blockade in early stages of virus replication. Thus, OAS3 pathway may represent a novel antialphaviral mechanism by which IFN-α/β controls CHIKV growth. HeLa cells expressing the truncated form of OAS3 were less resistant to CHIKV infection, raising the question on the involvement of OAS3 genetic polymorphism in human susceptibility to alphavirus infection.     

MicroRNA-mediated dysregulation of neural developmental genes in HPRT deficiency: clues for Lesch-Nyhan disease?

Mutations in the gene encoding the purine biosynthetic enzyme hypoxanthine-guanine phosphoribosyltransferase (HPRT) cause the intractable neurodevelopmental Lesch-Nyhan disease (LND) associated with aberrant development of brain dopamine pathways. In the current study, we have identified an increased expression of the microRNA miR181a in HPRT-deficient human dopaminergic SH-SY5Y neuroblastoma cells. Among the genes potentially regulated by miR181a are several known to be required for neural development, including Engrailed1 (En1), Engrailed2 (En2), Lmx1a and Brn2. We demonstrate that these genes are down-regulated in HPRT-deficient SH-SY5Y cells and that over-expression of miR181a significantly reduces endogenous expression of these genes and inhibits translation of luciferase plasmids bearing the En1/2 or Lmx1a 3'UTR miRNA-binding elements. Conversely, inhibition of miR181a increases the expression of these genes and enhances translation of luciferase constructs bearing the En1/2 and Lmx1a 3'UTR miRNA-binding sequences. We also demonstrate that key neurodevelopmental genes (e.g. Nurr1, Pitx3, Wnt1 and Mash1) known to be functional partners of Lmx1a and Brn2 are also markedly down-regulated in SH-SY5Y cells over-expressing miR181a and in HPRT-deficient cells. Our findings in SH-SY5Y cells demonstrate that HPRT deficiency is accompanied by dysregulation of some of the important pathways that regulate the development of dopaminergic neurons and dopamine pathways and that this defect is associated with and possibly due at least partly to aberrant expression of miR181a. Because aberrant expression of miR181a is not as apparent in HPRT-deficient LND fibroblasts, the relevance of the SH-SY5Y neuroblastoma cells to human disease remains to be proven. Nevertheless, we propose that these pleiotropic neurodevelopment effects of miR181a may play a role in the pathogenesis of LND.     

The RNA helicase, nucleotide 5'-triphosphatase, and RNA 5'-triphosphatase activities of Dengue virus protein NS3 are Mg2+-dependent and require a functional Walker B motif in the helicase catalytic core

The nonstructural protein 3 (NS3) of Dengue virus (DV) is a multifunctional enzyme carrying activities involved in viral RNA replication and capping: helicase, nucleoside 5'-triphosphatase (NTPase), and RNA 5'-triphosphatase (RTPase). Here, a 54-kDa C-terminal domain of NS3 (DeltaNS3) bearing all three activities was expressed as a recombinant protein. Structure-based sequence analysis in comparison with Hepatitis C virus (HCV) helicase indicates the presence of a HCV-helicase-like catalytic core domain in the N-terminal part of DeltaNS3, whereas the C-terminal part seems to be different. In this report, we show that the RTPase activity of DeltaNS3 is Mg2+-dependent as are both helicase and NTPase activities. Mutational analysis shows that the RTPase activity requires an intact NTPase/helicase Walker B motif in the helicase core, consistent with the fact that such motifs are involved in the coordination of Mg2+. The R513A substitution in the C-terminal domain of DeltaNS3 abrogates helicase activity and strongly diminishes RTPase activity, indicating that both activities are functionally coupled. DV RTPase seems to belong to a new class of Mg2+-dependent RTPases, which use the active center of the helicase/NTPase catalytic core in conjunction with elements in the C-terminal domain.     

Evidence that acute taurine treatment alters extracellular AMP hydrolysis and adenosine deaminase activity in zebrafish brain membranes

Taurine is one of the most abundant free amino acids in excitable tissues. In the brain, extracellular taurine may act as an inhibitory neurotransmitter, neuromodulator, and neuroprotector. Nucleotides are ubiquitous signaling molecules that play crucial roles for brain function. The inactivation of nucleotide-mediated signaling is controlled by ectonucleotidases, which include the nucleoside triphosphate diphosphohydrolase (NTPDase) family and ecto-5′-nucleotidase. These enzymes hydrolyze ATP/GTP to adenosine/guanosine, which exert a modulatory role controlling several neurotransmitter systems. The nucleoside adenosine can be inactivated in extracellular or intracellular milieu by adenosine deaminase (ADA). In this report, we tested whether acute taurine treatment at supra-physiological concentrations alters NTPDase, ecto-5′-nucleotidase, and ADA activities in zebrafish brain. Fish were treated with 42, 150, and 400 mg L⁻¹ taurine for 1 h, the brains were dissected and the enzyme assays were performed. Although the NTPDase activities were not altered, 150 and 400 mg L⁻¹ taurine increased AMP hydrolysis (128 and 153%, respectively) in zebrafish brain membranes and significantly decreased ecto-ADA activity (29 and 38%, respectively). In vitro assays demonstrated that taurine did not change AMP hydrolysis, whereas it promoted a significant decrease in ecto-ADA activity at 150 and 400 mg L⁻¹ (24 and 26%, respectively). Altogether, our data provide the first evidence that taurine exposure modulates the ecto-enzymes responsible for controlling extracellular adenosine levels in zebrafish brain. These findings could be relevant to evaluate potential beneficial effects promoted by acute taurine treatment in the central nervous system (CNS) of this species.     

The Flvr-encoded murine oligoadenylate synthetase 1b (Oas1b) suppresses 2-5A synthesis in intact cells

Resistance to flavivirus-induced disease in mice is conferred by the autosomal gene Flv, identified as 2′-5′ oligoadenylate synthetase 1b (Oas1b). Resistant mice express a full-length Oas1b protein while susceptible mice express the truncated Oas1btr. In this study, Oas1b was shown to be an inactive synthetase. Although the Oas/RNase L pathway was previously shown to have an antiviral role during flavivirus infections, Oas1b protein inhibited Oas1a in vitro synthetase activity in a dose-dependent manner and reduced 2-5A production in vivo in response to poly(I:C). These findings suggest that negative regulation of 2-5A by inactive Oas1 proteins may fine tune the RNase L response that if not tightly controlled could cause significant damage in cells. The results also indicate that flavivirus resistance conferred by Oas1b is not mediated by 2-5A. Instead, Oas1b inhibits flavivirus replication by an alternative mechanism that overrides the proviral effect of reducing 2-5A accumulation and RNase L activation.     

Japanese encephalitis virus infection of mouse cell lines: ability to prime mice for generation of virus specific cytotoxic T lymphocytes and differences in CTL recognisable viral determinants

Summary Ten different mouse cell lines were examined for Japanese encephalitis virus (JEV) infection in vitro and then tested for their ability to generate virus specific cytotoxic T lymphocytes (CTL). Among all cell lines examined, Neuro 2a (a neuroblastoma) was readily infected with JEV as examined by immunofluorescence and viral replication. Among other cells, P388D1, RAW 264.7 (Macrophage origin), Sp2/0 (B-cell Hybridoma), YAC-1 (T-cell lymphoma), and L929 (Fibroblast) were semipermissive to JEV infection. The cytopathic effects caused by progressive JEV infection varied from cell line to cell line. In the case of YAC-1 cells long-term viral antigen expression was observed without significant alterations in cell viability. Intermediate degrees of cytopathicity are seen in RAW 264.7 and L929 cells while infection of PS, Neuro 2a, P388D1 and Sp2/0 caused major viability losses. All infected cell lines were able to prime adult BALB/c (H-2^d) mice for the generation of secondary JEV specific CTL. In contrast to YAC-1, the permissive neuroblastoma cell line Neuro 2a (H-2K^kD^d) was found to be least efficient in its ability to stimulate anti-viral CTL generation. Cold target competition studies demonstrated that both Neuro 2a and YAC-1 (H-2K^kD^d) cells expressed similar viral determinants that are recognised by CTL, suggesting that the reason for the lower ability of Neuro 2a to stimulate anti-viral CTL was not due to lack of viral CTL determinants. These findings demonstrate that a variety of mouse cell lines can be infected with Japanese encephalitis virus, and that these infected cells could be utilised to generate virus specific CTL in BALB/c mice.     

Guanine nucleotide depletion induces differentiation and aberrant neurite outgrowth in human dopaminergic neuroblastoma lines: a model for basal ganglia dysfunction in Lesch-Nyhan disease

Lesch-Nyhan disease (LND), caused by complete deficiency of hypoxanthine guanine phosphoribosyltransferase (HPRT), is characterized by a neurological deficit, the etiology of which is unknown. Evidence has accumulated indicating that it might be related to dysfunction of the basal ganglia with a prominent loss of striatal dopamine fibers. Guanine nucleotide depletion has been shown to occur in cells from Lesch-Nyhan patients. In this study we demonstrate that chronic guanine nucleotide depletion induced by inhibition of inosine monophosphate dehydrogenase with low levels (50 nM) of mycophenolic acid (MPA) lead human neuroblastoma cell lines to differentiate toward the neuronal phenotype. The MPA-induced morphological changes were more evident in the dopaminergic line LAN5, than in the cholinergic line IMR32. MPA-induced differentiation, unlike that induced by retinoic acid, caused a less extensive neurite outgrowth and branching (similar to that observed in cultured HPRT-deficient dopaminergic neurons) and involved up-regulation of p53, p21 and bax, and bcl-2 down-regulation without p27 protein accumulation. These results suggest that guanine nucleotide depletion following HPRT deficiency, might lead to earlier and abnormal brain development mainly affecting the basal ganglia, displaying the highest HPRT activity, and could be responsible for the specific neurobehavioral features of LND.     

Immunohistochemical localization of d-serine dehydratase in chicken tissues

Chicken d-serine dehydratase (DSD) degrades d-serine to pyruvate and ammonia. The enzyme requires both pyridoxal 5′-phosphate and Zn²⁺ for its activity. d-Serine is a physiological coagonist that regulates the activity of the N-methyl-d-aspartate receptor (NMDAR) for l-glutamate. We have recently found in chickens that d-serine is degraded only by DSD in the brain, whereas it is also degraded to 3-hydroxypyruvate by d-amino acid oxidase (DAO) in the kidney and liver. In mammalian brains, d-serine is degraded only by DAO. It has not been clarified why chickens selectively use DSD for the control of d-serine concentrations in the brain. In the present study, we measured DSD activity in chicken tissues, and examined the cellular localization of DSD using a specific anti-chicken DSD antibody. The highest activity was found in kidney. Skeletal muscles and heart showed no activity. In chicken brain, cerebellum showed about 6-fold-higher activity (1.1 ± 0.3 U/g protein) than cerebrum (0.19 ± 0.03 U/g protein). At the cellular level DSD was demonstrated in proximal tubule cells of the kidney, in hepatocytes, in Bergmann-glia cells of the cerebellum and in astrocytes. The finding of DSD in glial cells seems to be important because d-serine is involved in NMDAR-dependent brain functions.     

The 5′CL-PCBP RNP complex, 3′ poly(A) tail and 2A are required for optimal translation of poliovirus RNA

In this study, we showed that the 5′CL-PCBP complex, 3′ poly(A) tail and viral protein 2A^pro are all required for optimal translation of PV RNA. The 2A^pro-mediated stimulation of translation was observed in the presence or absence of both the 5′CL and the 3′ poly(A) tail. Using protein-RNA tethering, we established that the 5′CL-PCBP complex is required for optimal viral RNA translation and identified the KH3 domain of PCBP2 as the functional region. We also showed that the 5′CL-PCBP complex and the 3′ poly(A) tail stimulate translation independent of each other. In addition to the independent function of each element, the 5′CL and the 3′ poly(A) tail function synergistically to stimulate and prolong translation. These results are consistent with a model in which the 5′CL-PCBP complex interacts with the 3′ poly(A)-PABP complex to form a 5′-3′ circular complex that facilitates ribosome reloading and stimulates PV RNA translation.