Shuqiong Niu

Changes in mRNA for CAPON and Dexras1 in adult rat following sciatic nerve transection.

Peripheral nerve transection has been implicated to cause a production of neuronal nitric oxide synthase (nNOS), which may influence a range of post-axotomy processes necessary for neuronal survival and nerve regeneration. Carboxy-terminal post synaptic density protein/Drosophila disc large tumor suppressor/zonula occuldens-1 protein (PDZ) ligand of neuronal nitric oxide synthase (CAPON), as an adaptor, interacts with nNOS via the PDZ domain helping regulate nNOS activity at postsynaptic sites in neurons. And Dexras1, a small G protein mediating multiple signal transductions, has been reported to form a complex with CAPON and nNOS. A role for the physiologic linkage by CAPON of nNOS to Dexras1 has suggested that NO-mediated activation of Dexras1 is markedly enhanced by CAPON. We investigated the changes in mRNA for CAPON, Dexras1 and nNOS in the sciatic nerve, dorsal root ganglia and lumbar spinal cord of adult rat following sciatic axotomy by TaqMan quantitative real-time PCR and in situ hybridization combined with immunofluorescence. Signals of mRNA for CAPON and Dexras1 were initially expressed in these neural tissues mentioned, transiently increased at certain time periods after sciatic axotomy and finally recovered to the basal level. It was also found that nNOS mRNA underwent a similar change pattern during this process. These results suggest that CAPON as well as Dexras1 may be involved in the different pathological conditions including nerve regeneration, neuron loss or survival and even pain process, possibly via regulating the nNOS activity or through the downstream targets of Dexras1.

The role of TNF-α and its receptors in the production of Src-suppressed C kinase substrate by rat primary type-2 astrocytes

Src-suppressed C kinase substrate (SSeCKS), an in vivo and in vitro protein kinase C substrate, is a major lipopolysaccharide (LPS) response protein which markedly upregulated in several organs, including brain, lung, heart, kidney, etc., indicating a possible role of SSeCKS in inflammatory process. In the central nervous system (CNS), astrocytes play a pivotal role in immunity as immunocompetent cells by secreting cytokines and inflammatory mediators, there are two types of astrocytes. Type-1 astrocytes can secrete TNF-alpha when stimulated with lipopolysaccharide (LPS), while the responses of type-2 astrocytes during inflammation are unknown. So we examined the expression change of SSeCKS mRNA in type-2 astrocytes after exposure to TNF-alpha and LPS. Real-time PCR showed that TNF-alpha or LPS affected SSeCKS mRNA expression in a time- and dose-dependent manner. Now that LPS induces SSeCKS expression in type-2 astrocytes and type-1 astrocytes are well known to play a pivotal role in immunity, we compared SSeCKS mRNA expression in type-1 astrocytes with type-2 astrocytes after LPS stimulation. Real-time PCR showed that SSeCKS mRNA level was higher in normal untreated type-2 astrocytes than that in normal untreated type-1 astrocytes, increased significantly after 0.1-100 ng/ml LPS stimulation in type-2 astrocytes, but increased weakly after 10-100 ng/ml LPS stimulation in type-1 astrocytes. By using siRNA knockdown of SSeCKS expression, LPS-induced TNF-alpha synthesis and secretion in type-2 astrocytes were partly inhibited, which indicated that SSeCKS played a role in the TNF-alpha biosynthesis in type-2 astrocytes during the stimulation with LPS. RT-PCR analysis revealed that TNFR1 and TNFR2 were present in normal untreated type-2 astrocytes and that TNF-alpha, TNFR1 and TNFR2 increased in type-2 astrocytes after exposure to TNF-alpha or LPS. Immunocytochemistry showed that TNFR1 was expressed in the cytoplasm, nucleus and processes of normal untreated type-2 astrocytes and distributed mainly in the cytoplasm and processes after exposure to LPS. TNFR2 was mainly expressed in the nucleus of normal untreated type-2 astrocytes and distributed mainly in the processes of type-2 astrocytes after exposure to LPS. Both anti-TNFR1 and anti-TNFR2 antibodies suppressed SSeCKS mRNA expression induced by TNF-alpha or LPS. From these results, we conclude that TNF-alpha signaling via both TNFR1 and TNFR2 translocated from nucleus to cytoplasm or processes is sufficient to induce SSeCKS mRNA. In addition, we observed that not only exogenous TNF-alpha but also TNF-alpha produced by type-2 astrocytes affected SSeCKS mRNA production in type-2 astrocytes. These results suggest that an autocrine loop involving TNF-alpha contributes to the production of SSeCKS mRNA in response to inflammation. In addition, SSeCKS production was also drastically suppressed by U0126 (ERK inhibitor), SB203580 (p38 inhibitor), or SP600125 (SAPK/JNK inhibitor), which indicated that type-2 astrocytes which regulated SSeCKS expression after LPS stimulation were via ERK, SAPK/JNK, and P38MAP kinase signal pathway.

Spatiotemporal patterns of postsynaptic density (PSD)-95 expression after rat spinal cord injury

Aims: Postsynaptic density (PSD)‐95 is a scaffolding protein linking the N‐methyl‐D‐aspartate receptor with neuronal nitric oxide synthase (nNOS), which contributes to many physiological and pathological actions. We here investigated whether PSD‐95 was involved in the secondary response following spinal cord injury (SCI). Methods: Spinal cord contusion (SCC) and spinal cord transection (SCT) models at thoracic (T) segment 9 (T9) were established in adults rats. Real‐time polymerase chain reaction, Western blot, immunohistochemistry and immunofluorescence were used to detect the temporal profile and spatial distribution of PSD‐95 after SCI. The association between PSD‐95 and nNOS in the injured cords was also assessed by coimmmunoprecipation and double immunofluorescent staining. Results: The mRNA and protein for PSD‐95 expression were significantly increased at 2 h or 8 h, and then gradually declined to the baseline level, ultimately up‐regulated again from 5 days to 7 days for its mRNA level and at 7 days or 14 days for its protein level after either SCC or SCT. PSD‐95 immunoreactivity was found in neurones, oligodendrocytes and synaptic puncta of spinal cord tissues within 5 mm from the lesion site. Importantly, injury‐induced expression of PSD‐95 was colabelled by active caspase‐3 (apoptotic marker), Tau‐1 (the marker for pathological oligodendrocytes) and nNOS. Conclusions: Accompanied by the spatio‐temporal changes for PSD‐95 expression, the association between PSD‐95 and nNOS undergoes substantial alteration after SCI. These two molecules are likely to form a complex on apoptotic neurones and pathological oligodendrocytes, which may in turn be involved in the secondary response after SCI.

Developmental regulation of PSD-95 and nNOS expression in lumbar spinal cord of rats.

Postsynaptic density (PSD)-95 is originally isolated from glutamatergic synapse where it serves as a physical tether to allow neuronal nitric oxide synthase (nNOS) signaling by N-methyl-D-aspartate receptor (NMDAR) activity. Considering the physiological importance of glutamate receptor and nitric oxide (NO) during development, we examined the spatiotemporal expression of PSD-95 and nNOS in the lumbar spinal cord at a postnatal stage. Temporally, both gene and protein levels of them gradually increased with age after birth, peaked at the postnatal day 14 (P14), and then decreased to an adult level. In addition, the enhanced coimmunoprecipitations between PSD-95 and nNOS were detected in developing spinal cord. Spatially, PSD-95 staining codistributed with nNOS in NeuN-positive motor neurons and sensory neurons at P14. These findings indicate that PSD-95 and nNOS might collectively participate in spinal cord development.

Altered β-1,4-galactosyltransferase I expression during early inflammation after spinal cord contusion injury

Post-traumatic inflammation has been implicated in secondary tissue damage after spinal cord injury (SCI). beta-1,4-Galactosyltransferase I (beta-1,4-GalT-I) is a key inflammatory mediator that plays a critical role in the initiation and maintenance of inflammatory reaction in diseases. The aim of the current study was to investigate whether beta-1,4-GalT-I is expressed in SCI. Spinal cord contusion model was established in adult rats. Real-time PCR and Western blot analysis were used to detect the spatio-temporal expression of beta-1,4-GalT-I after SCI. Lectin-fluorescent staining with RCA-I was used to detect the galactosylation of the membrane glycoproteins. The interaction and colocalization between beta-1,4-GalT-I and E-selectin in the injured spinal cords were also assessed by immunoprecipitation of E-selectin and double immunofluorescent staining, respectively. Real-time PCR revealed that beta-1,4-GalT-I mRNA reached the peak at 1d after spinal cord contusion. In situ hybridization indicated that beta-1,4-GalT-I mRNA was mainly distributed in the local inflammatory cells, adjacent to the center of injury. Double immunofluorescent staining showed that beta-1,4-GalT-I mostly overlapped with ED1-positive macrophages 1d after SCI, partly colocalized with microglia, neutrophils and a few with oligodendrocytes and astrocytes. The result of Lectin-fluorescent staining with RCA-I was similar to that of double immunofluorescent staining. Terminal galactosylation of E-selectin underwent obvious changes between sham and 3d after SCI by immunoprecipitation of E-selectin. Thus, the transient expression of high levels of beta-1,4-GalT-I may provide new insight into the early inflammation after SCI.

Spatiotemporal Expression of SSeCKS in Injured Rat Sciatic Nerve

SSeCKS (src suppressed C kinase substrate) functions in the control of cell signaling and cytoskeletal arrangement. It is expressed in brain and spinal cord, but little is known about its expression in peripheral nerves. In this study, in rats, real‐time polymerase chain reaction and Western blot analysis showed that expression of SSeCKS in crushed sciatic nerve reached its highest level 6 hr after crushing, whereas in a transection model, SSeCKS peaked at 2 days in the proximal stump and 12 hr in the distal stump. Immunohistochemical analysis demonstrated up‐regulation of SSeCKS protein surrounding the crush site and in the two stumps of the transected nerve. In addition, SSeCKS colocalized with growth‐associated protein 43 and with S100, which also changed with time after injury. These findings support the idea that SSeCKS participates in the adaptive response to peripheral nerve injury and may be associated with regeneration. Anat Rec, 291:527–537, 2008.

The Role of TNF-α and its Receptors in the Production of β-1,4 Galactosyltransferase I and V mRNAs by Rat Primary Astrocytes

Glycosylation is one of the most important post-translational modifications. It is clear that the single step of β-1,4-galactosylation is performed by a family of β-1, 4-galactosyltransferases (β-1,4-GalTs), and that each member of this family may play a distinct role in different tissues and cells. β-1,4-GalT I and V are involved in the biosynthesis of N-linked oligosaccharides. β-1,4-GalT I and V mRNAs are present in control astrocytes and affected by TNF-α and lipopolysaccharide (LPS) stimuli. In this study, we examined the regulatory mechanisms of tumor necrosis factor-α (TNF-α)-affected production of β-1,4-GalT I and V mRNAs. We show here that cultured astrocytes express TNF-α receptor 1 (TNFR1) and increased slightly after exposure to LPS. TNF-α and TNFR2 are not detected in control astrocytes and upregulated significantly with LPS stimulation and that activation of these receptors by TNF-α affects expressions of β-1,4-GalT I and V mRNAs. In addition, we observed that not only exogenous TNF-α but also TNF-α produced by astrocytes affected β-1,4-GalT I and V mRNAs production in astrocytes. These results suggest that an autocrine loop involving TNF-α contributes to the production of β-1,4-GalT I and V mRNA in response to inflammation.

Expression of β-1,4-Galactosyltransferase-I in Rat during Inflammation

Abstractβ-1,4-Galactosyltransferase-I (β-1,4-GalT-I) which is one of the best-studied glycosyltransferases, plays a key role in the synthesis of selectin ligands such as sialy Lewis (sLex) and sulfated sLex. Previous studies showed that inflammatory responses of β-1,4-GalT-I-deficient mice were impaired because of the defect in selectin-ligand biosynthesis. However, the expression of β-1,4-GalT-I during inflammation and its biological function remains to be elucidated. Real-time PCR showed that intraperitoneal administration of LPS strongly induced β-1,4-GalT-I mRNA expression in the lung, heart, liver, spleen, kidney, lymph node, hippocampus, and testis, as well as in the cerebral cortex. In the rat lung, liver and testis, LPS stimulation of β-1,4-GalT-I mRNA expression is time-dependent and biphasic. Lectin-fluorescent staining with RCA-I showed that LPS induced expression of galactose-containing glycans in rat lung and liver to the higher lever. Morphology analysis observed that galactose-containing glycans and β-1,4-GalT-I mRNA was mostly expressed in neutrophils, macrophages and endothelial cells. These findings indicated that β-1,4-GalT-I may play an important role in the inflammation reaction.

The Role of TNF-α and its Receptors in the Production of β-1,4-galactosyltransferase I mRNA by Rat Primary Type-2 Astrocytes

Abstractβ-1,4-galactosyltransferase I (β-1,4-GalT I) plays an important role in the synthesis of the backbone structure of adhesion molecules involved in leukocyte–endothelial cell interaction. The expression of β-1,4-GalT I mRNA increased in primary human endothelial cells after exposure to tumor necrosis factor-α (TNF-α). In the central nervous system (CNS), astrocytes play a pivotal role in immunity as immunocompetent cells by secreting cytokines and inflammatory mediators, there are two types of astrocytes. Type-1 astrocytes can secrete TNF-α when stimulated with Lipopolysaccharide (LPS), while the responses of type-2 astrocytes during inflammation are unknown. So we examined the expression change of β-1,4-GalT I mRNA in type-2 astrocytes after exposure to TNF-α and LPS. Real-time PCR showed that TNF-α or LPS affected β-1,4-GalT I mRNA expression in a time- and dose-dependent manner. RT-PCR analysis revealed that TNFR1 and TNFR2 were present in normal untreated type-2 astrocytes, and that TNF-α, TNFR1 and TNFR2 increased in type-2 astrocytes after exposure to TNF-α or LPS. Immunocytochemistry showed that TNFR1 was expressed in the cytoplasm, nucleus and processes of normal untreated type-2 astrocytes, and distributed mainly in the cytoplasm and processes after exposure to LPS. TNFR2 was mainly expressed in the nucleus of normal untreated type-2 astrocytes, and distributed mainly in the processes of type-2 astrocytes after exposure to LPS. Both anti-TNFR1 and anti-TNFR2 antibodies suppressed β-1,4-GalT I mRNA expression induced by TNF-α or LPS. From these results, we conclude that TNF-α signaling via both TNFR1 and TNFR2 translocated from nucleus to cytoplasm or processes is sufficient to induce β-1,4-GalT I mRNA. In addition, we observed that not only exogenous TNF-α but also TNF-α produced by type-2 astrocytes affected β-1,4-GalT I mRNA production in type-2 astrocytes. These results suggest that an autocrine loop involving TNF-α contributes to the production of β-1,4-GalT I mRNA in response to inflammation.

Effect of Peripheral Axotomy on Gene Expression of NIDD in Rat Neural Tissues

Peripheral nerve lesion-induced production of neuronal nitric oxide synthase (nNOS) was implicated to influence a range of postaxotomy processes necessary for neuronal survival and nerve regeneration (Zochodne et al., Neuroscience, 91:1515–1527, 1999; Keilhoff et al., Journal of Chemical Neuroanatomy, 24:181–187, 2002, Nitric Oxide, 10:101–111, 2004). Protein–protein interactions represent an important mechanism in the control of NOS spatial distribution or activity (Alderton et al., Biochemical Journal, 357:593–615, 2001; Dedio et al., FASEB Journal, 15:79–89, 2001; Zimmermann et al., Proceedings of the National Academy of Sciences, 99:17167–17172, 2002). As one of the nNOS-binding proteins, nNOS-interacting DHHC domain-containing protein with dendritic mRNA (NIDD) has recently been identified to increase nNOS enzyme activity by targeting nNOS to the synaptic plasma membrane in a postsynaptic density protein 95/discs-large/zona occlusens-1 domain dependent manner (Saitoh et al., Journal of Biological Chemistry, 279:29461–29468, 2004). In this paper, we established a rat model with peripheral axotomy to investigate the gene expression patterns of NIDD in neural tissues using TaqMan quantitative real-time polymerase chain reaction and in situ hybridization combined with immunofluorescence. It revealed that NIDD mRNA was upregulated after sciatic nerve transection with the similar expressing styles as that of the nNOS in the injured nerves, corresponding dorsal root ganglia, and lumbar spinal cord. These findings imply that NIDD may be involved in the different pathological conditions including nerve regeneration, neuron loss or survival, and even pain process, possibly via regulating the enzyme nNOS activity.