Mengling Chen

Temporal–spatial expressions of p27kip1 and its phosphorylation on Serine-10 after acute spinal cord injury in adult rat: Implications for post-traumatic glial proliferation

P27kip1, as a member of Cip/Kip family of cyclin-dependent kinase inhibitors, plays important roles in cell cycle regulation and neurogenesis in the developing central nervous system. Serine-10 is the major phosphorylation site of p27kip1, and post-translational regulation of p27kip1 by different phosphorylation events is critical for its function. To elucidate the expressions and possible functions of p27kip1 and its phosphorylation in central nervous system lesion and repair, we performed an acute spinal cord contusion injury model in adult rats. Our work studied the temporal-spatial expression patterns of p27kip1 and Serine-10 phosphorylated p27kip1 (p-p27s10). Western blot analysis showed p27kip1 level significantly decreased at day 3 after damage, while p-p27s10 was detected at a high-level at the same time reaching the uninjured level. Moreover, immunofluorescence double labeling suggested these changes were striking in microglia and astrocytes, which were largely proliferated. Immunohistochemical analysis revealed subcellular localization changes of p27kip1 and p-p27s10 staining between nucleus and cytoplasm after injury in about 20% of total positive cells including neurons and glial cells. We also investigated the increased interactions of p27kip1 and p-p27s10 with CRM1 3 days after injury by co-immunoprecipitation studies. Taken together, we hypothesized spinal cord injury stimulated mitogenic signals to induce a serine-threonine kinase KIS (kinase interacting stathmin) to phosphorylate p27kip1 on Serine-10, so that p27kip1 could bind to CRM1 and be exported from nuclei for degradation. Such an event facilitated cell cycle progression of glial cells, especially microglia and astrocytes which had a prevalent proliferation.

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.

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.

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.

Dynamic Changes of p27kip1 and Skp2 Expression in Injured Rat Sciatic Nerve

S phase kinase-associated protein 2 (Skp2), an F-box protein, is required for the ubiquitination and consequent degradation of p27kip1. Previous reports have showed that p27kip1 played important roles in cell cycle regulation and neurogenesis in the developing central nervous system. But the distribution and function of p27kip1 and Skp2 in nervous system lesion and regeneration remains unclear. In this study, we observed that they were expressed mainly in both Schwann cells and axons in adult rat sciatic nerve. Sciatic nerve crush and transection resulted in a significant up-regulation of Skp2 and a down-regulation of p27kip1. By immunochemistry, we found that in the distal stumps of transected nerve from the end to the edge, the appearance of Skp2 in the edge is coincided with the decrease in p27kip1 levels. Changes of them were inversely correlated. Results obtained by coimmunoprecipitation and double labeling further showed their interaction in the regenerating process. Thus, these results indicate that p27kip1 and Skp2 likely play an important role in peripheral nerve injury and regeneration.

Elevated expression of CAPON and neuronal nitric oxide synthase in the sciatic nerve of rats following constriction injury.

Neuronal nitric oxide synthase (nNOS) has been implicated in peripheral nerve lesions and regeneration. The CAPON adaptor protein interacts with the PDZ domain of nNOS, helping to regulate nNOS activity at post-synaptic sites in neurones, but it is not known whether its expression is altered in sciatic nerves after chronic nerve constriction injury. In the present study, the spatiotemporal expression of CAPON was determined in chronically constricted rat sciatic nerves. Similar to the level of protein expression, CAPON mRNA was significantly up-regulated for almost 5weeks following sciatic nerve injury. Immunohistochemistry demonstrated that increased CAPON was found mainly in S-100-positive Schwann cells. In addition, co-immunoprecipitation demonstrated an interaction between CAPON and nNOS in Schwann cells and the interaction was enhanced in injured sciatic nerves. CAPON may be involved in peripheral nerve regeneration through regulation of nNOS activity.