Changes in the BAG1 expression of Schwann cells after sciatic nerve crush.

Bcl-2-associated athanogene-1 (BAG1), a co-chaperone for Hsp70/Hsc70, is a multifunctional protein, which has been shown to suppress apoptosis and enhance neuronal differentiation. However, the expression and roles of BAG1 in peripheral system lesions and repair are still unknown. In this study, we investigated the dynamic changes in BAG1 expression in an acute sciatic nerve crush model in adult rats. Western blot analysis revealed that BAG1 was expressed in normal sciatic nerves. BAG1 expression increased progressively after sciatic nerve crush, reached a peak 2 weeks post-injury, and then returned to the normal level 4 weeks post-injury. Spatially, we observed that BAG1 was mainly expressed in Schwann cells and that BAG1 expression increased in Schwann cells after injury. In vitro, we found that BAG1 expression increased during the cyclic adenosine monophosphate (cAMP)-induced Schwann cell differentiation process. BAG1-specific siRNA inhibited cAMP-induced Schwann cell differentiation. In conclusion, we speculated that BAG1 was upregulated in the sciatic nerve after crush, which was associated with Schwann cell differentiation.

FOXO3a/p27kip1 expression and essential role after acute spinal cord injury in adult rat.

FOXO3a (Forkhead Class box O3a), as an important direct target of the phosphatidylinositol 3-kinase (PI3K)/protein B (Akt) pathway, which regulates the cell survival and the cell-cycle progression. Recent reports showed that FOXO3a could inhibit cell-cycle progression at the G1/S transition by controlling transcription of the cyclin-dependent kinase inhibitor p27(kip1) , which is also a key regulator of the mammalian neurogenesis. To elucidate the expression and role of FOXO3a in nervous system lesion and repair, we performed an acute spinal cord contusion injury (SCI) model in adult rats, which showed a temporal-spatial expression pattern of FOXO3a. Temporally, FOXO3a protein level significantly reduced day 3 after injury, and following FOXO3a down-regulation, p27(kip1) protein and mRNA levels were also decreased after injury. Spatially, decreased levels of FOXO3a and p27(kip1) were predominant in astrocytes, which were regenerating axons and largely proliferated after injury. Furthermore in vitro, Western blot analysis, RT-PCR, and immunofluorescence staining analysis demonstrated the relationship between FOXO3a and p27(kip1) in primary astrocytes. FOXO3a modulated the cell cycle by transcriptional regulation of p27(kip1) in astrocytes. Administration of the PI3K pharmacological inhibitor LY294002 abrogated this effect by regulating FOXO3a and p27(kip1) expression and subcellular localization. These results suggest that decreased levels of FOXO3a and p27(kip1) in spinal cord are involved in axonal regeneration and the proliferation of glial cells after SCI.

An upregulation of SENP3 after spinal cord injury: implications for neuronal apoptosis.

SENP3 (SUMO-specific proteases 3), a member of the small ubiquitin-like modifier specific protease family, was identified as a molecule that deconjugates SUMOylation of modified protein substrates and functions as an isopeptidase by disrupting SUMO homeostasis to facilitate cancer development and progression. However, its expression and function in nervous system injury and repair are still unclear. In this study, we employed an acute spinal cord injury (SCI) model in adult rats and investigated the dynamic changes of SENP3 expression in the spinal cord. Western blot analysis indicated a gradual increase in SENP3 expression, which peaked 3 days after SCI, and then declined over the following days. Immunohistochemistry results further confirmed that SENP3 was expressed at low levels in the gray and white matter in the non-injured condition and increased after SCI. Moreover, immunofluorescence double-labeling showed that SENP3 was co-expressed with the neuronal marker, NeuN. Furthermore, the SENP3-positive cells that were co-expressed with NeuN had also expressed active caspase-3 after injury. To investigate whether SENP3 plays a role in neuronal apoptosis, we applied H(2)O(2) to induce neuronal apoptosis in vitro. Western blot analysis showed a significant upregulation of SENP3 and active caspase-3 following H(2)O(2) stimulation. Taken together, these results suggest that SENP3 may play important roles in the pathophysiology of SCI.

Spatiotemporal patterns and essential role of TNF receptor-associated factor 5 expression after rat spinal cord Injury.

TRAF5 (TNF receptor-associated factor 5), which has a tumor necrosis factor receptor-associated factor (TRAF) domain in its carboxyl terminus, was identified CD40-associated factor. It was a signal transducer for NF-κB signal pathway and other pathway. To elucidate the expression and roles of TRAF5 in nervous system lesion and repair, we performed an acute spinal cord injury (SCI) model in adult rats and studied the dynamic changes of TRAF5 expression in spinal cord. Western blot and immunohistochemistry analysis revealed that TRAF5 was present in normal spinal cord. It gradually increased, reached a peak at 5 days after SCI, and then declined during the following days. Immunofluorescence double-labeling revealed that TRAF5 was co-expressed with NeuN and GFAP, respectively. Interesting, after injury, TRAF5 expression was increased predominantly in astrocytes, which highly expressed PCNA, a marker for proliferating cells. In conclusion, this is the first description of TRAF5 expression in spinal cord. Our data suggested that TRAF5 might play important roles in CNS pathophysiology after SCI.

ß-1,4-Galactosyltransferase I involved in Schwann cells proliferation and apoptosis induced by tumor necrosis factor-alpha via the activation of MAP kinases signal pathways.

ß-1,4-galactosyltransferase-I (ß-1,4-GalT-I) plays a critical role in the initiation and maintenance of peripheral nervous system inflammatory reaction. However, the exact function of ß-1,4-GalT-I in the regulation of SCs proliferation and apoptosis remains unclear. In this study, we found that low concentration of tumor necrosis factor-alpha (TNF-α) induced SCs proliferation, while high concentration of TNF-α induced SCs apoptosis. Meanwhile, the expressions of ß-1,4-GalT-I, TNFR1, and TNFR2 were changed following. When ß-1,4-GalT I overexpression, low concentration of TNF-α-induced SCs proliferation was partially repressed. Concurrently, the activity of ERK1/2 was decreased. While knocking down ß-1,4-GalT I expression, high concentration of TNF-α-induced SCs apoptosis was partially rescued. Consistent with this, the activity of P38 and JNK were decreased. We also found anti-TNFR2 antibody suppressed low concentration of TNF-α-induced SCs proliferation, while anti-TNFR1 antibody inhibited high concentration of TNF-α-induced SCs apoptosis. Thus, present data show that ß-1,4-GalT I may play an important role in SCs proliferation and apoptosis induced by TNF-α via different signal pathways and TNFR.

Dynamic changes of PIRH2 and p27kip1 expression in injured rat sciatic nerve.

p53-induced ring-H2 protein (PIRH2), a newly identified E3 ubiquitin ligase, has been reported to be interacted with p27Kip1 and promote ubiquitination of p27Kip1 independently of p53. p27kip1, a member of the Cip/Kip family of cyclin-dependent kinases inhibitors (CKIs), was shown to control cell cycle progression and promote cell proliferation. While the distribution and function of PIRH2 and p27kip1 in nervous system lesion and regeneration remains unclear. Here, we performed a sciatic nerve injury model in adult rats and studied the dynamic changes of PIRH2 and p27kip1 expression by western blot and RT-PCR in injured rat sciatic nerve. Sciatic nerve crush resulted in a significant up-regulation of PIRH2 and a down-regulation of p27kip1. Besides, we observed that they were expressed widely in both Schwann cells and axons in adult rat sciatic nerve by double immunofluorescence staining. Results obtained by coimmunoprecipitation and double labeling further showed their interaction in the regenerating process. Thus, these results indicate that PIRH2 and p27kip1 likely play an important role in peripheral nerve injury and regeneration.

The cyclin-dependent kinase inhibitor p27(Kip1) is a positive regulator of Schwann cell differentiation in vitro.

Schwann cell precursors differentiating into a myelinating phenotype are critical for peripheral nerve development and regeneration. However, little is known about the underlying molecular mechanisms of Schwann cell differentiation. In the present study, we performed a cyclic adenosine monophosphate-induced Schwann cell differentiation model in vitro. Western blot analysis showed that p27(Kip1) expression was upregulated during the differentiation of Schwann cell, while the inhibition of p27(Kip1) expression by short hairpin RNA-mediated knockdown significantly abolished the expression of promyelinating markers and the alteration of cellular morphology. In addition, immunofluorescence revealed a decrease of p27(Kip1) nuclear staining and a concomitant increase of cytoplasmic staining in differentiated Schwann cells. In summary, our data indicated that p27(Kip1) was a positive regulator of Schwann cell differentiation in vitro.

KPC1 expression and essential role after acute spinal cord injury in adult rat.

KPC1 (Kip1 ubiquitylation-promoting complex 1) is the catalytic subunit of the ubiquitin ligase KPC, which regulates the degradation of the cyclin-dependent kinase inhibitor p27(kip1) at the G1 phase of the cell cycle. To elucidate the expression and role of KPC1 in nervous system lesion and repair, we performed an acute spinal cord contusion injury (SCI) model in adult rats. Western blot analysis showed a significant up-regulation of KPC1 and a concomitant down-regulation of p27(kip1) following spinal injury. Immunohistochemistry and immunofluorescence revealed wide expression of KPC1 in the spinal cord, including expression in neurons and astrocytes. After injury, KPC1 expression was increased predominantly in astrocytes, which highly expressed PCNA, a marker for proliferating cells. Co-immunoprecipitation demonstrated increased interactions between p27(kip1) and KPC1 4 days after injury. To understand whether KPC1 plays a role in astrocyte proliferation, we applied LPS to induce astrocyte proliferation in vitro. Western blot analysis demonstrated that p27(kip1) expression was negatively correlated with KPC1 expression following LPS stimulation. Immunofluorescence analysis showed subcellular localizations of p27(kip1) and KPC1 were also changed following the stimulation of astrocytes with LPS. These results suggest that KPC1 is related to the down-regulation of p27(kip1); this event may be involved in the proliferation of astrocytes after SCI.