Expression of transforming growth factor-beta receptors in meningeal fibroblasts of the injured mouse brain.

The fibrotic scar which is formed after traumatic damage of the central nervous system (CNS) is considered as a major impediment for axonal regeneration. In the process of the fibrotic scar formation, meningeal fibroblasts invade and proliferate in the lesion site to secrete extracellular matrix proteins, such as collagen and laminin. Thereafter, end feet of reactive astrocytes elaborate a glia limitans surrounding the fibrotic scar. Transforming growth factor-beta1 (TGF-beta1), a potent scar-inducing factor, which is upregulated after CNS injury, has been implicated in the formation of the fibrotic scar and glia limitans. In the present study, expression of receptors to TGF-beta1 was examined by in situ hybridization histochemistry in transcortical knife lesions of the striatum in the mouse brain in combination with immunofluorescent staining for fibroblasts and astrocytes. Type I and type II TGF-beta receptor mRNAs were barely detected in the intact brain and first found in meningeal cells near the lesion 1 day postinjury. Many cells expressing TGF-beta receptors were found around the lesion site 3 days postinjury, and some of them were immunoreactive for fibronectin. After 5 days postinjury, many fibroblasts migrated from the meninges to the lesion site formed the fibrotic scar, and most of them expressed TGF-beta receptors. In contrast, few of reactive astrocytes expressed the receptors throughout the postinjury period examined. These results indicate that meningeal fibroblasts not reactive astrocytes are a major target of TGF-beta1 that is upregulated after CNS injury.

An in vitro model of the inhibition of axon growth in the lesion scar formed after central nervous system injury.

After central nervous system (CNS) injury, meningeal fibroblasts migrate in the lesion center to form a fibrotic scar which is surrounded by end feet of reactive astrocytes. The fibrotic scar expresses various axonal growth-inhibitory molecules and creates a major impediment for axonal regeneration. We developed an in vitro model of the scar using coculture of cerebral astrocytes and meningeal fibroblasts by adding transforming growth factor-beta1 (TGF-beta1), a potent fibrogenic factor. Addition of TGF-beta1 to this coculture resulted in enhanced proliferation of fibroblasts and the formation of cell clusters which consisted of fibroblasts inside and surrounded by astrocytes. The cell cluster in culture densely accumulated the extracellular matrix molecules and axonal growth-inhibitory molecules similar to the fibrotic scar, and remarkably inhibited the neurite outgrowth of cerebellar neurons. Therefore, this culture system can be available to analyze the inhibitory property in the lesion site of CNS.

Inflammatory disease and cancer with a decrease in Kupffer cell numbers in Nucling-knockout mice.

Nucling is a stress-inducible protein associated with apoptosomes. The cytochrome c-triggered formation of apoptosomes represents a key-initiating event in apoptosis. We have recently reported that Nucling regulates the apoptotic pathway by controlling the activation of NF-kappaB as well. Here we show that hepatocellular carcinoma (HCC) arising spontaneously against a background of hepatitis occurred more frequently in Nucling-knockout (KO) mice than wild-type (WT) mice. Biochemical serum testing revealed potential liver dysfunction with hypercholesterolemia in Nucling-KO males. In the background of Nucling-KO mice, we observed the up-regulation of TNFalpha, spontaneous NF-kappaB-activation and the induction of galectin-3 expression in liver. In addition, we observed a decrease in the number of Kupffer cells (KCs) in the KO mice. KCs are important for the hepatic immune system, acting as phagocytes or antigen-presenting cells (APCs). We found that KCs in Nucling-KO mice were apoptotic possibly through the up-regulation of TNFalpha. These observations indicate that Nucling is important for the regulation of NF-kappaB signals in liver. We propose that Nucling deficiency could be a powerful tool to reveal the NF-kappaB-related molecular networks leading to hepatitis and HCC development.

Regeneration of nigrostriatal dopaminergic axons after transplantation of olfactory ensheathing cells and fibroblasts prevents fibrotic scar formation at the lesion site.

The fibrotic scar formed after central nervous system injury has been considered an obstacle to axonal regeneration. The present study was designed to examine whether cell transplantation into a damaged central nervous system can reduce fibrotic scar formation and promote axonal regeneration. Nigrostriatal dopaminergic axons were unilaterally transected in rats and cultures of olfactory-ensheathing cells (OECs), and olfactory nerve fibroblasts were transplanted into the lesion site. In the absence of transplants, few tyrosine hydroxylase-immunoreactive axons extended across the lesion 2 weeks after the transection. Reactive astrocytes increased around the lesion, and a fibrotic scar containing type IV collagen deposits developed in the lesion center. The immunoreactivity of chondroitin sulfate side chains and core protein of NG2 proteoglycan increased in and around the lesion. One and 2 weeks after transection and simultaneous transplantation, dopaminergic axons regenerated across the transplanted tissues, which consisted of p75-immunoreactive OECs and fibronectin-immunoreactive fibroblasts. Reactive astrocytes and chondroitin sulfate immunoreactivity increased around the transplants, whereas the deposition of type IV collagen and fibrotic scar formation were completely prevented at the lesion site. Transplantation of meningeal fibroblasts similarly prevented the formation of the fibrotic scar, although its effect on regeneration was less potent than transplantation of OECs and olfactory nerve fibroblasts. The present results suggest that elimination of the inhibitory fibrotic scar is important for neural regeneration.

Attenuation of MPTP-induced neurotoxicity and locomotor dysfunction in Nucling-deficient mice via suppression of the apoptosome pathway.

1-Methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP)-induced neurotoxicity is one of the experimental models most commonly used to study the pathogenesis of Parkinson's disease (PD). Although the biochemical mechanisms underlying the cell death induced by MPTP remain to be clarified, it has been found that the mitochondrial apoptotic signaling pathway plays an important role in the neurotoxicity of MPTP. Nucling is a novel type of apoptosis-associated molecule, essential for cytochrome c, apoptosis protease activating factor 1 (Apaf-1), pro-caspase-9 apoptosome induction and caspase-9 activation following pro-apoptotic stress. Here we found that Nucling-deficient mice treated with MPTP did not exhibit locomotor dysfunction in an open-field test. The substantia nigra dopaminergic neurons of Nucling-deficient mice were resistant to the damaging effects of the neurotoxin MPTP. Up-regulated expression of apoptosome was attenuated in Nucling-deficient mice treated with MPTP. These results indicate an important role for Nucling in MPTP-induced neuronal degeneration and suggest that the suppression of Nucling would be of therapeutic benefit for the treatment of neurodegeneration in PD.

Nucling recruits Apaf-1/pro-caspase-9 complex for the induction of stress-induced apoptosis.

Nucling is a novel protein isolated from murine embryonal carcinoma cells with an up-regulated expression during cardiac muscle differentiation. We show here that Nucling was up-regulated by proapoptotic stimuli and important for the induction of apoptosis after cytotoxic stress. We further demonstrated that overexpressed Nucling was able to induce apoptosis. In Nucling-deficient cells, the expression levels of Apaf-1 and cytochrome c, which are the major components of an apoptosis-promoting complex named apoptosome, were both down-regulated under cellular stress. A deficiency of Nucling also conferred resistance to apoptotic stress on the cell. After UV irradiation, Nucling was shown to reside in an Apaf-1/pro-caspase-9 complex, suggesting that Nucling might be a key molecule for the formation and maintenance of this complex. Nucling induced translocation of Apaf-1 to the nucleus, thereby distributing the Nucling/Apaf-1/pro-caspase-9 complex to the nuclear fraction. These findings suggest that Nucling recruits and transports the apoptosome complex during stress-induced apoptosis.