Thioredoxin rod-derived cone viability factor protects against photooxidative retinal damage.

Rod-derived cone viability factor (RdCVF) is a trophic factor of the thioredoxins family that promotes the survival of cone photoreceptors. It is encoded by the nucleoredoxin-like gene 1 Nxnl1 which also encodes by alternative splicing a long form of RdCVF (RdCVFL), a thioredoxin enzyme that interacts with TAU. The known role of thioredoxins in the defense mechanism against oxidative damage led us to examine the retinal phenotype of the Nxnl1(-/-) mice exposed to photooxidative stress. Here we found that, in contrast to wild-type mice, the rod photoreceptors of Nxnl1(-/-) mice are more sensitive to light after exposure to 1700 or 2500 lx. The delivery of RdCVF by AAV to mice deficient of Nxnl1(-/-) protects rod photoreceptors from light damage. Interestingly, the RdCVF2L protein, encoded by the paralog gene Nxnl2, is able to reduce TAU phosphorylation, as does RdCVFL, but does not protect the rod from light damage. Our result shows that the Nxnl1 gene, through the thioredoxin RdCVFL, is part of an endogenous defense mechanism against photooxidative stress that is likely of great importance for human vision.

The disruption of the rod-derived cone viability gene leads to photoreceptor dysfunction and susceptibility to oxidative stress.

Rod-derived cone viability factor (RdCVF) is a thioredoxin-like protein, which has therapeutic potential for rod-cone dystrophies such as retinitis pigmentosa (RP). Cone loss in rodent models of RP is effectively reduced by RdCVF treatment. In this study, we investigate the physiological role of RdCVF in the retina by analyzing the phenotype of the mouse lacking the RdCVF gene, Nxnl1. Although the mice do not show an obvious developmental defect, an age-related reduction of both cone and rod function and a delay in the dark-adaptation of the retina are recorded by electroretinogram (ERG). This functional change is accompanied by a 17% reduction in cone density and a 20% reduction in thickness of the outer nuclear layer. The transcriptome of the retina reveals early changes in the expression of genes involved in programmed cell death, stress-response and redox-signaling, which is followed by a generalized injury response with increased microglial activation, GFAP, FGF2 and lipid peroxidation levels. Furthermore, cones of the mice lacking Nxnl1 are more sensitive to oxidative stress with a reduction of 65% in the cone flicker ERG amplitude measured under hyperoxic conditions. We show here that the RdCVF gene, in addition to therapeutic properties, has an essential role in photoreceptor maintenance and resistance to retinal oxidative stress.

Thrombin receptor-mediated increase of two matrix metalloproteinases, MMP-1 and MMP-3, in human endothelial cells.

Matrix metalloproteinases (MMPs) are responsible for the degradation of extracellular matrix components and are secreted by a variety of cells including human endothelial cells. Because alpha-thrombin is known to interact with matrix components and has been shown to activate latent MMP-2 in human umbilical vein endothelial cells, we investigated whether human alpha-thrombin could also regulate other MMPs secreted by the human saphenous vein or mammary artery endothelial cells (EC). After treatment of EC with increasing concentrations of thrombin for different periods of time, a significantly higher gelatinolytic activity of both MMP-1 and MMP-3 was observed in addition to MMP-2 activation. The effect of thrombin was time and dose-dependent, reaching a maximum at 24 hours. After treatment with 5 NIH U/ml thrombin for 24 hours, Western blotting revealed 9.5- and 4.4-fold increases over control values for MMP-3 and MMP-1, respectively. The synthetic thrombin receptor agonist peptide SFLLRNPNDKYEPF fully reproduced the action of thrombin, whereas chemical inactivation of the catalytic site of thrombin abolished its effect on MMP-1 and MMP-3. Thrombin and SFLLRNPNDKYEPF both induced MMP-3 mRNA synthesis but had no significant influence on constitutive MMP-1 mRNA levels. These results demonstrate that thrombin not only activates latent MMP-2 but also modulates MMP-1 and MMP-3 production in EC, this latter effect being mediated by the G-protein-coupled thrombin receptor. Hence, our present data provide evidence to support the suspected role of thrombin in tissue remodeling and angiogenesis.

PF4 inhibits thrombin-stimulated MMP-1 and MMP-3 metalloproteinase expression in human vascular endothelial cells.

We investigated whether PF4 could regulate the constitutive and thrombin-stimulated expression of metalloproteinases (MMPs) in endothelial cells (EC). PF4 inhibited the increase in the expression of MMP-1 and MMP-3 promoted by thrombin or the thrombin receptor agonist peptide SFLLRNPNDKYEPF (SFLL..) by 50% but did not modify the constitutive expression of these MMPs. This inhibitory effect was not mediated through a direct interaction of PF4 with thrombin or with the MMPs themselves. The interaction of PF4 with heparan sulfates at the surface of the EC appeared to be implicated in the inhibition mechanism of MMP-1 but not in that of MMP-3. MMP-1 transcription levels remained unchanged after PF4 treatment, whereas the increase in MMP-3 transcription induced by thrombin or SFLL.. was inhibited by approximately 50%. Expression of the tissue inhibitors of metalloproteinases TIMP-1 and TIMP-2 was not affected by PF4. The present data provide new evidence that the antiangiogenic properties of PF4 involve the inhibition of matrix breakdown and suggest that this property of PF4 could be especially relevant in the context of thrombin-regulated tissue remodelling.

Expression of the highly polysialylated neural cell adhesion molecule during postnatal myelination and following chemically induced demyelination of the adult mouse spinal cord.

We have investigated the expression of the highly polysialylated neural cell adhesion molecule in the mouse spinal cord during postnatal myelination and in the adult after chemically induced demyelination. By double immunohistochemistry, using a monoclonal antibody (anti-Men B) which specifically recognizes polysialic acid (PSA) units on neural cell adhesion molecule (N-CAM), and an anti-myelin basic protein, a caudorostral gradient of expression of PSA-NCAM was observed at postnatal day 1 (P1), which was inversely related to the gradient of myelination. At P7, PSA-NCAM labelling decreased relative to P1. In white matter, this decrease was correlated with the progression of myelination. PSA-NCAM immunoreactivity persisted in as yet unmyelinated structures, i.e. the corticospinal tract, the dorsomedial part of the ventral funiculus and the lateral funiculi, and decreased with the onset of myelination of these structures at P15. In the adult, PSA-NCAM expression remained in discrete structures, i.e. laminae I and II of the dorsal horn and lamina X around the central canal. The ependymal cells and the astrocyte endfeet under the meninges were also labelled. In addition, PSA-NCAM expression was reinduced on various cells and structures after lysolecithin-induced demyelination of the adult mouse spinal cord. At early times after demyelination, PSA-NCAM was expressed on glial cells around the lesion but also at a distance from this zone. Seven days after injection, cellular PSA-NCAM expression was found around but also within the lesion. This expression was totally abolished 15 days after injection. Double immunohistochemistry for PSA and cell-specific markers showed that the cells which expressed PSA-NCAM after demyelination were oligodendrocyte precursors, reactive astrocytes and Schwann cells. PSA-NCAM re-expression on all cell types was transient and ceased when myelin repair was accomplished. The spatial and temporal regulation of PSA-NCAM expression during development and after demyelination suggests a role for PSA-NCAM in glial plasticity during the myelination and remyelination processes.

Cellular expression of an HMGCR promoter-CAT fusion gene in transgenic mouse brain: evidence for a developmental regulation in oligodendrocytes.

The HMGCR gene encodes the 3-hydroxy-3-methylglutaryl coenzyme A reductase, which is the key enzyme for cholesterol synthesis. Mice transgenic for the prokaryotic chloramphenicol acetyl transferase (CAT) reporter gene fused with a 5' Bam H1 fragment including the promoter sequence for murine HMGCR gene have been obtained. Homozygote transgenic mice were derived from a particular line selected for similar regulation of endogenous HMGCR and the transgene expression by nutritional conditions in different tissue. In addition, high expression of the transgene was evidenced in the brain. Cellular expression of the CAT gene in the central nervous system (CNS) was investigated by immunohistochemistry (IHC). This study was performed on frozen sections of the developing and adult brain, using a rabbit anti-CAT antiserum especially raised for that purpose. CAT expression was observed in some rare individuals in different neural cell types including Purkinje cells and astrocytes. But the most outstanding observation was the high level of CAT expression correlated with differentiated pattern of oligodendrocyte (Ol) distribution observed in white-matter tracts. Double and triple labeling for CAT and stage-specific antigens were performed on transgenic Ol-enriched preparations and cultures. This study showed a normal sequence of differentiation in the transgenic oligodendroglial cell lineage and demonstrated a strict correlation between late differentiation and activation of the CAT gene in these cells: CAT expression started in transgenic Ols between galactocerebroside (GC)-positive and myelin basic protein (MBP)-positive stages and was detected in MBP-positive cells during the myelination period. After myelination, the number of CAT-positive Ols decreased in the adult brain. These observations demonstrate a developmental regulation of the CAT transgene in Ols during myelination in CNS and reinforce the hypothesis of endogenous synthesis as major source of cholesterol during myelination.

Transplanted transgenically marked oligodendrocytes survive, migrate and myelinate in the normal mouse brain as they do in the shiverer mouse brain.

The dye Hoechst 33342 was combined with an immunodetectable transgene product (chloramphenicol acetyltransferase, CAT) expressed in differentiated oligodendrocytes to trace their fate after transplantation in the normal and the shiverer mouse brain. In the shiverer brain, the technique allowed us to visualize grafted cells inside myelin basic protein-positive myelin patches. Most of these cells were CAT-positive/Hoechst 33342-negative, reinforcing our hypothesis that cell division probably follows migration of grafted oligodendrocytes. Correlation of their morphology and distribution with their location in the host CNS suggested a local effect on the cell division and morphogenesis of the grafted material. When compared with transplantation of fragments of normal newborn donor tissue into the newborn shiverer brain, no difference could be seen between the behaviour of normal and transgenic oligodendrocytes. In the normal brain, transgenic oligodendrocytes survived at least 150 days and successfully myelinated the host axons. The timing of differentiation of grafted cells was similar in both types of recipient brains. Migration occurred rostrally and caudally. Although migrating cells could be observed along the meninges and the blood vessels, migration occurred preferentially along white matter tracts. The extent of migration was influenced by the site of implantation, and grafted cells could be found up to 6 mm from the grafting point. No differences in the timing of differentiation or the pattern or extent of migration could thus be demonstrated when transgenic oligodendrocytes were transplanted in the normal or the shiverer brain. This validates our previous studies using the newborn shiverer mouse as recipient.

Pathways of migration of transplanted Schwann cells in the demyelinated mouse spinal cord.

We have studied the behavior of Schwann cells transplanted at a distance from an induced myelin lesion of the adult mouse spinal cord. These transplanted cells were mouse Schwann cells arising from an immortalized cell line (MSC80) which expresses several Schwann cell phenotypes including the ability to produce myelin. The behavior of MSC80 cells was compared to that of purified rat Schwann cells transplanted in the same conditions. Schwann cells were labeled in vitro with the nuclear fluorochrome Hoechst 33342 and were transplanted at distances of 2-8 mm from a lysolecithin-induced myelin lesion in the spinal cord of shiverer and normal mice. Our results show that transplanted MSC80 cells migrated toward the lesion, in both shiverer and normal mouse spinal cord, preferentially along the ependyma, meninges, and blood vessels. They also migrated along white matter tracts but traveled a longer distance in shiverer (8 mm) than in normal (2-3 mm) white matter. Using these different pathways, MSC80 cells arrived within the lesion of shiverer and normal mouse spinal cord at the average speed of 166 microns/hr (8 mm/48 hr). Migration was most efficient along the ependyma and the meninges where it attained up to 250 microns/hr. Migration was much slower in white matter tracts (95 microns/hr +/- 54 in the shiverer and only 38 microns/hr +/- 3 in the normal mouse). We also provide evidence for the specific attraction of MSC80 cells by the lysolecithin-induced lesion since 1) their number increased progressively with time in the lesion, and 2) MSC80 cells left their preferential pathways of migration specifically at the level of the lesion. Finally, combining the Hoechst Schwann cell labeling method with the immunohistochemical detection of the peripheral myelin protein, P0, we show that some of the MSC80 cells which have reached the lesion participate in myelin repair in both shiverer and normal lesioned mouse spinal cord. A series of control experiments performed with rat Schwann cells indicate that the migrating behavior of transplanted MSC80 cells was identical to that of purified but non-immortalized rat Schwann cells.

Hoechst 33342 a suitable fluorescent marker for Schwann cells after transplantation in the mouse spinal cord.

Purified rat Schwann cells labeled with Hoechst 33342 were transplanted into a lysophosphatidyl choline induced myelin lesion of the adult shiverer mouse spinal cord. Remyelination by grafted Schwann cells within the lesion was evidenced by codetection of Hoechst labeled Schwann cells and P0 (peripheral myelin protein) immunolabeled myelin on serial cryostat sections and confirmed on adjacent sec ions by electron microscopy. These data show that the Hoechst-shiverer model is an excellent model which can be used in intraspinal transplantation of myelin forming cells to demonstrate the origin of the newly formed myelin. Using this model, we bring the unquestionable evidence that cultured Schwann cells are capable after transplantation to participate with host oligodendrocytes in repair of a myelin lesion of the central nervous system.

Tracing transplanted oligodendrocytes during migration and maturation in the shiverer mouse brain.

Fragments of neural tissue from normal newborn mouse were stained with Hoechst 33342 dye before transplantation into the newborn shiverer mouse brain. Combination of this technique with immunohistochemistry demonstrated that, after transplantation, these cells are able to survive as long as unstained cells and to myelinate in the shiverer mouse host brain. Stained cells express the normal sequence of differentiation in terms of chronology of differentiation marker expression [04, galactocerebroside (GalC), myelin basic protein (MBP)], as normal cell do in situ. It has thus been possible by this technique to show the migration pathways of transplanted cells and to correlate them with the expression of specific markers: long distance migration along white matter axonal pathways occurs when cells are o4-positive, GalC-negative. By contrast, only GalC-positive cells are able to migrate across the grey matter in the absence of radial glia. Finally, it has been possible to propose a migration and differentiation sequence of these cells, suggesting that MBP-positive oligodendrocytes divide after migration in the target zone.