Gas6 increases myelination by oligodendrocytes and its deficiency delays recovery following cuprizone-induced demyelination

Abstract

Multiple sclerosis (MS) is a complex demyelinating disease of the central nervous system. Current research has shown that at least in some cases, the primary insult in MS could be directed at the oligodendrocyte, and that the earliest immune responses are primarily via innate immune cells. We have identified a family of receptor protein tyrosine kinases, known as the TAM receptors (Tyro3, Axl and Mertk), as potentially important in regulating both the oligodendrocyte and immune responses. We have previously shown that Gas6, a ligand for the TAM receptors, can affect the severity of demyelination in mice, with a loss of signalling via Gas6 leading to decreased oligodendrocyte survival and increased microglial activation during cuprizone-induced demyelination. We hypothesised TAM receptor signalling would also influence the extent of recovery in mice following demyelination. A significant effect of the absence of Gas6 was detected upon remyelination, with a lower level of myelination after 4 weeks of recovery in comparison with wild-type mice. The delay in remyelination was accompanied by a reduction in oligodendrocyte numbers. To understand the molecular mechanisms that drive the observed effects, we also examined the effect of exogenous Gas6 in in vitro myelination assays. We found that Gas6 significantly increased myelination in a dose-dependent manner, suggesting that TAM receptor signalling could be directly involved in myelination by oligodendrocytes. The reduced rate of remyelination in the absence of Gas6 could thus result from a lack of Gas6 at a critical time during myelin production after injury. These findings establish Gas6 as an important regulator of both CNS demyelination and remyelination.

Figure 1. LFB staining demonstrates a reduction in the extent of remyelination in the absence of Gas6.

A–F Wild-type and Gas6 KO mice were subjected to cuprizone challenge for 5 weeks followed by recovery in the absence of cuprizone for 0, 2 or 4 weeks. A. The corpus callosum was divided into three segments for image analysis: rostral (R), Middle (M) and Caudal (C) G–I Myelin density was assessed using image analysis as described in the Materials and Methods. G. A significant reduction in myelination was observed in the rostral segment of the corpus callosum (p = 0.039) and a strong trend towards reduction was observed in the middle segment (p = 0.052) (H). Scale bar = 1 mm.

Figure 2. EM analysis demonstrates a reduction in the number of myelinated axons during the course of remyelination in the absence of Gas6.

A–F Wild-type and Gas6 KO mice were subjected to cuprizone challenge for 5 weeks followed by recovery in the absence of cuprizone for 0, 2 or 4 weeks. The number of myelinated axons per mm² was quantified and shown in G–I. A significant reduction in myelination was observed in the caudal (I) segments of the corpus callosum in Gas6^−/− mice compared with Gas6^+/+ mice (p = 0.032). Scale bar = 2 µm.

Figure 3. EM analysis demonstrates that, following 10 weeks of recovery, Gas6 KO mice remyelinate to the same extent as Gas6 WT mice.

A–L Wild-type and Gas6 KO mice were subjected to cuprizone challenge for 5 weeks followed by recovery in the absence of cuprizone for 0 or 10 weeks. The number of myelinated axons/mm² was quantified in the rostral, middle and caudal segments of the corpus callosum (M–O). No significant differences were observed between Gas6^−/− or Gas6^+/+ mice at any time point or in segment (p>0.05). Scale bar = 2 µm.

Figure 4. Expression of oligodendrocyte lineage markers are altered in the absence of Gas6 during remyelination.

Wild-type and Gas6 KO mice were subjected to cuprizone challenge for 5 weeks followed by recovery in the absence of cuprizone for 0, 2, 4 or 10 weeks. A. Representative images of the middle segment of the corpus callosum, showing cells positive for APC(CC1) or double positive for Olig2/PDGFRα. B–G. A significant reduction in the number of APC(CC1) positive cells was observed in the caudal segment of the corpus callosum in Gas6^−/− mice compared with Gas6^+/+ mice (p = 0.0070) (D). The density of Olig2+/PDGFRα+ OPCs was significantly increased in Gas6^−/− mice compared with Gas6^+/+ mice following both 4 and 10 weeks of remyelination (4 weeks, p = 0.024; 10 weeks, p = 0.003) (E). Scale bar = 20 µm.

Figure 5. The number of IBA1 positive microglia is minimally altered by the absence of Gas6 during remyelination.

Wild-type and Gas6 KO mice were subjected to cuprizone challenge for 5 weeks followed by recovery in the absence of cuprizone for 0, 2, 4 or 10 weeks. A. Representative images of the middle segment of the corpus callosum, showing cells positive for IBA1. B–D The number of IBA1 positive cells/mm2 was quantified. A significant increase in the number of IBA1-positive microglia was observed in Gas6^−/− mice compared with Gas6^+/+ mice following 10 weeks of remyelination (p = 0.031). Scale bar = 20 µm.

Figure 6. Exogenous Gas6 increases the number of myelinated DRG neurons when co-cultured with oligodendrocytes.

A–D Oligodendrocytes and DRG neurons were co-cultured as described in the Materials and Methods. Exogenous Gas6 was added to the cultures at either 0 ng/ml (A), 1 ng/ml (B), 10 ng/ml (C) or 100 ng/ml (D) and the number of myelinated segments quantified, with the results shown in (E). Exogenous Gas6 significantly increased the number of myelinated segments in a dose-dependant manner (p<0.0001). F Western analysis showing exogenous Gas6 increased the expression of myelin compenents. Scale bar = 100 µm.