In contrast, at day 7–8 we noticed

the animals were weaker and by day 10, the P0-RafTR mice displayed severe impairment of coordination and positioning of their limbs, consistent with a demyelinating phenotype (Figure 2A). In some cases, the effects were so severe that the mice were unable to support their own body weight (Movie S1). Hind-paw prints from injected P0-RafTR mice were more elongated and had reduced “toespread” compared to injected WT littermates (Figure S2)—signs indicative of peripheral nerve damage (Crawley, 2008). Furthermore, a large impairment in motor coordination was observed Epacadostat datasheet as measured by the accelerating rotarod test (Movie S2); and quantified in Figure 2B. These results show that Raf-kinase activation in myelinated Schwann cells is sufficient to drive a rapid loss of peripheral nerve function in vivo, consistent with nerve demyelination. Our previous in vitro studies have shown that Raf/MEK/ERK driven Schwann cell dedifferentiation is associated with the downregulation

of myelin-specific gene expression Nintedanib chemical structure and the upregulation of genes expressed by dedifferentiated Schwann cells (Harrisingh et al., 2004). Quantitative RT-PCR analysis of nerves isolated from tamoxifen-injected P0-RafTR animals showed that by day 3 following the first injection (day 3) the expression of myelin genes were strongly downregulated. (Figure 2C). Conversely, markers of dedifferentiated Schwann cells in the adult, Krox-24 and p75 (also expressed by nonmyelinating Schwann cells), together with the proliferation marker cyclin D1, were strongly upregulated ( Figure 2C). However, analysis of sciatic nerves from these mice showed that at day 3, the structure of the nerves was indistinguishable from that of WT injected animals, with no differences in the degree of myelination ( Figures 2D and 2E), demonstrating that changes

in gene expression occurred prior to myelin breakdown. Moreover, axonal staining showed that the axons remained intact ( Figure 2D). The downregulation of myelin gene expression however observed on day 3 was sustained in the nerves of transgenic mice on day 10, when the motor dysfunction was severe (Figure 3A). However, when the structure of peripheral nerves was analyzed at this time, a dramatic change in histology was observed: most notably, there was widespread breakdown of myelin and increased cellularity in the intraneural spaces (Figures 3B and 3C). Quantification of the extent of demyelination confirmed a large decrease in the number of Schwann cell/axon units containing compact myelin (Figure 3D) and many of the remaining units displayed myelin infoldings and outfoldings together with vacuoles of degraded myelin protein, which are characteristic of demyelination in injured nerves. Immunostaining of the nerve showed a large increase in the number of p75-positive cells, confirming that these cells had dedifferentiated back to a progenitor-like state (Figure 3B).