Loss of the receptor tyrosine kinase Axl leads to enhanced inflammation in the CNS and delayed removal of myelin debris during Experimental Autoimmune Encephalomyelitis

<p>Abstract</p> <p>Background</p> <p>Axl, together with Tyro3 and Mer, constitute the TAM family of receptor tyrosine kinases. In the nervous system, Axl and its ligand Growth-arrest-specific protein 6 (Gas6) are expressed on multiple cell types. Axl functions in dampening the immune response, regulating cytokine secretion, clearing apoptotic cells and debris, and maintaining cell survival. Axl is upregulated in various disease states, such as in the cuprizone toxicity-induced model of demyelination and in multiple sclerosis (MS) lesions, suggesting that it plays a role in disease pathogenesis. To test for this, we studied the susceptibility of Axl-/- mice to experimental autoimmune encephalomyelitis (EAE), an animal model for multiple sclerosis.</p> <p>Methods</p> <p>WT and Axl-/- mice were immunized with myelin oligodendrocyte glycoprotein (MOG)_35-55peptide emulsified in complete Freund's adjuvant and injected with pertussis toxin on day 0 and day 2. Mice were monitored daily for clinical signs of disease and analyzed for pathology during the acute phase of disease. Immunological responses were monitored by flow cytometry, cytokine analysis and proliferation assays.</p> <p>Results</p> <p>Axl-/- mice had a significantly more severe acute phase of EAE than WT mice. Axl-/- mice had more spinal cord lesions with larger inflammatory cuffs, more demyelination, and more axonal damage than WT mice during EAE. Strikingly, lesions in Axl-/- mice had more intense Oil-Red-O staining indicative of inefficient clearance of myelin debris. Fewer activated microglia/macrophages (Iba1+) were found in and/or surrounding lesions in Axl-/- mice relative to WT mice. In contrast, no significant differences were noted in immune cell responses between naïve and sensitized animals.</p> <p>Conclusions</p> <p>These data show that Axl alleviates EAE disease progression and suggests that in EAE Axl functions in the recruitment of microglia/macrophages and in the clearance of debris following demyelination. In addition, these data provide further support that administration of the Axl ligand Gas6 could be therapeutic for immune-mediated demyelinating diseases.</p

GAS6 Enhances Repair Following Cuprizone-Induced Demyelination

Growth arrest-specific protein 6 (gas6) activities are mediated through the Tyro3, Axl, and Mer family of receptor tyrosine kinases. Gas6 is expressed and secreted by a wide variety of cell types, including cells of the central nervous system (CNS). In this study, we tested the hypothesis that administration of recombinant human Gas6 (rhGas6) protein into the CNS improves recovery following cuprizone withdrawal. After a 4-week cuprizone diet, cuprizone was removed and PBS or rhGas6 (400 ng/ml, 4 µg/ml and 40 µg/ml) was delivered by osmotic mini-pump into the corpus callosum of C57Bl6 mice for 14 days. Nine of 11 (82%) PBS-treated mice had abundant lipid-associated debris in the corpus callosum by Oil-Red-O staining while only 4 of 19 (21%) mice treated with rhGas6 had low Oil-Red-O positive droplets. In rhGas6-treated mice, SMI32-positive axonal spheroids and APP-positive deposits were reduced in number relative to PBS-treated mice. Compared to PBS, rhGas6 enhanced remyelination as revealed by MBP immunostaining and electron microscopy. The rhGas6-treated mice had more oligodendrocytes expressing Olig1 in the cytoplasm, indicative of oligodendrocyte progenitor cell maturation. Relative to PBS-treated mice, rhGas6-treated mice had fewer activated microglia in the corpus callosum by Iba1 immunostaining. The data show that rhGas6 treatment resulted in more efficient repair following cuprizone-induced injury

Gas6 Enhances Axonal Ensheathment by MBP Membranous Processes in Human DRG/OL Promyelinating Co-Cultures

The molecular requirements for human myelination are incompletely defined, and further study is needed to fully understand the cellular mechanisms involved during development and in demyelinating diseases. We have established a human co-culture model to study myelination. Our earlier observations showed that addition of human γ-carboxylated growth-arrest-specific protein 6 (Gas6) to human oligodendrocyte progenitor cell (OPC) cultures enhanced their survival and maturation. Therefore, we explored the effect of Gas6 in co-cultures of enriched OPCs plated on axons of human fetal dorsal root ganglia explant. Gas6 significantly enhanced the number of myelin basic protein-positive (MBP + ) oligodendrocytes with membranous processes parallel with and ensheathing axons relative to co-cultures maintained in defined medium only for 14 days. Gas6 did not increase the overall number of MBP + oligodendrocytes/culture; however, it significantly increased the length of MBP + oligodendrocyte processes in contact with and wrapping axons. Multiple oligodendrocytes were in contact with a single axon, and several processes from one oligodendrocyte made contact with one or multiple axons. Electron microscopy supported confocal Z-series microscopy demonstrating axonal ensheathment by MBP + oligodendrocyte membranous processes in Gas6-treated co-cultures. Contacts between the axonal and oligodendrocyte membranes were evident and multiple wraps of oligodendrocyte membrane around the axon were visible supporting a model system in which to study events in human myelination and aspects of non-compact myelin formation

EM demonstrates dystrophic axons and axonal spheroids in PBS- and rhGas6-treated mice.

<p>A. Axonal spheroids (>10 µm) are shown in PBS-treated mice (black arrow). Myelin debris (arrowhead) and lipid droplets (white arrow), are also noted. B – 400 ng/ml, C – 4 µg/ml of rhGas6 demonstrate dystrophic axons ranging in size from 3.0–9.5 µm in diameter (black arrow). Note microglial cell process (white arrow, B), and astrocytes in rhGas6-treated mice (gray arrow B and C), ×5000.</p

EM shows more myelinated axons in rhGas6-treated mice.

<p>Relative to PBS (A), the number of myelinated axons was significantly increased in mice treated with 400 ng/ml (B) and 4 µg/ml (C) rhGas6. Black arrows denote myelinated axons; white arrows show demyelinated, naked axons, ×5000.</p

Treatment with rhGas6 improves axonal integrity after cuprizone challenge.

<p>SMI32 and APP immunostaining of corpus callosum sections from mice treated for 14 days with PBS (A and E), 400 ng/ml (B and F), 4 µg/ml (C and G), 40 µg/ml of rhGas6 (D and H). Magnification ×50 (A–D), and ×400 (E–K). Increased number of SMI32- positive axonal swellings is observed in the PBS-treated mice (A–E) versus three doses of rhGas6-treated mice (B–H). Arrows show several axonal swellings indicating breakdown in axonal structure. I–K demonstrate APP immunostaining in mice treated with PBS (I), 400 ng/ml (J) and 4 µg/ml of rhGas6 (K). Arrows show APP positive deposits in the corpus callosum, ×400.</p

Similar numbers of Iba1-positive microglia are present in PBS- and rhGas6-treated mice.

<p>A. Following cuprizone ingestion for 4 weeks an extensive Iba1-positive microglial activation within the corpus callosum is observed. There was significant reduction of Iba1-postive microglia in both PBS- (B), and rhGas6-treated mice (C and D) at 14 days post treatment relative to 4 week cuprizone treated mice (p<0.02). Arrows show Iba1-positive microglia.</p

Number of Olig1-positive mature oligodendrocytes is increased in rhGas6-treated mice.

<p>Olig1-positive nuclear and cytoplasmic localization in cells (A–C). Increased numbers of cells with Olig1-positive cytoplasmic localization (arrows) in mice treated with 400 ng/ml (B), and 4 µg/ml rhGas6 (C) versus PBS-treated mice (A), are shown, ×400.</p