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

Nitrated α–Synuclein Immunity Accelerates Degeneration of Nigral Dopaminergic Neurons

The neuropathology of Parkinson's disease (PD) includes loss of dopaminergic neurons in the substantia nigra, nitrated alpha-synuclein (N-alpha-Syn) enriched intraneuronal inclusions or Lewy bodies and neuroinflammation. While the contribution of innate microglial inflammatory activities to disease are known, evidence for how adaptive immune mechanisms may affect the course of PD remains obscure. We reasoned that PD-associated oxidative protein modifications create novel antigenic epitopes capable of peripheral adaptive T cell responses that could affect nigrostriatal degeneration.Nitrotyrosine (NT)-modified alpha-Syn was detected readily in cervical lymph nodes (CLN) from 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP) intoxicated mice. Antigen-presenting cells within the CLN showed increased surface expression of major histocompatibility complex class II, initiating the molecular machinery necessary for efficient antigen presentation. MPTP-treated mice produced antibodies to native and nitrated alpha-Syn. Mice immunized with the NT-modified C-terminal tail fragment of alpha-Syn, but not native protein, generated robust T cell proliferative and pro-inflammatory secretory responses specific only for the modified antigen. T cells generated against the nitrated epitope do not respond to the unmodified protein. Mice deficient in T and B lymphocytes were resistant to MPTP-induced neurodegeneration. Transfer of T cells from mice immunized with N-alpha-Syn led to a robust neuroinflammatory response with accelerated dopaminergic cell loss.These data show that NT modifications within alpha-Syn, can bypass or break immunological tolerance and activate peripheral leukocytes in draining lymphoid tissue. A novel mechanism for disease is made in that NT modifications in alpha-Syn induce adaptive immune responses that exacerbate PD pathobiology. These results have implications for both the pathogenesis and treatment of this disabling neurodegenerative disease

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

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

Treatment with rhGas6 reduces Oil-Red-O positive deposits after cuprizone intoxication.

<p>A–F, corpus callosum of mouse brain after PBS treatment. Extensive deposition of Oil-Red- O positive droplets can be seen (arrows). Brain sections from mice treated with 400 ng/ml (G and J), 4 µg/ml (H and K), and 40 µg/ml (I and L) of rhGas6 demonstrate a beneficial effect for all tested rhGas6 doses. Arrows indicate the clearance of debris from the corpus callosum. Low and high magnification, ×50 and ×400.</p

Increased number of myelinated axons is observed in mice treated with rhGas6.

a<p>- data was collected from four photomicrographs, ×5000; 3 mice/group.</p>b<p>- 100 randomly selected axons were measured per mouse.</p><p>*- g-ratio of cuprizone untreated mice was 0.802±0.01 (mean ±s SD).</p><p>**- P<0.05.</p

MBP immunostaining shows that rhGas6 treatment enhances remyelination following cuprizone toxicity.

<p>Relative to untreated mice (A) there is demyelination of the corpus callosum of mice fed cuprizone for 4 weeks (B and C). A comparison of remyelination assessed by MBP staining of the PBS treated mice (D) versus rhGas6 treatment for 14 days following cuprizone withdrawal (E – 400 ng/ml; F - 4 µg/ml; G – 40 µg/ml of rhGas6) shows that the extent of remyelination is greater in rhGas6-treated mice, ×400.</p