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. 2018 Jun;13(2):265-276.
doi: 10.1007/s11481-018-9783-8. Epub 2018 Mar 17.

A Role for the Non-Receptor Tyrosine Kinase Abl2/Arg in Experimental Neuroinflammation

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Free PMC article

A Role for the Non-Receptor Tyrosine Kinase Abl2/Arg in Experimental Neuroinflammation

Freja Aksel Jacobsen et al. J Neuroimmune Pharmacol. .
Free PMC article

Abstract

Multiple sclerosis is a neuroinflammatory degenerative disease, caused by activated immune cells infiltrating the CNS. The disease etiology involves both genetic and environmental factors. The mouse genetic locus, Eae27, linked to disease development in the experimental autoimmune encephalomyelitis (EAE) model for multiple sclerosis, was studied in order to identify contributing disease susceptibility factors and potential drug targets for multiple sclerosis. Studies of an Eae27 congenic mouse strain, revealed that genetic variation within Eae27 influences EAE development. The Abl2 gene, encoding the non-receptor tyrosine kinase Arg, is located in the 4,1 megabase pair long Eae27 region. The Arg protein plays an important role in cellular regulation and is, in addition, involved in signaling through the B- and T-cell receptors, important for the autoimmune response. The presence of a single nucleotide polymorphism causing an amino acid change in a near actin-interacting domain of Arg, in addition to altered lymphocyte activation in the congenic mice upon immunization with myelin antigen, makes Abl2/Arg a candidate gene for EAE. Here we demonstrate that the non-synonymous SNP does not change Arg's binding affinity for F-actin but suggest a role for Abl kinases in CNS inflammation pathogenesis by showing that pharmacological inhibition of Abl kinases ameliorates EAE, but not experimental arthritis.

Keywords: Abl kinase; Arg; Eae27; Experimental autoimmune encephalomyelitis; Imatinib.

Conflict of interest statement

The authors declare that they have no conflict of interest.

Figures

Fig. 1
Fig. 1
Eae27 SNP analysis. SNP analysis revealed known SNPs within the Eae27 locus comparing the C57Bl/6 and RIIIS/J mouse strains. Identified SNPs were subsequently compared to next generation sequencing data for the B10.RIII strain and led to the identification of the restricted polymorphic Eae27, flanked by highly conserved regions
Fig. 2
Fig. 2
EAE development in BR.RIII-Eae27 congenic mice. Disease progression in mice homozygous for Eae27, inherited from the EAE susceptible B10.RIII background strain (B10.RIII; grey line) and the EAE resistant RIIIS/J donor strain (BR.RIII-Eae27; black line). Data are presented as mean clinical score ± SEM
Fig. 3
Fig. 3
Proliferation of spleen cells from mice immunized with MBP89–101 upon activation in vitro. 3H-thymidine incorporation in in vitro stimulated splenic lymphocytes from MBP89–101-immunized BR.RIIIS/J-Eae27 and B10.RIII mice. The graphs show T-cell proliferation in response to (a) anti-CD3/anti-CD28 (3 μg/ml) and (b) ConA, and B-cell proliferation in response to (c) LPS, and (d) anti-IgM stimulation. Data are reported as mean stimulation index ratio (SI) ± SEM. Results are based on data from six mice in each group and triplicate measurements for each point
Fig. 4
Fig. 4
The effect of imatinib on EAE and CIA development. Mice received imatinib or placebo 1 day prior to immunization and throughout the study. a EAE was induced by immunization with MBP89–101 (data represent mean clinical score ± SEM). b CIA was induced by immunization with Type II collagen (data represent fraction of mice with a clinical score below 10)
Fig. 5
Fig. 5
Inhibition of lymphocyte proliferation with Imatinib. Imatinib inhibits proliferation of splenic lymphocytes from naïve BR.RIII-Eae27 and B10.RIII mice upon in vitro stimulation with (a) LPS (10 μg/ml), (b) anti-IgM (40 μg/ml), or (c) anti-CD3/anti-CD28 (1 μg/ml and 3 μg/ml, respectively). Proliferation was measured as 3H-thymidine incorporation. Data represent mean CPM ± SEM
Fig. 6
Fig. 6
Arg expression in brain and spleen from BR.RIII-Eae27 and B10.RIII mice. Western blots showing (a) Arg expression in brain and spleen tissue from BR.RIIIS/J-Eae27 and B10.RIII mice. Molecular mass of Arg and Actin is 131 and 42 kDa, respectively. The two bands observed for Arg are believed to represent splice isoforms (Koleske et al., 1998). b The Arg:Actin integrated density is shown graphically. The Arg density is the sum of the two isoform bands
Fig. 7
Fig. 7
Domain structure of the Arg protein. The Arg kinase has two F-actin binding domains, one microtubule-binding domain, and three proline rich (PxxP) motifs in its C-terminal part. The N-terminal part of the protein expresses an SH3, SH2 and kinase domain, forming a domain structure common among tyrosine kinases. The missense SNP, rs30466582, is located within Arg’s microtubule-binding domain close to the C-terminal F-actin-binding domain
Fig. 8
Fig. 8
Arg and Arg V1030 bind with similar affinity to actin filaments. a Representative co-sedimentation assay gels showing increasing amounts of Arg/Arg V1030 M incubated with 1 μM actin. Supernatant (S) and pellet (P) samples for different concentrations are run side by side. b Binding of Arg/Arg V1030 to actin in the pellets was quantified from three trials and mean ± SEM is plotted vs. input concentration to calculate the dissociation constant (KD). No significant difference was noted in affinity of Arg/Arg V1030 M to actin
Fig. 9
Fig. 9
Alignment of nucleotide sequence 3067–3113 across species. Nucleotide sequence 3067–3113, flanking the identified missense SNP, rs30466582 (yellow*), in the mouse genome was aligned to human, rat, pig, and armadillo. Two missense SNPs in human are located close to the mouse SNP rs30466582 (ensemble.org)

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