Effects of isoxazolo-pyridinone 7e, a potent activator of the Nurr1 signaling pathway, on experimental autoimmune encephalomyelitis in mice

Abstract

Multiple sclerosis (MS) is an autoimmune chronic disease of the central nervous system (CNS) characterized by immune-mediated inflammation, demyelination and subsequent axonal damage. Gene expression profiling showed that Nurr1, an orphan nuclear receptor, is down-regulated in peripheral blood mononuclear cells of MS patients. Nurr1 exerts an anti-inflammatory role repressing the activity of the pro-inflammatory transcription factor NF-kB. Here, we report that the preventive treatment with isoxazolo-pyridinone 7e, an activator of Nurr1 signaling pathway, reduces the incidence and the severity of a MS murine model, i.e. experimental autoimmune encephalomyelitis (EAE). The compound is able to attenuate inflammation and neurodegeneration in spinal cords of EAE mice by an NF-kB pathway-dependent process.

Figure 1. Preventive administration of IP7e reduces the severity and the incidence of EAE.

The clinical course of EAE in IP7e (n = 9) and vehicle (n = 10) -treated EAE mice is compared. The analysis shows a decreased clinical score, i.e. median course (A), cumulative (C, bar represents median value) and maximum (D, bar represents median value) clinical score and a reduced weight loss (E) after the preventive administration of IP7e in EAE mice. IP7e leads also to an increase in percentage of disease-free mice (B). Arrows indicate the period of treatment (from 7^th to 23^th d.p.i.). Data are representative of 2 independent experiments. (Fig. A; Two Way ANOVA, treatment **P<0.01, Fig. E; Two Way ANOVA, treatment *P<0.05, Figure B, C, D; Student's t test, *: P<0.05; **: P<0.01). (d.p.i.; day post immunization, IP7e; isoxazolo-pyridinone 7e, IR; interquartile range, SEM; standard error of mean).

Figure 2. Preventive administration of IP7e reduces axonal damaged area in the spinal cord of mice with EAE.

Coronal sections of spinal cord of IP7e (B, D) and vehicle (A, C) –treated EAE mice. Axonal damage and demyelination are measured using respectively the Bielshowsky (A, B) and the Luxol fast Blue (C, D) staining in the spinal cord of IP7e (n = 5) and vehicle (n = 5) -treated EAE mice. Calibration bar, 250 µm. Quantification shows that the extent of neuronal damage (E) after IP7e treatment is significantly decreased, while the extent of demyelination (F) shows a tendency to decrease after IP7e treatment compared to vehicle- treated mice. (Student's t test, **: P<0.01). Data are expressed as mean ± SEM. (IP7e; isoxazolo-pyridinone 7e, SEM; standard error of mean).

Figure 3. Preventive administration of IP7e reduces the number of macrophages and T lymphocytes infiltrated in the spinal cord of mice with EAE.

Representative images of coronal sections of the spinal cord of IP7e (B, E, H) and vehicle (A, D, G) –treated EAE mice. The presence of perivascular inflammatory infiltrates (A, B), macrophages (D, E) and T lymphocytes (G, H) are evaluated using respectively Hematoxylin and Eosin staining and immunohistochemistry (IB4+ and CD3+ cells for macrophages and T lymphocytes, respectively) in the spinal cord of IP7e (n = 5) and vehicle (n = 5) -treated EAE mice. Calibration bars, 25 µm and 250 µm. Quantification shows that the number per section of perivascular inflammatory infiltrates shows a tendency to decrease after IP7etreatment (C). Furthermore, the number per section of macrophages (F) and T lymphocytes (I) after IP7e treatment results significantly decreased compared to vehicle-treated mice. (Student's t test, *: P<0.05; **: P<0.01). Data are expressed as mean ± SEM. (IP7e; isoxazolo-pyridinone 7e, SEM; standard error of mean).

Figure 4. Therapeutic administration of IP7e does not influence the severity of EAE.

The clinical course of EAE in IP7e (n = 8) and vehicle (n = 9) –treated EAE mice is compared. The analysis does not show any difference of disease severity i.e. median course (A), cumulative (B, bar represents median value) and maximum (C, bar represents median value) clinical score and weight loss (D) after the therapeutic administration of IP7e in EAE mice. Arrows indicate the period of treatment (from 21^th to 36^th d.p.i.). Data are representative of 2 independent experiments. (Two Way ANOVA, Student's t test). (d.p.i.; day post immunization, IP7e; isoxazolo-pyridinone 7e, IR; interquantile range, SEM; standard error of mean).

Figure 5. Therapeutic administration of IP7e does not influence EAE axonal loss, demyelination and, perivascular inflammatory infitration.

Representative images of coronal sections of the spinal cord of IP7e (B, E, H) and vehicle (A, D, G) –treated EAE mice. Axonal damage, demyelination, and perivascular inflammatory infiltrates are measured using respectively Bielshowsky (A, B), Luxol fast Blue (D, E) and Hematoxylin and Eosin (G, H) staining in the spinal cord of IP7e (n = 4) and vehicle (n = 5) -treated EAE mice. Calibration bars, 25 µm and 250 µm. Quantification shows no differences in axonal loss (C), demyelination (F) and perivascular infiltrates (I) after the therapeutic administration of IP7e in EAE mice (Student's t test). Data are expressed as mean ± SEM. (IP7e; isoxazolo-pyridinone 7e, SEM; standard error of mean).