Complexity of trophic factor signaling in experimental autoimmune encephalomyelitis: differential expression of neurotrophic and gliotrophic factors

Abstract

Soluble factors that promote survival and differentiation of glia and neurons during development are likely to play key roles in neurodegeneration and demyelinating diseases such as multiple sclerosis (MS) and have the potential to be important therapeutic targets. We examined the effect of TrkB signaling and the expression patterns of neurotrophic and gliotrophic factors in the mouse brain in MOG-induced experimental allergic encephalomyelitis (EAE). With induction of mild disease, TrkB heterozygous mice were more severely affected compared to their wild type littermates. However, with more potent disease induction, TrkB heterozygotes fared similar to their wild type littermates, suggesting complex modulatory roles for TrkB signaling. One possible explanation for this difference is that the expression patterns of neurotrophic factors correlate with disease severity in individual mice with mild disease, but not in more severe disease. With the less potent induction in C57BL/6 mice, we found that BDNF was consistently increased at EAE onset, while the soluble gliotrophic factor neuregulin (NRG1) was increased only in the chronic phase of the disease. Treatment of these animals with glatiramer acetate (GA) to decrease disease severity resulted in lower levels of both BDNF and NRG1 expression in some mice at 35days after immunization compared to those in untreated EAE mice, but had no direct effect on these factors in the absence of EAE. Our results suggest a complex interplay between neurotrophic and gliotrophic factors in EAE that is dependent on disease stage and severity. While signaling by BDNF through TrkB is protective in mild disease, this effect was not seen in more severe disease. The late induction of NRG1 in the chronic stage of disease could also worsen disease severity through its known ability to activate microglial, inflammatory pathways. While complex, these studies begin to define underlying axoglial trophic activities that are likely involved in both disease pathogenesis and repair.

Keywords: BDNF and TrkB tyrosine kinase receptor; Experimental autoimmune encephalomyelitis; Glatiramer acetate; Multiple sclerosis; Neuregulin1.

Figure 1. Decreased TrkB expression increases disease severity in less potent immunization conditions

Female trkB+/− and age- and gender-matched female wild type littermates (trkB+/+) were immunized with MOG_35-55(200μg)/CFA containing M. Tbc (1mg/ml or 5mg/ml)/PT(200X2 ng or 300X2 ng) using two immunization protocols resulting in mild (A) or severe (C) disease induction in trkB+/+ mice. Mice were scored daily for 48 days. Each line represents disease course for an individual mouse. There is increased disease incidence and disease severity in trkB+/− mice compared to trkB+/+ mice with the less potent immunization conditions (Figures 1A-1B). However, there is no difference in disease severity between the trkB+/− and trkB+/+ mice with the more potent immunization conditions (Figures 1C-1D).

Figure 2. Disease severity correlates with BDNF and NRG1 gene expression in trkB +/+ and +/− mice with less potent immunization conditions

Brain tissue was collected from individual mice 48 days after the induction of EAE (Figures 1 and Table 1). RNA isolation and qPCR were performed as described in the Methods. Each symbol represents value of gene expression and the cumulative clinical score for an individual mouse. Gene expression = RNA quantity (2^−ΔCt) (ΔCt = Ct for targeted gene – Ct for GAPDH). “R” value represents the linear correlation between the gene expression values and the disease severity for expression of each gene in trkB +/+ and +/− mice under less potent (A) and more potent (B) immunization protocols. The samples were considered to show better correlation if the “R” was closer to 1. *: p<0.05 by Test for Association/Correlation using cor.test {stats} software.

Figure 3. BDNF expression increases at EAE onset, while soluble type I NRG1 increases in chronic disease

C57BL/6 mice were immunized with MOG_35-55(200μg)/CFA containing M. Tbc (1mg/ml)/PT(200X2 ng), and were scored daily for disease. The mean score of EAE is shown (A). On the indicated day after immunization (onset day 12-15 or chronic phase day 35), brain tissues were collected from individual mice. RNA isolation and qPCR were performed from individual brain samples as described in the Methods. Each bar represents an individual mouse with indicated clinical score and the fold change in mRNA expression of types I (B) and III NRG1 (C), BDNF (D) and GDNF (E) at disease onset or chronic stage versus the average of the gene expression in naïve mice (n=4). Compared to naive mice (n=4), BDNF increased at disease onset and type I NRG1 expression increased in chronic stages (day 35 post-immunization). The samples were considered to show little change if the fold changes were less than 1.5 or were variable within the same group.

Figure 4. GA treatment reduces disease severity and blunts both BDNF and NRG1 induction

C57BL/6 mice were immunized with MOG_35-55(200 μg)/CFA containing M. Tbc (1mg/ml)/PT(200X2 ng), and were scored daily for EAE with GA treated and untreated groups. GA suppression: GA i.p started together with disease induction (8 daily injections). GA treatment: GA i.p started after the appearance of clinical symptoms (8 daily injections). RNA isolation and qPCR were performed at 35 days after immunization from individual brain samples as described in the Methods. Each bar represents an individual mouse with indicated clinical score and the fold change in mRNA expression of types I (B) and III NRG1 (C), BDNF (D) or GDNF (E) from GA suppression and GA treatment group versus the average of gene expression in untreated group (n=4 mice each group). Compared to untreated mice with EAE, type I NRG1 and BDNF expression was lower in mice treated with GA at disease onset.

Figure 5. GA has no direct effect on brain BDNF and NRG1 expression in untreated mice

Naïve C57BL/6 mice were treated with or without GA for 8 days. Brain tissues were collected from each group above. RNA isolation and qPCR were performed from individual brain samples as described in the Methods. Each bar represents the mean of fold change ±SD of gene expression from GA treated versus untreated group (n=5 mice each group). Compared to untreated mice, there is no significant change in mRNA expression for types I and III NRG1, BDNF or GDNF in GA treated mice.