Differential neuroprotective and antiinflammatory effects of estrogen receptor (ER)alpha and ERbeta ligand treatment

Abstract

Treatment with either estradiol or an estrogen receptor (ER)alpha ligand has been shown to be both antiinflammatory and neuroprotective in a variety of neurological disease models, but whether neuroprotective effects could be observed in the absence of an antiinflammatory effect has remained unknown. Here, we have contrasted effects of treatment with an ERalpha vs. an ERbeta ligand in experimental autoimmune encephalomyelitis, the multiple sclerosis model with a known pathogenic role for both inflammation and neurodegeneration. Clinically, ERalpha ligand treatment abrogated disease at the onset and throughout the disease course. In contrast, ERbeta ligand treatment had no effect at disease onset but promoted recovery during the chronic phase of the disease. ERalpha ligand treatment was antiinflammatory in the systemic immune system, whereas ERbeta ligand treatment was not. Also, ERalpha ligand treatment reduced CNS inflammation, whereas ERbeta ligand treatment did not. Interestingly, treatment with either the ERalpha or the ERbeta ligand was neuroprotective, as evidenced by reduced demyelination and preservation of axon numbers in white matter, as well as decreased neuronal abnormalities in gray matter. Thus, by using the ERbeta selective ligand, we have dissociated the antiinflammatory effect from the neuroprotective effect of estrogen treatment and have shown that neuroprotective effects of estrogen treatment do not necessarily depend on antiinflammatory properties. Together, these findings suggest that ERbeta ligand treatment should be explored as a potential neuroprotective strategy in multiple sclerosis and other neurodegenerative diseases, particularly because estrogen-related toxicities such as breast and uterine cancer are mediated through ERalpha.

Conflict of interest statement

The authors declare no conflict of interest.

Figures

Fig. 1.

Treatment with ERα- vs. ERβ-selective ligands has differential effects on chronic EAE. Ovariectomized C57BL/6 female mice were given daily s.c. injections of an ER ligand during active EAE and graded for clinical disease severity using the standard EAE grading scale. (Left) Mean clinical scores of ERα ligand-treated mice, as compared with vehicle-treated mice, were significantly reduced throughout disease, P < 0.0001. Each treatment group, n = 4; data are representative of a total of five repeated experiments. (Center) ERβ ligand-treated mice, as compared with vehicle-treated mice, were not significantly different early in disease (up to day 20 after disease induction) but then became significantly improved later during EAE (day 30 after disease induction), P < 0.001. Numbers of mice in each group were vehicle, n = 4; estradiol, n = 4; DPN, n = 8. Data are representative of experiments repeated twice. (Right) DPN treatment in vivo during EAE remains highly selective for ERβ. Clinical scores in ovariectomized ERβ KO C57BL/6 mice with active EAE were no different when comparing DPN-treated with vehicle-treated mice. Each treatment group, n = 4, and data are representative of experiments repeated twice. Estradiol-treated mice served as a positive control for a treatment effect in each experiment.

Fig. 2.

Treatment with ERα- vs. ERβ-selective ligands has differential effects on the systemic immune response. At day 19 (A and B) or day 40 (C and D) after disease induction, mice were killed, and cytokine production by autoantigen-stimulated splenocytes was determined. ERα ligand treatment significantly reduced TNFα, IFN-γ, and IL-6, and increased IL-5, during early EAE (A) and late EAE (C). In contrast, no significant differences with ERβ ligand treatment were seen in cytokine levels at either the early stage (B) or late stage (D) of EAE. Vehicle-treated and media-stimulated (first bar) and ER ligand-treated and media-stimulated (third bar) each served as negative controls for stimulations with autoantigen MOG 35–55 peptide (MOG) (second and fourth bars). Error bars indicate variability of cytokine values for individual mice within a given treatment group, with n = 4 mice for each treatment group. Data are representative of two to five experiments for each time point. No differences were observed with either ERα or ERβ ligand treatment, as compared with vehicle, for either IL-17 or IL-10, whereas IL-4 and IL-12 levels were too low to detect (not shown).

Fig. 3.

Treatment with an ERα ligand, not an ERβ ligand, reduced inflammation in spinal cords of mice with EAE. Consecutive thoracic spinal cord sections coimmunostained with NF200 (green) and CD45 (red) at ×10 magnification are shown from partial images (lateral funiculus, a portion of anterior funiculus and gray matter) from normal control, vehicle-treated EAE, ERα ligand-treated EAE, and ERβ ligand-treated EAE mice at day 19 (A) and day 40 (B) after disease induction. Vehicle-treated EAE cords had large areas of CD45+ cell staining in white matter as compared with the normal control, whereas ERα ligand-treated EAE mice had only occasional CD45 positivity. ERβ ligand-treated EAE mice had CD45+ cell staining, similar to that in vehicle-treated EAE. Consecutive sections from the same mice were also scanned at ×40 magnification (within ventral horn designated by the dotted line square area in normal image) to show the morphology of CD45+ cells in the gray matter. (C) Counting CD45+DAPI+ cells in the dorsal funiculi revealed that vehicle- and ERβ ligand-treated EAE mice had a significant increase compared with healthy controls, whereas the ERα ligand-treated groups did not. (D) Number of CD45+DAPI+ cells during later EAE was quantified as in C. Number of mice, three per treatment group; number of T1–T5 sections per mouse, six; total number of sections per treatment group, 18. Statistically significant compared with normals (*, P < 0.05; **, P < 0.001), 1 × 4 ANOVAs. Data are representative of experiments repeated in their entirety on another set of EAE mice with each of the treatments.

Fig. 4.

Treatment with ERα and ERβ ligands, each preserved MBP immunoreactivity in white matter of spinal cords of mice with EAE. At days 19 (A) and 40 (B) after disease induction, vehicle-treated EAE mice had reduced MBP immunoreactivity as compared with normal controls in dorsal columns of thoracic spinal cord sections imaged at ×10 magnification. In contrast, ERα and ERβ ligand-treated EAE mice showed relatively preserved MBP staining. Upon quantification (C and D), MBP immunoreactivity in the dorsal column was significantly lower in vehicle-treated EAE mice as compared with normal mice, whereas ERα and ERβ ligand-treated EAE mice each demonstrated no significant decreases. Myelin density is presented as percent of normal. Number of mice, three per treatment group; number of T1–T5 sections per mouse, six; total number of sections per treatment group, 18. Statistically significant compared with normal (*, P < 0.01; **, P < 0.005); 1 × 4 ANOVAs.

Fig. 5.

Treatment with ERα and ERβ ligands each preserved axonal densities in white matter of spinal cords of mice with EAE. Part of the anterior funiculus of thoracic spinal cord sections was imaged at ×40 after coimmunostaining with anti-NF200 (green) and anti-MBP (red). Distinct green axonal centers surrounded by red myelin sheaths can be seen in normal controls, and ERα and ERβ ligand-treated EAE mice at 19 days (A) and 40 days (B) after disease induction. In contrast, vehicle-treated EAE mice showed reduced axonal numbers and myelin, with focal demyelination (white asterisks). Upon quantification (C and D), neurofilament-stained axon numbers in white matter were significantly lower in vehicle-treated EAE mice as compared with normal mice, whereas ERα and ERβ ligand-treated EAE mice demonstrated no significant reduction in axon numbers as compared with normal controls. Axon number is presented as percent of normal. Number of mice, three per treatment group; number of T1–T5 sections per mouse, six; total number of sections per treatment group, 18. Statistically significant compared with normal (*, P < 0.01; **, P < 0.005); 1 × 4 ANOVAs.

Fig. 6.

Treatment with ERα and ERβ ligands each preserved neuronal staining in gray matter of spinal cords of mice with EAE. Split images of thoracic spinal cord sections stained with NeuN (red, i) and Nissl (ii) at ×4 magnification, derived from healthy controls, vehicle-treated EAE, ERα ligand-treated EAE, and ERβ ligand-treated EAE mice, killed at either day 19 (early; A) or day 40 (late; B) after disease induction are shown. iii is a merged confocal scan at ×40 of NeuN+ (red) and β3-tubulin+ (green) colabeled neurons from an area represented by the dotted white square area in i. iv is a ×40 magnification of Nissl-stained area in solid black square in ii. A decrease in NeuN+ immunostaining and Nissl staining was observed in the dorsal horn, intermediate zone, and ventral horn of vehicle-treated EAE mice as compared with normal control. White arrows in iii denote loss of NeuN+ staining. In contrast, EAE mice treated with either ERα or ERβ ligand had preserved NeuN and Nissl staining. Upon quantification of neurons in the entire delineated gray matter of T1–T5 sections, NeuN+ immunolabeled neurons were significantly decreased by nearly 41% in vehicle-treated EAE mice at day 19 (C) and nearly 31% at day 40 (D) as compared with normal controls, whereas ERα and ERβ ligand-treated EAE mice were not statistically different from normal controls. Number of mice, three per treatment group; number of T1–T5 sections per mouse, six; total number of sections per treatment group, 18. Statistically significant compared with normals (*, P < 0.05), 1 × 4 ANOVAs. Data are representative of experiments repeated in their entirety on another set of EAE mice with each of the treatments.

Fig. 7.

Treatment with an ERβ ligand results in recovery of motor function late during EAE. (Left) Ovariectomized C57BL/6 female mice with EAE were treated with ERβ ligand and assessed for motor performance on a rotarod apparatus. Although mean time on rotarod decreased abruptly at day 12 after disease induction in both the vehicle- and ERβ ligand-treated EAE mice, after day 30, the ERβ ligand-treated group demonstrated significant recovery of motor function, whereas the vehicle-treated did not improve. *, P < 0.01 and **, P < 0.005, ANOVA. Estradiol treatment served as a positive control for a treatment effect. Number of mice in each treatment group: vehicle, n = 4; DPN, n = 8; estradiol, n = 4. Data are representative of experiments repeated twice. (Right) In contrast to the improvement observed with ERβ ligand treatment of WT mice, no improvement was observed at the later phase of disease in ERβ ligand-treated ERβ KO mice. Again, vehicle served as a negative control, and estradiol served as a positive control, for a treatment effect. Number of mice in each treatment group: vehicle, n = 4; DPN, n = 4; estradiol, n = 4.