Neuroprotective and anti-inflammatory effects of estrogen receptor ligand treatment in mice

Abstract

Demyelination and neurodegeneration is a major contributor in the progression of disability in multiple sclerosis (MS). Thus, the development of therapies that are neuroprotective has elicited considerable interests. Estrogens and estrogen receptor (ER) ligand treatments are promising treatments to prevent MS-induced neurodegeneration and a multicenter phase II clinical trial of estriol as a beneficial therapy in MS is underway. Here, we discuss studies performed in our laboratory that examined the effects of ER ligands in the inflammatory/demyelinating disorder experimental autoimmune encephalomyelitis (EAE), a model of MS. Administration of estriol or 17beta-estradiol reduced clinical severity and this clinical disease improvement was associated with favorable changes in cytokine production. There was a significant decrease of neuronal pathology in gray matter along with myelin and axon preservation in white matter of spinal cords of mice with EAE. In subsequent experiments, we contrasted the results of ERalpha versus ERbeta ligand treatment. While ERalpha ligand treatment was anti-inflammatory, ERbeta ligand treatment was not. ERbeta ligand treatment nevertheless reduced demyelination and preserved axon numbers in white matter and prevented neuronal abnormalities in gray matter. Clinically, ERalpha ligand treatment abrogated the disease at the onset, while ERbeta ligand treatment had no effect at disease onset, but promoted recovery. Thus, unlike ERalpha ligand treatment, ERbeta ligand treatment was protective at the level of the target organ, independent of anti-inflammatory effects in the peripheral immune system. ERbeta ligand treatment should be considered as a potential neuroprotective agent for MS and other neurodegenerative diseases, particularly since breast and uterine cancer are mediated through ERalpha.

Figure 1

Differential effects with ER ligands on chronic EAE Ovariectomized C57BL/6 female mice were given daily subcutaneous injections of an ER ligand (ERα ligand-PPT and ERβ-DPN) during active EAE and graded using the standard EAE grading scale. (A) Mean clinical scores of ERα ligand treated mice (blue circle) as compared to vehicle treated mice were significantly reduced during the entire disease course. (B) Mean clinical scores of ERβ ligand treated mice (blue circle) as compared to vehicle treated mice, were not significantly different early in disease (up to day 20 after disease induction), but them became significantly improved later during EAE, (following day 30 after disease induction). (C) 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(21).

Figure 2

Even without anti-inflammatory effect ERβ ligand treatment is neuroprotective. (A–C) Treatment with an ERα ligand, not an ERβ ligand, reduced inflammation in spinal cords of mice with EAE. Representative H&E (A), anti-CD3 antibody (B), and anti-Mac 3 antibody (C) stained thoracic spinal cord sections (4X magnification) from healthy control, as well as vehicle, ERα ligand (PPT), and ERβ ligand (DPN) treated EAE mice, all killed at day 40 (late) after disease induction. Compared to controls, vehicle treated EAE spinal cords showed multifocal to coalescing areas of inflammation in the leptomeninges and white matter, around blood vessels, and in the parenchyma of the white matter. ERα ligand-treated spinal cords had reduced inflammation as compared to vehicle treated EAE, whereas ERβ ligand-treated did not have reduced levels of inflammation. Anti-CD3 antibody and anti-Mac 3 antibody staining revealed that the inflammation was composed of both T cells and macrophage lineage cells, respectively. Both T cells and macrophage lineage cell staining was reduced with ERα ligand, but not ERβ ligand treatment(21). (D) Treatment with an ERα ligand and an ERβ ligand each preserved myelin basic protein immunoreactivity and spared axonal pathology in white matter of spinal cords of mice with EAE. Part of the anterior funniculus of thoracic spinal cord sections was imaged at 40X co-immunostained with anti-NF200 (green) and anti-MBP (red). Distinct green axonal centers surrounded by red myelin sheaths can be seen in normal controls, PPT and DPN treated EAE mice from 40 day after disease induction. Vehicle treated mice show reduced axonal numbers and myelin, along with focal demyelination (white stars) and loss of axons(21). (E) Treatment with an ERα ligand and an ERβ ligand 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) in (i) and Nissl in (ii) at 4X magnification, derived from normal healthy control mice, vehicle treated EAE, ERα ligand (PPT) treated EAE and ERβ ligand (DPN) treated EAE mice, each sacrificed at day 40 after disease induction. Panel (iii) is a merged confocal scan at 40X of NeuN⁺ (red) and β3-tubulin⁺ (green) co-labeled neurons from an area represented by dotted white square area in (i). Panel (iv) is a 40X 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 to normal control. White arrows in panel (iii) denote loss of NeuN⁺ staining. In contrast, EAE mice treated with either PPT or DPN had preserved NeuN and Nissl staining(21).

Figure 3

ERβ ligand treatment results in recovery of motor function in EAE. (a) Mean time on rotarod decreased abruptly at day 12 after disease induction in both the vehicle and DPN treated EAE mice. But after day 30 the DPN treated group demonstrated significant recovery of motor function, while the vehicle treated did not improve. Estradiol treatment served as a positive control for a treatment effect. (b) In contrast to the improvement observed with DPN treatment of wild type mice, no improvement was observed at the later phase of disease in DPN treated ERβ KO mice(21).