Lentivirus-mediated estrogen receptor α overexpression in the central nervous system ameliorates experimental autoimmune encephalomyelitis in mice

Abstract

Abstract. Multiple sclerosis (MS) is an autoimmune disease characterized by inflammatory cell infiltration of the central nervous system (CNS) and multifocal demyelination. Clinical data and clinical indicators demonstrate that estrogen improves the relapse-remittance of MS patients. This study aimed to investigate the anti-inflammatory effects and the underlying mechanism(s) of action of estrogen and estrogen receptor α (ERα) in an experimental autoimmune encephalomyelitis (EAE) mouse model of MS. An ERα recombinant lentivirus was constructed. Mouse neurons were cultured in serum-free culture medium, and ERα recombinant lentivirus with a multiplicity of infection (MOI) of 5 was used to infect the neurons. Furthermore, neuronal ERα mRNA and protein expression were detected using real-time quantitative PCR and western blot analysis. We sterotaxically injected ERα recombinant lentivirus into the lateral ventricle of mouse brains, and successfully identified infected neurons using Flag immunofluorescence staining to determine the optimal dose. A total of 75 C57BL/6 mice were ovariectomized. After 2 weeks, EAE was induced with myelin oligodendrocyte glycoprotein (MOG) 35-55 peptide. The EAE mice were divided into 5 groups: the estrogen group (treatment with estradiol), the ERα agonist group (treatment with raloxifene), the ERα recombinant lentivirus group (ERα group, treatment with ERα recombinant lentivirus), the empty virus group and the normal saline (NS) group; clinical symptoms and body weight were compared among the groups. We assessed EAE-related parameters, detected pathological changes with immunohistochemistry and quantified the expression of myelin basic protein (MBP), matrix metalloproteinase-9 (MMP-9), and a subset of EAE-related cytokines using enzyme-linked immunosorbent assay (ELISA). We successfully constructed an ERα recombinant lentivirus. C57BL/6 mouse neurons can survive in culture for at least 8 weeks. During that period, the recombinant lentivirus was able to infect the neurons, while sustaining green fluorescence protein (GFP) expression. ERα recombinant lentivirus also infected the neurons at a MOI of 5. The ERα mRNA and protein expression levels were higher in the infected neurons compared to the uninfected ones. We successfully infected the CNS of C57BL/6 mice by stereotaxically injecting ERα recombinant lentivirus into the lateral ventricle of the mouse brains and induced EAE. The lentivirus-mediated overexpression of ERα reduced the incidence of EAE, ameliorated the clinical symptoms, inhibited inflammatory cell CNS infiltration, and reduced nerve fiber demyelination. MMP-9, tumor necrosis factor-α (TNF-α), interferon-γ (IFN-γ), interleukin (IL)-17 and IL-23 expression levels were decreased, while those of MBP and IL-4 were increased. These data demonstrate that it is possible to induce the overexpression of ERα using a recombinant lentivirus, and that this novel intervention ameliorates EAE in a mouse model. Mechanistically, estrogen and ERα inhibit inflammatory responses, and ERα allev...

Consistent induction of chronic experimental autoimmune encephalomyelitis in C57BL/6 mice for the longitudinal study of pathology and repair

“…A number of publications have used the Mann Whitney U test (Goldmann et al, 2013; Su et al, 2012; Zhang et al, 2015), the Friedman test (the non-parametric alternative to the one-way ANOVA with repeated measures (all the papers published from our group and others(Du et al, 2011; Hu and Qin, 2013; Spence et al, 2013; Wisdom et al, 2013)), or a two-way ANOVA followed by a Bonferroni post hoc test (Lélu et al, 2010; Rothhammer et al, 2011; Yu et al, 2015) to analyze EAE clinical scores. Unfortunately, multiple issues arise from employing these types of analyses.…”

RGS5 augments astrocyte activation and facilitates neuroinflammation via TNF signaling

“…To further examine whether RGS5-mediated inflammation contribute to neurodegeneration, we used A30P-mutant α-synuclein transgenic (Tg) mouse line, a widely used genetic animal model of PD, which develops a neurodegenerative syndrome that manifests in the spinal cord rather in the brain [ 28 , 41 ]. To achieve a higher transfection efficacy in astrocytes in the spinal cord as reported [ 46 ], the Tg mice were subjected to the injection of Lenti-GFAP-RGS5 (2 × 10 8 TU/ml; 20 µl) in the lateral ventricle (Fig. 2 F).…”

Raloxifene as Treatment for Various Types of Brain Injuries and Neurodegenerative Diseases: A Good Start

“…Estrogen has beneficial effects regarding relapse-remittance of MS patients as demonstrated by clinical data and clinical indicators. To better understand the mechanism(s) of action of estrogen and estrogen receptor ERα underlying these beneficial anti-inflammatory effects, Hu and Qin [ 70 ] studied an experimental autoimmune encephalomyelitis (EAE) mouse model of MS. In their study, an ERα recombinant lentivirus was used to infect neurons with ERα.…”

“…This overexpression of ERα apparently inhibited inflammatory cell CNS infiltration, and decreased expression levels of Matrix metallopeptidase 9 ( MMP-9 ), tumor necrosis factor-α (TNF-α), interferon-γ (IFN-γ), interleukin (IL)-17 and IL-23 expression levels, while expression of IL-4 was increased. In addition, overexpression of ERα reduced EAE incidence, and reduced nerve fiber demyelination, together with an increase in myelin basic protein (MBP) expression levels [ 70 ]. Moreover, the associated clinical symptoms were ameliorated.…”