Estrogen receptor beta in astrocytes modulates cognitive function in mid-age female mice.

Menopause is associated with cognitive deficits and brain atrophy, but the brain region and cell-specific mechanisms are not fully understood. Here, we identify a sex hormone by age interaction whereby loss of ovarian hormones in female mice at midlife, but not young age, induced hippocampal-dependent cognitive impairment, dorsal hippocampal atrophy, and astrocyte and microglia activation with synaptic loss. Selective deletion of estrogen receptor beta (ERß) in astrocytes, but not neurons, in gonadally intact female mice induced the same brain effects. RNA sequencing and pathway analyses of gene expression in hippocampal astrocytes from midlife female astrocyte-ERß conditional knock out (cKO) mice revealed Gluconeogenesis I and Glycolysis I as the most differentially expressed pathways. Enolase 1 gene expression was increased in hippocampi from both astrocyte-ERß cKO female mice at midlife and from postmenopausal women. Gain of function studies showed that ERß ligand treatment of midlife female mice reversed dorsal hippocampal neuropathology.

Decreased neurofilament light chain levels in estriol-treated multiple sclerosis.

Estrogens have neuroprotective actions depending on estrogen type, dose, and timing in both preclinical models and in women during health and disease. Serum neurofilament light chain is a putative biomarker of neurodegeneration in multiple sclerosis, aging, and other neurodegenerative diseases. Here, oral treatment with an estrogen unique to pregnancy (estriol) using an 8 mg dose to induce a mid-pregnancy blood estriol level reduced serum neurofilament light chain in nonpregnant MS women at mean age of 37 years. This is consistent with estriol-mediated protection from neuro-axonal injury and supports the use of serum neurofilament light chain as a biomarker in MS.

Axonal damage in spinal cord is associated with gray matter atrophy in sensorimotor cortex in experimental autoimmune encephalomyelitis.

BACKGROUND: Gray matter (GM) atrophy in brain is one of the best predictors of long-term disability in multiple sclerosis (MS), and recent findings have revealed that localized GM atrophy is associated with clinical disabilities. GM atrophy associated with each disability mapped to a distinct brain region, revealing a disability-specific atlas (DSA) of GM loss. OBJECTIVE: To uncover the mechanisms underlying the development of localized GM atrophy. METHODS: We used voxel-based morphometry (VBM) to evaluate localized GM atrophy and Clear Lipid-exchanged Acrylamide-hybridized Rigid Imaging-compatible Tissue-hYdrogel (CLARITY) to evaluate specific pathologies in mice with experimental autoimmune encephalomyelitis (EAE). RESULTS: We observed extensive GM atrophy throughout the cerebral cortex, with additional foci in the thalamus and caudoputamen, in mice with EAE compared to normal controls. Next, we generated pathology-specific atlases (PSAs), voxelwise mappings of the correlation between specific pathologies and localized GM atrophy. Interestingly, axonal damage (end-bulbs and ovoids) in the spinal cord strongly correlated with GM atrophy in the sensorimotor cortex of the brain. CONCLUSION: The combination of VBM with CLARITY in EAE can localize GM atrophy in brain that is associated with a specific pathology in spinal cord, revealing a PSA of GM loss.

Gene expression in oligodendrocytes during remyelination reveals cholesterol homeostasis as a therapeutic target in multiple sclerosis.

Regional differences in neurons, astrocytes, oligodendrocytes, and microglia exist in the brain during health, and regional differences in the transcriptome may occur for each cell type during neurodegeneration. Multiple sclerosis (MS) is multifocal, and regional differences in the astrocyte transcriptome occur in experimental autoimmune encephalomyelitis (EAE), an MS model. MS and EAE are characterized by inflammation, demyelination, and axonal damage, with minimal remyelination. Here, RNA-sequencing analysis of MS tissues from six brain regions suggested a focus on oligodendrocyte lineage cells (OLCs) in corpus callosum. Olig1-RiboTag mice were used to determine the translatome of OLCs in vivo in corpus callosum during the remyelination phase of a chronic cuprizone model with axonal damage. Cholesterol-synthesis gene pathways dominated as the top up-regulated pathways in OLCs during remyelination. In EAE, remyelination was induced with estrogen receptor-ß (ERß) ligand treatment, and up-regulation of cholesterol-synthesis gene expression was again observed in OLCs. ERß-ligand treatment in the cuprizone model further increased cholesterol synthesis gene expression and enhanced remyelination. Conditional KOs of ERß in OLCs demonstrated that increased cholesterol-synthesis gene expression in OLCs was mediated by direct effects in both models. To address this direct effect, ChIP assays showed binding of ERß to the putative estrogen-response element of a key cholesterol-synthesis gene (Fdps). As fetal OLCs are exposed in utero to high levels of estrogens in maternal blood, we discuss how remyelinating properties of estrogen treatment in adults during injury may recapitulate normal developmental myelination through targeting cholesterol homeostasis in OLCs.

Oestrogen receptor β ligand acts on CD11c+ cells to mediate protection in experimental autoimmune encephalomyelitis.

Oestrogen treatments are neuroprotective in a variety of neurodegenerative disease models. Selective oestrogen receptor modifiers are needed to optimize beneficial effects while minimizing adverse effects to achieve neuroprotection in chronic diseases. Oestrogen receptor beta (ERβ) ligands are potential candidates. In the multiple sclerosis model chronic experimental autoimmune encephalomyelitis, ERβ-ligand treatment is neuroprotective, but mechanisms underlying this neuroprotection remain unclear. Specifically, whether there are direct effects of ERβ-ligand on CD11c+ microglia, myeloid dendritic cells or macrophages in vivo during disease is unknown. Here, we generated mice with ERβ deleted from CD11c+ cells to show direct effects of ERβ-ligand treatment in vivo on these cells to mediate neuroprotection during experimental autoimmune encephalomyelitis. Further, we use bone marrow chimeras to show that ERβ in peripherally derived myeloid cells, not resident microglia, are the CD11c+ cells mediating this protection. CD11c+ dendritic cell and macrophages isolated from the central nervous system of wild-type experimental autoimmune encephalomyelitis mice treated with ERβ-ligand expressed less iNOS and T-bet, but more IL-10, and this treatment effect was lost in mice with specific deletion of ERβ in CD11c+ cells. Also, we extend previous reports of ERβ-ligand’s ability to enhance remyelination through a direct effect on oligodendrocytes by showing that the immunomodulatory effect of ERβ-ligand acting on CD11c+ cells is necessary to permit the maturation of oligodendrocytes. Together these results demonstrate that targeting ERβ signalling pathways in CD11c+ myeloid cells is a novel strategy for regulation of the innate immune system in neurodegenerative diseases. To our knowledge, this is the first report showing how direct effects of a candidate neuroprotective treatment on two distinct cell lineages (bone marrow derived myeloid cells and oligodendrocytes) can have complementary neuroprotective effects in vivo.awx315media15688130498001.

Bedside to bench to bedside research: Estrogen receptor beta ligand as a candidate neuroprotective treatment for multiple sclerosis.

Protective effects of pregnancy during MS have led to clinical trials of estriol, the pregnancy estrogen, in MS. Since estriol binds to estrogen receptor (ER) beta, ER beta ligand could represent a "next generation estriol" treatment. Here, ER beta ligand treatment was protective in EAE in both sexes and across genetic backgrounds. Neuroprotection was shown in spinal cord, sparing myelin and axons, and in brain, sparing neurons and synapses. Longitudinal in vivo MRIs showed decreased brain atrophy in cerebral cortex gray matter and cerebellum during EAE. Investigation of ER beta ligand as a neuroprotective treatment for MS is warranted.

Estriol combined with glatiramer acetate for women with relapsing-remitting multiple sclerosis: a randomised, placebo-controlled, phase 2 trial.

BACKGROUND: Relapses of multiple sclerosis decrease during pregnancy, when the hormone estriol is increased. Estriol treatment is anti-inflammatory and neuroprotective in preclinical studies. In a small single-arm study of people with multiple sclerosis estriol reduced gadolinium-enhancing lesions and was favourably immunomodulatory. We assessed whether estriol treatment reduces multiple sclerosis relapses in women. METHODS: We did a randomised, double-blind, placebo-controlled phase 2 trial at 16 academic neurology centres in the USA, between June 28, 2007, and Jan 9, 2014. Women aged 18-50 years with relapsing-remitting multiple sclerosis were randomly assigned (1:1) with a random permuted block design to either daily oral estriol (8 mg) or placebo, each in combination with injectable glatiramer acetate 20 mg daily. Patients and all study personnel, except for pharmacists and statisticians, were masked to treatment assignment. The primary endpoint was annualised relapse rate after 24 months, with a significance level of p=0.10. Relapses were confirmed by an increase in Expanded Disability Status Scale score assessed by an independent physician. Analysis was by intention to treat. The trial is registered with ClinicalTrials.gov, number NCT00451204. FINDINGS: We enrolled 164 patients: 83 were allocated to the estriol group and 81 were allocated to the placebo group. The annualised confirmed relapse rate was 0.25 relapses per year (95% CI 0.17-0.37) in the estriol group versus 0.37 relapses per year (0.25-0.53) in the placebo group (adjusted rate ratio 0.63, 95% CI 0.37-1.05; p=0.077). The proportion of patients with serious adverse events did not differ substantially between the estriol group and the placebo group (eight [10%] of 82 patients vs ten [13%] of 76 patients). Irregular menses were more common in the estriol group than in the placebo group (19 [23%] vs three [4%], p=0.0005), but vaginal infections were less common (one [1%] vs eight [11%], p=0.0117). There were no differences in breast fibrocystic disease, uterine fibroids, or endometrial lining thickness as assessed by clinical examination, mammogram, uterine ultrasound, or endometrial lining biopsy. INTERPRETATION: Estriol plus glatiramer acetate met our criteria for reducing relapse rates, and treatment was well tolerated over 24 months. These results warrant further investigation in a phase 3 trial. FUNDING: National Institutes of Health, National Multiple Sclerosis Society, Conrad N Hilton Foundation, Jack H Skirball Foundation, Sherak Family Foundation, and the California Community Foundation.

Astrocyte CCL2 sustains immune cell infiltration in chronic experimental autoimmune encephalomyelitis.

Chemokine (C-C motif) ligand 2 (CCL2), initially identified as monocyte chemoattractant protein-1 (MCP-1), recruits immune cells to the central nervous system (CNS) during autoimmune inflammation. CCL2 can be expressed by multiple cell types, but which cells are responsible for CCL2 function during acute and chronic phases of autoimmune disease is not known. We determined the role of CCL2 in astrocytes in vivo during experimental autoimmune encephalomyelitis (EAE) by using Cre-loxP gene deletion. Mice with a conditional gene deletion of CCL2 from astrocytes had less severe EAE late in disease while having a similar incidence and severity of disease at onset as compared to wild type (WT) control littermates. EAE mice devoid of CCL2 in astrocytes had less macrophage and T cell inflammation in the white matter of the spinal cord and less diffuse activation of astrocytes and microglia in both white and gray matter as well as less axonal loss and demyelination, compared to WT littermates. These findings demonstrate that CCL2 in astrocytes plays an important role in the continued recruitment of immune cells and activation of glial cells in the CNS during chronic EAE, thereby suggesting a novel cell specific target for neuroprotective treatments of chronic neuroinflammatory diseases.

XY sex chromosome complement, compared with XX, in the CNS confers greater neurodegeneration during experimental autoimmune encephalomyelitis.

Women are more susceptible to multiple sclerosis (MS) and have more robust immune responses than men. However, men with MS tend to demonstrate a more progressive disease course than women, suggesting a disconnect between the severity of an immune attack and the CNS response to a given immune attack. We have previously shown in an MS model, experimental autoimmune encephalomyelitis, that autoantigen-sensitized XX lymph node cells, compared with XY, are more encephalitogenic. These studies demonstrated an effect of sex chromosomes in the induction of immune responses, but did not address a potential role of sex chromosomes in the CNS response to immune-mediated injury. Here, we examined this possibility using XX versus XY bone marrow chimeras reconstituted with a common immune system of one sex chromosomal type. We found that experimental autoimmune encephalomyelitis mice with an XY sex chromosome complement in the CNS, compared with XX, demonstrated greater clinical disease severity with more neuropathology in the spinal cord, cerebellum, and cerebral cortex. A candidate gene on the X chromosome, toll-like receptor 7, was then examined. Toll-like receptor 7 expression in cortical neurons was higher in mice with XY compared with mice with XX CNS, consistent with the known neurodegenerative role for toll-like receptor 7 in neurons. These results suggest that sex chromosome effects on neurodegeneration in the CNS run counter to effects on immune responses, and may bear relevance to the clinical enigma of greater MS susceptibility in women but faster disability progression in men. This is a demonstration of a direct effect of sex chromosome complement on neurodegeneration in a neurological disease.

Estrogen receptor (ER) ß expression in oligodendrocytes is required for attenuation of clinical disease by an ERß ligand.

Treatment of experimental autoimmune encephalomyelitis (EAE) mice with the estrogen receptor (ER) ß ligand diarylpropionitrile (DPN) has been shown to have neuroprotective effects via stimulation of endogenous myelination. The direct cellular mechanisms underlying the effects of this ERß ligand on the central nervous system are uncertain because different cell types in both the peripheral immune system and central nervous system express ERs. ERß is the target molecule of DPN because DPN treatment fails to decrease EAE clinical symptoms in global ERß-null mice. Here we investigated the potential role of ERß expression in cells of oligodendrocyte (OL) lineage in ERß ligand-mediated neuroprotection. To this end, we selectively deleted ERß in OLs using the well-characterized Cre-loxP system for conditional gene knockout (CKO) in mice. The effects of this ERß CKO on ERß ligand-mediated neuroprotective effects in chronic EAE mice were investigated. ERß CKO in OLs prevented DPN-induced decrease in EAE clinical disease. DPN treatment during EAE did not attenuate demyelination, only partially improved axon conduction, and did not activate the phosphatidylinositol 3-kinase/serine-threonine-specific protein kinase/mammalian target of rapamycin signaling pathway in ERß CKO mice. However, DPN treatment significantly increased brain-derived neurotrophic factor levels in ERß CKO mice. These findings demonstrate that signaling through ERß in OLs is essential for the beneficial myelination effects of the ERß ligand DPN in chronic EAE mice. Further, these findings have important implications for neuroprotective therapies that directly target OL survival and myelination.

Estrogen mediates neuroprotection and anti-inflammatory effects during EAE through ERα signaling on astrocytes but not through ERß signaling on astrocytes or neurons.

Estrogens can signal through either estrogen receptor α (ERα) or ß (ERß) to ameliorate experimental autoimmune encephalomyelitis (EAE), the most widely used mouse model of multiple sclerosis (MS). Cellular targets of estrogen-mediated neuroprotection are still being elucidated. Previously, we demonstrated that ERα on astrocytes, but not neurons, was critical for ERα ligand-mediated neuroprotection in EAE, including decreased T-cell and macrophage inflammation and decreased axonal loss. Here, we determined whether ERß on astrocytes or neurons could mediate neuroprotection in EAE, by selectively removing ERß from either of these cell types using Cre-loxP gene deletion. Our results demonstrated that, even though ERß ligand treatment was neuroprotective in EAE, this neuroprotection was not mediated through ERß on either astrocytes or neurons and did not involve a reduction in levels of CNS inflammation. Given the differential neuroprotective and anti-inflammatory effects mediated via ERα versus ERß on astrocytes, we looked for molecules within astrocytes that were affected by signaling through ERα, but not ERß. We found that ERα ligand treatment, but not ERß ligand treatment, decreased expression of the chemokines CCL2 and CCL7 by astrocytes in EAE. Together, our data show that neuroprotection in EAE mediated via ERß signaling does not require ERß on either astrocytes or neurons, whereas neuroprotection in EAE mediated via ERα signaling requires ERα on astrocytes and reduces astrocyte expression of proinflammatory chemokines. These findings reveal important cellular differences in the neuroprotective mechanisms of estrogen signaling through ERα and ERß in EAE.

Estrogen receptor-ß ligand treatment after disease onset is neuroprotective in the multiple sclerosis model.

Multiple sclerosis (MS) is an autoimmune disease characterized by inflammation and neurodegeneration. Current MS treatments were designed to reduce inflammation in MS rather than directly to prevent neurodegeneration. Estrogen has well-documented neuroprotective effects in a variety of disorders of the CNS, including experimental autoimmune encephalomyelitis (EAE), the most widely used mouse model of MS. Treatment with an estrogen receptor-ß (ERß) ligand is known to ameliorate clinical disease effectively and provide neuroprotection in EAE. However, the protective effects of this ERß ligand have been demonstrated only when administered prior to disease (prophylactically). Here we tested whether ERß ligand treatment could provide clinical protection when treatment was initiated after onset of disease (therapeutically). We found that therapeutic treatment effectively ameliorated clinical disease in EAE. Specifically, ERß ligand-treated animals exhibited preserved axons and myelin compared with vehicle-treated animals. We observed no difference in the number of T lymphocytes, macrophages, or microglia in the CNS of vehicle- vs. ERß ligand-treated animals. Our findings show that therapeutically administered ERß ligand successfully treats clinical EAE, bearing translational relevance to MS as a candidate neuroprotective agent.

Formation of covalently modified folding intermediates of simian virus 40 Vp1 in large T antigen-expressing cells.

The folding and pentamer assembly of the simian virus 40 (SV40) major capsid protein Vp1, which take place in the infected cytoplasm, have been shown to progress through disulfide-bonded Vp1 folding intermediates. In this report, we further demonstrate the existence of another category of Vp1 folding or assembly intermediates: the nonreducible, covalently modified mdVp1s. These species were present in COS-7 cells that expressed a recombinant SV40 Vp1, Vp1ΔC, through plasmid transfection. The mdVp1s persisted under cell and lysate treatment and SDS-PAGE conditions that are expected to have suppressed the formation of artifactual disulfide cross-links. As shown through a pulse-chase analysis, the mdVp1s were derived from the newly synthesized Vp1ΔC in the same time frame as Vp1's folding and oligomerization. The apparent covalent modifications occurred in the cytoplasm within the core region of Vp1 and depended on the coexpression of the SV40 large T antigen (LT) in the cells. Analogous covalently modified species were found with the expression of recombinant polyomavirus Vp1s and human papillomavirus L1s in COS-7 cells. Furthermore, the mdVp1s formed multiprotein complexes with LT, Hsp70, and Hsp40, and a fraction of the largest mdVp1, md4, was disulfide linked to the unmodified Vp1ΔC. Both mdVp1 formation and most of the multiprotein complex formation were blocked by a Vp1 folding mutation, C87A-C254A. Our observations are consistent with a role for LT in facilitating the folding process of SV40 Vp1 by stimulating certain covalent modifications of Vp1 or by recruiting certain cellular proteins.

The XX sex chromosome complement in mice is associated with increased spontaneous lupus compared with XY.

OBJECTIVES: Many autoimmune diseases are characterised by a female predominance. This may be caused by sex hormones, sex chromosomes or both. This report uses a transgenic mouse model to investigate how sex chromosome complement, not confounded by differences in gonadal type, might contribute to lupus pathogenesis. METHODS: Transgenic NZM2328 mice were created by deletion of the Sry gene from the Y chromosome, thereby separating genetic from gonadal sex. Survival, renal histopathology and markers of immune activation were compared in mice carrying the XX versus the XY(-) sex chromosome complement, with each genotype being ovary bearing. RESULTS: Mice with XX sex chromosome complement compared with XY(-) exhibited poorer survival rates and increased kidney pathology. Splenic T lymphocytes from XX mice demonstrated upregulated X-linked CD40 ligand expression and higher levels of activation markers ex vivo. Increased MMP, TGF and IL-13 production was found, while IL-2 was lower in XX mice. An accumulation of splenic follicular B cells and peritoneal marginal zone B cells was observed, coupled with upregulated costimulatory marker expression on B cells in XX mice. CONCLUSION: These data show that the XX sex chromosome complement, compared with XY(-), is associated with accelerated spontaneous lupus.

Neuroprotection mediated through estrogen receptor-alpha in astrocytes.

Estrogen has well-documented neuroprotective effects in a variety of clinical and experimental disorders of the CNS, including autoimmune inflammation, traumatic injury, stroke, and neurodegenerative diseases. The beneficial effects of estrogens in CNS disorders include mitigation of clinical symptoms, as well as attenuation of histopathological signs of neurodegeneration and inflammation. The cellular mechanisms that underlie these CNS effects of estrogens are uncertain, because a number of different cell types express estrogen receptors in the peripheral immune system and the CNS. Here, we investigated the potential roles of two endogenous CNS cell types in estrogen-mediated neuroprotection. We selectively deleted estrogen receptor-α (ERα) from either neurons or astrocytes using well-characterized Cre-loxP systems for conditional gene knockout in mice, and studied the effects of these conditional gene deletions on ERα ligand-mediated neuroprotective effects in a well-characterized model of adoptive experimental autoimmune encephalomyelitis (EAE). We found that the pronounced and significant neuroprotective effects of systemic treatment with ERα ligand on clinical function, CNS inflammation, and axonal loss during EAE were completely prevented by conditional deletion of ERα from astrocytes, whereas conditional deletion of ERα from neurons had no significant effect. These findings show that signaling through ERα in astrocytes, but not through ERα in neurons, is essential for the beneficial effects of ERα ligand in EAE. Our findings reveal a unique cellular mechanism for estrogen-mediated CNS neuroprotective effects by signaling through astrocytes, and have implications for understanding the pathophysiology of sex hormone effects in diverse CNS disorders.

Association of simian virus 40 vp1 with 70-kilodalton heat shock proteins and viral tumor antigens.

Proper folding of newly synthesized viral proteins in the cytoplasm is a prerequisite for the formation of infectious virions. The major capsid protein Vp1 of simian virus 40 forms a series of disulfide-linked intermediates during folding and capsid formation. In addition, we report here that Vp1 is associated with cellular chaperones (HSP70) and a cochaperone (Hsp40) which can be coimmunoprecipitated with Vp1. Studies in vitro demonstrated the ATP-dependent interaction of Vp1 and cellular chaperones. Interestingly, viral cochaperones LT and ST were essential for stable interaction of HSP70 with the core Vp1 pentamer Vp1 (22-303). LT and ST also coimmunoprecipitated with Vp1 in vivo. In addition to these identified (co)chaperones, stable, covalently modified forms of Vp1 were identified for a folding-defective double mutant, C49A-C87A, and may represent a "trapped" assembly intermediate. By a truncation of the carboxyl arm of Vp1 to prevent the Vp1 folding from proceeding beyond pentamers, we detected several apparently modified Vp1 species, some of which were absent in cells transfected with the folding-defective mutant DNA. These results suggest that transient covalent interactions with known or unknown cellular and viral proteins are important in the assembly process.

Minor capsid proteins of simian virus 40 are dispensable for nucleocapsid assembly and cell entry but are required for nuclear entry of the viral genome.

We investigated the roles of simian virus 40 capsid proteins in the viral life cycle by analyzing point mutants in Vp1 and Vp2/3, as well as a deletion mutant lacking the Vp2/3 coding sequence. The Vp1 mutants (V243E and L245E) and the Vp2/3 mutants (F157E-I158E and P164R-G165E-G166R) were previously shown to be defective in Vp1-Vp2/3 interaction and to be noninfectious or poorly infectious, respectively. Here, we show that all these point mutants form stable particles following DNA transfection into cells. The Vp2/3-mutant particles contained very low levels of Vp2/3, whereas the Vp1 mutant particles contained no detectable Vp2/3. As expected, the deletion mutant also formed particles that were noninfectious. We further characterized the two Vp1 point mutants and the deletion mutant. All three mutant particles comprised Vp1 and histone-associated viral DNA, and all were able to enter cells. However, the mutant complexes failed to associate with host importins (owing to the loss of the Vp2/3 nuclear localization signal), and the mutant viral DNAs prematurely dissociated from the Vp1s, suggesting that the nucleocapsids did not enter the nucleus. Consistently, all three mutant particles failed to express large T antigen. Together, our results demonstrate unequivocally that Vp2/3 is dispensable for the formation of nucleocapsids. Further, the nucleocapsids' ability to enter cells implies that Vp1 contains the major determinants for cell attachment and entry. We propose that the major role of Vp2/3 in infectivity is to mediate the nuclear entry of viral DNA.