Seema K. Tiwari-Woodruff

Reactive Astrocytes Form Scar-Like Perivascular Barriers to Leukocytes during Adaptive Immune Inflammation of the CNS

Factors that regulate leukocyte entry and spread through CNS parenchyma during different types of CNS insults are incompletely understood. Reactive astrocytes have been implicated in restricting the spread of leukocytes from damaged into healthy parenchyma during the acute and local innate inflammatory events that follow CNS trauma, but the roles of reactive astrocytes during the chronic and widespread CNS inflammation associated with adaptive or acquired immune responses are uncertain. Here, we investigated the effects of transgenically targeted ablation of proliferating, scar-forming reactive astrocytes on the acquired immune inflammation associated with experimental autoimmune encephalitis (EAE). In wild-type mice with EAE, we found that reactive astrocytes densely surrounded perivascular clusters of leukocytes in a manner reminiscent of astrocyte scar formation after CNS trauma. Transgenically targeted ablation of proliferating astrocytes disrupted formation of these perivascular scars and was associated with a pronounced and significant increase in leukocyte entry into CNS parenchyma, including immunohistochemically identified macrophages, T lymphocytes and neutrophils. This exacerbated inflammation was associated with a substantially more severe and rapidly fulminant clinical course. These findings provide experimental evidence that reactive astrocytes form scar-like perivascular barriers that restrict the influx of leukocytes into CNS parenchyma and protect CNS function during peripherally initiated, acquired immune inflammatory responses in the CNS. The findings suggest that loss or disruption of astrocyte functions may underlie or exacerbate the inflammation and pathologies associated with autoimmune diseases of the CNS, including multiple sclerosis.

A role for sex chromosome complement in the female bias in autoimmune disease

Most autoimmune diseases are more common in women than in men. This may be caused by differences in sex hormones, sex chromosomes, or both. In this study, we determined if there was a contribution of sex chromosomes to sex differences in susceptibility to two immunologically distinct disease models, experimental autoimmune encephalomyelitis (EAE) and pristane-induced lupus. Transgenic SJL mice were created to permit a comparison between XX and XY within a common gonadal type. Mice of the XX sex chromosome complement, as compared with XY, demonstrated greater susceptibility to both EAE and lupus. This is the first evidence that the XX sex chromosome complement, as compared with XY, confers greater susceptibility to autoimmune disease.

Electrostatic interactions between transmembrane segments mediate folding of Shaker K+ channel subunits

In voltage-dependent Shaker K+ channels, charged residues E293 in transmembrane segment S2 and R365, R368, and R371 in S4 contribute significantly to the gating charge movement that accompanies activation. Using an intragenic suppression strategy, we have now probed for structural interaction between transmembrane segments S2, S3, and S4 in Shaker channels. Charge reversal mutations of E283 in S2 and K374 in S4 disrupt maturation of the protein. Maturation was specifically and efficiently rescued by second-site charge reversal mutations, indicating that electrostatic interactions exist between E283 in S2 and R368 and R371 in S4, and between K374 in S4 and E293 in S2 and D316 in S3. Rescued subunits were incorporated into functional channels, demonstrating that a native structure was restored. Our data indicate that K374 interacts with E293 and D316 within the same subunit. These electrostatic interactions mediate the proper folding of the protein and are likely to persist in the native structure. Our results raise the possibility that the S4 segment is tilted relative to S2 and S3 in the voltage-sensing domain of Shaker channels. Such an arrangement might provide solvent access to voltage-sensing residues, which we find to be highly tolerant of mutations.

Differential neuroprotective and antiinflammatory effects of estrogen receptor (ER)α and ERβ ligand treatment

Treatment with either estradiol or an estrogen receptor (ER)α 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 ERα vs. an ERβ ligand in experimental autoimmune encephalomyelitis, the multiple sclerosis model with a known pathogenic role for both inflammation and neurodegeneration. Clinically, ERα ligand treatment abrogated disease at the onset and throughout the disease course. In contrast, ERβ ligand treatment had no effect at disease onset but promoted recovery during the chronic phase of the disease. ERα ligand treatment was antiinflammatory in the systemic immune system, whereas ERβ ligand treatment was not. Also, ERα ligand treatment reduced CNS inflammation, whereas ERβ ligand treatment did not. Interestingly, treatment with either the ERα or the ERβ 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 ERβ 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 ERβ 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 ERα.

Treatment with an Estrogen Receptor α Ligand Is Neuroprotective in Experimental Autoimmune Encephalomyelitis

Multiple sclerosis is an inflammatory, neurodegenerative disease for which experimental autoimmune encephalomyelitis (EAE) is a model. Treatments with estrogens have been shown to decrease the severity of EAE through anti-inflammatory mechanisms. Here we investigated whether treatment with an estrogen receptor α (ERα) ligand could recapitulate the estrogen-mediated protection in clinical EAE. We then went on to examine both anti-inflammatory and neuroprotective mechanisms. EAE was induced in wild-type, ERα-, or ERβ-deficient mice, and each was treated with the highly selective ERα agonist, propyl pyrazole triol, to determine the effect on clinical outcomes, as well as on inflammatory and neurodegenerative changes. ERα ligand treatment ameliorated clinical disease in both wild-type and ERβ knock-out mice, but not in ERα knock-out mice, thereby demonstrating that the ERα ligand maintained ERα selectivity in vivo during disease. ERα ligand treatment also induced favorable changes in autoantigen-specific cytokine production in the peripheral immune system [decreased TNFα, interferon-γ, and interleukin-6, with increased interleukin-5] and decreased CNS white matter inflammation and demyelination. Interestingly, decreased neuronal staining [NeuN+ (neuronal-specific nuclear protein)/β3-tubulin+/Nissl], accompanied by increased immunolabeling of microglial/monocyte (Mac 3+) cells surrounding these abnormal neurons, was observed in gray matter of spinal cords of EAE mice at the earliest stage of clinical disease, 1–2 d after the onset of clinical signs. Treatment with either estradiol or the ERα ligand significantly reduced this gray matter pathology. In conclusion, treatment with an ERα ligand is highly selective in vivo, mediating both anti-inflammatory and neuroprotective effects in EAE.

Hippocampal CA1 atrophy and synaptic loss during experimental autoimmune encephalomyelitis, EAE

Over half of multiple sclerosis (MS) patients experience cognitive deficits, including learning and memory dysfunction, and the mechanisms underlying these deficits remain poorly understood. Neuronal injury and synaptic loss have been shown to occur within the hippocampus in other neurodegenerative disease models, and these pathologies have been correlated with cognitive impairment. Whether hippocampal abnormalities occur in MS models is unknown. Using experimental autoimmune encephalomyelitis (EAE), we evaluated hippocampal neurodegeneration and inflammation during disease. Hippocampal pathology began early in EAE disease course, and included decreases in CA1 pyramidal layer volume, loss of inhibitory interneurons and increased cell death of neurons and glia. It is interesting to note that these effects occurred in the presence of chronic microglial activation, with a relative paucity of infiltrating blood-borne immune cells. Widespread diffuse demyelination occurred in the hippocampus, but there was no significant decrease in axonal density. Furthermore, there was a significant reduction in pre-synaptic puncta and synaptic protein expression within the hippocampus, as well as impaired performance on a hippocampal-dependent spatial learning task. Our results demonstrate that neurodegenerative changes occur in the hippocampus during autoimmune-mediated demyelinating disease. This work establishes a preclinical model for assessing treatments targeted toward preventing hippocampal neuropathology and dysfunction in MS.

Oestrogen receptor β ligand: a novel treatment to enhance endogenous functional remyelination

Demyelinating diseases, such as multiple sclerosis, are characterized by inflammatory demyelination and neurodegeneration of the central nervous system. Therapeutic strategies that induce effective neuroprotection and enhance intrinsic repair mechanisms are central goals for future therapy of multiple sclerosis. Oestrogens and oestrogen receptor ligands are promising treatments to prevent multiple sclerosis-induced neurodegeneration. In the present study we investigated the capacity of oestrogen receptor β ligand treatment to affect callosal axon demyelination and stimulate endogenous myelination in chronic experimental autoimmune encephalomyelitis using electrophysiology, electron microscopy, immunohistochemistry and tract-tracing methods. Oestrogen receptor β ligand treatment of experimental autoimmune encephalomyelitis mice prevented both histopathological and functional abnormalities of callosal axons despite the presence of inflammation. Specifically, there were fewer demyelinated, damaged axons and more myelinated axons with intact nodes of Ranvier in oestrogen receptor β ligand-treated mice. In addition, oestrogen receptor β ligand treatment caused an increase in mature oligodendrocyte numbers, a significant increase in myelin sheath thickness and axon transport. Functional analysis of callosal axon conduction showed a significant improvement in compound action potential amplitudes, latency and in axon refractoriness. These findings show a direct neuroprotective effect of oestrogen receptor β ligand treatment on oligodendrocyte differentiation, myelination and axon conduction during experimental autoimmune encephalomyelitis.

Estrogen receptor β ligand therapy activates PI3K/Akt/mTOR signaling in oligodendrocytes and promotes remyelination in a mouse model of multiple sclerosis.

The identification of a drug that stimulates endogenous myelination and spares axon degeneration during multiple sclerosis (MS) could potentially reduce the rate of disease progression. Using experimental autoimmune encephalomyelitis (EAE), a mouse model of MS, we have previously shown that prophylactic administration of the estrogen receptor (ER) β ligand 2,3-bis(4-hydroxyphenyl)-propionitrile (DPN) decreases clinical disease, is neuroprotective, stimulates endogenous myelination, and improves axon conduction without altering peripheral cytokine production or reducing central nervous system (CNS) inflammation. Here, we assessed the effects of therapeutic DPN treatment during peak EAE disease, which represents a more clinically relevant treatment paradigm. In addition, we investigated the mechanism of action of DPN treatment-induced recovery during EAE. Given that prophylactic and therapeutic treatments with DPN during EAE improved remyelination-induced axon conduction, and that ER (α and β) and membrane (m)ERs are present on oligodendrocyte lineage cells, a direct effect of treatment on oligodendrocytes is likely. DPN treatment of EAE animals resulted in phosphorylated ERβ and activated the phosphatidylinositol 3-kinase (PI3K)/serine-threonine-specific protein kinase (Akt)/mammalian target of rapamycin (mTOR) signaling pathway, a pathway required for oligodendrocyte survival and axon myelination. These results, along with our previous studies of prophylactic DPN treatment, make DPN and similar ERβ ligands immediate and favorable therapeutic candidates for demyelinating disease.

Functional recovery of callosal axons following demyelination: a critical window.

Axonal dysfunction as a result of persistent demyelination has been increasingly appreciated as a cause of functional deficit in demyelinating diseases such as multiple sclerosis. Therefore, it is crucial to understand the ultimate causes of ongoing axonal dysfunction and find effective measures to prevent axon loss. Our findings related to functional deficit and functional recovery of axons from a demyelinating insult are important preliminary steps towards understanding this issue. Cuprizone diet for 3-6 wks triggered extensive corpus callosum (CC) demyelination, reduced axon conduction, and resulted in loss of axon structural integrity including nodes of Ranvier. Replacing cuprizone diet with normal diet led to regeneration of myelin, but did not fully reverse the conduction and structural deficits. A shorter 1.5 wk cuprizone diet also caused demyelination of the CC, with minimal loss of axon structure and nodal organization. Switching to normal diet led to remyelination and restored callosal axon conduction to normal levels. Our findings suggest the existence of a critical window of time for remyelination, beyond which demyelinated axons become damaged beyond the point of repair and permanent functional loss follows. Moreover, initiating remyelination early within the critical period, before prolonged demyelination-induced axon damage ensues, will improve functional axon recovery and inhibit disease progression.

Estrogen treatment decreases matrix metalloproteinase (MMP)-9 in autoimmune demyelinating disease through estrogen receptor alpha (ERα)

Matrix metalloproteinases (MMPs) have a crucial function in migration of inflammatory cells into the central nervous system (CNS). Levels of MMP-9 are elevated in multiple sclerosis (MS) and predict the occurrence of new active lesions on magnetic resonance imaging (MRI). This translational study aims to determine whether in vivo treatment with the pregnancy hormone estriol affects MMP-9 levels from immune cells in patients with MS and mice with experimental autoimmune encephalomyelitis (EAE). Peripheral blood mononuclear cells (PBMCs) collected from three female MS patients treated with estriol and splenocytes from EAE mice treated with estriol, estrogen receptor (ER) α ligand, ERβ ligand or vehicle were stimulated ex vivo and analyzed for levels of MMP-9. Markers of CNS infiltration were assessed using MRI in patients and immunohistochemistry in mice. Supernatants from PBMCs obtained during estriol treatment in female MS patients showed significantly decreased MMP-9 compared with pretreatment. Decreases in MMP-9 coincided with a decrease in enhancing lesion volume on MRI. Estriol treatment of mice with EAE reduced MMP-9 in supernatants from autoantigen-stimulated splenocytes, coinciding with decreased CNS infiltration by T cells and monocytes. Experiments with selective ER ligands showed that this effect was mediated through ERα. In conclusion, estriol acting through ERα to reduce MMP-9 from immune cells is one mechanism potentially underlying the estriol-mediated reduction in enhancing lesions in MS and inflammatory lesions in EAE.