Rory D. Spence

Neuroprotection mediated through estrogen receptor-α 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.

Neuroprotective effects of estrogens and androgens in CNS inflammation and neurodegeneration

Multiple sclerosis (MS) is a disease characterized by inflammation and demyelination. Currently, the cause of MS is unknown. Experimental autoimmune encephalomyelitis (EAE) is the most common mouse model of MS. Treatments with the sex hormones, estrogens and androgens, are capable of offering disease protection during EAE and are currently being used in clinical trials of MS. Beyond endogenous estrogens and androgens, treatments with selective estrogen receptor modulators (SERMs) for estrogen receptor alpha (ERα) and estrogen receptor beta (ERβ) are also capable of providing disease protection. This protection includes, but is not limited to, prevention of clinical disease, reduction of CNS inflammation, protection against demyelination, and protection against axonal loss. In EAE, current efforts are focused on using conditional cell specific knockouts of sex hormone receptors to identify the in vivo targets of these estrogens and androgens as well as downstream molecules responsible for disease protection.

Estrogen Mediates Neuroprotection and Anti-Inflammatory Effects during EAE through ERα Signaling on Astrocytes But Not through ERβ Signaling on Astrocytes or Neurons

Estrogens can signal through either estrogen receptor α (ERα) or β (ERβ) 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 ERβ on astrocytes or neurons could mediate neuroprotection in EAE, by selectively removing ERβ from either of these cell types using Cre-loxP gene deletion. Our results demonstrated that, even though ERβ ligand treatment was neuroprotective in EAE, this neuroprotection was not mediated through ERβ 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 ERβ on astrocytes, we looked for molecules within astrocytes that were affected by signaling through ERα, but not ERβ. We found that ERα ligand treatment, but not ERβ ligand treatment, decreased expression of the chemokines CCL2 and CCL7 by astrocytes in EAE. Together, our data show that neuroprotection in EAE mediated via ERβ signaling does not require ERβ 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 ERβ in EAE.

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.

Injury-Induced Regulation of Steroidogenic Gene Expression in the Cerebellum

Sex steroids assist adult neural tissue in the protection from and repair of damage resulting from neural injury; some steroids may be synthesized in the brain. Songbirds are especially useful models to explore steroidal neuroprotection and repair. First, the full suite of cholesterol transporters and steroidogenic enzymes are expressed in the zebra finch (ZF) brain. Second, estrogens promote recovery of behavioral function after damage to the adult ZF cerebellum. Third, the estrogen synthetic enzyme aromatase is rapidly upregulated in reactive glia following neural injury, including in the ZF cerebellum. We hypothesized that cerebellar injury would locally upregulate steroidogenic factors upstream of aromatase, providing the requisite substrate for neuroestrogen synthesis. We tested this hypothesis by lesioning the cerebellum of adult songbirds using both males and females that peripherally synthesize steroids in different amounts. We then used quantitative PCR to examine expression of mRNAs for the neurosteroidogenic factors TSPO, StAR, SCC, 3β-HSD, CYP17, and aromatase, at 2 and 8 days post-lesion. Compared to sham lesions, cerebellar lesions significantly upregulated mRNA levels of TSPO and aromatase. Sex differences in response to the lesions were detected for TSPO, StAR, and aromatase. All birds responded to experimental conditions by showing time-dependent changes in the expression of TSPO, SCC, and aromatase, suggesting that acute trauma or stress may impact neurosteroidogensis for many days. These data suggest that the cerebellum is an active site of steroid synthesis in the brain, and each steroidogenic factor likely provides neuroprotection and promotes repair.

Recovery of motor and cognitive function after cerebellar lesions in a songbird – role of estrogens

In addition to its key role in complex motor function, the cerebellum is increasingly recognized to have a role in cognition. Songbirds are particularly good models for the investigation of motor and cognitive processes but little is known about the role of the songbird cerebellum in these processes. To explore cerebellar function in a songbird, we lesioned the cerebellum of adult female zebra finches and examined the effects on a spatial working memory task and on motor function during this task. There is evidence for steroid synthesis in the songbird brain and neurosteroids may have an impact on some forms of neural plasticity in adult songbirds. We therefore hypothesized that neurosteroids would affect motor and cognitive function after a cerebellar injury. We found that cerebellar lesions produced deficits in motor and cognitive aspects of a spatial task. In line with our prediction, birds in which estrogen synthesis was blocked had impaired performance in our spatial task compared with those that had estrogen synthesis blocked but estrogen replaced. There was no clear effect of estrogen replacement on motor function. We also found that lesions induced expression of the estrogen synthetic enzyme aromatase in reactive astrocytes and Bergmann glia around a cerebellar lesion. These data suggest that the cerebellum of songbirds mediates both motor and cognitive function and that estrogens may improve the recovery of cognitive aspects of cerebellar function after injury.

Dynamic Development of Glucocorticoid Resistance during Autoimmune Neuroinflammation

CONTEXT Glucocorticoids (GC) are powerful endogenous and therapeutic modulators of inflammation and play a critical role for controlling autoimmunity. GC resistance can be seen in patients with cell-mediated autoimmune disorders, but it is unknown whether this represents a stable trait or a state. OBJECTIVE The objective of the study was to determine whether GC resistance of T cell responses is dynamically regulated in experimental autoimmune encephalomyelitis (EAE) and multiple sclerosis (MS). DESIGN This was a translational observational study. PATIENTS AND ANIMALS: EAE was induced in C57BL/6 mice. A cross-sectional sample of 25 patients with relapsing-remitting MS was included as well as four MS patients during pregnancy and postpartum. MAIN OUTCOME MEASURES Outcome measures included GC sensitivity of T cell proliferation and GC-mediated apoptosis. RESULTS GC resistance was seen in both autoantigen-specific and nonspecific responses of T cells obtained from mice with EAE. GC resistance preceded clinical symptoms and central nervous system infiltration of immune cells. T cells obtained during EAE were resistant to GC-induced apoptosis, and this was linked to down-regulation of GC receptor-α expression. GC resistance in T cells was also seen in MS patients with radiological evidence for ongoing inflammation. GC resistance was absent in the MS patients during pregnancy, when relapse risk is decreased, but recurred postpartum, a time of increased relapse risk. CONCLUSIONS These data demonstrate that GC resistance during autoimmune neuroinflammation is dynamically regulated. This has implications for the timing of steroid treatments and provides a putative pathway to explain the observed association between psychological stress and exacerbation of autoimmune diseases.

Estrogen receptor-β 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‐β (ERβ) ligand is known to ameliorate clinical disease effectively and provide neuroprotection in EAE. However, the protective effects of this ERβ ligand have been demonstrated only when administered prior to disease (prophylactically). Here we tested whether ERβ 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, ERβ 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. ERβ ligand‐treated animals. Our findings show that therapeutically administered ERβ ligand successfully treats clinical EAE, bearing translational relevance to MS as a candidate neuroprotective agent.

Bringing CLARITY to gray matter atrophy

Gray matter atrophy has been shown to be a strong correlate to clinical disability in multiple sclerosis (MS) and its most commonly used animal model, experimental autoimmune encephalomyelitis (EAE). However, the relationship between gray mater atrophy and the spinal cord pathology often observed in EAE has never been established. Here EAE was induced in Thy1.1-YFP mice and their brains imaged using in vivo magnetic resonance imaging (MRI). The brains and spinal cords were subsequently optically cleared using Clear Lipid-exchanged Acrylamide-hybridized Rigid Imaging-compatible Tissue-hYdrogel (CLARITY). Axons were followed 5mm longitudinally in three dimensions in intact spinal cords revealing that 61% of the axons exhibited a mean of 22 axonal ovoids and 8% of the axons terminating in axonal end bulbs. In the cerebral cortex, we observed a decrease in the mean number of layer V pyramidal neurons and a decrease in the mean length of the apical dendrites of the remaining neurons, compared to healthy controls. MRI analysis demonstrated decreased cortical volumes in EAE. Cross-modality correlations revealed a direct relationship between cortical volume loss and axonal end bulb number in the spinal cord, but not ovoid number. This is the first report of the use of CLARITY in an animal model of disease and the first report of the use of both CLARITY and MRI.