Background

Optic neuritis is an acute, demyelinating neuropathy of the optic nerve often representing the first appreciable symptom of multiple sclerosis. Wallerian degeneration of irreversibly damaged optic nerve axons leads to death of retinal ganglion cells, which is the cause of permanent visual impairment. Although the specific mechanisms responsible for triggering these events are unknown, it has been suggested that a key pathological factor is the activation of immune-inflammatory processes secondary to leukocyte infiltration. However, to date, there is no conclusive evidence to support such a causal role for infiltrating peripheral immune cells in the etiopathology of optic neuritis.

Methods

To dissect the contribution of the peripheral immune-inflammatory response versus the CNS-specific inflammatory response in the development of optic neuritis, we analyzed optic nerve and retinal ganglion cells pathology in wild-type and GFAP-IκBα-dn transgenic mice, where NF-κB is selectively inactivated in astrocytes, following induction of EAE.

Results

We found that, in wild-type mice, axonal demyelination in the optic nerve occurred as early as 8 days post induction of EAE, prior to the earliest signs of leukocyte infiltration (20 days post induction). On the contrary, GFAP-IκBα-dn mice were significantly protected and showed a nearly complete prevention of axonal demyelination, as well as a drastic attenuation in retinal ganglion cell death. This correlated with a decrease in the expression of pro-inflammatory cytokines, chemokines, adhesion molecules, as well as a prevention of NAD(P)H oxidase subunit upregulation.

Conclusions

Our results provide evidence that astrocytes, not infiltrating immune cells, play a key role in the development of optic neuritis and that astrocyte-mediated neurotoxicity is dependent on activation of a transcriptional program regulated by NF-κB. Hence, interventions targeting the NF-κB transcription factor in astroglia may be of therapeutic value in the treatment of optic neuritis associated with multiple sclerosis.

Tumour necrosis factor is linked to the pathophysiology of various neurodegenerative disorders including multiple sclerosis. Tumour necrosis factor exists in two biologically active forms, soluble and transmembrane. Here we show that selective inhibition of soluble tumour necrosis factor is therapeutic in experimental autoimmune encephalomyelitis. Treatment with XPro1595, a selective soluble tumour necrosis factor blocker, improves the clinical outcome, whereas non-selective inhibition of both forms of tumour necrosis factor with etanercept does not result in protection. The therapeutic effect of XPro1595 is associated with axon preservation and improved myelin compaction, paralleled by increased expression of axon-specific molecules (e.g. neurofilament-H) and reduced expression of non-phosphorylated neurofilament-H which is associated with axon damage. XPro1595-treated mice show significant remyelination accompanied by elevated expression of myelin-specific genes and increased numbers of oligodendrocyte precursors. Immunohistochemical characterization of tumour necrosis factor receptors in the spinal cord following experimental autoimmune encephalomyelitis shows tumour necrosis factor receptor 1 expression in neurons, oligodendrocytes and astrocytes, while tumour necrosis factor receptor 2 is localized in oligodendrocytes, oligodendrocyte precursors, astrocytes and macrophages/microglia. Importantly, a similar pattern of expression is found in post-mortem spinal cord of patients affected by progressive multiple sclerosis, suggesting that pharmacological modulation of tumour necrosis factor receptor signalling may represent an important target in affecting not only the course of mouse experimental autoimmune encephalomyelitis but human multiple sclerosis as well. Collectively, our data demonstrate that selective inhibition of soluble tumour necrosis factor improves recovery following experimental autoimmune encephalomyelitis, and that signalling mediated by transmembrane tumour necrosis factor is essential for axon and myelin preservation as well as remyelination, opening the possibility of a new avenue of treatment for multiple sclerosis.

The subventricular zone (SVZ) of the mammalian forebrain is a major source of multipotent stem cells during development, and contributes to neurogenesis throughout the lifespan of the organism. Several studies described molecules regulating adult neurogenesis, however, few of them have examined neurogenesis in the early postnatal period. Adult neurogenesis is regulated in part by ephrinB3 and its receptors, so we examined the role of EphB3 on neural stem/progenitor cells (NSPCs) proliferation in early postnatal development in the SVZ. To examine NSPCs proliferation, we used BrdU incorporation in both cultured NSPCs and neonatal gene-targeted knockout mice, as well as Ki67 immunostaining in EphB3−/− mice. We observed a significant increase in proliferation in cultured NSPCs derived from EphB3−/− mice and in the SVZ of EphB3−/− mice. These studies support an anti-proliferative role for EphB3 in regulating NSPCs numbers in the developing SVZ.

Ephrins and Eph receptor(s) have recently been implicated in regulating neurogenesis in the adult subventricular zone (SVZ) and rostral migratory stream (RMS). Here, we examined the role of ephrinB3-EphB3 signaling in mediating the SVZ response to traumatic brain injury (TBI). Analysis of EphB3 expression showed co-localization with glial fibrillary acidic protein (GFAP)-positive neural stem progenitor cells (NSPCs) and doublecortin-positive neuroblasts, while ephrinB3 was expressed outside the neurogenic region. TBI resulted in a significant reduction in EphB3 expression, which coincided with enhanced NSPC survival and proliferation at 3 and 7 days post-injury. Analysis of mice lacking either ephrinB3 (ephrinB3−/−) or EphB3 (EphB3−/−) showed a significant increase in bromodeoxyuridine (BrdU) incorporation and Ki67 immunoreactivity in the SVZ. Interestingly, cell death was dissimilar between knockout mice, where cell death was reduced in EphB3−/− but increased in ephrinB3−/− mice. Lateral ventricle infusion of soluble pre-clustered ephrinB3-Fc reversed the proliferative and cell death defects in ephrinB3−/− but not EphB3−/− mice and prevented TBI-induced proliferation in wild type NSPCs. Coincidently, tumor suppressor p53 expression was increased following EphB3 stimulation and is reduced in the absence of either EphB3 or ephrinB3. Furthermore, pharmacological inhibition and siRNA knockdown of p53 attenuated ephrinB3-Fc mediated growth suppression while having no effect on cell death in cultured NSPCs. These data demonstrate that EphB3 signaling suppresses NSPC proliferation in a p53-dependent manner, induces cell death in the absence of ligand stimulation and is transiently reduced in the SVZ to initiate the expansion and survival of endogenous adult NSPCs following TBI.

There is growing evidence that astrocytes play critical roles in neuron-glial interactions at the synapse. Astrocytes are believed to regulate pre- and post-synaptic structures and functions, in part, by the release of gliotransmitters such as glutamate, ATP and D-serine; however, little is known of how neurons and astrocytes communicate to regulate these processes. Here, we investigated a family of transmembrane proteins called ephrins and Eph receptors that are expressed in the synapse and are known to regulate synaptic transmission and plasticity. In addition to their presence on CA1 hippocampal neurons, we determined that ephrins and Eph receptors are also expressed on hippocampal astrocytes. Stimulation of hippocampal astrocytes with soluble ephrinB3, known to be expressed on CA1 post-synaptic dendrites, enhanced D-serine synthesis and release in culture. Conversely, ephrinB3 had no effect on D-serine release from astrocytes deficient in EphB3 and EphA4, which are the primary receptors for ephrinB3. Eph receptors mediate this response through interactions with PICK1 and by dephosphorylating PKCα to activate the conversion of L-serine to D-serine by serine racemase. These findings are supported in vivo, where reduced D-serine levels and synaptic transmissions are observed in the absence of EphB3 and EphA4. These data support a role for ephrins and Eph receptors in regulating astrocyte gliotransmitters, which may have important implications on synaptic transmission and plasticity.

The transcription factor nuclear factor kappa B (NF-κB) is a key regulator of inflammatory processes in reactive glial cells. We utilized a transgenic mouse model (GFAP-IκBα-dn) where the classical NF-κB pathway is inactivated by overexpression of a dominant negative (dn) form of the inhibitor of kappa B (IκBα) in glial fibrillary acidic protein (GFAP) expressing cells, which include astrocytes, Schwann cells, and satellite cells of the dorsal root ganglion (DRG) and sought to determine whether glial NF-κB inhibition leads to a reduction in pain behavior and inflammation following chronic constriction injury (CCI) of the sciatic nerve. As expected, following CCI nuclear translocation, and hence activation, of NF-κB was detected only in the in the sciatic nerve of wild type (WT) mice, and not in GFAP-IκBα-dn mice, while upregulation of GFAP was observed in the in sciatic nerve and DRGs of both WT and GFAP-IκBα-dn mice, indicative of glial activation. Following CCI, mechanical and thermal hyperalgesia were reduced in GFAP-IκBα-dn mice compared to WT, as well as gene and protein expression of CCL2, CCR2 and CXCL10 in the sciatic nerve. Additionally, gene expression of TNF, CCL2, and CCR2 was reduced in the DRGs of transgenic mice compared to WT after CCI. We can therefore conclude that transgenic inhibition of NF-κB in GFAP expressing glial cells attenuated pain and inflammation after peripheral nerve injury. These findings suggest that targeting the inflammatory response in Schwann cells and satellite cells may be important in treating neuropathic pain.

Reactive astrocytes have been implicated in neuronal loss following ischemic stroke. However, the molecular mechanisms associated with this process are yet to be fully elucidated. In this work, we tested the hypothesis that astroglial NF-κB, a key regulator of inflammatory responses, is a contributor to neuronal death following ischemic injury. We compared neuronal survival in the ganglion cell layer after retinal ischemia-reperfusion in wild type and in GFAP-IκBα-dn transgenic mice, where the NF-κB classical pathway is suppressed specifically in astrocytes. The GFAP-IκBα-dn mice showed significantly increased survival of neurons in the ganglion cell layer following ischemic injury as compared to WT littermates. Neuroprotection was associated with significantly reduced expression of pro-inflammatory genes, encoding Tnf-α, Ccl2 (Mcp1), Cxcl10 (IP10), Icam1, Vcam1, several subunits of NADPH oxidase and NO synthase in the retinas of GFAP-IκBα-dn mice. These data suggest that certain NF-κB-regulated pro-inflammatory and redox-active pathways are central to glial neurotoxicity induced by ischemic injury. The inhibition of these pathways in astrocytes may represent a feasible neuroprotective strategy for retinal ischemia and stroke.

Eph receptors have been implicated in regulating a diverse array of cellular functions in the developing nervous system. Recently, Eph receptors have been shown to promote cell death in adult germinal zones; however, their mechanisms of action remain ill-defined. In this study, we demonstrate that EphA4 is a new member of the dependence receptors family, which can initiate cell death in the absence of its ligand ephrinB3. Upon removal of its ligand, EphA4 triggers cell death that is dependent on caspase activation as caspase inhibitors prevent cell death. EphA4 itself is cleaved by caspase-3-like caspase in the intracellular domain at position D773/774, which is necessary for cell death initiation as mutation of the cleavage site abolishes apoptosis. In the adult subventricular zone, abolishing ephrinB3 results in increased cell death, while the absence of EphA4 results in excessive numbers of neuroblasts. Furthermore, infusion of soluble ephrinB3 into the lateral ventricle reduced cell death, and together these results support a dependence role for EphA4 in adult neurogenesis.

Chronic spinal cord injury (SCI) results in an accelerated trajectory of several cardiovascular disease (CVD) risk factors and related aging characteristics, however the molecular mechanisms that are activated have not been explored. Adipokines and leptin signaling are known to play a critical role in neuro-endocrine regulation of energy metabolism, and are now implicated in central inflammatory processes associated with CVD. Here, we examine hypothalamic adipokine gene expression and leptin signaling in response to chronic spinal cord injury and with advanced age. We demonstrate significant changes in fasting-induced adipose factor (FIAF), resistin (Rstn), long-form leptin receptor (LepRb) and suppressor of cytokine-3 (SOCS3) gene expression following chronic SCI and with advanced age. LepRb and Jak2/stat3 signaling is significantly decreased and the leptin signaling inhibitor SOCS3 is significantly elevated with chronic SCI and advanced age. In addition, we investigate endoplasmic reticulum (ER) stress and activation of the uncoupled protein response (UPR) as a biological hallmark of leptin resistance. We observe the activation of the ER stress/UPR proteins IRE1, PERK, and eIF2alpha, demonstrating leptin resistance in chronic SCI and with advanced age. These findings provide evidence for adipokine-mediated inflammatory responses and leptin resistance as contributing to neuro-endocrine dysfunction and CVD risk following SCI and with advanced age. Understanding the underlying mechanisms contributing to SCI and age related CVD may provide insight that will help direct specific therapeutic interventions.

In the central nervous system (CNS), the transcription factor nuclear factor (NF)-κB is a key regulator of inflammation and secondary injury processes. After trauma or disease, the expression of NF-κB–dependent genes is highly activated, leading to both protective and detrimental effects on CNS recovery. We demonstrate that selective inactivation of astroglial NF-κB in transgenic mice expressing a dominant negative (dn) form of the inhibitor of κBα under the control of an astrocyte-specific promoter (glial fibrillary acidic protein [GFAP]–dn mice) leads to a dramatic improvement in functional recovery 8 wk after contusive spinal cord injury (SCI). Histologically, GFAP mice exhibit reduced lesion volume and substantially increased white matter preservation. In parallel, they show reduced expression of proinflammatory chemokines and cytokines, such as CXCL10, CCL2, and transforming growth factor–β2, and of chondroitin sulfate proteoglycans participating in the formation of the glial scar. We conclude that selective inhibition of NF-κB signaling in astrocytes results in protective effects after SCI and propose the NF-κB pathway as a possible new target for the development of therapeutic strategies for the treatment of SCI.