Shaffiat Karmally

Inhibition of astroglial nuclear factor κB reduces inflammation and improves functional recovery after spinal cord injury

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.

Changes in GAD- and GABA- immunoreactivity in the spinal dorsal horn after peripheral nerve injury and promotion of recovery by lumbar transplant of immortalized serotonergic precursors.

We have utilized RN46A cells, an immortalized neuronal cell line derived from E13 brainstem raphe, as a model for transplant of bioengineered serotonergic cells. RN46A cells require brain-derived neurotrophic factor (BDNF) for increased survival and serotonin (5HT) synthesis in vitro and in vivo. RN46A cells were transfected with the rat BDNF gene, and the 46A-B14 cell line was subcloned. These cells survive longer than 7 weeks after transplantation into the subarachnoid space of the lumbar spinal cord and synthesize 5HT and BDNF. Chronic constriction injury (CCI) of the sciatic nerve was used to induce chronic neuropathic pain in the affected hindpaw in rats. Transplants of 46A-B14 cells placed 1 week after CCI alleviated chronic neuropathic pain, while transplants of 46A-V1 control cells, negative for 5HT and without the BDNF gene, had no effect on the induction of thermal and tactile nociception. When endogenous cells of the dorsal horn which contain the neurotransmitter gamma-aminobutyric acid (GABA) and its synthetic enzyme glutamate decarboxylase (GAD) were immunohistochemically quantified in the lumbar spinal cord 3 days and 1-8 weeks after CCI, the number of GABA- and GAD-immunoreactive (ir) cells decreased bilateral to the nerve injury as soon as 3 days after CCI. At 1 week after CCI, the number of GABA-ir cells continued to significantly decline bilaterally, returning to near normal numbers on the side contralateral to the nerve injury by 8 weeks after the nerve injury. The number of GAD-ir cells began to increase bilaterally to the nerve injury at 1 week after CCI and continued to significantly increase in numbers over normal values by 8 weeks after the nerve injury. When examined 2 and 8 weeks after CCI plus cell transplants, the transplants of 46A-B14 cells reversed the increase in GAD-ir cell numbers and the decrease in GABA-ir cells by 1 week after transplantation, while 46A-V1 control cell transplants after CCI had no effect on the changes in numbers of GAD-ir or GABA-ir cells. Collectively, these data suggest that altered 5HT levels, and perhaps BDNF secretion, related to the transplants ameliorate chronic pain and reverse the induction and maintenance of an endogenous pain mechanism in the dorsal horn. This induction mechanism is likely dependent on altered GAD regulation and GABA synthesis, initiated by CCI.

Transgenic Inhibition of Astroglial NF-κB Improves Functional Outcome in Experimental Autoimmune Encephalomyelitis by Suppressing Chronic Central Nervous System Inflammation

In the CNS, the transcription factor NF-κB is a key regulator of inflammation and secondary injury processes. Following trauma or disease, the expression of NF-κB-dependent genes is activated, leading to both protective and detrimental effects. In this study, we show that transgenic inactivation of astroglial NF-κB (glial fibrillary acidic protein-IκBα-dominant-negative mice) resulted in reduced disease severity and improved functional recovery following experimental autoimmune encephalomyelitis. At the chronic stage of the disease, transgenic mice exhibited an overall higher presence of leukocytes in spinal cord and brain, and a markedly higher percentage of CD8+CD122+ T regulatory cells compared with wild type, which correlated with the timing of clinical recovery. We also observed that expression of proinflammatory genes in both spinal cord and cerebellum was delayed and reduced, whereas the loss of neuronal-specific molecules essential for synaptic transmission was limited compared with wild-type mice. Furthermore, death of retinal ganglion cells in affected retinas was almost abolished, suggesting the activation of neuroprotective mechanisms. Our data indicate that inhibiting NF-κB in astrocytes results in neuroprotective effects following experimental autoimmune encephalomyelitis, directly implicating astrocytes in the pathophysiology of this disease.

Lumbar transplant of neurons genetically modified to secrete brain-derived neurotrophic factor attenuates allodynia and hyperalgesia after sciatic nerve constriction

&NA; Chronic delivery of anti‐nociceptive molecules by means of cell grafts near the pain processing centers of the spinal cord is a newly developing technique for the treatment of neuropathic pain. The rat neuronal cell line, RN33B, derived from E13 rat brainstem raphe and immortalized with the SV40 temperature‐sensitive allele of large T antigen (tsTag), was transfected with rat brain‐derived neurotrophic factor cDNA (BDNF), and the BDNF‐synthesizing cell line, 33BDNF.4, was isolated. The 33BDNF.4 cells synthesized mature BDNF protein at permissive temperature (33°C), when the cells were proliferating, and during differentiation at non‐permissive temperature (39°C) in vitro. The bio‐active BDNF protein was also secreted by the cells during both growth conditions, as measured by ELISA analysis of BDNF content and secretion. The bio‐activity of the BDNF in 33BDNF.4 cell conditioned media was assessed by neurite outgrowth from E15 dorsal root ganglion (DRG) cultures. A control cell line, 33V1, transfected with the vector alone, did not synthesize or secrete any significant BDNF at either growth condition. Both cell lines were used as grafts in a model of chronic neuropathic pain induced by unilateral chronic constriction injury (CCI) of the sciatic nerve. Pain‐related behaviors, including cold and tactile allodynia and thermal and tactile hyperalgesia, were evaluated after CCI in the affected hindpaw. When 33BDNF.4 and 33V1 cells were transplanted in the lumbar subarachnoid space of the spinal cord 1 week after CCI, they survived greater than 7 weeks on the pia mater around the spinal cord and the 33BDNF.4 cells continued to synthesize BDNF in vivo. Furthermore, the tactile and cold allodynia and tactile and thermal hyperalgesia induced by CCI was significantly reduced during the 2–7 week period after grafts of 33BDNF.4 cells. The maximal effect on chronic pain behaviors with the BDNF grafts occurred 2–3 weeks after transplant and the anti‐nociceptive effects of the BDNF cell grafts was permanent. Transplants of the control 33V1 cells had no effect on the allodynia and hyperalgesia induced by CCI and these cells did not synthesize BDNF in vivo. These data suggest that a chronically applied, low local dose of BDNF supplied by transplanted cells near the spinal dorsal horn was able to reverse the development of chronic neuropathic pain following CCI. The use of neural cell lines that are able to deliver anti‐nociceptive molecules, such as BDNF, in a model of chronic pain offers a novel approach to pain management and such ‘biologic minipumps’ can be developed for safe use in humans.

Inhibition of soluble tumour necrosis factor is therapeutic in experimental autoimmune encephalomyelitis and promotes axon preservation and remyelination.

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.

Transplants of neuronal cells bioengineered to synthesize GABA alleviate chronic neuropathic pain.

The use of cell lines utilized as biologic “minipumps” to provide antinociceptive molecules, such as GABA, in animal models of pain is a newly developing area in transplantation biology. The neuronal cell line, RN33B, derived from E13 brain stem raphe and immortalized with the SV40 temperature-sensitive allele of large T antigen (tsTag), was transfected with rat GAD67 cDNA (glutamate decarboxylase, the synthetic enzyme for GABA), and the GABAergic cell line, 33G10.17, was isolated. The 33G10.17 cells transfected with the GAD67 gene expressed GAD67 protein and synthesized low levels of GABA at permissive temperature (33°C), when the cells were proliferating, and increased GAD67 and GABA during differentiation at nonpermissive temperature (39°C) in vitro, because GAD67 protein expression was upregulated with differentiation. A control cell line, 33V1, transfected with the vector alone, contained no GAD67 or GABA at either temperature. These cell lines were used as grafts in a model of chronic neuropathic pain induced by unilateral chronic constriction injury (CCI) of the sciatic nerve. Pain-related behaviors, including cold and tactile allodynia and thermal and tactile hyperalgesia, were evaluated after CCI in the affected hind paw. When 33G10.17 and 33V1 cells were transplanted in the lumbar subarachnoid space of the spinal cord 1 week after CCI, they survived greater than 7 weeks on the pia mater around the spinal cord. Furthermore, the tactile and cold allodynia and tactile and thermal hyperalgesia induced by CCI was significantly reduced during the 2–7-week period after grafts of 33G10.17 cells. The maximal effect on chronic pain behaviors with the GABAergic grafts occurred 2–3 weeks after transplantation. Transplants of 33V1 control cells had no effect on the allodynia and hyperalgesia induced by CCI. These data suggest that a chronically applied, low local dose of GABA presumably supplied by transplanted cells near the spinal dorsal horn was able to reverse the development of chronic neuropathic pain following CCI. The use of neural cell lines that are able to deliver inhibitory neurotransmitters, such as GABA, in a model of chronic pain offers a novel approach to pain management.

A single intrathecal injection of GABA permanently reverses neuropathic pain after nerve injury

To investigate whether neuropathic pain is sensitive to spinal GABA levels, GABA was injected intrathecally after nerve injury and sensory behaviors were evaluated. Both thermal and tactile hypersensitivities were permanently reversed at the highest doses of GABA. However, if GABA was injected any later than 2-3 weeks after nerve injury, it was ineffective to prevent such hypersensitivity. This suggests that altered spinal GABA levels contribute to the induction phase of chronic neuropathic pain and that early intervention to restore GABA may prevent the development of that pain.

Astrocytes play a key role in EAE pathophysiology by orchestrating in the CNS the inflammatory response of resident and peripheral immune cells and by suppressing remyelination

Astrocytes respond to insult with a process of cellular activation known as reactive astrogliosis. One of the key signals regulating this phenomenon is the transcription factor nuclear factor‐kappa B (NF‐κB), which is responsible for modulating inflammation, cell survival, and cell death. In astrocytes, following trauma or disease, the expression of NF‐κB‐dependent genes is highly activated. We previously demonstrated that inactivation of astroglial NF‐κB in vivo (GFAP‐IκBα‐dn mice) leads to improved functional outcome in experimental autoimmune encephalomyelitis (EAE), and this is accompanied by reduction of pro‐inflammatory gene expression in the CNS. Here we extend our studies to show that recovery from EAE in GFAP‐IκBα‐dn mice is associated with reduction of peripheral immune cell infiltration into the CNS at the chronic phase of EAE. This is not dependent on a less permeable blood‐brain barrier, but rather on a reduced immune cell mobilization from the periphery. Furthermore, once inside the CNS, the ability of T cells to produce pro‐inflammatory cytokines is diminished during acute disease. In parallel, we found that the number of total and activated microglial cells is reduced, suggesting that functional improvement in GFAP‐IκBα‐dn mice is dependent upon reduction of the overall inflammatory response within the CNS sustained by both resident and infiltrating cells. This results in preservation of myelin compaction and enhanced remyelination, as shown by electron microscopy analysis of the spinal cord. Collectively our data indicate that astrocytes are key players in driving CNS inflammation and are directly implicated in the pathophysiology of EAE, since blocking their pro‐inflammatory capability results in protection from the disease. GLIA 2014;62:452–467

Nuclear Factor Kappa B Signaling Initiates Early Differentiation of Neural Stem Cells

Inflammatory mediators, many of which activate the signaling of nuclear factor kappa B (NFκB), have received increasing attention in the field of neurogenesis. NFκB signaling regulates neurite outgrowth and neural plasticity as well as the proliferation/apoptosis and terminal differentiation of neural stem cells (NSCs). Early neurogenesis from NSCs produces identical progeny through symmetric division and committed daughter cells through asymmetric division. Here, we show that NFκB signaling is required for NSC initial differentiation. The canonical IKKβ/IκBα/p65 pathway is activated during the initial stages of neural differentiation induced by treatment with TNFα or withdrawal of epidermal growth factor/basic fibroblast growth factor. NSC‐specific inhibition of NFκB in transgenic mice causes an accumulation of Nestin+/Sox2+/glial fibrillary acidic protein+ NSCs. Inhibition of NFκB signaling in vitro blocks differentiation and asymmetric division and maintains NSCs in an undifferentiated state. The induction of initial differentiation and asymmetry by NFκB signaling occurs through the inhibition of C/EBPβ expression. Our data reveal a novel function of NFκB signaling in early neurogenesis and provide insight into the molecular mechanisms underlying neurodevelopmental disorders and neurodegenerative diseases. STEM CELLS 2012;30:510–524

Neuronal glutamate transporter EAAT4 is expressed in astrocytes.

High‐affinity excitatory amino acid transporters (EAATs) are essential to terminate glutamatergic neurotransmission and to prevent excitotoxicity. To date, five distinct EAATs have been cloned from animal and human tissues: GLAST (EAAT1), GLT‐1 (EAAT2), EAAC1 (EAAT3), EAAT4, and EAAT5. EAAT1 and EAAT2 are commonly known as glial glutamate transporters, whereas EAAT3, EAAT4, and EAAT5 are neuronal. EAAT4 is largely expressed in cerebellar Purkinje cells. In this study, using immunohistochemistry and Western blotting, we found that EAAT4‐like immunoreactivity (ir) is enriched in the spinal cord and forebrain. Double‐labeled fluorescent immunostaining and confocal image analysis indicated that EAAT4‐like ir colocalizes with an astrocytic marker, glial fibrillary acidic protein (GFAP). The astrocytic localization of EAAT4 was further confirmed in astrocyte cultures by double‐labeled fluorescent immunocytochemistry and Western blotting. Reverse transcriptase‐polymerase chain reaction analysis demonstrated mRNA expression of EAAT4 in astrocyte cultures. Sequencing confirmed the specificity of the amplified fragment. These results demonstrate that EAAT4 is expressed in astrocytes. This astrocytic localization of neuronal EAAT4 may reveal a new function of EAAT4 in the central nervous system.