The transcription factor NF-B plays an important role in both physiological and pathological events in the central nervous system. Nevertheless, the mechanisms of NF-B-mediated regulation of gene expression, and the signaling molecules participating in the NF-B pathway in the central nervous system are, to date, poorly understood. To identify such molecules, we conducted a yeast two-hybrid screen of a human brain cDNA library using NIK as bait. As a result, we identified a novel NIK and IKK␤ binding protein designated NIBP that is mainly expressed in brain, muscle, heart, and kidney. Interestingly, low levels of expression were detected in immune tissues such as spleen, thymus, and peripheral blood leukocytes, where NF-B is known to modulate immune function. We demonstrated by immunohistochemistry that NIBP expression in the brain is localized to neurons. NIBP physically interacts with NIK, IKK␤, but not IKK␣ or IKK␥. NIBP overexpression potentiates tumor necrosis factor-␣-induced NF-B activation through increased phosphorylation of the IKK complex and its downstream IB␣ and p65 substrates. Finally, knockdown of NIBP expression by small interfering RNA reduces tumor necrosis factor-␣-induced NF-B activation, prevents nerve growth factor-induced neuronal differentiation, and decreases Bcl-xL gene expression in PC12 cells. Our data demonstrate that NIBP, by interacting with NIK and IKK␤, is a new enhancer of the cytokine-induced NF-B signaling pathway. Because of its neuronal expression, we propose that NIBP may be a potential target for modulating the NF-B signaling cascade in neuronal pathologies dependent upon abnormal activation of this pathway.

The extent of damage following spinal cord injury (SCI) can be reduced by various neuroprotective regimens that include maintaining levels of cyclic adenosine monophosphate (cyclic AMP), via administration of the phosphodiesterase 4 (PDE4) inhibitor Rolipram. The current study sought to determine the optimal neuroprotective dose, route and therapeutic window for Rolipram following contusive SCI in rat as well as its prominent PDE target and putative mechanism of protection. Rolipram or vehicle control (10% ethanol) was given subcutaneously (s.c.) daily for 2 wk post-injury (PI) after which the preservation of oligodendrocytes, neurons and central myelinated axons was stereologically assessed. Doses of 0.1 mg/kg to 1.0 mg/kg (given at 1 h PI) increased neuronal survival; 0.5 mg to 1.0 mg/kg protected oligodendrocytes and 1.0 mg/kg produced optimal preservation of central myelinated axons. Ethanol also demonstrated significant neuronal and oligo-protection; though the preservation provided was significantly less than Rolipram. Subsequent use of this optimal Rolipram dose, 1.0 mg/kg, via different routes (i.v., s.c. or oral, 1 h PI), demonstrated that i.v. administration produced the most significant and consistent cyto- and axo- protection, although all routes were effective. Examination of the therapeutic window for i.v. Rolipram (1.0 mg/kg), when initiated between 1 and 48 h after SCI, revealed maximal neuroprotection at 2 h post-SCI, although the protective efficacy of Rolipram could still be observed when administration was delayed for up to 48 h PI. Importantly, use of the optimal Rolipram regimen significantly improved locomotor function after SCI as measured by the BBB score. Lastly we show SCI-induced changes in PDE4A, B and D expression and phosphorylation as well as cytokine expression and immune cell infiltration. We demonstrate that Rolipram abrogates SCI-induced PDE4B1 and PDE4A5 production, PDE4A5 phosphorylation, MCP-1 expression and immune cell infiltration, while preventing post-injury reductions in IL-10. This work supports the use of Rolipram as an acute neuroprotectant following SCI and defines an optimal administration protocol and target for its therapeutic application.

Schwann cell (SC) implantation alone has been shown to promote the growth of propriospinal and sensory axons, but not long-tract descending axons, after thoracic spinal cord injury (SCI). In the current study, we examined if an axotomy close to the cell body of origin (so as to enhance the intrinsic growth response) could permit supraspinal axons to grow onto SC grafts. Adult female Fischer rats received a severe (C5) cervical contusion (1.1 mm displacement, 3 KDyn). At 1 week postinjury, 2 million SCs ex vivo transduced with lentiviral vector encoding enhanced green fluorescent protein (EGFP) were implanted within media into the injury epicenter; injury-only animals served as controls. Animals were tested weekly using the BBB score for 7 weeks postimplantation and received at end point tests for upper body strength: self-supported forelimb hanging, forearm grip force, and the incline plane. Following behavioral assessment, animals were anterogradely traced bilaterally from the reticular formation using BDA-Texas Red. Stereological quantification revealed a twofold increase in the numbers of preserved NeuN+ neurons rostral and caudal to the injury/graft site in SC implanted animals, corroborating previous reports of their neuroprotective efficacy. Examination of labeled reticulospinal axon growth revealed that while rarely an axon was present within the lesion site of injury-only controls, numerous reticulospinal axons had penetrated the SC implant/lesion milieu. This has not been observed following implantation of SCs alone into the injured thoracic spinal cord. Significant behavioral improvements over injury-only controls in upper limb strength, including an enhanced grip strength (a 296% increase) and an increased self-supported forelimb hanging, accompanied SC-mediated neuroprotection and reticulospinal axon growth. The current study further supports the neuroprotective efficacy of SC implants after SCI and demonstrates that SCs alone are capable of supporting modest supraspinal axon growth when the site of axon injury is closer to the cell body of the axotomized neuron.Key words: Axon regeneration; Axotomy; Cell body response; Intrinsic; Neuron; Neuroprotection; Supraspinal INTRODUCTIONapies aimed at the promotion of axon growth (59,86,134,142), remyelination (17,22,64,71,73) or neuroreplacement (37,126,137,150). Among the most successful The spinal cord is a critical communication pathway for facilitating the bilateral transmission of sensory and strategies for SCI repair are those that involve exogenous cell implantation (105,106), often when combined motor modalities between the brain and the periphery. Injury to this structure often results in permanent paralywith additional pharmacological or molecular therapies (105,106). sis and lifelong disability (92). Treatments to target spinal cord injury (SCI) in experimental models have fo-The implantation of peripheral nerve grafts into the spinal cord was first used to demonstrate that, contrary cused on (i) protecting the spinal cord from secondary injury t...

The mechanism of SCI induced oligodendrocyte apoptosis is poorly understood. Tumor necrosis factor alpha (TNF-α) is a pleiotropic cytokine that is involved in inflammatory mediated cell death and thought to play a pivotal role in secondary pathology after SCI, including oligodendrocyte cell death. Utilizing a contusive SCI model in rat, we characterized the spatial, temporal, and cellular profiles of tnf-α expression and production acutely post-injury. Temporally, tnf-α mRNA, as assessed by RT-PCR, increased within 1 h and remained elevated through 24 h, albeit to a much lesser extent. In situ hybridization performed at 1 h revealed tnf-α mRNA throughout a cross-sectional area of the spinal cord. Though the tnf-α mRNA was diffusely distributed in the white matter, it was predominantly expressed in the gray matter. On the other hand, TNF-α protein increased by 1 h post injury and peaked at 4 h as measured by ELISA. At 1 h, 4 h, or 24 h, TNF-α immunoreactivity was present both within and adjacent to the injury epicenter; in the ventral gray matter and throughout the majority of the white matter. Using a cell specific marker (CC1) we found that oligodendrocytes were the principle TNF-α immunoreactive cell type after SCI as analyzed by stereological methods. These oligodendrocytes were also TUNEL positive, suggesting that TNF-α might be cytotoxic to oligodendrocytes acutely following cervical SCI.