TNF-alpha-induced neuroregeneration through an NF-kappaB-dependent pathway: a new mechanism involving EphB2 in the context of HIV-1 neuroinflammation

The use of highly active antiretroviral therapy (HAART) has significantly decreased the mortality rate of HIV-1 patients, however the increased survival has led to the development of complications associated with the persistence of the viral infection. Nearly half of HIV-1-infected individuals develop HIV-associated neurocognitive disorders (HAND) as the effects of the chronic infection leads to neuronal injury and synaptic loss in the central nervous system (CNS). The neurotoxicity of HIV-1 has largely been attributed to the inflammation caused by viral replication and the altered signaling of astrocytes, microglia, and macrophages. Although HAART has improved the control of viral replication, the effects from inflammation remain a concern, particularly those of the pro-inflammatory cytokine, tumor necrosis factor alpha (TNF-alpha).;TNF-alpha signaling has largely been considered to be neurotoxic but has been able to regulate neurite outgrowth in the context of neural development. Since TNF-alpha is upregulated in various neurodegenerative conditions, we considered potential outcomes of TNF-alpha on neurite outgrowth following injury. Initially, most would assume that TNF-alpha would prevent neurite outgrowth as apoptosis is a common outcome of TNF-alpha-induced signaling. If TNF-alpha signaling strictly prevents neurite outgrowth, anti-TNF-alpha therapies could be considered to reverse this effect. Anti-TNF-alpha therapies have been considered for HIV-1-infected patients to reduce the chronic inflammation, however inhibiting TNF-alpha signaling could have side-effects that could prevent neuronal recovery from HIV-1 effects.;Targeting pathways downstream of TNF-alpha signaling would be more advantageous to mediate the beneficial role of TNF-alpha in the CNS. We investigated the transcriptional effects of TNF-alpha treatment on neurons to uncover a potential pathway to promote neurite outgrowth. One pathway we have discovered to be beneficial in primary human fetal neurons is TNF-alpha-induced Ephrin B2 upregulation. Ephrin B2 (EphB2) receptors are important mediators of neuronal development and synaptic plasticity, however little has been established in regards to their role in HIV and inflammation, particularly in the CNS. EphB2 can mediate axonal development by providing retractive cues to assist the axon to reach the target, but EphB2 can also promote dendritic branching to improve learning and memory, which would be particularly beneficial for HAND patients that experience cognitive deficits. We observed a correlation between the upregulation of EphB2 in response to TNF-alpha and neurite outgrowth, which provides a potential pathway to repair damaged neurons and re-establish lost neuronal connections. Dendritic pruning and neuronal loss has been observed in HAND patients, so this ability to promote repair could prevent, improve, or recover the cognitive deficits experienced by HIV-patients with HAND. TNF-alpha, although primarily known to induce neurotoxicity, strongly activates the nuclear factor-kappaB (NF-kappaB) pathway, which can have a very wide range of transcriptional effects. Therefore, our hypothesis is that the TNF-alpha-induced neurite regrowth occurs through an upregulation EphB2 in an NF-kappaB-dependent pathway.;TNF-alpha has been well established to induce NF-kappaB signaling, mostly by promoting the translocation of the NF-kappaB p65 DNA binding factor to the nucleus for transcriptional regulatory effects. NF-kappaB can regulate neuronal growth and process development of both dendrites and axons, which would correlate to the neurite regrowth observed following TNF-alpha upon induced injury. The regulation of EphB2 by NF-kappaB has not been extensively studied, but EphB2 can be negatively regulated by an NF-kappaB family member, c-Rel. We analyzed the EphB2 promoter and identified three NF-kappaB p65 binding sites upstream from the transcriptional start site, which provided insight to our hypothesis. We established that p65 directly binds to and can regulate EphB2 promoter activity in response to TNF-alpha. Since the dual role of TNF-alpha can be dependent on the receptor through which the signaling proceeds, either TNF-alpha receptor 1 (TNFR1) or TNF-alpha receptor 1 (TNFR2), we investigated if this upregulation of EphB2 is receptor dependent and determined EphB2 is induced primarily through activation of TNFR2.;Taken together, our studies characterize a novel mechanism for neurite outgrowth following injury in neurons: TNF-alpha/TNFR2-induced EphB2 signaling in an NF-kappaB p65-dependent manner. In addition to the established mechanism, we developed a technique to assess the effects of EphB2 knockout and overexpression in the context of neurite outgrowth: EphB2-targeted-Cas9n and EphB2 inducible construct. The strategies created provide a valuable toolset to demonstrate the direct effects of modulating EphB2 signaling, not only in neurons for effects on neuronal health and synaptic plasticity, but also in other disease models, such as glioblastoma, in which EphB2 was demonstrated to promote invasion and migration of tumor cells. These observations and the usefulness of the modulatory strategies likely extend to multiple neurodegenerative diseases that demonstrate cognitive deficits that correlate to neuroinflammation. (Abstract shortened by ProQuest.).