Lentiviral vector-mediated stable expression of sTNFR-Fc in human macrophage and neuronal cells as a potential therapy for neuroAIDS.

BACKGROUND: Human immunodeficiency virus type 1 (HIV-1) infection frequently causes neurologic disease, which is the result of viral replication and activation of macrophages and microglia in the CNS, and subsequent secretion of high levels of neurotoxic products, including tumor necrosis factor-α (TNF-α). We therefore hypothesized that a soluble TNF-α antagonist might have potential utility as a neuroprotective effecter molecule, and conducted proof-of-concept studies to test this hypothesis. METHODS: To develop novel therapeutics for the treatment of neuroAIDS, we constructed and characterized a soluble TNF receptor (sTNFR)-Fc fusion protein with the goal of neutralizing TNF-α, and tested the stability of expression of this gene following delivery by a lentiviral vector. RESULTS: High-titer lentiviral vectors were prepared, allowing efficient transduction of macrophage/glial and neuronal cell lines, as well as primary rat cerebellar neurons. Efficient, stable secretion of sTNFR-Fc was demonstrated in supernatants from transduced cell lines over 20 passages, using both western blot and ELISA. Biological activity of the secreted sTNFR-Fc was confirmed by TNF-specific in vitro protein binding and functional blocking assays. Finally, the secreted protein was shown to protect neuronal cells from TNF-α, HIV-1 Tat-, and gp120-mediated neurotoxicity. CONCLUSIONS: These results demonstrate that lentiviral vector mediated expression of sTNFR-Fc may have potential as a novel therapy for neuroAIDS.

Ankyrin-rich membrane spanning protein plays a critical role in nuclear factor-kappa B signaling.

Activation of nuclear factor-kappaB (NF-kappaB), a key feature of the neurotrophin signaling, has been shown to be critical for neuronal survival under pathologic settings. However, the precise mechanism by which neurotrophins activate NF-kappaB is not well understood. Here we report that the Ankyrin-rich Membrane Spanning (ARMS/Kidins220) protein, a novel transmembrane substrate of tropomyosin receptor kinase B (TrkB), plays an important role in NF-kappaB signaling elicited by brain-derived neurotrophic factor (BDNF). Accordingly, depletion of ARMS by specific RNA interference, or disruption of ARMS-TrkB interaction with expression of dominant-negative ARMS mutant, abolished BDNF-induced signaling to NF-kappaB. Our data further suggests that ARMS may promote NF-kappaB signaling via activation of mitogen-activated kinase (MAPK) and IkappaB kinase (IKK), thereby facilitating phosphorylation of RelA (major NF-kappaB subunit) at an IKK-sensitive site. The results shown here identify ARMS as a major factor that links neurotrophin signaling to NF-kappaB.

Glycogen synthase kinase 3beta-mediated apoptosis of primary cortical astrocytes involves inhibition of nuclear factor kappaB signaling.

Recent studies have revealed a positive correlation between astrocyte apoptosis and rapid disease progression in persons with neurodegenerative diseases. Glycogen synthase kinase 3beta (GSK-3beta) is a molecular regulator of cell fate in the central nervous system and a target of the phosphatidylinositol 3-kinase (PI-3K) pathway. We have therefore examined the role of the PI-3K pathway, and of GSK-3beta, in regulating astrocyte survival. Our studies indicate that inhibition of PI-3K leads to apoptosis in primary cortical astrocytes. Furthermore, overexpression of a constitutively active GSK-3beta mutant (S9A) is sufficient to cause astrocyte apoptosis, whereas an enzymatically inactive GSK-3beta mutant (K85M) has no effect. In light of reports on the interplay between GSK-3beta and nuclear factor kappaB (NF-kappaB), and because of the antiapoptotic activity of NF-kappaB, we examined the effect of GSK-3beta overexpression on NF-kappaB activation. These experiments revealed strong inhibition of NF-kappaB activation in astrocytes upon overexpression of the S9A, but not the K85M, mutant of GSK-3beta. This was accompanied by stabilization of the NF-kappaB-inhibitory protein, IkappaBalpha and down-regulation of IkappaB kinase (IKK) activity. These findings therefore implicate GSK-3beta as a regulator of NF-kappaB activation in astrocytes and suggest that the pro-apoptotic effects of GSK-3beta may be mediated at least in part through the inhibition of NF-kappaB pathway.

Neurotrophin signaling through tropomyosin receptor kinases contributes to survival and proliferation of non-Hodgkin lymphoma.

OBJECTIVE: Neurotrophin receptor signaling has been increasingly recognized as an important factor in the development and progression of a variety of malignancies. In order to analyze the potential contribution of neurotrophin signaling to lymphoma cell survival, we investigated the role of a neurotrophin axis in promoting survival and proliferation of non-Hodgkin lymphoma (NHL) cells. MATERIALS AND METHODS: The role of neurotrophins in the survival and proliferation of NHL cells was determined by exposing cells to the Trk-specific inhibitor, K252a, and then performing (3)H-thymidine incorporation and Annexin-V/propidium iodide staining. The involvement of nuclear factor-kappaB (NF-kappaB) in this process was studied using Western blot, electrophoretic mobility shift assay, and immunofluorescence assays. RESULTS: Here we demonstrate that both primary NHL cells and diffuse large B-cell lymphoma cell lines express Trk receptors and their neurotrophin ligands. Furthermore, these cells are sensitive to the Trk-specific inhibitor, K252a, as evidenced by the inhibition of proliferation and/or induction of apoptosis. Analysis of the mechanism into the effects of K252a revealed that, in the OCI-LY3 cell line, K252a induced a subnuclear distribution of NF-kappaB resulting in the sequestration of RelA in the nucleolus, thereby inhibiting NF-kappaB-dependent gene transcription. This results in the loss of interleukin-6 production; a known survival-promoting signal for OCI-LY3, as well as many primary diffuse large B-cell lymphomas. CONCLUSION: Thus, Trk receptors represent a novel therapeutic target for the treatment of NHL.

Transposon excision from an atypical site: a mechanism of evolution of novel transposable elements.

The role of transposable elements in sculpting the genome is well appreciated but remains poorly understood. Some organisms, such as humans, do not have active transposons; however, transposable elements were presumably active in their ancestral genomes. Of specific interest is whether the DNA surrounding the sites of transposon excision become recombinogenic, thus bringing about homologous recombination. Previous studies in maize and Drosophila have provided conflicting evidence on whether transposon excision is correlated with homologous recombination. Here we take advantage of an atypical Dissociation (Ds) element, a maize transposon that can be mobilized by the Ac transposase gene in Arabidopsis thaliana, to address questions on the mechanism of Ds excision. This atypical Ds element contains an adjacent 598 base pairs (bp) inverted repeat; the element was allowed to excise by the introduction of an unlinked Ac transposase source through mating. Footprints at the excision site suggest a micro-homology mediated non-homologous end joining reminiscent of V(D)J recombination involving the formation of intra-helix 3' to 5' trans-esterification as an intermediate, a mechanism consistent with previous observations in maize, Antirrhinum and in certain insects. The proposed mechanism suggests that the broken chromosome at the excision site should not allow recombinational interaction with the homologous chromosome, and that the linked inverted repeat should also be mobilizable. To test the first prediction, we measured recombination of flanking chromosomal arms selected for the excision of Ds. In congruence with the model, Ds excision did not influence crossover recombination. Furthermore, evidence for correlated movement of the adjacent inverted repeat sequence is presented; its origin and movement suggest a novel mechanism for the evolution of repeated elements. Taken together these results suggest that the movement of transposable elements themselves may not directly influence linkage. Possibility remains, however, for novel repeated DNA sequences produced as a consequence of transposon movement to influence crossover in subsequent generations.

Involvement of nonclassical MHC class Ib molecules in heat shock protein-mediated anti-tumor responses.

Nonclassical MHC class Ib (class Ib) genes are found in all jawed vertebrates, and their products are hypothesized to be indicators of intracellular stress and malignancy. They may be involved in immune recognition of classical MHC class Ia (class Ia)-low or -negative tumor cells through their interaction with T cell receptors and/or non-T cell inhibitory or triggering receptors expressed by NK cells and T cells. In the frog Xenopus, the molecular chaperone gp96 mediates a potent immune response involving antigen-specific classical class Ia-unrestricted CD8+ CTL (CCU-CTL) against a transplantable thymic tumor (15/0) that does not express class Ia molecules. We hypothesized that Xenopus nonclassical class Ib gene products (XNC) are involved in gp96-mediated CCU-CTL anti-tumor responses. To investigate the involvement of class Ib gene products in Xenopus anti-tumor responses, we generated, for the first time in ectothermic vertebrates, stable tumor transfectants expressing short hairpin RNA (shRNA) to silence either XNC directly or beta2m to prevent class Ib surface expression. Both types of 15/0 transfectants are more resistant to CCU-CTL killing, more tumorigenic and more susceptible to NK-like cell killing. This study provides in vitro and in vivo evidence of the evolutionary conservation of class Ib involvement in anti-tumor CD8+ T cell responses.

Functional synergy between CD40 ligand and HIV-1 Tat contributes to inflammation: implications in HIV type 1 dementia.

HIV type 1 (HIV-1)-associated dementia (HAD) is believed to occur due to aberrant activation of monocyte-derived macrophages and brain-resident microglial cells by viral proteins as well as by the proinflammatory mediators released by infected cells. To investigate the inflammatory aspects of the disease, we examined the levels of soluble CD40L (sCD40L) in paired samples of plasma and cerebrospinal fluid obtained from 25 HIV-infected individuals. A significantly higher level of sCD40L was detected in both cerebrospinal fluid and plasma from HIV-infected patients with cognitive impairment, compared with their nonimpaired counterparts. The contribution of sCD40L to the pathogenesis of HAD was then examined by in vitro experiments. rCD40L synergized with HIV-1 Tat to increase TNF-alpha release from primary human monocytes and microglia, in an NF-kappaB-dependent manner. The mechanistic basis for this synergism was attributed to a Tat-mediated up-regulation of CD40 in monocytes and microglia. Finally, the CD40L-mediated increase in TNF-alpha production by monocytes was shown to be biologically important; immunodepletion experiments revealed that TNF-alpha was essential for the neurotoxic effects of conditioned medium recovered from Tat/CD40L-treated monocytes. Taken together, our results show that CD40 signaling in microglia and monocytes can synergize with the effects of Tat, further amplifying inflammatory processes within the CNS and influencing neuronal survival.

Activation of the adenosine A1 receptor inhibits HIV-1 tat-induced apoptosis by reducing nuclear factor-kappaB activation and inducible nitric-oxide synthase.

Human immunodeficiency virus dementia (HIV-D) is a nonfocal central nervous system manifestation characterized by cognitive, behavioral, and motor abnormalities. The pathophysiology of neuronal damage in HIV-D includes a direct toxic effect of viral proteins on neuronal cells and an indirect effect caused by the release of inflammatory mediators and neurotoxins by activated macrophages/microglia and astrocytes, culminating into neuronal apoptosis. Previous studies have documented that the nucleoside adenosine mediates neuroprotection by activating adenosine A(1) receptor subtype (A(1)AR) linked to suppression of neuronal excitability. In this study, we show that A(1)AR activation protects against HIV-1 Tat-induced toxicity in primary cultures of rat cerebellar granule neurons and in rat pheochromocytoma (PC12) cell. In PC12 cells, HIV-1 Tat increased [Ca(2+)](i) levels, release of nitric oxide (NO), and expression of inducible nitric-oxide synthase (iNOS) and A(1)AR. Activation of A(1)AR suppressed Tat-mediated increases in [Ca(2+)](i) and NO. Furthermore, A(1)AR agonists inhibited iNOS expression in a nuclear factor-kappaB (NF-kappaB)-dependent manner. It is noteworthy that activation of the A(1)AR or inhibition of NOS protected against Tat-induced apoptosis in PC12 cells and cerebellar granule cells. Moreover, activation of the A(1)AR-inhibited Tat-induced increases in the levels of proapoptotic proteins Bax and caspase-3. Taken together, our results demonstrate that the A(1)AR protects against HIV-1 toxicity by inhibiting NF-kappaB, thereby reducing the expression of iNOS and NO radicals and neuronal apoptosis.

Human immunodeficiency virus-encoded Tat activates glycogen synthase kinase-3beta to antagonize nuclear factor-kappaB survival pathway in neurons.

The pathogenesis of human immunodeficiency virus type 1 (HIV-1)-associated dementia is mediated by neuronal dysfunction and death, brought about by the action of soluble neurotoxic factors that are released by virally infected macrophages and microglia. Paradoxically, many candidate HIV-1 neurotoxins also possess the ability to activate nuclear factor-kappa B (NF-kappaB), which has a potent pro-survival effect in primary neurons. The present study explored this conundrum and investigated why NF-kappaB might fail to protect neurons that are exposed to candidate HIV-1 neurotoxins. Here, we evaluated the ability of virus-depleted conditioned medium produced by HIV-1-infected human macrophages (HIV-MCMs) to modulate NF-kappaB activity in neurons. We demonstrated that HIV-MCMs inhibit the normal signaling pathways that lead to NF-kappaB activation in neurons. This inhibitory effect of HIV-MCM is dependent upon the presence of HIV-1 Tat, which activates glycogen synthase kinase (GSK)-3beta in neurons. Activation of GSK-3beta, in turn, results in modification of the NF-kappaB subunit RelA at serine 468, thereby regulating the physical interaction of RelA with histone deacetylase-3 corepressor molecules. Furthermore, neutralization of Tat or inhibition of GSK-3beta activity prevents neuronal apoptosis induced by HIV-MCM. We conclude that HIV-1 Tat may compromise neuronal function and fate by interfering with normal survival pathways subserved by NF-kappaB. These findings may have important therapeutic implications for the management of HIV-1-associated dementia.

Mechanism of NF-kappaB inactivation induced by survival signal withdrawal in cerebellar granule neurons.

Activity of the transcription factor nuclear factor-kappaB (NF-kappaB) has been shown to be necessary for maintaining neuronal viability. In cultured rat cerebellar granule neurons, trophic factor withdrawal induces NF-kappaB inactivation, resulting in cell death. The exact mechanism of this inactivation, however, has not been revealed. Here we report that trophic factor deprivation in cultured cerebellar granule neurons leads to a rapid and sustained increase in the level of IkappaBalpha and IkappaBbeta, the inhibitory proteins of NF-kappaB, causing prolonged NF-kappaB inactivation. Transient NF-kappaB activation resulting in new IkappaBalpha mRNA and protein synthesis gives rise to the rapid increase of IkappaBalpha level. The importance of elevated IkappaB level in neuronal apoptosis was confirmed in transfection experiments. Ectopic expression of a stabilized form of IkappaBalpha protein promoted neuronal death. Our findings suggest a novel mode of initiation of neuronal apoptosis wherein survival signal withdrawal induces NF-kappaB to lethally turn itself off.