Recent studies have revealed a positive correlation between astrocyte apoptosis and rapid disease progression in persons with neurodegenerative diseases. Glycogen synthase kinase 3␤ (GSK-3␤) 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-3␤, 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-3␤ mutant (S9A) is sufficient to cause astrocyte apoptosis, whereas an enzymatically inactive GSK-3␤ mutant (K85M) has no effect. In light of reports on the interplay between GSK-3␤ and nuclear factor B (NF-B), and because of the antiapoptotic activity of NF-B, we examined the effect of GSK-3␤ overexpression on NF-B activation. These experiments revealed strong inhibition of NF-B activation in astrocytes upon overexpression of the S9A, but not the K85M, mutant of GSK-3␤. This was accompanied by stabilization of the NF-B-inhibitory protein, IB␣ and down-regulation of IB kinase (IKK) activity. These findings therefore implicate GSK-3␤ as a regulator of NF-B activation in astrocytes and suggest that the pro-apoptotic effects of GSK-3␤ may be mediated at least in part through the inhibition of NF-B pathway.

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-α release from primary human monocytes and microglia, in an NF-κB-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-α production by monocytes was shown to be biologically important; immunodepletion experiments revealed that TNF-α 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.

The HIV-1 gene products Tat and gp120 are toxic to neurons and can activate cells of myeloid origin, properties that are thought to contribute to the clinical manifestations of HIV-1-associated dementia (HAD). To investigate the intracellular signaling mechanisms involved in these events, the effect of Tat and gp120 on mixed lineage kinase (MLK) 3 activation was examined. Tat and gp120 were shown to induce autophosphorylation of MLK3 in primary rat neurons; this was abolished by the addition of an inhibitor of MLK3 (CEP1347). CEP1347 also enhanced survival of both rat and human neurons and inhibited the activation of human monocytes after exposure to Tat and gp120. Furthermore, overexpression of wild-type MLK3 led to the induction of neuronal death, whereas expression of a dominant negative MLK3 mutant protected neurons from the toxic effects of Tat. MLK3-dependent downstream signaling events were implicated in the neuroprotective and monocyte-deactivating pathways triggered by CEP1347. Thus, the inhibition of p38 MAPK and JNK protected neurons from Tat-induced apoptosis, whereas the inhibition of p38 MAPK, but not of JNK, was sufficient to prevent Tat- and gp120-mediated activation of monocytes. These results suggest that the normal function of MLK3 is compromised by HIV-1 neurotoxins (Tat, gp120), resulting in the activation of downstream signaling events that result in neuronal death and monocyte activation (with release of inflammatory cytokines). In aggregate, our data define MLK3 as a promising therapeutic target for intervention in HAD.

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.

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 β2m 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.