MECHANISMS OF NEURONAL APOPTOSIS
Apoptosis may occur via two major pathways, the “extrinsic pathway” induced by the ligation of certain transmembrane proteins with cytoplasmic death domains and the “intrinsic pathway” resulting from depolarization of mitochondrial membranes and the release of cytochrome c from the mitochondria.
Both pathways involve the activation of cellular caspases, and both are in no way mutually exclusive. These pathways are well defined in neurons and seem to be perturbed in several neurodegenerative diseases, including Alzheimer’s disease, amyotrophic lateral sclerosis, and stroke (reviewed in Friedlander160).Extrinsic Pathway
The binding of TNF-α to TNF-α receptor-1 (TNFR1) activates TNFR1-associated death domain protein (TRADD), which, in turn, interacts with Fas-associated death domain protein (FADD) to induce apoptosis.161 The finding that TNFR1 is expressed on some neurons, coupled with the fact that HIV-1-infected and gp120-stimulated microglia release TNF-α, suggests a role for TNF-α- mediated apoptosis of neurons in HIV-1 infection. Expression of TNF-α and its receptor is elevated in brains from patients with HAD,162 and TNF-α is capable of stimulating apoptosis in human neurons.163 TNF-α was also implicated in mediating gp120-induced apoptosis of sensory neurons.164 In this study, gp120 ligation of CXCR4 on Schwann cells induced their production of RANTES, which, in turn, induced TNF-α production by DRG neurons to mediate neurotoxicity in an autocrine manner.
In contrast to the evidence presented above, TNF-α was shown to have a neuroprotective role. Pretreatment of hippocampal, cortical, or septal neurons with TNF-α was shown to protect from glucose-deprivation neurotoxicity by maintaining Ca2+ homeostasis.165 The neuroprotection afforded by TNF-α seems to be mediated through its activation of the transcription factor NF-κB and the subsequent upregulation of antiapoptotic proteins (reviewed in Saha and Pahan166).
In particular, TNF-α was shown to induce the expression of the chemokine CX3CL1 (fractalkine) in astrocytes,167 which, in turn, protects neurons from HIV-1 gp120-mediated toxicity.168 It is thought that the timing and the duration of exposure to TNF-α are important factors in determining its neurotoxic or neuroprotective role. For example, TNF-α was shown to be neuroprotective when applied before ischemic stress, but it becomes neurotoxic when applied after the same insult.169 In addition, the role of TNF-α may be affected by the expression of other elements within the diseased brain. For example, in combination with IL-1β, TNF-α induces expression of iNOS in glial cells.170 In contrast, when given alone, TNF-α has limited effect on the expression of the same gene.171Two recent studies suggest the contribution of TNF-related apoptosis-inducing ligand (TRAIL) expressed on HIV-1-infected macrophages to neuronal apoptosis.172,173 TRAIL is a member of the TNF superfamily of proteins, showing homology to TNF and Fas ligand (CD95L). TRAIL is capable of inducing apoptosis in target cells by binding to receptors that contain conserved death domains (e.g., TRAIL-R1 and TRAIL-R2). Although originally thought to induce apoptosis only in tumor or transformed cells, TRAIL was later shown to induce apoptosis in virally infected cells. TRAIL mediates activation-induced cell death in peripheral blood mononuclear cells (PBMCs) from HIV- 1-infected individuals,174 and lymphocytes from HIV-1-infected patients seem to be more sensitive to TRAIL-induced apoptosis compared to uninfected controls.175,176 Furthermore, large numbers of HIV-1-uninfected CD4+ T cells undergo TRAIL-mediated apoptosis in HIV-infected lymphoid organs.177 In addition to secreting neurotoxic agents, HIV-1-exposed macrophages were recently shown to induce neuronal apoptosis via a TRAIL-mediated process.172,173 TRAIL-R1 and TRAIL-R2 were detected in both human fetal neuron cultures and on neurons in brain tissue from HIVE patients.172 In agreement with earlier studies,178,179 Ryan et al.
demonstrated that exposure of human macrophages to HIV-1 resulted in their increased expression of TRAIL.172 Although not previously detected in normal brain tissue,180 TRAIL-expressing macrophages were detected in brain tissue from HIVE patients and were spatially associated with caspase-3-expressing neurons. In a series of in vitro experiments using human fetal neuron cultures, Ryan et al. demonstrated that recombinant human TRAIL protein-induced apoptosis could be prevented by inhibition of either caspase-8 or caspase-9, suggesting that both the extrinsic pathway and amplification of this by the intrinsic pathway were required for this process.172 Similar observations were seen in a murine model in which human PBMCs were transplanted into nonobese diabetic severe combined immunodeficiency mice.173 HIV-1 infection in combination with LPS administration induced infiltration of infected human cells into the perivascular region of the brain and neuronal apoptosis, which frequently colocalized with HIV-1-infected macrophages expressing TRAIL. In both studies, the specificity of TRAIL-induced apoptosis was demonstrated using a neutralizing antibody against human TRAIL.A role for the Fas/Fas ligand system in mediating neuronal apoptosis in HIV-1 infection remains to be determined. Aquaro et al. demonstrated that anti-Fas antibody could reverse the programmed cell death observed in astrocytes exposed to supernatants from HIV-infected mac- rophages,181 and elevated levels of Fas and Fas ligand were reported in the brains of HAD patients.182 However, further studies are required to elucidate the extent to which this system may play a role in HAD.
Intrinsic Pathway
Dysregulation of neuronal cell Ca2+ homeostasis may play a central role in both HIV-1 gp120 and Tat-induced apoptosis of neurons through the intrinsic pathway. As discussed previously, HIV-1 gp120-induced neurotoxicity may occur through a direct action of neurons or through an indirect mechanism involving toxic products released from gp120-stimulated microglia and macrophages. One such toxic product is arachidonic acid, which facilitates NMDA-evoked currents in neurons183 and stimulates glutamate release from astrocytes184 while simultaneously inhibiting its uptake by glial cells.184-186 Thus, the net result is a hyperactivation of NMDA receptors, resulting in potentially lethal levels of Ca2+ influx.
In support, blockade of NMDA receptors but not non-NMDA receptors was shown to inhibit HIV-1 gp120-induced neurotoxicity.187 HIV-1 gp120 also stimulates Na+∕H+ exchange in astrocytes, resulting in increased intracellular pH.188 Intracellular alkalinization can, in turn, activate ion-motive transporters, resulting in increased extracellular K+ levels, inhibition of glutamate uptake, enhanced glutamate release, and depolarization of neuronal membranes,189 thus again priming NMDA receptors on neurons for hyperactivation by an increased pool of extracellular glutamate. An additional mechanism of HIV-1 gp120-induced neurotoxicity is the direct binding of the viral protein to CXCR4 expressed on neurons. Recently, Bachis et al. demonstrated that HIV-1 gp120-mediated apoptosis of cerebellar granule cells could be prevented by the CXCR4 inhibitor AMD3100 but not by the glutamate receptor antagonist MK801, suggesting that in their system, the toxic mechanism of gp120 primarily involves activation of the CXCR4 receptor.190 CXCR4 is a G protein-coupled receptor that, upon ligand activation, triggers a transient increase in cytosolic free Ca2+,104 suggesting again that dysregulation of Ca2+ homeostasis may be a key event in HIV-1 gp120-induced neurotoxicity. HIV-1 Tat protein interacts directly with neurons, inducing phosphatidylinositol 3-kinase activity,191 increased IP3 levels, and subsequent release of Ca2+ from IP3-sensitive ER stores.192 This ability of HIV-1 Tat to induce IP3-sensitive release of Ca2+ in neurons led to the speculation that exogenous HIV-1 Tat interacts with CXCR4, a G protein-linked receptor that is coupled to inositol hydrolysis and IP3-mediated Ca2+ release. Indeed, neurons express CXCR4, and interaction of HIV-1 Tat with CXCR4 was demonstrated in human PBMCs,193 though this remains to be demonstrated in neurons. Inhibition of IP3-mediated Ca2+ release from the ER was shown to inhibit Tat-induced neurotoxicity, underscoring the importance of inositol signaling in this process.192 In addition, HIV-1 Tat induces Ca2+ influx from the extracellular environment through at least four types of neuronal calcium channels, including L- and N-type voltage-gated channels, and the NMDA and non-NMDA ionotropic glutamate receptors.194-198 The elevation of cytosolic Ca2+ is an early event in the neuronal apoptosis cascade and seems to trigger mitochondrial calcium uptake, production of reactive oxygen species, and mitochondrial stress, resulting in mitochondrial membrane depolarization and subsequent release of apoptotic factors.199,200Experimental data from both in vitro and in vivo studies from the Bagetta laboratory led to the suggestion that an underlying mechanism of neuronal cell death triggered by HIV-1 gp120 involves the chemokine receptor-mediated enhancement of brain IL-1β and subsequent enhancement of cyclooxygenase (COX-2) expression.201 Intracerebroventricular (i.c.v.) injection of recombinant HIV-1 gp120 IIIB protein in adult rats induced apoptosis of neurons in the brain neocortex, which was preceded by enhanced IL-1β expression.202 Double-labeling immunofluorescence experiments established that the main sources of gp120-enhanced IL-1β expression in the rat neocortex were neurons and microglia.203 Subchronic i.c.v. administration of recombinant IL-1β induced apoptosis in the neocortex of rat brain, whereas combined treatment with HIV-1 gp120 and an inhibitor of caspase-1 (the enzyme that converts pro-IL-1β into biologically active IL-1β) resulted in a reduction in neuronal cell death compared with animals treated with HIV-1 gp120 alone, implicating a role for IL-1β in neocortical cell death. Pretreatment with a nonneurotoxic dose of SDF-1 reduced both gp120-induced IL-1β production and neuronal apoptosis, implicating the role of CXCR4 receptor stimulation in mediating gp120 neurotoxicity.202 The role of IL-1β in mediating neurotoxicity induced by HIV-1 gp120 is also supported by in vitro data, when exposure of human CHP100 neuroblastoma cells to the viral protein enhanced IL-1β secretion and cell death.204 Again, inhibition of IL-1β production and blockade of the IL-1β receptor both protected the neuroblastoma cells from HIV-1 gp120-induced cell death.
In addition to IL-1β, i.c.v. injection of HIV-1 gp120 caused early enhancement of functionally active COX-2 expression in the neocortex of rat, resulting in increased prostaglandin E2 (PGE2) production.205,206 HIV-1 gp120 induced accumulation of PGE2 in human CHP100 neuroblastoma cells by enhancing the COX activity of the arachidonate-metab- olizing enzyme prostaglandin H synthase, whereas inhibitors of this process provided protection against the cell death triggered by HIV-1 gp120.207 Interestingly, inhibitors of IL-1β production also prevented the enhanced COX-2 expression otherwise induced by HIV-1 gp120, suggesting that IL-1β may be the signal by which the viral protein enhanced COX-2 expression.204 Products of the arachidonic cascade, especially PGE2, stimulate the release of glutamate from astrocytes in a Camdependent manner.208 Thus, it was suggested201 that HIV-1 gp120, through an IL-1β-dependent mechanism, induces COX-2 expression in neuronal cells to convert arachidonic acid into PGE2, which accumulates. Elevated PGE2 levels, in turn, increase synaptic glutamate levels by stimulating its release from astrocyes, triggering neuronal cell oxidative stress and apoptotic death through an excitotoxic pathway involving stimulation of the NMDA receptor.