Mechanisms of Neuroimmunity and Neurodegeneration Associated with HIV-1 Infection and AIDS
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- Kaul, M. & Lipton, S.A. Jrnl NeuroImmune Pharm (2006) 1: 138. doi:10.1007/s11481-006-9011-9
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Infection with the human immunodeficiency virus-1 (HIV-1) and acquired immunodeficiency syndrome (AIDS) are a persistent health problem worldwide. HIV-1 seems to enter the brain very soon after peripheral infection and can induce severe and debilitating neurological problems that include behavioral abnormalities, motor dysfunction, and frank dementia. Infected peripheral immune-competent cells, in particular macrophages, appear to infiltrate the CNS and provoke a neuropathological response involving all cell types in the brain. The course of HIV-1 disease is strongly influenced by viral and host factors, such as the viral strain and the response of the host's immune system. In addition, HIV-1-dependent disease processes in the periphery have a substantial effect on the pathological changes in the central nervous system (CNS), although the brain eventually harbors a distinctive viral population of its own. In the CNS, HIV-1 also incites activation of chemokine receptors, inflammatory mediators, extracellular matrix-degrading enzymes, and glutamate receptor-mediated excitotoxicity, all of which can initiate numerous downstream signaling pathways and disturb neuronal and glial function. Although there have been many major improvements in the control of viral infection in the periphery, an effective therapy for HIV-1-associated dementia (HAD) is still not available. This article addresses recently uncovered pathologic neuroimmune and degenerative mechanisms contributing to neuronal damage induced by HIV-1 and discusses experimental and potentially future therapeutic approaches.
Infection with the human immunodeficiency virus-1 (HIV-1) and acquired immunodeficiency syndrome (AIDS) are a persistent health problem worldwide. HIV-1 cannot only destroy the immune system of its host and lead to AIDS, the virus can also induce a variety of neurological problems that culminate in frank dementia. The worldwide development of HIV-related disease is alarming, with estimated numbers growing from more than 35 million existing infections in 2001 to 38 million in 2003, and more than 20 million deaths since 1981 (UNAIDS, 2004). In the absence of treatment more often than in the presence of medication, AIDS opportunistic infections may affect the central nervous system (CNS), but HIV infection itself can also induce a number of neurological syndromes (Petito et al., 1986). Furthermore, anemia in HIV-1 infection seems to be an early predictor for a high risk of neuropsychological impairment (McArthur et al., 1993). Neuropathological conditions directly triggered by HIV-1 include peripheral neuropathies, vacuolar myelopathy, and a syndrome of cognitive and motor dysfunction that has been designated HIV-associated dementia (HAD) (Kaul et al., 2001; Power et al., 2002; Gendelman et al., 2005). A mild form of HAD is termed minor cognitive motor disorder (MCMD) (Ellis et al., 1997; Kaul et al., 2001; Gendelman et al., 2005).
The contributing mechanisms of MCMD and HAD remain incompletely understood, but the discovery in the brain of cellular binding sites for HIV-1, the chemokine receptors, and recent progress in understanding neuroinflammation and neural stem cell biology keep providing new and hitherto unexpected insights (Lavi et al., 1998; Miller and Meucci, 1999; Kaul et al., 2001, 2005; Kramer-Hammerle et al., 2005; Minghetti, 2005; Gonzalez-Scarano and Martin-Garcia, 2005).
HIV-1 infection, AIDS, and HAD before and in the era of highly active antiretroviral therapy
HIV-1 productively infects macrophages and lymphocytes, first in the periphery and then in the brain, after binding of the viral envelope protein gp120 to one of several possible chemokine receptors in conjunction with CD4. Depending on the primary sequence of their gp120, different HIV-1 strains may use CCR5 (CD195) and CCR3, or CXCR4 (CD184), or a combination of these chemokine receptors to enter target cells (Dragic et al., 1996; Oberlin et al., 1996; He et al., 1997).
Interestingly, some individuals exposed to HIV-1 remain uninfected or never show seroconversion. Although one reason for this can be the absence of a functional CCR5 molecule (Liu et al., 1996), some people seem to mount an unconventional and very effective humoral immune response that includes IgA antibodies against viral glycoprotein 41 (gp41) and IgG recognizing a CD4–gp120 complex (Lopalco et al., 2005). Other individuals who become infected but remain asymptomatic in the long term and do not progress to AIDS have been found to express high levels of certain CCR5-binding β-chemokines (Paxton et al., 1996).
Unfortunately, the majority of individuals exposed to HIV-1 become infected, and the prevalence of HAD was estimated to be 20–30% in individuals with low CD4 T cell counts and advanced HIV disease (McArthur et al., 1993). Furthermore, anemia associated with HIV-1 infection happened to be an early predictor for a high risk of neuropsychological impairment (McArthur et al., 1993). The introduction of highly active antiretroviral therapy (HAART) has increased the life expectancy of individuals infected with HIV-1 and resulted in an at least temporary decrease in the incidence of HAD to as low as 10.5% (McArthur et al., 2003). However, although improvements in control of peripheral viral replication and the treatment of opportunistic infections continue to extend survival times, HAART failed to provide complete protection from HAD or to reverse the disease in most cases (Cunningham et al., 2000). In fact, HAD constitutes a significant independent risk factor for death due to AIDS, and it is assumed to be the most common cause of dementia worldwide among individuals age 40 and below (Ellis et al., 1997). This might at least be partially attributable to poor penetration into the CNS of HIV protease inhibitors and several of the nucleoside analogs; moreover, distinct viral drug resistance patterns in plasma and cerebrospinal fluid (CSF) compartments have recently been reported (Cunningham et al., 2000; Kaul et al., 2005; Kramer-Hammerle et al., 2005). Also, HIV seems to penetrate into the CNS right after infection in the periphery, and then resides primarily in perivascular macrophages and microglia (Ho et al., 1985; Gartner, 2000; Gonzalez-Scarano and Martin-Garcia, 2005); however, current therapeutic guidelines for AIDS suggest to start HAART only once the numbers of CD4+ T-cells begin to decline. Because this might occur years after peripheral infection, HAART is unlikely to prevent the entry of HIV-1 into the CNS (Kramer-Hammerle et al., 2005). Consequently, as people live longer with HIV-1 and AIDS, the prevalence of dementia might be rising, and in recent years the incidence of HAD as an AIDS-defining illness has actually increased (Lipton, 1997; Cunningham et al., 2000; Kaul et al., 2001, 2005; McArthur et al., 2003; Kramer-Hammerle et al., 2005). Furthermore, the proportion of new cases of HAD displaying a CD4 cell count greater then 200 μl−1 is growing (McArthur et al., 2003), and MCMD may be more prevalent than frank dementia in the HAART era (Ellis et al., 1997). However, since a peripheral immune response and inflammatory processes can trigger and influence the activation of all cell types in the CNS (Turrin and Rivest, 2004; Chakravarty and Herkenham, 2005), the effects of HIV-1 infection in the brain should always be considered in conjunction with systemic conditions. Altogether, a better understanding of the pathogenesis of HAD, including viral and host factors, is needed to identify additional therapeutic targets for the prevention and treatment of this neurodegenerative disease.
From neuropathology of HIV infection to development of MCMD/HAD
The pathological features characteristic for HIV infection in the brain are commonly referred to as HIV encephalitis, and include widespread reactive astrocytosis, myelin pallor, microglial nodules, activated resident microglia, multinucleated giant cells, and infiltration predominantly by monocytoid cells, including blood-derived macrophages (Petito et al., 1986). Surprisingly, measures of cognitive function do not correlate well with numbers of HIV-infected cells, multinucleated giant cells, or viral antigens in CNS tissue (Glass et al., 1995; Masliah et al., 1997). In contrast, increased numbers of microglia (Glass et al., 1995), elevated TNF-α mRNA in microglia and astrocytes (Wesselingh et al., 1997), evidence of excitotoxins (Heyes et al., 1991), decreased synaptic and dendritic density, and selective neuronal loss (Masliah et al., 1997) constitute the pathological features most closely associated with the clinical signs of HAD. Furthermore, signs of neuronal apoptosis have been linked to HAD (Petito and Roberts, 1995; Adle-Biassette et al., 1999), although this finding is not clearly associated with viral burden or a history of dementia (Adle-Biassette et al., 1999). The localization of apoptotic neurons is correlated with evidence of structural atrophy and closely associated with signs of microglial activation, especially within subcortical deep gray structures (Adle-Biassette et al., 1999).
Since the introduction of HAART, however, HIV neuropathology has been shifting. The number of opportunistic infections appears to be reduced, but somehow (surprisingly) the prevalence of HIV-associated inflammatory encephalitis seems to be higher at autopsy (Langford et al., 2003). Postmortem specimen from HIV patients who failed HAART showed even more encephalitis and severe leukencephalopathy. Another postmortem study found increased macrophage/microglia infiltration and activation in hippocampus and basal ganglia of HAART-treated patients as compared to samples from the time before HAART (Anthony et al., 2005).
Histological studies on specimens from HIV-1-infected humans found that lymphocytes and monocytes enter the brain (Petito et al., 1986). The pathophysiological relevance of CNS invading lymphocytes in HAD is not clearly established (Petito etal., 1986; Asensio and Campbell, 1999; Anthony etal., 2005). However, infiltrating lymphocytes and activated microglia in brains with HIV-1 encephalitis might show strong immunoreactivity for IL-16, a natural ligand of CD4. Because this cytokine inhibits HIV-1 propagation, lymphocytes might contribute to an innate antiviral immune response in the CNS in addition to microglia (Zhao et al., 2004). Furthermore, IL-4 and interferon (IFN)-γ, two cytokines with important roles in immune regulation, are produced by different lymphocyte populations and seem to induce in microglia a cytoprotective phenotype (Butovsky et al., 2005). In contrast, lipopolysaccharide (LPS) and aggregated β-amyloid appear to provoke a cytotoxic phenotype, perhaps comparable to what HIV-1/gp120 achieves.
The blood–brain barrier (BBB) also plays a crucial role in HIV infection of the CNS (Nottet et al., 1996; Gartner, 2000). Microglia and astrocytes produce chemokines—cell migration/chemotaxis inducing cytokines—such as monocyte chemoattractant protein (MCP)-1, which appears to regulate the migration of peripheral blood mononuclear cells through the BBB (Asensio and Campbell, 1999). In fact, a mutant MCP-1 allele that causes increased infiltration of mononuclear phagocytes into tissues was recently implicated in an increased risk of HAD (Gonzalez et al., 2002). As an alternative to entry via infected macrophages, it was suggested that the inflammatory cytokine, tumor necrosis factor-alpha (TNF-α), promotes a paracellular route for HIV-1 across the BBB (Fiala et al., 1997). It seems that a vicious cycle of immune dysregulation, inflammation, and BBB dysfunction might be required on the side of the host to achieve sufficient entry of infected or activated immune cells into the brain to cause neuronal injury (Kaul et al., 2001, 2005; Minghetti, 2005; Allan et al., 2005; Gonzalez-Scarano and Martin-Garcia, 2005). Variations in envelope protein gp120 on the part of the virus might also influence the timing and extent of events allowing viral entry into the CNS and subsequent neuronal injury (Power et al., 1998).
Chemokine receptors and cells of the immune system in HIV-1 infection and HAD
Chemokine receptors belong to the large family of seven transmembrane-spanning domain, G-protein coupled receptors, and as such can trigger multiple intracellular signaling events (Locati and Murphy, 1999). Although chemokines and their receptors were originally found to mediate leukocyte trafficking and to intimately contribute to the organization of inflammatory responses of the immune system, they are now known to contribute to far more physiological and pathological processes (Dragic et al., 1996; Oberlin etal., 1996; Locati and Murphy, 1999; Tran and Miller, 2003). Additional functions include the intricate control of organogenesis, including hematopoiesis, angiogenesis, and development of heart and brain (Ma et al., 1998; Locati and Murphy, 1999). Furthermore, chemokines and their receptors are essential for maintenance, maturation, and migration of hematopoietic and neural stem cells (Lapidot and Petit, 2002; Tran and Miller, 2003). However, the most prominent pathological function of certain chemokine receptors seems to be the mediation of HIV-1 infection (Dragic et al., 1996; Oberlin et al., 1996; Locati and Murphy, 1999).
Infection of macrophages and lymphocytes by HIV-1, systemically and in the brain, can occur after binding of the viral envelope protein gp120 to one of several possible chemokine receptors in conjunction with CD4. Depending on the exact type of gp120, different HIV-1 strains may use CCR5 (CD195) and CCR3, or CXCR4 (CD184), or a combination of these chemokine receptors to enter target cells (Dragic et al., 1996; Oberlin et al., 1996; He et al., 1997). Generally, T cells are infected by “T-tropic” viruses via the α-chemokine receptor CXCR4 and/or the β-chemokine receptor CCR5. In contrast, macrophages and microglia are infected by “M-tropic” HIV-1 primarily via CCR5 and CCR3, but the α-chemokine receptor CXCR4 may also be involved (He et al., 1997; Ohagen et al., 1999; Michael and Moore, 1999; Chen et al., 2002). The crucial role of chemokine receptors on several levels in HIV-1 disease has become increasingly obvious in recent years (Verani and Lusso, 2002). Usually, CCR5-preferring HIV-1 strains (R5-tropic) are transmitted between humans, and individuals lacking CCR5 are highly resistant to primary HIV infection (Liu et al., 1996). CXCR4-using viruses (X4-tropic) occur in about 50% of infected individuals later in the course of HIV-1 disease, and indicate progression to AIDS (Michael and Moore, 1999). X4-tropic HIV-1 strains might be inhibited in mucosal transmission as a result of the high expression of stromal cell-derived factor-1 (SDF-1, CXCL12), the natural ligand for CXCR4, in genital and rectal epithelium (Agace et al., 2000). Furthermore, the CCR5 ligands MIP-1α (CCL-3), MIP-1β (CCL-4), and RANTES (CCL-5) are prominently produced by T lymphocytes and suppress HIV-1 infection (Cocchi et al., 1995). In fact, individuals exposed to HIV-1 who remain yet uninfected or become infected but remain asymptomatic in the long term and do not progress to AIDS have been found to express high levels of the same CCR5-binding β-chemokines (Paxton et al., 1996).
The HIV coreceptors CCR5 and CXCR4, among other chemokine receptors, are also present in neurons and astrocytes (Miller and Meucci, 1999; Asensio and Campbell, 1999), although these cells are not thought to harbor productive infection. However, several in vitro studies strongly suggest that CXCR4 is directly involved in HIV-associated neuronal damage, whereas CCR5 may additionally play a protective role (Hesselgesser et al., 1998; Meucci et al., 1998; Kaul and Lipton, 1999; Meucci et al., 2000).
In cerebrocortical neurons and neuronal cell lines from humans and rodents, picomolar concentrations of HIV-1 gp120, as well as intact virus, can induce neuronal death via CXCR4 and CCR5 receptors (Hesselgesser et al., 1998; Meucci et al., 1998; Kaul and Lipton, 1999; Ohagen et al., 1999; Chen et al., 2002; Garden et al., 2004).
Using mixed neuronal/glial cerebrocortical cultures from rat and mouse, we have further investigated the role of chemokine receptors in the neurotoxicity of gp120. We found that gp120 from CXCR4 (X4)-preferring as well as CCR5 (R5)-preferring and dual tropic HIV-1 strains were all able to trigger neuronal death. Although gp120 from one out of two X4-preferring HIV-1 strains no longer showed neurotoxicity in CXCR4-deficient cerebrocortical cultures, dual tropic gp120SF2 showed surprisingly even greater neurotoxicity in CCR5 knockout cultures compared to wild-type or CXCR4-deficient cultures (Kaul et al., 2005). These findings are consistent with a primarily neurotoxic effect of CXCR4 activation by gp120. In contrast, activation of CCR5 might at least in part be neuroprotective depending on the HIV-1 strain from which a given gp120 originated. Furthermore, we observed earlier that the CCR5 ligands MIP-1β and RANTES protect neurons against gp120-induced toxicity (Kaul and Lipton, 1999).
In mixed neuronal/glial cerebrocortical cultures that mimic the cellular composition of the intact brain, HIV/gp120-induced apoptotic death appears to be predominantly mediated via the release of microglial toxins rather than by direct neuronal damage (Brenneman et al., 1988; Giulian et al., 1990; Kaul and Lipton, 1999; Chen et al., 2002; Garden et al., 2004). However, nanomolar concentrations of SDF-1α/β interacting with CXCR4 can induce apoptotic death of neurons independent of microglial activation, suggesting a possible direct interaction with neurons while interaction with astrocytes can also occur (Kaul and Lipton, 1999; Zheng et al., 1999; Bezzi et al., 2001). In contrast to these findings, it has been reported that SDF-1α can also provide neuroprotection from X4-preferring gp120-induced damage of isolated hippocampal or cortical neurons and mixed glial/neuronal cerebrocortical cells (Meucci et al., 1998, 2000; Khan et al., 2005). Although the reasons for the apparently contradictory findings remain to be elucidated, SDF-1 was found to activate Akt and MAPKs (Khan et al., 2004) and regulate the expression and localization of cell cycle proteins (Khan et al., 2003, 2005). It increased the acetylation of p53 and p21 as well as the expression of retinoblastoma protein (Rb) while reducing the amount of phosphorylated Rb in the nucleus. Together with a reduction of the activity of the transcription factor E2F1, an overall antiapoptotic effect was observed. The envelope protein of HIV-1IIIB, however, induced an opposite effect to SDF-1 in the nucleus, triggered the activation of Apaf-1, and promoted cell death. In human neurons, CXCR4 has been reported to mediate the toxic effect of gp120, a process involving the synthesis of ceramide and NADPH-dependent production of superoxide radicals (Jana and Pahan, 2004). Besides these in vitro findings, one group has observed deviations from the normal expression pattern of the same cell cycle proteins in postmortem brains derived from nonhuman primates with SIV encephalitis and humans with HIV encephalitis (Jordan-Sciutto et al., 2002). Interestingly, these changes to cell cycle proteins correlated with the presence of activated microglia and macrophages.
Because in vitro inhibition of microglial activation is sufficient to prevent neuronal death after gp120 exposure, it seems likely that stimulation of CXCR4 in macrophages/microglia is a prerequisite for the neurotoxicity of gp120 (Kaul and Lipton, 1999; Ohagen et al., 1999). In contrast, SDF-1 might directly activate CXCR4 in astrocytes and neurons to trigger neuronal death, for example, by reversing glutamate uptake in astrocytes (Hesselgesser et al., 1998; Kaul and Lipton, 1999; Kaul et al., 2001; Bezzi et al., 2001). SDF-1 is produced by astrocytes, macrophages, neurons, and Schwann cells (Zheng et al., 1999; McGrath et al., 1999). An increase in SDF-1 mRNA has been detected in HIV encephalitis (Zheng et al., 1999; Asensio and Campbell, 1999), and protein expression of SDF-1 also appears to be elevated in the brains of HIV patients (Langford et al., 2002). To what degree the increased expression of SDF-1 aggravates neuronal damage by HIV-1 remains to be shown. We had previously reported that intact SDF-1 can be toxic to mature neurons in a CXCR4-dependent manner, at least in culture (Kaul and Lipton, 1999; Zheng et al., 1999; Kaul et al., 2005). Additionally, it was recently reported that cleavage of SDF-1 by matrix metalloproteinases (MMPs) may contribute to neuronal injury and thus HAD via a non-CXCR4-mediated mechanism (Zhang et al., 2003). Importantly, increased expression and activation of MMPs, including MMP-2 and MMP-9, were detected in HIV-infected macrophages and also in postmortem brain specimens from AIDS patients compared with uninfected controls (Johnston et al., 2000). As elegantly demonstrated by Power and colleagues, MMP-2 released from HIV-infected macrophages is able to proteolytically remove four amino acids from the N terminus of SDF-1. The truncated α-chemokine is an even more powerful neurotoxin than full-length SDF-1, but it no longer binds CXCR4 (Zhang et al., 2003).
Transgenic mice (tg) expressing HIV-1/gp120 in their CNS manifest neuropathological features that are similar to the findings in brains of AIDS patients, such as reactive astrocytosis, increased number and activation of microglia, reduction of synaptodendritic complexity, loss of large pyramidal neurons (Toggas et al., 1994), and induction of MMP-2 (Marshall et al., 1998). In addition, these gp120 tg mice display significant behavioral deficits, such as extended escape latency, and reduced swimming velocity and spatial retention (D'hooge et al., 1999). These findings are in line with the hypothesis that the HIV-1 surface glycoprotein and engagement of the host's viral receptors may well be sufficient to initiate neuronal injury and behavioral alterations.
Effect of chemokines and HIV/gp120 on neural stem and progenitor cells
CXCR4 and its ligand SDF-1 are important components in the physiological functions of hematopoietic and neural stem cells (Asensio and Campbell, 1999; Tran and Miller, 2003). This indicates that HIV-1 could also directly interfere with the biological functions of the neural stem and progenitor cells.
In cultures of primary mouse and human neural progenitor cells obtained during the fetal period, cells stain positively for the neural stem cell marker nestin and readily undergo cell division. After several rounds of proliferation, the progenitors exit the cell cycle and express neuronal markers such as βIII-tubulin (TuJ1). Our immunocytochemical studies showed that the progenitors are positive for CXCR4 and CCR5. Treatment with HIV-1/gp120 reduced the number of progenitors and differentiating neurons. Accounting for these observations, we found that gp120 inhibited the proliferation of neural progenitor cells without producing apoptosis. The resulting decrease in neural stem cell proliferation engendered by gp120 also means that there are fewer progenitor cells present to differentiate in neurons, thus impairing neurogenesis (Okamoto, McKercher, Kaul, Lipton, unpublished data). These findings were recently complemented and extended by other groups using commercially generated human neural progenitor cells (Krathwohl and Kaiser, 2004a,b). In those experiments, chemokines promoted the quiescence and survival of human neural progenitor cells via stimulation of CXCR4 and CCR3 through a mechanism that involves down-regulation of extracellularly regulated kinase-1 and -2 (ERK-1/2) and simultaneous up-regulation of the neuronal glycoprotein reelin (Krathwohl and Kaiser, 2004a). Exposure to HIV-1 caused quiescence of neural progenitors, also through the engagement of CXCR4 and CCR3. The coat protein HIV-1/gp120 reportedly down-regulated ERK-1/2 but had no effect on Reelin (Krathwohl and Kaiser, 2004b). The effects of both the chemokines and HIV-1/gp120 were reversible and could be inhibited with recombinant Apolipoprotein E3 (ApoE3), but not ApoE4. Although it is widely accepted that HIV-1 fails to productively infect neurons, it has been reported that neural progenitor cells are permissive to the virus (Mattson et al., 2005). The recent findings that HIV-1/gp120 can interfere with the normal function of neural progenitor cells suggested the possibility that HAD might develop not only as a consequence of injury and death of existing neurons, but also due to viral disturbance of potential repair mechanisms in the CNS (Fig. 1B).
Mechanisms of neuronal injury and death in HIV-1 infection and HAD
Despite the progress being made in uncovering the pathologic processes, how exactly HIV infection results in neuronal injury as well as neurocognitive and motor impairment remains a controversial topic (Kaul et al., 2005; Kramer-Hammerle et al., 2005; Mattson et al., 2005; Gonzalez-Scarano and Martin-Garcia, 2005). Although there is a general agreement that HIV does not infect neurons, the primary cause of the neuronal damage remains in question. There is evidence to support multiple hypotheses for neuronal injury by various viral proteins—including Tat, Nef, Vpr, and the Env proteins gp120 and gp41 (Brenneman et al., 1988; Adamson et al., 1996; New et al., 1997; Piller et al., 1998; Koedel et al., 1999; Kaul et al., 2001, 2005; Mattson et al., 2005). These findings have led to at least two different hypotheses on how HIV results in neuronal injury in the brain. The hypotheses can be described as the “direct injury” hypothesis and the “indirect” or “bystander effect” hypothesis. These two hypotheses are not mutually exclusive, and the available data support a role for both, although an indirect form of neurotoxicity seems to predominate (Gartner, 2000; Kaul et al., 2001, 2005; Mattson et al., 2005; Gonzalez-Scarano and Martin-Garcia, 2005).
The hypothesis that HIV proteins can directly injure neurons without requiring the intermediary function of nonneuronal cells (microglia and/or astrocytes) is supported by experiments showing that viral envelope proteins are toxic in serum-free primary neuronal cultures (Meucci et al., 1998, 2000) and in neuroblastoma cell lines (Hesselgesser et al., 1998). In these experimental paradigms, the impact of neurotoxic cytokines and EAAs secreted from nonneuronal cells is minimized because serum-free neuronal cultures contain few, if any, nonneuronal cells, and neuroblastoma lines do not contain cells of other phenotypes. The HIV coat protein gp120 interacts with several members of the chemokine receptor family (see above), and the direct form of HIV-induced neuronal injury may be mediated by chemokine receptor signaling. Indeed, experiments aimed at blocking chemokine receptor signaling can, in some cases, prevent HIV/gp120-induced neuronal apoptosis (Meucci et al., 1998, 2000; Kaul and Lipton, 1999; Zheng et al., 1999). Additionally, nanomolar concentrations of gp120 have been reported to interact with the glycine binding site of the N-methyl-d-aspartate-type glutamate receptor (NMDAR) (Fontana et al., 1997), suggesting another mechanism by which HIV/gp120 may have a direct effect on neuronal cell death. HIV-protein Tat (HIV/Tat) can be taken up into PC12 cells by a receptor-mediated mechanism (Liu et al., 2000) and may also have a direct effect on neurons by potentiating the response to excitotoxic stimuli (reviewed in Mattson et al., 2005). Experiments using cultured hippocampal neurons revealed that the HIV protein Vpr (HIV/Vpr) may be directly neurotoxic through the formation of a cation-permeable channel (Piller et al., 1998). However, interpretation of most of these in vitro findings must account for the fact that the experimental results were obtained in the absence of nonneuronal cells and therefore a predominantly indirect effect would not be detected. Although the absence of nonneuronal cells allows the study of potential direct effects of viral proteins on neurons, the pathophysiological relevance of these results remain unclear because neurons in the brain never encounter the potential toxins in the absence of glial cells.
Apoptotic neurons do not colocalize with infected microglia in HAD patients (Ohagen et al., 1999; Adle-Biassette et al., 1999), supporting the hypothesis that HIV infection causes neurodegeneration through the release of soluble factors. Therefore, the propensity for cell–cell interactions mandates that disease pathogenesis in vitro be approached in a “mixed” neuronal/glial primary culture system that recapitulates the type and proportion of cells normally found in the intact brain (Fig. 1). Systems designed to study the effect of soluble factors released from microglia have included mixed cerebrocortical cultures from human fetal brain directly infected with HIV (Ohagen et al., 1999), severe combined immunodeficiency (SCID) mice inoculated with HIV-infected human monocytes (Xiong et al., 2000), gp120 transgenic mice (Toggas et al., 1996), and mixed rodent cerebrocortical cultures exposed to picomolar concentrations of the envelope protein HIV/gp120 (Brenneman et al., 1988; Dreyer et al., 1990; Giulian et al., 1993; Meucci and Miller, 1996; Kaul and Lipton, 1999).
Using such in vitro and in vivo models, we and others have found evidence for a predominantly indirect neurotoxic effect that occurs as a result of the response of nonneuronal cells to HIV infection or shed HIV proteins, as previously described. Much of the data supporting the hypothesis of indirect neuronal injury stems from experiments designed to examine the toxicity of HIV envelope proteins or supernatants of infected macrophages (Dreyer et al., 1990; Giulian et al., 1990). Picomolar concentrations of HIV/gp120 induce injury and apoptosis in primary rodent and human neurons (Brenneman et al., 1988; Dreyer et al., 1990; Zheng et al., 1999; Ohagen et al., 1999). In our hands, the predominant mode of HIV/gp120 neurotoxicity to cerebrocortical neurons requires the presence of macrophages/microglia (Giulian et al., 1990; Kaul and Lipton, 1999; Kaul et al., 2001). Indeed, HIV-1-infected or gp120-stimulated mononuclear phagocytes release neurotoxins that stimulate the NMDAR, as described earlier. NMDAR antagonists can ameliorate neuronal cell death in vitro because of HIV-infected macrophages or purified recombinant gp120 (Dreyer et al., 1990; Chen et al., 2002), and in vivo in gp120 transgenic mice (Toggas et al., 1996).
Excessive stimulation of the NMDAR induces several detrimental intracellular signals that contribute to neuronal cell injury and subsequent death by apoptosis or necrosis, depending on the intensity of the initialinsult (Nicotera et al., 1997). If the initial excitotoxic insult is fulminant, for example, in the ischemic core of a stroke, the cells die early from loss of ionic homeostasis, resulting in acute swelling and lysis (necrosis). If the insult is more mild, as seen in severalneurodegenerative disorders including HAD, neurons enter a delayed death pathway known as apoptosis (Nicotera et al., 1997). Neuronal apoptosis after excitotoxic insult involves Ca2+ overload, activation of p38 MAP kinase and p53, release of cytochrome c and other molecules such as apoptosis-inducing factor (AIF) from mitochondria, and activation of caspases, free radical formation, lipid peroxidation, and chromatin condensation (Tenneti et al., 1998; Asensio and Campbell, 1999; Garden et al., 2002, 2004). Activated caspase-3 and p53 are prominently detected in neurons of brains from HAD patients, and, in vitro, p53 is indispensable in neurons (and microglia) for HIV-1/gp120 to cause neurotoxicity (Garden et al., 2002, 2004). It has been suggested that CXCR4 and p53 are connected through signaling pathways mediating toxic or protective mechanisms depending on whether gp120 or SDF-1 acts as the ligand (Khan et al., 2005).
Excessive intracellular Ca2+ overstimulates nNOS and protein kinase cascades with consequent generation of deleterious levels of free radicals, including reactive oxygen species (ROS) and nitric oxide (NO) (Nicotera et al., 1997). NO can react with ROS to form cytotoxic peroxynitrite (ONOO−) (Nicotera et al., 1997). Oxidativeprocesses and cellular distress are also reflected by alterations to the cellular lipid metabolism, and an increase in ceramide, sphingomyelin, and hydroxynonenal has been implicated in the neurotoxic pathways associated with HAD (Mattson et al., 2005).
In addition to the intracellular effects of NO and oxidative stress, we have recently identified an extracellular proteolytic pathway to neuronal injury mediated by these effectors. In this pathway, S-nitrosylation (transfer of NO to a critical cysteine thiol group) and subsequent oxidation serve to activate MMP-9 and possibly other MMPs (Gu et al., 2002). Proteolytically active MMP-9 induces and promotes neuronal death presumably by disrupting the cellular mechanism(s) that allow essential attachment to the extracellular matrix and neighboring neurons.
In addition to chemokines, MMPs, and EAAs, HIV-infected or gp120-activated microglia also release inflammatory cytokines, including TNF-α and IL-1β (Wesselingh et al., 1997; Persidsky et al., 1997). Among other actions, both of these cytokines stimulate the release of l-cysteine from macrophages, and pharmacologic blockade of IL-1β or antibody neutralization of TNF-α prevents this release (Yeh et al., 2000). Under physiological or pathophysiological conditions, l-cysteine can stimulate NMDARs and lead to neuronal apoptosis (Yeh et al., 2000). TNF-α is capable of stimulating apoptosis in human neurons (Talley et al., 1995), but an indirect route of injury cannot be excluded, such as auto- or paracrine inflammatory stimulation of macrophages and microglia to produce neurotoxins. Expression of TNF-α and its receptor are elevated in the brain of patients with HAD (Wesselingh et al., 1997). Experiments aimed at addressing the question of interactions between neurotoxins associated with HAD revealed that TNF-α and HIV/Tat synergize to promote neuronal death, and this effect is prevented by antioxidants (Ohagen etal., 1999). It remains possible that TNF-α can activate caspases within neurons via TNF-α receptor-1 (TNFR1), because TNFR1 is found on at least some neurons, and it can trigger caspase-8 activation. Indeed, we have found that antibody neutralization of TNF-α or inhibition of caspase-8 prevents the neurotoxicity of HIV/gp120 in cultured cerebrocortical neurons (Garden et al., 2002); and caspase-8 activity can directly or indirectly activate caspase-3, leading to apoptosis. Another member of this enzyme family, caspase-1, converts inactive pro-IL-1β into mature, active IL-1β, thereby generating a factor with a complex role in potential promotion and limitation of neuroinflammation and neuronal injury (Allan et al., 2005). These findings suggest that inflammatory cytokines, including TNF-α and IL-1β, may have important regulatory roles in HIV-associated neuropathology (Talley et al., 1995; Yeh et al., 2000; Kaul et al., 2001, 2005; Kramer-Hammerle et al., 2005; Allan et al., 2005; Gonzalez-Scarano and Martin-Garcia, 2005).
Transgenic (tg) mice expressing HIV-1/gp120 in their CNS manifest neuropathological features that are similar to the findings in brains of AIDS patients, including reactive astrocytosis, increased number and activation of microglia, reduction of synaptodendritic complexity, loss of large pyramidal neurons (Toggas et al., 1994), and induction of MMP-2 (Marshall et al., 1998). In addition, these gp120 tg mice display significant behavioral deficits, such as extended escape latency, and reduced swimming velocity and spatial retention (D'hooge et al., 1999). In gp120 tg mice, neuronal damage is ameliorated by the NMDAR antagonist memantine (Toggas et al., 1996). Memantine-treated gp120 tg and non-tg control mice retain a density of presynaptic terminals and dendrites that is similar to untreated non-tg/wild-type controls but significantly higher than in untreated gp120 tg animals (Toggas et al., 1996). This finding confirms the hypothesis that the HIV-1 surface glycoprotein is sufficient to initiate excitotoxic neuronal injury and death. It also shows that an antagonist of NMDAR overstimulation can ameliorate HIV-associated neuronal damage in vivo (Dreyer et al., 1990; Toggas et al., 1996).
Experimental and potential approaches for prevention or therapy of HAD
An effective pharmacotherapy for HAD is not presently available. Previous approaches to cope with HAD reflect the challenging complexity inherent in the treatment of patients with AIDS (reviewed by Melton et al., 1997 and Turchan et al., 2003). Previous and current therapeutic approaches include various antiretroviral compounds, alone or in combination, such as Zidovudine, Didanosine, Zalcitabine, and Stavudine. Of these, only Zidovudine has been found to cross the blood–brain barrier to a certain extent. Although Zidovudine has a beneficial effect on HAD, the effect is not lasting. The other antiretroviral drugs may not penetrate the brain sufficiently to eradicate the virus in the CNS. Thus, an adjunctive treatment besides antiretroviral drugs is needed.
Based on the evolving pathogenesis of HAD described above, several potential therapeutic strategies to attenuate neuronal damage are worth exploring. Among others, agents warranting consideration include NMDAR blockers, cytokines, chemokines, chemokine and cytokine receptor antagonists, p38 MAPK inhibitors, caspase inhibitors, and antioxidants (free radical scavengers or other inhibitors of excessive nitric oxide or reactive oxygen species).
NMDAR antagonists have been shown to attenuate neuronal damage due to either HIV-infected macrophages or HIV/gp120, both in vitro and in vivo. The open-channel blocker, memantine, prevents excessive NMDAR activity while sparing physiological function (Lipton, 1997). Also, unlike other NMDAR antagonists tested in clinical trials to date, memantine has proven both safe and effective in a number of phase-III clinical trials for Alzheimer's disease and vascular dementia. The results of a large, multicenter NIH-sponsored clinical trial using this agent in patients with HAD has suggested some benefit, and improved second-generation drugs are currently under development. Previous, small clinical trials of a voltage-activated calcium channel blocker, nimodipine, and a PAF inhibitor suggested some therapeutic benefit, but were not conclusive (Turchan et al., 2003). An additional clinical trial using the antioxidant drug selegiline is aimed at combating the effects of excitotoxicity by minimizing the impact of free radicals (Turchan et al., 2003).
Mood changes reaching the level of disorders are one of many problems associated with HIV-1 disease. Thus, lithium has been suggested as a treatment for HAD because it also affects the cytoprotective phosphoinositol-3 kinase (PI3K)/Akt (protein kinase B)/GSK-3β pathway (Everall et al., 2002).
Previously, we have shown that the cytokine erythropoietin (EPO) may not only be effective in treating anemia but also for protecting neurons, because it prevents NMDAR-mediated and HIV-1/gp120-induced neuronal death in mixed cerebrocortical cultures (Digicaylioglu et al., 2004). Because EPO is already clinically approved for the treatment of anemia, human trials of EPO as a neuroprotectant from HIV-associated dementia may be expedited (Digicaylioglu et al., 2004).
Chemokine receptors allow HIV-1 to enter cells and as such are major potential therapeutic targets in the fight against AIDS (Michael and Moore, 1999). Antagonists of CXCR4 and CCR5 inhibit HIV-1 entry and are being assessed in clinical trials (Michael and Moore, 1999). However, the benefit of inhibitors of chemokine receptors for HIV-associated neurological complications, although likely, remains to be shown (Gartner, 2000; Kaul et al., 2001, 2005). Interestingly, as alluded to above, certain chemokines have been shown to protect neurons, even though the virus does not productively infect neurons. In particular, β-chemokines (acting on CCR5 receptors) and fractalkine prevent gp120-induced neuronal apoptosis in vitro (Kaul and Lipton, 1999; Bruno et al., 2000; Meucci etal., 2000), and, similarly, some β-chemokines can ameliorate NMDAR-mediated neurotoxicity (Bruno et al., 2000). Additionally, HIV-infected patients with higher CSF concentrations of the β-chemokines MIP-1α/β and RANTES performed better on neuropsychological measures then those with low or undetectable levels (Letendre et al., 1999). These findings support the hypothesis that selected β-chemokines may represent a potential treatment modality for HAD.
Neuronal apoptosis appears to be one of the hallmarks of neurodegenerative diseases including HAD (Adle-Biassette et al., 1999). Because caspases carry out the apoptotic program, caspase inhibitors may be helpful in preventing detrimental neuronal loss (Garden et al., 2002). As detailed above, caspases have been implicated in HIV-related neuronal damage. However, caspase inhibitors are not currently available in a form deliverable to the CNS or targeted to degenerating neurons. Although further advances in the caspase field might eventually produce such drugs, care must be exerted to avoid inhibitors that disturb physiologic turnover of cells or even promote oncogenic processes.
Finally, the pharmaceutical industry is currently developing p38 inhibitors for a variety of inflammatory- and stress-related conditions, such as arthritis, and this may expedite trials for CNS indications including HAD. The rationale is that p38 MAPK inhibitors have been shown to reduce or abrogate neuronal apoptosis due to excitotoxicity, HIV/gp120 exposure, or α-chemokine (SDF-1) toxicity (Kaul and Lipton, 1999).
The most recent experimental evidence regarding the pathologic mechanism of HAD indicates that synergy between excitatory and inflammatory pathways to neuronal injury and death may, at least in part, be shared with other CNS disorders including stroke, spinal cord injury, and Alzheimer's disease. Development of new therapeutic strategies for HAD will therefore likely impact several other neurodegenerative diseases and possibly vice versa.
M.K. and S.A.L. are supported by the National Institutes of Health, R01 NS050621 (to M.K.), P01 HD029587, R01 EY09024, R01 NS046994, R01 EY05477, and R01 NS41207 (to S.A.L.). S.A.L. is/has been a consultant to Allergan, Alcon, Forest Laboratories, NeuroMolecular Pharmaceuticals, Inc., and Neurobiological Technologies, Inc. in the field of neuroprotective agents.