Kurt F. Hauser

Neurotoxicity and dysfunction of dopaminergic systems associated with AIDS dementia

Infection with the human immunodefiency virus (HIV) selectively targets the basal ganglia resulting in loss of dopaminergic neurons. Although frequently asymptomatic, some patients may develop signs of dopamine deficiency de novo. Accordingly, they are highly susceptible to drugs that act on dopaminergic systems. Both neuroleptics and psychostimulants may exacerbate these symptoms. Experimental evidence suggests that viral proteins such as gp120 and Tat can cause toxicity to dopaminergic neurons, and this toxicity is synergistic with compounds such as methamphetamine and cocaine that also act on the dopaminergic system. In addition, other neurotransmitters that modulate dopaminergic function, such as glutamate and opioids, may also modify the susceptibility of the dopamine system to HIV. Therefore, a thorough understanding of the mechanisms that lead to this selective neurotoxicity of dopaminergic neurons would also likely lead to the development of therapeutic modalities for patients with HIV dementia.

Synergistic increases in intracellular Ca2+, and the release of MCP-1, RANTES, and IL-6 by astrocytes treated with opiates and HIV-1 Tat

Recent evidence suggests that injection drug users who abuse heroin are at increased risk of CNS complications from human immunodeficiency virus (HIV) infection. Opiate drugs may intrinsically alter the pathogenesis of HIV by directly modulating immune function and by directly modifying the CNS response to HIV. Despite this, the mechanisms by which opiates increase the neuropathogenesis of HIV are uncertain. In the present study, we describe the effect of morphine and the HIV‐1 protein toxin Tat1‐72 on astroglial function in cultures derived from ICR mice. Astroglia maintain the blood‐brain barrier and influence inflammatory signaling in the CNS. Astrocytes can express μ‐opioid receptors, and are likely targets for abused opiates, which preferentially activate μ‐opioid receptors. While Tat alone disrupts astrocyte function, when combined with morphine, Tat causes synergistic increases in [Ca2+]i. Moreover, astrocyte cultures treated with morphine and Tat showed exaggerated increases in chemokine release, including monocyte chemoattractant protein‐1 (MCP‐1) and regulated on activation, normal T cell expressed and secreted (RANTES), as well as interleukin‐6 (IL‐6). Morphine‐Tat interactions were prevented by the μ‐opioid receptor antagonist β‐funaltrexamine, or by immunoneutralizing Tat1‐72 or substituting a nontoxic, deletion mutant (TatΔ31‐61). Our findings suggest that opiates may increase the vulnerability of the CNS to viral entry (via recruitment of monocytes/macrophages) and ensuing HIV encephalitis by synergistically increasing MCP‐1 and RANTES release by astrocytes. The results further suggest that astrocytes are key intermediaries in opiate‐HIV interactions and disruptions in astroglial function and inflammatory signaling may contribute to an accelerated neuropathogenesis in HIV‐infected individuals who abuse opiates.

Neurotoxicity of HIV-1 proteins gp120 and Tat in the rat striatum

HIV-associated dementia complex is a serious disabling disease characterized by cognitive, behavioral and motor dysfunction. Basal ganglia involvement in HIV-1 infection may be responsible for some of the psychomotor symptoms associated with HIV dementia. The objectives of the present study were to determine: (1) whether gp120 and Tat produce striatal toxicity, and (2) whether gp120 and Tat show synergistic toxicity in the striatum. In these studies, the recombinant proteins gp120, Tat, or saline (0.9%) were stereotaxically injected in the striatum of adult male rats. The striatal sections were evaluated for area of tissue loss (Cresyl-violet stained sections) and the number of GFAP immunoreactive cells 7 days after the injections. Doses of gp120 250 ng/microl or higher and Tat 5 microg/microl or higher produced a significant area of tissue loss and significantly increased the number of GFAP reactive cells. We found no toxicity in animals treated with immunoabsorbed gp120 or Tat. Combined gp120 (100 ng/microl)+Tat (1 microg/microl) injections into the rat striatum significantly increased the area of tissue loss and altered morphology and increased number of GFAP reactive cells, as compared to controls. Thus, the present results suggest the involvement of gp120 and Tat in striatal toxicity and provide a model for further studies to fully characterize their role in HIV-1 toxicity and to develop therapeutic strategies for HIV-1 associated dementia complex.

Synergistic neurotoxicity of opioids and human immunodeficiency virus-1 Tat protein in striatal neurons in vitro

Human immunodeficiency virus (HIV) infection selectively targets the striatum, a region rich in opioid receptor-expressing neural cells, resulting in gliosis and neuronal losses. Opioids can be neuroprotective or can promote neurodegeneration. To determine whether opioids modify the response of neurons to human immunodeficiency virus type 1 (HIV-1) Tat protein-induced neurotoxicity, neural cell cultures from mouse striatum were initially characterized for mu and/or kappa opioid receptor immunoreactivity. These cultures were continuously treated with morphine, the opioid antagonist naloxone, and/or HIV-1 Tat (1-72) protein, a non-neurotoxic HIV-1 Tat deletion mutant (TatDelta31-61) protein, or immunoneutralized HIV-1 Tat (1-72) protein. Neuronal and astrocyte viability was examined by ethidium monoazide exclusion, and by apoptotic changes in nuclear heterochromatin using Hoechst 33342. Morphine (10nM, 100nM or 1microM) significantly increased Tat-induced (100 or 200nM) neuronal losses by about two-fold at 24h following exposure. The synergistic effects of morphine and Tat were prevented by naloxone (3microM), indicating the involvement of opioid receptors. Furthermore, morphine was not toxic when combined with mutant Tat or immunoneutralized Tat. Neuronal losses were accompanied by chromatin condensation and pyknosis. Astrocyte viability was unaffected. These findings demonstrate that acute opioid exposure can exacerbate the neurodegenerative effect of HIV-1 Tat protein in striatal neurons, and infer a means by which opioids may hasten the progression of HIV-associated dementia.

Endogenous opioids regulate dendritic growth and spine formation in developing rat brain.

Continuous blockade of endogenous opioid-opioid receptor interaction by opioid antagonists from birth to day 10 increased neuronal maturation in the rat brain. The lengths of oblique dendrites of pyramidal cells in the cerebral cortex and basilar dendrites of the hippocampus were increased from controls by 136 and 51%, respectively, whereas the concentrations of spines in these cells were increased 183 and 69%, respectively. Total dendritic length of spiny branches of cerebellar Purkinje neurons was 65% greater than controls, and spine concentration of granule cells in the dentate gyrus was increased by 76%. Thus, endogenous opioids exert a remarkable influence on the timetable and magnitude of dendritic elaboration and spine formation, and serve as an important trophic influence in the regulation of neuro-ontogeny.

HIV-1 Tat and opiate-induced changes in astrocytes promote chemotaxis of microglia through the expression of MCP-1 and alternative chemokines.

Opiates exacerbate human immunodeficiency virus type 1 (HIV‐1) Tat1‐72‐induced release of key proinflammatory cytokines by astrocytes, which may accelerate HIV neuropathogenesis in opiate abusers. The release of monocyte chemoattractant protein‐1 (MCP‐1, also known as CCL2), in particular, is potentiated by opiate–HIV Tat interactions in vitro. Although MCP‐1 draws monocytes/macrophages to sites of CNS infection, and activated monocytes/microglia release factors that can damage bystander neurons, the role of MCP‐1 in neuro‐acquired immunodeficiency syndrome (neuroAIDS) progression in opiate abusers, or nonabusers, is uncertain. Using a chemotaxis assay, N9 microglial cell migration was found to be significantly greater in conditioned medium from mouse striatal astrocytes exposed to morphine and/or Tat1‐72 than in vehicle‐, μ‐opioid receptor (MOR) antagonist‐, or inactive, mutant TatΔ31‐61‐treated controls. Conditioned medium from astrocytes treated with morphine and Tat caused the greatest increase in motility. The response was attenuated using conditioned medium immunoneutralized with MCP‐1 antibodies, or medium from MCP‐1−/− astrocytes. In the presence of morphine (time‐release, subcutaneous implant), intrastriatal Tat increased the proportion of neural cells that were astroglia and F4/80+ macrophages at 7 days post‐injection. This was not seen after treatment with Tat alone, or with morphine plus inactive TatΔ31‐61 or naltrexone. Glia displayed increased MOR and MCP‐1 immunoreactivity after morphine and/or Tat exposure. The findings indicate that MCP‐1 underlies most of the response of microglia, suggesting that one way in which opiates exacerbate neuroAIDS is by increasing astroglial‐derived proinflammatory chemokines at focal sites of CNS infection and promoting macrophage entry and local microglial activation. Importantly, increased glial expression of MOR can trigger an opiate‐driven amplification/positive feedback of MCP‐1 production and inflammation.

Estrogen protects against the synergistic toxicity by HIV proteins, methamphetamine and cocaine

BackgroundHuman immunodeficiency virus (HIV) infection continues to increase at alarming rates in drug abusers, especially in women. Drugs of abuse can cause long-lasting damage to the brain and HIV infection frequently leads to a dementing illness.To determine how these drugs interact with HIV to cause CNS damage, we used an in vitro human neuronal culture characterized for the presence of dopaminergic receptors, transporters and estrogen receptors. We determined the combined effects of dopaminergic drugs, methamphetamine, or cocaine with neurotoxic HIV proteins, gp120 and Tat.ResultsAcute exposure to these substances resulted in synergistic neurotoxic responses as measured by changes in mitochondrial membrane potential and neuronal cell death. Neurotoxicity occurred in a sub-population of neurons. Importantly, the presence of 17beta-estradiol prevented these synergistic neurotoxicities and the neuroprotective effects were partly mediated by estrogen receptors.ConclusionOur observations suggest that methamphetamine and cocaine may affect the course of HIV dementia, and additionally suggest that estrogens modify the HIV-drug interactions.

Opioid-dependent growth of glial cultures: Suppression of astrocyte DNA synthesis by met-enkephalin

The action of met-enkephalin on the growth of astrocytes in mixed-glial cultures was examined. Primary, mixed-glial cultures were isolated from 1 day-old mouse cerebral hemispheres and continuously treated with either basal growth media (controls), 1 microM met-enkephalin, 1 microM met-enkephalin plus the opioid antagonist naloxone (3 microM), or naloxone alone (3 microM). Absolute numbers of neural cells were counted in unstained preparations, while combined [3H]-thymidine autoradiography and glial fibrillary acid protein (GFAP) immunocytochemistry was performed to identify specific changes in astrocytes. When compared to control and naloxone treated cultures, met-enkephalin caused a significant decrease in both total cell numbers, and in [3H]-thymidine incorporation by GFAP-positive cells with flat morphology. These results indicate that met-enkephalin suppresses astrocyte growth in culture.

Apoptotic death of striatal neurons induced by human immunodeficiency virus-1 Tat and gp120: Differential involvement of caspase-3 and endonuclease G.

Human immunodeficiency virus-1 (HIV-1) infection affects the striatum, resulting in gliosis and neuronal losses. To determine whether HIV-1 proteins induce striatal neurotoxicity through an apoptotic mechanism, mouse striatal neurons isolated on embryonic day 15 and the effects of HIV-1 Tat1–72 and gp120 on survival were assessed in vitro. Mitochondrial release of cytochrome c, caspase-3 activation, and neuron survival, as well as an alternative apoptotic pathway involving endonuclease G (endo G), were assessed at 4 h, 24 h, 48 h, and/or 72 h using enzyme assays and immunoblotting. Both HIV-1 Tat and gp120 significantly increased caspase-3 activation in a concentration-dependent manner in striatal neurons at 4 h following continuous exposure in vitro. Tat1–72 and gp120 caused significant neuronal losses at 48 h and/or 72 h. Tat1–72 increased cytochrome c release, and caspase-3 and endo G activation at 4 h, 24 h, and/or 72 h. By contrast, gp120 increased caspase-3 activation, but failed to increase cytochrome c or endo G levels in the cytoplasm at 4 h, 24 h, and/or 72 h. The cell permeant caspase inhibitor Z-DEVD-FMK significantly attenuated gp120-induced, but not Tat1–72-induced, neuronal death, suggesting that gp120 acts in large part through the activation of caspase(s), whereas Tat1–72-induced neurotoxicity was accompanied by activating an alternative pathway involving endo G. Thus, although Tat1–72 and gp120 induced significant neurotoxicity, the nature of the apoptotic events preceding death differed. Collectively, our findings suggest that HIV-1 proteins are intrinsically toxic to striatal neurons and the pathogenesis is mediated through separate actions involving both caspase-3 and endo G.

μ-Opioid receptor-induced Ca2+ mobilization and astroglial development: morphine inhibits DNA synthesis and stimulates cellular hypertrophy through a Ca2+-dependent mechanism

Morphine, a preferential μ-opioid receptor agonist, alters astroglial development by inhibiting cell proliferation and by promoting cellular differentiation. Although morphine affects cellular differentiation through a Ca2+-dependent mechanism, few studies have examined whether Ca2+ mediates the effect of opioids on cell proliferation, or whether a particular Ca2+ signal transduction pathway mediates opioid actions. Moreover, it is uncertain whether one or more opioid receptor types mediates the developmental effects of opioids. To address these questions, the present study examined the role of μ-opioid receptors and Ca2+ mobilization in morphine-induced astrocyte development. Morphine (1 gmM) and non-morphine exposed cultures enriched in murine astrocytes were incubated in Ca2+-free media supplemented with < 0.005, 0.3, 1.0, or 3.0 mM Ca2+ ([Ca2+]o), or in unmodified media containing Ca2+ ionophore (A23187), nifedipine (1 μM), dantrolene (10 μM), thapsigargin (100 nM), or l-glutamate (100 μM) for 0-72 h. μ-Opioid receptor expression was examined immunocytochemically using specific (MOR1) antibodies. Intracellular Ca2+ ([Ca2+]i) was measured by microfluorometric analysis using fura-2. Astrocyte morphology and bromodeoxyuridine (BrdU) incorporation (DNA synthesis) were assessed in glial fibrillary acidic protein (GFAP) immunoreactive astrocytes. The results showed that morphine inhibited astroglial growth by activating μ-opioid receptors. Astrocytes expressed MOR1 immunoreactivity and morphines actions were mimicked by the selective μ, agonist PL017. In addition, morphine inhibited DNA synthesis by mobilizing [Ca2+]i in developing astroglia. At normal [Ca2+]o, morphine attenuated DNA synthesis by increasing [Ca2+]i; low [Ca2+]o (0.3 mM) blocked this effect, while treatment with Ca2+ ionophore or glutamate mimicked morphines actions. At extremely low [Ca2+]o (< 0.005 mM), morphine paradoxically increased BrdU incorporation. Although opioids can increase [Ca2+]i in astrocytes through several pathways, not all affect DNA synthesis or cellular morphology. Nifedipine (which blocks L-type Ca2+ channels) did not prevent morphine-induced reductions in BrdU incorporation or cellular differentiation, while thapsigargin (which depletes IP3-sensitive Ca2+ stores) severely affected inhibited DNA synthesis and cellular differentiation-irrespective of morphine treatment. However, dantrolene (an inhibitor of Ca2+-dependent Ca2+ release) selectively blocked the effects of morphine. Collectively, the findings suggest that opioids suppress astroglial DNA synthesis and promote cellular hypertrophy by inhibiting Ca2+-dependent Ca2+ release from dantrolene-sensitive intracellular stores. This implies a fundamental mechanism by which opioids affect central nervous system maturation.