Pamela E. Knapp

Activation of the caspase-3 apoptotic cascade in traumatic spinal cord injury.

Traumatic spinal cord injury often results in complete loss of voluntary motor and sensory function below the site of injury. The long-term neurological deficits after spinal cord trauma may be due in part to widespread apoptosis of neurons and oligodendroglia in regions distant from and relatively unaffected by the initial injury. The caspase family of cysteine proteases regulates the execution of the mammalian apoptotic cell death program. Caspase-3 cleaves several essential downstream substrates involved in the expression of the apoptotic phenotype in vitro, including gelsolin, PAK2, fodrin, nuclear lamins and the inhibitory subunit of DNA fragmentation factor. Caspase-3 activation in vitro can be triggered by upstream events, leading to the release of cytochrome c from the mitochondria and the subsequent transactivation of procaspase-9 by Apaf-1 (refs. 13, 14, 15). We report here that these upstream and downstream components of the caspase-3 apoptotic pathway are activated after traumatic spinal cord injury in rats, and occur early in neurons in the injury site and hours to days later in oligodendroglia adjacent to and distant from the injury site. Given these findings, targeting the upstream events of the caspase-3 cascade has therapeutic potential in the treatment of acute traumatic injury to the spinal cord.

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.

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.

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.

Anti-death properties of TNF against metabolic poisoning: mitochondrial stabilization by MnSOD

The cytokine tumor necrosis factor (TNF) is toxic to some mitotic cells, but protects cultured neurons from a variety of insults by mechanisms that are unclear. Pretreatment of neurons or astrocytes with TNF caused significant increases in MnSOD activity, and also significantly attenuated 3-nitropropionic acid (3-NP) induced superoxide accumulation and loss of mitochondrial transmembrane potential. In oligodendrocytes, however, MnSOD activity was not increased, and 3-NP toxicity was unaffected by TNF. Genetically engineered PC6 cells that overexpress MnSOD also were resistant to 3-NP-induced damage. TNF pretreatment and MnSOD overexpression prevented 3-NP induced apoptosis, and shifted the mode of death from necrosis to apoptosis in response to high levels of 3-NP. Mitochondria isolated from either MnSOD overexpressing PC6 cells or TNF-treated neurons maintained resistance to 3-NP-induced loss of transmembrane potential and calcium homeostasis, and showed attenuated release of caspase activators. Overall, these results indicate that MnSOD activity directly stabilizes mitochondrial transmembrane potential and calcium buffering ability, thereby increasing the threshold for lethal injury. Additional studies showed that levels of oxidative stress and striatal lesion size following 3-NP administration in vivo are increased in mice lacking TNF receptors.

Active, phosphorylated fingolimod inhibits histone deacetylases and facilitates fear extinction memory

FTY720 (fingolimod), an FDA-approved drug for treatment of multiple sclerosis, has beneficial effects in the CNS that are not yet well understood, independent of its effects on immune cell trafficking. We show that FTY720 enters the nucleus, where it is phosphorylated by sphingosine kinase 2 (SphK2), and that nuclear FTY720-P binds and inhibits class I histone deacetylases (HDACs), enhancing specific histone acetylations. FTY720 is also phosphorylated in mice and accumulates in the brain, including the hippocampus, inhibits HDACs and enhances histone acetylation and gene expression programs associated with memory and learning, and rescues memory deficits independently of its immunosuppressive actions. Sphk2−/− mice have lower levels of hippocampal sphingosine-1-phosphate, an endogenous HDAC inhibitor, and reduced histone acetylation, and display deficits in spatial memory and impaired contextual fear extinction. Thus, sphingosine-1-phosphate and SphK2 play specific roles in memory functions and FTY720 may be a useful adjuvant therapy to facilitate extinction of aversive memories.

Morphine causes rapid increases in glial activation and neuronal injury in the striatum of inducible HIV‐1 tat transgenic mice

HIV encephalitis (HIVE) is accompanied by brain inflammation, leukocyte infiltration, and glial activation, and HIV patients who abuse opiates are more likely to develop HIVE. To better understand how opiates could alter HIV‐related brain inflammation, the expression of astrocyte (GFAP immunoreactivity) and macrophage/microglial (F4/80 or Mac1 immunoreactivity) markers in the striatum, and the percentage of 3‐nitrotyrosine (3‐NT) positive macrophages/microglia, was determined following a 2‐day exposure to morphine (5 mg/kg/day via time‐release, subcutaneous implant) and doxycycline in GFAP‐driven, doxycycline‐inducible HIV‐1 Tat transgenic mice. Data show that both morphine and Tat induction via doxycycline increased astrocyte activation, with significant additive increases achieved with combined morphine and doxycycline exposure. By contrast, combined Tat induction and morphine exposure, but neither manipulation alone, significantly increased the proportion of macrophages/microglia present in the striatum of transgenic mice, although morphine exposure was necessary to elevate 3‐NT co‐detection in Mac1‐positive macrophages/microglia. Finally, Tat induction increased the percentage of neurons expressing active caspase‐3, and this was even more significantly elevated by co‐administration of morphine. In spite of elevations in caspase‐3, neuronal TUNEL reactivity was unchanged in all groups, even after 10 days of Tat induction. Importantly, co‐administration of naltrexone completely antagonized the effects of morphine. These findings indicate that morphine rapidly and significantly increases the activation of astrocytes and macrophages/microglia in the brains of inducible Tat transgenic mice, supporting the theory that early inflammatory changes in glia could underlie the development of HIVE in opiate‐abusing AIDS patients.

Prodynorphin Mutations Cause the Neurodegenerative Disorder Spinocerebellar Ataxia Type 23

Spinocerebellar ataxias (SCAs) are dominantly inherited neurodegenerative disorders characterized by progressive cerebellar ataxia and dysarthria. We have identified missense mutations in prodynorphin (PDYN) that cause SCA23 in four Dutch families displaying progressive gait and limb ataxia. PDYN is the precursor protein for the opioid neuropeptides, α-neoendorphin, and dynorphins A and B (Dyn A and B). Dynorphins regulate pain processing and modulate the rewarding effects of addictive substances. Three mutations were located in Dyn A, a peptide with both opioid activities and nonopioid neurodegenerative actions. Two of these mutations resulted in excessive generation of Dyn A in a cellular model system. In addition, two of the mutant Dyn A peptides induced toxicity above that of wild-type Dyn A in cultured striatal neurons. The fourth mutation was located in the nonopioid PDYN domain and was associated with altered expression of components of the opioid and glutamate system, as evident from analysis of SCA23 autopsy tissue. Thus, alterations in Dyn A activities and/or impairment of secretory pathways by mutant PDYN may lead to glutamate neurotoxicity, which underlies Purkinje cell degeneration and ataxia. PDYN mutations are identified in a small subset of ataxia families, indicating that SCA23 is an infrequent SCA type (∼0.5%) in the Netherlands and suggesting further genetic SCA heterogeneity.

Opioid system diversity in developing neurons, astroglia, and oligodendroglia in the subventricular zone and striatum: Impact on gliogenesis in vivo

Accumulating evidence, obtained largely in vitro, indicates that opioids regulate the genesis of neurons and glia and their precursors in the nervous system. Despite this evidence, few studies have assessed opioid receptor expression in identified cells within germinal zones or examined opioid effects on gliogenesis in vivo. To address this question, the role of opioids was explored in the subventricular zone (SVZ) and/or striatum of 2–5‐day‐old and/or adult ICR mice. The results showed that subpopulations of neurons, astrocytes, and oligodendrocytes in the SVZ and striatum differentially express μ‐, δ‐, and/or κ‐receptor immunoreactivity in a cell type‐specific and developmentally regulated manner. In addition, DNA synthesis was assessed by examining 5‐bromo‐2′‐deoxyuridine (BrdU) incorporation into glial and nonglial precursors. Morphine (a preferential μ‐agonist) significantly decreased the number of BrdU‐labeled GFAP+ cells compared with controls or mice co‐treated with naltrexone plus morphine. Alternatively, in S100β+ cells, morphine did not significantly decrease BrdU incorporation; however, significant differences were noted between mice treated with morphine and those treated with morphine plus naltrexone. Most cells were GFAP−/S100β−. When BrdU incorporation was assessed within the total population (glia and nonglia), morphine had no net effect, but naltrexone alone markedly increased BrdU incorporation. This finding suggests that DNA synthesis in GFAP−/S100β− cells is tonically suppressed by endogenous opioids. Assuming that S100β and GFAP, respectively, distinguish among younger and older astroglia, this implies that astroglial replication becomes increasingly sensitive to morphine during maturation, and suggests that opioids differentially regulate the development of distinct subpopulations of glia and glial precursors. GLIA 36:78–88, 2001.

Morphine Exacerbates HIV-1 Tat-Induced Cytokine Production in Astrocytes through Convergent Effects on [Ca2+]i, NF-κB Trafficking and Transcription

Astroglia are key cellular sites where opiate drug signals converge with the proinflammatory effects of HIV-1 Tat signals to exacerbate HIV encephalitis. Despite this understanding, the molecular sites of convergence driving opiate-accelerated neuropathogenesis have not been deciphered. We therefore explored potential points of interaction between the signaling pathways initiated by HIV-1 Tat and opioids in striatal astrocytes. Profiling studies screening 152 transcription factors indicated that the nuclear factor-kappa B (NF-κB) subunit, c-Rel, was a likely candidate for Tat or Tat plus opiate-induced increases in cytokine and chemokine production by astrocytes. Pretreatment with the NF-κB inhibitor parthenolide provided evidence that Tat±morphine-induced release of MCP-1, IL-6 and TNF-α by astrocytes is NF-κB dependent. The nuclear export inhibitor, leptomycin B, blocked the nucleocytoplasmic shuttling of NF-κB; causing p65 (RelA) accumulation in the nucleus, and significantly attenuated cytokine production in Tat±morphine exposed astrocytes. Similarly, chelating intracellular calcium ([Ca2+]i) blocked Tat±morphine-evoked MCP-1 and IL-6 release, while artificially increasing the concentration of extracellular Ca2+ reversed this effect. Taken together, these results demonstrate that: 1) exposure to Tat±morphine is sufficient to activate NF-κB and cytokine production, 2) the release of MCP-1 and IL-6 by Tat±morphine are highly Ca2+-dependent, while TNF-α appears to be less affected by the changes in [Ca2+]i, and 3) in the presence of Tat, exposure to opiates augments Tat-induced NF-κB activation and cytokine release through a Ca2+-dependent pathway.