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Biomedical subjects

P E Knapp

Publications and source records attributed to P E Knapp.

At least 19 recordsLinked to original sources
PubMed2005

Differential involvement of p38 and JNK MAP kinases in HIV-1 Tat and gp120-induced apoptosis and neurite degeneration in striatal neurons.

The role of p38 and c-jun-N-terminal kinases 1/2, members of the mitogen-activated protein kinase family, in mediating the toxic effects of human immunodeficiency virus-1 transactivator of transcription (Tat) and gp120 were explored in primary mouse striatal neurons in vitro. Both Tat and gp120 caused significant increases in p38 and c-jun-N-terminal kinase mitogen-activated protein kinase phosphorylation, caspase-3 activity, neurite losses and cell death in striatal neurons. Tat-induced increases in caspase-3 activity were significantly attenuated by an inhibitor of c-jun-N-terminal kinase (anthra[1,9-cd]pyrazol-6(2H)-one), but not by an inhibitor of p38 ([4-(4-fluorophenyl)-2-(4-methylsul-finylphenyl)-5-(4-pyridyl)1 H-imidazole]), mitogen-activated protein kinase. However, despite preventing increases in caspase-3 activity, c-jun-N-terminal kinase inhibition failed to avert Tat-induced neuronal losses suggesting that the reductions in caspase-3 activity were insufficient to prevent cell death caused by Tat. Alternatively, gp120-induced increases in caspase-3 activity, neurite losses and neuronal death were prevented by p38, but not c-jun-N-terminal kinase, mitogen-activated protein kinase inhibition. Our findings suggest that gp120 induces neuronal dysfunction and death through actions at p38 mitogen-activated protein kinase, while Tat kills neurons through actions that are independent of p38 or c-jun-N-terminal kinase mitogen-activated protein kinase, or through the concurrent activation of multiple proapoptotic pathways.

Animals↗
PubMed2005

Molecular targets of opiate drug abuse in neuroAIDS.

Opiate drug abuse, through selective actions at mu-opioid receptors (MOR), exacerbates the pathogenesis of human immunodeficiency virus-1 (HIV-1) in the CNS by disrupting glial homeostasis, increasing inflammation, and decreasing the threshold for pro-apoptotic events in neurons. Neurons are affected directly and indirectly by opiate-HIV interactions. Although most opiates drugs have some affinity for kappa (KOR) and/or delta (DOR) opioid receptors, their neurotoxic effects are largely mediated through MOR. Besides direct actions on the neurons themselves, opiates directly affect MOR-expressing astrocytes and microglia. Because of their broad-reaching actions in glia, opiate abuse causes widespread metabolic derangement, inflammation, and the disruption of neuron-glial relationships, which likely contribute to neuronal dysfunction, death, and HIV encephalitis. In addition to direct actions on neural cells, opioids modulate inflammation and disrupt normal intercellular interactions among immunocytes (macrophages and lymphocytes), which on balance further promote neuronal dysfunction and death. The neural pathways involved in opiate enhancement of HIV-induced inflammation and cell death, appear to involve MOR activation with downstream effects through PI3-kinase/Akt and/or MAPK signaling, which suggests possible targets for therapeutic intervention in neuroAIDS.

AIDS Dementia Complex↗
PubMed2003

Dynorphin A (1-17) induces apoptosis in striatal neurons in vitro through alpha-amino-3-hydroxy-5-methylisoxazole-4-propionate/kainate receptor-mediated cytochrome c release and caspase-3 activation.

Dynorphin A (1-17), an endogenous opioid neuropeptide, can have pathophysiological consequences at high concentrations through actions involving glutamate receptors. Despite evidence of excitotoxicity, the basic mechanisms underlying dynorphin-induced cell death have not been explored. To address this question, we examined the role of caspase-dependent apoptotic events in mediating dynorphin A (1-17) toxicity in embryonic mouse striatal neuron cultures. In addition, the role of opioid and/or glutamate receptors were assessed pharmacologically using dizocilpine maleate (MK(+)801), a non-equilibrium N-methyl-D-aspartate (NMDA) antagonist; 6-cyano-7-nitroquinoxaline-2,3-dione, a competitive alpha-amino-3-hydroxy-5-methylisoxazole-4-propionate (AMPA)/kainate antagonist; or (-)-naloxone, a general opioid antagonist. The results show that dynorphin A (1-17) (>or=10 nM) caused concentration-dependent increases in caspase-3 activity that were accompanied by mitochondrial release of cytochrome c and the subsequent death of cultured mouse striatal neurons. Moreover, dynorphin A-induced neurotoxicity and caspase-3 activation were significantly attenuated by the cell permeable caspase inhibitor, caspase-3 inhibitor-II (z-DEVD-FMK), further suggesting an apoptotic cascade involving caspase-3. AMPA/kainate receptor blockade significantly attenuated dynorphin A-induced cytochrome c release and/or caspase-3 activity, while NMDA or opioid receptor blockade typically failed to prevent the apoptotic response. Last, dynorphin-induced caspase-3 activation was mimicked by the ampakine CX546 [1-(1,4-benzodioxan-6-ylcarbonyl)piperidine], which suggests that the activation of AMPA receptor subunits may be sufficient to mediate toxicity in striatal neurons. These findings provide novel evidence that dynorphin-induced striatal neurotoxicity is mediated by a caspase-dependent apoptotic mechanism that largely involves AMPA/kainate receptors.

Animals↗
PubMed2001

Endogenous opioids and oligodendroglial function: possible autocrine/paracrine effects on cell survival and development.

Previous work has shown that oligodendrocytes (OLs) express both micro- and kappa-opioid receptors. In developing OLs, micro receptor activation increases OL proliferation, while the kappa-antagonist nor-binaltorphimine (NorBNI) affects OL differentiation. Because exogenous opioids were not present in our defined culture medium, we hypothesized that NorBNI blocked endogenous opioids produced by the OLs themselves. To test this, intact and partially processed proenkephalin and prodynorphin-derived peptides were assessed in OLs using immunocytochemistry or Western blot analysis, or both. Immature OLs possessed large amounts of intact and partially processed proenkephalin precursors, as well as posttranslational products of prodynorphin including dynorphin A (1-17). With maturation, however, intact or partially processed proenkephalin was expressed by only about 50% of OLs, while dynorphin A (1-17) was undetectable. To assess the function of OL-derived opioids, the effect of kappa-agonists/antagonists on OL differentiation and death was explored. kappa-Agonists alone had no effect. In contrast, NorBNI significantly increased OL death. Additive OL losses were evident when NorBNI was paired with toxic levels of glutamate, suggesting that kappa-receptor blockade alone is sufficient to induce OL death. Thus, the results indicate that OLs express proenkephalin and prodynorphin peptides in a developmentally regulated manner, and further suggest that opioids produced by OLs modulate OL maturation and survival through local (i.e., autocrine and/or paracrine) mechanisms.

Aging↗
PubMed2001

Structure-activity analysis of dynorphin A toxicity in spinal cord neurons: intrinsic neurotoxicity of dynorphin A and its carboxyl-terminal, nonopioid metabolites.

Dynorphin A [dynorphin A (1-17)] is an endogenous opioid peptide that is antinociceptive at physiological concentrations. Levels of dynorphin A increase markedly following spinal cord trauma and may contribute to secondary neurodegeneration. Both kappa opioid and N-methyl-d-aspartate (NMDA) receptor antagonists can modulate the effects of dynorphin, suggesting that dynorphin is acting through kappa opioid and/or NMDA receptor types. Despite these findings, few studies have critically examined the mechanisms of dynorphin A neurotoxicity at the cellular level. To better understand how dynorphin affects cell viability, structure-activity studies were performed examining the effects of dynorphin A and dynorphin A-derived peptide fragments on the survival of mouse spinal cord neurons coexpressing kappa opioid and NMDA receptors in vitro. Time-lapse photography was used to repeatedly follow the same neurons before and during experimental treatments. Dynorphin A caused significant neuronal losses that were dependent on concentration (> or = 1 microM) and duration of exposure. Moreover, exposure to an equimolar concentration of dynorphin A fragments (100 microM) also caused a significant loss of neurons. The rank order of toxicity was dynorphin A (1-17) > dynorphin A (1-13) congruent with dynorphin A (2-13) congruent with dynorphin A (13-17) (least toxic) > dynorphin A (1-5) ([Leu(5)]-enkephalin) or dynorphin A (1-11). Dynorphin A (1-5) or dynorphin A (1-11) did not cause neuronal losses even following 96 h of continuous exposure, while dynorphin A (3-13), dynorphin A (6-17), and dynorphin A (13-17) were neurotoxic. The NMDA receptor antagonist MK-801 (dizocilpine) (10 microM) significantly attenuated the neurotoxic effects of dynorphin A and/or dynorphin-derived fragments except dynorphin A (13-17), suggesting that the neurotoxic effects of dynorphin were largely mediated by NMDA receptors. Thus, toxicity resides in the carboxyl-terminal portion of dynorphin A and this minimally includes dynorphin A (3-13) and (13-17). Our findings suggest that dynorphin A and/or its metabolites may contribute significantly to neurodegeneration during spinal cord injury and that alterations in dynorphin A biosynthesis, metabolism, and/or degradation may be important in determining injury outcome.

Amino Acid Sequence↗
PubMed2001

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 mu-, delta-, and/or kappa-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 mu-agonist) significantly decreased the number of BrdU-labeled GFAP(+) cells compared with controls or mice co-treated with naltrexone plus morphine. Alternatively, in S100beta(+) 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(-)/S100beta(-). 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(-)/S100beta(-) cells is tonically suppressed by endogenous opioids. Assuming that S100beta 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.

Aging↗
PubMed2000

Neuronal interaction determines the expression of the alpha-2 isoform of Na, K-ATPase in oligodendrocytes.

Na,K-ATPase is an integral membrane enzyme responsible for maintenance of the transmembrane Na+/K+ gradient which generates membrane excitability. Previous studies showed that oligodendrocytes within the CNS robustly expressed the alpha2 isoform of the Na,K-ATPase while oligodendrocytes in isolated cultures did not. We tested whether the levels of this isoform might be modulated by interactions with neurons. Western blots showed alpha2 protein expression was very low in rat optic nerve immediately after birth, but that expression was greatly increased by days 5 and 14. In adult optic nerves, levels were barely detectable. Since the first myelinated axons are observed in rat optic nerve at day 5, and the next 2 weeks are considered the period of peak myelination, this timing suggested a relationship between oligodendrocyte-neuron contact, myelination onset and the upregulation of the alpha2 isoform. In further experiments we plated oligodendrocytes in isolation or in co-culture with neurons dissociated from cerebral cortex at the day of birth. After 6 days in vitro, 45% of oligodendrocytes co-cultured with neurons expressed abundant alpha2 protein which was detected by immunohistochemistry, a six-fold increase over cells expressing alpha2 protein in isolated cultures. Conditioned medium from neuronal cultures did not affect alpha2 levels in oligodendrocytes. These results suggest that neurons may play a role in upregulating glial expression of the alpha2 isoform during peak periods of myelination, and that the effect is likely to be dependent on contact.

Animals↗
PubMed1999

Abnormal Ca(2+) regulation in oligodendrocytes from the dysmyelinating jimpy mouse.

Jimpy (jp) is a point mutation in the gene on the X chromosome which codes for the major myelin proteolipid protein. Most oligodendrocytes (OLs) in the jp mouse undergo cell death at the time when they should be actively myelinating. Loss of mature OLs results in severe CNS dysmyelination. Dying jp OLs have the morphology of apoptotic cells but it is not clear how the mutation activates biochemical pathways which lead to programmed death of OLs in jp CNS. There is compelling evidence from a number of systems that high levels of intracellular Ca(2+) ([Ca2+]i) can activate downstream processes which result in both apoptotic and necrotic cell death. To determine whether [Ca2+](i) dysregulation might be involved in the death of jp OLs, we used ratiometric imaging to determine levels of [Ca2+](i) in OLs cultured from jp and normal CNS and in immortalized cell lines derived from jp and normal OLs. Immortalized jp OLs and OLs isolated directly from jp brain both showed a similar elevation in [Ca2+](i) ranging from 60% to 150% over control values. A higher baseline [Ca2+](i) in jp OLs might increase their vulnerability to other insults due to abnormal protein processing or changes in signaling pathways which act as a final trigger for cell death.

Animals↗
PubMed1999

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.

Adenosine Triphosphate↗
PubMed1999

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. 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.

Animals↗
PubMed1999

Programmed cell death without DNA fragmentation in the jimpy mouse: secreted factors can enhance survival.

Jimpy is one of many related mutations affecting the myelin proteolipid protein gene that causes severe hypomyelination in the central nervous system (CNS). Underlying the hypomyelination is a failure of oligodendrocytes (OLs) to differentiate, and the premature death of large numbers of OLs during the developmental period. Previous light and electron microscopic evidence suggested that jimpy OLs die in a manner consistent with programmed cell death. We have used TUNEL staining as a biochemical marker for apoptosis in conjunction with immunostaining for OL and myelin markers. At 13 - 14 days postnatal, a time when the number of dying OLs in jimpy CNS is increased more than five times normal, there are only modest increases (70% in spinal cord; 20% in cerebral cortex) in TUNEL labeled cells in mutant CNS tissues. The results in vitro are similar, and only a small per cent of TUNEL labeled cells have the antigenic phenotype of OLs. The discrepancy between numbers of dying and TUNEL labeled cells suggests either that most jimpy OLs do not undergo programmed cell death or that the biochemical pathways leading to their death do not involve DNA fragmentation which is detected by the TUNEL method. We also present evidence that jimpy OLs show increased survival and enhanced differentiation when they are grown in vitro in medium conditioned by cells lines which express products of the proteolipid protein gene. Cell lines expressing proteolipid protein and the alternatively spliced DM20 protein have differential effects on cell numbers and production of myelin-like membranes.

Animals↗
PubMed1998

Endogenous opioid system in developing normal and jimpy oligodendrocytes: mu and kappa opioid receptors mediate differential mitogenic and growth responses.

The early development of both neurons and neuroglia may be modulated by signaling through opioid mediated pathways. Neurons and astroglia not only express specific types of opiate receptors, but also respond functionally to opioids with altered rates of proliferation and growth. The present study was undertaken to determine if opioids also modulate development of the other major CNS macroglial cell, the oligodendrocyte (OL). Using well-characterized polyclonal antibodies specific for delta-, kappa-, and mu-opiate receptors, OLs grown in vitro were shown to express mu-receptors at a very immature stage prior to expression of kappa-receptors. This developmentally regulated sequence differs from the pattern of expression in neurons and astroglia. delta-receptors are apparently absent from cultured OLs. OLs also have physiologic responses to selective mu- and kappa-receptor agonists and antagonists. Exposure of relatively immature O4+ OLs to the mu-receptor agonist PL017 [H-Tyr-Pro-Phe(N-Me)-D-Pro-NH2] resulted in a significant enhancement in the rate of DNA synthesis. This effect, which was not observed in more mature MBP+ OLs, was entirely blocked by the antagonist naloxone. Although the kappa-receptor pathway appeared to be uninvolved in controlling proliferation, the kappa-receptor antagonist nor-binaltorphimine significantly increased the size of myelin-like membranes produced by the cultured OLs. Interestingly, OLs derived from the jimpy mouse, a mutant characterized by an almost complete lack of CNS myelin and premature death of OLs, were found to be deficient in kappa-opiate receptors. Our findings clearly show that OLs not only express specific opiate receptors, but also respond to changes in their level of stimulation in ways that could profoundly impact nervous system morphology and function. If opiate receptors are expressed by OLs in vivo, their pharmacological manipulation might provide a novel pathway for modulating OL and myelin production both during development and in demyelinated conditions.

Animals↗
PubMed1997

Injury stimulates outgrowth and motility of oligodendrocytes grown in vitro.

Oligodendrocytes which form myelin within the CNS develop from small, highly motile cells that are largely bipolar into mature cells which extend many processes and which produce myelin membranes around multiple axons. The production of myelin sheaths is thought to anchor mature oligodendrocytes (OLs), limiting their motility. When the brain sustains an injury, OLs do not make a significant effort to remyelinate, a fact attributed to both their lack of proliferation and their inability to migrate or extend processes into areas of injury. To test the motility and growth potential of mature OLs, we have designed an in vitro system in which individual cells can undergo long-term observation. Additionally, cells can be mechanically injured by transection of processes using a low-power laser beam. Both control and injured OLs undergo several types of structural change, including extension and retraction of processes and membranes, as well as changes in process caliber. Some OLs exhibit a high degree of motility, moving several hundred micrometers within days. Rather than interfering with the cells' ability to undergo structural change, injury actually stimulated outgrowth of new processes and motility. Neither injury nor addition of basic fibroblast growth factor (bFGF) increased the rate of OL division. However, bFGF paradoxically caused an increase in uptake of the DNA synthesis marker bromodeoxyuridine and had negative effects on OL survival. The unexpected findings that OLs with a mature phenotype are motile and undergo constant structural modification in vitro and that injury induces certain behaviors suggest that myelin-forming OLs in the brain may be capable of a high degree of plasticity under certain conditions.

Animals↗
PubMed1996

mu-Opioid receptor activation enhances DNA synthesis in immature oligodendrocytes.

Opioids disrupt nervous system development by inhibiting the proliferation of neuronal and glial progenitors. These studies explored the hypothesis that mu opioid receptors are expressed by immature oligodendrocytes (OLs) and are functionally related to growth. Antibodies identifying the cloned mu opioid receptor demonstrated that cultured OLs expressed mu opioid receptor immunoreactivity very early during development. Cultures were treated with the selective mu opioid receptor agonist H-Tyr-Pro-Phe (N-Me)-D-Pro-NH2 (PL017; 1 microM), or PL017 (1 microM) plus the antagonist naloxone (3 microM). Opioid-dependent changes in DNA synthesis were assessed by determining the proportion of bromodeoxyuridine (BrdU)-labeled O4-immunoreactive OLs. Treatment with PL017 caused a 311% increase in the proportion of O4-immunoreactive OLs incorporating BrdU compared to untreated controls, and these effects were prevented by co-administering naloxone. These preliminary results indicate that (i) immature OLs express mu opioid receptors and that (ii) the activation of this receptor type is functionally coupled to DNA synthesis and the cell division cycle. The expression of opioid receptors by OLs suggests that the endogenous opioid system is widely distributed among glial types.

Animals↗
PubMed1996

Epigenetic factors up-regulate expression of myelin proteins in the dysmyelinating jimpy mutant mouse.

Proteolipid protein (PLP) is a major structural component of central nervous system (CNS) myelin. Evidence exists that PLP or the related splice variant DM-20 protein may also play a role in early development of oligodendrocytes (OLs), the cells that form CNS myelin. There are several naturally occurring mutations of the PLP gene that have been used to study the roles of PLP both in myelination and in OL differentiation. The PLP mutation in the jimpy (jp) mouse has been extensively characterized. These mutants produce no detectable PLP and exhibit an almost total lack of CNS myelin. Additionally, most OLs in affected animals die prematurely, before producing myelin sheaths. We have studied cultures of jp CNS in order to understand whether OL survival and myelin formation require production of normal PLP. When grown in primary cultures, jp OLs mimic the relatively undifferentiated phenotype of jp OLs in vivo. They produce little myelin basic protein (MBP), never immunostain for PLP, and rarely elaborate myelin-like membranes. We report here that jp OLs grown in medium conditioned by normal astrocytes synthesize MBP and incorporate it into membrane expansions. Some jp OLs grown in this way stain with PLP antibodies, including an antibody to a peptide sequence specific for the mutant jp PLP. This study shows that: (1) an absence of PLP does not necessarily lead to dysmyelination or OL death; (2) OLs are capable of translating at least a portion of the predicted jp PLP; (3) the abnormal PLP made in the cultured jp cells is not toxic to OLs. These results also highlight the importance of environmental factors in controlling OL phenotype.

Animals↗
PubMed1996

Proteolipid protein: is it more than just a structural component of myelin?

Proteolipid protein (PLP) is the most abundant protein of central nervous system (CNS) myelin. Because of its predicted topography, PLP has been assumed to function as a structural component of myelin, providing stability and maintaining the compact lamellar structure. However, developmental studies have shown that the PLP gene is active long before myelination begins. This and other evidence from various PLP mutants and transgenic models has fueled speculation that PLP or other products of the gene have additional, nonstructural roles both within and outside the CNS. PLP is structurally related to a family of ion channel proteins which includes the connexins, synaptophysins and various neurotransmitter receptors, and there is some experimental evidence which supports a role for PLP in ion gating. Other provocative ideas are that the PLP gene may influence autocrine signaling within oligodendrocytes or that PLP mRNAs have a function apart from protein coding.

Animals↗