APOE-mimetic peptides reduce behavioral deficits, plaques and tangles in Alzheimer's disease transgenics.

BACKGROUND: After age, the second largest risk factor for Alzheimer's disease (AD) is apolipoprotein E (APOE) genotype, where APOE4 is associated with lower apoE protein levels, more severer brain pathology, enhanced inflammation and disease. Small peptides corresponding to the receptor-binding region of apoE mimic the anti-inflammatory activity of the apoE holoprotein. These apoE mimetics greatly improve behavioral outcomes and neuronal survival in head trauma models that display AD pathology and neuronal loss. OBJECTIVE: To determine whether apoE mimetics change behavior, inflammation and pathology in CVND-AD (SwDI-APP/NOS2(-/-)) transgenic mice. METHODS: Starting at 9 months, apoE peptides were subcutaneously administered 3 times per week for 3 months followed by behavioral, histochemical and biochemical testing. RESULTS: Treatment with apoE mimetics significantly improved behavior while decreasing the inflammatory cytokine IL-6, neurofibrillary tangle-like and amyloid plaque-like structures. Biochemical measures matched the visible pathological results. CONCLUSIONS: Treatment with apoE mimetics significantly improved behavior, reduced inflammation and reduced pathology in CVND-AD mice. These improvements are associated with apoE-mimetic-mediated increases in protein phosphatase 2A activity. Testing in additional AD models showed similar benefits, reinforcing this novel mechanism of action of apoE mimetics. These data suggest that the combination of anti-inflammatory and neuroprotective activities of apoE mimetics represents a new generation of potential therapeutics for AD.

Quantitative measurement of postural sway in mouse models of human neurodegenerative disease.

Detection of motor dysfunction in genetic mouse models of neurodegenerative disease requires reproducible, standardized and sensitive behavioral assays. We have utilized a center of pressure (CoP) assay in mice to quantify postural sway produced by genetic mutations that affect motor control centers of the brain. As a positive control for postural instability, wild type mice were injected with harmaline, a tremorigenic agent, and the average areas of the 95% confidence ellipse, which measures 95% of the CoP trajectory values recorded in a single trial, were measured. Ellipse area significantly increased in mice treated with increasing doses of harmaline and returned to control values after recovery. We also examined postural sway in mice expressing mutations that mimic frontotemporal dementia with Parkinsonism linked to chromosome 17 (FTDP-17) (T-279, P301L or P301L-nitric oxide synthase 2 (NOS2)(-/-) mice) and that demonstrate motor symptoms. These mice were then compared with a mouse model of Alzheimer's disease (APPSwDI mice) that demonstrates cognitive, but not motor deficits. T-279 and P301L-NOS2(-/-) mice demonstrated a significant increase in CoP ellipse area compared with appropriate wild type control mice or to mice expressing the P301L mutation alone. In contrast, postural instability was significantly reduced in APPSwDI mice that have cognitive deficits but do not have associated motor deficits. The CoP assay provides a simple, sensitive and quantitative tool to detect motor deficits resulting from postural abnormalities in mice and may be useful in understanding the underlying mechanisms of disease.

Y+ and y+ L arginine transporters in neuronal cells expressing tyrosine hydroxylase.

Arginine is a semi-essential amino acid that serves as sole substrate for enzymes involved in diverse cell processes including redox balance via nitric oxide synthase (NOS) and cell proliferation via arginase. Neurons that express nNOS require intracellular arginine to generate nitric oxide (NO). Using a TH+ neuronal cell line (CAD cells), we show that neuronal NO production is largely dependent on extracellular arginine. Although a small intracellular pool exists in CAD cells, the lack of mRNA for argininosuccinate synthase (AS), a rate limiting enzyme for arginine recycling, suggests that intracellular pools are not re-supplied by this mechanism in this sub-class of neurons. Rather, arginine is taken up from the extracellular media by two primary transport systems, the y+ and the y+ L systems. The expression of CAT1, CAT3, y+ LAT1 and y+ LAT2 mRNAs supports the presence of each system. CAD cell arginine transport is depressed by increased extracellular K+ levels and demonstrates that variations in membrane potential control neuronal arginine uptake. Short term exposure to the oxidizing agents, rotenone and Angeli's salt, but not FeSO4, increases arginine transport. The regulation of arginine uptake by physiological factors suggests that arginine supply adapts in a moment-to-moment fashion to the changing needs of the neuron.

Disrupted spermine homeostasis: a novel mechanism in polyglutamine-mediated aggregation and cell death.

Our data suggest a novel mechanism whereby pathological-length polyglutamine (polyQ) proteins promote the spermine synthetic pathway, increasing polyQ-aggregation and cell death. As detected in a cell-free turbidity assay, spermine promotes aggregation of thio-polyQ62 in a dose-dependent manner. Using a stable neuronal cell line expressing pathological-length [polyQ57-yellow fluorescent protein (YFP) (Q57)] or non-pathological-length [polyQ19-YFP (Q19)] polyglutamine protein, we show that multiple steps in the production of polyamines are affected in Q57 cells, suggesting dysfunctional spermine homeostasis. As the building block for spermine synthesis, arginine transport is significantly increased in neuronal cell lines stably expressing Q57. Q57 lines displayed upregulated basal and inducible arginase I activities that were not seen in polyQ19-YFP lines. Normal induction of spermidine/spermine N-acetyltransferase in Q19 lines regulating back-conversion of spermine, thereby reducing spermine levels, however, was not observed in Q57 lines. Pharmacological activation of ornithine decarboxylase (ODC), a key enzyme of the polyamine synthetic pathway, increased cellular aggregates and increased cell death in Q57 cells not observed in Q19 cells. Inhibition of ODC by difluoromethylornithine prevented basal and induced cell death in Q57 cells, demonstrating a central role for polyamines in this process.

Glutamate acting at NMDA receptors stimulates embryonic cortical neuronal migration.

During cortical development, embryonic neurons migrate from germinal zones near the ventricle into the cortical plate, where they organize into layers. Mechanisms that direct neuronal migration may include molecules that act as chemoattractants. In rats, GABA, which localizes near the target destination for migrating cortical neurons, stimulates embryonic neuronal migration in vitro. In mice, glutamate is highly localized near the target destinations for migrating cortical neurons. Glutamate-induced migration of murine embryonic cortical cells was evaluated in cell dissociates and cortical slice cultures. In dissociates, the chemotropic effects of glutamate were 10-fold greater than the effects of GABA, demonstrating that for murine cortical cells, glutamate is a more potent chemoattractant than GABA. Thus, cortical chemoattractants appear to differ between species. Micromolar glutamate stimulated neuronal chemotaxis that was mimicked by microM NMDA but not by other ionotropic glutamate receptor agonists (AMPA, kainate, quisqualate). Responding cells were primarily derived from immature cortical regions [ventricular zone (vz)/subventricular zone (svz)]. Bromodeoxyuridine (BrdU) pulse labeling of cortical slices cultured in NMDA antagonists (microM MK801 or APV) revealed that antagonist exposure blocked the migration of BrdU-positive cells from the vz/svz into the cortical plate. PCR confirmed the presence of NMDA receptor expression in vz/svz cells, whereas electrophysiology and Ca2+ imaging demonstrated that vz/svz cells exhibited physiological responses to NMDA. These studies indicate that, in mice, glutamate may serve as a chemoattractant for neurons in the developing cortex, signaling cells to migrate into the cortical plate via NMDA receptor activation.

Apolipoprotein E acts to increase nitric oxide production in macrophages by stimulating arginine transport.

Previous studies have shown that apolipoprotein E (apoE) plays a role in immune function by modulating tissue redox balance. Using a mouse macrophage cell line (RAW 264.7), we have examined the mechanism by which apoE regulates nitric oxide (NO) production in macrophages. ApoE potentiates NO production in immune activated RAW cells in combination with lipopolysaccharide or polyinosinic:polycytidylic acid (PIC), agents known to induce expression of inducible nitric oxide synthase mRNA and protein. The effect is not observed with apolipoprotein B or heat-inactivated apoE. The combination of PIC plus apoE produced more NO than the level expected from an additive effect of PIC and apoE alone. Furthermore, this increase was observed at submaximal extracellular arginine concentrations, suggesting that apoE altered arginine (substrate) availability. Examination of [(3)H]arginine uptake across the cell membrane demonstrated that arginine uptake was increased by PIC but further increased by PIC plus apoE. Treatment of RAW cells with apoE was associated with an increased apparent V(max) and decreased affinity for arginine as well as a switch in the induction of mRNA for subtypes of cationic amino acid transporters (CAT). Treatment of RAW cells with PIC plus apoE resulted in the loss of detectable CAT1 mRNA and expression of CAT2 mRNA. Regulation of arginine availability is a novel action of apoE on the regulation of macrophage function and the immune response.

Polyribonucleotides induce nitric oxide production by human monocyte-derived macrophages.

Cytokine-mediated activation of inducible nitric oxide synthase (iNOS) in monocytes or macrophages is species specific. In contrast to rat or mouse, human macrophages do not produce measurable levels of nitric oxide (NO) when induced by inflammatory mediators. Exposure to noncytokine mediators such as tumor cells or viruses, however, has recently been shown to activate human iNOS. NO production in response to these mediators is much lower than that seen for rat or mouse cells and often requires several days of stimulation. We have found that the synthetic, doublestranded polyribonucleotide polyinosinic-polycytidilic acid (Poly I:C), commonly used to mimic viral exposure, activated iNOS in human monocyte-derived macrophages (MDM). The production of NO, measured by nitrite accumulation, was detected after 24 h of stimulation with Poly I:C. The single-stranded polyribonucleotide Poly I, but not Poly C, also increased NO production. Nitrite production was enhanced when the MDM were primed (pretreated) with gamma or alpha interferon or other immune mediators such as IL-4 and was reduced by the iNOS inhibitor, N-methyl-L-arginine (L-NMMA). The use of Poly I:C to initiate NO production in human macrophages provides a useful tool to study the differences between the commonly used animal models and human cells and may provide insight into the pathophysiological significance of these differences.

Production of superoxide anions by a CNS macrophage, the microglia.

Microglia have been implicated in both physiological and pathological processes of the brain. Their possible roles have been compared to those of macrophages and granulocytes. Here we demonstrate that specific ability of microglia to secrete the superoxide radical ion in response to a complement activated agent, opsonized zymosan, and to phorbol myristate acetate. As in other organs, this endogenously produced reactive oxygen intermediate could have both beneficial and deleterious effects.

The action of hydrogen peroxide on paired pulse and long-term potentiation in the hippocampus.

The action of a reactive oxygen intermediate, that is, hydrogen peroxide (H2O2) on modulation of synaptic transmission was examined in the hippocampal brain slice preparation. Microinjection of H2O2 into the apical dendritic region of the CA1 pyramidal cells produced no change in either the pattern or amplitude of paired pulse facilitation compared to saline injection (control). Long term potentiation (LTP), induced by high frequency stimulation of homosynaptic inputs, however, was blocked by microinjection of H2O2 into the dendritic tree. LTP was seen in only 2 out of 10 slices investigated when treated with H2O2 while LTP was seen in 4 out of 5 slices when saline injected. The results suggest that a reactive oxygen intermediate can selectively modify synaptic mechanisms in the hippocampus.

Location-dependent artifact for no measurement using multiwell plates.

The Griess reaction is widely used to measure the cellular production of NO by detecting the supernatant levels of nitrite. Ordinarily, background levels of nitrite in the media are subtracted from the levels of nitrite produced by the cells by preparing a "blank" during the determination of the standard curve. Although this method is adequate for most experimental conditions, it cannot be used when cell supernatants are collected from multiwell dishes, particularly when low amounts of NO are produced and when long incubation periods are required to induce NO production. Our data show that a highly variable level of nitrite is found in the absence of cells in the media from wells at the edges of the 96-well plate while media from interior wells shows no detectable nitrite accumulation. The most likely source of this noncellular NO is from nitric oxides (NOx) found in the ambient air and reduction of air exchange or regulation of the gaseous environment eliminates this "border effect."

Microglial function in human APOE3 and APOE4 transgenic mice: altered arginine transport.

The APOE4 genotype is a known risk factor for Alzheimer's disease (AD) and is associated with poorer outcomes after neuropathological insults. To understand APOE's function, we have examined microglia, the CNS specific macrophage, in transgenic mice expressing the human APOE3 and APOE4 gene allele. Our data demonstrate that arginine uptake is enhanced in APOE4 microglia compared to APOE3 microglia. The increased arginine uptake in APOE4 Tg microglia is associated with an increased expression of mRNA for cationic amino acid transporter-1 (Cat1), a constuitively expressed member of the arginine selective transport system (the y+ transport system) found in most cells. The macrophage-associated transporter, cationic amino acid transporter 2B (Cat2B) did not demonstrate a change in mRNA expression. This change in microglial arginine transport suggests a potential impact of the APOE4 gene allele on those biochemical pathways such as NO production or cell proliferation to which arginine contributes.

Regulation of microglial function by interferons.

Cultured neonatal rat microglia were pretreated with varying doses of either purified interferon (IFN) alpha/beta or recombinant IFN gamma for 24 or 48 h and the following functional parameters examined; superoxide anion production, interleukin-1 secretion and chemotaxis. IFN gamma produced a marked increase in superoxide anion levels when PMA was used to initiate superoxide anion production but had no effect in OPZ-stimulated microglia. Treatment with IFN alpha/beta potentiated superoxide anion production in both PMA and OPZ-stimulated cells. Interleukin-1 activity was decreased by treatment with IFN gamma for 24 h while IFN alpha/beta increased IL-1 activity at 48 h. Removal of serum from the treatment media prevented the action of IFN alpha/beta on IL-1 production. Treatment with IFN alpha/beta or gamma decreased chemotaxis of microglia in response to zymosan activated serum. The data indicate that IFN gamma and alpha/beta can regulate microglial function and that this regulation may differ from that seen for other monocytically derived macrophages.

Lectin staining of cultured CNS microglia.

Carbohydrate binding proteins, known as lectins, bind to specific sugar groups on most membranes. We used fluorescent and light microscopy to study the interaction of various lectins with the membranes of microglia cultured from neonatal rat or fetal mouse cerebral cortices. Microglia stained intensely with GS-1, RCA, WGA, and ConA and slightly with DBA, UEA, BPA, and SBA. No staining was seen with GS-2, MPA, or PNA. Staining was specific for microglia in the mixed glial cultures and was dose dependent. In addition, microglial lectin binding could be reduced or blocked by competitive inhibition using specific sugars. Treatment of the microglia with agents such as dimethylsulfoxide (DMSO), interleukin-1 (IL-1), interferon (IFN), or lipopolysaccharide (LPS) did not eliminate lectin staining, although the degree of staining was altered. Positive staining of the microglia was also associated with a functional change for at least one lectin, i.e., ConA. Superoxide anion production by microglia was increased in the presence of ConA. Overall, binding of the lectins GS-1, RCA, WGA, and ConA can be used as an identifying tool for microglia in glial cultures, but intensity of staining varies depending on their functional state.

Inhibition of microglial superoxide anion production by isoproterenol and dexamethasone.

Microglia, like other tissue macrophages, are a component of the hypothalamic-pituitary endocrine-immune axis and, as such, are responsive to both neural and endocrine factors. Using cultured neonatal hamster microglia, we have examined the effect of isoproterenol, a beta-adrenergic agonist, and dexamethasone, a synthetic glucocorticoid, on superoxide anion production. For these experiments, microglia were pretreated with isoproterenol or dexamethasone and then induced to produce superoxide anion by exposure of the cells to phorbol myristate acetate (PMA). Our study demonstrates that the PMA-stimulated production of superoxide anion was decreased by acute (30 min) and chronic (24 h) pretreatment of the microglia with isoproterenol and was blocked by the beta-adrenergic receptor antagonist, propranolol. Since a rise in intracellular cAMP may be a prime factor in the inhibition of superoxide anion production in isoproterenol-treated cells, we used forskolin, a known activator of the adenylate cyclase in place of isoproterenol and re-investigate superoxide anion production. Short term exposures to forskolin produced a lower amount of superoxide anion than PMA-stimulated alone and, thus, mimicked the effect of isoproterenol. However, treatment with the same concentration of forskolin for 24 h prior to the induction of the NADPH oxidase did not significantly change PMA-stimulated superoxide anion production from untreated values. Thus, chronic exposure to forskolin produced a different effect than chronic exposure to isoproterenol. Isoproterenol and forskolin both increased immunoreactivity for the protein products of the early response genes, c-fos and c-jun. Pretreatment with dexamethasone for 24 h also inhibited superoxide anion production and was blocked by the protein synthesis inhibitor, cycloheximide. The simultaneous addition of varying concentrations of dexamethasone and 5 microM isoproterenol did not produce a greater inhibition in superoxide anion production than either agent alone. The down-regulation of microglial function by adrenergic agonists and by glucocorticoids provides a way in which the cytotoxicity of these immune cells can be reduced and may be a factor in the paracrine regulation of microglia.

Microglial contribution to oxidative stress in Alzheimer's disease.

Microglia are the CNS macrophage and are a primary cellular component of plaques in Alzheimer's disease (AD) that may contribute to the oxidative stress associated with chronic neurodegeneration. We now report that superoxide anion production in microglia or macrophages from 3 different species is increased by long term exposure (24 hours) to A beta peptides. Since A beta competes for the uptake of opsonized latex beads and for the production of superoxide anion by opsonized zymosan, a likely site of action are membrane receptors associated with the uptake of opsonized particles or fibers. The neurotoxic fibrillar peptides A beta (1-42) and human amylin increase radical production whereas a non-toxic, non-fibrillar peptide, rat amylin, does not. We also report that the effect of A beta peptides on superoxide anion production is not associated with a concomitant increase in nitric oxide (NO) production in either human monocyte derived macrophages (MDM) or hamster microglia from primary cultures. Since NO is known to protect membrane lipids and scavenge superoxide anion, the lack of A beta-mediated induction of NO production in human microglia and macrophages may be as deleterious as the over-production of superoxide anion induced by chronic exposure to A beta peptides.

Activated human microglia produce the excitotoxin quinolinic acid.

We aimed to determine the relative role of quinolinic acid synthesis in purified human microglia, monocyte-derived macrophages and astrocytes in the human brain following immune stimulation. Microglia and macrophages significantly increased quinolinic acid synthesis from tryptophan following activation by either lipopolysaccharide or interferon-gamma. Quinolinic acid synthesis by individual microglia was heterogeneous, and its production by activated macrophages was approximately 32-fold greater than its microglial synthesis. Quinolinic acid synthesis by astrocytes was undetectable. Microglia may, therefore, be the primary endogenous cell type responsible for quinolinic acid synthesis in the brain parenchyma. However, under pathological conditions which precipitate blood-brain barrier compromise and/or leukocytic infiltration, intracerebral quinolinic acid may be derived chiefly from cells of the peripheral immune system such as activated macrophages.

The action of oxygen and oxygen at high pressure on inhibitory transmission.

The effect of 100% oxygen at ambient pressure, 100% oxygen at 1.7 Atmospheres Absolute (ATA), 100% oxygen at 5.1 ATA, helium at 1.7 ATA and helium at 5.1 ATA on inhibitory synaptic transmission was studied using the lobster walking leg neuromuscular preparation. Exposure to 100% oxygen at ambient pressure, at 1.7 ATA or at 5.1 ATA produced a decrease in inhibitory transmission manifest as a fall in inhibitory synaptic conductance (Ginh). The largest decrease in Ginh was seen in 100% oxygen at ambient pressure, while a progressively smaller decrease was seen in 100% oxygen at 1.7 ATA and 5.1 ATA, respectively. Also associated with 100% oxygen at ambient pressure was the disappearance of inhibitory junction potentials. Pressurization with helium produced a fall in Ginh at 5.1 ATA but no change or a slight increase at 1.7 ATA. The action of either 100% oxygen at ambient and at 1.7 or 5.1 ATA or helium at 1.7 or 5.1 ATA was shown to be on presynaptic parameters since the percent decrease in Ro induced by exogenous application of gamma-aminobutyric acid (GABA), the inhibitory transmitter, was the same in either 100% oxygen at ambient pressure, 100% oxygen or helium at 1.7 ATA and 5.1 ATA. The similarity in action of oxygen to the action of isoniazid, a known glutamic acid decarboxylase (the enzyme that catalyzes the production of GABA) inhibitor in the same preparation suggests that one possible site of oxygen action is on GABA production.

An electrophysiological analysis of oxygen and pressure on synaptic transmission.

The effect of oxygen at high pressure (OHP), helium at 150 PSIG and 100% oxygen at ambient pressure on excitatory synaptic transmission was studied using the lobster walking leg neuromuscular preparation. Both 100% oxygen at 150 PSIG (7135 mm Hg oxygen) and helium at 150 PSIG (7000 mm Hg helium plus 135 mm Hg oxygen) produced a significant decrease in the amplitude of the junction potential (Vejp). The decrease in Vejp induced by OHP, however, was greater than with pressure alone. OHP also produced a significant decrease in short term facilitation. Exposure to 100% oxygen at ambient pressure produced a transient increase in Vejp and a large increase in frequency of miniature junction potentials. In each case the change in Vejp was due to changes in presynaptic release of transmitter since quantal content per fiber (M') was shown to decrease for OHP and helium at 150 PSIG and to transiently rise with 100% oxygen at ambient pressure. In addition, the response to exogenously applied glutamate (the putative neurotransmitter) was not affected by OHP, 150 PSIG helium or 100% oxygen at ambient pressure. This further indicates a presynaptic site of action.

Trigeminal responses to thermal stimulation of the oral cavity in rattlesnakes (Crotalus viridis) before and after bilateral anesthetization of the facial pit organs.

Multiunit neural responses from the superficial maxillary branch of the trigeminal nerve in prairie rattlesnakes were elicited by intraoral thermal stimulation. The responses to oral stimulation were shown to be independent of responses obtained by thermal stimulation of the loreal pits. Histological examination of the dorsal lip, palate, and fang sheath regions revealed dense ramifying neurons in the epidermal layers of the fang sheaths that were morphologically similar to suspected infrared sensitive neurons in the pit membranes.

Characterization of interleukin-1 production by microglia in culture.

The production of interleukin-1 (IL-1) by cultured neonatal rat microglia was studied using the D10 cell assay. The results show that IL-1 was secreted in response to lipopolysaccharide (LPS) in a dose- and time-dependent fashion. IL-1 production was specific to microglia and was not induced in astrocytes. Indomethacin, which is known to modulate the release of IL-1 from monocytes, had no effect on LPS-stimulated microglia. Aging of the microglia from two weeks to 4 weeks in culture, however, reduced the release of IL-1 in response to LPS. Our data indicate that microglia are a major source of IL-1 and that the release of IL-1 depends on the presence of inflammatory mediators such as LPS and the age of the culture.