Cholesterol reduction by methyl-beta-cyclodextrin attenuates the delta opioid receptor-mediated signaling in neuronal cells but enhances it in non-neuronal cells

Opioid receptors have been shown to be located in and regulated by lipid rafts/caveolae in caveolin-rich non-neuronal cells. Here, we found that caveolin-1 level was very low in rat brain and undetectable in NG108-15 cells, which endogenously express delta opioid receptors (DOR). Rat caudate putamen (CPu) membranes, NG108-15 cells and CHO cells stably transfected with FLAG-mouse-DOR (CHO-FLAG-mDOR) were homogenized, sonicated in a detergent-free 0.5M Na(2)CO(3) buffer and fractionated through discontinuous or continuous sucrose density gradients. About 70% of opioid receptors in CPu and DOR in both cell lines were present in low-density (5-20% sucrose) membrane domains enriched in cholesterol and ganglioside M1 (GM1), characteristics of lipid rafts in plasma membranes. In both cells, stimulation with permeable or non-permeable full agonists, but not with partial or inverse agonists, for 30min shifted approximately 25% of DORs out of rafts, by a naloxone-reversible and pertussis toxin-insensitive mechanism, which may undergo internalization. Methyl-beta-cyclodextrin (MCD) treatment greatly reduced cholesterol and shifted DOR to higher density fractions and decreased DPDPE affinities. MCD treatment attenuated DPDPE-induced [(35)S]GTPgammaS binding in CPu and NG108-15 cells, but enhanced it in CHO-FLAG-mDOR cells. In CHO-FLAG-mDOR cells, G(alphai) co-immunoprecipitated with caveolin-1, which was shown to inhibit G(alphai/o), and MCD treatment dramatically reduced the association leading to disinhibition. Thus, although localization in rafts and agonist-induced shift of DOR are independent of caveolin-1, lipid rafts sustain DOR-mediated signaling in caveolin-deficient neuronal cells, but appear to inhibit it in caveolin-enriched non-neuronal cells. Cholesterol-dependent association of caveolin-1 with and the resulting inhibition of G proteins may be a contributing factor.     

Total ganglioside ablation at mouse motor nerve terminals alters neurotransmitter release level

Neuronal membrane gangliosides, forming a large family of sialylated glycosphingolipids, have been hypothesized to play important roles in synaptic transmission. We studied the ex vivo electrophysiological function of neuromuscular junctions of GM2/GD2‐synthase*GD3‐synthase compound null‐mutant mice after acute removal of GM3, the only remaining ganglioside in this mouse, by in vitro treatment with neuraminidase. We found 16% enhancement of the acetylcholine release per nerve impulse at low‐rate (0.3 Hz) nerve stimulation. Conversely, the treatment reduced the acetylcholine release evoked by high‐rate (40 Hz) nerve stimulation. Also, 25 ms paired‐pulse facilitation of endplate potentials was reduced by the neuraminidase‐treatment. These effects may indicate a modest modulatory influence of the negative electrical charges carried by the sialic acid molecules of gangliosides on the function of presynaptic Ca_v2.1 channels, affecting the magnitude and kinetics of the Ca²⁺ influx that induces neurotransmitter release from the motor nerve terminal. Our results show that gangliosides are to some extent involved in neurotransmission at the neuromuscular junction, but that their presence is not an absolute requirement in this process. Synapse 64:335–338, 2010. © 2009 Wiley‐Liss, Inc.     

Identification of mouse type-2-like astrocytes: demonstration of glutamate and GABA transmitter activated responses.

We have identified mouse type-2-like astrocytes and examined some of their electrophysiological properties. Cultures were prepared from P4 mouse neopallia. We demonstrate that mouse type-2-like astrocytes can be identified using the following criteria: presence of glial fibrillary acidic protein (GFAP), presence of chondroitin sulfate polysaccharide, and presence of gamma-aminobutyric acid (GABA). A2B5-binding is not a sufficient criterion to identify O2A lineage cells in mouse neopallial glial cultures since the monoclonal antibody A2B5 binds not only to O2A lineage cells but also to a subpopulation of large, flat type-1-like astrocytes. Mouse type-2-like astrocytes have resting membrane potentials of -76.2 +/- 2.1 mV-i.e., similar to that of mouse type-1-like astrocytes. The input resistance of 44.2 +/- 0.5 M omega is an order of magnitude greater than that of type-1-like astrocytes suggesting the type-2-like astrocytes are not extensively electrically coupled either to each other or to type-1-like astrocytes. Glutamate application caused an 8.8 +/- 1.7 mV depolarization of type-2-like astrocytes. Application of glutamate to barium treated astrocytes caused a fast depolarization with a peak amplitude of 21.4 +/- 1.8 mV; the cells repolarized from this peak by about 10 mV and upon removal of glutamate returned to its pre-glutamate value. Application of GABA caused a transient depolarization of 14.0 +/- 1.7 mV. The presence of barium resulted in a steady-state GABA-induced depolarization of 10.3 +/- 2.0 mV. Neither SITS nor beta-alanine interfered with the amplitude of the glutamate and GABA responses.     

Patterns of Ganglioside Expression in B Cell Neoplasms

Twenty seven B cell neoplasms were examined by high performance thin layer chromatography (HPTLC) and immune thin layer chromatography (ITLC) to determine ganglioside expression. Patterns of expression in the cells were compared with conventional morphology, genotype, and glycoprotein immunophenotype. Patterns of ganglioside expression were found for each of the tumor types analyzed (5 acute lymphoblastic lymphomas (ALL), 5 Burkitt's Lymphomas (BL), 4 chronic lymphocytic leukemias (CLL), 3 diffuse poorly differentiated lymphomas (DPDL), 7 diffuse histiocytic lymphomas (DHL), and 3 multiple myelomas (MM). GM3 was the predominant ganglioside found in all B cell neoplasms except multiple myeloma where GM2 was equivalent to GM3. GM1 was detected by ITLC in all B cell tumors, but significant amounts were found by HPTLC only in ALL, CLL, and DHL. Small amounts of GD3 and GD2 were found in several B cell neoplasms. Significant amounts of other gangliosides were not found. The expression of GM2 on the MM cell lines, a cell type derived from outside of the nervous system, is unusual. This high level of expression was also seen in metabolic labeling studies. GM2 was readily detectable in the SKMM1 human multiple myeloma cell line by flow cytometry and served as a target for human complement-mediated cytotoxicity. Although the functions of gangliosides are largely unknown, the patterns of gangliosides found for this system of human B cell malignancies may serve to provide targets for specific immunotherapy and clues to their functions.     

The effect of GM1 ganglioside on coerulospinal, noradrenergic, adult neurons and on fetal monoaminergic neurons transplanted into the transected spinal cord of the adult rat

GM₁ ganglioside, thyroxine and hydrocortisone were tested for their ability to improve the survival and growth of fetal focus coeruleus noradrenergic neurons in the transected, adult spinal cord. GM₁ alone was also tested for its effect on fetal mesencephalic dopaminergic neurons implanted into a small dorsolateral cavity at the L2 region of the cord previously transected at the T9–T10 region. None of the substances tested had any measurable effect on either of the fetal implants. However, in the GM₁- and thyroxine-treated animals the somatic dendrites of the axotomized, noradrenergic, coerulospinal neurons appeared more robust, and more intensely fluorescent, compared to their appropriate controls. GM₁ also caused a pronounced sprouting of the axotomized monoaminergic (catecholaminergic and serotonergic) fibres in the rostral region of the cord adjacent to the transection site. All of the mesencephalic dopaminergic implants survived in both the GM₁-treated animals and their saline-injected controls. However, their development was apparently not influenced by GM₁. The results indicate that GM₁ and thyroxine can enhance those aspects of the reactive mechanisms of mature, axotomized, noradrenergic coerulospinal neurons that promote their regeneration. As such, GM₁ could become a useful tool in current attempts to foster the regeneration of damaged monoaminergic neurons in the mammalian CNS.     

CD4+ T Cell–mediated Granulomatous Pathology in Schistosomiasis Is Downregulated by a B Cell–dependent Mechanism Requiring Fc Receptor Signaling

The effector functions of CD4⁺ T lymphocytes are generally thought to be controlled by distinct populations of regulatory T cells and their soluble products. The role of B cells in the regulation of CD4-dependent host responses is less well understood. Hepatic egg granuloma formation and fibrosis in murine schistosomiasis are dependent on CD4⁺ lymphocytes, and previous studies have implicated CD8⁺ T cells or cross-regulatory cytokines produced by T helper (Th) lymphocytes as controlling elements of this pathologic process. In this report, we demonstrate that B cell–deficient (μMT) mice exposed to Schistosoma mansoni develop augmented tissue pathology and, more importantly, fail to undergo the spontaneous downmodulation in disease normally observed during late stages of infection. Unexpectedly, B cell deficiency did not significantly alter T cell proliferative response or cause a shift in the Th1/Th2 balance. Since schistosome-infected Fc receptor–deficient (FcR γ chain knockout) mice display the same exacerbated egg pathology as that observed in infected μMT mice, the B cell– dependent regulatory mechanism revealed by these experiments appears to require receptor-mediated cell triggering. Together, the data demonstrate that humoral immune response/FcR interactions can play a major role in negatively controlling inflammatory disease induced by CD4⁺ T cells.     

神经节苷脂对脑外伤大鼠大脑皮质和海马区GAP-43表达的影响

目的观察神经节苷脂（GM1）对脑外伤大鼠大脑皮质和海马区神经生长相关蛋白-43（GAP-43）表达的影响,探讨GM1促进脑外伤神经修复的可能机制。方法选择出生10周雄性SD大鼠200只,随机分为假手术组60只,无脑外伤,术前腹腔注射生理盐水3 d。脑外伤模型140只,分为模型组70只,术前腹腔注射生理盐水3 d;GM1组70只,术前腹腔注射GM13 d。应用酶联免疫法检测大鼠大脑顶叶皮质和海马区GAP-43于0～24 h不同时间点的表达情况,并在术后1～5 d不同时间点进行行为学检测。结果 （1）GM1组大鼠大脑顶叶皮质0～16 h和海马区0～24 h GAP-43的表达均明显低于模型组（P〈0.01）,而与假手术组比较,大鼠顶叶皮质区8～24 h GAP-43的表达,差异有统计学意义（P〈0.01）。（2）GM1组大鼠大脑顶叶皮质GAP-43的表达,术后随着时间的增加而升高,至24 h已达模型组水平。（3）假手术组的行为学表现1～5 d均优于模型组和GM1组（P〈0.01）,GM1组在术后2～5 d运动能力逐步增强,行为学表现优于模型组（P〈0.05）。结论预防性使用GM1有效地促进大鼠大脑皮质GAP-43的表达升高,降低脑外伤大鼠脑损伤程度,从而发挥其对实验性脑外伤大鼠的神经保护作用。     

单唾液酸四己糖神经节苷脂对重型颅脑损伤患者血清超敏 C 反应蛋白、白介素-6 和肿瘤坏死因子-α 水平的影响及疗效观察

目的 探讨单唾液酸四己糖神经节苷脂对重型颅脑损伤患者血清超敏C反应蛋白（hs-CRP）、白介素（IL）-6和肿瘤坏死因子（TNF）-α水平的影响及疗效观察.方法 选取2011年1月～2013年2月浙江省天台县人民医院ICU住院治疗的重型颅脑损伤患者78例,随机分为观察组39例和对照组39例.两组均予以降颅压、抗感染、亚低温治疗和预防并发症等常规治疗.观察组加用单唾液酸四己糖神经节苷脂静滴100 mg/次,1次/d,连用14 d.观察两组治疗前和治疗14 d后血清hs-CRP、IL-6和TNF-α水平的变化,并随访治疗后2个月的临床效果.结果 两组治疗前血清hs-CRP、IL-6和TNF-α水平比较差异无统计学意义（t=0.08、0.09、0.13,P＞0.05）.治疗14 d后,两组血清hs-CRP、IL-6和TNF-α水平[对照组（16.38±4.23）μg/L、（32.16±8.76）μg/L、（14.57±4.12）μg/L;观察组（11.45±3.12）μg/L、（21.15±7.24）μg/L、（9.14±2.76） μg/L]均较治疗前[对照组（23.12±6.15）μg/L、（47.86±10.16）μg/L、（19.82±5.68） μg/L;观察组（22.94±6.48） μg/L、（48.07±9.79）μg/L、（20.05±6.04）μg/L]明显下降,差异有统计学意义（P＜ 0.05或P＜0.01）;且观察组治疗14 d后hs-CRP、IL-6和TNF-α水平明显低于对照组,差异均有统计学意义（P＜0.05）.随访2个月,观察组临床总有效率（89.74％）明显高于对照组（69.23％）,差异有统计学意义（x2=5.03,P＜ 0.05）.结论 单唾液酸四己糖神经节苷脂用于重型颅脑损伤具有较好效果,作用与其降低血清hs-CRP、IL-6和TNF-α水平密切相关.     

单唾液酸四己糖神经节苷脂治疗新生儿缺氧缺血性脑病的疗效及安全性

目的探讨单唾液酸四己糖神经节苷脂治疗新生儿缺氧缺血性脑病(HIE)疗效及安全性。方法 86例HIE患儿随机分为实验组和对照组。两组均予以常规治疗,实验组加用单唾液酸四己糖神经节苷脂钠针20 mg静滴,1次/d,连用10 d。结果治疗10 d后,实验组意识恢复时间、吸吮能力恢复时间、肌张力恢复时间、原始反射恢复时间均短于对照组(P<0.05或P<0.01);两组患儿NANB评分均较前明显改善(P<0.01),且实验组改善幅度优于对照组(P<0.05);治疗10 d后,实验组临床总有效率明显优于对照组(χ2=4.44,P<0.05),两组患儿均未出现明显不良反应。结论单唾液酸四己糖神经节苷脂治疗HIE能明显改善临床症状,提高治疗效果,安全性好。     

综合疗法联合神经节苷脂治疗中老年重症脑卒中的疗效

目的探讨综合疗法联合神经节苷脂治疗中老年重症脑卒中的疗效。方法选择2010年9月至2012年9月在重庆市第五人民医院确诊为中老年重症脑卒中患者150例,利用随机数字表法分为试验组和对照组,每组75例,试验组采用综合疗法联合神经节苷脂法,对照组采用综合疗法。比较两组患者治疗前后的血清参数-神经元特异性烯醇化酶(NSE)、超氧化物岐化酶(SOD)以及血流动力学参数,并分析神经功能缺损程度(NDS)、日常生活活动量(ADL)等临床指标。结果试验组经治疗后的血清中NSE、ODS的浓度显著低于对照组[(13.96±1.18)μg/L vs(25.16±0.45)μg/L,(8.39±0.63)μmol/L vs(14.71±0.66)μmol/L,P<0.05];全血高切黏度、全血低切黏度显著低于对照组[(3.68±1.05)m Pa·s vs(5.67±1.34)m Pa·s,(6.95±0.72)m Pa·s vs(9.73±0.86)m Pa·s,P<0.05],红细胞沉降率显著高于对照组[(26.2±1.1)mm/h vs(17.9±1.2)mm/h,P<0.05];NDS评分显著低于对照组[(12.2±2.1)分vs(23.5±1.8)分,P<0.05];ADL评分显著高于对照组[(75.5±1.2)分vs(57.4±1.4)分,P<0.05]。结论综合疗法联合神经节苷脂能够改善病变的血管,修复受损的神经系统,临床应用价值高。