Yaar M, Zhai S, Panova I, Fine R E, Eisenhauer P B, Blusztajn J K, Lopez-Coviella I, Gilchrest B A
Department of Dermatology, Boston University School of Medicine, Boston, MA 02118-2394, USA.
Neuropathol Appl Neurobiol. 2007 Oct;33(5):533-43. doi: 10.1111/j.1365-2990.2007.00844.x. Epub 2007 Jun 27.
The current study determined the ability of a p75(NTR) antagonistic cyclic peptide to rescue cells from beta amyloid (Abeta) (1-40)-induced death. p75(NTR)-, p140(trkA)-NIH-3T3 cells or E17 foetal rat cortical neurones were incubated with 125I-NGF or 125I-Abeta (1-40) and increasing concentrations of the cyclic peptide (CATDIKGAEC). Peptide ability to displace 125I-NGF or 125I-Abeta (1-40) binding was determined. Duplicate cultures were preincubated with CATDIKGAEC (250 nM) or diluent and then stimulated with Abeta (1-40). Peptide ability to displace Abeta (1-40) binding, interfere with Abeta (1-40)-induced signalling and rescue cells from Abeta-mediated toxicity was determined by immunoprecipitation and autoradiography, Northern blotting, JNK activation, MTT and trypan blue assays. The peptide inhibited NGF and Abeta (1-40) binding to p75(NTR), but not to p140(trkA). Abeta (1-40) induced c-jun transcription (57.3% +/- 0.07%) in diluent-treated p75(NTR)-cells, but not in cells preincubated with the cyclic peptide. Also, at 250 nM, the peptide reduced Abeta (1-40)-induced phosphorylation of JNK by 71.8% +/- 0.03% and protected neurones against Abeta-induced toxicity as determined by: trypan blue exclusion assay (53% +/- 11% trypan blue-positive cells in diluent pretreated cultures vs. 28% +/- 5% in cyclic peptide-pretreated cultures); MTT assay (0.09 +/-0.03 units in diluent-pretreated cells vs. 0.12 +/- 0.004 units in cyclic peptide-pretreated cells); and visualization of representative microscopic fields. Our data suggest that a cyclic peptide homologous to amino acids 28-36 of NGF known to mediate binding to p75(NTR) can interfere with Abeta (1-40) signalling and rescue neurones from Abeta (1-40)-induced toxicity.
本研究确定了一种p75（神经营养因子受体p75亚型，NTR）拮抗环肽将细胞从β淀粉样蛋白（Aβ）（1 - 40）诱导的死亡中拯救出来的能力。将p75（NTR）-、p140（酪氨酸激酶受体A，trkA）-NIH - 3T3细胞或E17胎鼠皮质神经元与125I - 神经生长因子（NGF）或125I - Aβ（1 - 40）以及浓度递增的环肽（CATDIKGAEC）一起孵育。测定该肽取代125I - NGF或125I - Aβ（1 - 40）结合的能力。将重复培养物用CATDIKGAEC（250 nM）或稀释剂预孵育，然后用Aβ（1 - 40）刺激。通过免疫沉淀和放射自显影、Northern印迹、应激活化蛋白激酶（JNK）激活、噻唑蓝（MTT）和台盼蓝测定法，确定该肽取代Aβ（1 - 40）结合、干扰Aβ（1 - 40）诱导的信号传导以及将细胞从Aβ介导的毒性中拯救出来的能力。该肽抑制NGF和Aβ（1 - 40）与p75（NTR）的结合，但不抑制与p140（trkA）的结合。在稀释剂处理的p75（NTR）细胞中，Aβ（1 - 40）诱导c - jun转录（57.3%±0.07%），但在与环肽预孵育的细胞中未诱导。同样，在250 nM时，该肽使Aβ（1 - 40）诱导的JNK磷酸化降低了71.8%±0.03%，并保护神经元免受Aβ诱导的毒性，这通过以下方法确定：台盼蓝排斥试验（稀释剂预处理培养物中53%±11%的台盼蓝阳性细胞，而环肽预处理培养物中为28%±5%）；MTT试验（稀释剂预处理细胞中为0.09±0.03单位，环肽预处理细胞中为0.12±0.004单位）；以及代表性显微镜视野的观察。我们的数据表明，一种与已知介导与p75（NTR）结合的NGF的28 - 36位氨基酸同源的环肽可以干扰Aβ（1 - 40）信号传导，并将神经元从Aβ（1 - 40）诱导的毒性中拯救出来。
