Nogo-66 Receptor As A New Drug Target To Treat Alzheimer's Disease

ObjectivesTo find out whether Nogo-66 receptor has a dual role in the inhibition of amyloid P protein (A) generation and promotion of regeneration neurite, whether it can be a potential target for drug therapy of Alzheimer's disease (AD). To search AD therapy drugs which have a dual role in inhibition of Aβproduction and promotion of neurite regeneration.Methods(1)The neurons from cerebral cortex of newborn rat were cultured in B27-supplemented Neurobasal, a serum-free medium combination, and identified by using NSE and MAP2 immunocytochemical staining.(2)The rat cortical neurons were cultured in different cell densities and different concentrations of B27,to observe its effect on neuron growth.(3)The rat cortical neurons were cultured with various concenstration (3.5μM,7μM,14μM) of Nogo-P4 (Nogo-66 receptor agonist).The cell viability of neuron was determined by the MTT method.To observe the effect of Nogo-P4 on the neurite growth, the convert phase microscope was used to analyse the number of cell with neurites, as well as the average length of neurite. To observe the effect of Nogo-P4 on AP42 secretion of cortical neurons, enzyme-linked immunoadsordent assay (ELISA) was carried out to analyze AP42 production. (4) The mechanism of Nogo-P4 on cortical neurite regeneration and Aβ42 secretion were studied by adding Nogo-66 receptor blocker NEP1-40 and receptor downstream signaling molecule ROCK inhibitor Y-27632 and PKC inhibitor GO6976.(5)We search the effective compounds from traditional Chinese medicine by using Nogo-P4 treated cell model for drug screening.Results(1)Most of the neurons were stained to be positive with two different neuron-specific markers NSE and MAP-2 immunocytochemical, suggesting the majority of cultured cells were neurons.(2)Appropriate cell density (1,200 cells/mm2) and appropriate concentration of B27 (2%B27) were beneficial on the growth of neurons.(3) The MTT assay showed that Nogo-P4 at different concentrations didn't have any toxic effect on cortical neuron survival. Different concentraion of Nogo-P4 could reduce the number of cell with neurite and the average length of neurite.Moreover,Nogo-P4 significantly increased the production of Aβ42.(4)Nogo-66 receptor antagonists could promote neuronal regeneration, but could not reduce the generation of AP42.Use of PKC inhibitors could promote neuronal regeneration, and increased A042 generation.Use of ROCK inhibitors could not only promote neuron regeneration, but also reduce the generation of AP42.(5)Through drug screening, an effective compound SQ was found. SQ could antagonize Nogo-P4 to increase the number of cell with neurites and the average length of neurite. SQ did not reduce neuronal secretion of Aβ42.Conclusion(1) Nogo-P4 inhibits neurite regeneration of rat cortical neurons, while Nogo-P4 promotes Aβ42 secretion of rat cortical neurons.(2)Nogo-P4 inhibits neurite regeneration by directly activating Nogo-66 receptor and downstream signaling molecules ROCK and PKC.The Nogo-P4 promotes the Aβ42 secretion by directly activating downstream signaling molecules ROCK.(3)Nogo-66 receptor can be used as target promoting neurite regeneration, but not as a target for inhibition of Aβ42 generation, it can be used as target for AD drug therapy, but is not the best target. PKC can be used as target promoting neurite regeneration, but also to promote Aβ42 generation.So PKC is not appropriate to use as a target for AD drug therapy. ROCK is a target which can truly inhibit AP42 production and promote neurite regeneration, is an ideal drug target for AD therapy. Drugs targeting ROCK will be more effective. (4)SQ, the compound from traditional Chinese medicine, can promote neurite regeneration, but can not reduce AP42 production.The pharmacological target of SQ possibly is Nogo-66 receptor.