Furthermore, the expression of genes that are activated by c Myc and MycN decreases Inhibitors,Modulators,Libraries after NGF deprivation, for example id2 and ptma. Ptma can act as a repressor of the apoptosome so it will be interesting to determine whether Ptma pro tein levels also decrease after NGF withdrawal. Conver sely, the expression of genes repressed by c Myc and MycN increases after NGF deprivation, for example, ndrg1. Conclusions The sympathetic neuron model is one of the best stu died models of neuronal apoptosis. For the first time, we now have a global overview of the changes occurring at the transcriptional level in NGF deprived sympathetic neurons. In the future, it will be interesting to determine how Inhibitors,Modulators,Libraries the regulated genes identified in this study contri bute to the NGF withdrawal induced death pathway.
This may lead to the identification of new targets for the development of neuroprotective drugs that inhibit neuronal death following acute injuries to the nervous system or in neurodegenerative diseases. Methods Cell culture Animal experiments were performed according to the Animals Act 1986 under a license reviewed AV-951 and approved by the Biological Services Unit at University College London. Sympathetic neurons were isolated from the superior cervical ganglia of 1 day old Sprague Dawley rats and cultured in Dulbeccos modified Eagle medium supplemented with 10% fetal calf serum, 2 mM glutamine and penicillin streptomycin as described previously. To limit the proliferation of non neuro nal cells, the antimitotic agents fluorodeoxyuridine and uridine were Inhibitors,Modulators,Libraries added to the SCG medium at a final concentration of 20 uM.
For some experi ments, 2. 5S NGF was also added to SCG medium at a final concentration of 50 ng ml. Neu rons Inhibitors,Modulators,Libraries were plated on 13 mm diameter glass coverslips coated with poly L lysine and laminin placed in 3. 5 cm diameter dishes containing 2 ml of SCG medium and NGF for 5 7 days. In NGF withdrawal experiments, neu rons were washed twice in SCG medium lacking NGF and then refed with SCG medium supplemented with a neutralising anti NGF antibody at 100 ng ml. The MLK inhibitor, CEP 11004 was dissolved in DMSO and used at a final concentration of 400 nM. RNA extraction Total RNA was isolated from sympathetic neurons cul tured for 7 days using an RNeasy mini kit. An on column DNase digestion was performed to eliminate genomic DNA contamination using DNase I according to the manufacturers instructions.
RNA con centrations were determined using a NanoDrop spectro photometer. RNA was further analysed for integrity and quality on an Agilent Bioanalyser. Array hybridisation Up to 2 ug of total RNA was processed and labelled using the Affymetrix GeneChip Whole Transcript Sense Target Labelling Assay as outlined in the manufacturers instructions. Hybridisation to Affymetrix Rat Exon 1.