The trophic effect of nerve growth factor in primary cultures of rat hippocampal neurons is associated to an anti-inflammatory and immunosuppressive transcriptional program

Nerve growth factor, the prototype of a family of neurotrophins, elicits differentiation and survival of peripheral and central neuronal cells. Although its neural mechanisms have been studied extensively, relatively little is known about the transcriptional regulation governing its effects. We have previously observed that in primary cultures of rat hippocampal neurons treatment with nerve growth factor for 72 hours increases neurite outgrowth and cell survival. To obtain a comprehensive view of the underlying transcriptional program, we performed whole-genome expression analysis by microarray technology. We identified 541 differentially expressed genes and characterized dysregulated pathways related to innate immunity: the complement system and neuro-inflammatory signaling. The exploitation of such genes and pathways may help interfering with the intracellular mechanisms involved in neuronal survival and guide novel therapeutic strategies for neurodegenerative diseases. Whole genome expression profiling by microarray technology was performed in HNs following treatment with vehicle (Control) or Ngf (100 ng/ml) for 72h. Total RNA was extracted with Trizol (Life Technologies, Monza, Italy) from four biological replicates (derived from the same litter) for each of the two experimental conditions examined: vehicle and Ngf treated HNs. RNA integrity was confirmed by using a RNA chip and a 2100 Bioanalyzer (Agilent Technologies), with the protocol outlined by the manufacturer. Complementary RNAs (cRNAs) labeled with Cy3-CTP were synthesized from 1 μg of total RNA of each sample using the Low RNA Input Fluorescent Linear Amplification Kit (Agilent Technologies), following the manufacturer's protocol. Aliquots (750 ng) of Cy3-labeled cRNA targets were hybridized on 8 ×64K Whole Rat Genome Oligo expression microarrays (Agilent Technologies). Microarray hybridization and washing were performed using reagents and instruments (hybridization chambers and rotating oven) as previously indicated (Cavallaro, 2015b). Significant changes in gene expression levels were evaluated by a Moderated T-Test followed by the Westfall-Young procedure as a multiple testing correction. Genes with a corrected P value < 0.05 were considered as differentially expressed.