Analysis of neuronal transcriptome and functional studies of memory related-genes in Aplysia

Abstract

To study the molecular and cellular mechanism of learning and memory, marine mollusk Aplysia has been used for a long time. While extensive research has been performed, however, lack of its large size genomic database is the major limiting factor for investigation of the molecular mechanism of learning and memory in Aplysia. Especially, it has been established that activation of cAMP-PKA pathway is critical for memory consolidation in Aplysia. However, the 5-HT receptor which activates adenylyl cyclase has not been cloned. To overcome this limitation, high-throughput analysis based on the EST database of closely related species, Aplysia kurodai and Aplysia californica, was performed in the present study. Furthermore, a number of novel learning-related genes were cloned and characterized based on the high-throughput transcriptome analysis. Firstly, some putative learning related genes were identified based on the 5-HT induced expression profile. It is well known that 5-HT not only activates several signal transduction pathways such as PKA and PKC, but also regulates expression of several learning-related genes in Aplysia neurons. To identify 5-HT regulated genes, microarray was performed as a primary screening and the hits were validated by quantitative RT-PCR analysis. Based on the screen, a number of candidate genes involved in the molecular mechanism of learning and memory were discovered. Secondly, to investigate the more efficient way to identify learning-related genes, evolutionary analysis based on the expressed sequence tag (EST) databases of A. kurodai and A. californica was performed. Calculated evolutionary rate of each gene suggests that eliminating genes with extremely low evolutionary rates can be an effective way to find candidate genes for the learning and memory study, because many housekeeping genes show relatively low evolutionary rates. However, the probability of neuronal expression of certain genes could not be predicted by calculating the Ka/Ks value of the genes. Thirdly, the relationship between the AU-rich element(ARE) binding protein, ApAUF1, and the well-studied transcription factor, ApC/EBP was examined. Overexpressed ApAUF1 was localized to the cytosol and neurites. Data obtained from microarray and EST databases suggested that ApAUF1 may regulate ApC/EBP mRNA. ApC/EBP mRNA has several AREs in its 3UTR, and the ApAUF1 is specifically bound to the region. These results indicated that the ApAUF1 may function as a negative regulator of ApC/EBP mRNA. Finally, the 5-HT receptor which is positively coupled to adenylyl cyclase, was cloned based on the EST database and sequence analysis. The cloned receptor, 5-HTapAC1, has strong homology to other invertebrate 5-HT7 receptors. It is expressed in the sensory and motor neurons, and localized to plasma membrane and the synaptic region of sensory neurons. 5-HTapAC1 is specifically coupled to Gs, but not Gq, and also has an important role in the activation of the cAMP-PKA pathway by 5-HT treatment. Moreover, knock-down of the receptor blocks the induction of synaptic facilitation in non-depressed synapses as well as the reversal of synaptic depression called dishabituation in moderately depressed synapses.