Long non-coding RNAs in formation of memory through long-term potentiation Jesper L. V. Mååg, Karin Wibrand, Debabrata Panja, Clive R. Bramham & Marcel Dinger Control (log FPKM) Stimulated (log FPKM) Log2(Stimulated/Control) F i g u r e 8 : T h e t o p 2 0 0 d i ff e r e n t i a l l y e x p r e s s e d g e n e s b e t w e e n H F S & c o n t r o l s . Activation Signal Activation Signal Repeated stimulation The mechanisms of Long-term potentiation (LTP). Increased stimulation over time leads to increased connectivity between the synaptic cleft. This strengthens the signal between two neurons as they get more receptors and signalling molecules. The process is thought to be involved in memory formation. 1 1 2 2 2 Genome Informatics, The Kinghorn Cancer Centre, Garvan Institute of Medical Research. Department of Biomedicine, K.G. Jebsen Centre for Research on Neuropsychiatric Disorders, University of Bergen 1 2 Figure 1. Long-term potentiation Figure 2. Methodology for LTP induction: Four rats were anesthetised with urethane 1.5g/kg. Z Z Z High frequency stimulation (HFS) in the right Dentate Gyrus (DG). 400Hz x 8, 4 times in 10s interval. Repeated 3 times with 5min inbetween. DG DG from the right brain half was surgically removed and RNA was extracted. The left DG was used as a control. RNA-seq was performed on the samples. Rat 1 Rat 2 Rat 3 Rat 4 0 30 60 90 120 Animals Fold change HFS/Control Animals Rat 1 Rat 2 Rat 3 Rat 4 Arc mRNA expression in HFS samples Arc is a protein that is upregulated after HFS. qPCR of Arc was used as a positive control for successful stimulation. One rat failed, and was subsequently removed. Control Control Control Stimulated Stimulated Stimulated Multidimensional Scaling (MDS) visualises the similiarities between samples. As seen here Stimulated samples and Controls clusters closer to respective group. Figure 3. Clustering of samples: Stimulated 1 Stimulated 2 Stimulated 3 Control 1 Control 2 Control 3 Log(FPKM+1) 10 5 0 Differential expression analysi s was conducted with the Tophat/Cufflinks suite as well as de novo transcriptional assembly with Trinity. The heatmap shows the 733 Differentiallyexpressed genes according to Cuffdiff. Known protein coding genes, and miRNA, that have previously been shown to play a role in LTP were shown to be differentially expressed between control and stimulated samples. Novel LncRNAs were among the differentially expressed genes. These lacked coding potential according to CPC. EdgeR DESeq Cuffdiff Figure 4. Differential expression: Figure 5. Comparison between different programs: Overlap of DE genes between EdgeR, DESeq, and Cuffdiff. Most samples found in DESeq were also covered in EdgeR. Cuffdiff showed similarities to the two. Around ~1/3 were unique to Cuffdiff or EdgeR. es Stimulated Control ESTs Refseq Arc Mir212 Mir132 Figure 6. Examples of differentially expressed genes Examples of differentially expressed genes. Top two: Arc, and Mir 212, Mir 132 have previously been associated with long-term potentiation. Bottom two : Examples of novel lncRNAs identified in this study. 0 2 4 2h HFS+ 2h CPP 2h HFS CA 2h HFS DG Condition Fold change HFS/Control LncRNA1 LncRNA2 qPCR of novel LncRNA Figure 7. qPCR of two LncRNA qPCR of the lncRNA shown in figure 6. The figure represent different conditions. Stimualtion in Dentate Gyrus , in Cornu Ammonis , and DG stimulation plus CPP. The stimulation is localised to DG, with only minimal effect in CA. CPP, a NMDA-R antagonist known to supress LTP, inhibits stimulations in the DG. (n=5) The top 200 differentially expressed genes identified by using the Tophat/Cufflink suite. As seen in the green circle, the majority of all genes were upregulated in the HFS samples compared to control. Conclusio n : LncRNA s have been shown to have a wide variety of biological functions in vivo. Mercer, T.R. et al., 2007. Noncoding RNAs in Long-Term Memory Formation. The Neuroscientist Roberts, A. et al., 2012. Differential gene and transcript expression analysis of RNA-seq experiments with TopHat and Cufflinks. Nature protocol Shepherd, J.D. & Bear, M.F., 2011. New views of Arc, a master regulator of synaptic plasticity. Nature Neuroscience Wibrand, K. et al., 2006. Identification of genes co-upregulated with Arcduring BDNF-induced long-term potentiation in adult rat dentate gyrus in vivo. European Journal of Neuroscience References: LncRNA1 LncRNA2 * 6 * 432 223 358 215 72 Here we showed that lncRNAs potentially play a role in the formation of long term potentiation, which is used as a model for memory formation. This was the first study to examine the global transcription patterns after LTP. We identified over 700 transcripts that were DE in this model. Further studies will correlate how these transcripts function in the brain, and how these could be linked to neurodegenerative disorders such as Alzheimer’s and Parkinson’s Disease.