Long non-coding RNAs in formation of memory through long-term potentiationJesper L. V. Mååg, Karin Wibrand, Debabrata Panja, Clive R. Bramham & Marcel Dinger

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he top 200 di�erentially expressed genes between HFS & controls.

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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.

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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 Bergen1 2

Figure 1. Long-term potentiation

Figure 2. Methodology for LTP induction:

Four rats were anesthetisedwith urethane1.5g/kg.

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High frequency stimulation (HFS) in the right Dentate Gyrus (DG).400Hz x 8, 4 times in 10s interval.Repeated 3 times with 5min inbetween.

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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.

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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 successfulstimulation. One rat failed, and was subsequently removed.

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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:

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Di�erential expressionanalysiswas conducted with the Tophat/Cu�inks suite as well as de novo transcriptionalassembly with Trinity. The heatmap shows the 733 Di�erentiallyexpressed genes according to Cu�di�. Known protein coding genes,and miRNA, that have previously been shown to playa role in LTP were shown to be di�erentially expressed betweencontrol and stimulated samples.Novel LncRNAs were among thedi�erentially expressed genes.These lacked coding potentialaccording to CPC.

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Figure 4. Di�erential expression:

Figure 5. Comparison between di�erent programs:

Overlap of DE genes between EdgeR, DESeq, and Cu�di�. Most samples found in DESeq were also covered in EdgeR. Cu�di� showed similarities tothe two. Around ~1/3 were unique to Cu�di� or EdgeR.

RefSeq Genes

Rat ESTs That Have Been SplicedRefSeq Genes

Rat mRNAs from GenBankoverlay of RNAseq Tracks from rat brain potentiation study controls

overlay of RNAseq Tracks from rat brain potentiation study HFS

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Mir212 Mir132

Figure 6. Examples of di�erentially expressed genes

Examples of di�erentially expressed genes. Top two: Arc, and Mir 212, Mir 132 have previously been associated with long-term potentiation. Bottom two : Examples of novel lncRNAs identi�ed in this study.

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qPCR of novel LncRNAFigure 7. qPCR of two LncRNA

qPCR of the lncRNA shown in �gure 6. The �gure representdi�erent conditions. Stimualtion in Dentate Gyrus, in Cornu Ammonis, and DG stimulation plus CPP. The stimulation is localised to DG, with only minimal e�ect in CA. CPP, a NMDA-Rantagonist known to supress LTP, inhibitsstimulations in the DG.(n=5)

The top 200 di�erentially expressed genes identi�ed by using the Tophat/Cu�ink suite. As seen in the green circle, the majority of all genes were upregulated in the HFSsamples compared to control.

Conclusion:LncRNAs 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 NeuroscientistRoberts, A. et al., 2012. Di�erential gene and transcript expression analysis of RNA-seq experiments with TopHat and Cu�inks. Nature protocolShepherd, J.D. & Bear, M.F., 2011. New views of Arc, a master regulator of synaptic plasticity. Nature NeuroscienceWibrand, K. et al., 2006. Identi�cation of genes co-upregulated with Arcduring BDNF-induced long-term potentiation in adult rat dentate gyrus in vivo. European Journal of Neuroscience

References:

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Here we showed that lncRNAspotentially play a role in the formation of long termpotentiation, which is used as a model for memory formation.

This was the �rst study toexamine the global transcription patterns after LTP. We identi�ed over 700transcripts 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 disorderssuch as Alzheimer’s and Parkinson’s Disease.