RNA‐Seq Analysis Reveals Ectopic Expression of Truncated Non‐Functional Serotonin Transporter in the Hippocampus of SERT‐KO Mice

The serotonergic system innervates the cortex, entorhinal cortex, hippocampus (HPC), nucleus accumbens, amygdala and anterior hypothalamus via serotonergic neurons arising from raphe nuclei in the mid and hindbrain. These structures are part of the limbic system, which is involved in emotions and motivation. Termini of the serotonergic neurons that innervate these limbic regions contain the serotonin transporter (SERT), which is a therapeutic target for many mood disorders. To date, insight into the link between SERT and its impact on these psychiatric conditions has been afforded by the use of a SERT‐deficient mouse line (SERT‐KO). Numerous studies have used this murine model to investigate serotonergic function in the HPC and links to neuropsychiatric disorders. In the present study, RNA‐Seq was performed on isolated HPC from 9 week old male C57BL6/J (WT) and B6.129(Cg)‐ Slc6a4 tm1Kpl /J (SERT‐KO) mice yielding 28–34 million mappable reads, indicating exceptional sequencing depth of the mouse genome. Reads were aligned to the mouse genome (mm10) using Tophat v2.0.13 and fragments were assigned to genes using Feature Counts v1.4.6. Differential gene expression analysis was performed with R/Bioconductor package DESeq2 v1.10.1 and analyzed with Ingenuity Pathway Analysis (IPA, Qiagen). Unexpectedly, an mRNA species capable of coding for amino acids 115 to 630 of SERT was detected in KO HPC at levels significantly higher than full‐length SERT in WT HPC samples (219 vs. 2 fragment counts, respectively). The truncated SERT transcript parallels the genetic mutation used to create the SERT‐KO mouse where exon 2 of the promoter region was replaced with a PGK neomycin‐polyA cassette. The altered 5′‐UTR region in the SERT‐KO mice appears to allow for ectopic expression of truncated SERT mRNA in the cell bodies of pyramidal, granule and/or glial cells residing in the HPC. Expression of SERT mRNA in the HPC of WT and KO mice was normalized to the reference gene Beta‐2‐microglobulin (B2M), revealing a 155‐fold increase in SERT mRNA in the KO mouse vs WT. IPA analysis of genes showing significant expression changes identified a number of genes involved in the “Unfolded Protein Response (UPR)” and “Endoplasmic Reticulum Stress” pathways consistent with ectopic expression of the truncated SERT mRNA which does not code for transmembrane domain 1 of SERT and likely result in mis‐ or unfolded protein. In addition, IPA identified the “Circadian Rhythm Signaling” pathway in line with previous reports looking at changes in circadian rhythm and REM sleep in SERT‐KO mice. IPA “upstream” analysis also suggests that expression changes of several genes were linked to brain‐derived neurotrophic factor (BDNF) and whereas BDNF expression changes were detected between WT and SERT‐KO samples, the difference did not reach significance. However, this analysis does not discriminate isoforms of BDNF, an area of future focus. Finally, although our study focuses on the HPC, other tissues including those outside the central nervous system could be affected by inappropriate SERT expression. Support or Funding Information NIH‐NIGMS P20 GM104360