5.2.1 RNA sequencing
As TCF4 is a transcription factor, we expect the phenotype changes to be driven primarily by changes in gene expression on RNA level. Therefore we performed RNAseq. We found up-regulation of Mov10and downregulation of Adora2a, Penk, Tac1, Drd1a, Pde10a, Pde1band Foxp2in PFC ofTcf4tg mice. In hippocampus,Top3b, BC1andPlxna1were up- andMov10was downregulated.
Genes downregulated in PFC ofTcf4tg mice:
Adora2aencodes the A2A receptor of adenosine — a widespread inhibitory neuromodulator in the brain, important for fine-tuning and synchronization of neuronal activity, sleep homeostasis, hypoxia, sensorimotor gating and cognition (reviewed in242–244). According to the
adenosine-hypofunction hypothesis of schizophrenia242, A2A receptors are proposed as a target for anty-psychotic drugs, since they have antagonistic actions to dopamine D2 receptors244. Adenosine receptors are blocked by caffeine243, which in high doses has prosychotic242,244and anxiogenic245 effects. ADORA2Ais also linked to panic disorder245 andAdora2a-/-mice show increased anxi-ety243.
Penkencodes proenkephalin A, an endogenous opioid polypeptide hormone that after proteolytic cleavage gives rise to enkephalins246. Enkephalins bind toδ-opioid receptors and both are involved in analgesia, reward and anxiety (reviewed in247). Downregulation ofPenkin PFC has been linked to schizophrenia248,249and postpartum psychosis250.
Drd1a encodes the dopamine receptor D1, which regulates AMPAR phosphorylation and trafficking to the membrane, and thus is a prominent modulator of synaptic plasticity in PFC (reviewed in101). Alterations in D1 activity during adolescence may have a particularly strong effect on synaptic plasticity and cortical development101.
Tac1encodes Protachykinin-1, a precursor of several peptides, e.g. Substance P, associated with pain perception and neuropsychiatric disorders (reviewed in251).TAC1is downregulated in the PFC of psychotic patients252.
Pde10aand Pde1b, both highly expressed in striatum, encode cAMP and cAMP-inhibited cGMP 3’,5’-cyclic phosphodiesterases. They regulate cAMP/PKA and cGMP/PKG signalling and inhibitors of PDE10A are proposed as drugs for schizophrenia8,253.
Foxp2 encodes a transcription factor crucial for human speech254, singing in birds255 and sensorimotor integration in mice (reviewed in256). As one of theTCF4risk alleles influences verbal memory70,Foxp2is a highly interesting candidate. Many targets and partners of FOXP2 have been associated with psychiatric diseases, including schizophrenia (reviewed in256).
Mov10(Putative helicase MOV-10) was upregulated in PFC and downregulated in the hippo-campus of tg mice. It is an element of the RNA-induced silencing complex (RISC) that silences mRNA expression, after binding microRNAs (reviewed in257).
Genes upregulated in the hippocampus ofTcf4tg mice:
BC1, Brain cytoplasmic RNA, is a small non-coding RNA. Its transcription is regulated through an E-box258, which can be a target of TCF4. Our RNA isolation protocol captures only mRNAs, butBC1was detected probably due to its polyA region259. BC1represses translation predominantly in dendritic synapses260,261. Upon neuronal activityBC1inhibits activity-induced increases of Fragile X mental retardation protein 1 (FMRP) and PSD95 in mouse hippocampus262. BC1 -/-mice appear healthy, breed well263 and show normal spatial learning264. However, they display high anxiety264, increasedγ-oscillations in EEG, neuronal hyperexcitability and startle-induced seizures262. BC1is present only in rodents, thus it has low relevance for human patients.
Top3β, encoding DNA topoisomerase 3-β-1, was upegulated in hippocampus ofTcf4tg mice.
In humansTOP3β is associated with schizophrenia and cognitive impairment265. It is coupled to FMRP265, a cytoplasmatic modulator of microRNA-RISC complexes257.
Plxna1encodes Plexin-A1, a coreceptor for class 3 semaphorins, which is expressed in neurons of hippocampal CA1–CA3 regions, sensory cortex266 and cortical subplate50. It plays a role in
axon guidance267, regeneration268 and pruning (reviewed in108,269). Plexins induce changes in cytoskeletal architecture and synapse elimination269. Because of their developmental function, they can be involved in autims and schizophrenia269.
Mov10was described above in the PFC section.
5.2.2 Proteomic analysis
We analysed cytosolic and synaptosomal proteome in PFC of 4 weeks oldTcf4tg and wt mice. In the cytosol we observed consistent downregulation of several ribosomal proteins and GTPases and several cell growth & maintenance proteins. β-tubulins and actin-binding proteins were upregulated, which may be in line with increased frequency of synaptic spines, observed in STED microscopy.
Numerous signalling proteins were upregulated in synaptosomes, mainly Ca2+binding proteins, GTPases and Ser/Thr kinases (including three CaMKII subunits). Several energy metabolism proteins (oxydoreductases and phosphotransferases), few ribosomal proteins and ion channels were also upregulated. Additionally, we validated upregulation of CaMKII, HOMER1, VAMP2 in synaptosomes as well as VAMP1 and 2 in cytosol. VAMPs are Vesicle-associated membrane proteins (known also as Synaptobrevins). HOMER1 is a scaffolding protein that is upregulated during LTP and seizures. CaMKII (Calcium/calmodulin-dependent protein kinase type II) promotes LTP and LTD and formation of immature spines, by acting of actin and actin-binding proteins and CDK5 (also upregulated in Tcf4tg)270,271. It is also involved in cognition and psychiatric disorders270.
5.2.3 Expression analyses — conclusions
There was no overlap between differentially expressed genes found by RNAseq and proteomics.
This lack of coherence could be explained by deregulation of microRNAs and other non-coding RNAs (e.g. small RNAs or circRNAs). InTcf4tg mice,BC1andTop3b, both involved in suppression of translation initiation, were upregulated in hippocamus. AdditionallyMov10, associated with the RISC complex, was upregulated in PFC and downregulated in hippocampus. All three candidates closely interact with Fragile X mental retardation protein 1 (FMRP). The Fragile X syndrome is characterized by, among others, intellectual disability and distinct facial features272. FMRP is an RNA-binding protein that modulates microRNA-RISC complexes and is important for RNA transport and translation repression257. FMRP-deficiency in mice and humans leads to immature (long and thin) spine morphology and increase of dendritic spine number in the cortex, that are characteristic for early developmental stages273,274. Based on our RNA sequencing data, regulatory RNAs seem to be a promising candidate explaining the inconsistency between RNAseq and proteomic data. In future experiments we will adapt our protocol for microRNA and perform microRNAs sequencing.
Murine Adora2a, Foxp2, Drd1a and Tac1 are highly expressed in striatum, moderately in the isocortex and low or undetectable in the hippocampal formation50. Downregulation of these
genes in PFC ofTcf4 Tcf4tg mice may be an artefact caused by striatal contamination of cortical preparation. To validate this result we need to analyse cortical samples obtained, e.g. by laser capture microdissection. The results may be also influenced by a gender bias in our sample.
Unfortunately, our attempt to validate the candidate genes by RT-qPCR was unsuccessful due to the low number of replicates.
Upregulation of synaptic proteins that promote plasticity, e.g. HOMER1 and CaMKII, may be in line with enhanced LTP in the Tcf4tg mice and increased levels of cytosolic β-tubulins and actin-binding proteins could reflect increase of synaptic spine frequency observed in STED microscopy.