15 Mar 2016
March 16, 2016. Thanks to a novel technique that allows researchers to probe gene expression in a subpopulation of genetically altered cells, a new study provides insights into how a schizophrenia risk gene regulates the excitability of neurons in the rodent prefrontal cortex. The study of transcription factor 4 (TCF4), which has been repeatedly associated with schizophrenia, was led by Brady Maher of the Lieber Institute for Brain Development and was published online on March 10 in Neuron.
This study provides "intriguing data that the schizophrenia risk gene, Transcription Factor 4 (TCF4), normally functions to repress the expression of two ion channels: KCNQ1 and Nav1.8 (SCN10a) that are not naturally expressed in brain," wrote Amy Arnsten of Yale University, who was not involved in the study, in a comment to SRF.
TCF4 has been linked to several neurodevelopment disorders, including a type of autism called Pitt-Hopkins syndrome (PTHS), 18q syndrome, and schizophrenia. The genomic region containing TCF4 was one of the earliest identified in genomewide association studies (GWAS) as containing single nucleotide polymorphisms (SNPs) associated with schizophrenia. And unlike other previous GWAS hits, TCF4 SNPs were found to be strongly significant also in the most recent GWAS conducted by the Schizophrenia Working Group of the Psychiatric Genomics Consortium (see SRF related news report).
Specific TCF4 polymorphisms have been associated with differences in cognitive function and sensorimotor gating in people with schizophrenia and controls (see SRF related news report and Zhu et al., 2013).
TCF4 is a transcription factor that can either repress or activate the transcription of genes and is highly expressed in the human central nervous system during development. However, little is known about the genes regulated by TCF4 and how changes in expression of TCF4 could lead to its associated disorders.
In this study, Maher and colleagues explored these questions by using two rodent models of PTHS, which is caused by TCF4 mutations that lead to protein deficiency. For their first model, the researchers used in-utero electroporation (IUE) to insert shRNAs or CRISPR-Cas9 constructs into the rat prefrontal cortex to knock down expression of TCF4 just before neurogenesis. The researchers chose this time point after determining that TCF4 mRNA expression peaks in the late prenatal period in both rats and humans. The IUE technique targeted neurons in the 2/3 cortical layer.
When Maher and colleagues then used whole-cell electrophysiology to record action potentials in IUE-transfected neurons, they found that knocking down TCF4 protein severely decreased the frequency of action potentials. This result suggests that TCF4 regulates the intrinsic excitability of these neurons. The researchers determined that the decrease in firing was due to an increase in the after-hyperpolarization current, which makes it harder for neurons to repeatedly fire.
"If such changes occurred in neurons in humans with mutations to TCF4, it could have devastating effects on prefrontal cortical function," wrote Arnsten.
These findings suggested that knocking down TCF4 changed the expression of one or more ion channels. To determine which ion channels were affected, Maher and colleagues needed to look at the expression of ion channels in only the cells that had been electroporated with the TCF4 knockdown constructs. In order to do this, they developed a novel technique that they named iTRAP, where they used translating ribosome affinity purification (TRAP) in IUE-transfected neurons.
"It allowed us to purify RNA only from the cells that we transfected, thus allowing us to compare the RNA between TCF4 knockdown cells and control cells," Maher told SRF.
The team determined that the expression of two ion channels—KCNQ1 and SCN10a—was significantly upregulated in the cells where TCF4 had been knocked down. Follow-up experiments showed that TCF4 binds directly to the KCNQ1 and SCN10a genes, suggesting that TCF4 could be directly repressing the expression of these genes.
"Both of these ion channels, KCNQ1 and SCN10a, are primarily thought to be expressed in the peripheral nervous system," said Maher. "We think what's happening is that normally, TCF4 represses the expression of these ion channels in the central nervous system. And in people who are lacking TCF4, they're having ectopic expression of these peripheral ion channels in the central nervous system."
Next, the researchers looked at action potential frequency and SCN10a and KCNQ1 levels in a transgenic mouse model of PTHS, which has a truncated copy of TCF4 on one allele. Whole-cell recordings from neurons in the 2/3 layer of the prefrontal cortex also showed a decrease in action potential frequency. Intriguingly, there was increased expression of SCN10a RNA in the frontal cortex in these mice; however, levels of KCNQ1 were actually lower. These findings were mirrored in a pharmacological experiment that found that SCN10a antagonists could rescue action potential output, but KCNQ1 antagonists could not.
TCF4 and schizophrenia
The relevance of these findings for increasing our understanding of the biology of schizophrenia is unclear at this point—primarily because it is not known how, or even if, the schizophrenia-risk SNPs in TCF4 change the expression or the function of the protein.
"We've been trying to find an association—we're still working on it—between the schizophrenia GWAS positive SNPs and TCF4 expression. The TCF4 locus has been associated with risk for schizophrenia, and the overlapping symptomatology between autism and schizophrenia adds to the link, but we don't know the specific mechanism in the case of schizophrenia," said Maher. "The mechanism could be anything at this point. It could be too much TCF4, or too little, or involve a previously uncharacterized isoform or function of TCF4. We also don't know when during development it could matter." Maher says his team is exploring this question using RNA sequencing from postmortem human brains across development and in various clinical samples.
However, expression of KCNQ1 and/or SCN10a in the prefrontal cortex could hypothetically explain some of the cognitive deficits seen in schizophrenia. "[I]f KCNQ1 is expressed on dlPFC [dorsal lateral prefrontal cortex] neurons in subjects with TCF4 mutations, it could result in weaker dlPFC network connectivity, reduced firing, and impaired cognitive abilities," wrote Arnsten. "It is also interesting to speculate that increased expression of Nav1.8 [SCN10a] in brain could result in nonspecific increases in excitability under conditions of inflammation, as research on the schizophrenia prodrome indicates that inflammation heralds the descent into illness."
Arnsten also notes that "a larger picture is emerging whereby schizophrenia is increasingly associated with genetic insults to ion channels (e.g., Cav1.2, KCNH2, Nicotinic-_7, NMDAR, HCN), or to regulators of cAMP-PKA or calcium signaling that regulate the open state of those channels (e.g., mGluR3, DISC1 anchoring of PDE4A, VIPR2). These genetic alterations would change neuronal excitability, particularly under conditions such as stress, where calcium-cAMP signaling is increased and defects may be most evident. Such genetic alterations to neuronal excitability may be most devastating in the newly evolved layer III dlPFC circuits that must generate precisely patterned and precisely timed representations needed for healthy cognitive function."—Summer E. Allen.
Rannals MD, et al. Psychiatric Risk Gene Transcription Factor 4 Regulates Intrinsic Excitability of Prefrontal Neurons via Repression of SCN10a and KCNQ1. Neuron. Published online 2016 March 10. Abstract