Rescue of behavioral and electrophysiological phenotypes in a Pitt-Hopkins syndrome mouse model by genetic restoration of Tcf4 expression.

Pitt-Hopkins syndrome (PTHS) is a neurodevelopmental disorder caused by monoallelic mutation or deletion in the transcription factor 4 (TCF4) gene. Individuals with PTHS typically present in the first year of life with developmental delay and exhibit intellectual disability, lack of speech, and motor incoordination. There are no effective treatments available for PTHS, but the root cause of the disorder, TCF4 haploinsufficiency, suggests that it could be treated by normalizing TCF4 gene expression. Here, we performed proof-of-concept viral gene therapy experiments using a conditional Tcf4 mouse model of PTHS and found that postnatally reinstating Tcf4 expression in neurons improved anxiety-like behavior, activity levels, innate behaviors, and memory. Postnatal reinstatement also partially corrected EEG abnormalities, which we characterized here for the first time, and the expression of key TCF4-regulated genes. Our results support a genetic normalization approach as a treatment strategy for PTHS, and possibly other TCF4-linked disorders.

Isoform-Specific Reduction of the Basic Helix-Loop-Helix Transcription Factor TCF4 Levels in Huntington's Disease.

Huntington's disease (HD) is an inherited neurodegenerative disorder with onset of characteristic motor symptoms at midlife, preceded by subtle cognitive and behavioral disturbances. Transcriptional dysregulation emerges early in the disease course and is considered central to HD pathogenesis. Using wild-type (wt) and HD knock-in mouse striatal cell lines we observed a HD genotype-dependent reduction in the protein levels of transcription factor 4 (TCF4), a member of the basic helix-loop-helix (bHLH) family with critical roles in brain development and function. We characterized mouse Tcf4 gene structure and expression of alternative mRNAs and protein isoforms in cell-based models of HD, and in four different brain regions of male transgenic HD mice (R6/1) from young to mature adulthood. The largest decrease in the levels of TCF4 at mRNA and specific protein isoforms were detected in the R6/1 mouse hippocampus. Translating this finding to human disease, we found reduced expression of long TCF4 isoforms in the postmortem hippocampal CA1 area and in the cerebral cortex of HD patients. Additionally, TCF4 protein isoforms showed differential synergism with the proneural transcription factor ASCL1 in activating reporter gene transcription in hippocampal and cortical cultured neurons. Induction of neuronal activity increased these synergistic effects in hippocampal but not in cortical neurons, suggesting brain region-dependent differences in TCF4 functions. Collectively, this study demonstrates isoform-specific changes in TCF4 expression in HD that could contribute to the progressive impairment of transcriptional regulation and neuronal function in this disease.

The Intellectual Disability and Schizophrenia Associated Transcription Factor TCF4 Is Regulated by Neuronal Activity and Protein Kinase A.

Transcription factor 4 (TCF4 also known as ITF2 or E2-2) is a basic helix-loop-helix (bHLH) protein associated with Pitt-Hopkins syndrome, intellectual disability, and schizophrenia (SCZ). Here, we show that TCF4-dependent transcription in cortical neurons cultured from embryonic rats of both sexes is induced by neuronal activity via soluble adenylyl cyclase and protein kinase A (PKA) signaling. PKA phosphorylates TCF4 directly and a PKA phosphorylation site in TCF4 is necessary for its transcriptional activity in cultured neurons and in the developing brain in vivo We also demonstrate that Gadd45g (growth arrest and DNA damage inducible gamma) is a direct target of neuronal-activity-induced, TCF4-dependent transcriptional regulation and that TCF4 missense variations identified in SCZ patients alter the transcriptional activity of TCF4 in neurons. This study identifies a new role for TCF4 as a neuronal-activity-regulated transcription factor, offering a novel perspective on the association of TCF4 with cognitive disorders.SIGNIFICANCE STATEMENT The importance of the basic helix-loop-helix transcription factor transcription factor 4 (TCF4) in the nervous system is underlined by its association with common and rare cognitive disorders. In the current study, we show that TCF4-controlled transcription in primary cortical neurons is induced by neuronal activity and protein kinase A. Our results support the hypotheses that dysregulation of neuronal-activity-dependent signaling plays a significant part in the etiology of neuropsychiatric and neurodevelopmental disorders.

Sumoylation regulates the transcriptional activity of different human NFAT isoforms in neurons.

In the nervous system, four calcium/calcineurin-regulated members of the nuclear factor of activated T-cells (NFAT) family of transcription factors, NFATc1-c4, are involved in many developmental and functional processes, such as corticogenesis, synaptogenesis, synaptic plasticity and neurotransmission, that all need precise gene regulation. Therefore it is important to understand molecular events that contribute to the regulation of the transcriptional activity of specific NFAT isoforms. Previously, we have shown that there are a number of alternative splice variants of NFAT genes expressed in the brain and that neuronal activity leads to isoform-specific transactivation capacities of different human NFAT proteins. Here we looked at the effect of sumoylation as a possible regulator of the transcriptional activity of different human NFAT isoforms in rat primary cortical and hippocampal neurons in response to membrane depolarization and compared the results to those obtained from non-neuronal HEK293-FT and BHK-21 cells in response to calcium signaling. Our results show that in primary hippocampal neurons, sumoylation represses the transcriptional activity of NFATc1, NFATc2, and NFATc3 isoforms, whereas in cortical neurons, transactivation capacity of only NFATc1 and NFATc2 is repressed by sumoylation. In non-neuronal cells, however, transcriptional activity of all four NFAT isoforms is repressed by sumoylation in HEK293-FT cells, while only NFATc1 and NFATc2 isoforms are affected by sumoylation in BHK-21 cells. Altogether, our results show that sumoylation represses the transcription activation capacities of NFAT isoforms and that the effect is cell type-specific.

Regulation of different human NFAT isoforms by neuronal activity.

Nuclear factor of activated T-cells (NFAT) is a family of transcription factors comprising four calcium-regulated members: NFATc1, NFATc2, NFATc3, and NFATc4. Upon activation by the calcium-dependent phosphatase calcineurin (CaN), NFATs translocate from cytosol to the nucleus and regulate their target genes, which in the nervous system are involved in axon growth, synaptic plasticity, and neuronal survival. We have shown previously that there are a number of different splice variants of NFAT genes expressed in the brain. Here, we studied the subcellular localizations and transactivation capacities of alternative human NFAT isoforms in rat primary cortical or hippocampal neurons in response to membrane depolarization and compared the induced transactivation levels in neurons to those obtained from HEK293 cells in response to calcium signaling. We confirm that in neurons the translocation to the nucleus of all NFAT isoforms is reliant on the activity of CaN. However, our results suggest that both the regulation of subcellular localization and transcriptional activity of NFAT proteins in neurons is isoform specific. We show that in primary hippocampal neurons NFATc2 isoforms have very fast translocation kinetics, whereas NFATc4 isoforms translocate relatively slowly to the nucleus. Moreover, we demonstrate that the strongest transcriptional activators in HEK293 cells are NFATc1 and NFATc3, but in neurons NFATc3 and NFATc4 lead to the highest induction, and NFATc2 and NFATc1 display isoform-specific transcription activation capacities. Altogether, our results indicate that the effects of calcium signaling on the action of NFAT proteins are isoform-specific and can differ between cell types. We show that the effects of calcium signaling on the action of NFAT proteins are isoform-specific and differ between cell types. Although nuclear localization of all NFAT isoforms in neurons requires calcineurin, the subcellular distributions, neuronal activity-induced nuclear translocation extent and kinetics, and transcription activation capacities of alternative NFAT proteins vary.

Alternative splicing and expression of human and mouse NFAT genes.

Four members of the nuclear factor of activated T cells (NFAT) family (NFATC1, NFATC2, NFATC3, and NFATC4) are Ca(2+)-regulated transcription factors that regulate several processes in vertebrates, including the development and function of the immune, cardiovascular, musculoskeletal, and nervous systems. Here we describe the structures and alternative splicing of the human and mouse NFAT genes, including novel splice variants for NFATC1, NFATC2, NFATC3, and NFATC4, and show the expression of different NFAT mRNAs in various mouse and human tissues and brain regions by RT-PCR. Our results show that alternatively spliced NFAT mRNAs are expressed differentially and could contribute to the diversity of functions of the NFAT proteins. Since NFAT family members are Ca(2+)-regulated and have critical roles in neuronal gene transcription in response to electrical activity, we describe the expression of NFATC1, NFATC2, NFATC3, and NFATC4 mRNAs in the adult mouse brain and in the adult human hippocampus using in situ hybridization and show that all NFAT mRNAs are expressed in the neurons of the mouse brain with specific patterns for each NFAT.