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

Abstract

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.

Expression of alternative transcription factor 4 mRNAs and protein isoforms in the developing and adult rodent and human tissues

“…Western blot analysis revealed that TCF4 protein signal was detectable in both adult human brain and SH-SY5Y cell line (Figure 9). We also detected a possible non-specific signal located between the long and medium TCF4 isoforms in both SH-SY5Y and human brain lysates (Figure 9) since this signal was not detected using other TCF4 antibodies (data not shown) validated by us before (Nurm et al, 2021). Different to SH-SY5Y cell line, we detected all three TCF4 isoform groups in the adult human brain, however expression level of longer TCF4 isoforms was higher compared to the medium and short isoforms (Figure 9; Supplementary Figure S8).…”

“…The use of numerous alternative 5′ exons results in the expression of many transcripts from the Tcf4 gene, resulting in a variety of TCF4 protein isoforms with different expression patterns between tissue types ( Sepp et al, 2011 ; Nurm et al, 2021 ). We have previously described transcripts from the mouse Tcf4 gene based on available mRNA and expressed sequence tag data from various tissues ( Nurm et al, 2021 ) but characterizing all the isoform encoding transcripts using short-read sequencing is complicated. Here, we did long-read direct RNA-sequencing (RNA-seq) on the Oxford Nanopore Technologies platform from postnatal day 3 (P3) mouse cerebral cortex.…”

“…Before RNA-seq library preparation, 20 μg of P3 BALB/c cortical RNA was enriched for Tcf4 transcripts in wash/binding buffer (0.5 M NaCl, 20 mM Tris–HCl pH 7.5, and 1 mM EDTA) with 8 μM each of three 5′ biotinylated oligonucleotides (Microsynth AG)—two oligonucleotides were complementary to the 3` untranslated region of Tcf4, and one was complementary to the bHLH region of Tcf4 ( Supplementary Table S1 ). As there are no known Tcf4 transcripts that lack the bHLH region or exon 21, we expect that our Tcf4 mRNA enrichment strategy is unbiased and enriches all possible TCF4 transcripts ( Sepp et al, 2011 ; Nurm et al, 2021 ). First the mixture was incubated at 70°C for 2 min and then cooled to room temperature in about 30 min in a heating block.…”

“…Membranes were blocked with 5% skimmed milk (Sigma-Aldrich) in 1x Tris Buffered Saline with 0.1% Tween-20 (TBST, Sigma Aldrich) before incubating with primary [mouse monoclonal anti-ITF-2 (TCF4); C-8, 1:1,000 dilution, Santa Cruz] and secondary (goat polyclonal anti-mouse IgG HRP-conjugated antibody 1:5,000 dilution, Thermo Scientific) antibody in 2.5% skimmed milk-TBST solution overnight at 4°C and 1 h at room temperature, respectively. Specificity of the anti-ITF-2 (TCF4; C-8, Santa Cruz) antibody has been previously validated using tissue lysates from Tcf4 knockout mice ( Nurm et al, 2021 ). For peripheral tissues, mouse IgG kappa binding protein conjugated to HRP was used as a secondary antibody (1:5,000 dilution, Santa Cruz).…”

“…To date, none of the previous studies of TCF4 mRNA ( Ma et al, 2018 ; Li et al, 2019 ; Phan et al, 2020 ) and protein ( Jung et al, 2018 ; Kim et al, 2020 ) expression have described expression of the variety of TCF4 isoforms. Expression of the mouse and human TCF4 gene results in many different transcripts encoding N-terminally distinct protein isoforms which vary in their intracellular localization, transactivation capability ( Sepp et al, 2011 , 2017 ; Nurm et al, 2021 ) and possibly mediate their function depending on dosage ( Ravanpay and Olson, 2008 ). Here, we investigated the complex expression dynamics of different TCF4 mRNAs and protein isoforms in the developing and adult rodent and human tissues.…”