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.
Numerical data used to generate all figures are provided in the Figure Source Data files that correspond to figure labels. Single-cell transcriptomic data from the neonatal mouse cortex and the adult mouse nervous system were obtained from GEO accession GSE123335 and from http://mousebrain.org/downloads.html. All code to reproduce the plots is provided at https://github.com/jeremymsimon/Kim_TCF4.
- Hyojin Kim
- Benjamin Philpot
- Hyojin Kim
- Benjamin Philpot
- Hanna Vihma
- Andrew J Kennedy
The funder (Ben Philpot) had a role in the conceptualization, supervision, data curation, manuscript writing, and the decision to submit the work for publication. The funder (Hyojin Kim) had a role in the investigation, project administration, data curation, analysis, and manuscript writing. Other funders (Hanna Vihma and Andrew J Kennedy) had roles in data acquisition.
Animal experimentation: All research procedures using mice were approved by the Institutional Animal Care and Use Committee at the University of North Carolina at Chapel Hill (IACUC protocol# 20-156.0) and Institutional Animal Care and Use Committee at Bates College (IACUC protocol# 21-05) and conformed to National Institutes of Health guidelines.
- Joseph G Gleeson, Howard Hughes Medical Institute, The Rockefeller University, United States
© 2022, Kim et al.
This article is distributed under the terms of the Creative Commons Attribution License permitting unrestricted use and redistribution provided that the original author and source are credited.
Eukaryotic genes are interrupted by introns that are removed from transcribed RNAs by splicing. Patterns of splicing complexity differ between species, but it is unclear how these differences arise. We used inter-species association mapping with Saccharomycotina species to correlate splicing signal phenotypes with the presence or absence of splicing factors. Here we show that variation in 5' splice site sequence preferences correlate with the presence of the U6 snRNA N6-methyladenosine methyltransferase METTL16 and the splicing factor SNRNP27K. The greatest variation in 5' splice site sequence occurred at the +4 position and involved a preference switch between adenosine and uridine. Loss of METTL16 and SNRNP27K orthologs, or a single SNRNP27K methionine residue, was associated with a preference for +4U. These findings are consistent with splicing analyses of mutants defective in either METTL16 or SNRNP27K orthologs and models derived from spliceosome structures, demonstrating that inter-species association mapping is a powerful orthogonal approach to molecular studies. We identified variation between species in the occurrence of two major classes of 5' splice sites, defined by distinct interaction potentials with U5 and U6 snRNAs, that correlates with intron number. We conclude that variation in concerted processes of 5' splice site selection by U6 snRNA is associated with evolutionary changes in splicing signal phenotypes.
Insufficient insulin secretion to meet metabolic demand results in diabetes. The intracellular flux of Ca2+ into β-cells triggers insulin release. Since genetics strongly influences variation in islet secretory responses, we surveyed islet Ca2+ dynamics in eight genetically diverse mouse strains. We found high strain variation in response to four conditions: (1) 8 mM glucose; (2) 8 mM glucose plus amino acids; (3) 8 mM glucose, amino acids, plus 10 nM glucose-dependent insulinotropic polypeptide (GIP); and (4) 2 mM glucose. These stimuli interrogate β-cell function, α- to β-cell signaling, and incretin responses. We then correlated components of the Ca2+ waveforms to islet protein abundances in the same strains used for the Ca2+ measurements. To focus on proteins relevant to human islet function, we identified human orthologues of correlated mouse proteins that are proximal to glycemic-associated single-nucleotide polymorphisms in human genome-wide association studies. Several orthologues have previously been shown to regulate insulin secretion (e.g. ABCC8, PCSK1, and GCK), supporting our mouse-to-human integration as a discovery platform. By integrating these data, we nominate novel regulators of islet Ca2+ oscillations and insulin secretion with potential relevance for human islet function. We also provide a resource for identifying appropriate mouse strains in which to study these regulators.