Gain-of function mutations in some genes underlie neurodegenerative conditions whereas loss-of-function mutations have distinct phenotypes. Such appears to be the case with the protein ataxin 1 (ATXN1), which forms a transcriptional repressor complex with capicua (CIC). Gain-of-function of the complex leads to neurodegeneration, but ATXIN1-CIC is also essential for survival. We set out to understand the functions of ATXN1-CIC in the developing forebrain and found that losing the complex results in hyperactivity, impaired learning and memory, and abnormal maturation and maintenance of upper layer cortical neurons. We also found that CIC modulates social interactions in the hypothalamus and medial amygdala. Informed by these neurobehavioral features in mouse mutants, we identified five patients with de novo heterozygous truncating mutations in CIC that share similar clinical features, including intellectual disability, attention deficit/hyperactivity disorder (ADHD), and autism spectrum disorder. Our study demonstrates that loss of ATXN1-CIC complexes causes a spectrum of neurobehavioral phenotypes.
Spina bifida is one of the most common neural tube defects (NTDs) with a complex etiology. Variants in planar cell polarity (PCP) genes have been associated with NTDs including spina bifida in both animal models and human cohorts. In this study, we sequenced all exons of CELSR1 in 192 spina bifida patients from a California population to determine the contribution of CELSR1 mutations in the studied population. Novel and rare variants identified in these patients were subsequently genotyped in 190 ethnically matched control individuals. Six missense mutations not found in controls were predicted to be deleterious by both SIFT and PolyPhen. Two TG dinucleotide repeat variants were individually detected in 2 spina bifida patients but not detected in controls. In vitro functional analysis showed that the two TG dinucleotide repeat variants not only changed subcellular localization of the CELSR1 protein, but also impaired the physical association between CELSR1 and VANGL2, and thus diminished the ability to recruit VANGL2 for cell-cell contact. In total, 3% of our spina bifida patients carry deleterious or predicted to be deleterious CELSR1 mutations. Our findings suggest that CELSR1 mutations contribute to the risk of spina bifida in a cohort of spina bifida patients from California.
Apurinic/apyrimidinic endonuclease 1/redox effector factor-1 (APE1/Ref-1) is a ubiquitous multifunctional protein that possesses both DNA-repair and redox regulatory activities. Although it was originally identified as a DNA-repair enzyme, accumulating evidence supports a role of APE1/Ref-1 in tumor development. To investigate association between APE1/Ref-1 polymorphisms and lung cancer risk in Chinese populations, we first genotyped three variants of APE1/Ref-1 and found a -141 T-to-G variant (rs1760944) in the promoter associated with decreased risk of lung cancer [odds ratio (OR) = 0.62 for GG; P=0.043]. Similar results were obtained in a follow-up replication study. Combined data from the two studies comprising a total of 1072 lung cancer patients and 1064 cancer-free control participants generated a more significant association (P=0.002). We observed lower APE1/Ref-1 mRNA levels in the presence of the protective G allele in human peripheral blood mononuclear cells and normal lung tissues. The -141G-allele-promoter construct exhibited decreased luciferase reporter gene expression. Electrophoretic mobility shift assays and surface plasmon resonance analysis showed that the -141G allele impaired the binding affinity of some transcription factor, accounting for lower APE1/Ref-1-promoter activity. Supershift assays further revealed that the protein of interest was octamer-binding transcription factor-1 (Oct-1). Chromatin immunoprecipitation reconfirmed binding of Oct-1 to the APE1/Ref-1 -141-promoter region. We also found that Oct-1 conferred attenuated transactivation capacity toward the -141G variant by exogenously introducing Oct-1. These data indicate that genetic variations in APE1/Ref-1 may modify susceptibility to lung cancer and provide new insights into an unexpected effect of APE1/Ref-1 on lung carcinogenesis.
Tissues achieve their complex spatial organization through an interplay between gene regulatory networks, cell-cell communication, and physical interactions mediated by mechanical forces. Current strategies to generate in-vitro tissues have largely failed to implement such active, dynamically coordinated mechanical manipulations, relying instead on extracellular matrices which respond to, rather than impose mechanical forces. Here, we develop devices that enable the actuation of organoids. We show that active mechanical forces increase growth and lead to enhanced patterning in an organoid model of the neural tube derived from single human pluripotent stem cells (hPSC). Using a combination of single-cell transcriptomics and immunohistochemistry, we demonstrate that organoid mechanoregulation due to actuation operates in a temporally restricted competence window, and that organoid response to stretch is mediated extracellularly by matrix stiffness and intracellularly by cytoskeleton contractility and planar cell polarity. Exerting active mechanical forces on organoids using the approaches developed here is widely applicable and should enable the generation of more reproducible, programmable organoid shape, identity and patterns, opening avenues for the use of these tools in regenerative medicine and disease modelling applications.
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