Deficiency of the Fanconi anemia core complex protein FAAP100 results in severe Fanconi anemia

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Abstract

Fanconi anemia (FA) is a rare genetic disease characterized by loss-of-function variants in any of the 22 previously identified genes (FANCA-FANCW) that encode proteins participating in the repair of DNA interstrand crosslinks (ICLs). Patient phenotypes are variable, but may include developmental abnormalities, early onset pancytopenia, and predisposition to hematologic and solid tumors. Here, we describe two unrelated families with multiple pregnancy losses and offspring presenting with severe developmental and hematologic abnormalities leading to death in utero or in early life. Homozygous loss-of-function variants in FAAP100 were identified in affected children of both families. The FAAP100 protein associates with FANCB and FANCL, the E3 ubiquitin ligase responsible for the monoubiquitination of FANCD2 and FANCI, which is necessary for FA pathway function. Patient-derived cells exhibited phenotypes consistent with FA. Expression of the wild-type FAAP100 cDNA, but not the patient-derived variants, rescued the observed cellular phenotypes. This establishes FAAP100 deficiency as a cause of Fanconi anemia, with FAAP100 gaining an alias as FANCX. The extensive developmental malformations of individuals with FAAP100 loss-of-function variants are among the most severe across previously described FA phenotypes, indicating that the FA pathway is essential for human development.

Authors

Benjamin A. Harrison, Emma Mizrahi-Powell, John Pappas, Kristen Thomas, Subrahmanya Vasishta, Shripad Hebbar, Anju Shukla, Shalini S. Nayak, Tina K. Truong, Amy Woroch, Yara Kharbutli, Bruce D. Gelb, Cassie S. Mintz, Gilad D. Evrony, Agata Smogorzewska

Aberrant ERK signaling in astrocytes impairs learning and memory in RASopathy-associated BRAF mutant mouse models

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Abstract

RAS/MAPK pathway mutations often induce RASopathies with overlapping features, such as craniofacial dysmorphology, cardiovascular defects, dermatologic abnormalities, and intellectual disabilities. Although BRAF gene mutations are associated with cardio-facio-cutaneous (CFC) syndrome and Noonan syndrome, it remains unclear how these mutations impair cognition. Here, we investigated the underlying neural mechanisms using several mouse models harboring a gain-of-function BRAF mutation (K499E) discovered in RASopathy patients. We found expressing BRAF K499E (KE) in neural stem cells under the control of a Nestin-Cre promoter (Nestin;BRAFKE/+) induced hippocampal memory deficits, but expressing it in excitatory or inhibitory neurons did not. BRAF KE expression in neural stem cells led to aberrant reactive astrogliosis, increased astrocytic Ca2+ fluctuations, and reduced hippocampal long-term depression (LTD) in mice. Consistently, 3D human cortical spheroids expressing BRAF KE also showed reactive astrogliosis. Astrocyte-specific AAV-BRAF KE delivery induced memory deficits, reactive astrogliosis, and increased astrocytic Ca2+ fluctuations. Notably, reducing ERK activity in astrocytes rescued the memory deficits and altered astrocytic Ca2+ activity of Nestin;BRAFKE/+ mice. Furthermore, reducing astrocyte Ca2+ activity rescued the spatial memory impairments of BRAF KE-expressing mice. Our results demonstrate that ERK hyperactivity contributes to astrocyte dysfunction associated with Ca2+ dysregulation, leading to the memory deficits of BRAF-associated RASopathies.

Authors

Minkyung Kang, Jihye Choi, Jeongho Han, Toshiyuki Araki, Soo-Whee Kim, Hyun-Hee Ryu, Min-Gyun Kim, Seoyeon Kim, Hanbyul Jang, Sun Yong Kim, Kyoung-Doo Hwang, Soobin Kim, Myeongjong Yoo, Jaegeon Lee, Kitae Kim, Pojeong Park, Ja Eun Choi, Dae Hee Han, Yujin Kim, Jeongyeon Kim, Sunghoe Chang, Bong-Kiun Kaang, Jung Min Ko, Keun-Ah Cheon, Joon-Yong An, Sang Jeong Kim, Hyungju Park, Benjamin G. Neel, Chul Hoon Kim, Yong-Seok Lee

A whole-body imaging technique for tumor-specific diagnostics and screening of B7-H3-targeted therapies

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Abstract

BACKGROUND. B7-H3 or CD276 is notably overexpressed in various malignant tumor cells in humans, with extremely high expression rates. The development of a radiotracer that targets B7-H3 may provide a universal tumor-specific imaging agent and allow the noninvasive assessment of the whole-body distribution of B7-H3-expressing lesions. METHODS. We enhanced and optimized the structure of an affibody (ABY) that targets B7-H3 to create the radiolabeled radiotracer [68Ga]Ga-B7H3-BCH, and then, we conducted both foundational experiments and clinical translational studies. RESULTS. [68Ga]Ga-B7H3-BCH exhibited high affinity (Kd = 4.5 nM), and it was taken up in large amounts by B7-H3-transfected cells (A549CD276 and H1975CD276 cells); these phenomena were inhibited by unlabeled precursors. Moreover, PET imaging of multiple xenograft models revealed extensive [68Ga]Ga-B7H3-BCH uptake by tumors. In a clinical study including 20 patients with malignant tumors, the [68Ga]Ga-B7H3-BCH signal aggregated in both primary and metastatic lesions, surpassing 18F-FDG in overall diagnostic efficacy for tumors (85.0% vs 81.7%), including differentiated hepatocellular and metastatic gastric cancers. A strong correlation between B7-H3 expression and [68Ga]Ga-B7H3-BCH uptake in tumors was observed, and B7-H3 expression was detected with 84.38% sensitivity and 100% specificity when an SUVmax of 3.85 was set as the cutoff value. Additionally, B7-H3-specific PET imaging is expected to predict B7H3 expression levels in tumor cells, intratumoral stroma and peritumoral tissues. CONCLUSION. In summary, [68Ga]Ga-B7H3-BCH has potential for the noninvasive identification of B7H3 expression in systemic lesions in patients with malignant tumors. This agent has prospects for improving pretreatment evaluation, predicting therapeutic responses, and monitoring resistance to therapy in patients with malignancies. TRIAL REGISTRATION. ClinicalTrials.gov NCT06454955. FUNDING. This research was financially supported by the Natural Science Foundation of Beijing Municipality (No. 7242266), the National Natural Science Foundation of China (No. 82202201), and the Young Elite Scientists Sponsorship Program by CAST (No. YESS20220230).

Authors

Lei Xia, Yan Wu, Yanan Ren, Zhen Wang, Nina Zhou, Wenyuan Zhou, Lixin Zhou, Ling Jia, Chengxue He, Xiangxi Meng, Hua Zhu, Zhi Yang

Elevated microRNA-187 causes cardiac endothelial dysplasia to promote congenital heart disease through inhibition of NIPBL

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Abstract

Cardiac endothelial cells are essential for heart development, and disruption of this process can lead to congenital heart disease (CHD). However, how miRNAs influence cardiac endothelial cells in CHD remains unclear. This study identified elevated miR-187 expression in embryonic heart endothelial cells from CHD fetuses. Using a conditional knock-in model, we showed that increased miR-187 levels in embryonic endothelial cells induce CHD in homozygous fetal mice, closely mirroring human CHD. Mechanistically, miR-187 targets NIPBL, which is responsible for recruiting the cohesin complex and facilitating chromatin accessibility. Consequently, the endothelial cell-specific upregulation of miR-187 inhibited NIPBL, leading to reduced chromatin accessibility and impaired gene expression, which hindered endothelial cell development and ultimately caused heart septal defects and reduced heart size both in vitro and in vivo. Importantly, exogenous miR-187 expression in human cardiac organoids mimicked developmental defects in the cardiac endothelial cells, reversible by NIPBL replenishment. Our findings establish the miR-187/NIPBL axis as a potent regulator that inhibits cardiac endothelial cell development by attenuating the transcription of numerous endothelial genes, with our mouse and human cardiac organoid models effectively replicating severe defects from minor perturbations. This discovery suggests that targeting the miR-187/NIPBL pathway could offer a promising therapeutic approach for CHD.

Authors

Chao Li, Zizheng Tan, Hongdou Li, Xiaoying Yao, Chuyue Peng, Yue Qi, Bo Wu, Tong-Jin Zhao, Chengtao Li, Jianfeng Shen, Hongyan Wang

Transcription factor KROX20 marks epithelial stem cells for hair follicle formation

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Abstract

Epidermal stem cells control homeostasis and regeneration of skin and hair. In the hair follicle (HF) bulge of mammals, populations of slow-cycling stem cells regenerate the HF during cyclical rounds of anagen (growth), telogen (quiescence), and catagen (regression). Multipotent epidermal cells are also present in the HF above the bulge area, contributing to the formation and maintenance of sebaceous gland and upper and middle portions of the HF. Here, we report that the transcription factor Krox20 is enriched in an epidermal stem cell population located in the upper/ middle HF. Expression analyses and lineage tracing using inducible Krox20-CreERT showed that Krox20-lineage cells migrate out of this HF region and contribute to the formation of bulge in the HF, serving as ancestors of bulge stem cells. In vivo depletion of these cells arrests HF morphogenesis. This study identifies a novel marker for an epidermal stem cell population that is indispensable for hair homeostasis.

Authors

Elnaz Ghotbi, Edem Tchegnon, Zhiguo Chen, Stephen Li, Tracey Shipman, Yong Wang, Jenny Raman, Yumeng Zhang, Renee M. McKay, Chung-Ping Liao, Lu Q. Le

Hif-2α programmes oxygen chemosensitivity in chromaffin cells

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Abstract

The study of transcription factors that determine specialised neuronal functions has provided invaluable insights into the physiology of the nervous system. Peripheral chemoreceptors are neurone-like electro-physiologically excitable cells that link the oxygen content of arterial blood to the neuronal control of breathing. In the adult, this oxygen chemosensitivity is exemplified by the Type I cells of the carotid body and recent work has revealed one isoform of the transcription factor HIF, HIF-2α, to have a non-redundant role in the development and function of that organ. Here we show that the activation of HIF-2α, including isolated overexpression alone, is sufficient to induce oxygen chemosensitivity in the otherwise unresponsive adult adrenal medulla. This phenotypic change in the adrenal medulla was associated with retention of extra-adrenal paraganglioma-like tissues that resemble the foetal organ of Zuckerkandl and also manifest oxygen chemosensitivity. Acquisition of chemosensitivity was associated with changes in the adrenal medullary expression of classes of genes that are ordinarily characteristic of the carotid body, including G-protein regulators and atypical subunits of mitochondrial cytochrome oxidase. Overall, the findings suggest that, at least in certain tissues, HIF-2α acts as a phenotypic driver for cells that display oxygen chemosensitivity, thus linking two major oxygen sensing systems.

Authors

Maria Prange-Barczynska, Holly A. Jones, Yoichiro Sugimoto, Xiaotong Cheng, Joanna D.C.C Lima, Indrika Ratnayaka, Gillian Douglas, Keith J. Buckler, Peter J. Ratcliffe, Thomas P. Keeley, Tammie Bishop

De novo monoallelic Reelin missense variants act in a dominant-negative manner causing neuronal migration disorders

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Abstract

Reelin (RELN) is a secreted glycoprotein essential for cerebral cortex development. In humans, recessive RELN variants cause cortical and cerebellar malformations, while heterozygous variants were associated to epilepsy, autism and mild cortical abnormalities. However, their functional effects remain unknown. We identified inherited and de novo RELN missense variants in heterozygous patients with neuronal migration disorders (NMDs) as diverse as pachygyria and polymicrogyria. We investigated in culture and in the developing mouse cerebral cortex how different variants impacted RELN function. Polymicrogyria-associated variants behaved as gain-of-function showing an enhanced ability to induce neuronal aggregation, while those linked to pachygyria as loss-of-function leading to defective neuronal aggregation/migration. The pachygyria-associated de novo heterozygous RELN variants acted as dominant-negative by preventing wild-type RELN secretion in culture, animal models and patients, thereby causing dominant NMDs. We demonstrated how mutant RELN proteins in vitro and in vivo predict cortical malformation phenotypes, providing valuable insights into the pathogenesis of such disorders.

Authors

Martina Riva, Sofia Ferreira, Kotaro Hayashi, Yoann Saillour, Vera P. Medvedeva, Takao Honda, Kanehiro Hayashi, Claire Altersitz, Shahad Albadri, Marion Rosello, Julie Dang, Malo Serafini, Frédéric Causeret, Olivia J. Henry, Charles-Joris Roux, Céline Bellesme, Elena Freri, Dragana Josifova, Elena Parrini, Renzo Guerrini, Filippo Del Bene, Kazunori Nakajima, Nadia Bahi-Buisson, Alessandra Pierani

The mechanosensory channel PIEZO1 functions upstream of angiopoietin/TIE/FOXO1 signaling in lymphatic development

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Abstract

Lymphedema is a debilitating disease with no effective cure and affects an estimated 250 million individuals worldwide. Prior studies have identified mutations in piezo-type mechanosensitive ion channel component 1 (PIEZO1), angiopoietin 2 (ANGPT2), and tyrosine kinase with Ig-like and EGF-like domains 1 (TIE1) in patients with primary lymphedema. Here, we identified crosstalk between these molecules and showed that activation of the mechanosensory channel PIEZO1 in lymphatic endothelial cells (LECs) caused rapid exocytosis of the TIE ligand ANGPT2, ectodomain shedding of TIE1 by disintegrin and metalloproteinase domain–containing protein 17 (ADAM17), and increased TIE/PI3K/AKT signaling, followed by nuclear export of the transcription factor FOXO1. These data establish a functional network between lymphedema-associated genes and provide what we believe to be the first molecular mechanism bridging channel function with vascular signaling and intracellular events culminating in transcriptional regulation of genes expressed in LECs. Our study provides insights into the regulation of lymphatic function and molecular pathways involved in human disease.

Authors

Jing Du, Pan Liu, Yalu Zhou, Sol Misener, Isha Sharma, Phoebe Leeaw, Benjamin R. Thomson, Jing Jin, Susan E. Quaggin

The long non-coding RNA CARDINAL attenuates cardiac hypertrophy by modulating protein translation

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Abstract

One of the features of pathological cardiac hypertrophy is enhanced translation and protein synthesis. Translational inhibition has been shown to be an effective means of treating cardiac hypertrophy, although system-wide side effects are common. Regulators of translation, such as cardiac-specific long non-coding RNAs (lncRNAs), could provide new, more targeted, therapeutic approaches to inhibit cardiac hypertrophy. Therefore, we generated mice lacking a previously identified lncRNA named CARDINAL to examine its cardiac function. We demonstrate that CARDINAL is a cardiac-specific, ribosome associated lncRNA and show that its expression is induced in the heart upon pathological cardiac hypertrophy; its deletion in mice exacerbates stress-induced cardiac hypertrophy and augments protein translation. In contrast, overexpression of CARDINAL attenuates cardiac hypertrophy in vivo and in vitro, and suppresses hypertrophy-induced protein translation. Mechanistically, CARDINAL interacts with developmentally regulated GTP binding protein 1 (DRG1) and blocks its interaction with DRG family regulatory protein 1 (DFRP1); as a result, DRG1 is downregulated, thereby modulating the rate of protein translation in the heart in response to stress. This study provides evidence for the therapeutic potential of targeting cardiac-specific lncRNAs to suppress disease-induced translational changes and to treat cardiac hypertrophy and heart failure.

Authors

Xin He, Tinqun Yang, Yao Wei Lu, Gengze Wu, Gang Dai, Qing Ma, Mingming Zhang, Huimin Zhou, Tianxin Long, Youchen Yan, Zhuomin Liang, Chen Liu, William T. Pu, Yugang Dong, Jingsong Ou, Hong Chen, John D. Mably, Jiangui He, Da-Zhi Wang, Zhan-Peng Huang

BAP1 is required prenatally for differentiation and maintenance of postnatal murine enteric nervous system

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Abstract

Epigenetic regulatory mechanisms are underappreciated, yet are critical for enteric nervous system (ENS) development and maintenance. We discovered that fetal loss of the epigenetic regulator Bap1 in the ENS lineage caused severe postnatal bowel dysfunction and early death in Tyrosinase-Cre Bap1fl/fl mice. Bap1-depleted ENS appeared normal in neonates; however, by P15, Bap1-deficient enteric neurons were largely absent from the small and large intestine of Tyrosinase-Cre Bap1fl/fl mice. Bowel motility became markedly abnormal with disproportionate loss of cholinergic neurons. Single-cell RNA sequencing at P5 showed that fetal Bap1 loss in Tyrosinase-Cre Bap1fl/fl mice markedly altered the composition and relative proportions of enteric neuron subtypes. In contrast, postnatal deletion of Bap1 did not cause enteric neuron loss or impaired bowel motility. These findings suggest that BAP1 is critical for postnatal enteric neuron differentiation and for early enteric neuron survival, a finding that may be relevant to the recently described human BAP1-associated neurodevelopmental disorder.

Authors

Sabine Schneider, Jessica B. Anderson, Rebecca P. Bradley, Katherine Beigel, Christina M. Wright, Beth A. Maguire, Guang Yan, Deanne M. Taylor, J. William Harbour, Robert O. Heuckeroth