Impaired glycine neurotransmission causes adolescent idiopathic scoliosis

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Abstract

Adolescent idiopathic scoliosis (AIS) is the most common form of spinal deformity affecting millions of adolescents worldwide, but it lacks a defined theory of etiopathogenesis. As such, treatment of AIS is limited to bracing and/or invasive surgery post onset. Pre-onset diagnosis or preventive treatment remains unavailable. Here we performed a genetic analysis of a large multi-center AIS cohort and identified disease-causing and predisposing variants of SLC6A9 in multi-generation families, trios, and sporadic patients. Variants of SLC6A9, which encodes glycine transporter 1 (GLYT1), reduced glycine uptake activity in cells, leading to an increased extracellular glycine level and aberrant glycinergic neurotransmission. Slc6a9 mutant zebrafish exhibited discoordination of spinal neural activities and pronounced lateral spinal curvature, a phenotype resembling human patients. The penetrance and severity of curvature was sensitive to the dosage of functional glyt1. Administration of a glycine receptor antagonist or a clinically-used glycine neutralizer (sodium benzoate) partially rescued the phenotype. Our results indicate a neuropathic origin for “idiopathic” scoliosis, involving the dysfunction of synaptic neurotransmission and central pattern generators (CPGs), potentially a common cause of AIS. Our work further suggests avenues for early diagnosis and intervention of AIS in preadolescents.

Authors

Xiaolu Wang, Ming Yue, Jason Pui Yin Cheung, Prudence Wing Hang Cheung, Yanhui Fan, Meicheng Wu, Xiaojun Wang, Sen Zhao, Anas M. Khanshour, Jonathan J. Rios, Zheyi Chen, Xiwei Wang, Wenwei Tu, Danny Chan, Qiuju Yuan, Dajiang Qin, Guixing Qiu, Zhihong Wu, Jianguo Zhang, Shiro Ikegawa, Nan Wu, Carol A. Wise, Yong Hu, Keith Dipp Kei Luk, You-Qiang Song, Bo Gao

Spliceosome malfunction causes neurodevelopmental disorders with overlapping features

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Abstract

Pre-mRNA splicing is a highly coordinated process. While its dysregulation has been linked to neurological deficits, our understanding of the underlying molecular and cellular mechanisms remains limited. We implicated pathogenic variants in U2AF2 and PRPF19, encoding spliceosome subunits in neurodevelopmental disorders (NDDs), by identifying 46 unrelated individuals with 23 de novo U2AF2 missense variants (including seven recurrent variants in 30 individuals) and six individuals with de novo PRPF19 variants. Eight U2AF2 variants dysregulated splicing of a model substrate. Neuritogenesis was reduced in human neurons differentiated from human pluripotent stem cells carrying two U2AF2 hyper-recurrent variants. Neural loss of function of the Drosophila orthologs, U2af50 and Prp19, led to lethality, abnormal mushroom body (MB) patterning, and social deficits, differentially rescued by wild-type and mutant U2AF2 or PRPF19. Transcriptome profiling revealed splicing substrates or effectors (including Rbfox1, a third splicing factor), which rescued MB defects in U2af50 deficient flies. Upon re-analysis of negative clinical exomes followed by data sharing, we further identified six NDD patients carrying RBFOX1 missense variants which, by in vitro testing, showed loss of function. Our study implicates three splicing factors as NDD causative genes and establishes a genetic network with hierarchy underlying human brain development and function.

Authors

Dong Li, Qin Wang, Allan Bayat, Mark R. Battig, Yijing Zhou, Daniëlle G.M. Bosch, Gijs van Haaften, Leslie Granger, Andrea K. Petersen, Luis A. Pérez-Jurado, Gemma Aznar-Laín, Anushree Aneja, Miroslava Hancarova, Sarka Bendova, Martin Schwarz, Radka Kremlíková Pourová, Zdenek Sedlacek, Beth A. Keena, Michael E. March, Cuiping Hou, Nora O'Connor, Elizabeth J. Bhoj, Margaret H. Harr, Gabrielle Lemire, Kym M. Boycott, Meghan C. Towne, Megan Li, Mark Tarnopolsky, Lauren Brady, Michael J. Parker, Hanna Faghfoury, Lea Kristin Parsley, Emanuele Agolini, Maria Lisa Dentici, Antonio Novelli, Meredith S. Wright, Rachel Palmquist, Khanh Lai, Marcello Scala, Pasquale Striano, Michele Iacomino, Federico Zara, Annina Cooper, Timothy J. Maarup, Melissa Byler, Robert Roger Lebel, Tugce B. Balci, Raymond J. Louie, Michael J. Lyons, Jessica Douglas, Catherine B. Nowak, Alexandra Afenjar, Juliane Hoyer, Boris Keren, Saskia M. Maas, Mahdi M. Motazacker, Julian A. Martinez-Agosto, Ahna M. Rabani, Elizabeth M. McCormick, Marni Falk, Sarah M. Ruggiero, Ingo Helbig, Rikke S. Møller, Lino Tessarollo, Francesco Tomassoni-Ardori, Mary Ellen Palko, Tzung-Chien Hsieh, Peter M. Krawitz, Mythily Ganapathi, Bruce D. Gelb, Vaidehi Jobanputra, Ashley Wilson, John Greally, Sébastien Jacquemont, Khadijé Jizi, Bruel Ange-Line, Chloé Quelin, Vinod K. Misra, Erika Chick, Corrado Romano, Donatella Greco, Alessia Arena, Manuela Morleo, Vincenzo Nigro, Rie Seyama, Yuri Uchiyama, Naomichi Matsumoto, Ryoji Taira, Katsuya Tashiro, Yasunari Sakai, Gökhan Yigit, Bernd Wollnik, Michael Wagner, Barbara Kutsche, Anna C.E. Hurst, Michelle L. Thompson, Ryan J. Schmidt, Linda M. Randolph, Rebecca C. Spillmann, Vandana Shashi, Edward J. Higginbotham, Dawn Cordeiro, Amanda Carnevale, Gregory Costain, Tayyaba Khan, Benoît Funalot, Frederic Tran Mau-Them, Luis Fernandez Garcia Moya, Sixto García-Miñaúr, Matthew Osmond, Lauren Chad, Nada Quercia, Diana Carrasco, Chumei Li, Amarilis Sanchez-Valle, Meghan Kelley, Mathilde Nizon, Brynjar O. Jensson, Patrick Sulem, Kari Stefansson, Svetlana Gorokhova, Tiffany Busa, Marlène Rio, Hamza Hadj Abdallah, Marion Lesieur-Sebellin, Jeanne Amiel, Véronique Pingault, Sandra Mercier, Marie Vincent, Christophe Philippe, Clemence Fatus-Fauconnier, Kathryn Friend, Rebecca K. Halligan, Sunita Biswas, Jane M.R. Rosser, Cheryl Shoubridge, Mark A. Corbett, Christopher Barnett, Jozef Gecz, Kathleen A. Leppig, Anne Slavotinek, Carlo Marcelis, Rolph Pfundt, Bert B.A. de Vries, Marjon A. van Slegtenhorst, Alice S. Brooks, Benjamin Cogne, Thomas Rambaud, Zeynep Tümer, Elaine H. Zackai, Naiara Akizu, Yuanquan Song, Hakon Hakonarson

Hypomorphic variants of SEL1L-HRD1 ER-associated degradation are associated with neurodevelopmental disorders

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Abstract

Recent studies using cell type-specific knockout mouse models have improved our understanding of the pathophysiological relevance of SEL1L-HRD1 endoplasmic reticulum (ER)-associated degradation (ERAD); however, its importance in humans remains unclear as no disease variant has been identified. Here we report the identification of three bi-allelic missense variants of SEL1L and HRD1 (or SYVN1) in six children from three independent families presenting with developmental delay, intellectual disability, microcephaly, facial dysmorphisms, hypotonia and/or ataxia. These SEL1L (p.Gly585Asp, p.Met528Arg) and HRD1 (p.Pro398Leu) variants were hypomorphic and impaired ERAD function at distinct steps of ERAD including substrate recruitment (SEL1L p.Gly585Asp), SEL1L-HRD1 complex formation (SEL1L p.Met528Arg), and HRD1 activity (HRD1 p.Pro398Leu). Our study not only provide new insights into the structure-function relationship of SEL1L-HRD1 ERAD, but also establish the importance of SEL1L-HRD1 ERAD in humans.

Authors

Huilun Helen Wang, Liangguang Leo Lin, Zexin Jason Li, Xiaoqiong Wei, Omar Askander, Gerarda Cappuccio, Mais O. Hashem, Laurence Hubert, Arnold Munnich, Mashael Alqahtani, Qi Pang, Margit Burmeister, You Lu, Karine Poirier, Claude Besmond, Shengyi Sun, Nicola Brunetti-Pierri, Fowzan S. Alkuraya, Ling Qi

Biallelic Cys141Tyr variant of SEL1L is associated with neurodevelopmental disorders, agammaglobulinemia and premature death

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Abstract

SEL1L-HRD1 ERAD plays a critical role in many physiological processes in mice, including immunity, water homeostasis and energy metabolism; however, its relevance and importance in humans remain unclear as no disease variant has been identified. Here we report a bi-allelic SEL1L variant (p. Cys141Tyr) in five patients from a consanguineous Slovakian family. These patients presented with not only ERAD-associated neurodevelopmental disorders with onset infancy (ENDI) syndromes, but infantile-onset agammaglobulinemia with no mature B cells, resulting in frequent infections and early death. This variant disrupted the formation of a disulfide bond in the luminal fibronectin II domain of SEL1L, largely abolishing the function of SEL1L-HRD1 ERAD complex in part via proteasomal-mediated self-destruction by HRD1. This study reports a new disease entity termed the “ENDI-agammaglobulinemia” (ENDI-A) syndrome and establishes an inverse correlation between SEL1L-HRD1 ERAD functionality and disease severity in humans.

Authors

Denisa Weis, Liangguang Leo Lin, Huilun Helen Wang, Zexin Jason Li, Katarína Kušíková, Peter Ciznar, Hermann Maximillian Wolf, Alexander Leiss-Piller, Zhihong Wang, Xiaoqiong Wei, Serge Weis, Katarina Skalicka, Gabriela Hrckova, Lubos Danisovic, Andrea Soltysova, Tingxuan Tina Yang, René Günther Feichtinger, Johannes Adalbert Mayr, Ling Qi

Truncated titin protein in dilated cardiomyopathy incorporates into the sarcomere and transmits force

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Abstract

Authors

Quentin McAfee, Matthew A. Caporizzo, Keita Uchida, Kenneth C. Bedi Jr., Kenneth B. Margulies, Zolt Arany, Benjamin L. Prosser

PP2A inhibition causes synthetic lethality in BRCA2-mutated prostate cancer models via reactivating spindle assembly checkpoint

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Abstract

Mutations in the BRCA2 tumor suppressor gene have been associated with an increased risk of developing prostate cancer. One of the paradoxes concerning BRCA2 is the fact that its inactivation affects genetic stability and is deleterious for cellular and organismal survival, while BRCA2-mutated cancer cells adapt to this detriment and malignantly proliferate. Therapeutic strategies for tumors arising from BRCA2 mutations may be discovered by understanding these adaptive mechanisms. In this study, we conducted forward genetic synthetic viability screenings in C. elegans brc-2 (Cebrc-2) mutants and found that Ceubxn-2 inactivation rescued the viability of Cebrc-2 mutants. Moreover, loss of NSFL1C, the mammalian ortholog of CeUBXN-2, suppressed the spindle assembly checkpoint (SAC) activation and promoted the survival of BRCA2-deficient cells. Mechanistically, NSFL1C recruited USP9X to inhibit the polyubiquitination of AURKB and reduce the removal of AURKB from the centromeres by VCP, which is essential for SAC activation. SAC inactivation is common in BRCA2-deficient prostate cancer patients, but PP2A inhibitors could reactivate the SAC and achieve BRCA2-deficient prostate tumor synthetic lethality. Our research reveals the survival adaptation mechanism of BRCA2-deficient prostate tumor cells and provides different angles for exploring synthetic lethal inhibitors in addition to targeting DNA damage repair pathways.

Authors

Jian Wang, Yuke Chen, Shiwei Li, Wanchang Liu, Xiao Albert Zhou, Yefei Luo, Zhanzhan Xu, Yundong Xiong, Kaiqi Cheng, Mingjian Ruan, Wei Yu, Xiaoman Li, Weibin Wang, Jiadong Wang

A narrow T cell receptor repertoire instructs thymic differentiation of MHC class Ib-restricted CD8⁺ regulatory T-cells

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Abstract

Although most CD8+ T-cells are equipped to kill infected or transformed cells, a subset may regulate immune responses and preserve self-tolerance. Here we describe a CD8 lineage that is instructed to differentiate into CD8 T regulatory cells (Treg) by a surprisingly restricted set of T-cell receptors (TCR) that recognize MHC-E (mouse Qa-1) and several dominant self-peptides. Recognition and elimination of pathogenic target cells that express these Qa-1self-peptide complexes selectively inhibits pathogenic antibody responses without generalized immune suppression. Immunization with synthetic agonist peptides that mobilize CD8 Treg in vivo efficiently inhibit anti-graft antibody responses and markedly prolong heart and kidney organ graft survival. Definition of TCR-dependent differentiation and target recognition by this lineage of CD8 Treg may open the way to new therapeutic approaches to inhibit pathogenic antibody responses.

Authors

Hye-Jung Kim, Hidetoshi Nakagawa, John Y. Choi, Xuchun Che, Andrew Divris, Qingshi Liu, Andrew E. Wight, Hengcheng Zhang, Anis Saad, Zhabiz Solhjou, Christa Deban, Jamil R. Azzi, Harvey Cantor

Durable responses to ATR inhibition with ceralasertib in tumors with genomic defects and high inflammation

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Abstract

BACKGROUND. PATRIOT was the first-in-human phase I study of the oral ATR (ataxia telangiectasia and Rad3-related) inhibitor ceralasertib (AZD6738) in advanced solid tumors. METHODS. Primary objective was safety. Secondary objectives included assessment of anti-tumor responses, pharmacokinetic (PK) and pharmacodynamic (PD) studies. Sixty-seven patients received ceralasertib 20-240 mg BD continuously or intermittently (14 of a 28-day cycle). RESULTS. Intermittent dosing was better tolerated than continuous, which was associated with dose-limiting hematological toxicity. The recommended phase 2 dose of ceralasertib was 160 mg twice daily for 2 weeks in a 4-weekly cycle. Modulation of target and increased DNA damage were identified in tumor and surrogate PD. There were 5 (8%) confirmed partial responses (PR, 40-240 mg BD), 34 (52%) stable disease (SD) including 1 unconfirmed partial response, and 27 (41%) progressive disease. Durable responses were seen in tumors with loss of AT-rich interactive domain-containing protein 1A (ARID1A) and DNA damage response defects. Treatment modulated tumor and systemic immune markers and responding tumors were more immune-inflamed than non-responding. CONCLUSION. Ceralasertib monotherapy was tolerated at 160 mg BD intermittent and associated with anti-tumor activity. TRIAL REGISTRATION. Clinicaltrials.gov: NCT02223923, EudraCT: 2013-003994-84. FUNDING. Cancer Research UK, AstraZeneca, UK Department of Health (National Institute for Health Research), Rosetrees Trust, Experimental Cancer Medicine Centre. FUNDING. AstraZeneca provided funding for components of the clinical conduct of PATRIOT and drug supply and labelling.

Authors

Magnus T. Dillon, Jeane Guevara, Kabir Mohammed, Emmanuel Christian Patin, Simon A. Smith, Emma Dean, Gemma N. Jones, Sophie E. Willis, Marcella Petrone, Carlos Silva, Khin Thway, Catey Bunce, Ioannis Roxanis, Pablo Nenclares, Anna Wilkins, Martin McLaughlin, Adoracion Jayme-Laiche, Sarah Benafif, Georgios Nintos, Vineet Kwatra, Lorna Grove, David C. Mansfield, Paula Proszek, Philip Martin, Luiza Moore, Karen E. Swales, Udai Banerji, Mark P. Saunders, James Spicer, Martin D. Forster, Kevin J. Harrington

CDKL5 regulates p62-mediated selective autophagy and confers protection against neurotropic viruses

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Abstract

Virophagy, the selective autophagosomal engulfment and lysosomal degradation of viral components, is crucial for neuronal cell survival and antiviral immunity. However, the mechanisms leading to viral antigen recognition and capture by autophagic machinery remain poorly understood. Here, we identified cyclin-dependent kinase-like 5 (CDKL5), known to function in neurodevelopment, as an essential regulator of virophagy. Loss of function mutations in CDKL5 are associated with a severe neurodevelopmental encephalopathy. We found deletion of CDKL5 or expression of a clinically-relevant pathogenic mutant of CDKL5 reduced virophagy of Sindbis virus (SINV), a neurotropic RNA virus, and increased intracellular accumulation of SINV capsid protein aggregates and cellular cytotoxicity. CDKL5 knockout mice displayed increased viral antigen accumulation and neuronal cell death after SINV infection and enhanced lethality after infection with several neurotropic viruses. Mechanistic studies demonstrated that CDKL5 directly binds the canonical selective autophagy receptor p62 and phosphorylates p62 at T269/S272 to promote its interaction with viral capsid aggregates. We found that CDKL5-mediated phosphorylation of p62 facilitated the formation of large p62 inclusion bodies that captured viral capsids to initiate capsid targeting to autophagic machinery. Overall, these findings identify a cell-autonomous innate immune mechanism for autophagy activation to clear intracellular toxic viral protein aggregates during infection.

Authors

Josephine W. Thinwa, Zhongju Zou, Emily Parks, Salwa Sebti, Kelvin K. Hui, Yongjie Wei, Mohammad Goodarzi, Vibha Singh, Greg Urquhart, Jenna L. Jewell, Julie K. Pfeiffer, Beth Levine, Tiffany A. Reese, Michael U. Shiloh

In situ vaccination via tissue-targeted cDC1 expansion enhances the immunogenicity of chemoradiation and immunotherapy

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Abstract

Even with the prolific clinical use of next-generation cancer therapeutics, many tumors remain unresponsive or become refractory to therapy, creating a medical need. In cancer, DCs are indispensable to T cell activation, so there is a restriction on cytotoxic T cell immunity if DCs are not present in sufficient numbers in the tumor and draining lymph nodes to uptake and present relevant cancer antigens. To address this bottleneck, we developed a Flt3L-based therapeutic named Alb-Flt3L that demonstrated superior pharmacokinetic properties compared to Flt3L, including significantly longer half-life, accumulation in tumor and lymph node, and cross-presenting DCs expansion following a single injection. We demonstrated that Alb-Flt3L, in combination with standard-of-care chemotherapy and radiation therapy, serves as an in situ vaccination strategy capable of engendering polyclonal tumor neoantigen-specific immunity spontaneously. In addition, Alb-Flt3L-mediated tumor control synergized with immune checkpoint blockade delivered as anti-PD-L1. The mechanism of action of Alb-Flt3L treatment revealed a dependency on Batf3, type-I-interferons, and plasmacytoid DCs. Finally, the ability of Alb-Flt3L to expand human DC was explored in humanized mice. We observed significant expansion of human cross-presenting DC subsets, supporting the notion that Alb-Flt3L could be used clinically to modulate human DC populations in future cancer therapeutic regimens.

Authors

Brandon Lam, Yu Jui Kung, John Lin, Ssu-Hsueh Tseng, Hsin-Fang Tu, Claire Huang, Brandon Lee, Esteban Velarde, Ya Chea Tsai, Rafael Villasmil, Sung Taek Park, Deyin Xing, Chien-Fu Hung, T.-C. Wu