Hypertrophic cardiomyopathy (HCM) is the most prominent cause of sudden cardiac death in young individuals. Due to heterogeneity in the clinical manifestations, conventional HCM drugs have limitations for mitochondrial hypertrophic cardiomyopathy. Discovering more effective compounds would be of substantial benefit for further elucidating the pathogenic mechanisms of HCM and treating patients with this condition. We previously reported the MT-RNR2 variant associated with HCM that results in mitochondrial dysfunction. Here, we screened a mitochondria-associated compound library by quantifying the mitochondrial membrane potential of HCM cybrids and the survival rate of HCM induced pluripotent stem cell-derived cardiomyocytes (iPSC-CMs) in galactose media. 1-Deoxynojirimycin (DNJ) was identified to rescue mitochondrial function by targeting optic atrophy protein 1 (OPA1) to promote its oligomerization, leading to reconstruction of the mitochondrial cristae. DNJ treatment further recovered the physiological properties of HCM iPSC-CMs by improving Ca2+ homeostasis and electrophysiological properties. An angiotensin II-induced cardiac hypertrophy mouse model further verified the efficacy of DNJ in promoting cardiac mitochondrial function and alleviating cardiac hypertrophy in vivo. These results demonstrated that DNJ could be a potential mitochondrial rescue agent for mitochondrial hypertrophic cardiomyopathy. Our findings will help elucidate the mechanism of HCM and provide a potential therapeutic strategy.
Qianqian Zhuang, Fengfeng Guo, Lei Fu, Yufei Dong, Shaofang Xie, Xue Ding, Shuangyi Hu, Xuanhao D. Zhou, Yangwei Jiang, Hui Zhou, Yue Qiu, Zhaoying Lei, Mengyao Li, Huajian Cai, Mingjie Fan, Lingjie Sang, Yong Fu, Dong Zhang, Aifu Lin, Xu Li, Tilo Kunath, Ruhong Zhou, Ping Liang, Zhong Liu, Qingfeng Yan
Patient-derived induced pluripotent stem cells (iPSCs) provide a powerful tool for identifying cellular and molecular mechanisms of disease. Macular telangiectasia type 2 (MacTel) is a rare, late-onset degenerative retinal disease with an extremely heterogeneous genetic architecture, lending itself to the use of iPSCs. Whole-exome sequencing screens and pedigree analyses have identified rare causative mutations that account for less than 5% of cases. Metabolomic surveys of patient populations and GWAS have linked MacTel to decreased circulating levels of serine and elevated levels of neurotoxic 1-deoxysphingolipids (1-dSLs). However, retina-specific, disease-contributing factors have yet to be identified. Here, we used iPSC-differentiated retinal pigmented epithelial (iRPE) cells derived from donors with or without MacTel to screen for novel cell-intrinsic pathological mechanisms. We show that MacTel iRPE cells mimicked the low serine levels observed in serum from patients with MacTel. Through RNA-Seq and gene set enrichment pathway analysis, we determined that MacTel iRPE cells are enriched in cellular stress pathways and dysregulation of central carbon metabolism. Using respirometry and mitochondrial stress testing, we functionally validated that MacTel iRPE cells had a reduction in mitochondrial function that was independent of defects in serine biosynthesis and 1-dSL accumulation. Thus, we identified phenotypes that may constitute alternative disease mechanisms beyond the known serine/sphingolipid pathway.
Kevin T. Eade, Brendan Robert E. Ansell, Sarah Giles, Regis Fallon, Sarah Harkins-Perry, Takayuki Nagasaki, Simone Tzaridis, Martina Wallace, Elizabeth A. Mills, Samaneh Farashi, Alec Johnson, Lydia Sauer, Barbara Hart, Elena D. Rubio, Melanie Bahlo, Christian Metallo, Rando Allikmets, Marin L. Gantner, Paul S. Bernstein, Martin Friedlander
Glioblastoma ranks among the most aggressive and lethal of all human cancers. Self-renewing, highly tumorigenic glioblastoma stem cells (GSCs) contribute to therapeutic resistance and maintain cellular heterogeneity. Here, we interrogated superenhancer landscapes of primary glioblastoma specimens and patient-derived GSCs, revealing a kelch domain-containing gene (KLHDC8A) with a previously unknown function as an epigenetically-driven oncogene. Targeting KLHDC8A decreased GSC proliferation and self-renewal, induced apoptosis, and impaired in vivo tumor growth. Transcription factor control circuitry analyses revealed that the master transcriptional regulator SOX2 stimulated KLHDC8A expression. Mechanistically, KLHDC8A bound Chaperonin-Containing TCP1 (CCT) to promote assembly of primary cilia to activate Hedgehog signaling. KLHDC8A expression correlated with Aurora B/C Kinase inhibitor activity, which induced primary cilia and Hedgehog signaling. Combinatorial targeting of Aurora B/C Kinase and Hedgehog displayed augmented benefit against GSC proliferation. Collectively, superenhancer-based discovery revealed KLHDC8A as a novel molecular target of cancer stem cells that promotes ciliogenesis to activate the Hedgehog pathway, offering insights into therapeutic vulnerabilities for glioblastoma treatment.
Derrick Lee, Ryan C. Gimple, Xujia Wu, Briana C. Prager, Zhixin Qiu, Qiulian Wu, Vikas Daggubati, Aruljothi Mariappan, Jay Gopalakrishnan, Matthew R. Sarkisian, David R. Raleigh, Jeremy N. Rich
Preterm birth is the leading cause of death in children under 5 years of age. Premature infants who receive life-saving oxygen therapy often develop bronchopulmonary dysplasia (BPD), a chronic lung disease. Infants with BPD are at a high risk of abnormal neurodevelopment, including motor and cognitive difficulties. While neural progenitor cells (NPCs) are crucial for proper brain development, it is unclear whether they play a role in BPD-associated neurodevelopmental deficits. Here, we showed that hyperoxia-induced experimental BPD in newborn mice led to life-long impairments in cerebrovascular structure and function, as well as impairments in NPC self-renewal and neurogenesis. A neurosphere assay utilizing non-human primate preterm baboon NPCs confirmed impairment in NPC function. Moreover, gene expression profiling revealed that genes involved in cell proliferation, angiogenesis, vascular autoregulation, neuronal formation, and neurotransmission were dysregulated following neonatal hyperoxia. These impairments were associated with motor and cognitive decline in aging hyperoxia-exposed mice, reminiscent of deficits observed in patients with BPD. Altogether, our findings established a relationship between BPD and abnormal neurodevelopmental outcomes and identified molecular and cellular players of neonatal brain injury that persist throughout adulthood, that may be targeted for early intervention to aid this vulnerable patient population.
Marissa A. Lithopoulos, Xavier Toussay, Shumei Zhong, Liqun Xu, Shamimunisa B. Mustafa, Julie Ouellette, Moises Freitas-Andrade, Cesar C. Comin, Hayam A. Bassam, Adam N. Baker, Yiren Sun, Michael Wakem, Alvaro G. Moreira, Cynthia L. Blanco, Arul Vadivel, Catherine Tsilfidis, Steven R. Seidner, Ruth S. Slack, Diane C. Lagace, Jing Wang, Baptiste Lacoste, Bernard Thébaud
A fundamental issue in regenerative medicine is whether there exist endogenous regulatory mechanisms that limit the speed and efficiency of the repair process. We report the existence of a maturation checkpoint during muscle regeneration which pauses myofibers at a neonatal stage. This checkpoint is regulated by the mitochondrial protein mitofusin 2 (Mfn2), whose expression is activated in response to muscle injury. Mfn2 is required for growth and maturation of regenerating myofibers; in the absence of Mfn2, new myofibers arrested at a neonatal stage, characterized by centrally nucleated myofibers and loss of H3K27me3 repressive marks at the neonatal myosin heavy chain gene. A similar arrest at the neonatal stage was observed in infantile cases of human centronuclear myopathy. Mechanistically, Mfn2 upregulation suppressed expression of Hypoxia-induced Factor 1α (Hif1α), which is induced in the setting of muscle damage. Sustained Hif1α signaling blocked maturation of new myofibers at the neonatal-to-adult fate transition, revealing the existence of a checkpoint that delays muscle regeneration. Correspondingly, inhibition of Hif1α allowed myofibers to bypass the checkpoint, thereby accelerating the repair process. We conclude that skeletal muscle contains a regenerative checkpoint which regulates the speed of myofiber maturation in response to Mitofusin 2 and Hif1α activity.
Xun Wang, Yuemeng Jia, Jiawei Zhao, Nicholas P. Lesner, Cameron J. Menezes, Spencer D. Shelton, Siva Sai Krishna Venigalla, Jian Xu, Chunyu Cai, Prashant Mishra
The stomach corpus epithelium is organized into anatomical units that consist of glands and pits and contain different specialized secretory cells. Acute and chronic injury of the corpus are associated with characteristic changes of cellular differentiation and proliferation. Processes that control cellular differentiation under homeostatic conditions and upon injury are not well understood. R-spondin 3 (Rspo3) is a Wnt signalling enhancer secreted by gastric stromal cells, which controls stem cell homeostasis in different organs. Here we investigated the function of Rspo3 in the corpus during homeostasis, acute injury, and H. pylori infection. Using organoid culture and conditional mouse models, we demonstrate that RSPO3 is a critical driver of secretory cell differentiation in the corpus gland towards parietal and chief cells, while its absence promoted pit cell differentiation. Acute loss of chief and parietal cells induced by high dose tamoxifen - or merely the depletion of LGR5+ chief cells – caused an upregulation of RSPO3 expression, which was required for the initiation of a coordinated regenerative response via the activation of yes-associated protein (YAP) signaling. This response enabled a rapid recovery of the injured secretory gland cells. However, in the context of chronic H. pylori infection, the R-spondin-driven regeneraton was maintained long-term, promoing severe glandular hyperproliferation and the development of premalignant metaplasia.
Anne-Sophie Fischer, Stefanie Müllerke, Alexander Arnold, Julian Heuberger, Hilmar Berger, Manqiang Lin, Hans-Joachim Mollenkopf, Jonas Wizenty, David Horst, Frank Tacke, Michael Sigal
The basis of immune evasion, a hallmark of cancer, can differ even when cancers arise from one cell type such as in the human skin keratinocyte carcinomas: basal and squamous cell carcinoma. Here we showed that the basal cell carcinoma tumor initiating cell surface protein CD200, through ectodomain shedding, was responsible for the near absence of NK cells within the basal cell carcinoma tumor microenvironment. In situ, CD200 underwent ectodomain shedding by metalloproteinases MMP3 and MMP11, which released biologically active soluble CD200 into the basal cell carcinoma microenvironment. CD200 bound its cognate receptor on NK cells, to suppress MAPK pathway signaling that in turn blocked indirect (gamma interferon release) and direct cell killing. In addition, reduced ERK phosphorylation relinquished negative regulation of PPARγ regulated gene transcription and lead to membrane accumulation of the Fas/FADD death receptor and its ligand, FasL that resulted in activation-induced apoptosis. Blocking CD200 inhibition of MAPK or PPARγ signaling restored NK cell survival and tumor cell killing, with relevance to many cancer types. Our results thus uncover a paradigm for CD200 as a potentially novel and targetable NK cell specific immune checkpoint, which is responsible for NK cell associated poor outcomes in many cancers.
Huw J. Morgan, Elise Rees, Simone Lanfredini, Kate A. Powell, Jasmine Gore, Alex Gibbs, Charlotte Lovatt, Gemma E. Davies, Carlotta Olivero, Boris Y. Shorning, Giusy Tornillo, Alex Tonks, Richard Darley, Eddie C.Y. Wang, Girish K. Patel
Defining mechanism(s) that maintain tissue stem quiescence is important for improving tissue regeneration, cell therapies, aging, and cancer. We report here that genetic ablation of Id2 in adult hematopoietic stem cells (HSCs) promotes increased HSC activation and differentiation, which results in HSC exhaustion and bone marrow failure over time. Id2Δ/Δ HSCs showed increased cycling, ROS production, mitochondrial activation, ATP production, and DNA damage compared with Id2+/+ HSCs, supporting the conclusion that Id2Δ/Δ HSCs are less quiescent. Mechanistically, HIF-1α expression was decreased in Id2Δ/Δ HSCs, and stabilization of HIF-1α in Id2Δ/Δ HSCs restored HSC quiescence and rescued HSC exhaustion. Inhibitor of DNA binding 2 (ID2) promoted HIF-1α expression by binding to the von Hippel-Lindau (VHL) protein and interfering with proteasomal degradation of HIF-1α. HIF-1α promoted Id2 expression and enforced a positive feedback loop between ID2 and HIF-1α to maintain HSC quiescence. Thus, sustained ID2 expression could protect HSCs during stress and improve HSC expansion for gene editing and cell therapies.
Brad L. Jakubison, Tanmoy Sarkar, Kristbjorn O. Gudmundsson, Shweta Singh, Lei Sun, Holly M. Morris, Kimberly D. Klarmann, Jonathan R. Keller
Human pluripotent stem cell (hPSC)-based replacement therapy holds great promise in treating Parkinson’s disease (PD). However, the heterogeneity of hPSC-derived donor cells and the low yield of midbrain dopaminergic (mDA) neurons after transplantation hinder its broad clinical application. Here, we depicted the single-cell molecular landscape during mDA neuron differentiation. We found that this process recapitulated the development of multiple but adjacent fetal brain regions including ventral midbrain, isthmus, and ventral hindbrain, resulting in heterogenous donor cell population. We reconstructed the differentiation trajectory of mDA lineage and identified CLSTN2 and PTPRO as specific surface markers of mDA progenitors, which were predictive of mDA neuron differentiation and could facilitate highly enriched mDA neurons (up to 80%) following progenitor sorting and transplantation. Marker sorted progenitors exhibited higher therapeutic potency in correcting motor deficits of PD mice. Different marker sorted grafts had a strikingly consistent cellular composition, in which mDA neurons were enriched, while off-target neuron types were mostly depleted, suggesting stable graft outcomes. Our study provides a better understanding of cellular heterogeneity during mDA neuron differentiation, and establishes a strategy to generate highly purified donor cells to achieve stable and predictable therapeutic outcomes, raising the prospect of hPSC-based PD cell replacement therapies.
Peibo Xu, Hui He, Qinqin Gao, Yingying Zhou, Ziyan Wu, Xiao Zhang, Linyu Sun, Gang Hu, Qian Guan, Zhiwen You, Xinyue Zhang, Wenping Zheng, Man Xiong, Yuejun Chen
Fanconi Anemia (FA) is the most prevalent inherited bone marrow failure (BMF) syndrome. Nevertheless, the pathophysiological mechanisms of BMF in FA have not been fully elucidated. Since FA cells are defective in DNA repair, we hypothesized that FA hematopoietic stem and progenitor cells (HSPCs) might express DNA damage-associated stress molecules such as Natural Killer group 2 member D ligands (NKG2D-Ls). These ligands could then interact with the activating NKG2D receptor expressed in cytotoxic NK or CD8+ T cells which may result in progressive HSPCs depletion. Our results indeed demonstrated upregulated levels of NKG2D-Ls in cultured FA fibroblasts and T cells, which were further exacerbated by mitomycin C or formaldehyde. Notably, a high proportion of BM CD34+ HSPCs from FA patients also expressed increased levels of NKG2D-Ls, which correlated inversely with the percentage of CD34+ cells in BM. Remarkably, the reduced clonogenic potential characteristic of FA HSPCs was improved by blocking NKG2D/NKG2D-L interactions. Moreover, the in vivo blockage of these interactions in a BMF FA mouse model ameliorated the anemia in these animals. Our study demonstrates the involvement of NKG2D/NKG2D-L interactions in FA HSPC functionality, suggesting an unexpected role of the immune system in the progressive BMF characteristic of FA.
Jose A. Casado, Antonio Valeri, Rebeca Sanchez-Domínguez, Paula Vela, Andrea Lopez, Susana Navarro, Omaira Alberquilla, Helmut Hanenberg, Roser Pujol, Jose C. Segovia, Jordi Minguillón, Jordi Surrallés, Cristina Diaz-de-Heredia, Julián Sevilla, Paula Rio, Juan A. Bueren