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- Volume 125, Issue 7
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On the cover: Separating pulmonary and systemic circulation
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Review
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Abstract
Human pluripotent stem cells are known to have the capacity to renew indefinitely, being intrinsically able to differentiate into many different cell types. These characteristics have generated tremendous enthusiasm about the potential applications of these cells in regenerative medicine. However, major challenges remain with the development and testing of novel experimental stem cell therapeutics in the field. In this Review, we focus on the nature of the preclinical challenges and discuss potential solutions that could help overcome them. Furthermore, we discuss the use of allogeneic versus autologous stem cell products, including a review of their respective advantages and disadvantages, major clinical requirements, quality standards, time lines, and costs of clinical grade development.
Authors
Evgenios Neofytou, Connor Galen O’Brien, Larry A. Couture, Joseph C. Wu
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Commentaries
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Abstract
μ-Opioid agonists mediate their analgesic effect through GPCRs that are generated via alternate splicing of the
Oprm1 transcript. While the majority of μ-opioids interact with receptors comprising the canonical 7 transmembrane (7TM) domain, a recently identified class of μ-opioids appears to require a 6TM domain variant. In this issue of theJCI , Lu and colleagues provide an in vivo proof-of-concept demonstration that a 6TM isoform of the μ-opioid receptor can support functional analgesia inOprm1 -deficent animals. The 6TM isoform was pharmacologically distinct from the canonical 7TM μ-opioid receptor, and 6TM agonists had a reduced side effect profile, which confers a strong therapeutic advantage over standard opioid analgesics. The observations of Lu et al. extend the reach of opioid-receptor neurobiology and pharmacology into a new era of analgesic discovery. This advance emerges from a series of fundamental research analyses in which elements of the endogenous opioid system were frequently in the vanguard.Authors
Michael J. Iadarola, Matthew R. Sapio, Andrew J. Mannes
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Systemic lupus erythematosus (SLE) is a multisystem autoimmune disease with a broad spectrum of clinical presentations involving multiple organ systems. An abnormal response to self-antigens is thought to drive the development of SLE; however, the factors that underlie this dysfunction are not clear. In this issue of the
JCI , Li and colleagues present compelling evidence to show that type I interferons (IFNs) produced by plasmacytoid dendritic cells inhibit the clearance of apoptotic cells (ACs) by marginal zone macrophages. Specifically, type I IFNs increase the translocation of marginal zone (MZ) B cells to the follicular region of the spleen, thereby disrupting interactions between these B cells and MZ macrophages (MZMs), which in turn disrupts megakaryoblastic leukemia 1–mediated (MKL1-mediated) mechanosensing and inhibits AC phagocytosis by MZMs. The results of this study provide important insight into factors that inhibit AC clearance and promote the development of SLE.Authors
Claudia Mauri, Madhvi Menon
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Pancreatic β cells support glucose homeostasis with great precision by matching insulin release to the metabolic needs of the moment. Previous gene-expression analysis indicates that adult β cells not only produce cell-specific proteins, but also repress a small set of housekeeping genes — such as those encoding lactate dehydrogenase A (LDHA), solute transporter MCT1, and hexokinase 1 (HK1) — that would otherwise interfere with normal β cell function. In this issue of the JCI, Dhawan et al. elucidate a molecular mechanism involved in β cell–specific repression of
Ldha andHk1 that is mediated by induction of the de novo DNA methyltransferase DNMT3A during the first weeks after birth. Failure to induce DNMT3A-dependent methylation disrupts normal glucose-induced insulin release in adult life. The results of this study reinforce the idea that the phenotype of adult β cells has two faces and that failure to achieve selective gene repression undermines β cell support of normal glucose homeostasis.Authors
Frans Schuit
×Abstract
Multiple processes are capable of activating the onset of parturition; however, the specific contributions of the mother and the fetus to this process are not fully understood. In this issue of the
JCI , Gao and colleagues present evidence that steroid receptor coactivators 1 and 2 (SRC-1 and SRC-2) regulate surfactant protein-A (SP-A) and platelet-activating factor (PAF) expression, which increases in the developing fetal lung. WT dams crossed with males deficient for both SRC-1 and SRC-2 had suppressed myometrial inflammation, increased serum progesterone, and delayed parturition, which could be reconciled by injection of either SP-A or PAF into the amnion. Together, the results of this study demonstrate that the fetal lungs produce signals to initiate labor in the mouse. This work underscores the importance of the fetus as a contributor to the onset of murine, and potentially human, parturition.Authors
Erin L. Reinl, Sarah K. England
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Synaptic disorganization is a prominent feature of many neurological diseases of the CNS, including Parkinson’s disease, intellectual development disorders, and autism. Although synaptic plasticity is critical for learning and memory, it is unclear whether this innate property helps restore synaptic function in disease once the primary cause of disease is abrogated. An answer to this question may come from a recent investigation in X-linked retinoschisis, a currently untreatable retinopathy. In this issue of the
JCI , Ou, Vijayasarathy, and colleagues showed progressive disorganization of key functional elements of the synapse between photoreceptors and ON-bipolar cells in a retinoschisin-deficient mouse model. Moreover, they demonstrated that adeno-associated virus–mediated (AAV-mediated) delivery of the retinoschisin gene restores structure and function to the photoreceptor to ON–bipolar cell synapse in mouse models, even in adults at advanced stages of the disease. The results of this study hold promise that AAV-based supplemental gene therapy will benefit patients with X-linked retinoschisis in a forthcoming clinical trial.Authors
Timm Schubert, Bernd Wissinger
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Activation of estrogen receptor α (ERα) in the brain prevents obesity as the result of increased energy expenditure and decreased food intake. While ERα is present on several neural populations, it is not clear how different regions of the brain mediate the weight-regulating effects of ERα activation. In this issue of the
JCI , Xu and colleagues provide extensive evidence that ERα is abundant on neurons expressing single-minded–1 (SIM1) in the medial amygdala (MeA) and that loss of ERα in these cells enhances weight gain in both male and female mice, as the result of reduced physical activity. Moreover, focal deletion of ERα from the MeA recapitulated these alterations in energy homeostasis. Conversely, overexpression of ERα in the MeA partially prevented mice from diet-induced obesity, while chemogenetic activation of SIM1-expressing neurons in the MeA transiently promoted physical activity. The results of this study provide important insight into the regions of the brain that mediate ERα-dependent energy homeostasis.Authors
Chia Li, Michael J. Krashes
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Research Articles
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Abstract
Myeloproliferative neoplasms (MPNs) are characterized by the clonal expansion of one or more myeloid cell lineage. In most cases, proliferation of the malignant clone is ascribed to defined genetic alterations. MPNs are also associated with aberrant expression and activity of multiple cytokines; however, the mechanisms by which these cytokines contribute to disease pathogenesis are poorly understood. Here, we reveal a non-redundant role for steady-state IL-33 in supporting dysregulated myelopoiesis in a murine model of MPN. Genetic ablation of the IL-33 signaling pathway was sufficient and necessary to restore normal hematopoiesis and abrogate MPN-like disease in animals lacking the inositol phosphatase SHIP. Stromal cell–derived IL-33 stimulated the secretion of cytokines and growth factors by myeloid and non-hematopoietic cells of the BM, resulting in myeloproliferation in SHIP-deficient animals. Additionally, in the transgenic JAK2V617F model, the onset of MPN was delayed in animals lacking IL-33 in radio-resistant cells. In human BM, we detected increased numbers of IL-33–expressing cells, specifically in biopsies from MPN patients. Exogenous IL-33 promoted cytokine production and colony formation by primary CD34+ MPN stem/progenitor cells from patients. Moreover, IL-33 improved the survival of JAK2V617F-positive cell lines. Together, these data indicate a central role for IL-33 signaling in the pathogenesis of MPNs.
Authors
Lukas F. Mager, Carsten Riether, Christian M. Schürch, Yara Banz, Marie-Hélène Wasmer, Regula Stuber, Alexandre P. Theocharides, Xiaohong Li, Yu Xia, Hirohisa Saito, Susumu Nakae, Gabriela M. Baerlocher, Markus G. Manz, Kathy D. McCoy, Andrew J. Macpherson, Adrian F. Ochsenbein, Bruce Beutler, Philippe Krebs
×Abstract
The integrated stress response (ISR) is a critical mediator of cancer cell survival, and targeting the ISR inhibits tumor progression. Here, we have shown that activating transcription factor 4 (ATF4), a master transcriptional effector of the ISR, protects transformed cells against anoikis — a specialized form of apoptosis — following matrix detachment and also contributes to tumor metastatic properties. Upon loss of attachment, ATF4 activated a coordinated program of cytoprotective autophagy and antioxidant responses, including induced expression of the major antioxidant enzyme heme oxygenase 1 (HO-1). HO-1 upregulation was the result of simultaneous activation of ATF4 and the transcription factor NRF2, which converged on the
HO1 promoter. Increased levels of HO-1 ameliorated oxidative stress and cell death. ATF4-deficient human fibrosarcoma cells were unable to colonize the lungs in a murine model, and reconstitution of ATF4 or HO-1 expression in ATF4-deficient cells blocked anoikis and rescued tumor lung colonization. HO-1 expression was higher in human primary and metastatic tumors compared with noncancerous tissue. Moreover, HO-1 expression correlated with reduced overall survival of patients with lung adenocarcinoma and glioblastoma. These results establish HO-1 as a mediator of ATF4-dependent anoikis resistance and tumor metastasis and suggest ATF4 and HO-1 as potential targets for therapeutic intervention in solid tumors.Authors
Souvik Dey, Carly M. Sayers, Ioannis I. Verginadis, Stacey L. Lehman, Yi Cheng, George J. Cerniglia, Stephen W. Tuttle, Michael D. Feldman, Paul J.L. Zhang, Serge Y. Fuchs, J. Alan Diehl, Constantinos Koumenis
×Abstract
Subarachnoid hemorrhage (SAH) carries a 50% mortality rate. The extravasated erythrocytes that surround the brain contain heme, which, when released from damaged red blood cells, functions as a potent danger molecule that induces sterile tissue injury and organ dysfunction. Free heme is metabolized by heme oxygenase (HO), resulting in the generation of carbon monoxide (CO), a bioactive gas with potent immunomodulatory capabilities. Here, using a murine model of SAH, we demonstrated that expression of the inducible HO isoform (HO-1, encoded by
Hmox1 ) in microglia is necessary to attenuate neuronal cell death, vasospasm, impaired cognitive function, and clearance of cerebral blood burden. Initiation of CO inhalation after SAH rescued the absence of microglial HO-1 and reduced injury by enhancing erythrophagocytosis. Evaluation of correlative human data revealed that patients with SAH have markedly higher HO-1 activity in cerebrospinal fluid (CSF) compared with that in patients with unruptured cerebral aneurysms. Furthermore, cisternal hematoma volume correlated with HO-1 activity and cytokine expression in the CSF of these patients. Collectively, we found that microglial HO-1 and the generation of CO are essential for effective elimination of blood and heme after SAH that otherwise leads to neuronal injury and cognitive dysfunction. Administration of CO may have potential as a therapeutic modality in patients with ruptured cerebral aneurysms.Authors
Nils Schallner, Rambhau Pandit, Robert LeBlanc III, Ajith J. Thomas, Christopher S. Ogilvy, Brian S. Zuckerbraun, David Gallo, Leo E. Otterbein, Khalid A. Hanafy
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The generation of potent opioid analgesics that lack the side effects of traditional opioids may be possible by targeting truncated splice variants of the μ-opioid receptor. μ-Opioids act through GPCRs that are generated from the
Oprm1 gene, which undergoes extensive alternative splicing. The most abundant set ofOprm1 variants encode classical full-length 7 transmembrane domain (7TM) μ-opioid receptors that mediate the actions of the traditional μ-opioid drugs morphine and methadone. In contrast, 3-iodobenzoyl-6β-naltrexamide (IBNtxA) is a potent analgesic against thermal, inflammatory, and neuropathic pain that acts independently of 7TM μ-opioid receptors but has no activity in mice lacking a set of 6TM truncated μ-opioid receptor splice variants. Unlike traditional opioids, IBNtxA does not depress respiration or result in physical dependence or reward behavior, suggesting it acts through an alternative μ-opioid receptor target. Here we demonstrated that a truncated 6TM splice variant, mMOR-1G, can rescue IBNtxA analgesia in a μ-opioid receptor–deficient mouse that lacks allOprm1 splice variants, ablating μ-opioid activity in these animals. Intrathecal administration of lentivirus containing the 6TM variant mMOR-1G restored IBNtxA, but not morphine, analgesia inOprm1 -deficient animals. Together, these results confirm that a truncated 6TM GPCR is both necessary and sufficient for IBNtxA analgesia.Authors
Zhigang Lu, Jin Xu, Grace C. Rossi, Susruta Majumdar, Gavril W. Pasternak, Ying-Xian Pan
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The selective pressure that drives antigenic changes in influenza viruses is thought to originate from the human immune response. Here, we have characterized the B cell repertoire from a previously vaccinated donor whose serum had reduced neutralizing activity against the recently evolved clade 6B H1N1pdm09 viruses. While the response was markedly polyclonal, 88% of clones failed to recognize clade 6B viruses; however, the ability to neutralize A/USSR/90/1977 influenza, to which the donor would have been exposed in childhood, was retained. In vitro selection of virus variants with representative monoclonal antibodies revealed that a single amino acid replacement at residue K163 in the Sa antigenic site, which is characteristic of the clade 6B viruses, was responsible for resistance to neutralization by multiple monoclonal antibodies and the donor serum. The K163 residue lies in a part of a conserved surface that is common to the hemagglutinins of the 1977 and 2009 H1N1 viruses. Vaccination with the 2009 hemagglutinin induced an antibody response tightly focused on this common surface that is capable of selecting current antigenic drift variants in H1N1pdm09 influenza viruses. Moreover, amino acid replacement at K163 was not highlighted by standard ferret antisera. Human monoclonal antibodies may be a useful adjunct to ferret antisera for detecting antigenic drift in influenza viruses.
Authors
Kuan-Ying A. Huang, Pramila Rijal, Lisa Schimanski, Timothy J. Powell, Tzou-Yien Lin, John W. McCauley, Rodney S. Daniels, Alain R. Townsend
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Patients with inflammatory bowel disease are at increased risk for colon cancer due to augmented oxidative stress. These patients also have compromised antioxidant defenses as the result of nutritional deficiencies. The micronutrient selenium is essential for selenoprotein production and is transported from the liver to target tissues via selenoprotein P (SEPP1). Target tissues also produce SEPP1, which is thought to possess an endogenous antioxidant function. Here, we have shown that mice with
Sepp1 haploinsufficiency or mutations that disrupt either the selenium transport or the enzymatic domain of SEPP1 exhibit increased colitis-associated carcinogenesis as the result of increased genomic instability and promotion of a protumorigenic microenvironment. Reduced SEPP1 function markedly increased M2-polarized macrophages, indicating a role for SEPP1 in macrophage polarization and immune function. Furthermore, compared with partial loss, complete loss of SEPP1 substantially reduced tumor burden, in part due to increased apoptosis. Using intestinal organoid cultures, we found that, compared with those from WT animals,Sepp1 -null cultures display increased stem cell characteristics that are coupled with increased ROS production, DNA damage, proliferation, decreased cell survival, and modulation of WNT signaling in response to H2O2-mediated oxidative stress. Together, these data demonstrate that SEPP1 influences inflammatory tumorigenesis by affecting genomic stability, the inflammatory microenvironment, and epithelial stem cell functions.Authors
Caitlyn W. Barrett, Vishruth K. Reddy, Sarah P. Short, Amy K. Motley, Mary K. Lintel, Amber M. Bradley, Tanner Freeman, Jefferson Vallance, Wei Ning, Bobak Parang, Shenika V. Poindexter, Barbara Fingleton, Xi Chen, Mary K. Washington, Keith T. Wilson, Noah F. Shroyer, Kristina E. Hill, Raymond F. Burk, Christopher S. Williams
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In mammals, the outflow tract (OFT) of the developing heart septates into the base of the pulmonary artery and aorta to guide deoxygenated right ventricular blood into the lungs and oxygenated left ventricular blood into the systemic circulation. Accordingly, defective OFT septation is a life-threatening condition that can occur in both syndromic and nonsyndromic congenital heart disease. Even though studies of genetic mouse models have previously revealed a requirement for VEGF-A, the class 3 semaphorin SEMA3C, and their shared receptor neuropilin 1 (NRP1) in OFT development, the precise mechanism by which these proteins orchestrate OFT septation is not yet understood. Here, we have analyzed a complementary set of ligand-specific and tissue-specific mouse mutants to show that neural crest–derived SEMA3C activates NRP1 in the OFT endothelium. Explant assays combined with gene-expression studies and lineage tracing further demonstrated that this signaling pathway promotes an endothelial-to-mesenchymal transition that supplies cells to the endocardial cushions and repositions cardiac neural crest cells (NCCs) within the OFT, 2 processes that are essential for septal bridge formation. These findings elucidate a mechanism by which NCCs cooperate with endothelial cells in the developing OFT to enable the postnatal separation of the pulmonary and systemic circulation.
Authors
Alice Plein, Amélie Calmont, Alessandro Fantin, Laura Denti, Naomi A. Anderson, Peter J. Scambler, Christiana Ruhrberg
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BACKGROUND. Graft-versus-host disease (GVHD) is a major cause of morbidity and mortality following allogeneic hematopoietic stem cell transplantation (HCT). In mice, naive T cells (TN) cause more severe GVHD than memory T cells (TM). We hypothesized that selective depletion of TN from human allogeneic peripheral blood stem cell (PBSC) grafts would reduce GVHD and provide sufficient numbers of hematopoietic stem cells and TM to permit hematopoietic engraftment and the transfer of pathogen-specific T cells from donor to recipient, respectively.METHODS. In a single-arm clinical trial, we transplanted 35 patients with high-risk leukemia with TN-depleted PBSC grafts following conditioning with total body irradiation, thiotepa, and fludarabine. GVHD prophylactic management was with tacrolimus immunosuppression alone. Subjects received CD34-selected PBSCs and a defined dose of TM purged of CD45RA+ TN. Primary and secondary objectives included engraftment, acute and chronic GVHD, and immune reconstitution.RESULTS. All recipients of TN-depleted PBSCs engrafted. The incidence of acute GVHD was not reduced; however, GVHD in these patients was universally corticosteroid responsive. Chronic GVHD was remarkably infrequent (9%; median follow-up 932 days) compared with historical rates of approximately 50% with T cell–replete grafts. TM in the graft resulted in rapid T cell recovery and transfer of protective virus-specific immunity. Excessive rates of infection or relapse did not occur and overall survival was 78% at 2 years.CONCLUSION. Depletion of TN from stem cell allografts reduces the incidence of chronic GVHD, while preserving the transfer of functional T cell memory.TRIAL REGISTRATION. ClinicalTrials.gov (NCT 00914940).FUNDING. NIH, Burroughs Wellcome Fund, Leukemia and Lymphoma Society, Damon Runyon Cancer Research Foundation, and Richard Lumsden Foundation.Authors
Marie Bleakley, Shelly Heimfeld, Keith R. Loeb, Lori A. Jones, Colette Chaney, Stuart Seropian, Ted A. Gooley, Franziska Sommermeyer, Stanley R. Riddell, Warren D. Shlomchik
×Abstract
Current stem cell–based strategies for tissue regeneration involve ex vivo manipulation of these cells to confer features of the desired progenitor population. Recently, the concept that endogenous stem/progenitor cells could be used for regenerating tissues has emerged as a promising approach that potentially overcomes the obstacles related to cell transplantation. Here we applied this strategy for the regeneration of injured tendons in a rat model. First, we identified a rare fraction of tendon cells that was positive for the known tendon stem cell marker CD146 and exhibited clonogenic capacity, as well as multilineage differentiation ability. These tendon-resident CD146+ stem/progenitor cells were selectively enriched by connective tissue growth factor delivery (CTGF delivery) in the early phase of tendon healing, followed by tenogenic differentiation in the later phase. The time-controlled proliferation and differentiation of CD146+ stem/progenitor cells by CTGF delivery successfully led to tendon regeneration with densely aligned collagen fibers, normal level of cellularity, and functional restoration. Using siRNA knockdown to evaluate factors involved in tendon generation, we demonstrated that the FAK/ERK1/2 signaling pathway regulates CTGF-induced proliferation and differentiation of CD146+ stem/progenitor cells. Together, our findings support the use of endogenous stem/progenitor cells as a strategy for tendon regeneration without cell transplantation and suggest this approach warrants exploration in other tissues.
Authors
Chang H. Lee, Francis Y. Lee, Solaiman Tarafder, Kristy Kao, Yena Jun, Guodong Yang, Jeremy J. Mao
×Abstract
Despite the wide availability of antiretroviral drugs, more than 250,000 infants are vertically infected with HIV-1 annually, emphasizing the need for additional interventions to eliminate pediatric HIV-1 infections. Here, we aimed to define humoral immune correlates of risk of mother-to-child transmission (MTCT) of HIV-1, including responses associated with protection in the RV144 vaccine trial. Eighty-three untreated, HIV-1–transmitting mothers and 165 propensity score–matched nontransmitting mothers were selected from the Women and Infants Transmission Study (WITS) of US nonbreastfeeding, HIV-1–infected mothers. In a multivariable logistic regression model, the magnitude of the maternal IgG responses specific for the third variable loop (V3) of the HIV-1 envelope was predictive of a reduced risk of MTCT. Neutralizing Ab responses against easy-to-neutralize (tier 1) HIV-1 strains also predicted a reduced risk of peripartum transmission in secondary analyses. Moreover, recombinant maternal V3–specific IgG mAbs mediated neutralization of autologous HIV-1 isolates. Thus, common V3-specific Ab responses in maternal plasma predicted a reduced risk of MTCT and mediated autologous virus neutralization, suggesting that boosting these maternal Ab responses may further reduce HIV-1 MTCT.
Authors
Sallie R. Permar, Youyi Fong, Nathan Vandergrift, Genevieve G. Fouda, Peter Gilbert, Robert Parks, Frederick H. Jaeger, Justin Pollara, Amanda Martelli, Brooke E. Liebl, Krissey Lloyd, Nicole L. Yates, R. Glenn Overman, Xiaoying Shen, Kaylan Whitaker, Haiyan Chen, Jamie Pritchett, Erika Solomon, Emma Friberg, Dawn J. Marshall, John F. Whitesides, Thaddeus C. Gurley, Tarra Von Holle, David R. Martinez, Fangping Cai, Amit Kumar, Shi-Mao Xia, Xiaozhi Lu, Raul Louzao, Samantha Wilkes, Saheli Datta, Marcella Sarzotti-Kelsoe, Hua-Xin Liao, Guido Ferrari, S. Munir Alam, David C. Montefiori, Thomas N. Denny, M. Anthony Moody, Georgia D. Tomaras, Feng Gao, Barton F. Haynes
×Abstract
Estrogen receptor–negative (ER-negative) breast cancers are extremely aggressive and associated with poor prognosis. In particular, effective treatment strategies are limited for patients diagnosed with triple receptor–negative breast cancer (TNBC), which also carries the worst prognosis of all forms of breast cancer; therefore, extensive studies have focused on the identification of molecularly targeted therapies for this tumor subtype. Here, we sought to identify molecular targets that are capable of suppressing tumorigenesis in TNBCs. Specifically, we found that death-associated protein kinase 1 (DAPK1) is essential for growth of p53-mutant cancers, which account for over 80% of TNBCs. Depletion or inhibition of DAPK1 suppressed growth of p53-mutant but not p53-WT breast cancer cells. Moreover, DAPK1 inhibition limited growth of other p53-mutant cancers, including pancreatic and ovarian cancers. DAPK1 mediated the disruption of the TSC1/TSC2 complex, resulting in activation of the mTOR pathway. Our studies demonstrated that high DAPK1 expression causes increased cancer cell growth and enhanced signaling through the mTOR/S6K pathway; evaluation of multiple breast cancer patient data sets revealed that high DAPK1 expression associates with worse outcomes in individuals with p53-mutant cancers. Together, our data support targeting DAPK1 as a potential therapeutic strategy for p53-mutant cancers.
Authors
Jing Zhao, Dekuang Zhao, Graham M. Poage, Abhijit Mazumdar, Yun Zhang, Jamal L. Hill, Zachary C. Hartman, Michelle I. Savage, Gordon B. Mills, Powel H. Brown
×Abstract
Multiple convergent lines of evidence implicate both α-synuclein (encoded by
SCNA ) and mitochondrial dysfunction in the pathogenesis of sporadic Parkinson’s disease (PD). Occupational exposure to the mitochondrial complex I inhibitor rotenone increases PD risk; rotenone-exposed rats show systemic mitochondrial defects but develop specific neuropathology, including α-synuclein aggregation and degeneration of substantia nigra dopaminergic neurons. Here, we inhibited expression of endogenous α-synuclein in the adult rat substantia nigra by adeno-associated virus–mediated delivery of a short hairpin RNA (shRNA) targeting the endogenous ratSnca transcript. Knockdown of α-synuclein by ~35% did not affect motor function or cause degeneration of nigral dopaminergic neurons in control rats. However, in rotenone-exposed rats, progressive motor deficits were substantially attenuated contralateral to α-synuclein knockdown. Correspondingly, rotenone-induced degeneration of nigral dopaminergic neurons, their dendrites, and their striatal terminals was decreased ipsilateral to α-synuclein knockdown. These data show that α-synuclein knockdown is neuroprotective in the rotenone model of PD and indicate that endogenous α-synuclein contributes to the specific vulnerability of dopaminergic neurons to systemic mitochondrial inhibition. Our findings are consistent with a model in which genetic variants influencing α-synuclein expression modulate cellular susceptibility to environmental exposures in PD patients. shRNA targeting theSNCA transcript should be further evaluated as a possible neuroprotective therapy in PD.Authors
Alevtina D. Zharikov, Jason R. Cannon, Victor Tapias, Qing Bai, Max P. Horowitz, Vipul Shah, Amina El Ayadi, Teresa G. Hastings, J. Timothy Greenamyre, Edward A. Burton
×Abstract
Type 2 diabetes mellitus (T2DM) is a worldwide heath problem that is characterized by insulin resistance and the eventual loss of β cell function. As recent studies have shown that loss of ribosomal protein (RP) S6 kinase 1 (S6K1) increases systemic insulin sensitivity, S6K1 inhibitors are being pursued as potential agents for improving insulin resistance. Here we found that S6K1 deficiency in mice also leads to decreased β cell growth, intrauterine growth restriction (IUGR), and impaired placental development. IUGR is a common complication of human pregnancy that limits the supply of oxygen and nutrients to the developing fetus, leading to diminished embryonic β cell growth and the onset of T2DM later in life. However, restoration of placental development and the rescue of IUGR by tetraploid embryo complementation did not restore β cell size or insulin levels in
S6K1–/– embryos, suggesting that loss of S6K1 leads to an intrinsic β cell lesion. Consistent with this hypothesis, reexpression of S6K1 in β cells ofS6K1–/– mice restored embryonic β cell size, insulin levels, glucose tolerance, and RPS6 phosphorylation, without rescuing IUGR. Together, these data suggest that a nutrient-mediated reduction in intrinsic β cell S6K1 signaling, rather than IUGR, during fetal development may underlie reduced β cell growth and eventual development of T2DM later in life.Authors
Sung Hee Um, Melanie Sticker-Jantscheff, Gia Cac Chau, Kristina Vintersten, Matthias Mueller, Yann-Gael Gangloff, Ralf H. Adams, Jean-Francois Spetz, Lynda Elghazi, Paul T. Pfluger, Mario Pende, Ernesto Bernal-Mizrachi, Albert Tauler, Matthias H. Tschöp, George Thomas, Sara C. Kozma
×Abstract
Macrophages clear millions of apoptotic cells daily and, during this process, take up large quantities of cholesterol. The membrane transporter ABCA1 is a key player in cholesterol efflux from macrophages and has been shown via human genetic studies to provide protection against cardiovascular disease. How the apoptotic cell clearance process is linked to macrophage ABCA1 expression is not known. Here, we identified a plasma membrane–initiated signaling pathway that drives a rapid upregulation of
ABCA1 mRNA and protein. This pathway involves the phagocytic receptor brain-specific angiogenesis inhibitor 1 (BAI1), which recognizes phosphatidylserine on apoptotic cells, and the intracellular signaling intermediates engulfment cell motility 1 (ELMO1) and Rac1, as ABCA1 induction was attenuated in primary macrophages from mice lacking these molecules. Moreover, this apoptotic cell–initiated pathway functioned independently of the liver X receptor (LXR) sterol–sensing machinery that is known to regulate ABCA1 expression and cholesterol efflux. When placed on a high-fat diet, mice lacking BAI1 had increased numbers of apoptotic cells in their aortic roots, which correlated with altered lipid profiles. In contrast, macrophages from engineered mice with transgenic BAI1 overexpression showed greater ABCA1 induction in response to apoptotic cells compared with those from control animals. Collectively, these data identify a membrane-initiated pathway that is triggered by apoptotic cells to enhance ABCA1 within engulfing phagocytes and with functional consequences in vivo.Authors
Aaron M. Fond, Chang Sup Lee, Ira G. Schulman, Robert S. Kiss, Kodi S. Ravichandran
×Abstract
Ischemic heart disease is the leading cause of heart failure. Both clinical trials and experimental animal studies demonstrate that chronic hypoxia can induce contractile dysfunction even before substantial ventricular damage, implicating a direct role of oxygen in the regulation of cardiac contractile function. Prolyl hydroxylase domain (PHD) proteins are well recognized as oxygen sensors and mediate a wide variety of cellular events by hydroxylating a growing list of protein substrates. Both PHD2 and PHD3 are highly expressed in the heart, yet their functional roles in modulating contractile function remain incompletely understood. Here, we report that combined deletion of
Phd2 andPhd3 dramatically decreased expression of phospholamban (PLN), resulted in sustained activation of calcium/calmodulin-activated kinase II (CaMKII), and sensitized mice to chronic β-adrenergic stress–induced myocardial injury. We have provided evidence that thyroid hormone receptor-α (TR-α), a transcriptional regulator of PLN, interacts with PHD2 and PHD3 and is hydroxylated at 2 proline residues. Inhibition of PHDs increased the interaction between TR-α and nuclear receptor corepressor 2 (NCOR2) and suppressedPln transcription. Together, these observations provide mechanistic insight into how oxygen directly modulates cardiac contractility and suggest that cardiac function could be modulated therapeutically by tuning PHD enzymatic activity.Authors
Liang Xie, Xinchun Pi, W.H. Davin Townley-Tilson, Na Li, Xander H.T. Wehrens, Mark L. Entman, George E. Taffet, Ashutosh Mishra, Junmin Peng, Jonathan C. Schisler, Gerhard Meissner, Cam Patterson
×Abstract
Therapeutic strategies that target disease-associated transcripts are being developed for a variety of neurodegenerative syndromes. Protein levels change as a function of their half-life, a property that critically influences the timing and application of therapeutics. In addition, both protein kinetics and concentration may play important roles in neurodegeneration; therefore, it is essential to understand in vivo protein kinetics, including half-life. Here, we applied a stable isotope-labeling technique in combination with mass spectrometric detection and determined the in vivo kinetics of superoxide dismutase 1 (SOD1), mutation of which causes amyotrophic lateral sclerosis. Application of this method to human SOD1-expressing rats demonstrated that SOD1 is a long-lived protein, with a similar half-life in both the cerebral spinal fluid (CSF) and the CNS. Additionally, in these animals, the half-life of SOD1 was longest in the CNS when compared with other tissues. Evaluation of this method in human subjects demonstrated successful incorporation of the isotope label in the CSF and confirmed that SOD1 is a long-lived protein in the CSF of healthy individuals. Together, the results of this study provide important insight into SOD1 kinetics and support application of this technique to the design and implementation of clinical trials that target long-lived CNS proteins.
Authors
Matthew J. Crisp, Kwasi G. Mawuenyega, Bruce W. Patterson, Naveen C. Reddy, Robert Chott, Wade K. Self, Conrad C. Weihl, Jennifer Jockel-Balsarotti, Arun S. Varadhachary, Robert C. Bucelli, Kevin E. Yarasheski, Randall J. Bateman, Timothy M. Miller
×Abstract
Emixustat is a visual cycle modulator that has entered clinical trials as a treatment for age-related macular degeneration (AMD). This molecule has been proposed to inhibit the visual cycle isomerase RPE65, thereby slowing regeneration of 11-
cis -retinal and reducing production of retinaldehyde condensation byproducts that may be involved in AMD pathology. Previously, we reported that all-trans -retinal (atRAL) is directly cytotoxic and that certain primary amine compounds that transiently sequester atRAL via Schiff base formation ameliorate retinal degeneration. Here, we have shown that emixustat stereoselectively inhibits RPE65 by direct active site binding. However, we detected the presence of emixustat-atRAL Schiff base conjugates, indicating that emixustat also acts as a retinal scavenger, which may contribute to its therapeutic effects. Using agents that lack either RPE65 inhibitory activity or the capacity to sequester atRAL, we assessed the relative importance of these 2 modes of action in protection against retinal phototoxicity in mice. The atRAL sequestrant QEA-B-001-NH2 conferred protection against phototoxicity without inhibiting RPE65, whereas an emixustat derivative incapable of atRAL sequestration was minimally protective, despite direct inhibition of RPE65. These data indicate that atRAL sequestration is an essential mechanism underlying the protective effects of emixustat and related compounds against retinal phototoxicity. Moreover, atRAL sequestration should be considered in the design of next-generation visual cycle modulators.Authors
Jianye Zhang, Philip D. Kiser, Mohsen Badiee, Grazyna Palczewska, Zhiqian Dong, Marcin Golczak, Gregory P. Tochtrop, Krzysztof Palczewski
×Abstract
BACKGROUND . TGF-β has potent profibrotic activity in vitro and has long been implicated in systemic sclerosis (SSc), as expression of TGF-β–regulated genes is increased in the skin and lungs of patients with SSc. Therefore, inhibition of TGF-β may benefit these patients.METHODS . Patients with early, diffuse cutaneous SSc were enrolled in an open-label trial of fresolimumab, a high-affinity neutralizing antibody that targets all 3 TGF-β isoforms. Seven patients received two 1 mg/kg doses of fresolimumab, and eight patients received one 5 mg/kg dose of fresolimumab. Serial mid-forearm skin biopsies, performed before and after treatment, were analyzed for expression of the TGF-β–regulated biomarker genes thrombospondin-1 (THBS1 ) and cartilage oligomeric protein (COMP ) and stained for myofibroblasts. Clinical skin disease was assessed using the modified Rodnan skin score (MRSS).RESULTS . In patient skin,THBS1 expression rapidly declined after fresolimumab treatment in both groups (P = 0.0313 at 7 weeks andP = 0.0156 at 3 weeks), and skin expression ofCOMP exhibited a strong downward trend in both groups. Clinical skin disease dramatically and rapidly decreased (P < 0.001 at all time points). Expression levels of other TGF-β–regulated genes, includingSERPINE1 andCTGF , declined (P = 0.049 andP = 0.012, respectively), and a 2-gene, longitudinal pharmacodynamic biomarker of SSc skin disease decreased after fresolimumab treatment (P = 0.0067). Dermal myofibroblast infiltration also declined in patient skin after fresolimumab (P < 0.05). Baseline levels ofTHBS1 were predictive of reducedTHBS1 expression and improved MRSS after fresolimumab treatment.CONCLUSION . The rapid inhibition of TGF-β–regulated gene expression in response to fresolimumab strongly implicates TGF-β in the pathogenesis of fibrosis in SSc. Parallel improvement in the MRSS indicates that fresolimumab rapidly reverses markers of skin fibrosis.TRIAL REGISTRATION . Clinicaltrials.gov NCT01284322.FUNDING . This study was supported by the Boston University Clinical and Translational Science Institute, the NIH’s National Center for Advancing Translational Sciences Clinical and Translational Sciences, the National Institute of Arthritis Musculoskeletal and Skin Disease: Scleroderma Core Centers and Scleroderma Center of Research Translation, the Boston University Medical Campus Microarray Core, the Kellen Foundation at Hospital for Special Surgery, the Scleroderma Research Foundation, the Dr. Ralph and Marian Falk Medical Research Trust, and Novartis.Authors
Lisa M. Rice, Cristina M. Padilla, Sarah R. McLaughlin, Allison Mathes, Jessica Ziemek, Salma Goummih, Sashidhar Nakerakanti, Michael York, Giuseppina Farina, Michael L. Whitfield, Robert F. Spiera, Romy B. Christmann, Jessica K. Gordon, Janice Weinberg, Robert W. Simms, Robert Lafyatis
×Steroid receptor coactivators 1 and 2 mediate fetal-to-maternal signaling that initiates parturition
Abstract
The precise mechanisms that lead to parturition are incompletely defined. Surfactant protein-A (SP-A), which is secreted by fetal lungs into amniotic fluid (AF) near term, likely provides a signal for parturition; however, SP-A–deficient mice have only a relatively modest delay (~12 hours) in parturition, suggesting additional factors. Here, we evaluated the contribution of steroid receptor coactivators 1 and 2 (SRC-1 and SRC-2), which upregulate SP-A transcription, to the parturition process. As mice lacking both SRC-1 and SRC-2 die at birth due to respiratory distress, we crossed double-heterozygous males and females. Parturition was severely delayed (~38 hours) in heterozygous dams harboring SRC-1/-2–deficient embryos. These mothers exhibited decreased myometrial NF-κB activation, PGF2α, and expression of contraction-associated genes; impaired luteolysis; and elevated circulating progesterone. These manifestations also occurred in WT females bearing SRC-1/-2 double-deficient embryos, indicating that a fetal-specific defect delayed labor. SP-A, as well as the enzyme lysophosphatidylcholine acyltransferase-1 (LPCAT1), required for synthesis of surfactant dipalmitoylphosphatidylcholine, and the proinflammatory glycerophospholipid platelet-activating factor (PAF) were markedly reduced in SRC-1/-2–deficient fetal lungs near term. Injection of PAF or SP-A into AF at 17.5 days post coitum enhanced uterine NF-κB activation and contractile gene expression, promoted luteolysis, and rescued delayed parturition in SRC-1/-2–deficient embryo-bearing dams. These findings reveal that fetal lungs produce signals to initiate labor when mature and that SRC-1/-2–dependent production of SP-A and PAF is crucial for this process.
Authors
Lu Gao, Elizabeth H. Rabbitt, Jennifer C. Condon, Nora E. Renthal, John M. Johnston, Matthew A. Mitsche, Pierre Chambon, Jianming Xu, Bert W. O’Malley, Carole R. Mendelson
×Abstract
T cell proliferation is critical for immune responses; however, the molecular mechanisms that mediate the proliferative response are poorly understood. MicroRNAs (miRs) regulate various molecular processes, including development and function of the immune system. Here, utilizing multiple complementary genetic and molecular approaches, we investigated the contribution of a hematopoietic-specific miR, miR-142, in regulating T cell responses. T cell development was not affected in animals with a targeted deletion of
Mir142 ; however, T cell proliferation was markedly reduced following stimulation both in vitro and in multiple murine models of graft-versus-host disease (GVHD). miR-142–deficient T cells demonstrated substantial cell-cycling defects, and microarray and bioinformatics analyses revealed upregulation of genes involved in cell cycling. Moreover, 2 predicted miR-142 target genes, the atypical E2F transcription factorsE2f7 andE2f8 , were most highly upregulated in miR-142–deficient cells. Clustered regularly interspaced short palindromic repeat interference–mediated (CRISPRi-mediated) silencing of E2F7 and E2F8 in miR-142–deficient T cells ameliorated cell-cycling defects and reduced GVHD, and overexpression of these factors in WT T cells inhibited the proliferative response. Together, these results identify a link between hematopoietic-specific miR-142 and atypical E2F transcription factors in the regulation of mature T cell cycling and suggest that targeting this interaction may be relevant for mitigating GVHD.Authors
Yaping Sun, Katherine Oravecz-Wilson, Nathan Mathewson, Ying Wang, Richard McEachin, Chen Liu, Tomomi Toubai, Julia Wu, Corinne Rossi, Thomas Braun, Thomas Saunders, Pavan Reddy
×Abstract
Increasing evidence indicates that the gut microbiota can be altered to ameliorate or prevent disease states, and engineering the gut microbiota to therapeutically modulate host metabolism is an emerging goal of microbiome research. In the intestine, bacterial urease converts host-derived urea to ammonia and carbon dioxide, contributing to hyperammonemia-associated neurotoxicity and encephalopathy in patients with liver disease. Here, we engineered murine gut microbiota to reduce urease activity. Animals were depleted of their preexisting gut microbiota and then inoculated with altered Schaedler flora (ASF), a defined consortium of 8 bacteria with minimal urease gene content. This protocol resulted in establishment of a persistent new community that promoted a long-term reduction in fecal urease activity and ammonia production. Moreover, in a murine model of hepatic injury, ASF transplantation was associated with decreased morbidity and mortality. These results provide proof of concept that inoculation of a prepared host with a defined gut microbiota can lead to durable metabolic changes with therapeutic utility.
Authors
Ting-Chin David Shen, Lindsey Albenberg, Kyle Bittinger, Christel Chehoud, Ying-Yu Chen, Colleen A. Judge, Lillian Chau, Josephine Ni, Michael Sheng, Andrew Lin, Benjamin J. Wilkins, Elizabeth L. Buza, James D. Lewis, Yevgeny Daikhin, Ilana Nissim, Marc Yudkoff, Frederic D. Bushman, Gary D. Wu
×Abstract
Pancreatic β cells secrete insulin in response to postprandial increases in glucose levels to prevent hyperglycemia and inhibit insulin secretion under fasting conditions to protect against hypoglycemia. β cells lack this functional capability at birth and acquire glucose-stimulated insulin secretion (GSIS) during neonatal life. Here, we have shown that during postnatal life, the de novo DNA methyltransferase DNMT3A initiates a metabolic program by repressing key genes, thereby enabling the coupling of insulin secretion to glucose levels. In a murine model, β cell–specific deletion of
Dnmt3a prevented the metabolic switch, resulting in loss of GSIS. DNMT3A bound to the promoters of the genes encoding hexokinase 1 (HK1) and lactate dehydrogenase A (LDHA) — both of which regulate the metabolic switch — and knockdown of these two key DNMT3A targets restored the GSIS response in islets from animals with β cell–specificDnmt3a deletion. Furthermore, DNA methylation–mediated repression of glucose-secretion decoupling genes to modulate GSIS was conserved in human β cells. Together, our results reveal a role for DNA methylation to direct the acquisition of pancreatic β cell function.Authors
Sangeeta Dhawan, Shuen-Ing Tschen, Chun Zeng, Tingxia Guo, Matthias Hebrok, Aleksey Matveyenko, Anil Bhushan
×Abstract
Estrogen receptor–α (ERα) activity in the brain prevents obesity in both males and females. However, the ERα-expressing neural populations that regulate body weight remain to be fully elucidated. Here we showed that single-minded–1 (SIM1) neurons in the medial amygdala (MeA) express abundant levels of ERα. Specific deletion of the gene encoding ERα (
Esr1 ) from SIM1 neurons, which are mostly within the MeA, caused hypoactivity and obesity in both male and female mice fed with regular chow, increased susceptibility to diet-induced obesity (DIO) in males but not in females, and blunted the body weight–lowering effects of a glucagon-like peptide-1–estrogen (GLP-1–estrogen) conjugate. Furthermore, selective adeno-associated virus-mediated deletion ofEsr1 in the MeA of adult male mice produced a rapid body weight gain that was associated with remarkable reductions in physical activity but did not alter food intake. Conversely, overexpression of ERα in the MeA markedly reduced the severity of DIO in male mice. Finally, an ERα agonist depolarized MeA SIM1 neurons and increased their firing rate, and designer receptors exclusively activated by designer drug–mediated (DREADD-mediated) activation of these neurons increased physical activity in mice. Collectively, our results support a model where ERα signals activate MeA neurons to stimulate physical activity, which in turn prevents body weight gain.Authors
Pingwen Xu, Xuehong Cao, Yanlin He, Liangru Zhu, Yongjie Yang, Kenji Saito, Chunmei Wang, Xiaofeng Yan, Antentor Othrell Hinton Jr., Fang Zou, Hongfang Ding, Yan Xia, Chunling Yan, Gang Shu, San-Pin Wu, Bin Yang, Yuxin Feng, Deborah J. Clegg, Richard DeMarchi, Sohaib A. Khan, Sophia Y. Tsai, Francesco J. DeMayo, Qi Wu, Qingchun Tong, Yong Xu
×Abstract
Systemic lupus erythematosus (SLE) is a severe autoimmune disease that is associated with increased circulating apoptotic cell autoantigens (AC-Ags) as well as increased type I IFN signaling. Here, we describe a pathogenic mechanism in which follicular translocation of marginal zone (MZ) B cells in the spleens of BXD2 lupus mice disrupts marginal zone macrophages (MZMs), which normally clear AC debris and prevent follicular entry of AC-Ags. Phagocytosis of ACs by splenic MZMs required the megakaryoblastic leukemia 1 (MKL1) transcriptional coactivator–mediated mechanosensing pathway, which was maintained by MZ B cells through expression of membrane lymphotoxin-α1β2 (mLT). Specifically, type I IFN–induced follicular shuttling of mLT-expressing MZ B cells disengaged interactions between these MZ B cells and LTβ receptor–expressing MZMs, thereby downregulating MKL1 in MZMs. Loss of MKL1 expression in MZMs led to defective F-actin polymerization, inability to clear ACs, and, eventually, MZM dissipation. Aggregation of plasmacytoid DCs in the splenic perifollicular region, follicular translocation of MZ B cells, and loss of MKL1 and MZMs were also observed in an additional murine lupus model and in the spleens of patients with SLE. Collectively, the results suggest that lupus might be interrupted by strategies that maintain or enhance mechanosensing signaling in the MZM barrier to prevent follicular entry of AC-Ags.
Authors
Hao Li, Yang-Xin Fu, Qi Wu, Yong Zhou, David K. Crossman, PingAr Yang, Jun Li, Bao Luo, Laurence M. Morel, Janusz H. Kabarowski, Hideo Yagita, Carl F. Ware, Hui-Chen Hsu, John D. Mountz
×Abstract
Strategies aimed at invoking synaptic plasticity have therapeutic potential for several neurological conditions. The human retinal synaptic disease X-linked retinoschisis (XLRS) is characterized by impaired visual signal transmission through the retina and progressive visual acuity loss, and mice lacking retinoschisin (RS1) recapitulate human disease. Here, we demonstrate that restoration of RS1 via retina-specific delivery of adeno-associated virus type 8-
RS1 (AAV8-RS1 ) vector rescues molecular pathology at the photoreceptor–depolarizing bipolar cell (photoreceptor-DBC) synapse and restores function in adultRs1 -KO animals. Initial development of the photoreceptor-DBC synapse was normal in theRs1 -KO retina; however, the metabotropic glutamate receptor 6/transient receptor potential melastatin subfamily M member 1–signaling (mGluR6/TRPM1-signaling) cascade was not properly maintained. Specifically, the TRPM1 channel and G proteins Gαo, Gβ5, and RGS11 were progressively lost from postsynaptic DBC dendritic tips, whereas the mGluR6 receptor and RGS7 maintained proper synaptic position. This postsynaptic disruption differed from other murine night-blindness models with an electronegative electroretinogram response, which is also characteristic of murine and human XLRS disease. Upon AAV8-RS1 gene transfer to the retina of adult XLRS mice, TRPM1 and the signaling molecules returned to their proper dendritic tip location, and the DBC resting membrane potential was restored. These findings provide insight into the molecular plasticity of a critical synapse in the visual system and demonstrate potential therapeutic avenues for some diseases involving synaptic pathology.Authors
Jingxing Ou, Camasamudram Vijayasarathy, Lucia Ziccardi, Shan Chen, Yong Zeng, Dario Marangoni, Jodie G. Pope, Ronald A. Bush, Zhijian Wu, Wei Li, Paul A. Sieving
Copyright © 2024 American Society for Clinical Investigation
ISSN: 0021-9738 (print), 1558-8238 (online)