Research
Citation Information: J Clin Invest. 2021. https://doi.org/10.1172/JCI140903.
Abstract
DREAM is a transcriptional repressor complex that regulates cell proliferation and its loss causes neonatal lethality in mice. To investigate DREAM function in adult mice, we utilized an assembly defective p107 protein and conditional deletion of its redundant family member p130. In the absence of DREAM assembly, mice displayed shortened survival characterized by systemic amyloidosis, but no evidence of excessive cellular proliferation. Amyloid deposits were found in the heart, liver, spleen, and kidneys, but not the brain or bone marrow. Using laser capture microdissection followed by mass spectrometry, we identified apolipoproteins as the most abundant components of amyloids. Intriguingly, apoA-IV was the most detected amyloidogenic protein in amyloid deposits, suggesting AApoAIV amyloidosis. AApoAIV is a recently described form whereby wildtype apoA-IV has been shown to predominate in amyloid plaques. We determined that DREAM directly regulates Apoa4 by chromatin immunoprecipitation and that the histone variant H2AZ is reduced from the Apoa4 gene body in DREAM’s absence, leading to overexpression. Collectively, we describe a mechanism by which epigenetic misregulation causes apolipoprotein overexpression and amyloidosis, potentially explaining the origins of non-genetic amyloid subtypes.
Authors
Pirunthan Perampalam, Haider M. Hassan, Grace E. Lilly, Daniel T. Passos, Joseph Torchia, Patti K. Kiser, Andrea Bozovic, Vathany Kulasingam, Frederick A. Dick
Citation Information: J Clin Invest. 2021. https://doi.org/10.1172/JCI141380.
Abstract
Mutations affecting mitochondrial coenzyme Q (CoQ) biosynthesis lead to kidney failure due to selective loss of podocytes, essential cells of the kidney filter. Curiously, neighboring tubular epithelial cells are spared early in disease, despite higher mitochondrial content. We sought to illuminate non-canonical, cell-specific roles for CoQ, independent of the electron transport chain (ETC). Here we demonstrate that CoQ depletion caused by Pdss2 enzyme deficiency in podocytes results in perturbations in polyunsaturated fatty acid (PUFA) metabolism and the Braf/Mapk pathway, rather than ETC dysfunction. Single nucleus RNA sequencing from kidneys of Pdss2kd/kd mice with nephrotic syndrome and global CoQ-deficiency identified a podocyte-specific perturbation of the Braf/Mapk pathway. Treatment with GDC-0879, a Braf/Mapk-targeting compound ameliorated kidney disease in Pdss2kd/kd mice. Mechanistic studies in Pdss2-depleted podocytes revealed a previously unknown perturbation in PUFA metabolism that was confirmed in vivo. Gpx4, an enzyme that protects against PUFA-mediated lipid peroxidation, was elevated in disease and restored after GDC-0879 treatment. We demonstrate broader human disease relevance by uncovering patterns of GPX4 and Braf/Mapk pathway gene expression in tissue from patients with kidney diseases. Our studies reveal ETC-independent roles for CoQ in podocytes and point to Braf/Mapk as a candidate pathway for the treatment of kidney diseases.
Authors
Eriene-Heidi Sidhom, Choah Kim, Maria Kost-Alimova, May Theng Ting, Keith Keller, Julian Avila-Pacheco, Andrew J.B. Watts, Katherine A. Vernon, Jamie L. Marshall, Estefanía Reyes-Bricio, Matthew Racette, Nicolas Wieder, Giulio Kleiner, Elizabeth J. Grinkevich, Fei Chen, Astrid Weins, Clary B. Clish, Jillian L. Shaw, Catarina M. Quinzii, Anna Greka
Citation Information: J Clin Invest. 2021. https://doi.org/10.1172/JCI131698.
Abstract
In order to sustain proficient life-long hematopoiesis, hematopoietic stem cells (HSCs) must possess robust mechanisms to preserve their quiescence and genome integrity. DNA-damaging stress can perturb HSC homeostasis by affecting their survival, self-renewal and differentiation. Ablation of the kinase ATM, a master regulator of the DNA damage response, impairs HSC fitness. Paradoxically, we show here that loss of a single allele of Atm enhances HSC functionality in mice. To explain this observation, we explored a possible link between ATM and the tumor suppressor PTEN, which also regulates HSC function. We generated and analyzed a knock-in mouse line (PtenS398A/S398A), in which PTEN cannot be phosphorylated by ATM. Similar to Atm+/-, PtenS398A/S398A HSCs have enhanced hematopoietic reconstitution ability, accompanied by resistance to apoptosis induced by genotoxic stress. Single-cell transcriptomic analyses and functional assays revealed that dormant PtenS398A/S398A HSCs aberrantly tolerate elevated mitochondrial activity and the accumulation of reactive oxygen species, which are normally associated with HSC priming for self-renewal or differentiation. Our results unveil a molecular connection between ATM and PTEN, which couples the response to genotoxic stress and dormancy in HSC.
Authors
Jerome Fortin, Christian Bassi, Parameswaran Ramachandran, Wanda Y. Li, Ruxiao Tian, Ida Zarrabi, Graham Hill, Bryan E. Snow, Jillian Haight, Chantal Tobin, Kelsey Hodgson, Andrew Wakeham, Vuk Stambolic, Tak W. Mak
Citation Information: J Clin Invest. 2021. https://doi.org/10.1172/JCI140853.
Abstract
Lymphatic filariasis is the major global cause of non-hereditary lymphoedema. We demonstrate the filarial nematode, Brugia malayi, induces lymphatic remodelling and impaired lymphatic drainage following parasitism of limb lymphatics in a mouse model. Lymphatic insufficiency was associated with elevated circulating lymphangiogenic mediators, including vascular endothelial growth factor C. Lymphatic insufficiency was dependent on type-2 adaptive immunity, interleukin-4 receptor, recruitment of C-C chemokine receptor-2 monocytes and alternatively-activated macrophages with pro-lymphangiogenic phenotype. Oral treatments with second-generation tetracyclines improved lymphatic function, while other classes of antibiotic had no significant effect. Second-generation tetracyclines directly targeted lymphatic endothelial cell proliferation and modified type-2 pro-lymphangiogenic macrophage development. Doxycycline treatment impeded monocyte recruitment, inhibited polarisation of alternatively-activated macrophages and suppressed T cell adaptive immune responses following infection. Our results determine a mechanism-of-action for the anti-morbidity effects of doxycycline in filariasis and supports clinical evaluation of second-generation tetracyclines as affordable, safe therapeutics for lymphoedemas of chronic inflammatory origin.
Authors
Julio Furlong-Silva, Stephen D. Cross, Amy E. Marriott, Nicolas Pionnier, John Archer, Andrew Steven, Stefan Schulte-Merker, Matthias Mack, Young-Kwon Hong, Mark J. Taylor, Joseph D. Turner
Citation Information: J Clin Invest. 2021. https://doi.org/10.1172/JCI145476.
Abstract
Characterization of the T cell response in individuals who recover from SARS-CoV-2 infection is critical to understand its contribution to protective immunity. A multiplexed peptide-MHC tetramer approach was used to screen 408 SARS-CoV-2 candidate epitopes for CD8+ T cell recognition in a cross-sectional sample of 30 COVID-19 convalescent individuals. T cells were evaluated using a 28-marker phenotypic panel, and findings were modelled against time from diagnosis, humoral and inflammatory responses. There were 132 SARS-CoV-2-specific CD8+ T cell responses detected across six different HLAs, corresponding to 52 unique epitope reactivities. CD8+ T cell responses were detected in almost all convalescent individuals and were directed against several structural and non-structural target epitopes from the entire SARS-CoV-2 proteome. A unique phenotype for SARS-CoV-2-specific T cells was observed that was distinct from other common virus-specific T cells detected in the same cross-sectional sample and characterized by early differentiation kinetics. Modelling demonstrated a coordinated and dynamic immune response characterized by a decrease in inflammation, increase in neutralizing antibody titer, and differentiation of a specific CD8+ T cell response. Overall, T cells exhibited distinct differentiation into stem-cell and transitional memory states, subsets, which may be key to developing durable protection.
Authors
Hassen Kared, Andrew D. Redd, Evan M. Bloch, Tania S. Bonny, Hermi R. Sumatoh, Faris Kairi, Daniel Carbajo, Brian Abel, Evan W. Newell, Maria Bettinotti, Sarah E. Benner, Eshan U. Patel, Kirsten Littlefield, Oliver Laeyendecker, Shmuel Shoham, David Sullivan, Arturo Casadevall, Andrew Pekosz, Alessandra Nardin, Michael Fehlings, Aaron AR Tobian, Thomas C. Quinn
Citation Information: J Clin Invest. 2020. https://doi.org/10.1172/JCI143173.
Abstract
Lysosomal dysfunction caused by mutations in lysosomal genes results in lysosomal storage disorder (LSD), characterized by accumulation of damaged proteins and organelles in cells and functional abnormalities in major organs, including the heart, skeletal muscle and liver. In LSD, autophagy is inhibited at the lysosomal degradation step and accumulation of autophagosomes is observed. Enlargement of the left ventricle (LV) and contractile dysfunction were observed in RagA/B cardiac-specific knockout (cKO) mice, a mouse model of LSD in which lysosomal acidification is impaired irreversibly. YAP, a downstream effector of the Hippo pathway, was accumulated in RagA/B cKO mouse hearts. Inhibition of YAP ameliorated cardiac hypertrophy and contractile dysfunction and attenuated accumulation of autophagosomes without affecting lysosomal function, suggesting that YAP plays an important role in mediating cardiomyopathy in RagA/B cKO mice. Cardiomyopathy was also alleviated by downregulation of Atg7, an intervention to inhibit autophagy, whereas it was exacerbated by stimulation of autophagy. YAP physically interacted with transcription factor EB (TFEB), a master transcription factor that controls autophagic and lysosomal gene expression, thereby facilitating accumulation of autophagosomes without degradation. These results indicate that accumulation of YAP in the presence of LSD promotes cardiomyopathy by stimulating accumulation of autophagosomes through activation of TFEB.
Authors
Shohei Ikeda, Jihoon Nah, Akihiro Shirakabe, Peiyong Zhai, Shin-ichi Oka, Sebastiano Sciarretta, Kun-Liang Guan, Hiroaki Shimokawa, Junichi Sadoshima
Citation Information: J Clin Invest. 2020. https://doi.org/10.1172/JCI137681.
Abstract
Human metabolic incorporation of non-human sialic acid (Sia) N-glycolylneuraminic acid into endogenous glycans generates inflammation via pre-existing antibodies, likely contributing to red-meat-induced atherosclerosis acceleration. Exploring if this mechanism affects atherosclerosis in end-stage renal disease (ESRD), we instead found serum accumulation of 2-keto-3-deoxy-D-glycero-D-galacto-2-nonulosonic acid (Kdn), a Sia prominently expressed in cold-blooded vertebrates. Levels of Kdn precursor mannose also increased but within normal range in ESRD patients. Mannose ingestion by healthy volunteers raised urinary mannose and Kdn. Kdn production pathways remain conserved in mammals but were diminished by a M42T substitution in a key biosynthetic enzyme, N-acetylneuraminate synthase. Remarkably, reversion to the ancestral methionine then occurred independently in two lineages, including humans. However, mammalian glycan databases contain no Kdn-glycans. We hypothesize that potential toxicities of excess mannose in mammals is partly buffered by conversion to free Kdn. Thus, mammals likely conserved Kdn biosynthesis and modulated it in lineage-specific manner, not for glycosylation, but to control physiological mannose intermediates/metabolites. However, human cells can be forced to express Kdn-glycans, via genetic mutations enhancing Kdn utilization, or by transfection with fish enzymes producing CMP-Kdn. Antibodies against Kdn-glycans occur in pooled human immunoglobulins. Pathological conditions that elevate Kdn levels could therefore result in antibody-mediated inflammatory pathologies.
Authors
Kunio Kawanishi, Sudeshna Saha, Sandra Diaz, Michael Vaill, Aniruddha Sasmal, Shoib S. Siddiqui, Biswa P. Choudhury, Kumar Sharma, Xi Chen, Ian C. Schoenhofen, Chihiro Sato, Ken Kitajima, Hudson H. Freeze, Anja Münster-Kühnel, Ajit Varki
Citation Information: J Clin Invest. 2020. https://doi.org/10.1172/JCI138935.
Abstract
Previous studies have shown that nitric oxide (NO) supplements may prevent bone loss and fractures in preclinical models of estrogen deficiency. However, the mechanisms by which NO modulates bone anabolism remain largely unclear. Argininosuccinate lyase (ASL) is the only mammalian enzyme capable of synthesizing arginine, the sole precursor for nitric oxide synthase (NOS)-dependent NO synthesis. Moreover, ASL is also required for channeling extracellular arginine to NOS for NO production. ASL deficiency (ASLD) is thus a model to study cell-autonomous, NOS-dependent NO deficiency. Here, we report that loss of ASL led to decreased NO production and impairment of osteoblast differentiation. Mechanistically, the bone phenotype was at least in part driven by the loss of NO-mediated activation of the glycolysis pathway in osteoblasts that led to decreased osteoblast differentiation and function. Heterozygous deletion of Caveolin-1, a negative regulator of NO synthesis, restored NO production, osteoblast differentiation, glycolysis, and bone mass in a hypomorphic mouse model of ASLD. The translational significance of these preclinical studies was further reiterated by studies conducted in induced pluripotent stem cells (iPSCs) from an individual with ASLD. Taken together, our findings suggest that ASLD is a unique genetic model for studying NO-dependent osteoblast function and that the NO-glycolysis pathway may be a new target to modulate bone anabolism.
Authors
Zixue Jin, Jordan Kho, Brian Dawson, Ming-Ming Jiang, Yuqing Chen-Evenson, Saima Ali, Lindsay C. Burrage, Monica Grover, Donna J. Palmer, Dustin L. Turner, Philip Ng, Sandesh C.S. Nagamani, Brendan Lee
Citation Information: J Clin Invest. 2020. https://doi.org/10.1172/JCI140147.
Abstract
Autosomal dominant "sterile alpha motif domain containing 9 (Samd9) and Samd9L (Samd9/9L) syndromes" are a large subgroup of currently established inherited bone marrow failure syndromes that include MIRAGE, ataxia pancytopenia, and familial monosomy 7 syndromes. Samd9/9L genes are located in tandem on chromosome 7 and have been known to be the genes responsible for myeloid malignancies associated with monosomy 7. Additionally, as interferon-inducible genes, Samd9/9L are crucial for protection against viruses. Samd9/9L syndromes are caused by gain-of-function mutations and develop into infantile myelodysplastic syndromes associated with monosomy 7 (MDS/-7) at extraordinarily high frequencies. We generated mice expressing Samd9LD764N, which mimic the MIRAGE syndrome presenting with growth retardation, a short life, bone marrow failure, and multi-organ degeneration. In hematopoietic cells, Samd9LD764N downregulates the endocytosis of transferrin and c-Kit resulting in a rare cause of anemia and a low bone marrow reconstitutive potential that ultimately causes MDS/-7. By contrast, in non-hematopoietic cells we tested, Samd9LD764N upregulated the endocytosis of EGFR by Ship2 phosphatase translocation to the cytomembrane and activated lysosomes, resulting in the reduced expression of surface receptors and signaling. Thus Samd9/9L is a downstream regulator of interferon that controls receptor metabolism, with constitutive activation leading to multi-organ dysfunction.
Authors
Akiko Nagamachi, Akinori Kanai, Megumi Nakamura, Hiroshi Okuda, Akihiko Yokoyama, Satoru Shinriki, Hirotaka Matsui, Toshiya Inaba
Citation Information: J Clin Invest. 2020. https://doi.org/10.1172/JCI143301.
Abstract
Chronic pancreatitis affects over 250,000 people in the US and millions worldwide. It is associated with chronic debilitating pain, pancreatic exocrine failure, high-risk of pancreatic cancer, and usually progresses to diabetes. Treatment options are limited and ineffective. We developed a new potential therapy, wherein a pancreatic ductal infusion of 1-2% acetic acid in mice and non-human primates resulted in a non-regenerative, near-complete ablation of the exocrine pancreas, with complete preservation of the islets. Pancreatic ductal infusion of acetic acid in a mouse model of chronic pancreatitis led to resolution of chronic inflammation and pancreatitis-associated pain. Furthermore, acetic acid-treated animals showed improved glucose tolerance and insulin secretion. The loss of exocrine tissue in this procedure would not typically require further management in patients with chronic pancreatitis because they usually have pancreatic exocrine failure requiring dietary enzyme supplements. Thus, this procedure, which should be readily translatable to humans through an endoscopic retrograde cholangiopancreatography (ERCP), may offer a potential innovative non-surgical therapy for chronic pancreatitis that relieves pain and prevents the progression of pancreatic diabetes.
Authors
Mohamed Saleh, Kartikeya Sharma, Ranjeet S. Kalsi, Joseph C. Fusco, Anuradha Sehrawat, Jami L. Saloman, Ping Guo, Ting Zhang, Nada Mohamed, Yan Wang, Krishna Prasadan, George Gittes
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