Disrupting the DREAM transcriptional repressor complex induces apolipoprotein overexpression and systemic amyloidosis in mice

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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

Targeting a Braf/Mapk pathway rescues podocyte lipid peroxidation in CoQ deficiency kidney disease

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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

NOD2 drives early IL-33-dependent expansion of group 2 innate lymphoid cells during Crohn's disease-like ileitis

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Abstract

Innate lymphoid cells (ILCs) are enriched at barrier surfaces, including the gastrointestinal tract. While most studies have focused on the balance between pathogenic group 1 ILCs (ILC1s) and protective ILC3s in maintaining gut homeostasis and during chronic intestinal inflammation, such as Crohn’s disease (CD), less is known regarding ILC2s. Using an established murine model of CD-like ileitis, i.e., SAMP1/YitFc (SAMP) strain, we showed that ILC2s, compared to ILC1s and ILC3s, were increased within draining mesenteric lymph nodes and ilea of SAMP vs. AKR (parental control) mice early, during the onset of disease. Gut-derived ILC2s from Crohn’s patients vs. healthy controls were also increased and expand, similar to ILC1s, in greater proportion compared to ILC3s. Importantly, we report that the intracellular bacterial-sensing protein, nucleotide-binding oligomerization domaining-containing protein-2, encoded by NOD2, the first and strongest susceptibility gene identified for CD, promoted ILC2 expansion, which was dramatically reduced in SAMP lacking NOD2 and SAMP raised under germ-free conditions. Furthermore, these effects occurred through a mechanism involving the IL-33/ST2 ligand-receptor pair. Collectively, our results indicate a functional link between NOD2 and ILC2s, regulated by the IL-33/ST2 axis, that mechanistically may contribute to early events leading to CD pathogenesis.

Authors

Carlo De Salvo, Kristine-Ann Buela, Brecht Creyns, Daniele Corridoni, Nitish Rana, Hannah L. Wargo, Chiara Cominelli, Peter G. Delaney, Fabio Cominelli, Alexander Rodriguez-Palacios, Séverine Vermeire, Theresa T. Pizarro

COVID-19 vaccine testing in pregnant females is necessary

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Abstract

Limited evidence exists regarding the use of the currently approved COVID-19 mRNA vaccines (Pfizer-BioNtech BNT162b2 and Moderna mRNA-1273) during pregnancy. In this Viewpoint, Klein et al. discuss gaps in knowledge and make recommendations to incorporate age, sex, and pregnancy in the preclinical and clincal vaccine development pipeline.

Authors

Sabra L. Klein, Patrick S. Creisher, Irina Burd

Concerted roles of PTEN and ATM in controlling hematopoietic stem cell fitness and dormancy

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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

Tetracyclines improve experimental lymphatic filariasis pathology by disrupting interleukin-4 receptor-mediated lymphangiogenesis

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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

SARS-CoV-2-specific CD8+ T cell responses in convalescent COVID-19 individuals

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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

CRISPR/Cas9 directed to the Ube3a antisense transcript improves Angelman syndrome phenotype in mice

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Abstract

Gene editing holds the potential to correct mutations and cure devastating genetic disorders. The technology has not yet proven efficacious for therapeutic use in central nervous system (CNS) diseases with ubiquitous neuronal defects. Angelman syndrome (AS), a severe neurodevelopmental disorder, is caused by a lack of maternal expression of the UBE3A gene. Due to genomic imprinting, only neurons are affected. One therapeutic approach focuses on the intact paternal UBE3A copy in AS patients that is silenced by an antisense transcript (UBE3A-ATS). We show here that gene editing of Ube3a-ATS in the mouse brain results in the formation of base pair insertions/deletions (indels) in neurons and the subsequent unsilencing of the paternal Ube3a allele in neurons, which partially corrects the behavior phenotype of a murine AS model. This study provides compelling evidence to further investigate editing of the homologous region of the human UBE3A-ATS, since this may provide a lasting therapeutic effect for AS patients.

Authors

Ralf S. Schmid, Xuefeng Deng, Priyalakshmi Panikker, Msema Msackyi, Camilo Breton, James M. Wilson

Turbinmicin inhibits Candida biofilm growth by disrupting fungal vesicle-mediated trafficking

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Abstract

The emergence of drug-resistant fungi has prompted an urgent threat alert from the Centers for Disease Control. Biofilm assembly by these pathogens further impairs effective therapy. We recently identifed an antifungal, turbinmicin, that inhibits the fungal vesicle-mediated trafficking pathway and demonstrates broad-spectrum activity against planktonically growing fungi. During biofilm growth, vesicles with unique features play a critical role in the delivery of the biofilm extracellular matrix components. As these components are largely responsible for the drug resistance associated with biofilm growth, we explored the utility of turbinmicin in the biofilm setting. We found that turbinmicin disrupts extracellular vesicle delivery during biofilm growth, and this impairs the subsequent assembly of the biofilm matrix. We demonstrated that elimination of the extracellular matrix renders the drug-resistant biofilm communities susceptible to fungal killing by turbinmicin. Furthermore, the addition of turbinmicin to otherwise ineffective antifungal therapy potentiated the activity of these drugs. The underlying role of vesicles explains this dramatic activity and was supported by phenotype reversal with the addition of exogenous biofilm extracellular vesicles. This striking capacity to cripple biofilm assembly mechanisms reveals a new approach to eradicating biofilms and sheds light on turbinmicin as a promising anti-biofilm drug.

Authors

Miao Zhao, Fan Zhang, Robert Zarnowski, Kenneth J. Barns, Ryley Jones, Jen L. Fossen, Hiram Sanchez, Scott R. Rajski, Anjon Audhya, Tim S. Bugni, David R. Andes

YAP plays a crucial role in the development of cardiomyopathy in lysosomal storage diseases

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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