TPP1 mutagenesis screens unravel shelterin interfaces and functions in hematopoiesis

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Abstract

Telomerase catalyzes chromosome end replication in stem cells and other long-lived cells. Mutations in telomerase or telomere-related genes result in diseases known as telomeropathies. Telomerase is recruited to chromosome ends by the ACD/TPP1 protein (TPP1 hereafter), a component of the shelterin complex that protects chromosome ends from unwanted end-joining. TPP1 facilitates end-protection by binding shelterin proteins POT1 and TIN2. TPP1 variants have been associated with telomeropathies, but remain poorly characterized in vivo. Disease variants and mutagenesis scans provide efficient avenues to interrogate the distinct physiological roles of TPP1. Here, we conduct mutagenesis in the TIN2- and POT1-binding domains of TPP1 to discover mutations that dissect TPP1’s functions. Our results extend upon current structural data to reveal that the TPP1-TIN2 interface is more extensive than previously thought, and highlight the robustness of the POT1-TPP1 interface. Introduction of separation-of-function mutants alongside known TPP1 telomeropathy mutations in mouse hematopoietic stem cells (mHSCs) lacking endogenous TPP1 demonstrated a clear phenotypic demarcation. TIN2- and POT1-binding mutants were unable to rescue mHSC failure resulting from end-deprotection. In contrast, TPP1 telomeropathy mutations sustained mHSC viability, consistent with them selectively impacting end-replication. These results highlight the power of scanning mutagenesis in revealing structural interfaces and dissecting multifunctional genes.

Authors

Sherilyn Grill, Shilpa Padmanaban, Ann Friedman, Eric Perkey, Frederick Allen, Valerie M. Tesmer, Jennifer Chase, Rami Khoriaty, Catherine E. Keegan, Ivan Maillard, Jayakrishnan Nandakumar

Clinical application of intrathecal gadobutrol for assessment of cerebrospinal fluid tracer clearance to blood

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Abstract

BACKGROUND. Methodology for estimation of cerebrospinal fluid (CSF) tracer clearance could have wide clinical application in predicting excretion of intrathecal drugs and metabolic solutes from brain metabolism, and for diagnostic work-up of cerebrospinal fluid disturbances. METHODS. The magnetic resonance imaging (MRI) contrast agent gadobutrol (Gadovist) was utilized as CSF tracer and injected into the lumbar CSF. Gadobutrol is contained outside blood vessels of the central nervous system (CNS) and is thus eliminated along extra-vascular pathways, analogous to many CNS metabolites and intrathecal drugs. Tracer enrichment was verified and assessed in CSF by MRI at level of the cisterna magna in parallel with obtaining blood samples through 48 hours. RESULTS. In a reference patient cohort (REF; n=29), both enrichment within CSF and blood coincided in time. Blood concentration profiles of gadobutrol through 48 hours varied between patients diagnosed with CSF leakage (n=4), idiopathic normal pressure hydrocephalus dementia (iNPH; n=7), pineal cysts (n=8), and idiopathic intracranial hypertension (IIH; n=4). CONCLUSION. Assessment of CSF tracer clearance is clinically feasible and may provide a way to predict extra-vascular clearance of intrathecal drugs and endogenous metabolites from the CNS. The peak concentration in blood (at about 10 hrs) preceded by far peak tracer enhancement at MRI in extra-cranial lymphatic structures (at about 24 hrs) as shown in previous studies, indicating a major role of the spinal canal in CSF clearance capacity. FUNDING. The work was supported by Department of Neurosurgery, Oslo university hospital, and Norwegian Institute for Air Research, Kjeller, Norway, and University of Oslo.

Authors

Per K. Eide, Espen Mariussen, Hilde Uggerud, Are H. Pripp, Aslan Lashkarivand, Bjørnar Hassel, Hege Christensen, Markus Herberg Hovd, Geir Ringstad

SARS-CoV-2 vaccines: a triumph of science and collaboration

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Abstract

Roughly one year after the first case of COVID-19 was identified and less than one year after the sequencing of SARS-CoV-2, multiple SARS-CoV-2 vaccines with demonstrated safety and efficacy in phase III clinical trials are available. The most promising vaccines have targeted the surface glycoprotein (S-protein) of SARS-CoV-2 and achieved an approximate 85-95% reduction in the risk of symptomatic COVID-19, while retaining excellent safety profiles and modest side effects in the phase III clinical trials. The mRNA, replication-incompetent viral vector, and protein subunit vaccine technologies have all been successfully employed. Some novel SARS-CoV-2 variants evade but do not appear to fully overcome the potent immunity induced by these vaccines. Emerging real-world effectiveness data add evidence for protection from severe COVID-19. This is an impressive first demonstration of the effectiveness of the mRNA vaccine and vector vaccine platforms. The success of SARS-CoV-2 vaccine development should be credited to open science, industry partnerships, harmonization of clinical trials, and the altruism of study participants. The manufacturing and distribution of the emergency use-authorized SARS-CoV-2 vaccines are ongoing challenges. What remains now is to ensure broad and equitable global vaccination against COVID-19.

Authors

Jonathan L. Golob, Njira Lugogo, Adam S. Lauring, Anna S. Lok

Natural mucosal barriers and COVID-19 in children

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Abstract

Background: COVID-19 is more benign in children compared to adults for unknown reasons. This contrasts with other respiratory viruses where disease manifestations are often more severe in children. We hypothesize that a more robust early innate immune response to SARS-CoV-2 protects against severe disease. Methods: Clinical outcomes, SARS-CoV-2 viral copies and cellular gene expression were compared in nasopharyngeal swabs obtained at the time of presentation to the Emergency Department from 12 children and 27 adults using bulk RNA sequencing and quantitative reverse transcription PCR. Total protein, cytokines and anti-SARS-CoV-2 IgG and IgA were quantified in nasal fluid. Results: SARS-CoV-2 copies, ACE2 and TMPRSS2 gene expression were similar in children and adults, but children displayed higher expression of genes associated with interferon signaling, NLRP3 inflammasome, and other innate pathways. Higher levels of IFN-α2, IFN-γ, IP-10, IL-8, and IL-1β protein were detected in nasal fluid in children versus adults. Children also expressed higher levels of genes associated with immune cells whereas expression of those associated with epithelial cells did not differ in children versus adults. Anti-SARS-CoV-2 IgA and IgG were detected at similar levels in nasal fluid from both groups. None of the children required supplemental oxygen whereas 7 adults did (p=0.03); four adults died. Conclusions: These findings provide direct evidence of a more vigorous early mucosal immune response in children compared to adults and suggest that this contributes to favorable clinical outcomes.

Authors

Carl A. Pierce, Sharlene Sy, Benjamin Galen, Doctor Y. Goldstein, Erika P. Orner, Marla J. Keller, Kevan C. Herold, Betsy C. Herold

Mitochondria-dependent phase separation of disease-relevant proteins drives pathological features of age-related macular degeneration

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Abstract

Age-related macular degeneration (AMD) damages the retinal pigment epithelium (RPE), the tissue that safeguards photoreceptor health, leading to irreversible vision loss. Polymorphisms in cholesterol and complement genes are implicated in AMD, yet mechanisms linking risk variants to RPE injury remain unclear. We sought to determine how allelic variants in the apolipoprotein E cholesterol transporter modulate RPE homeostasis and function. Using live-cell imaging, we show that inefficient cholesterol transport by the AMD risk-associated ApoE2 increases RPE ceramide, leading to autophagic defects and complement-mediated mitochondrial damage. Mitochondrial injury drives redox state-sensitive cysteine-mediated phase separation of ApoE2, forming biomolecular condensates that could nucleate drusen. The protective ApoE4 isoform lacks these cysteines and is resistant to phase separation and condensate formation. In Abca4-/- Stargardt macular degeneration mice, mitochondrial dysfunction induces liquid-liquid phase separation of p62/SQSTM1, a multifunctional protein that regulates autophagy. Drugs that decrease RPE cholesterol or ceramide prevent mitochondrial injury and phase separation in vitro and in vivo. In AMD donor RPE, mitochondrial fragmentation correlates with ApoE and p62 condensates. Our studies demonstrate that major AMD genetic and biological risk pathways converge upon RPE mitochondria, and identify mitochondrial stress-mediated protein phase separation as an important pathogenic mechanism and promising therapeutic target in AMD.

Authors

Nilsa La Cunza, Li Xuan Tan, Thushara Thamban, Colin J Germer, Gurugirijha Rathnasamy, Kimberly Toops, Aparna Lakkaraju

Corrigendum: Adrenergic-mediated increases in INHBA drive CAF phenotype and collagens

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Abstract

Archana S. Nagaraja, Robert L. Dood, Guillermo Armaiz-Pena, Yu Kang, Sherry Y. Wu, Julie K. Allen, Nicholas B. Jennings, Lingegowda S. Mangala, Sunila Pradeep, Yasmin Lyons, Monika Haemmerle, Kshipra M. Gharpure, Nouara C. Sadaoui, Cristian Rodriguez-Aguayo, Cristina Ivan, Ying Wang, Keith Baggerly, Prahlad Ram, Gabriel Lopez-Berestein, Jinsong Liu, Samuel C. Mok, Lorenzo Cohen, Susan K. Lutgendorf, Steve W. Cole, and Anil K. Sood Original citation: JCI Insight. 2017;2(16):e93076. https://doi.org/10.1172/jci.insight.93076. Citation for this corrigendum: JCI Insight. 2021;6(7):e149895. https://doi.org/10.1172/jci.insight.149895. The Editors previously posted an Expression of Concern for this article regarding images in Figure 5E that appeared to be the same (siControl Stress and siNHBA Control). An institutional review committee concluded that the error in Figure 5E was inadvertent and recommended correction. The authors provided the correct image for the siControl Stress sample from the original study. The authors regret the error.

Authors

JCI Insight Staff

Corrigendum: Improving vascular maturation using noncoding RNAs increases antitumor effect of chemotherapy

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Abstract

Lingegowda S. Mangala, Hongyu Wang, Dahai Jiang, Sherry Y. Wu, Anoma Somasunderam, David E. Volk, Ganesh L. R. Lokesh, Xin Li, Sunila Pradeep, Xianbin Yang, Monika Haemmerle, Cristian Rodriguez-Aguayo, Archana S Nagaraja, Rajesha Rupaimoole, Emine Bayraktar, Recep Bayraktar, Li Li, Takemi Tanaka, Wei Hu, Cristina Ivan, Kshipra M Gharpure, Michael H. McGuire, Varatharasa Thiviyanathan, Xinna Zhang, Sourindra N. Maiti, Nataliya Bulayeva, Hyun-Jin Choi, Piotr L. Dorniak, Laurence J.N. Cooper, Kevin P. Rosenblatt, Gabriel Lopez-Berestein, David G. Gorenstein, and Anil K. Sood Original citation: JCI Insight. 2016;1(17):e87754. https://doi.org/10.1172/jci.insight.87754. Citation for this corrigendum: JCI Insight. 2021;6(7):e149896. https://doi.org/10.1172/jci.insight.149896. The Editors previously posted an Expression of Concern for this article regarding images in Supple-mental 4B that appeared similar (Endo40 and Endo42 samples) and images in Supplemental Figure 9 that appeared to be the same (CH and CH/Endo28-NPs for heart tissue). No changes were necessary for Supplemental Figure 4B, as the Endo40 and Endo42 samples were determined to be derived from adjacent tumor sections. An institutional review committee concluded that the error in Supplemental Figure 9 was inadvertent and recommended correction. The authors provided the correct image for the CH/Endo28-NPs sample from the original study. An updated supplemental file that includes the correct version of Supplemental Figure 9 is now posted. The authors regret the error.

Authors

JCI Insight Staff

Inhibition of HIF-prolyl hydroxylases improves healing of intestinal anastomoses

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Abstract

Anastomotic leakage (AL) accounts for a major part of in-house mortality in patients undergoing colorectal surgery. Local ischemia and abdominal sepsis are common risk factors contributing to AL and are characterized by upregulation of the hypoxia-inducible factor (HIF) pathway. The HIF pathway is critically regulated by HIF-prolyl hydroxylases (PHDs). Here, we investigated the significance of PHDs and the effects of pharmacologic PHD inhibition (PHI) during anastomotic healing. Ischemic or septic colonic anastomoses were created in mice by ligation of mesenteric vessels or lipopolysaccharide-induced abdominal sepsis, respectively. Genetic PHD-deficiency (Phd1-/-, Phd2+/-, and Phd3-/-) or PHI were applied to manipulate PHD activity. Pharmacologic PHI and genetic PHD2-haplodeficiency (Phd2+/-) significantly improved healing of ischemic or septic colonic anastomoses, as indicated by increased bursting pressure and reduced AL rates. Only Phd2+/- (but not PHI or Phd1-/-) protected from sepsis-related mortality. Mechanistically, PHI and Phd2+/- induced immuno-modulatory (M2) polarization of macrophages, resulting in increased collagen content and attenuated inflammation-driven immune cell recruitment. We conclude that PHI improves healing of colonic anastomoses in ischemic or septic conditions by Phd2+/--mediated M2 polarization of macrophages, conferring a favourable microenvironment for anastomotic healing. Patients with critically perfused colorectal anastomosis or abdominal sepsis could benefit from pharmacologic PHI.

Authors

Moritz J. Strowitzki, Gwendolyn Kimmer, Julian S. Wehrmann, Alina S. Ritter, Praveen Radhakrishnan, Vanessa M. Opitz, Christopher Tuffs, Marvin Biller, Julia Kugler, Ulrich Keppler, Jonathan M. Harnoss, Johannes Klose, Thomas Schmidt, Alfonso Blanco, Cormac T. Taylor, Martin Schneider

Ciclopirox olamine induces ferritinophagy and reduces cyst burden in polycystic kidney disease

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Abstract

Despite the recent launch of Tolvaptan, the search for safer polycystic kidney disease (PKD) drugs continues. Ciclopirox (CPX) or its olamine salt (CPX-O) are contained in number of commercially available antifungal agents. CPX is also reported to possess anticancer activity. Several mechanisms of action have been proposed including chelation of iron and inhibition of iron dependent enzymes. Here, we show that CPX-O inhibited in vitro cystogenesis of primary human PKD cyst-lining epithelial cells cultured in a 3D collagen matrix. To assess in vivo role of CPX-O, we treated PKD mice with CPX-O. CPX-O reduced the kidney- to-body weight ratios of PKD mice. This was also associated with decreased cell proliferation, decreased cystic area and improved renal function. Ferritin levels were significantly elevated in cystic kidneys of PKD mice, and CPX-O treatment reduced renal ferritin levels. The reduction in ferritin was associated with increased ferritinophagy marker, NCOA4 which reversed upon CPX-O treatment in PKD mice. Interestingly, these effects on ferritin appeared independent of iron. These data suggest that CPX-O can induce ferritin degradation via ferritinophagy which is associated with decreased cyst growth progression in PKD mice. Most importantly these data indicate that CPX-O has the potential to treat autosomal dominant PKD.

Authors

Priyanka S. Radadiya, Mackenzie M. Thornton, Rajni V. Puri, Sireeesha Yerrathota, Johnny Dinh-Phan, Brenda Magenheimer, Dharmalingam Subramaniam, Pamela V. Tran, Hao Zhu, Subhashini Bolisetty, James P. Calvet, Darren P. Wallace, Madhulika Sharma

An inducible Cre mouse for studying roles of the RPE in retinal physiology and disease

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Abstract

The retinal pigment epithelium (RPE) provides vital metabolic support for retinal photoreceptor cells and also is an important player in numerous retinal diseases. Gene manipulation in mice using the Cre-LoxP system is an invaluable tool for studying the genetic basis of these retinal diseases. However, existing RPE-targeted Cre mouse lines have critical limitations that restrict their reliability for studies of disease pathogenesis and treatment, including mosaic Cre expression, inducer-independent activity, off-target Cre expression, and intrinsic toxicity. Here, we report the generation and characterization of a knock-in mouse line in which a P2A-CreERT2 coding sequence is fused with the native RPE-specific 65 kDa protein (Rpe65) gene for co-translational expression of CreERT2. Cre+/- mice were able to recombine a stringent Cre reporter allele with >99% efficiency and absolute RPE specificity upon tamoxifen induction at both post-natal days (PD) 21 and 50. Tamoxifen-independent Cre activity was negligible at PD64. Moreover, tamoxifen-treated Cre+/- mice displayed no signs of structural or functional retinal pathology up to 4 months of age. Despite weak RPE65 expression from the knock-in allele, visual cycle function was normal in Cre+/- mice. These data indicate that Rpe65CreERT2 mice are well-suited for studies of gene function and pathophysiology in the RPE.

Authors

Elliot H. Choi, Susie Suh, David E. Einstein, Henri Leinonen, Zhiqian Dong, Sriganesh Ramachandra Rao, Steven J. Fliesler, Seth Blackshaw, Minzhong Yu, Neal S. Peachey, Krzysztof Palczewski, Philip D. Kiser