Publications by authors named "Yoav Hadas"

24 Publications

  • Page 1 of 1

Spinal lumbar dI2 interneurons contribute to stability of bipedal stepping.

Elife 2021 Aug 16;10. Epub 2021 Aug 16.

Department of Medical Neurobiology, IMRIC, Hebrew University - Hadassah Medical School, Jerusalem, Israel.

Peripheral and intraspinal feedback is required to shape and update the output of spinal networks that execute motor behavior. We report that lumbar dI2 spinal interneurons in chicks receive synaptic input from afferents and premotor neurons. These interneurons innervate contralateral premotor networks in the lumbar and brachial spinal cord, and their ascending projections innervate the cerebellum. These findings suggest that dI2 neurons function as interneurons in local lumbar circuits, are involved in lumbo-brachial coupling, and that part of them deliver peripheral and intraspinal feedback to the cerebellum. Silencing of dI2 neurons leads to destabilized stepping in posthatching day 8 hatchlings, with occasional collapses, variable step profiles, and a wide-base walking gait, suggesting that dI2 neurons may contribute to the stabilization of the bipedal gait.
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http://dx.doi.org/10.7554/eLife.62001DOI Listing
August 2021

Direct Reprogramming Induces Vascular Regeneration Post Muscle Ischemic Injury.

Mol Ther 2021 Jul 28. Epub 2021 Jul 28.

Cardiovascular Research Institute, Icahn School of Medicine at Mount Sinai, New York, NY, USA, 10029; Department of Genetics and Genomic Sciences, Icahn School of Medicine at Mount Sinai, New York, NY, USA, 10029; Black Family Stem Cell Institute, Icahn School of Medicine at Mount Sinai, New York, NY, USA, 10029. Electronic address:

Reprogramming non-cardiomyocytes (non-CMs) into cardiomyocyte (CM)-like cells is a promising strategy for cardiac regeneration in conditions such as ischemic heart disease. Here, we used a modified mRNA (modRNA) gene delivery platform to deliver a cocktail of four cardiac-reprogramming genes (Gata4 (G), Mef2c (M), Tbx5 (T) and Hand2 (H)) together with three reprogramming-helper genes (Dominant Negative (DN)-TGFβ, DN-Wnt8a and Acid ceramidase (AC)), termed 7G-modRNA, to induce CM-like cells. We showed that 7G-modRNA reprogrammed 57% of CM-like cells in vitro. Through a lineage-tracing model, we determined that delivering the 7G-modRNA cocktail at the time of myocardial infarction reprogrammed ∼25% of CM-like cells in the scar area and significantly improved cardiac function, scar size, long-term survival and capillary density. Mechanistically, we determined that while 7G-modRNA cannot create de-novo beating CMs in vitro or in vivo, it can significantly upregulate pro-angiogenic mesenchymal stromal cells markers and transcription factors. We also demonstrated that our 7G-modRNA cocktail leads to neovascularization in ischemic-limb injury, indicating CM-like cells importance in other organs besides the heart. modRNA is currently being used around the globe for vaccination against COVID-19, and this study proves this is a safe, highly efficient gene delivery approach with therapeutic potential to treat ischemic diseases.
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http://dx.doi.org/10.1016/j.ymthe.2021.07.014DOI Listing
July 2021

Therapeutic Delivery of Pip4k2c-Modified mRNA Attenuates Cardiac Hypertrophy and Fibrosis in the Failing Heart.

Adv Sci (Weinh) 2021 05 12;8(10):2004661. Epub 2021 Mar 12.

Cardiovascular Research Center Icahn School of Medicine at Mount Sinai New York NY 10029 USA.

Heart failure (HF) remains a major cause of morbidity and mortality worldwide. One of the risk factors for HF is cardiac hypertrophy (CH), which is frequently accompanied by cardiac fibrosis (CF). CH and CF are controlled by master regulators mTORC1 and TGF-, respectively. Type-2-phosphatidylinositol-5-phosphate-4-kinase-gamma (Pip4k2c) is a known mTORC1 regulator. It is shown that Pip4k2c is significantly downregulated in the hearts of CH and HF patients as compared to non-injured hearts. The role of Pip4k2c in the heart during development and disease is unknown. It is shown that deleting Pip4k2c does not affect normal embryonic cardiac development; however, three weeks after TAC, adult Pip4k2c mice has higher rates of CH, CF, and sudden death than wild-type mice. In a gain-of-function study using a TAC mouse model, Pip4k2c is transiently upregulated using a modified mRNA (modRNA) gene delivery platform, which significantly improve heart function, reverse CH and CF, and lead to increased survival. Mechanistically, it is shown that Pip4k2c inhibits TGF1 via its N-terminal motif, Pip5k1, phospho-AKT 1/2/3, and phospho-Smad3. In sum, loss-and-gain-of-function studies in a TAC mouse model are used to identify Pip4k2c as a potential therapeutic target for CF, CH, and HF, for which modRNA is a highly translatable gene therapy approach.
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http://dx.doi.org/10.1002/advs.202004661DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8132051PMC
May 2021

In Vitro Synthesis of Modified RNA for Cardiac Gene Therapy.

Methods Mol Biol 2021 ;2158:281-294

Cardiovascular Research Center, Icahn School of Medicine at Mount Sinai, New York, NY, USA.

Modified mRNA (modRNA) is a promising new gene therapy approach that has safely and effectively delivered genes into different tissues, including the heart. Current efforts to use DNA-based or viral gene therapy to induce cardiac regeneration postmyocardial infarction (MI) or in heart failure (HF) have encountered key challenges, e.g., genome integration and delayed and noncontrolled expression. By contrast, modRNA is a transient, safe, non-immunogenic, and controlled gene delivery method that is not integrated into the genome. For most therapeutic applications, especially in regenerative medicine, the ability to deliver genes to the heart transiently and with control is vital for achieving therapeutic effect. Additionally, modRNA synthesis is comparatively simple and inexpensive compared to other gene delivery methods (e.g., protein), though a simple, clear in vitro transcription (IVT) protocol for synthesizing modRNA is needed for it to be more widely used. Here, we describe a simple and improved step-by-step IVT protocol to synthesize modRNA for in vitro or in vivo applications.
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http://dx.doi.org/10.1007/978-1-0716-0668-1_21DOI Listing
March 2021

Delivery of Modified mRNA in a Myocardial Infarction Mouse Model.

J Vis Exp 2020 06 11(160). Epub 2020 Jun 11.

Cardiovascular Research Center, Icahn School of Medicine at Mount Sinai; Department of Genetics and Genomic Sciences, Icahn School of Medicine at Mount Sinai; Black Family Stem Cell Institute, Icahn School of Medicine at Mount Sinai;

Myocardial infarction (MI) is a leading cause of morbidity and mortality in the Western world. In the past decade, gene therapy has become a promising treatment option for heart disease, owing to its efficiency and exceptional therapeutic effects. In an effort to repair the damaged tissue post-MI, various studies have employed DNA-based or viral gene therapy but have faced considerable hurdles due to the poor and uncontrolled expression of the delivered genes, edema, arrhythmia, and cardiac hypertrophy. Synthetic modified mRNA (modRNA) presents a novel gene therapy approach that offers high, transient, safe, nonimmunogenic, and controlled mRNA delivery to the heart tissue without any risk of genomic integration. Due to these remarkable characteristics combined with its bell-shaped pharmacokinetics in the heart, modRNA has become an attractive approach for the treatment of heart disease. However, to increase its effectiveness in vivo, a consistent and reliable delivery method needs to be followed. Hence, to maximize modRNA delivery efficiency and yield consistency in modRNA use for in vivo applications, an optimized method of preparation and delivery of modRNA intracardiac injection in a mouse MI model is presented. This protocol will make modRNA delivery more accessible for basic and translational research.
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http://dx.doi.org/10.3791/60832DOI Listing
June 2020

Optimization of 5' Untranslated Region of Modified mRNA for Use in Cardiac or Hepatic Ischemic Injury.

Mol Ther Methods Clin Dev 2020 Jun 31;17:622-633. Epub 2020 Mar 31.

Cardiovascular Research Center, Icahn School of Medicine at Mount Sinai, New York, NY 10029, USA.

Modified mRNA (modRNA) is a gene-delivery platform for transiently introducing a single gene or several genes of interest to different cell types and tissues. modRNA is considered to be a safe vector for gene transfer, as it negligibly activates the innate immune system and does not compromise the genome integrity. The use of modRNA in basic and translational science is rising, due to the clinical potential of modRNA. We are currently using modRNA to induce cardiac regeneration post-ischemic injury. Major obstacles in using modRNA for cardiac ischemic disease include the need for the direct and single administration of modRNA to the heart and the inefficient translation of modRNA due to its short half-life. Modulation of the 5' untranslated region (5' UTR) to enhance translation efficiency in ischemic cardiac disease has great value, as it can reduce the amount of modRNA needed per delivery and will achieve higher and longer protein production post-single delivery. Here, we identified that 5' UTR, from the fatty acid metabolism gene carboxylesterase 1D (Ces1d), enhanced the translation of firefly luciferase (Luc) modRNA by 2-fold in the heart post-myocardial infarction (MI). Moreover, we identified, in the Ces1d, a specific RNA element (element D) that is responsible for the improvement of modRNA translation and leads to a 2.5-fold translation increment over Luc modRNA carrying artificial 5' UTR, post-MI. Importantly, we were able to show that 5' UTR Ces1d also enhances modRNA translation in the liver, but not in the kidney, post-ischemic injury, indicating that Ces1d 5' UTR and element D may play a wider role in translation of protein under an ischemic condition.
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http://dx.doi.org/10.1016/j.omtm.2020.03.019DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7150433PMC
June 2020

Altering Sphingolipid Metabolism Attenuates Cell Death and Inflammatory Response After Myocardial Infarction.

Circulation 2020 03 29;141(11):916-930. Epub 2020 Jan 29.

Cardiovascular Research Center (Y.H., E.Y., M.M.Ż., E.C., N.S., M.T.K.S., R.K., A.A.K., K.K., A.M., N.H., L.Z., A.F, M.G.K.), Icahn School of Medicine at Mount Sinai, New York.

Background: Sphingolipids have recently emerged as a biomarker of recurrence and mortality after myocardial infarction (MI). The increased ceramide levels in mammalian heart tissues during acute MI, as demonstrated by several groups, is associated with higher cell death rates in the left ventricle and deteriorated cardiac function. Ceramidase, the only enzyme known to hydrolyze proapoptotic ceramide, generates sphingosine, which is then phosphorylated by sphingosine kinase to produce the prosurvival molecule sphingosine-1-phosphate. We hypothesized that Acid Ceramidase (AC) overexpression would counteract the negative effects of elevated ceramide and promote cell survival, thereby providing cardioprotection after MI.

Methods: We performed transcriptomic, sphingolipid, and protein analyses to evaluate sphingolipid metabolism and signaling post-MI. We investigated the effect of altering ceramide metabolism through a loss (chemical inhibitors) or gain (modified mRNA [modRNA]) of AC function post hypoxia or MI.

Results: We found that several genes involved in de novo ceramide synthesis were upregulated and that ceramide (C16, C20, C20:1, and C24) levels had significantly increased 24 hours after MI. AC inhibition after hypoxia or MI resulted in reduced AC activity and increased cell death. By contrast, enhancing AC activity via AC modRNA treatment increased cell survival after hypoxia or MI. AC modRNA-treated mice had significantly better heart function, longer survival, and smaller scar size than control mice 28 days post-MI. We attributed the improvement in heart function post-MI after AC modRNA delivery to decreased ceramide levels, lower cell death rates, and changes in the composition of the immune cell population in the left ventricle manifested by lowered abundance of proinflammatory detrimental neutrophils.

Conclusions: Our findings suggest that transiently altering sphingolipid metabolism through AC overexpression is sufficient and necessary to induce cardioprotection post-MI, thereby highlighting the therapeutic potential of AC modRNA in ischemic heart disease.
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http://dx.doi.org/10.1161/CIRCULATIONAHA.119.041882DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7135928PMC
March 2020

Effects of genetic transfection on calcium cycling pathways mediated by double-stranded adeno-associated virus in postinfarction remodeling.

J Thorac Cardiovasc Surg 2020 05 30;159(5):1809-1819.e3. Epub 2019 Sep 30.

Department of Cardiology, Cardiovascular Research Center, Icahn School of Medicine at Mount Sinai, New York, NY.

Objective: Restoring calcium sensor protein (S100A1) activity in failing hearts poses a promising therapeutic strategy. We hypothesize that cardiac overexpression of the S100A1 gene mediated by a double-stranded adeno-associated virus (scAAV) results in better functional and molecular improvements compared with the single-stranded virus (ssAAV).

Methods: Heart failure was induced by coronary artery ligation. Then, intramyocardial injections of saline, AAV9 empty capsid, scAAV9.S100A1, and ssAAV9.S100A1 were performed. Ten weeks postinfarction, all rats received cardiac evaluation; serum and tissue were collected for genetic analysis, cytokine profiling, and assessments of mitochondrial function and structure.

Results: Overexpression of AAV9.S100A1 improved systolic and diastolic function. Compared with control, ejection fraction was greater in treated groups (54.8% vs 32.3%, P < .05). Similarly, end-diastolic volume index was significantly less in the treated group than in control (1.14 vs 1.59 mL/cm), whereas fractional shortening was greater in treated groups than control (26% vs 38%, P < .05). Interestingly, cardiac mechanics were significantly better when treated with double-stranded virus compared with single-stranded. Quantitative polymerase chain reaction demonstrated robust transfection of myocardium with the S100A1 gene, with more infection in the self-complimentary group compared with the single-stranded group (5.68 ± 0.44 vs 4.09 ± 0.25 log genome copies per 100 ng of DNA; P < .0001). Concentrations of the inflammatory cytokines were elevated in the ssAAV9/S100A1 group compared with the scAAV9/S100A1. Assessment of mitochondrial respiration and morphology demonstrated that injection of self-complementary vector saved both mitochondrial structure and function.

Conclusions: Gene therapy of S100A1 can prevent pathologic postmyocardial infarction remodeling and decrease inflammatory response in ischemic heart failure.
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http://dx.doi.org/10.1016/j.jtcvs.2019.08.089DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7103560PMC
May 2020

Optimizing Modified mRNA Synthesis Protocol for Heart Gene Therapy.

Mol Ther Methods Clin Dev 2019 Sep 30;14:300-305. Epub 2019 Jul 30.

Cardiovascular Research Center, Icahn School of Medicine at Mount Sinai, New York, NY 10029, USA.

Synthetic modified RNA (modRNA) is a novel vector for gene transfer to the heart and other organs. modRNA can mediate strong, transient protein expression with minimal induction of the innate immune response and risk for genome integration. modRNA is already being used in several human clinical trials, and its use in basic and translational science is growing. Due to the complexity of preparing modRNA and the high cost of its reagents, there is a need for an improved, cost-efficient protocol to make modRNA. Here we show that changing the ratio between anti-reverse cap analog (ARCA) and N1-methyl-pseudouridine (N1mΨ), favoring ARCA over N1mΨ, significantly increases the yield per reaction, improves modRNA translation, and reduces its immunogenicity . This protocol will make modRNA preparation more accessible and financially affordable for basic and translational research.
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http://dx.doi.org/10.1016/j.omtm.2019.07.006DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6722299PMC
September 2019

[GENE THERAPY POTENTIAL AS A TREATMENT FOR HEART FAILURE].

Harefuah 2018 Feb;157(2):112-116

Cardiovascular Research Center, Mount Sinai School of Medicine, New York, NY, USA.

Introduction: Advances in understanding the molecular biology of heart failure, the evolution of vector technology, as well as defining the targets for therapeutic interventions has placed heart failure within the reach of gene-based therapy. During the last decade the concept of delivering cDNA encoding a therapeutic gene to failing cardiomyocytes has moved from hypothesis to the bench of preclinical applications and clinical trials. However, despite significant promise, several obstacles exist, which are described in this review. We anticipate that advances in the field will improve gene therapy in heart failure in future clinical approaches.
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February 2018

MPI depletion enhances O-GlcNAcylation of p53 and suppresses the Warburg effect.

Elife 2017 06 23;6. Epub 2017 Jun 23.

Department of Pediatrics, Icahn School of Medicine at Mount Sinai, New York, United States.

Rapid cellular proliferation in early development and cancer depends on glucose metabolism to fuel macromolecule biosynthesis. Metabolic enzymes are presumed regulators of this glycolysis-driven metabolic program, known as the Warburg effect; however, few have been identified. We uncover a previously unappreciated role for Mannose phosphate isomerase (MPI) as a metabolic enzyme required to maintain Warburg metabolism in zebrafish embryos and in both primary and malignant mammalian cells. The functional consequences of MPI loss are striking: glycolysis is blocked and cells die. These phenotypes are caused by induction of p53 and accumulation of the glycolytic intermediate fructose 6-phosphate, leading to engagement of the hexosamine biosynthetic pathway (HBP), increased O-GlcNAcylation, and p53 stabilization. Inhibiting the HBP through genetic and chemical methods reverses p53 stabilization and rescues the Mpi-deficient phenotype. This work provides mechanistic evidence by which MPI loss induces p53, and identifies MPI as a novel regulator of p53 and Warburg metabolism.
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http://dx.doi.org/10.7554/eLife.22477DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5495572PMC
June 2017

Optimizing Cardiac Delivery of Modified mRNA.

Mol Ther 2017 06 4;25(6):1306-1315. Epub 2017 Apr 4.

Cardiovascular Research Center, Icahn School of Medicine at Mount Sinai, New York, NY 10029, USA; Department of Genetics and Genomic Sciences, Icahn School of Medicine at Mount Sinai, New York, NY 10029, USA; Black Family Stem Cell Institute, Icahn School of Medicine at Mount Sinai, New York, NY 10029, USA. Electronic address:

Modified mRNA (modRNA) is a new technology in the field of somatic gene transfer that has been used for the delivery of genes into different tissues, including the heart. Our group and others have shown that modRNAs injected into the heart are robustly translated into the encoded protein and can potentially improve outcome in heart injury models. However, the optimal compositions of the modRNA and the reagents necessary to achieve optimal expression in the heart have not been characterized yet. In this study, our aim was to elucidate those parameters by testing different nucleotide modifications, modRNA doses, and transfection reagents both in vitro and in vivo in cardiac cells and tissue. Our results indicate that optimal cardiac delivery of modRNA is with N1-Methylpseudouridine-5'-Triphosphate nucleotide modification and achieved using 0.013 μg modRNA/mm/500 cardiomyocytes (CMs) transfected with positively charged transfection reagent in vitro and 100 μg/mouse heart (1.6 μg modRNA/μL in 60 μL total) sucrose-citrate buffer in vivo. We have optimized the conditions for cardiac delivery of modRNA in vitro and in vivo. Using the described methods and conditions may allow for successful gene delivery using modRNA in various models of cardiovascular disease.
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http://dx.doi.org/10.1016/j.ymthe.2017.03.016DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5474881PMC
June 2017

Modified mRNA as a therapeutic tool to induce cardiac regeneration in ischemic heart disease.

Wiley Interdiscip Rev Syst Biol Med 2017 01 2;9(1). Epub 2016 Dec 2.

Cardiovascular Research Center, Icahn Institute for Genomics and Multiscale Biology, Icahn School of Medicine at Mount Sinai, New York, NY, USA.

Ischemic heart disease (IHD) is a leading cause of morbidity and mortality in developed countries. Current pharmacological and interventional therapies provide significant improvement in the life quality of patient; however, they are mostly symptom-oriented and not curative. A high disease and economic burden of IHD requires the search for new therapeutic strategies to significantly improve patients' prognosis and quality of life. One of the main challenges during IHD is the massive loss of cardiomyocytes that possess minimal regenerative capacity. Recent understanding of the pathophysiological mechanisms underlying IHD, as well as new therapeutic approaches provide new hope for patients suffering from IHD. Synthetic modified mRNA (modRNA) is a new gene delivery vector that is increasingly used in in vivo applications. modRNA is a relatively stable, non-immunogenic, highly-expressed molecule that has been shown to mediate high and transient expression of proteins in different type of cells and tissues including cardiomyocytes. modRNA properties, together with its expression kinetics in the heart make it an attractive option for the treatment of IHD, especially after myocardial infarction. In this review we discuss the role of gene therapy in cardiac regeneration as an approach to treat IHD; traditional and innovative gene delivery methods; and focus specifically on modRNA structure, mode of delivery, and its use for the induction of endogenous regenerative capacity, mainly in the context of IHD. WIREs Syst Biol Med 2017, 9:e1367. doi: 10.1002/wsbm.1367 For further resources related to this article, please visit the WIREs website.
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http://dx.doi.org/10.1002/wsbm.1367DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5880206PMC
January 2017

Insulin-Like Growth Factor 1 Receptor-Dependent Pathway Drives Epicardial Adipose Tissue Formation After Myocardial Injury.

Circulation 2017 Jan 1;135(1):59-72. Epub 2016 Nov 1.

From Cardiovascular Research Center, Department of Genetics and Genomic Sciences, and Black Family Stem Cell Institute, Icahn School of Medicine at Mount Sinai, New York (L.Z., N.S., Y.H.); Department of Cardiology, Boston Children's Hospital, MA (L.Z., M.S.O., L.Y.Y., Q.M., W.T.P.); Cardiovascular and Metabolic Diseases Innovative Medicine Biotech Unit, AstraZeneca, Möllndal, Sweden (D.S., Q.-D.W.); The State Key Laboratory of Cell Biology, Institute of Biochemistry and Cell Biology, Shanghai Institutes for Biological Sciences, Chinese Academy of Sciences (B.Z.); Department of Genetics (W.L.C.), Harvard Stem Cell Institute (W.E., W.T.P.), Harvard Medical School, and Cardiovascular Research Center, Massachusetts General Hospital (M.A.), Harvard Medical School, Boston, MA; and Department of Cell and Molecular Biology and Medicine, Karolinska Institutet, Stockholm, Sweden (K.R.C.).

Background: Epicardial adipose tissue volume and coronary artery disease are strongly associated, even after accounting for overall body mass. Despite its pathophysiological significance, the origin and paracrine signaling pathways that regulate epicardial adipose tissue's formation and expansion are unclear.

Methods: We used a novel modified mRNA-based screening approach to probe the effect of individual paracrine factors on epicardial progenitors in the adult heart.

Results: Using 2 independent lineage-tracing strategies in murine models, we show that cells originating from the Wt1 mesothelial lineage, which includes epicardial cells, differentiate into epicardial adipose tissue after myocardial infarction. This differentiation process required Wt1 expression in this lineage and was stimulated by insulin-like growth factor 1 receptor (IGF1R) activation. IGF1R inhibition within this lineage significantly reduced its adipogenic differentiation in the context of exogenous, IGF1-modified mRNA stimulation. Moreover, IGF1R inhibition significantly reduced Wt1 lineage cell differentiation into adipocytes after myocardial infarction.

Conclusions: Our results establish IGF1R signaling as a key pathway that governs epicardial adipose tissue formation in the context of myocardial injury by redirecting the fate of Wt1 lineage cells. Our study also demonstrates the power of modified mRNA -based paracrine factor library screening to dissect signaling pathways that govern progenitor cell activity in homeostasis and disease.
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http://dx.doi.org/10.1161/CIRCULATIONAHA.116.022064DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5195872PMC
January 2017

Dysregulation of miRNA-9 in a Subset of Schizophrenia Patient-Derived Neural Progenitor Cells.

Cell Rep 2016 05 21;15(5):1024-1036. Epub 2016 Apr 21.

Department of Psychiatry, Icahn School of Medicine at Mount Sinai, 1425 Madison Avenue, New York, NY 10029, USA; Friedman Brain Institute, Icahn School of Medicine at Mount Sinai, 1425 Madison Avenue, New York, NY 10029, USA. Electronic address:

Converging evidence indicates that microRNAs (miRNAs) may contribute to disease risk for schizophrenia (SZ). We show that microRNA-9 (miR-9) is abundantly expressed in control neural progenitor cells (NPCs) but also significantly downregulated in a subset of SZ NPCs. We observed a strong correlation between miR-9 expression and miR-9 regulatory activity in NPCs as well as between miR-9 levels/activity, neural migration, and diagnosis. Overexpression of miR-9 was sufficient to ameliorate a previously reported neural migration deficit in SZ NPCs, whereas knockdown partially phenocopied aberrant migration in control NPCs. Unexpectedly, proteomic- and RNA sequencing (RNA-seq)-based analysis revealed that these effects were mediated primarily by small changes in expression of indirect miR-9 targets rather than large changes in direct miR-9 targets; these indirect targets are enriched for migration-associated genes. Together, these data indicate that aberrant levels and activity of miR-9 may be one of the many factors that contribute to SZ risk, at least in a subset of patients.
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http://dx.doi.org/10.1016/j.celrep.2016.03.090DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4856588PMC
May 2016

A 'tool box' for deciphering neuronal circuits in the developing chick spinal cord.

Nucleic Acids Res 2014 Oct 21;42(19):e148. Epub 2014 Aug 21.

Department of Medical Neurobiology, IMRIC, Hebrew University Medical School, Jerusalem, Israel

The genetic dissection of spinal circuits is an essential new means for understanding the neural basis of mammalian behavior. Molecular targeting of specific neuronal populations, a key instrument in the genetic dissection of neuronal circuits in the mouse model, is a complex and time-demanding process. Here we present a circuit-deciphering 'tool box' for fast, reliable and cheap genetic targeting of neuronal circuits in the developing spinal cord of the chick. We demonstrate targeting of motoneurons and spinal interneurons, mapping of axonal trajectories and synaptic targeting in both single and populations of spinal interneurons, and viral vector-mediated labeling of pre-motoneurons. We also demonstrate fluorescent imaging of the activity pattern of defined spinal neurons during rhythmic motor behavior, and assess the role of channel rhodopsin-targeted population of interneurons in rhythmic behavior using specific photoactivation.
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http://dx.doi.org/10.1093/nar/gku750DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4231727PMC
October 2014

Electroporation of the hindbrain to trace axonal trajectories and synaptic targets in the chick embryo.

J Vis Exp 2013 May 29(75):e50136. Epub 2013 May 29.

Koret School of Veterinary Medicine, The Hebrew University of Jerusalem.

Electroporation of the chick embryonic neural tube has many advantages such as being quick and efficient for the expression of foreign genes into neuronal cells. In this manuscript we provide a method that demonstrates uniquely how to electroporate DNA into the avian hindbrain at E2.75 in order to specifically label a subset of neuronal progenitors, and how to follow their axonal projections and synaptic targets at much advanced stages of development, up to E14.5. We have utilized novel genetic tools including specific enhancer elements, Cre/Lox - based plasmids and the PiggyBac-mediated DNA transposition system to drive GFP expression in a subtype of hindbrain cells (the dorsal most subgroup of interneurons, dA1). Axonal trajectories and targets of dA1 axons are followed at early and late embryonic stages at various brainstem regions. This strategy contributes advanced techniques for targeting cells of interest in the embryonic hindbrain and for tracing circuit formation at multiple stages of development.
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http://dx.doi.org/10.3791/50136DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3724688PMC
May 2013

Distinct cis regulatory elements govern the expression of TAG1 in embryonic sensory ganglia and spinal cord.

PLoS One 2013 26;8(2):e57960. Epub 2013 Feb 26.

Dept. of medical neurobiology, IMRIC, Hebrew University-Hadassah Medical School, Jerusalem, Israel.

Cell fate commitment of spinal progenitor neurons is initiated by long-range, midline-derived, morphogens that regulate an array of transcription factors that, in turn, act sequentially or in parallel to control neuronal differentiation. Included among these are transcription factors that regulate the expression of receptors for guidance cues, thereby determining axonal trajectories. The Ig/FNIII superfamily molecules TAG1/Axonin1/CNTN2 (TAG1) and Neurofascin (Nfasc) are co-expressed in numerous neuronal cell types in the CNS and PNS - for example motor, DRG and interneurons - both promote neurite outgrowth and both are required for the architecture and function of nodes of Ranvier. The genes encoding TAG1 and Nfasc are adjacent in the genome, an arrangement which is evolutionarily conserved. To study the transcriptional network that governs TAG1 and Nfasc expression in spinal motor and commissural neurons, we set out to identify cis elements that regulate their expression. Two evolutionarily conserved DNA modules, one located between the Nfasc and TAG1 genes and the second directly 5' to the first exon and encompassing the first intron of TAG1, were identified that direct complementary expression to the CNS and PNS, respectively, of the embryonic hindbrain and spinal cord. Sequential deletions and point mutations of the CNS enhancer element revealed a 130bp element containing three conserved E-boxes required for motor neuron expression. In combination, these two elements appear to recapitulate a major part of the pattern of TAG1 expression in the embryonic nervous system.
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http://journals.plos.org/plosone/article?id=10.1371/journal.pone.0057960PLOS
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3582508PMC
August 2013

Axonal patterns and targets of dA1 interneurons in the chick hindbrain.

J Neurosci 2012 Apr;32(17):5757-71

Koret School of Veterinary Medicine, Robert H. Smith Faculty of Agriculture, Food and Environment, The Hebrew University of Jerusalem, Rehovot 76100, Israel.

Hindbrain dorsal interneurons that comprise the rhombic lip relay sensory information and coordinate motor outputs. The progenitor dA1 subgroup of interneurons, which is formed along the dorsal-most region of the caudal rhombic lip, gives rise to the cochlear and precerebellar nuclei. These centers project sensory inputs toward upper-brain regions. The fundamental role of dA1 interneurons in the assembly and function of these brainstem nuclei is well characterized. However, the precise en route axonal patterns and synaptic targets of dA1 interneurons are not clear as of yet. Novel genetic tools were used to label dA1 neurons and trace their axonal trajectories and synaptic connections at various stages of chick embryos. Using dA1-specific enhancers, two contralateral ascending axonal projection patterns were identified; one derived from rhombomeres 6-7 that elongated in the dorsal funiculus, while the other originated from rhombomeres 2-5 and extended in the lateral funiculus. Targets of dA1 axons were followed at later stages using PiggyBac-mediated DNA transposition. dA1 axons were found to project and form synapses in the auditory nuclei and cerebellum. Investigation of mechanisms that regulate the patterns of dA1 axons revealed a fundamental role of Lim-homeodomain (HD) proteins. Switch in the expression of the specific dA1 Lim-HD proteins Lhx2/9 into Lhx1, which is typically expressed in dB1 interneurons, modified dA1 axonal patterns to project along the routes of dB1 subgroup. Together, the results of this research provided new tools and knowledge to the assembly of trajectories and connectivity of hindbrain dA1 interneurons and of molecular mechanisms that control these patterns.
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http://dx.doi.org/10.1523/JNEUROSCI.4231-11.2012DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6703607PMC
April 2012

Motor and dorsal root ganglion axons serve as choice points for the ipsilateral turning of dI3 axons.

J Neurosci 2010 Nov;30(46):15546-57

Department of Medical Neurobiology, Institute for Medical Research Israel-Canada, Hebrew University-Hadassah Medical School, Jerusalem, Israel.

The axons of the spinal intersegmental interneurons are projected longitudinally along various funiculi arrayed along the dorsal-ventral axis of the spinal cord. The roof plate and the floor plate have a profound role in patterning their initial axonal trajectory. However, other positional cues may guide the final architecture of interneuron tracks in the spinal cord. To gain more insight into the organization of specific axonal tracks in the spinal cord, we focused on the trajectory pattern of a genetically defined neuronal population, dI3 neurons, in the chick spinal cord. Exploitation of newly characterized enhancer elements allowed specific labeling of dI3 neurons and axons. dI3 axons are projected ipsilaterally along two longitudinal fascicules at the ventral lateral funiculus (VLF) and the dorsal funiculus (DF). dI3 axons change their trajectory plane from the transverse to the longitudinal axis at two novel checkpoints. The axons that elongate at the DF turn at the dorsal root entry zone, along the axons of the dorsal root ganglion (DRG) neurons, and the axons that elongate at the VLF turn along the axons of motor neurons. Loss and gain of function of the Lim-HD protein Isl1 demonstrate that Isl1 is not required for dI3 cell fate. However, Isl1 is sufficient to impose ipsilateral turning along the motor axons when expressed ectopically in the commissural dI1 neurons. The axonal patterning of dI3 neurons, revealed in this study, highlights the role of established axonal cues-the DRG and motor axons-as intermediate guidepost cues for dI3 axons.
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http://dx.doi.org/10.1523/JNEUROSCI.2380-10.2010DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6633670PMC
November 2010

Deciphering axonal pathways of genetically defined groups of neurons in the chick neural tube utilizing in ovo electroporation.

J Vis Exp 2010 May 2(39). Epub 2010 May 2.

Department of Medical Neurobiology, Institute for Medical Research Israel Canada, Hebrew University-Hadassah Medical School.

Employment of enhancer elements to drive expression of reporter genes in neurons is a widely used paradigm for tracking axonal projection. For tracking axonal projection of spinal interneurons in vertebrates, germ line-targeted reporter genes yield bilaterally symmetric labeling. Therefore, it is hard to distinguish between the ipsi- and contra-laterally projecting axons. Unilateral electroporation into the chick neural tube provides a useful means to restrict expression of a reporter gene to one side of the central nervous system, and to follow axonal projection on both sides. This video demonstrates first how to handle the eggs prior to injection. At HH stage 18-20, DNA is injected into the sacral level of the neural tube, then tungsten electrodes are placed parallel to the embryo and short electrical pulses are administered with a pulse generator. The egg is sealed with tape and placed back into an incubator for further development. Three days later (E6) the spinal cord is removed as an open book preparation from embryo, fixed, and processed for whole mount antibody staining. The stained spinal cord is mounted on slide and visualized using confocal microscopy.
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http://dx.doi.org/10.3791/1792DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC2902355PMC
May 2010

Transcriptional control of axonal guidance and sorting in dorsal interneurons by the Lim-HD proteins Lhx9 and Lhx1.

Neural Dev 2009 Jun 19;4:21. Epub 2009 Jun 19.

Department of Medical Neurobiology, IMRIC, Hebrew University, Hadassah Medical School, Jerusalem, Israel.

Background: Lim-HD proteins control crucial aspects of neuronal differentiation, including subtype identity and axonal guidance. The Lim-HD proteins Lhx2/9 and Lhx1/5 are expressed in the dorsal spinal interneuron populations dI1 and dI2, respectively. While they are not required for cell fate acquisition, their role in patterning the axonal trajectory of dI1 and dI2 neurons remains incompletely understood.

Results: Using newly identified dI1- and dI2-specific enhancers to trace axonal trajectories originating from these interneurons, we found that each population is subdivided into several distinct groups according to their axonal pathways. dI1 neurons project axons rostrally, either ipsi- or contra-laterally, while dI2 are mostly commissural neurons that project their axons rostrally and caudally. The longitudinal axonal tracks of each neuronal population self-fasciculate to form dI1- and dI2-specific bundles. The dI1 bundles are spatially located ventral relative to dI2 bundles. To examine the functional contribution of Lim-HD proteins to establishment of dI axonal projections, the Lim-HD code of dI neurons was altered by cell-specific ectopic expression. Expression of Lhx1 in dI1 neurons caused a repression of Lhx2/9 and imposed caudal projection to the caudal commissural dI1 neurons. Complementarily, when expressed in dI2 neurons, Lhx9 repressed Lhx1/5 and triggered a bias toward rostral projection in otherwise caudally projecting dI2 neurons, and ventral shift of the longitudinal axonal fascicule.

Conclusion: The Lim-HD proteins Lhx9 and Lhx1 serve as a binary switch in controlling the rostral versus caudal longitudinal turning of the caudal commissural axons. Lhx1 determines caudal turning and Lhx9 triggers rostral turning.
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http://dx.doi.org/10.1186/1749-8104-4-21DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC2704203PMC
June 2009

Involvement of Gluconic Acid and Glucose Oxidase in the Pathogenicity of Penicillium expansum in Apples.

Phytopathology 2007 Mar;97(3):384-90

ABSTRACT The contribution of gluconic acid secretion to the colonization of apple tissue by Penicillium expansum was analyzed by modulation (increase or decrease) of gluconic acid accumulation at the infection court. P. expansum isolates that express the most gox2 transcripts and concomitant glucose oxidase (GOX) activity and that secrete the most gluconic acid cause disease of apple at the fastest rate. Cultures grown under reduced oxygen concentration generated fewer gox2 transcripts, produced less gluconic acid, and led to a 15% reduction in disease. Furthermore, the detection of significantly high levels of transcripts of gox2 and GOX activity at the edge of the decaying tissue emphasize the involvement of GOX in tissue acidification of the decaying tissue. Taken together, these results emphasize the importance of GOX in the production of the gluconic acid that leads, in turn, to host tissue acidification. This acidification enhanced the expression of pectolytic enzymes and the establishment of conditions for necrotrophic development of P. expansum.
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http://dx.doi.org/10.1094/PHYTO-97-3-0384DOI Listing
March 2007
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