deciphering-axonal-pathways-genetically-defined-groups-neurons-chick

Deciphering Axonal Pathways of Genetically Defined Groups of Neurons in the Chick Neural Tube Utilizing <em>in ovo</em> Electroporation

Employment of enhancer elements to drive expression of reporter genes in neurons is a widely used paradigm for tracking axonal projection. For tracking ...

electroporation-hindbrain-to-trace-axonal-trajectories-synaptic

Electroporation of the Hindbrain to Trace Axonal Trajectories and Synaptic Targets in the Chick Embryo

Electroporation of  the chick embryonic neural tube has many advantages such as being quick and efficient  for the expression of foreign genes into ...

delivery-of-modified-mrna-in-a-myocardial-infarction-mouse-model

Delivery of Modified mRNA in a Myocardial Infarction Mouse Model

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 ...

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.

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

ABSTRAKT war der Beitrag der Glucons&auml;ure S&auml;ureerzeugung an der Kolonisierung der Apfel Tissue von Penicillium Expansum durch Modulation (Zunahme oder Abnahme) der Glucons&auml;ure saure Anh&auml;ufung am Hof Infektion analysiert. P. Expansum Isolate &auml;u&szlig;ern, die meisten gox2 Abschriften und damit einhergehende Glukose-Oxidase (GOX) Aktivit&auml;t und sezernieren, die am meisten Glucons&auml;ure verursachen Krankheit von Apple mit der schnellsten Rate. Kulturen, die unter reduzierter Sauerstoffkonzentration gewachsen generiert weniger gox2 Abschriften, produziert weniger Glucons&auml;ure S&auml;ure und f&uuml;hrte zu einer Reduktion von 15 % bei Krankheit. Dar&uuml;ber hinaus die Erkennung von signifikant hohe Abschriften von GOX Aktivit&auml;ten am Rande des verfallenden Gewebes und gox2 betonen die Beteiligung von GOX in Gewebe Versauerung des verfallenden Gewebes. Zusammengenommen, unterstreichen diese Ergebnisse die Bedeutung von GOX in der Produktion von der Glucons&auml;ure, die wiederum zur Host Gewebe Versauerung f&uuml;hrt. Diese Versauerung verst&auml;rkt Ausdruck der Pektolytische Enzyme und die Schaffung der Voraussetzungen f&uuml;r die Entwicklung der Necrotrophic von p. Expansum.

Einbeziehung der Glucons&auml;ure und Glucose-Oxidase in der Pathogenit&auml;t von Penicillium Expansum in &Auml;pfeln.

Lim-HD Proteine Steuern entscheidende Aspekte der neuronale Differenzierung, einschlie&szlig;lich Subtyp Identit&auml;t und axonale Anleitung. Die Lim-HD-Proteine, Lhx2/9 und Lhx1/5 werden jeweils in der dorsalen Wirbels&auml;ule Interneuron Populationen dI1 und dI2, ausgedr&uuml;ckt. Obwohl sie nicht f&uuml;r Zelle Schicksal Erwerb erforderlich sind, bleibt ihre Rolle bei der Musterung des axonalen gemalten dI1 und dI2 Neuronen unvollst&auml;ndig verstanden.

Transkriptionelle Kontrolle der axonalen Anleitung und Sortieren in dorsalen Interneuronen durch die Lim-HD-Proteine, Lhx9 und Lhx1.

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.

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

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.

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

Die Axone der spinalen intersegmental Interneuronen werden Longitudinal entlang verschiedener Funiculi gekleidet entlang der Achse dorsal-Ventral des R&uuml;ckenmarks projiziert. Die Dach-Platte und der Bodenplatte haben eine profunde Rolle bei der Musterung ihrer anf&auml;nglichen axonale Flugbahn. Jedoch k&ouml;nnen andere positionelle Hinweise die endg&uuml;ltige Architektur Interneuron Production im R&uuml;ckenmark f&uuml;hren. Um mehr Einblick in die Organisation von spezifischen axonale Tracks im R&uuml;ckenmark, konzentrierten wir uns auf die Flugbahn-Muster einer genetisch definierten neuronalen Population, dI3 Neuronen im R&uuml;ckenmark K&uuml;ken. Verwertung neu gekennzeichnete Enhancer-Elemente erlaubt bestimmte Kennzeichnung von dI3 Neuronen und Axone. dI3 Axone sind ipsilaterally entlang zwei l&auml;ngs Fascicules an der ventralen seitlichen Funiculus (VLF) und der dorsalen Funiculus (DF) projiziert. dI3 Axone &auml;ndern ihre Flugbahn-Flugzeug von der quer zur L&auml;ngsachse an zwei neuartige Checkpoints. Die Axone, die zum Jahreswechsel DF in der Spinalganglien Eintrag Zone entlang der Axone der Ganglienzellen der Spinalganglien (DRG) Neuronen zu verl&auml;ngern und die Axone, die zum Jahreswechsel VLF entlang der Axone von Motoneuronen verl&auml;ngern. Verlust und Gewinn der Funktion des Proteins Lim-HD Isl1 zeigen, dass Isl1 f&uuml;r dI3 Zelle Schicksal nicht ben&ouml;tigt. Allerdings reicht Isl1 ipsilateral drehen entlang die motorischen Axone ectopically in den Neuronen kommissurale dI1 ausgedr&uuml;ckt zu verh&auml;ngen. Die axonale Musterung dI3 Neuronen, offenbart in dieser Studie unterstreicht die Rolle von etablierten axonale Signale-die DRG und motorischen Axone-als fortgeschrittene Stilfragen Cues f&uuml;r dI3 Axone.

Motor und Dorsal Root Ganglion Axone dienen als Wahl f&uuml;r die ipsilaterale drehen dI3 Axone.

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.

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

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.

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

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.

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

-Epicardial adipose tissue (EAT) 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 EAT's formation and expansion are unclear.

An IGF1R-Dependent Pathway Drives Epicardial Adipose Tissue Formation After Myocardial Injury.

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.

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

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.

Optimizing Cardiac Delivery of Modified mRNA.

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.

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

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.

Optimizing Modified mRNA  Synthesis Protocol for Heart Gene Therapy.

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).

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

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.

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

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.

ArticleTotal : 3	Year
Publication title Cited by 12	2010
Publication title Cited by 9	2013
Publication title Cited by 7	2020

Article	Year
Involvement of Gluconic Acid and Glucose Oxidase in the Pathogenicity of Penicillium expansum in Apples. Phytopathology\| PubMed ID: 18943660	2007
Transcriptional control of axonal guidance and sorting in dorsal interneurons by the Lim-HD proteins Lhx9 and Lhx1. Neural development\| PubMed ID: 19545367	2009
Motor and dorsal root ganglion axons serve as choice points for the ipsilateral turning of dI3 axons. The Journal of neuroscience : the official journal of the Society for Neuroscience\| PubMed ID: 21084609	2010
Axonal patterns and targets of dA1 interneurons in the chick hindbrain. The Journal of neuroscience : the official journal of the Society for Neuroscience\| PubMed ID: 22539838	2012
Distinct cis regulatory elements govern the expression of TAG1 in embryonic sensory ganglia and spinal cord. PloS one\| PubMed ID: 23469119	2013
Dysregulation of miRNA-9 in a Subset of Schizophrenia Patient-Derived Neural Progenitor Cells. Cell reports\| PubMed ID: 27117414	2016
An IGF1R-Dependent Pathway Drives Epicardial Adipose Tissue Formation After Myocardial Injury. Circulation\| PubMed ID: 27803039	2016
Modified mRNA as a therapeutic tool to induce cardiac regeneration in ischemic heart disease. Wiley interdisciplinary reviews. Systems biology and medicine\| PubMed ID: 27911047	2017
Optimizing Cardiac Delivery of Modified mRNA. Molecular therapy : the journal of the American Society of Gene Therapy\| PubMed ID: 28389322	2017
MPI depletion enhances O-GlcNAcylation of p53 and suppresses the Warburg effect. eLife\| PubMed ID: 28644127	2017
Optimizing Modified mRNA Synthesis Protocol for Heart Gene Therapy. Molecular therapy. Methods & clinical development\| PubMed ID: 31508439	2019
Effects of genetic transfection on calcium cycling pathways mediated by double-stranded adeno-associated virus in postinfarction remodeling. The Journal of thoracic and cardiovascular surgery\| PubMed ID: 31679707	2020
Altering Sphingolipid Metabolism Attenuates Cell Death and Inflammatory Response After Myocardial Infarction. Circulation\| PubMed ID: 31992066	2020
Optimization of 5' Untranslated Region of Modified mRNA for Use in Cardiac or Hepatic Ischemic Injury. Molecular therapy. Methods & clinical development\| PubMed ID: 32300609	2020

Yoav Hadas

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