Publications by authors named "Jacqueline Simonet"

12 Publications

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CTP synthase polymerization in germline cells of the developing egg supports egg production.

Biol Open 2020 07 21;9(7). Epub 2020 Jul 21.

Cancer Biology Program, Fox Chase Cancer Center, 333 Cottman Avenue, Philadelphia, PA 19111, USA

Polymerization of metabolic enzymes into micron-scale assemblies is an emerging mechanism for regulating their activity. CTP synthase (CTPS) is an essential enzyme in the biosynthesis of the nucleotide CTP and undergoes regulated and reversible assembly into large filamentous structures in organisms from bacteria to humans. The purpose of these assemblies is unclear. A major challenge to addressing this question has been the inability to abolish assembly without eliminating CTPS protein. Here we demonstrate that a recently reported point mutant in CTPS, Histidine 355A (H355A), prevents CTPS filament assembly and dominantly inhibits the assembly of endogenous wild-type CTPS in the ovary. Expressing this mutant in ovarian germline cells, we show that disruption of CTPS assembly in early stage egg chambers reduces egg production. This effect is exacerbated in flies fed the glutamine antagonist 6-diazo-5-oxo-L-norleucine, which inhibits CTP synthesis. These findings introduce a general approach to blocking the assembly of polymerizing enzymes without eliminating their catalytic activity and demonstrate a role for CTPS assembly in supporting egg production, particularly under conditions of limited glutamine metabolism.This article has an associated First Person interview with the first author of the paper.
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http://dx.doi.org/10.1242/bio.050328DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7390647PMC
July 2020

Freedom of assembly: metabolic enzymes come together.

Mol Biol Cell 2020 06;31(12):1201-1205

Cancer Biology Program, Fox Chase Cancer Center, Philadelphia, PA 19111.

Many different enzymes in intermediate metabolism dynamically assemble filamentous polymers in cells, often in response to changes in physiological conditions. Most of the enzyme filaments known to date have only been observed in cells, but in a handful of cases structural and biochemical studies have revealed the mechanisms and consequences of assembly. In general, enzyme polymerization functions as a mechanism to allosterically tune enzyme kinetics, and it may play a physiological role in integrating metabolic signaling. Here, we highlight some principles of metabolic filaments by focusing on two well-studied examples in nucleotide biosynthesis pathways-inosine-5'-monophosphate (IMP) dehydrogenase and cytosine triphosphate (CTP) synthase.
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http://dx.doi.org/10.1091/mbc.E18-10-0675DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7353150PMC
June 2020

Reconstituted IMPDH polymers accommodate both catalytically active and inactive conformations.

Mol Biol Cell 2017 Aug 9. Epub 2017 Aug 9.

Cancer Biology Program, Fox Chase Cancer Center, 333 Cottman Avenue, Philadelphia, PA 19111

Several metabolic enzymes undergo reversible polymerization into macromolecular assemblies. The function of these assemblies is often unclear but in some cases they regulate enzyme activity and metabolic homeostasis. The guanine nucleotide biosynthetic enzyme inosine monophosphate dehydrogenase (IMPDH) forms octamers that polymerize into helical chains. In mammalian cells, IMPDH filaments can associate into micron-length assemblies. Polymerization and enzyme activity are regulated in part by binding of purine nucleotides to an allosteric regulatory domain. ATP promotes octamer polymerization, whereas GTP promotes a compact, inactive conformation whose ability to polymerize is unknown. Also unclear is whether polymerization directly alters IMPDH catalytic activity. To address this, we identified point mutants of human IMPDH2 that either prevent or promote polymerization. Unexpectedly, we found that polymerized and non-assembled forms of recombinant IMPDH have comparable catalytic activity, substrate affinity, and GTP sensitivity and validated this finding in cells. Electron microscopy revealed that substrates and allosteric nucleotides shift the equilibrium between active and inactive conformations in both the octamer and the filament. Unlike other metabolic filaments, which selectively stabilize active or inactive conformations, recombinant IMPDH filaments accommodate multiple states. These conformational states are finely tuned by substrate availability and purine balance, while polymerization may allow cooperative transitions between states.
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http://dx.doi.org/10.1091/mbc.E17-04-0263DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5620369PMC
August 2017

Osterix/Sp7 limits cranial bone initiation sites and is required for formation of sutures.

Dev Biol 2016 05 16;413(2):160-72. Epub 2016 Mar 16.

Department of Cell and Developmental Biology, Perelman School of Medicine at the University of Pennsylvania, Philadelphia, PA, USA. Electronic address:

During growth, individual skull bones overlap at sutures, where osteoblast differentiation and bone deposition occur. Mutations causing skull malformations have revealed some required genes, but many aspects of suture regulation remain poorly understood. We describe a zebrafish mutation in osterix/sp7, which causes a generalized delay in osteoblast maturation. While most of the skeleton is patterned normally, mutants have specific defects in the anterior skull and upper jaw, and the top of the skull comprises a random mosaic of bones derived from individual initiation sites. Osteoblasts at the edges of the bones are highly proliferative and fail to differentiate, consistent with global changes in gene expression. We propose that signals from the bone itself are required for orderly recruitment of precursor cells and growth along the edges. The delay in bone maturation caused by loss of Sp7 leads to unregulated bone formation, revealing a new mechanism for patterning the skull and sutures.
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http://dx.doi.org/10.1016/j.ydbio.2016.03.011DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5469377PMC
May 2016

Arx together with FoxA2, regulates Shh floor plate expression.

Dev Biol 2014 Sep 23;393(1):137-48. Epub 2014 Jun 23.

Department of Pathology, Brigham and Women׳s Hospital, Harvard Medical School, USA; Department of Pathology and Laboratory Medicine, Children׳s Hospital of Philadelphia, USA. Electronic address:

Mutations in the Aristaless related homeodomain transcription factor (ARX) are associated with a diverse set of X-linked mental retardation and epilepsy syndromes in humans. Although most studies have been focused on its function in the forebrain, ARX is also expressed in other regions of the developing nervous system including the floor plate (FP) of the spinal cord where its function is incompletely understood. To investigate the role of Arx in the FP, we performed gain-of-function studies in the chick using in ovo electroporation, and loss-of-function studies in Arx-deficient mice. We have found that Arx, in conjunction with FoxA2, directly induces Sonic hedgehog (Shh) expression through binding to a Shh floor plate enhancer (SFPE2). We also observed that FoxA2 induces Arx through its transcriptional activation domain whereas Nkx2.2, induced by Shh, abolishes this induction. Our data support a feedback loop model for Arx function; through interactions with FoxA2, Arx positively regulates Shh expression in the FP, and Shh signaling in turn activates Nkx2.2, which suppresses Arx expression. Furthermore, our data are evidence that Arx plays a role as a context dependent transcriptional activator, rather than a primary inducer of Shh expression, potentially explaining how mutations in ARX are associated with diverse, and often subtle, defects.
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http://dx.doi.org/10.1016/j.ydbio.2014.06.012DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4681535PMC
September 2014

Conditional Loss of Arx From the Developing Dorsal Telencephalon Results in Behavioral Phenotypes Resembling Mild Human ARX Mutations.

Cereb Cortex 2015 Sep 2;25(9):2939-50. Epub 2014 May 2.

Division of Child Neurology, Children's Hospital of Philadelphia, Department of Neurology and Pediatrics, Perelman School of Medicine at the University of Pennsylvania, Philadelphia, PA, USA Current address: Division of Child Neurology, Children's Hospital of Philadelphia, 3615 Civic Center Boulevard, Philadelphia, PA 19072, USA.

Mutations in the Aristaless-Related Homeobox (ARX) gene cause structural anomalies of the brain, epilepsy, and neurocognitive deficits in children. During forebrain development, Arx is expressed in both pallial and subpallial progenitor cells. We previously demonstrated that elimination of Arx from subpallial-derived cortical interneurons generates an epilepsy phenotype with features overlapping those seen in patients with ARX mutations. In this report, we have selectively removed Arx from pallial progenitor cells that give rise to the cerebral cortical projection neurons. While no discernable seizure activity was recorded, these mice exhibited a peculiar constellation of behaviors. They are less anxious, less social, and more active when compared with their wild-type littermates. The overall cortical thickness was reduced, and the corpus callosum and anterior commissure were hypoplastic, consistent with a perturbation in cortical connectivity. Taken together, these data suggest that some of the structural and behavioral anomalies, common in patients with ARX mutations, are specifically due to alterations in pallial progenitor function. Furthermore, our data demonstrate that some of the neurobehavioral features found in patients with ARX mutations may not be due to on-going seizures, as is often postulated, given that epilepsy was eliminated as a confounding variable in these behavior analyses.
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http://dx.doi.org/10.1093/cercor/bhu090DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4537438PMC
September 2015

Arx is required for specification of the zona incerta and reticular nucleus of the thalamus.

J Neuropathol Exp Neurol 2014 Mar;73(3):253-61

From the Department of Pathology, Children's Hospital of Philadelphia (CNS); Penn-PORT Program (CNS), and Department of Cell and Developmental Biology (JCS), University of Pennsylvania; and Department of Neurology, Children's Hospital of Philadelphia (EDM), Philadelphia, Pennsylvania; and Department of Pathology, Brigham Women and Children's Hospital, Boston, Massachusetts (JAG).

Mutations in the aristaless-related homeobox (ARX) gene result in a spectrum of structural and functional nervous system disorders including lissencephaly, movement disorders, intellectual disabilities, and epilepsy. Some patients also have symptoms indicating hypothalamic dysfunction, but little is known about the role of ARX in diencephalic development. To begin evaluating diencephalic defects, we examined the expression of a panel of known genes and gene products that label specific diencephalic nuclei in 2 different Arx mutant mouse lines at E18.5. Male mice engineered to have a polyalanine expansion mutation (Arx) revealed no expression differences in any diencephalic nucleus when compared with wild-type littermates. In contrast, mice null for Arx (Arx) lost expression of specific markers of the thalamic reticular nucleus and zona incerta (ZI) while retaining expression in other thalamic nuclei and in the hypothalamus. Tyrosine hydroxylase, a marker of the dopaminergic A13 subnucleus of ZI, was among those lost, suggesting a requirement for Arx in normal thalamic reticular nucleus and ZI development and, specifically, for A13 dopaminergic fate. Because the ZI and A13 regions make connections to several hypothalamic nuclei, such misspecification may contribute to the "hypothalamic dysfunction" observed in some patients.
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http://dx.doi.org/10.1097/NEN.0000000000000048DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4038102PMC
March 2014

ARX regulates cortical intermediate progenitor cell expansion and upper layer neuron formation through repression of Cdkn1c.

Cereb Cortex 2015 Feb 22;25(2):322-35. Epub 2013 Aug 22.

Department of Neuroscience, San Raffaele Scientific Institute, Milan 20132, Italy.

Mutations in the Aristaless-related homeobox (ARX) gene are found in a spectrum of epilepsy and X-linked intellectual disability disorders. During development Arx is expressed in pallial ventricular zone (VZ) progenitor cells where the excitatory projection neurons of the cortex are born. Arx(-/Y) mice were shown to have decreased proliferation in the cortical VZ resulting in smaller brains; however, the basis for this reduced proliferation was not established. To determine the role of ARX on cell cycle dynamics in cortical progenitor cells, we generated cerebral cortex-specific Arx mouse mutants (cKO). The loss of pallial Arx resulted in the reduction of cortical progenitor cells, particularly the proliferation of intermediate progenitor cells (IPCs) was affected. Later in development and postnatally cKO brains showed a reduction of upper layer but not deeper layer neurons consistent with the IPC defect. Transcriptional profile analysis of E14.5 Arx-ablated cortices compared with control revealed that CDKN1C, an inhibitor of cell cycle progression, is overexpressed in the cortical VZ and SVZ of Arx KOs throughout corticogenesis. We also identified ARX as a direct regulator of Cdkn1c transcription. Together these data support a model where ARX regulates the expansion of cortical progenitor cells through repression of Cdkn1c.
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http://dx.doi.org/10.1093/cercor/bht222DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4351425PMC
February 2015

Late-emigrating trunk neural crest cells in turtle embryos generate an osteogenic ectomesenchyme in the plastron.

Dev Dyn 2013 Nov 6;242(11):1223-35. Epub 2013 Sep 6.

Biology Department, Millersville University, Millersville, Pennsylvania.

Background: The turtle plastron is composed of a keratinized epidermis overlying nine dermal bones. Its developmental origin has been controversial; recent evidence suggests that the plastral bones derive from trunk neural crest cells (NCCs).

Results: This study extends the observations that there is a turtle-specific, second wave of trunk NCC delamination and migration, after the original NCCs have reached their destination and differentiated. This second wave was confirmed by immunohistochemistry in whole-mounts and serial sections, by injecting DiI (1,1', di-octadecyl-3,3,3',3',-tetramethylindo-carbocyanine perchlorate) into the lumen of the neural tube and tracing labeled cells into the plastron, and by isolating neural tubes from older turtle embryos and observing delaminating NCCs. This later migration gives rise to a plastral ectomesenchyme that expresses NCC markers and can be induced to initiate bone formation.

Conclusions: The NCCs of this second migration have properties similar to those of the earlier NCCs, but also express markers characteristic of cranial NCCs. The majority of the cells of the plastron mesenchyme express neural crest markers, and have osteogenic differentiation capabilities that are similar or identical to craniofacial ectomesenchyme. Our evidence supports the contention that turtle plastron bones are derived from a late emigrating population of cells derived from the trunk neural crest.
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http://dx.doi.org/10.1002/dvdy.24018DOI Listing
November 2013

Differential effects of a polyalanine tract expansion in Arx on neural development and gene expression.

Hum Mol Genet 2012 Mar 22;21(5):1090-8. Epub 2011 Nov 22.

Neuroscience Graduate Group, University of Pennsylvania School of Medicine, Philadelphia, PA 19104, USA.

Polyalanine (poly-A) tracts exist in 494 annotated proteins; to date, expansions in these tracts have been associated with nine human diseases. The pathogenetic mechanism by which a poly-A tract results in these various human disorders remains uncertain. To understand the role of this mutation type, we investigated the change in functional properties of the transcription factor Arx when it has an expanded poly-A tract (Arx(E)), a mutation associated with infantile spasms and intellectual disabilities in humans. We found that although Arx(E) functions normally in the dorsal brain, its function in subpallial-derived populations of neurons is compromised. These contrasting functions are associated with the misregulation of Arx targets through the loss of the ability of Arx(E) to interact with the Arx cofactor Tle1. Our data demonstrate a novel mechanism for poly-A expansion diseases: the misregulation of a subset of target genes normally regulated by a transcription factor.
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http://dx.doi.org/10.1093/hmg/ddr538DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3277309PMC
March 2012

Leading process branch instability in Lis1+/- nonradially migrating interneurons.

Cereb Cortex 2010 Jun 27;20(6):1497-505. Epub 2009 Oct 27.

University of Pennsylvania School of Medicine, Philadelphia, PA 19104, USA.

Mammalian forebrain development requires extensive migration, yet the mechanisms through which migrating neurons sense and respond to guidance cues are not well understood. Similar to the axon growth cone, the leading process and branches of neurons may guide migration, but the cytoskeletal events that regulate branching are unknown. We have previously shown that loss of microtubule-associated protein Lis1 reduces branching during migration compared with wild-type neurons. Using time-lapse imaging of Lis1(+/-) and Lis1(+/+) cells migrating from medial ganglionic eminence explant cultures, we show that the branching defect is not due to a failure to initiate branches but a defect in the stabilization of new branches. The leading processes of Lis1(+/-) neurons have reduced expression of stabilized, acetylated microtubules compared with Lis1(+/+) neurons. To determine whether Lis1 modulates branch stability through its role as the noncatalytic beta regulatory subunit of platelet-activating factor (PAF) acetylhydrolase 1b, exogenous PAF was applied to wild-type cells. Excess PAF added to wild-type neurons phenocopies the branch instability observed in Lis1(+/-) neurons, and a PAF antagonist rescues leading process branching in Lis1(+/-) neurons. These data highlight a role for Lis1, acting through the PAF pathway, in leading process branching and microtubule stabilization.
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http://dx.doi.org/10.1093/cercor/bhp211DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC2871376PMC
June 2010

Control of large, established tumor xenografts with genetically retargeted human T cells containing CD28 and CD137 domains.

Proc Natl Acad Sci U S A 2009 Mar 11;106(9):3360-5. Epub 2009 Feb 11.

Abramson Family Cancer Research Institute, University of Pennsylvania School of Medicine, Philadelphia, PA 19104, USA.

Mesothelin is a cell-surface molecule over-expressed on a large fraction of carcinomas, and thus is an attractive target of immunotherapy. A molecularly targeted therapy for these cancers was created by engineering T cells to express a chimeric receptor with high affinity for human mesothelin. Lentiviral vectors were used to express a single-chain variable fragment that binds mesothelin and that is fused to signaling domains derived from T-cell receptor zeta, CD28, and CD137 (4-1BB). When stimulated by mesothelin, lentivirally transduced T cells were induced to proliferate, express the antiapoptotic gene Bcl-X(L), and secrete multiple cytokines, all features characteristic of central memory T cells. When transferred intratumorally or intravenously into NOD/scid/IL2rgamma(-/-) mice engrafted with large pre-established tumors, the engineered T cells reduced the tumor burden, and in some cases resulted in complete eradication of the tumors at low effector-to-target ratios. Incorporation of the CD137 signaling domain specifically reprogrammed cells for multifunctional cytokine secretion and enhanced persistence of T cells. These findings have important implications for adoptive immunotherapy of cancer, especially in the context of poorly immunogenic tumors. Genetically redirected T cells have promise of targeting T lymphocytes to tumor antigens, confer resistance to the tumor microenvironment, and providing immunosurveillance.
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http://dx.doi.org/10.1073/pnas.0813101106DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC2651342PMC
March 2009