Analysis of the intracellular traffic of IgG in the context of Down syndrome (trisomy 21)

Persons with Down syndrome (DS, trisomy 21) have widespread cellular protein trafficking defects. There is a paucity of data describing the intracellular transport of IgG in the context of endosomal-lysosomal alterations linked to trisomy 21. In this study, we analyzed the intracellular traffic of IgG mediated by the human neonatal Fc receptor (FcRn) in fibroblast cell lines with trisomy 21. Intracellular IgG trafficking studies in live cells showed that fibroblasts with trisomy 21 exhibit higher proportion of IgG in lysosomes (~10% increase), decreased IgG content in intracellular vesicles (~9% decrease), and a trend towards decreased IgG recycling (~55% decrease) in comparison to diploid cells. Amyloid-beta precursor protein (APP) overexpression in diploid fibroblasts replicated the increase in IgG sorting to the degradative pathway observed in cells with trisomy 21. The impact of APP on the expression of FCGRT (alpha chain component of FcRn) was investigated by APP knock down and overexpression of the APP protein. APP knock down increased the expression of FCGRT mRNA by ~60% in both diploid and trisomic cells. Overexpression of APP in diploid fibroblasts and HepG2 cells resulted in a decrease in FCGRT and FcRn expression. Our results indicate that the intracellular traffic of IgG is altered in cells with trisomy 21. This study lays the foundation for future investigations into the role of FcRn in the context of DS.

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Analysis of the intracellular traffic of IgG in the context of Down syndrome (trisomy 21)

Scientific Reports ◽

10.1038/s41598-021-90469-z ◽

2021 ◽

Vol 11 (1) ◽

Author(s):

R. B. Cejas ◽

M. Tamaño-Blanco ◽

J. G. Blanco

Keyword(s):

Down Syndrome ◽

Trisomy 21 ◽

Intracellular Transport ◽

Live Cells ◽

Intracellular Traffic ◽

Diploid Fibroblasts ◽

Knock Down ◽

Diploid Cells ◽

The Impact ◽

In Cells

AbstractPersons with Down syndrome (DS, trisomy 21) have widespread cellular protein trafficking defects. There is a paucity of data describing the intracellular transport of IgG in the context of endosomal-lysosomal alterations linked to trisomy 21. In this study, we analyzed the intracellular traffic of IgG mediated by the human neonatal Fc receptor (FcRn) in fibroblast cell lines with trisomy 21. Intracellular IgG trafficking studies in live cells showed that fibroblasts with trisomy 21 exhibit higher proportion of IgG in lysosomes (~ 10% increase), decreased IgG content in intracellular vesicles (~ 9% decrease), and a trend towards decreased IgG recycling (~ 55% decrease) in comparison to diploid cells. Amyloid-beta precursor protein (APP) overexpression in diploid fibroblasts replicated the increase in IgG sorting to the degradative pathway observed in cells with trisomy 21. The impact of APP on the expression of FCGRT (alpha chain component of FcRn) was investigated by APP knock down and overexpression of the APP protein. APP knock down increased the expression of FCGRT mRNA by ~ 60% in both diploid and trisomic cells. Overexpression of APP in diploid fibroblasts and HepG2 cells resulted in a decrease in FCGRT and FcRn expression. Our results indicate that the intracellular traffic of IgG is altered in cells with trisomy 21. This study lays the foundation for future investigations into the role of FcRn in the context of DS.

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Ca2+ signals promote GLUT4 exocytosis and reduce its endocytosis in muscle cells

AJP Endocrinology and Metabolism ◽

10.1152/ajpendo.00045.2014 ◽

2014 ◽

Vol 307 (2) ◽

pp. E209-E224 ◽

Cited By ~ 23

Author(s):

Q. Li ◽

X. Zhu ◽

S. Ishikura ◽

D. Zhang ◽

J. Gao ◽

...

Keyword(s):

Cell Surface ◽

Muscle Cells ◽

Live Cells ◽

Intracellular Traffic ◽

Pkc Inhibitor ◽

Compound C ◽

Calmodulin Kinase ◽

Intracellular Ca ◽

Tissue Changes ◽

The Impact

Elevating cytosolic Ca2+ stimulates glucose uptake in skeletal muscle, but how Ca2+ affects intracellular traffic of GLUT4 is unknown. In tissue, changes in Ca2+ leading to contraction preclude analysis of the impact of individual, Ca2+-derived signals. In L6 muscle cells stably expressing GLUT4 myc, the Ca2+ ionophore ionomycin raised cytosolic Ca2+ and caused a gain in cell surface GLUT4 myc. Extra- and intracellular Ca2+ chelators (EGTA, BAPTA-AM) reversed this response. Ionomycin activated calcium calmodulin kinase II (CaMKII), AMPK, and PKCs, but not Akt. Silencing CaMKIIδ or AMPKα1/α2 partly reduced the ionomycin-induced gain in surface GLUT4 myc, as did peptidic or small molecule inhibitors of CaMKII (CN21) and AMPK (Compound C). Compared with the conventional isoenzyme PKC inhibitor Gö6976, the conventional plus novel PKC inhibitor Gö6983 lowered the ionomycin-induced gain in cell surface GLUT4 myc. Ionomycin stimulated GLUT4 myc exocytosis and inhibited its endocytosis in live cells. siRNA-mediated knockdown of CaMKIIδ or AMPKα1/α2 partly reversed ionomycin-induced GLUT4 myc exocytosis but did not prevent its reduced endocytosis. Compared with Gö6976, Gö6983 markedly reversed the slowing of GLUT4 myc endocytosis triggered by ionomycin. In summary, rapid Ca2+ influx into muscle cells accelerates GLUT4 myc exocytosis while slowing GLUT4 myc endocytosis. CaMKIIδ and AMPK stimulate GLUT4 myc exocytosis, whereas novel PKCs reduce endocytosis. These results identify how Ca2+-activated signals selectively regulate GLUT4 exocytosis and endocytosis in muscle cells.

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Human iPSC-derived Down syndrome astrocytes display genome-wide perturbations in gene expression, an altered adhesion profile, and increased cellular dynamics

Human Molecular Genetics ◽

10.1093/hmg/ddaa003 ◽

2020 ◽

Vol 29 (5) ◽

pp. 785-802 ◽

Cited By ~ 5

Author(s):

Blandine Ponroy Bally ◽

W Todd Farmer ◽

Emma V Jones ◽

Selin Jessa ◽

J Benjamin Kacerovsky ◽

...

Keyword(s):

Extracellular Matrix ◽

Down Syndrome ◽

Cell Adhesion ◽

Brain Development ◽

Trisomy 21 ◽

Chromatin Accessibility ◽

Chromosome 21 ◽

Genome Wide ◽

And Function ◽

The Impact

Abstract Down syndrome (DS), caused by the triplication of human chromosome 21, leads to significant alterations in brain development and is a major genetic cause of intellectual disability. While much is known about changes to neurons in DS, the effects of trisomy 21 on non-neuronal cells such as astrocytes are poorly understood. Astrocytes are critical for brain development and function, and their alteration may contribute to DS pathophysiology. To better understand the impact of trisomy 21 on astrocytes, we performed RNA-sequencing on astrocytes from newly produced DS human induced pluripotent stem cells (hiPSCs). While chromosome 21 genes were upregulated in DS astrocytes, we found consistent up- and down-regulation of genes across the genome with a strong dysregulation of neurodevelopmental, cell adhesion and extracellular matrix molecules. ATAC (assay for transposase-accessible chromatin)-seq also revealed a global alteration in chromatin state in DS astrocytes, showing modified chromatin accessibility at promoters of cell adhesion and extracellular matrix genes. Along with these transcriptomic and epigenomic changes, DS astrocytes displayed perturbations in cell size and cell spreading as well as modifications to cell-cell and cell-substrate recognition/adhesion, and increases in cellular motility and dynamics. Thus, triplication of chromosome 21 is associated with genome-wide transcriptional, epigenomic and functional alterations in astrocytes that may contribute to altered brain development and function in DS.

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Restoration of keratinocytic phenotypes in autonomous trisomy-rescued cells

Stem Cell Research & Therapy ◽

10.1186/s13287-021-02448-w ◽

2021 ◽

Vol 12 (1) ◽

Author(s):

Akiko Tanuma-Takahashi ◽

Momoko Inoue ◽

Kazuhiro Kajiwara ◽

Ryo Takagi ◽

Ayumi Yamaguchi ◽

...

Keyword(s):

Stem Cells ◽

Down Syndrome ◽

Trisomy 21 ◽

Kinase Inhibitor ◽

Rho Kinase ◽

Feeder Cells ◽

Metabolic Systems ◽

Rho Kinase Inhibitor ◽

The Impact ◽

3D Skin

Abstract Background An extra copy of chromosome 21 in humans can alter cellular phenotypes as well as immune and metabolic systems. Down syndrome is associated with many health-related problems and age-related disorders including dermatological abnormalities. However, few studies have focused on the impact of trisomy 21 (T21) on epidermal stem cells and progenitor cell dysfunction. Here, we investigated the differences in keratinocytic characteristics between Down syndrome and euploid cells by differentiating cells from trisomy 21-induced pluripotent stem cells (T21-iPSCs) and autonomous rescued disomy 21-iPSCs (D21-iPSCs). Methods Our protocol for keratinocytic differentiation of T21-iPSCs and D21-iPSCs was employed. For propagation of T21- and D21-iPSC-derived keratinocytes and cell sheet formation, the culture medium supplemented with Rho kinase inhibitor on mouse feeder cells was introduced as growth rate decreased. Before passaging, selection of a keratinocytic population with differential dispase reactivity was performed. Three-dimensional (3D) air-liquid interface was performed in order to evaluate the ability of iPSC-derived keratinocytes to differentiate and form stratified squamous epithelium. Results Trisomy-rescued disomy 21-iPSCs were capable of epidermal differentiation and expressed keratinocytic markers such as KRT14 and TP63 upon differentiation compared to trisomy 21-iPSCs. The lifespan of iPSC-derived keratinocytes could successfully be extended on mouse feeder cells in media containing Rho kinase inhibitor, to more than 34 population doublings over a period of 160 days. Dispase-based purification of disomy iPSC-derived keratinocytes contributed epidermal sheet formation. The trisomy-rescued disomy 21-iPSC-derived keratinocytes with an expanded lifespan generated 3D skin in combination with a dermal fibroblast component. Conclusions Keratinocytes derived from autonomous trisomy-rescued iPSC have the ability of stratification for manufacturing 3D skin with restoration of keratinocytic functions.

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Myeloproliferative Disease in a Mouse Model of Down Syndrome.

Blood ◽

10.1182/blood.v106.11.4946.4946 ◽

2005 ◽

Vol 106 (11) ◽

pp. 4946-4946

Author(s):

Gina Mundschau ◽

Sarah Jilani ◽

Hui Liu ◽

Elizabeth Davis ◽

Michelle Le Beau ◽

...

Keyword(s):

Stem Cells ◽

Bone Marrow ◽

Down Syndrome ◽

Mouse Model ◽

Trisomy 21 ◽

Extramedullary Hematopoiesis ◽

Similar Process ◽

Stem Cell Population ◽

Myeloproliferative Disease ◽

The Impact

Abstract Trisomy 21 causes human Down syndrome (DS), a heterogeneous group of phenotypes including marked predisposition to leukemia. Children with Down syndrome are 500 times more likely to develop acute megakaryoblastic leukemia (AMKL) than other children. Furthermore, non-DS children with AMKL often have acquired trisomy 21 in their leukemic clones, suggesting that trisomy 21 in hematopoietic cells contributes to leukemic transformation. To better understand the impact of trisomy 21 on blood cell homeostasis and leukemia, we studied hematopoiesis in the Ts65Dn mouse model of Down syndrome. Ts65Dn mice harbor a segmental trisomy for mouse chromosome 16, homologous to human chromosome 21, and display many of the phenotypes associated with human DS, including craniofacial anomalies and learning deficits. To define the hematopoietic parameters for this strain, we performed monthly complete blood counts for a cohort of trisomic mice and their disomic littermates and discovered the development of progressive thrombocytosis and mild anemia in trisomic animals. Increased numbers of CD41+ megakaryocytes with lower modal ploidy were detected in the bone marrow and spleen of Ts65Dn mice as early as three months of age. Over time, expansion of the megakaryocyte population was accompanied by a decrease in TER119+ cells in the bone marrow, myelofibrosis, splenomegaly, and extramedullary hematopoiesis. Colony forming assays demonstrated increased colony forming ability in the spleens of trisomic mice along with variable decreased hematopoiesis in the bone marrow. Further, characterization of stem cells in the bone marrow indicated a hyperproliferative stem cell population. Importantly, the mice did not develop malignant leukemia by the age of 18 months and no mutations were found in the blood transcription factor GATA1, which is commonly affected in human AMKL. While Ts65Dn mice do not develop the AMKL seen in humans with DS, our results indicate that, trisomy 16 can cause hyperproliferation of the myeloid lineages, extramedullary hematopoiesis, and bone marrow fibrosis in mice. Additionally, these results suggest that trisomy 21 in humans may initiate a similar process in hematopoietic stem cells, which may contribute to leukemogenesis. Unexpectedly, this phenotype also bears significant resemblance to the human myeloproliferative disease chronic idiopathic myelofibrosis (CIMF). These findings may provide insight into the origins and progression of human myeloid diseases, including AMKL and CIMF.

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Characterization Of Oncogenes On Chromosome 21 Identified By shRNA-Based Viability Screening

Blood ◽

10.1182/blood.v122.21.1201.1201 ◽

2013 ◽

Vol 122 (21) ◽

pp. 1201-1201

Author(s):

Lena Stachorski ◽

Veera Raghavan Thangapandi ◽

Dirk Reinhardt ◽

Jan-Henning Klusmann

Keyword(s):

Transcription Factor ◽

Cell Viability ◽

Progenitor Cells ◽

Cell Lines ◽

Trisomy 21 ◽

Fold Increase ◽

Hematopoietic Stem ◽

Megakaryocytic Differentiation ◽

Knock Down ◽

The Impact

Abstract Children with trisomy 21 (Down syndrome, DS) are predisposed to develop acute megakaryoblastic leukemia (DS-AMKL) as well as the antecedent transient leukemia (DS-TL). Mutations in the hematopoietic transcription factor GATA1 have been found in nearly all children with DS-AMKL and DS-TL, but not in other malignancies. Recent whole genome sequencing efforts strongly supported the hypothesis that the triad of fetal origin, trisomy 21 and GATA1s-mutation are sufficient to cause DS-TL. Thus, the presence of an extra copy of hsa21 perturbs fetal hematopoiesis to provide a GATA1s sensitive background during leukemogenesis. To decipher the deregulated oncogenic gene network on hsa21, we conducted a shRNA-based viability screening. GATA1s-mutated DS-AMKL cell line CMK as well as non-DS-AML cell lines (K562, M-07) as control were lentivirally transduced and the effect of the knock-down was evaluated by cell viability and proliferation assays. Upon knock-down we found 42 genes conferring a profound selective growth disadvantage in DS-AMKL cell lines. Interestingly, 31 out of those candidate genes are located in one particular chromosomal region (21q22.1-21q22.3) and in addition 11 (out of 14 tested) are overexpressed in DS-AMKL compared to non-DS-AMKL. In a secondary functional validation screening the effects of the knock-down on the cell lines were analyzed by competition assays, apoptosis assays and cell viability assays as well as colony forming assays. Furthermore, differentiation and morphology were characterized using immunophenotyping and cytospins, respectively. We could demonstrate that the potential oncogenes participate in different cellular processes affecting proliferation, cell viability, apoptosis or differentiation. To further delineate the impact of 11 selected candidates on normal hematopoiesis, we characterized their effects in gain- and loss-of-function studies (confirmed by qRT-PCR) using CD34+ hematopoietic stem and progenitor cells (HSPCs). Four of those genes (USP25 [ubiquitin specific peptidase 25], BACH1 [BTB and CNC homology 1, basic leucine zipper transcription factor 1], U2AF1 [U2 small nuclear RNA auxiliary factor 1] and C21orf33) inhibited megakaryocytic and erythroid in vitro differentiation upon knockdown. The fraction of cells expressing early and late megakaryocytic markers CD41 and CD42b or early erythroid marker CD36 was reduced by 2-20-fold (P<0.001). Inversely, the knock-down of those four genes and two other genes (ATP5O [ATP synthase, H+ transporting, mitochondrial F1 complex, O subunit] and C21orf45) enhanced the myeloid differentiation propensity of CD34+-HSPCs (2.1-13.4-fold increase of CD14+-monocytic cells, P<0.001). The opposite effect was observed in gain-of-function studies. Ectopic expression of six genes (hU2AF1, mC21orf33, hIFNGR2 [interferon gamma receptor 2], hWDR4 [WD repeat domain 4] or mGABPA [GA binding protein transcription factor, alpha subunit 60kDa]) resulted in a radical switch in lineage commitment with a drastic change from erythroid to megakaryocytic differentiation (CD41+ 1.7-2.4-fold increase, P<0.001, CD235a+ [late erythroid marker] 3-300-fold reduction, P<0.001). Thus, we found a remarkable number of genes regulating erythroid and megakaryocytic differentiation as well as proliferation in normal hematopoiesis. Given the genetic background during trisomy 21-mediated leukemogenesis, we propose a complex interactive network located in one particular region on hsa21. Deregulation of this network might result in synergistic effects on hematopoietic differentiation, which promotes transformation of GATA1s-mutated fetal hematopoietic progenitor cells. Disclosures: No relevant conflicts of interest to declare.

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‘The Stuff of Slow Constitution’: Reading Down Syndrome for Race, Disability, and the Timing that Makes Them So

Somatechnics ◽

10.3366/soma.2016.0193 ◽

2016 ◽

Vol 6 (2) ◽

pp. 235-248 ◽

Cited By ~ 2

Author(s):

Mel Y. Chen

Keyword(s):

Down Syndrome ◽

Trisomy 21 ◽

Historical Perspective ◽

Early History ◽

Present Moment ◽

History Of ◽

Living Being

In this paper I would like to bring into historical perspective the interrelation of several notions such as race and disability, which at the present moment seem to risk, especially in the fixing language of diversity, being institutionalised as orthogonal in nature to one another rather than co-constitutive. I bring these notions into historical clarity primarily through the early history of what is today known as Down Syndrome or Trisomy 21, but in 1866 was given the name ‘mongoloid idiocy’ by English physician John Langdon Down. In order to examine the complexity of these notions, I explore the idea of ‘slow’ populations in development, the idea of a material(ist) constitution of a living being, the ‘fit’ or aptness of environmental biochemistries broadly construed, and, finally, the germinal interarticulation of race and disability – an ensemble that continues to commutatively enflesh each of these notions in their turn.

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Characterization of Caenorhabditis elegans Homologs of the Down Syndrome Candidate Gene DYRK1A

Genetics ◽

10.1093/genetics/163.2.571 ◽

2003 ◽

Vol 163 (2) ◽

pp. 571-580 ◽

Cited By ~ 1

Author(s):

William B Raich ◽

Celine Moorman ◽

Clay O Lacefield ◽

Jonah Lehrer ◽

Dusan Bartsch ◽

...

Keyword(s):

Down Syndrome ◽

Caenorhabditis Elegans ◽

Trisomy 21 ◽

Gene Dosage ◽

Inducible Promoters ◽

C Elegans ◽

Dual Specificity ◽

Specificity Protein

Abstract The pathology of trisomy 21/Down syndrome includes cognitive and memory deficits. Increased expression of the dual-specificity protein kinase DYRK1A kinase (DYRK1A) appears to play a significant role in the neuropathology of Down syndrome. To shed light on the cellular role of DYRK1A and related genes we identified three DYRK/minibrain-like genes in the genome sequence of Caenorhabditis elegans, termed mbk-1, mbk-2, and hpk-1. We found these genes to be widely expressed and to localize to distinct subcellular compartments. We isolated deletion alleles in all three genes and show that loss of mbk-1, the gene most closely related to DYRK1A, causes no obvious defects, while another gene, mbk-2, is essential for viability. The overexpression of DYRK1A in Down syndrome led us to examine the effects of overexpression of its C. elegans ortholog mbk-1. We found that animals containing additional copies of the mbk-1 gene display behavioral defects in chemotaxis toward volatile chemoattractants and that the extent of these defects correlates with mbk-1 gene dosage. Using tissue-specific and inducible promoters, we show that additional copies of mbk-1 can impair olfaction cell-autonomously in mature, fully differentiated neurons and that this impairment is reversible. Our results suggest that increased gene dosage of human DYRK1A in trisomy 21 may disrupt the function of fully differentiated neurons and that this disruption is reversible.

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