The Experts below are selected from a list of 237 Experts worldwide ranked by ideXlab platform
Katja G Weinacht - One of the best experts on this subject based on the ideXlab platform.
-
Adenylate Kinase 2 Links Energy Metabolism and Cell Fate in Hematopoietic Stem and Progenitor Cells
Blood, 2019Co-Authors: Wenqing Wang, Avni Awani, Daniel P. Dever, Matthew H. Porteus, Daniel Thomas, Andrew W Devilbiss, Thomas P. Mathews, Sean J. Morrison, Katja G WeinachtAbstract:While hematopoietic stem and progenitor cells (HSPCs) were thought to rely mainly on glycolysis for energy supply, emerging evidence suggests that defects in mitochondrial functions can impact HSPC development with respect to self-renewal, differentiation and aging. The exact mechanisms underlying metabolic reprogramming and cell fate decisions during human hematopoiesis, however, remain elusive. Biallelic mutations in the mitochondrial enzyme adenylate kinase 2 (AK2), cause Reticular Dysgenesis (RD), one of the most profound forms of severe combined immunodeficiency (SCID). AK2 catalyzes the interconversion between adenine nucleotides and thereby controls the availability of ADP for oxidative phosphorylation. Clinically, RD patients not only present with profound lymphopenia, typical for classic SCID, but also suffer from severe congenital neutropenia. The developmental arrest across the T, NK and granulocytic lineages suggests that AK2 deficiency causes a metabolic defect with global impact on hematopoiesis. Our prior work in induced pluripotent stem cells (iPSCs) from RD patients has shown that maturation-arrested iPSC-derived HSPCs exhibit increased oxidative stress and an energy-depleted adenine nucleotide profile, suggesting that AK2-regulated mitochondrial bioenergetics play an integral role in HSPC differentiation. Therefore, RD serves as an excellent model to study the impact of mitochondrial metabolism during human HSPC development. Methods: Since iPSCs do not recapitulate definitive hematopoiesis, we developed an AK2 biallelic knock-out model in primary human HSPCs using CRISPR/Cas9 gene editing. Employing a homologous recombination-mediated dual color reporter strategy, we were able to select for HSPCs with biallelic AK2 knock-out. HSPCs edited at the safe harbor AAVS1 site were used as a control. FACS purified AK2-/- and AAVS1-/- HSPCs were in vitro differentiated along the granulocytic lineage, and cells at various differentiation stages were sorted for RNA-seq and metabolomics analysis. Results: We analyzed the myeloid differentiation potential of AK2-/- HSPCs in vitro. Compared to AAVS1-/- controls, AK2-/- HSPCs displayed a severely decreased colony forming potential of both myeloid and erythroid lineages. In addition, AK2-/- HSPCs showed a granulocytic maturation arrest at the HLA-DR-, CD117+ promyelocyte stage, consistent with the characteristic phenotype observed in RD patients. We then performed RNA-seq studies on in vitro differentiated promyelocyte and neutrophil subpopulations derived from AK2-/- and control HSPCs. The RNA-seq analysis showed differential gene expression in glutathione metabolism and IL-10 signaling pathways, suggesting an increase in oxidative stress and inflammation, respectively, caused by AK2 deficiency. In addition, genes implicated in antimicrobial function and granule synthesis were downregulated in AK2-/- neutrophils, suggesting a functional defect. Liquid chromatography-mass spectrometry (LC-MS/MS) studies to delineate differences in metabolite profile conferred by AK2 deficiency at different stages of HSPC development are currently in progress. Conclusions: We have established the first cell-traceable biallelic AK2 CRISPR knock-out model in primary human HSPCs that recapitulates the myeloid phenotype of RD patients. This model allows us to profile AK2 knock-out cells at different developmental stages. AK2-/- granulocyte precursors showed a transcriptional signature suggestive of worsening oxidative stress, inflammation and defective effector cell functions during maturation. To understand the mechanistic underpinnings for these observations we are now using a global metabolomics approach to profile the changes in energy metabolites that occur during development in AK2-deficient and control HSPC subpopulations. Understanding how metabolism governs differentiation and self-renewal of human HSPCs has important translational implications to improve hematopoietic stem cell products and transplantation outcomes. Disclosures Morrison: Frequency Therapeutics: Consultancy, Membership on an entity's Board of Directors or advisory committees; OncoMed Pharmaceuticals: Equity Ownership; GI Therapeutics: Equity Ownership, Membership on an entity's Board of Directors or advisory committees; Kolon Gene Therapeutics: Consultancy; Protein Fluidics: Other: Stock Options.
-
An Engineered Cell-Traceable Model of Reticular Dysgenesis in Human Hematopoietic Stem Cells Linking Metabolism and Differentiation
Blood, 2018Co-Authors: Wenqing Wang, Avni Awani, Daniel P. Dever, Matthew H. Porteus, Lauren Reich, Yusuke Nakauchi, Daniel Thomas, Katja G WeinachtAbstract:Abstract Hematopoietic stem cell (HSC) differentiation is accompanied by a metabolic shift from glycolysis to oxidative phosphorylation (OXPHOS) to meet the increasing energy demand during proliferation and differentiation. However, the role of mitochondrial metabolism in HSC differentiation goes beyond ATP production. Metabolites generated during mitochondrial metabolism may impact in HSC fate decisions through stable epigenetic modifications. Despite some progress in understanding mitochondrial communication during HSC development, their role in human hematopoiesis remains largely elusive, where the lack of appropriate model systems poses a major obstacle. Reticular Dysgenesis (RD), a rare and particularly severe form of severe combined immunodeficiency (SCID), offers an attractive model for studying the role of mitochondrial metabolism in hematopoiesis. RD is an autosomal recessive disease caused by biallelic mutations of the mitochondrial enzyme Adenylate Kinase 2 (AK2). AK2 catalyzes the reversible phosphorylation of adenosine monophosphate (AMP) to adenosine diphosphate (ADP), which serves as the substrate for the ATP synthase. In addition to defective lymphocyte development typical of classic SCID, RD patients also suffer from impaired myeloid development, suggestive of a global defect in hematopoiesis. In a human induced pluripotent stem cell (iPSC) model for RD, hematopoietic stem and progenitor cells (HSPCs) recapitulate a profound maturation arrest of the myeloid lineage, increased oxidative stress and an energy-depleted metabolite and transcriptional profile. We hypothesize that AK2 defects drive hematopoietic cell fate decisions through changes in metabolites that regulate the activities of DNA/histone modifying enzymes and result in stable epigenetic modifications. Methods: Since iPSCs are not suitable to model the epigenetic characteristics of definitive hematopoiesis, we developed a novel model system in which we deleted AK2 in primary human HSCs using CRISPR/Cas9 gene editing technique. We found a highly effective single-guide RNA (sgRNA) targeting the catalytic LID domain of the AK2 gene to introduce directed DNA double stranded breaks (DSBs), and use a homologous recombination (HR)-mediated dual reporter system to track and isolate cells with biallelic AK2 disruption. Results: Our single-color GFP reporter system consistently produces a >60% GFP+ population of AK2-targeted CD34+ umbilical cord blood (UCB) cells. With dual GFP/BFP reporters, we were able to achieve 6% GFP/BFP double positive cells with confirmed biallelic AK2 knock-out. Since HR events on one allele are biologically linked to CRISPR/Cas9 mediated DSBs on the other, we assessed insertion and/or deletion (INDEL) frequency and protein expression in a single reporter (GFP+) population of AK2-targeted UCBs. We detected an INDEL frequency of over 90% on the non-HR alleles along with nearly absent AK2 protein expression by Western Blot. These results indicated that the highly efficient single-color reporter system with >60% targeting efficiency is sufficient to achieve an AK2 biallelic knock-out population in primary HSCs. in vitro myeloid differentiation of these AK2-targeted HSCs recapitulates the RD phenotype with impaired neutrophil but preserved monocyte development. Conclusion: This novel disease model for RD will now allow us to examine the cellular and molecular impact of perturbations in metabolism on human HSC development. We will investigate the effect on differentiation potential, metabolite profile, transcriptome and epigenome in vitro as well as in a xenograft mouse model. Elucidating how metabolism governs differentiation and self-renewal of human HSCs will not only advance our basic understanding of many blood and immune diseases, but has important translational implications for improving the use of HSCs in hematopoietic stem cell transplantation, gene and cell therapy. Disclosures Porteus: CRISPR Therapeutics: Consultancy, Membership on an entity's Board of Directors or advisory committees.
-
Reticular Dysgenesis associated ak2 protects hematopoietic stem and progenitor cell development from oxidative stress
Journal of Experimental Medicine, 2015Co-Authors: Alberto Rissone, Elisa Giocaliere, Jayashree Jagadeesh, Kerstin Felgentreff, Katja G Weinacht, Kerry Dobbs, Kevin Bishop, Giancarlo La Marca, Waleed AlherzAbstract:Adenylate kinases (AKs) are phosphotransferases that regulate the cellular adenine nucleotide composition and play a critical role in the energy homeostasis of all tissues. The AK2 isoenzyme is expressed in the mitochondrial intermembrane space and is mutated in Reticular Dysgenesis (RD), a rare form of severe combined immunodeficiency (SCID) in humans. RD is characterized by a maturation arrest in the myeloid and lymphoid lineages, leading to early onset, recurrent, and overwhelming infections. To gain insight into the pathophysiology of RD, we studied the effects of AK2 deficiency using the zebrafish model and induced pluripotent stem cells (iPSCs) derived from fibroblasts of an RD patient. In zebrafish, Ak2 deficiency affected hematopoietic stem and progenitor cell (HSPC) development with increased oxidative stress and apoptosis. AK2-deficient iPSCs recapitulated the characteristic myeloid maturation arrest at the promyelocyte stage and demonstrated an increased AMP/ADP ratio, indicative of an energy-depleted adenine nucleotide profile. Antioxidant treatment rescued the hematopoietic phenotypes in vivo in ak2 mutant zebrafish and restored differentiation of AK2-deficient iPSCs into mature granulocytes. Our results link hematopoietic cell fate in AK2 deficiency to cellular energy depletion and increased oxidative stress. This points to the potential use of antioxidants as a supportive therapeutic modality for patients with RD.
-
Reticular Dysgenesis–associated AK2 protects hematopoietic stem and progenitor cell development from oxidative stress
Journal of Experimental Medicine, 2015Co-Authors: Alberto Rissone, Waleed Al-herz, Elisa Giocaliere, Jayashree Jagadeesh, Kerstin Felgentreff, Katja G Weinacht, Kerry Dobbs, Kevin Bishop, Giancarlo La Marca, Marypat JonesAbstract:Adenylate kinases (AKs) are phosphotransferases that regulate the cellular adenine nucleotide composition and play a critical role in the energy homeostasis of all tissues. The AK2 isoenzyme is expressed in the mitochondrial intermembrane space and is mutated in Reticular Dysgenesis (RD), a rare form of severe combined immunodeficiency (SCID) in humans. RD is characterized by a maturation arrest in the myeloid and lymphoid lineages, leading to early onset, recurrent, and overwhelming infections. To gain insight into the pathophysiology of RD, we studied the effects of AK2 deficiency using the zebrafish model and induced pluripotent stem cells (iPSCs) derived from fibroblasts of an RD patient. In zebrafish, Ak2 deficiency affected hematopoietic stem and progenitor cell (HSPC) development with increased oxidative stress and apoptosis. AK2-deficient iPSCs recapitulated the characteristic myeloid maturation arrest at the promyelocyte stage and demonstrated an increased AMP/ADP ratio, indicative of an energy-depleted adenine nucleotide profile. Antioxidant treatment rescued the hematopoietic phenotypes in vivo in ak2 mutant zebrafish and restored differentiation of AK2-deficient iPSCs into mature granulocytes. Our results link hematopoietic cell fate in AK2 deficiency to cellular energy depletion and increased oxidative stress. This points to the potential use of antioxidants as a supportive therapeutic modality for patients with RD.
-
linking oxidative stress to cell fate ipsc based disease modeling identifies new therapeutic target in Reticular Dysgenesis
Blood, 2014Co-Authors: Katja G WeinachtAbstract:Reticular Dysgenesis (RD) is one of the most serious forms of severe combined immune deficiency (SCID). It is characterized by complete absence of circulating lymphocytes and neutrophils. In addition, patients suffer from sensorineural hearing loss. Before newborn screening for SCID was implemented, the majority of patients succumbed to infection long before hematopoietic cell transplantation (HCT) could be attempted. To this date, the prognosis for RD remains grim. RD is caused by mutations in the mitochondrial ADP-generator Adenylate Kinase 2 (AK2). AK1 is a cytosolic protein that may compensate in various tissues for the lack of AK2. However, AK1 is not expressed in leukocytes and the stria vascularis of the inner ear [1]. While this observation may explain where AK2 defects manifest, the molecular mechanisms how AK2 defects take effect, remain largely obscure. Significant obstacles to elucidating disease pathology have been the lack of a suitable animal models and the unavailability of patient specimens. Using skin fibroblasts from an RD-patient we have recently identified at Boston Children’s Hospital [2], we have generated induced pluripotent stem cells (iPSC) with homozygous loss of function mutation in AK2. In-vitro myeloid differentiation of AK2-mutated iPSCs recapitulates the characteristic maturation arrest at the promyelocyte stage observed in-vivo in patients with this condition. AK2 is expressed in the intermitochondrial space and serves as primary mitochondrial ADP generator by promoting the reversible reaction AMP + ATP = 2 ADP. Maintenance of adequate levels of ADP is critical to support ATP synthase activity. Using Mass Spectrometry, we have shown that decreased AK2 activity leads to an increase in the AMP/ADP ratio in iPS-derived myeloid cells, indicating that AK2 is indispensable in maintaining ADP supply in the myeloid lineage. We have also performed transcriptome analysis of AK2- mutated myeloid cells compared to control and found a significant down regulation of ATP-dependant transporters. Based on this data, we hypothesized that in patients with RD, ADP-depletion in myeloid progenitors leads to stage 4 respiration, a well defined state in mitochondrial biology, in which the ATP-synthase lacks substrate and decreases its activity. This causes a reduction in proton flux from the intermitochondrial space back into the matrix, transient rise in membrane potential, and an escalation in the formation of reactive oxygen species (ROS). The cell responds by activating “inducible uncoupling”, the opening of alternative proton pores, which allows proton flux back into the matrix, bypassing the ATP-synthase and foregoing the use of energy stored in the proton gradient. While this represents a cellular rescue mechanism in response to acute oxidative stress, extended oxidative-stress-induced uncoupling eventually leads to a decline in proton gradient and membrane potential and ultimately in demise of the cell. To test this hypothesis, we have added Glutathione, the primary endogenous cellular antioxidant, to the culture conditions. We also tested G-CSF and all-trans-retinoic acid (ATRA), agents known to promote promyelocyte differentiation to mature neutrophils in other conditions. While G-CSF had no, and ATRA clearly deleterious effects on myeloid maturation in AK2-mutated cells, Glutathione led to a significant improvement in differentiation, allowing development of mature neutrophils in-vitro . Our results suggest that cell fate in RD is linked to oxidative stress and identify antioxidants as a possible therapeutic approach that may help reduce early mortality due to severe infections in patients with RD. Disclosures No relevant conflicts of interest to declare.
Donna A. Wall - One of the best experts on this subject based on the ideXlab platform.
-
Umbilical Cord Blood Stem Cell Transplantation in Severe T-Cell Immunodeficiency Disorders
Pediatric Asthma Allergy & Immunology, 2005Co-Authors: Alan P. Knutsen, Michael E. Kelly, Donna A. WallAbstract:Hematopoietic stem cell transplantation is the treatment of choice for severe primary T-cell immunodeficiencies. When an HLA-identical sibling as the donor is not available, an alternative donor stem cell source is needed. In primary T-cell immunodeficiencies, T-cell–depleted HLA-haploidentical bone marrow transplantation has been particularly successful in reconstituting the immune system in many but not all of the severe T-cell immune deficiency disorders. This study reports the use of umbilical cord blood (UCB) stem cell transplantation in 16 patients with severe T cell immune deficiency disorders. UCB was evaluated as a stem cell source for immune reconstitution in children with severe primary T-cell immunodeficiency disorders, such as severe combined immunodeficiency syndrome (SCID), Reticular Dysgenesis, thymic dysplasia, combined immunodeficiency disease (CID), and Wiskott-Aldrich syndrome (WAS) when an HLA-matched sibling donor was unavailable. From 1996 through 2004, 16 children diagnosed with se...
-
Umbilical Cord Blood Transplantation in Severe T-Cell Immunodeficiency Disorders: Two-Year Experience
Journal of Clinical Immunology, 2000Co-Authors: Alan P. Knutsen, Donna A. WallAbstract:Hematopoietic stem cell transplantation is the treatment of choice for severe primary T-cell immunodeficiencies. When an HLA-identical sibling as the donor is not available, an alternative donor stem cell source is needed. In primary T-cell immunodeficiencies, T-cell-depleted HLA-haploidentical bone marrow transplantation has been particularly successful in reconstituting the immune system in many but not all of the severe T-cell immune deficiency disorders. This study reports the use of umbilical cord blood (UCB) stem cell transplantation in severe T-cell immune deficiency. Umbilical cord blood was evaluated as a stem cell source for immune reconstitution in children with severe primary T-cell immunodeficiency disorders, such as severe combined immunodeficiency syndrome (SCID), Reticular Dysgenesis, thymic dysplasia, combined immunodeficiency disease (CID), and Wiskott–Aldrich syndrome (WAS) when a matched sibling donor was unavailable. From 1/96 through 5/98, eight children received unrelated cord blood stem cell transplantation following a preparative regimen for the treatment of combined immunodeficiency diseases. The patients ranged in age from 2 weeks to 8 years. The cord blood units were 3/6 HLA antigen matches in two children, 4/6 in four children, and 5/6 in two child, with molecular HLA-DR mismatch in three of the children. The average time for neutrophil engraftment (absolute neutrophil count >500/mm^3) was 12 days (range 10–15 days) and the average time for platelet engraftment (platelet count >20,000/mm^3) was 36 days (range 24–50 days). A patient with Reticular Dysgenesis failed to engraft following her first transplant, but fully engrafted after a second unrelated donor cord blood transplantation. Five of six patients exhibited grade I graft-versus-host disease (GvHD), while one child had grade IV skin and gut GvHD. Immunologic reconstitution demonstrated that cord blood stem cell transplantation resulted in consistent and stable T-, B- and natural killer (NK) cell development. The kinetics of development were such that T-cell development occurred between 60 to 100 days. Initial T-cell engraftment consisted predominantly of CD45RO+, CD3+, and CD4+ T cells, and at 12 to 24 months changed to CD45RA+, CD3+, and CD4+ T cells, indicating de novo maturation of T cells. NK cell development occurred at approximately 180 days. B cells engrafted early, and study of functional B-cell antibody responses revealed that five of six patients in whom intravenous immune globulin has been discontinued have low detectable antibody responses to tetanus and diphtheria toxoid immunizations at 18 to 24 months posttransplantation. Unrelated umbilical donor cord blood is an alternative source of stem cells for transplantation in children with severe T-cell immune deficiency disorders when a suitable HLA-matched donor is not available and when a T-depleted haploidentical preparation is not beneficial. Benefits of UCB include rapid and reliable recovery of immune function, low risk of GvHD, and low viral transmission rate.
-
Association of Reticular Dysgenesis (thymic alymphoplasia and congenital aleukocytosis) with bilateral sensorineural deafness.
The Journal of pediatrics, 1999Co-Authors: Trudy N. Small, Donna A. Wall, Joanne Kurtzberg, Morton J. Cowan, Richard J. O'reilly, Wilhelm FriedrichAbstract:Reticular Dysgenesis is a rare congenital disorder characterized by severe combined immunodeficiency and profound neutropenia, curable to date, only by bone marrow transplantation. This report describes the association of bilateral sensorineural deafness with this disease.
-
Kinetics of T-cell development of umbilical cord blood transplantation in severe T-cell immunodeficiency disorders ☆ ☆☆
Journal of Allergy and Clinical Immunology, 1999Co-Authors: Alan P. Knutsen, Donna A. WallAbstract:Abstract Background: Hematopoietic stem-cell transplantation is the treatment of choice for severe primary T-cell immunodeficiencies. When an HLA-identical sibling donor is not available, an alternative donor stem-cell source is needed. In primary T-cell immunodeficiencies, T-cell–depleted HLA-haploidentical bone marrow transplantation has been particularly successful in reconstituting the T-cell immune system in many of the severe combined immunodeficiency syndrome types. However, there are some problems associated with this preparation as a stem donor source, such as increased resistance to engraftment, a long period of time for T-cell engraftment to occur, and failure to engraft B cells and B-cell functions. These problems can be especially troublesome if the patient is infected before the transplantation. Objective: Umbilical cord blood was evaluated as a stem-cell source for immune reconstitution in children with severe primary T-cell immunodeficiency disorders, such as severe combined immunodeficiency syndrome, Reticular Dysgenesis, thymic dysplasia, and combined immunodeficiency disease, when a matched sibling donor was unavailable. Methods: From January 1996 through July 1997, 6 children received unrelated cord blood stem-cell transplantation after a preparative regimen for the treatment of combined immunodeficiency diseases. The patients ranged in age from 2 weeks to 6 years. The cord blood units were 3 of 6 HLA antigen matches in 2 children, 4 of 6 HLA antigen matches in 3 children, and 5 of 6 HLA antigen matches in 1 child, with molecular HLA-DR mismatch in 3 of the children. Results: The average time for neutrophil engraftment (absolute neutrophil count, >500/mm 3 ) was 12 days (range, 10 to 15 days), and the average time for platelet engraftment (platelet count, >20,000/mm 3 ) was 36 days (range, 24 to 50 days). In a patient with Reticular Dysgenesis, the first transplant failed to engraft but fully engrafted after a second unrelated donor cord blood transplantation. Five of 6 patients exhibited grade I graft-versus-host disease (GvHD), although 1 child experienced grade IV skin and gut GvHD. Immunologic reconstitution demonstrated that cord blood stem-cell transplantation resulted in consistent and stable T-cell, B-cell, and natural killer–cell development. The kinetics of recovery of phenotypic expression and function of T cells occurred between 60 to 100 days and that of natural killer cells at approximately 180 days. B cells engrafted early, and a study of functional B-cell antibody responses revealed that 2 of 2 patients in whom intravenous immune globulin was discontinued have low detectable antibody responses to tetanus and diphtheria toxoid immunizations more than 1 year after the transplantation. Conclusions: Unrelated umbilical donor cord blood is an excellent source of stem cells for transplantation of children with immune deficiency disorders. Benefits include rapid and reliable recovery of immune function, low risk of GvHD, and low viral transmission rate. (J Allergy Clin Immunol 1999;103:823-32.)
Alberto Rissone - One of the best experts on this subject based on the ideXlab platform.
-
a model for Reticular Dysgenesis shows impaired sensory organ development and hair cell regeneration linked to cellular stress
Disease Models & Mechanisms, 2019Co-Authors: Alberto Rissone, Raman Sood, Erin Jimenez, Blake Carrington, Claire Slevin, Stephen Wincovitch, Fabio Candotti, Kevin Bishop, Shawn M BurgessAbstract:ABSTRACT Mutations in the gene AK2 are responsible for Reticular Dysgenesis (RD), a rare and severe form of primary immunodeficiency in children. RD patients have a severely shortened life expectancy and without treatment die, generally from sepsis soon after birth. The only available therapeutic option for RD is hematopoietic stem cell transplantation (HSCT). To gain insight into the pathophysiology of RD, we previously created zebrafish models for Ak2 deficiencies. One of the clinical features of RD is hearing loss, but its pathophysiology and causes have not been determined. In adult mammals, sensory hair cells of the inner ear do not regenerate; however, their regeneration has been observed in several non-mammalian vertebrates, including zebrafish. Therefore, we used our RD zebrafish models to determine whether Ak2 deficiency affects sensory organ development and/or hair cell regeneration. Our studies indicated that Ak2 is required for the correct development, survival and regeneration of sensory hair cells. Interestingly, Ak2 deficiency induces the expression of several oxidative stress markers and it triggers an increased level of cell death in the hair cells. Finally, we show that glutathione treatment can partially rescue hair cell development in the sensory organs in our RD models, pointing to the potential use of antioxidants as a therapeutic treatment supplementing HSCT to prevent or ameliorate sensorineural hearing deficits in RD patients.
-
a model for Reticular Dysgenesis shows impaired sensory organ development and hair cell regeneration linked to cellular stress
bioRxiv, 2019Co-Authors: Alberto Rissone, Raman Sood, Erin Jimenez, Blake Carrington, Claire Slevin, Stephen Wincovitch, Fabio Candotti, Kevin Bishop, Shawn M BurgessAbstract:Mutations in the gene AK2 are responsible for Reticular Dysgenesis (RD), a rare and severe form of primary immunodeficiency in children. RD patients have a severely shortened life expectancy and without treatment die a few weeks after birth. The only available therapeutic option for RD is bone marrow transplantation. To gain insight into the pathophysiology of RD, we previously created zebrafish models for an AK2 deficiency. One of the clinical features of RD is hearing loss, but its pathology and causes have not been determined. In adult mammals, sensory hair cells of the inner ear do not regenerate; however, their regeneration has been observed in several non-mammalian vertebrates, including zebrafish. Therefore, we use our RD zebrafish models to determine if AK2 deficiency affects sensory organ development and/or hair cell regeneration. Our studies indicated that AK2 is required for the correct development, survival and regeneration of sensory hair cells. Interestingly, AK2 deficiency induces the expression of several oxidative stress markers and it triggers an increased level of cell death in the hair cells. Finally, we show that glutathione treatment can partially rescue hair cell development in the sensory organs in our RD models, pointing to the potential use of antioxidants as a supportive therapeutic modality for RD patients, not only to increase their chances of survival, but to prevent or ameliorate their sensorineural hearing deficits.
-
Reticular Dysgenesis associated ak2 protects hematopoietic stem and progenitor cell development from oxidative stress
Journal of Experimental Medicine, 2015Co-Authors: Alberto Rissone, Elisa Giocaliere, Jayashree Jagadeesh, Kerstin Felgentreff, Katja G Weinacht, Kerry Dobbs, Kevin Bishop, Giancarlo La Marca, Waleed AlherzAbstract:Adenylate kinases (AKs) are phosphotransferases that regulate the cellular adenine nucleotide composition and play a critical role in the energy homeostasis of all tissues. The AK2 isoenzyme is expressed in the mitochondrial intermembrane space and is mutated in Reticular Dysgenesis (RD), a rare form of severe combined immunodeficiency (SCID) in humans. RD is characterized by a maturation arrest in the myeloid and lymphoid lineages, leading to early onset, recurrent, and overwhelming infections. To gain insight into the pathophysiology of RD, we studied the effects of AK2 deficiency using the zebrafish model and induced pluripotent stem cells (iPSCs) derived from fibroblasts of an RD patient. In zebrafish, Ak2 deficiency affected hematopoietic stem and progenitor cell (HSPC) development with increased oxidative stress and apoptosis. AK2-deficient iPSCs recapitulated the characteristic myeloid maturation arrest at the promyelocyte stage and demonstrated an increased AMP/ADP ratio, indicative of an energy-depleted adenine nucleotide profile. Antioxidant treatment rescued the hematopoietic phenotypes in vivo in ak2 mutant zebrafish and restored differentiation of AK2-deficient iPSCs into mature granulocytes. Our results link hematopoietic cell fate in AK2 deficiency to cellular energy depletion and increased oxidative stress. This points to the potential use of antioxidants as a supportive therapeutic modality for patients with RD.
-
Reticular Dysgenesis–associated AK2 protects hematopoietic stem and progenitor cell development from oxidative stress
Journal of Experimental Medicine, 2015Co-Authors: Alberto Rissone, Waleed Al-herz, Elisa Giocaliere, Jayashree Jagadeesh, Kerstin Felgentreff, Katja G Weinacht, Kerry Dobbs, Kevin Bishop, Giancarlo La Marca, Marypat JonesAbstract:Adenylate kinases (AKs) are phosphotransferases that regulate the cellular adenine nucleotide composition and play a critical role in the energy homeostasis of all tissues. The AK2 isoenzyme is expressed in the mitochondrial intermembrane space and is mutated in Reticular Dysgenesis (RD), a rare form of severe combined immunodeficiency (SCID) in humans. RD is characterized by a maturation arrest in the myeloid and lymphoid lineages, leading to early onset, recurrent, and overwhelming infections. To gain insight into the pathophysiology of RD, we studied the effects of AK2 deficiency using the zebrafish model and induced pluripotent stem cells (iPSCs) derived from fibroblasts of an RD patient. In zebrafish, Ak2 deficiency affected hematopoietic stem and progenitor cell (HSPC) development with increased oxidative stress and apoptosis. AK2-deficient iPSCs recapitulated the characteristic myeloid maturation arrest at the promyelocyte stage and demonstrated an increased AMP/ADP ratio, indicative of an energy-depleted adenine nucleotide profile. Antioxidant treatment rescued the hematopoietic phenotypes in vivo in ak2 mutant zebrafish and restored differentiation of AK2-deficient iPSCs into mature granulocytes. Our results link hematopoietic cell fate in AK2 deficiency to cellular energy depletion and increased oxidative stress. This points to the potential use of antioxidants as a supportive therapeutic modality for patients with RD.
-
Adenylate Kinase 2 Regulates Zebrafish Primitive and Definitive Hematopoiesis
Blood, 2012Co-Authors: Alberto Rissone, Trevor Blake, Raman Sood, Jaya Jagadeesh, Simon Karen, Kevin Bishop, Fabio CandottiAbstract:Abstract 1208 Introduction: The adenylate kinase (AK) gene family consists of 7 different members that contribute to energy cell metabolism by converting ATP + AMP to 2 molecules of ADP. AKs are critical players in ensuring cellular energy homeostasis in all tissues and are generally involved in a broad range of cellular functions. Among AKs, AK2 has unique features such as its location in the mitochondrial intermembrane space and critical role in human lymphopoiesis and granulopoiesis. Indeed, mutations of the AK2 gene cause Reticular Dysgenesis (RD), an autosomal recessive form of severe combined immunodeficiency (SCID) characterized by an early differentiation arrest in the granulocyte lineage and impaired lymphoid maturation. The mechanisms underlying the pathophysiology of RD remain unclear. The phenotype of AK2 deficient animals has not been reported in the literature, but murine lines carrying homozygous inactivating retroviral insertions are embryonically lethal (our personal observations). Objectives: To study the role of AK2 in hematopoietic system development and define the effects of AK2 deficiency, we set out to generate a zebrafish model of RD. Methods: We injected zebrafish embryos with morpholino oligomers specific for the two zebrafish AK2 isoforms and analyzed the serial expression pattern of several hematopoietic markers in developing AK2 morphants. To confirm our observations in AK2 knockdown embryos, we screened a zebrafish DNA library of ENU-induced mutations and recovered a mutant fish line carrying a T371C/L124P missense mutation within the exon 4 of AK2 gene that is predicted to be deleterious for protein stability and function. Results: The downregulation of zebrafish AK2 expression phenocopied the human disease and resulted in strong reduction of developing lymphocytes. In addition, in situ hybridization for GATA1, alpha-globin 1, L-plastin and Odianisidine staining indicated that erythroid development was affected in AK2 morphants during primitive hematopoiesis, while myeloid development was conserved. Furthermore, in situ hybridization studies of the expression of markers of zebrafish definitive hematopoiesis showed abnormalities distributed among all hematopoietic lineages suggesting a broad role of AK2 in zebrafish hematopoiesis. Importantly, the ENU-induced Ak2 mutant recapitulated all the primitive and definitive hematopoietic phenotypes observed in AK2 morphants. Finally, preliminary data suggest that AK2 deficiency (both in morphant and mutant embryos) induces an increased level of reactive oxygen species (ROS) triggering oxidative stress and consequent apoptosis in hematopoietic progenitor cells. Conclusions: Our data provide new insights into the AK2 function and indicate that zebrafish represents a good model for studying the molecular mechanisms involved in RD. To date, our mutant line represents the first example of animal model of this rare and unique human disease. Disclosures: No relevant conflicts of interest to declare.
Kevin Bishop - One of the best experts on this subject based on the ideXlab platform.
-
a model for Reticular Dysgenesis shows impaired sensory organ development and hair cell regeneration linked to cellular stress
Disease Models & Mechanisms, 2019Co-Authors: Alberto Rissone, Raman Sood, Erin Jimenez, Blake Carrington, Claire Slevin, Stephen Wincovitch, Fabio Candotti, Kevin Bishop, Shawn M BurgessAbstract:ABSTRACT Mutations in the gene AK2 are responsible for Reticular Dysgenesis (RD), a rare and severe form of primary immunodeficiency in children. RD patients have a severely shortened life expectancy and without treatment die, generally from sepsis soon after birth. The only available therapeutic option for RD is hematopoietic stem cell transplantation (HSCT). To gain insight into the pathophysiology of RD, we previously created zebrafish models for Ak2 deficiencies. One of the clinical features of RD is hearing loss, but its pathophysiology and causes have not been determined. In adult mammals, sensory hair cells of the inner ear do not regenerate; however, their regeneration has been observed in several non-mammalian vertebrates, including zebrafish. Therefore, we used our RD zebrafish models to determine whether Ak2 deficiency affects sensory organ development and/or hair cell regeneration. Our studies indicated that Ak2 is required for the correct development, survival and regeneration of sensory hair cells. Interestingly, Ak2 deficiency induces the expression of several oxidative stress markers and it triggers an increased level of cell death in the hair cells. Finally, we show that glutathione treatment can partially rescue hair cell development in the sensory organs in our RD models, pointing to the potential use of antioxidants as a therapeutic treatment supplementing HSCT to prevent or ameliorate sensorineural hearing deficits in RD patients.
-
a model for Reticular Dysgenesis shows impaired sensory organ development and hair cell regeneration linked to cellular stress
bioRxiv, 2019Co-Authors: Alberto Rissone, Raman Sood, Erin Jimenez, Blake Carrington, Claire Slevin, Stephen Wincovitch, Fabio Candotti, Kevin Bishop, Shawn M BurgessAbstract:Mutations in the gene AK2 are responsible for Reticular Dysgenesis (RD), a rare and severe form of primary immunodeficiency in children. RD patients have a severely shortened life expectancy and without treatment die a few weeks after birth. The only available therapeutic option for RD is bone marrow transplantation. To gain insight into the pathophysiology of RD, we previously created zebrafish models for an AK2 deficiency. One of the clinical features of RD is hearing loss, but its pathology and causes have not been determined. In adult mammals, sensory hair cells of the inner ear do not regenerate; however, their regeneration has been observed in several non-mammalian vertebrates, including zebrafish. Therefore, we use our RD zebrafish models to determine if AK2 deficiency affects sensory organ development and/or hair cell regeneration. Our studies indicated that AK2 is required for the correct development, survival and regeneration of sensory hair cells. Interestingly, AK2 deficiency induces the expression of several oxidative stress markers and it triggers an increased level of cell death in the hair cells. Finally, we show that glutathione treatment can partially rescue hair cell development in the sensory organs in our RD models, pointing to the potential use of antioxidants as a supportive therapeutic modality for RD patients, not only to increase their chances of survival, but to prevent or ameliorate their sensorineural hearing deficits.
-
Reticular Dysgenesis associated ak2 protects hematopoietic stem and progenitor cell development from oxidative stress
Journal of Experimental Medicine, 2015Co-Authors: Alberto Rissone, Elisa Giocaliere, Jayashree Jagadeesh, Kerstin Felgentreff, Katja G Weinacht, Kerry Dobbs, Kevin Bishop, Giancarlo La Marca, Waleed AlherzAbstract:Adenylate kinases (AKs) are phosphotransferases that regulate the cellular adenine nucleotide composition and play a critical role in the energy homeostasis of all tissues. The AK2 isoenzyme is expressed in the mitochondrial intermembrane space and is mutated in Reticular Dysgenesis (RD), a rare form of severe combined immunodeficiency (SCID) in humans. RD is characterized by a maturation arrest in the myeloid and lymphoid lineages, leading to early onset, recurrent, and overwhelming infections. To gain insight into the pathophysiology of RD, we studied the effects of AK2 deficiency using the zebrafish model and induced pluripotent stem cells (iPSCs) derived from fibroblasts of an RD patient. In zebrafish, Ak2 deficiency affected hematopoietic stem and progenitor cell (HSPC) development with increased oxidative stress and apoptosis. AK2-deficient iPSCs recapitulated the characteristic myeloid maturation arrest at the promyelocyte stage and demonstrated an increased AMP/ADP ratio, indicative of an energy-depleted adenine nucleotide profile. Antioxidant treatment rescued the hematopoietic phenotypes in vivo in ak2 mutant zebrafish and restored differentiation of AK2-deficient iPSCs into mature granulocytes. Our results link hematopoietic cell fate in AK2 deficiency to cellular energy depletion and increased oxidative stress. This points to the potential use of antioxidants as a supportive therapeutic modality for patients with RD.
-
Reticular Dysgenesis–associated AK2 protects hematopoietic stem and progenitor cell development from oxidative stress
Journal of Experimental Medicine, 2015Co-Authors: Alberto Rissone, Waleed Al-herz, Elisa Giocaliere, Jayashree Jagadeesh, Kerstin Felgentreff, Katja G Weinacht, Kerry Dobbs, Kevin Bishop, Giancarlo La Marca, Marypat JonesAbstract:Adenylate kinases (AKs) are phosphotransferases that regulate the cellular adenine nucleotide composition and play a critical role in the energy homeostasis of all tissues. The AK2 isoenzyme is expressed in the mitochondrial intermembrane space and is mutated in Reticular Dysgenesis (RD), a rare form of severe combined immunodeficiency (SCID) in humans. RD is characterized by a maturation arrest in the myeloid and lymphoid lineages, leading to early onset, recurrent, and overwhelming infections. To gain insight into the pathophysiology of RD, we studied the effects of AK2 deficiency using the zebrafish model and induced pluripotent stem cells (iPSCs) derived from fibroblasts of an RD patient. In zebrafish, Ak2 deficiency affected hematopoietic stem and progenitor cell (HSPC) development with increased oxidative stress and apoptosis. AK2-deficient iPSCs recapitulated the characteristic myeloid maturation arrest at the promyelocyte stage and demonstrated an increased AMP/ADP ratio, indicative of an energy-depleted adenine nucleotide profile. Antioxidant treatment rescued the hematopoietic phenotypes in vivo in ak2 mutant zebrafish and restored differentiation of AK2-deficient iPSCs into mature granulocytes. Our results link hematopoietic cell fate in AK2 deficiency to cellular energy depletion and increased oxidative stress. This points to the potential use of antioxidants as a supportive therapeutic modality for patients with RD.
-
ak2 deficiency in zebrafish recapitulates human Reticular Dysgenesis an autosomal recessive form of severe combined immunodeficiency
Blood, 2013Co-Authors: Jaya Jagadeesh, Karen L Simon, Raman Sood, Kevin Bishop, Fabio CandottiAbstract:The adenylate kinase (AK) gene family consists of 7 different members that contribute to energy cell metabolism by converting ATP+AMP to 2ADP. AKs are critical players in ensuring cellular energy homeostasis in all tissues. Mutations in the AK2 gene are responsible for Reticular Dysgenesis (RD), an autosomal recessive form of severe combined immunodeficiency (SCID). RD is characterized by an early differentiation arrest in the granulocyte lineage and impaired lymphoid maturation and it represents less than 2% of total SCID. Affected children succumb to overwhelming infections early in life unless their immune system is successfully restored with allogeneic hematopoietic stem cells transplant (HSCT). The mechanisms underlying the pathophysiology of RD remain unclear. The phenotype of AK2 deficient animals has never been reported in the literature, but murine lines carrying homozygous inactivating retroviral insertions are embryonically lethal (our personal observations). We used the zebrafish model to perform a comprehensive study of the effects of AK2 deficiency using Morpholino oligomers injections and two different kinds of AK2 mutants (a ENU-induced T371C/L124P missense mutant and two null mutant lines generated using zinc-finger nuclease technology). In situ hybridization analyses of AK2-deficient embryos indicated that only erythroid development was affected during primitive hematopoiesis. Conversely, during definitive hematopoiesis, the loss of function of AK2 resulted in abnormalities distributed along all hematopoietic lineages suggesting an impairment of hematopoietic stem cell (HSC) development. Moreover, we observed that the AK2 deficiency induced oxidative stress and consequent apoptosis in both primitive erythroid cells and definitive HSCs. Importantly, antioxidant treatment of AK2 mutant embryos rescued the hematopoietic phenotypes as indicated by the recovered expression of HSC and lymphoid markers (such as c-myb and rag1). Overall, our data indicate that zebrafish represents a good model for studying the molecular mechanisms involved in RD and testing of new therapeutic interventions. To date, our mutant lines remain the only animal model of this rare and lethal human disease. Disclosures: No relevant conflicts of interest to declare.
Ulrich Pannicke - One of the best experts on this subject based on the ideXlab platform.
-
Recent advances in understanding the pathogenesis and management of Reticular Dysgenesis.
British journal of haematology, 2017Co-Authors: Manfred Hoenig, Ulrich Pannicke, Hubert B. Gaspar, Klaus SchwarzAbstract:Reticular Dysgenesis is a rare immunodeficiency which is clinically characterized by the combination of Severe Combined Immunodeficiency (SCID) with agranulocytosis and sensorineural deafness. Mutations in the gene encoding adenylate kinase 2 (AK2) were identified to cause this phenotype. In this review, we will demonstrate important clinical differences between Reticular Dysgenesis and other SCID entities and summarize recent concepts in the understanding of the pathophysiology of the disease and the management strategies for this difficult condition.
-
Reticular Dysgenesis international survey on clinical presentation transplantation and outcome
Blood, 2017Co-Authors: Manfred Hoenig, Chantal Lagreslepeyrou, Andrew R. Gennery, Luigi D. Notarangelo, Ulrich Pannicke, Fulvio Porta, Mary SlatterAbstract:Reticular Dysgenesis (RD) is a rare congenital disorder defined clinically by the combination of severe combined immunodeficiency (SCID), agranulocytosis, and sensorineural deafness. Mutations in the gene encoding adenylate kinase 2 were identified to cause the disorder. Hematopoietic stem cell transplantation (HSCT) is the only option to cure this otherwise fatal disease. Retrospective data on clinical presentation, genetics, and outcome of HSCT were collected from centers in Europe, Asia, and North America for a total of 32 patients born between 1982 and 2011. Age at presentation was <4 weeks in 30 of 32 patients (94%). Grafts originated from mismatched family donors in 17 patients (55%), from matched family donors in 6 patients (19%), and from unrelated marrow or umbilical cord blood donors in 8 patients (26%). Thirteen patients received secondary or tertiary transplants. After transplantation, 21 of 31 patients were reported alive at a mean follow-up of 7.9 years (range: 0.6-23.6 years). All patients who died beyond 6 months after HSCT had persistent or recurrent agranulocytosis due to failure of donor myeloid engraftment. In the absence of conditioning, HSCT was ineffective to overcome agranulocytosis, and inclusion of myeloablative components in the conditioning regimens was required to achieve stable lymphomyeloid engraftment. In comparison with other SCID entities, considerable differences were noted regarding age at presentation, onset, and type of infectious complications, as well as the requirement of conditioning prior to HSCT. Although long-term survival is possible in the presence of mixed chimerism, high-level donor myeloid engraftment should be targeted to avoid posttransplant neutropenia.
-
Reticular Dysgenesis: international survey on clinical presentation, transplantation, and outcome
Blood, 2017Co-Authors: Manfred Hoenig, Chantal Lagresle-peyrou, Andrew R. Gennery, Luigi D. Notarangelo, Ulrich Pannicke, Morton J. Cowan, Fulvio Porta, Mary Slatter, Polina Stepensky, Hamoud Al-mousaAbstract:Reticular Dysgenesis (RD) is a rare congenital disorder defined clinically by the combination of severe combined immunodeficiency (SCID), agranulocytosis, and sensorineural deafness. Mutations in the gene encoding adenylate kinase 2 were identified to cause the disorder. Hematopoietic stem cell transplantation (HSCT) is the only option to cure this otherwise fatal disease. Retrospective data on clinical presentation, genetics, and outcome of HSCT were collected from centers in Europe, Asia, and North America for a total of 32 patients born between 1982 and 2011. Age at presentation was
-
Reticular Dysgenesis (aleukocytosis) is caused by mutations in the gene encoding mitochondrial adenylate kinase 2
Nature Genetics, 2009Co-Authors: Ulrich Pannicke, Manfred Hönig, Isabell Hess, Claudia Friesen, Karlheinz Holzmann, Eva-maria Rump, Thomas F Barth, Markus T Rojewski, Ansgar Schulz, Thomas BoehmAbstract:Human severe combined immunodeficiencies (SCID) are phenotypically and genotypically heterogeneous diseases^ 1 . Reticular Dysgenesis is the most severe form of inborn SCID. It is characterized by absence of granulocytes and almost complete deficiency of lymphocytes in peripheral blood, hypoplasia of the thymus and secondary lymphoid organs, and lack of innate and adaptive humoral and cellular immune functions, leading to fatal septicemia within days after birth^ 2 , 3 , 4 , 5 , 6 , 7 , 8 . In bone marrow of individuals with Reticular Dysgenesis, myeloid differentiation is blocked at the promyelocytic stage, whereas erythro- and megakaryocytic maturation is generally normal. These features exclude a defect in hematopoietic stem cells but point to a unique aberration of the myelo-lymphoid lineages. The dramatic clinical course of Reticular Dysgenesis and its unique hematological phenotype have spurred interest in the unknown genetic basis of this syndrome. Here we show that the gene encoding the mitochondrial energy metabolism enzyme adenylate kinase 2 (AK2) is mutated in individuals with Reticular Dysgenesis. Knockdown of zebrafish ak2 also leads to aberrant leukocyte development, stressing the evolutionarily conserved role of AK2. Our results provide in vivo evidence for AK2 selectivity in leukocyte differentiation. These observations suggest that Reticular Dysgenesis is the first example of a human immunodeficiency syndrome that is causally linked to energy metabolism and that can therefore be classified as a mitochondriopathy. Klaus Schwarz and colleagues report mutations in the gene encoding mitochondrial adenylate kinase 2 in Reticular Dysgenesis, the most severe form of inborn combined immunodeficiency, characterized by the absence of granulocytes and almost complete deficiency of lymphocytes in peripheral blood.
-
Reticular Dysgenesis (aleukocytosis) is caused by mutations in the gene encoding mitochondrial adenylate kinase 2
Nature genetics, 2008Co-Authors: Ulrich Pannicke, Manfred Hönig, Isabell Hess, Claudia Friesen, Karlheinz Holzmann, Eva-maria Rump, Thomas F Barth, Markus T Rojewski, Ansgar Schulz, Thomas BoehmAbstract:Klaus Schwarz and colleagues report mutations in the gene encoding mitochondrial adenylate kinase 2 in Reticular Dysgenesis, the most severe form of inborn combined immunodeficiency, characterized by the absence of granulocytes and almost complete deficiency of lymphocytes in peripheral blood.
