Role Of Wiskott-Aldrich Syndrome Protein Research Articles

Wiskott Aldrich syndrome protein regulates non-selective autophagy and mitochondrial homeostasis in human myeloid cells.

The actin cytoskeletal regulator Wiskott Aldrich syndrome protein (WASp) has been implicated in maintenance of the autophagy-inflammasome axis in innate murine immune cells. Here, we show that WASp deficiency is associated with impaired rapamycin-induced autophagosome formation and trafficking to lysosomes in primary human monocyte-derived macrophages (MDMs). WASp reconstitution in vitro and in WAS patients following clinical gene therapy restores autophagic flux and is dependent on the actin-related protein complex ARP2/3. Induction of mitochondrial damage with CCCP, as a model of selective autophagy, also reveals a novel ARP2/3-dependent role for WASp in formation of sequestrating actin cages and maintenance of mitochondrial network integrity. Furthermore, mitochondrial respiration is suppressed in WAS patient MDMs and unable to achieve normal maximal activity when stressed, indicating profound intrinsic metabolic dysfunction. Taken together, we provide evidence of new and important roles of human WASp in autophagic processes and immunometabolic regulation, which may mechanistically contribute to the complex WAS immunophenotype.

CRISPR/Cas9-mediated deletion of the Wiskott-Aldrich syndrome locus causes actin cytoskeleton disorganization in murine erythroleukemia cells.

Wiskott-Aldrich syndrome (WAS) is a recessive X-linked inmmunodeficiency caused by loss-of-function mutations in the gene encoding the WAS protein (WASp). WASp plays an important role in the polymerization of the actin cytoskeleton in hematopoietic cells through activation of the Arp2/3 complex. In a previous study, we found that actin cytoskeleton proteins, including WASp, were silenced in murine erythroleukemia cells defective in differentiation. Here, we designed a CRISPR/Cas9 strategy to delete a 9.5-kb genomic region encompassing the Was gene in the X chromosome of murine erythroleukemia (MEL) cells. We show that Was-deficient MEL cells have a poor organization of the actin cytoskeleton that can be recovered by restoring Was expression. We found that whereas the total amount of actin protein was similar between wild-type and Was knockout MEL cells, the latter exhibited an altered ratio of monomeric G-actin to polymeric F-actin. We also demonstrate that Was overexpression can mediate the activation of Bruton’s tyrosine kinase. Overall, these findings support the role of WASp as a key regulator of F-actin in erythroid cells.

Constitutive activation of WASp in X-linked neutropenia renders neutrophils hyperactive.

Congenital neutropenia is characterized by low absolute neutrophil numbers in blood, leading to recurrent bacterial infections, and patients often require life-long granulocyte CSF (G-CSF) support. X-linked neutropenia (XLN) is caused by gain-of-function mutations in the actin regulator Wiskott-Aldrich syndrome protein (WASp). To understand the pathophysiology in XLN and the role of WASp in neutrophils, we here examined XLN patients and 2 XLN mouse models. XLN patients had reduced myelopoiesis and extremely low blood neutrophil number. However, their neutrophils had a hyperactive phenotype and were present in normal numbers in XLN patient saliva. Murine XLN neutrophils were hyperactivated, with increased actin dynamics and migration into tissues. We provide molecular evidence that the hyperactivity of XLN neutrophils is caused by WASp in a constitutively open conformation due to contingent phosphorylation of the critical tyrosine-293 and plasma membrane localization. This renders WASp activity less dependent on regulation by PI3K. Our data show that the amplitude of WASp activity inside a cell could be enhanced by cell-surface receptor signaling even in the context in which WASp is already in an active conformation. Moreover, these data categorize XLN as an atypical congenital neutropenia in which constitutive activation of WASp in tissue neutrophils compensates for reduced myelopoiesis.

N-WASP is required for B-cell–mediated autoimmunity in Wiskott-Aldrich syndrome

AbstractMutations of the Wiskott-Aldrich syndrome gene (WAS) are responsible for Wiskott-Aldrich syndrome (WAS), a disease characterized by thrombocytopenia, eczema, immunodeficiency, and autoimmunity. Mice with conditional deficiency of Was in B lymphocytes (B/WcKO) have revealed a critical role for WAS protein (WASP) expression in B lymphocytes in the maintenance of immune homeostasis. Neural WASP (N-WASP) is a broadly expressed homolog of WASP, and regulates B-cell signaling by modulating B-cell receptor (BCR) clustering and internalization. We have generated a double conditional mouse lacking both WASP and N-WASP selectively in B lymphocytes (B/DcKO). Compared with B/WcKO mice, B/DcKO mice showed defective B-lymphocyte proliferation and impaired antibody responses to T-cell-dependent antigens, associated with decreased autoantibody production and lack of autoimmune kidney disease. These results demonstrate that N-WASP expression in B lymphocytes is required for the development of autoimmunity of WAS and may represent a novel therapeutic target in WAS.

T Cell Receptor-Associated Actin Patches Generated by Wiskott-Aldrich Syndrom Protein

T cell activation involves massive cytoskeletal remodeling, including dynamic actin polymerization and centripetal flow proximal to T cell-antigen presenting surface synapse (IS). F-actin is required for the formation of signaling T cell receptor microclusters (MCs) and for on-going signaling. Wiskott-Aldrich Syndrome Protein (WASP) is an actin nucleation-promoting factor that is critical for appropriate T cell function. Patients with mutations in WASP experience a severe immunodeficiency and autoimmunity. The role of WASP in actin polymerization at the immunological synapse has been surprisingly cryptic as WASP deficient T cells still display dramatic increases in F-actin at the immunological synapse. Here, with the aid of live cell as well as super-resolution imaging in primary T cells, we identify WASP dependent F-actin patches closely associated with TCR microclusters. The patches also contain phosphorylated HS-1, a cortactin homolog, and phosphorylated Cas-L a mechanotransduction protein, which are recruited down-stream of WASP. Closely related proteins N-WASP and WAVE2 do not participate in TCR-associated F-actin patches. Super-resolution imaging reveals that the patches are composed of multiple actin arrays that are oriented to diverge from TCR microclusters. We conclude that F-actin patches represent a unique mode of actin polymerization in the immunological synapse that integrate the well documented functional defects in WASP deficient T cells to F-actin nucleation activity.

Systemic autoimmunity and defective Fas ligand secretion in the absence of the Wiskott-Aldrich syndrome protein

AbstractAutoimmunity is a surprisingly common complication of primary immunodeficiencies, yet the molecular mechanisms underlying this clinical observation are not well understood. One widely known example is provided by Wiskott-Aldrich syndrome (WAS), an X-linked primary immunodeficiency disorder caused by mutations in the gene encoding the WAS protein (WASp) with a high incidence of autoimmunity in affected patients. WASp deficiency affects T-cell antigen receptor (TCR) signaling and T-cell cytokine production, but its role in TCR-induced apoptosis, one of the mechanisms of peripheral immunologic tolerance, has not been investigated. We find that WASp-deficient mice produce autoantibodies and develop proliferative glomerulonephritis with immune complex deposition as they age. We also find that CD4+ T lymphocytes from WASp-deficient mice undergo reduced apoptosis after restimulation through the TCR. While Fas-induced cell death is normal, WASp deficiency affects TCR-induced secretion of Fas ligand (FasL) and other components of secretory granules by CD4+ T cells. These results describe a novel role of WASp in regulating TCR-induced apoptosis and FasL secretion and suggest that WASp-deficient mice provide a good model for the study of autoimmune manifestations of WAS and the development of more specific therapies for these complications.

Role of WASP in cell polarity and podosome dynamics of myeloid cells

The integrin-dependent migration of myeloid cells requires tight coordination between actin-based cell membrane protrusion and integrin-mediated adhesion to form a stable leading edge. Under this mode of migration, polarised myeloid cells including dendritic cells, macrophages and osteoclasts develop podosomes that sustain the extending leading edge. Podosome integrity and dynamics vary in response to changes in the physical and biochemical properties of the cell environment. In the current article we discuss the role of various factors in initiation and stability of podosomes and the roles of the Wiskott Aldrich Syndrome Protein (WASP) in this process. We discuss recent data indicating that in a cellular context WASP is crucial not only for localised actin polymerisation at the leading edge and in podosome cores but also for coordination of integrin clustering and activation during podosome formation and disassembly.

The Mechanism of CSF-1-induced Wiskott-Aldrich Syndrome Protein Activation in Vivo: A ROLE FOR PHOSPHATIDYLINOSITOL 3-KINASE AND Cdc42

A role for Wiskott-Aldrich syndrome protein (WASP) in chemotaxis to various agents has been demonstrated in monocyte-derived cell types. Although WASP has been shown to be activated by multiple mechanisms in vitro, it is unclear how WASP is regulated in vivo. A WASP biosensor (WASPbs), which uses intramolecular fluorescence resonance energy transfer to report WASP activation in vivo, was constructed, and following transfection of macrophages, activation of WASPbs upon treatment with colony-stimulating factor-1 (CSF-1) was detected globally as early as 30 s and remained localized to protrusive regions at later time points. Similar results were obtained when endogenous WASP activation was determined using conformation-sensitive antibodies. In vivo CSF-1-induced WASP activation was fully Cdc42-dependent. Activation of WASP in response to treatment with CSF-1 was also shown to be phosphatidylinositol 3-kinase-dependent. However, treatment with the Src family kinase inhibitors PP2 or SU6656 or disruption of the major tyrosine phosphorylation site of WASPbs (Y291F mutation) did not reduce the level of CSF-1-induced WASP activation. Our results indicate that WASP activation downstream of CSF-1R is phosphatidylinositol 3-kinase- and Cdc42-dependent consistent with an involvement of these molecules in macrophage migration. However, although tyrosine phosphorylation of WASP has been proposed to stimulate WASP activity, we found no evidence to indicate that this occurs in vivo.

A WAVE2-Abi1 complex mediates CSF-1-induced F-actin-rich membrane protrusions and migration in macrophages

Colony-stimulating factor 1 (CSF-1) is an important physiological chemoattractant for macrophages. The mechanisms by which CSF-1 elicits the formation of filamentous actin (F-actin)-rich membrane protrusions and induces macrophage migration are not fully understood. In particular, very little is known regarding the contribution of the different members of the Wiskott-Aldrich Syndrome protein (WASP) family of actin regulators in response to CSF-1. Although a role for WASP itself in macrophage chemotaxis has been previously identified, no data was available regarding the function of WASP family verprolin-homologous (WAVE) proteins in this cell type. We found that WAVE2 was the predominant isoform to be expressed in primary macrophages and in cells derived from the murine monocyte/macrophage RAW264.7 cell line (RAW/LR5). CSF-1 treatment of macrophages resulted in WAVE2 accumulation in F-actin-rich protrusions induced by CSF-1. Inhibition of WAVE2 function by expressing a dominant-negative mutant or introducing anti-WAVE2 antibodies in RAW/LR5 cells, as well as reduction of endogenous WAVE2 expression by RNA-mediated interference (RNAi), resulted in a significant reduction of CSF-1-elicited F-actin protrusions. WAVE2 was found in a protein complex together with Abelson kinase interactor 1 (Abi1) in resting or stimulated cells. Both WAVE2 and Abi1 were recruited to and necessary for the formation of F-actin protrusions in response to CSF-1. Reducing the levels of WAVE2, directly or by targeting Abi1, resulted in an impaired cell migration to CSF-1. Altogether these data identify a WAVE2-Abi1 complex crucial for the normal actin cytoskeleton reorganization and migration of macrophages in response to CSF-1.

A Role for Wiskott-Aldrich Syndrome Protein in T-cell Receptor-mediated Transcriptional Activation Independent of Actin Polymerization

Wiskott-Aldrich syndrome protein (WASP) plays a key role in cytoskeletal rearrangement and transcriptional activation in T-cells. Recent evidence links WASP and related proteins to actin polymerization by the Arp2/3 complex. To study whether the role of WASP in actin polymerization is coupled to T-cell receptor (TCR)-mediated transcriptional activation, we made a series of WASP deletion mutants and tested them for actin co-localization, actin polymerization, and transcriptional activation of NFAT. A WASP mutant with a deletion in the C-terminal region (WASPDeltaC) that is defective in actin polymerization potentiated NFAT transcription following TCR activation by anti-CD3 and anti-CD3/CD28 antibodies, but not by phorbol 12-myristate 13-acetate/ionomycin. Furthermore, cotransfection of a dominant-active mutant (WASP-WH2-C) for Arp2/3 polymerization did not inhibit NFAT activation. Finally, by analyzing a series of WASP double-domain deletion mutants, we determined that the WASP homology-1 domain is responsible for NFAT transcriptional activation. Our results suggest that WASP activates transcription following TCR stimulation in a manner that is independent of its role in Arp2/3-directed actin polymerization.

A case of Wiskott‐Aldrich syndrome with cilial immotility

We report the case history of a 25-year-old man who was diagnosed as having Wiskott-Aldrich syndrome in infancy. He had the typical features of severe eczema, thrombocytopenia and recurrent sinopulmonary infection. By his late teenage years he had developed bronchiectasis despite having normal levels of immunoglobulin and IgG subclasses. His functional antibody responses to tetanus toxoid and pneumococcal vaccine were suboptimal (15% and 52% of normal respectively). Regular intravenous immunoglobulin therapy resulted in a reduced frequency and severity of infection but despite this treatment he continued to get respiratory infections. In April 1999 he presented with three episodes of acute gastrointestinal haemorrhage. CT scan of the abdomen and arteriography showed multiple arterial aneurysms with evidence of active haemorrhage within the liver and upper small bowel. Inflammatory markers (ESR and C-reactive protein) were markedly elevated and he was diagnosed as having acute inflammatory vasculitis as a complication of his primary immune disorder.1 Prednisone and cyclophosphamide therapy were deemed necessary to treat the vasculitis and prevent further life-threatening haemorrhage. Before immunosuppressive therapy was started, seminal fluid samples were obtained for storage. Analysis revealed not only a low sperm count (<2 × 106/ml) but also the fact that all spermatozoa were immotile. In order to investigate cilial function more fully, nasal brush biopsy samples were obtained and cilial activity was assessed by a standard method on fresh samples. No evidence of cilial movement was observed in any of the tissue samples. The role of Wiskott-Aldrich Syndrome Protein (WASP) in transmitting signals from CDC42HS to the actin cytoskeleton of cells To our knowledge, this is the first report of cilial immotility as a feature of this condition. Wiskott-Aldrich syndrome is an X-linked primary immunodeficiency disorder characterised by thrombocytopenia, eczema and recurrent infection.2 The gene defect has been identified at the Xp11.22 position on the short arm of the X chromosome and designated the WAS gene.3 A variety of mutations have been described, all of which result in the absence of WAS protein or the production of a functionally defective protein. In normal individuals WAS protein plays a central role in the control of cellular morphology and the organisation of the cytoskeleton of cells.4 Defects in some of these, including actin polymerisation (Figure 1), have been identified and help to explain many of the clinical manifestations of WAS.5 In the past, the increased frequency of infection in this disorder was attributed to impaired antibody production. Some patients, however, including this case, develop sinopulmonary infection and bronchiectasis at an early age which cannot be explained by a defect in antibody production alone and may represent some other defect in the antibacterial defence system of the respiratory tract. It is not clear whether or not cilial immotility occurs in all WAS patients or only in certain genetic variants of the disorder.