ALBERT

Description: Proper development of the CNS axon-glia unit requires bi-directional communication between axons and oligodendrocytes (OLs). We show that the signaling lipid phosphatidylinositol-3,5-bisphosphate [PI(3,5)P2] is required in neurons and in OLs for normal CNS myelination. In mice, mutations of Fig4, Pikfyve or Vac14, encoding key components of the PI(3,5)P2 biosynthetic complex, each lead to impaired OL maturation, severe CNS hypomyelination and delayed propagation of compound action potentials. Primary OLs deficient in Fig4 accumulate large LAMP1+ and Rab7+ vesicular structures and exhibit reduced membrane sheet expansion. PI(3,5)P2 deficiency leads to accumulation of myelin-associated glycoprotein (MAG) in LAMP1+perinuclear vesicles that fail to migrate to the nascent myelin sheet. Live-cell imaging of OLs after genetic or pharmacological inhibition of PI(3,5)P2 synthesis revealed impaired trafficking of plasma membrane-derived MAG through the endolysosomal system in primary cells and brain tissue. Collectively, our studies identify PI(3,5)P2 as a key regulator of myelin membrane trafficking and myelinogenesis.

Description: Key Points Different isoforms of PIP5KIγ fulfill unique functions in platelets.

Description: Platelets release different types of secretory granules into their local environment, and this allows them to contribute to a variety of physiologic processes. These platelet granules include alpha granules, dense granules, and lysosomes, which all derive from the endosomal-lysosomal system. However, the mechanism of the biogenesis of each type of granule is not completely understood. Phosphatidylinositol-3,5-bisphosphate [PtdIns(3,5)P2] is a membrane phosphoinositide that is essential for the regulation of membrane homeostasis, as well as for vesicle trafficking and cargo transport along the endolysosomal system in mammals. PtdIns(3,5)P2 is synthesized on endosomes by the lipid kinase PIKfyve. Given the role of PIKfyve-mediated PtdIns(3,5)P2 production in the endolysosomal pathway in other mammalian cells, we hypothesized that PtdIns(3,5)P2 was an essential regulator for the biogenesis of granules within platelets. To analyze the contribution of PtdIns(3,5)P2 to platelet granule biogenesis, we generated mice lacking PIKfyve kinase activity specifically in their platelets and in their megakaryocytes (PIKfyveflox/flox Pf4-Cre). We found that when compared with the control mice, PIKfyveflox/flox Pf4-Cre mice contained higher levels of the lysosomal enzyme, β-hexosaminidase, within their platelets and within their plasma. The PIKfyve-ablated platelets also released excessive β-hexosaminidase ex vivo upon stimulation with ADP, collagen or thrombin. However, the percentage of the total cellular β-hexosaminidase that was released from the PIKfyve-ablated platelets was comparable with that of control platelets. This suggests that the increased release of β-hexosaminidase from the PIKfyve-ablated platelets is due to the excessive storage of this enzyme within these platelets and not because of increased efficiency of lysosome secretion. In addition, we observed that PIKfyve-ablated platelets expressed increased amounts of Lysosomal Associated Membrane Protein 1 (LAMP-1), a marker of late endosomes and lysosomes. However, PIKfyve-ablated platelets expressed normal amounts of Early Endosome Antigen 1 (EEA-1), a marker of early endosomes. These results suggest that PIKfyve is critical for a component of platelet lysosome biology that occurs after the maturation of early endosomes. Together, these data demonstrate that PIKfyve is essential for the homeostasis of the endolysosomal system in platelets. Notably, the generation and secretion of the alpha granule components were intact in the PIKFyve-ablated platelets. This was shown by the normal expression of von Willebrand factor, platelet basic protein, and platelet factor 4. Likewise, secretion of ATP stored in the dense granules was similar between the PIKfyveflox/flox Pf4-Cre mice and their control littermates. Together, these results suggest that PIKfyve plays an essential regulatory role along the endolysosomal pathway in platelets, and the loss of PIKfyve in platelets can lead to an abnormality of lysosomal storage. Unexpectedly, we also found that the loss of PIKfyve exclusively within platelets triggers an inappropriate inflammatory response. This is shown by the massive tissue infiltration of aberrant vacuolated macrophages. In turn, this leads to multiple organ defects that impair development, body mass, and survival in mice. Moreover, mice lacking PIKfyve within their platelets developed accelerated arterial thrombosis in vivo, despite having normal platelet aggregation ex vivo. It is also remarkable that mice lacking PIKfyve in their platelets attenuated their organ defects when the secretion of their platelet lysosomes were inhibited in vivo. Collectively, our study demonstrates that PIKfyve is an essential regulator of platelet lysosome biogenesis. This study also highlights the previously unrecognized and important contributions of platelet lysosomal storage to inflammation, arterial thrombosis, and macrophage biology. Disclosures: No relevant conflicts of interest to declare.

Description: Abstract 697 Phosphatidylinositol kinases are crucial in the generation of diverse membrane phosphoinositides that regulate the intracellular signaling for essential cellular processes including membrane trafficking. PIKFyve is a kinase that generates phosphatidylinostol 3,5 bisphosphate (PtdIns(3,5)P2) from PtdIns(3)P. Yeast mutants lacking the PIKFyve homologue were unable to synthesize PtdIns(3,5)P2 and exhibited enlarged vacuoles, defective membrane recycling and abnormal protein sorting to multivesicular bodies. In human platelets, PtdIns(3,5)P2 synthesis increased following thrombin stimulation, and our data indicated that murine platelets express PIKFyve mRNA. We hypothesized that PIKFyve and its phospholipid product, PtdIns(3,5)P2 would be critical in the membrane trafficking during the biogenesis of platelet granules. To test this hypothesis, we engineered mice to lack PIKFyve specifically within their platelets and megakaryocytes. The resulting PIKFyvefl/fl mice contained a conditional targeting mutation within two exons encoding for a critical component of its kinase domain. The conditionally targeted mice were crossed with transgenic mice expressing Cre recombinase under the control of platelet factor 4 (PF4) promoter. As expected, RT-PCR analysis confirmed a complete loss of normal PIKFyve mRNA in platelets derived from PIKFyvefl/fl PF4 Cre+ mice. Surprisingly, by 3 weeks of age, PIKFyvefl/fl PF4 Cre+ mice started to display a robust pleomorphic phenotype characterized by dorsal hair loss and progressive weight gain due to generalized body swelling. By 5–6 months of age, the body weight of the PIKFyvefl/fl PF4 Cre+ mice was about 30% higher than that of the control littermate mice (PIKFyvefl/fl PF4 Cre- or PIKFyvefl/+ PF4 Cre+) and appeared acutely ill with decreased ambulation and feeding. A DEXA scan revealed that PIKFyvefl/fl PF4 Cre+ mice had approximately 50% lower body fat content compared to their control littermates. Necropsy of PIKFyvefl/fl PF4 Cre+ mice at about 6 months of age demonstrated that their weight gain was due to massive infiltration of largely vacuolated F4/80 expressing macrophages in multiple organs including skin, muscle, lung, heart, liver, pancreas, small and large intestines, kidney, genital organs, thymus and bone marrow. The vacuoles within these macrophages occupied the entire cytoplasm and were reminiscent of PIKFyve null yeast mutant cells. Unexpectedly, PCR analysis of genomic DNA from tissues infiltrated by these vacuolated macrophages revealed that PIKFyve deletion had occurred within these cells. This indicated that the PF4 Cre was expressed in these macrophages and led to targeting of PIKFyve and ultimately to the development of vacuolated macrophages that infiltrated multiple organs. Despite the pleomorphic phenotype, the platelet counts of the PIKFyvefl/fl PF4 Cre+ mice were normal. Furthermore, platelets lacking PIKFyve normally exposed P-selectin from alpha granules and released ATP from dense granules in response to agonist stimulation. Nevertheless, the PIKFyvefl/fl PF4 Cre+ platelets exhibited a three-fold increase of basal b-hexosaminidase, a lysosomal hydrolase. This data suggests a role for PIKFyve in the biogenesis of platelet lysosomes. Furthermore, in vivo platelet thrombus formation analysis using the ferric chloride induced carotid artery injury model revealed that PIKFyvefl/fl PF4 Cre+ mice made an occluding thrombus significantly faster than the control littermate mice demonstrating that they are prothrombotic. Histological analysis of the carotid artery specimen at the site of thrombus demonstrated that the intravascular side did not contain vacuolated macrophages. This suggests that the enhanced thrombosis formation was due to the increased lysosomal content within the platelets, and not due to infiltration by abnormal macrophages. We conclude that PF4 promoter driven selective deletion of PIKFyve kinase is associated with defective biogenesis of platelet lysosomes and a prothrombotic effect in vivo. Furthermore, PIKFyvefl/fl PF4 Cre+ mice display a pleomorphic phenotype associated with vacuolated macrophage accumulation in multiple organs and suggest that PF4 promoter driven Cre expression is not restricted to megakaryocytes and platelets. This study also highlights a potential problem in previous and current work using the PF4 Cre transgenic model. Disclosures: No relevant conflicts of interest to declare.

Description: In response to agonist stimulation, platelets undergo a rapid reorganization of their actin cytoskeleton. This process involves simultaneous disassembly and assembly of filamentous actin, and is one of the earliest events of platelet activation. Ex vivo flow models support the hypothesis that actin assembly is essential for stable in vivo platelet adhesion and thrombus formation under the shear conditions found within the arterial system. On the cellular level, the reorganization of actin that occurs during platelet activation involves the formation of an actin network similar to that found in lamellipodia at the leading edge of the locomoting fibroblasts. During lamellipod formation in fibroblasts, the predominant actin isoform of the cell leading edge is beta-actin. In these cells, beta-actin undergoes posttranslational arginylation (i.e., addition of arginine onto the N-terminus). We estimate that up to 40% of actin at the leading edge of fibroblasts is arginylated, and that the absence of this modification results in actin filament aggregation in vitro. Arginylation of actin is mediated by the arginyltransferase, ATE1. Embryos lacking ATE1 die between E12 and E17 due to severe cardiovascular defects and massive hemorrhage. Fibroblasts derived from knockout embryos collapse their extending lamella, potentially because their non-arginylated actin aggregates at the extending edge. The beta-isoform of actin is over 85% of the total actin in platelets. To gain insight into whether actin could undergo arginylation in platelets, we analyzed actin from resting and thrombin- or PMA-stimulated platelets fractionated on 2D gels. Our analysis indicates that platelet activation results in a prominent shift of a significant fraction of actin protein into the basic pI range, consistent with the addition of arginine onto the actin molecule. This result suggests that actin might be arginylated in platelets, and that this arginylation is activation-dependent. We hypothesized that if platelet actin reorganization indeed occurs by the mechanisms similar to those seen in fibroblasts, it should also be dependent on the arginylation. To test this hypothesis, we reconstituted the hematopoietic system of lethally irradiated mice with hematopoietic precursor cells collected from the livers of ATE1 null or wildtype embryos at day 13.5 of development. Platelets derived from ATE1 knockout radiation chimeric mice exhibited normal binding to fluorescently labeled fibrinogen in response to stimulation of the thrombin receptor or by PMA. However, in contrast to platelets derived from radiation chimeric mice rescued with normal hematopoietic cells, platelets derived from chimeric mice with ATE1 null hematopoietic cells spread poorly to immobilized fibrinogen (decreased 35.6%). When blood harvested from radiation chimeric mice was allowed to adhere to immobilized collagen under flow, wild type platelets adhered firmly, and rarely formed unstable adhesions (15 ± 10%). In contrast, ATE1 null platelets adhered less well, and frequently failed to form stable adhesions (50% ±15%). Together, these results suggest that beta-actin is rapidly arginylated in agonist-stimulated platelets, and that arginylation is required for actin reorganization during platelet adhesion and lamellipodia formation. We hypothesize that ATE1-mediated arginylation of platelet beta-actin is also essential for in vivo platelet adhesion and thrombosis.

Description: Chemokines acting through G-protein coupled receptors play an essential role in both the immune and inflammatory responses. Phosphatidylinositol 3-kinase (PI3K) and phospholipase C (PLC) are two distinct signaling molecules that have been proposed as potential candidates in the regulation of this process. Studies with knockout mice have demonstrated a critical role for PI3Kγ, but not PLCβ, in Gαi-coupled receptor-mediated neutrophil chemotaxis. We compared the chemotactic response of peripheral T-cells derived from wild type mice with mice containing loss-of-function mutations of either PI3Kγ, or both of the two predominant lymphocyte PLCβ isoforms (PLCβ2 and PLCβ3). In contrast to neutrophils, loss of PI3Kγ did not significantly impair T-cell migration in vitro, although PI3K pharmacologic inhibitor experiments suggest that another isoform of this enzyme might contribute to T-cell migration. However, loss of PLCβ2β3 decreased chemokine-stimulated T-cell migration in vitro. Chelation of intracellular calcium by BAPTA-AM and Quin-2 AM decreased the chemotactic response of wild type lymphocytes, but pharmacologic inhibition of PKC isoforms by GF109203x did not impair T-cell migration. This suggests that the T-cell migration defect seen in the PLCβ2β3-null T-cells may be due to an impaired ability to increase the cytoplasmic calcium concentration, while there appears to be little requirement for PKC activity. Indeed, SDF-1α-induced calcium efflux was not detected in the PLCβ2β3-null lymphocytes. Compared to fluorescently labeled wild type T-cells, labeled PLCβ2β3 knockout T-cells migrated less efficiently into secondary lymphoid organs of recipient mice. This demonstrates that PLCβ is also required for migration in vivo. PLCβ2β3-null mice develop spontaneous skin ulcers starting around 3 months of age. Histological examination of the lesions revealed a dense inflammatory infiltrate composed of neutrophils, macrophages, and plasma cells, consistent with acute and chronic inflammation. Remarkably, lymphocytes, typical of chronic inflammation, were rare to absent by histology and by paraffin immunohistochemistry for CD3, also consistent with an in vivo migratory defect of T-cells. These results show that phospholipid second messengers generated by PLCβ and isoforms of PI3K, other than PI3Kγ, play a critical role in lymphocyte chemotaxis. Collectively, our data demonstrate that although PLCβ-mediated signaling plays no role in neutrophil chemotaxis, it makes a substantial contribution to this process within T-lymphocytes.

Description: Pleckstrin, the platelet and leukocyte C kinase substrate, is a prominent substrate of PKC in platelets, monocytes, macrophages, lymphocytes, and granulocytes. Pleckstrin accounts for 1% of the total protein in these cells, but it is best known for containing the 2 prototypic Pleckstrin homology, or PH, domains. Overexpressed pleckstrin can affect polyphosphoinositide second messenger–based signaling events; however, its true in vivo role has been unknown. Here, we describe mice containing a null mutation within the pleckstrin gene. Platelets lacking pleckstrin exhibit a marked defect in exocytosis of δ and α granules, αIIbβ3 activation, actin assembly, and aggregation after exposure to the PKC stimulant, PMA. Pleckstrin-null platelets aggregate normally in response to thrombin, but they fail to aggregate in response to thrombin in the presence of PI3K inhibitors, suggesting that a PI3K-dependent signaling pathway compensates for the loss of pleckstrin. Although pleckstrin-null platelets merged their granules in response to stimulation of PKC, they failed to empty their contents into the open canalicular system. This might be attributable to impaired actin assembly present in cells lacking pleckstrin. These data show that pleckstrin regulates the fusion of granules to the cell membrane and is an essential component of PKC-mediated exocytosis.

Description: Following thrombin stimulation, platelet PIP5KI synthesizes phosphatidylinositol 4,5-bisphosphate (PIP2), which can be hydrolyzed by phospholipase C to generate second messengers such as IP3. PIP2 also regulates cytoskeletal dynamics by directly interacting with actin-binding proteins. Three isoforms of PIP5KI (α, β, and γ) are all capable of phosphorylating PI4P to synthesize PIP2. However, these isoforms have different primary structures, expression levels in various tissues, and intracellular localization. We have generated and characterized murine lines lacking PIP5KIβ or PIP5KIγ, which are the two predominant platelet isoforms. We also phenotyped platelets lacking PIP5KIα, which is the least abundant isoform. PIP5KIβ-null mice appeared developmentally normal and had normal platelet counts, however they had small defects in aggregation following exposure to all agonists. In contrast, platelets lacking PIP5KIα aggregated normally. Although platelets lacking PIP5KIβ have only a moderate deficiency of PIP2 under basal conditions, they have a striking deficiency in PIP2 synthesis and IP3 formation following thrombin stimulation. We have also observed that platelets lacking both PIP5KIα and PIP5KIβ have a complete loss of thrombin-induced IP3 synthesis, even though they still contain PIP5KIγ, which is the predominant PIP5KI isoform in platelets. Additionally, we found when using a carotid injury model that PIP5KIβ-null platelets failed to properly form arterial thrombi in vivo. This demonstrates that PIP5KIβ, like PIP5KIα, contributes to the rapid synthesis of a pool of PIP2 that is required for second messenger formation and in vivo platelet adhesion. This contrasts the PIP5KIγ-synthesized pool of PIP2 that does not contribute to these processes. We have found that loss of PIP5KIγ null mutation impairs cardiac development and leads to embryonic lethality. PIP5KIγ null megakaryocytes derived from yolk sac progenitor cells have a defect in anchoring their cell membranes to the underlying actin cytoskeleton. To understand the role of this PIP5KI isoform in platelet biology, we conditionally rescued the PIP5KIγ null mutation within myocardiocytes allowing us to obtain living mice. Platelets from these animals lacked PIP5KIγ, yet aggregated normally when exposed to all agonists. To analyze these cells for a failure to anchor their cell membranes, we used laser tweezers to pull the cell membrane apart from the cytoskeleton. Wild type cells had rigid membranes that resisted stretching by trapped fibrinogen-coated beads that were pulled by laser tweezers. In contrast, the PIP5KIγ-null platelets had flexible membranes that were easily stretched, and ultimately allowed membrane tethers to form. Together, these results demonstrate that following stimulation of a G-protein coupled receptor, IP3 is completely derived from a rapidly synthesized discrete pool of PIP2 that is synthesized by PIP5KIα and PIP5KIβ. In contrast, the pool of PIP2 synthesized by PIP5KIγ contributes to preserving the integrity of the membrane cytoskeleton. In conclusion, this work demonstrates that spatially restricted PIP2 synthesis by individual PIP5KI isoforms differentially controls second messenger formation and the integrity of the actin cytoskeleton.