The Group VIA Phospholipase A 2 (iPLA 2 ␤) is the first recognized cytosolic Ca 2؉ -independent PLA 2 and has been proposed to participate in arachidonic acid (20:4) incorporation into glycerophosphocholine lipids, cell proliferation, exocytosis, apoptosis, and other processes. To study iPLA 2 ␤ functions, we disrupted its gene by homologous recombination to generate mice that do not express iPLA 2 ␤. Heterozygous iPLA 2 ␤ ؉/؊ breeding pairs yield a Mendelian 1:2:1 ratio of iPLA 2 ␤ ؉/؉ , iPLA 2 ␤ ؉/؊ , and iPLA 2 ␤ ؊/؊ pups and a 1:1 male:female gender distribution of iPLA 2 ␤ ؊/؊ pups. Several tissues of wild-type mice express iPLA 2 ␤ mRNA, immunoreactive protein, and activity, and testes express the highest levels. Testes or other tissues of iPLA 2 ␤ ؊/؊ mice express no iPLA 2 ␤ mRNA or protein, but iPLA 2 ␤ ؊/؊ testes are not deficient in 20:4-containing glycerophosphocholine lipids, indicating that iPLA 2 ␤ does not play an obligatory role in formation of such lipids in that tissue. Spermatozoa from iPLA 2 ␤ ؊/؊ mice have reduced motility and impaired ability to fertilize mouse oocytes in vitro and in vivo, and inhibiting iPLA 2 ␤ with a bromoenol lactone suicide substrate reduces motility of wild-type spermatozoa in a time-and concentration-dependent manner. Mating iPLA 2 ␤ ؊/؊ male mice with iPLA 2 ␤ ؉/؉ , iPLA 2 ␤ ؉/؊ , or iPLA 2 ␤ ؊/؊ female mice yields only about 7% of the number of pups produced by mating pairs with an iPLA 2 ␤ ؉/؉ or iPLA 2 ␤ ؉/؊ male, but iPLA 2 ␤ ؊/؊ female mice have nearly normal fertility. These findings indicate that iPLA 2 ␤ plays an important functional role in spermatozoa, suggest a target for developing male contraceptive drugs, and complement reports that disruption of the Group IVA PLA 2 (cPLA 2 ␣) gene impairs female reproductive ability.

Early atherosclerosis depends upon responses by immune cells resident in the intimal aortic wall. Specifically, the healthy intima is thought to be populated by vascular dendritic cells (DC) that, during hypercholesterolemia, initiate atherosclerosis by being the first to accumulate cholesterol. Whether these cells remain key players in later stages of disease is unknown. Using murine lineage tracing models and gene expression profiling, we reveal that myeloid cells present in the intima of the aortic arch are not DCs but instead specialized aorta intima resident macrophages (Mac AIR ) that depend upon Csf-1 and sustained by local proliferation. Although Mac AIR comprise the earliest foam cells in plaques, their proliferation during plaque progression is limited. After months of hypercholesterolemia, their presence in plaque is overtaken by recruited monocytes, which induce Mac AIR -defining genes. These data redefine the lineage of intimal phagocytes and suggest that proliferation is insufficient to sustain generations of macrophages during plaque progression.

Studies involving pharmacologic or molecular biologic manipulation of Group VIA phospholipase A 2 (iPLA 2 ␤) activity in pancreatic islets and insulinoma cells suggest that iPLA 2 ␤ participates in insulin secretion. It has also been suggested that iPLA 2 ␤ is a housekeeping enzyme that regulates cell 2-lysophosphatidylcholine (LPC) levels and arachidonate incorporation into phosphatidylcholine (PC). We have generated iPLA 2 ␤-null mice by homologous recombination and have reported that they exhibit reduced male fertility and defective motility of spermatozoa. Here we report that pancreatic islets from iPLA 2 ␤-null mice have impaired insulin secretory responses to D-glucose and forskolin. Electrospray ionization mass spectrometric analyses indicate that the abundance of arachidonate-containing PC species of islets, brain, and other tissues from iPLA 2 ␤-null mice is virtually identical to that of wild-type mice, and no iPLA 2 ␤ mRNA was observed in any tissue from iPLA 2 ␤-null mice at any age. Despite the insulin secretory abnormalities of isolated islets, fasting and fed blood glucose concentrations of iPLA 2 ␤-null and wild-type mice are essentially identical under normal circumstances, but iPLA 2 ␤-null mice develop more severe hyperglycemia than wild-type mice after administration of multiple low doses of the ␤-cell toxin streptozotocin, suggesting an impaired islet secretory reserve. A high fat diet also induces more severe glucose intolerance in iPLA 2 ␤-null mice than in wild-type mice, but PLA 2 ␤-null mice have greater responsiveness to exogenous insulin than do wild-type mice fed a high fat diet. These and previous findings thus indicate that iPLA 2 ␤-null mice exhibit phenotypic abnormalities in pancreatic islets in addition to testes and macrophages.

Mutations in the PLA2G6 gene, which encodes group VIA calcium-independent phospholipase A2 (iPLA 2 ␤), were recently identified in patients with infantile neuroaxonal dystrophy (INAD) and neurodegeneration with brain iron accumulation. A pathological hallmark of these childhood neurodegenerative diseases is the presence of distinctive spheroids in distal axons that contain accumulated membranes. We used iPLA 2 ␤-KO mice generated by homologous recombination to investigate neurodegenerative consequences of PLA2G6 mutations. iPLA 2 ␤-KO mice developed age-dependent neurological impairment that was evident in rotarod, balance, and climbing tests by 13 months of age. The primary abnormality underlying this neurological impairment was the formation of spheroids containing tubulovesicular membranes remarkably similar to human INAD. Spheroids were strongly labeled with anti-ubiquitin antibodies. Accumulation of ubiquitinated protein in spheroids was evident in some brain regions as early as 4 months of age, and the onset of motor impairment correlated with a dramatic increase in ubiquitin-positive spheroids throughout the neuropil in nearly all brain regions. Furthermore accumulating ubiquitinated proteins were observed primarily in insoluble fractions of brain tissue, implicating protein aggregation in this pathogenic process. These results indicate that loss of iPLA 2 ␤ causes age-dependent impairment of axonal membrane homeostasis and protein degradation pathways, leading to age-dependent neurological impairment. iPLA 2 ␤-KO mice will be useful for further studies of pathogenesis and experimental interventions in INAD and neurodegeneration with brain iron accumulation.

A cytosolic 84-kDa group VIA phospholipase A 2 (iPLA 2 ␤) that does not require Ca 2؉ for catalysis has been cloned from several sources, including rat and human pancreatic islet ␤-cells and murine P388D1 cells. Many potential iPLA 2 ␤ functions have been proposed, including a signaling role in ␤-cell insulin secretion and a role in generating lysophosphatidylcholine acceptors for arachidonic acid incorporation into P388D1 cell phosphatidylcholine (PC). Proposals for iPLA 2 ␤ function rest in part on effects of inhibiting iPLA 2 ␤ activity with a bromoenol lactone (BEL) suicide substrate, but BEL also inhibits phosphatidate phosphohydrolase-1 and a group VIB phospholipase A 2 . Manipulation of iPLA 2 ␤ expression by molecular biologic means is an alternative approach to study iPLA 2 ␤ functions, and we have used a retroviral construct containing iPLA 2