The pathogenesis of nonalcoholic steatohepatitis (NASH) is unclear, despite epidemiological data implicating FFAs. We studied the pathogenesis of NASH using lipoapoptosis models. Palmitic acid (PA) induced classical apoptosis of hepatocytes. PA-induced lipoapoptosis was inhibited by acyl-CoA synthetase inhibitor but not by ceramide synthesis inhibitors, suggesting that conversion products other than ceramide are involved. Phospholipase A 2 (PLA 2 ) inhibitors blocked PA-induced hepatocyte death, suggesting an important role for PLA 2 and its product lysophosphatidylcholine (LPC). Small interfering RNA for Ca 21 -independent phospholipase A 2 (iPLA 2 ) inhibited the lipoapoptosis of hepatocytes. PA increased LPC content, which was reversed by iPLA 2 inhibitors. Pertussis toxin or dominant-negative Ga i mutant inhibited hepatocyte death by PA or LPC acting through G-protein-coupled receptor (GPCR)/Ga i . PA decreased cardiolipin content and induced mitochondrial potential loss and cytochrome c translocation. Oleic acid inhibited PA-induced hepatocyte death by diverting PA to triglyceride and decreasing LPC content, suggesting that FFAs lead to steatosis or lipoapoptosis according to the abundance of saturated/unsaturated FFAs. LPC administration induced hepatitis in vivo. LPC content was increased in the liver specimens from NASH patients. These results demonstrate that LPC is a death effector in the lipoapoptosis of hepatocytes and suggest potential therapeutic values of PLA 2 inhibitors or GPCR/ Ga i inhibitors in

2 ␤) is considered to play a role in signal transduction and maintenance of homeostasis or remodeling of membrane phospholipids. A role of iPLA 2 ␤ has been suggested in various physiological and pathological processes, including immunity, chemotaxis, and cell death, but the details remain unclear. Accordingly, we investigated mice with targeted disruption of the iPLA 2 ␤ gene. iPLA 2 ␤ Ϫ/Ϫ mice developed normally and grew to maturity, but all showed evidence of severe motor dysfunction, including a hindlimb clasping reflex during tail suspension, abnormal gait, and poor performance in the hanging wire grip test. Neuropathological examination of the nervous system revealed widespread degeneration of axons and/or synapses, accompanied by the presence of numerous spheroids (swollen axons) and vacuoles. These findings provide evidence that impairment of iPLA 2 ␤ causes neuroaxonal degeneration, and indicate that the iPLA 2 ␤ Ϫ/Ϫ mouse is an appropriate animal model of human neurodegenerative diseases associated with mutations of the iPLA 2 ␤ gene, such as infantile neuroaxonal dystrophy and neurodegeneration with brain iron accumulation.

Infantile neuroaxonal dystrophy (INAD) is a fatal neurodegenerative disease characterized by the widespread presence of axonal swellings (spheroids) in the CNS and PNS and is caused by gene abnormality in PLA2G6[calcium-independent phospholipase A 2 ␤ (iPLA 2 ␤)], which is essential for remodeling of membrane phospholipids. To clarify the pathomechanism of INAD, we pathologically analyzed the spinal cords and sciatic nerves of iPLA 2 ␤ knock-out (KO) mice, a model of INAD. At 15 weeks (preclinical stage), periodic acid-Schiff (PAS)-positive granules were frequently observed in proximal axons and the perinuclear space of large neurons, and these were strongly positive for a marker of the mitochondrial outer membrane and negative for a marker of the inner membrane. By 100 weeks (late clinical stage), PAS-positive granules and spheroids had increased significantly in the distal parts of axons, and ultrastructural examination revealed that these granules were, in fact, mitochondria with degenerative inner membranes. Collapse of mitochondria in axons was accompanied by focal disappearance of the cytoskeleton. Partial membrane loss at axon terminals was also evident, accompanied by degenerative membranes in the same areas. Imaging mass spectrometry showed a prominent increase of docosahexaenoic acidcontaining phosphatidylcholine in the gray matter, suggesting insufficient membrane remodeling in the presence of iPLA 2 ␤ deficiency. Prominent axonal degeneration in neuroaxonal dystrophy might be explained by the collapse of abnormal mitochondria after axonal transportation. Insufficient remodeling and degeneration of mitochondrial inner membranes and presynaptic membranes appear to be the cause of the neuroaxonal dystrophy in iPLA 2 ␤-KO mice.

Apoptosis is defined on the basis of morphological changes like nuclear fragmentation and chromatin condensation, which are dependent on caspases. Many forms of caspase-independent cell death have been reported, but the mechanisms are still poorly understood. We found that hypoxic cell death was independent of caspases and was associated with significant nuclear shrinkage. Neither Bcl-2 nor Apaf-1 deficiency prevented hypoxic nuclear shrinkage. To understand the molecular mechanism of the nuclear shrinkage, we developed an in vitro system using permeabilized cells, which allowed us to purify a novel member of the phospholipase A2 (PLA2) family that induced nuclear shrinkage. Purified PLA2 induced nuclear shrinkage in our permeabilized cell system. PLA2 inhibitors prevented hypoxic nuclear shrinkage in cells and cell death. Hypoxia caused elevation of PLA2 activity and translocation of intracellular PLA2s to the nucleus. Knockdown of the Ca2+-independent PLA2 delayed nuclear shrinkage and cell death. These results indicate that Ca2+-independent PLA2 is crucial for a caspase-independent cell death signaling pathway leading to nuclear shrinkage.

To clarify the role of α-synuclein (αSyn) in neuronal membrane remodeling, we analyzed the expression of αSyn in neurons with a dysfunction of PLA2G6, which is indispensable for membrane remodeling. αSyn/phosphorylated-αSyn (PαSyn) distribution and neurodegeneration were quantitatively estimated in PLA2G6-knockout (KO) mice, which demonstrate marked mitochondrial membrane degeneration. We also assessed the relationship between αSyn deposits and mitochondria in brain tissue from patients with PLA2G6-associated neurodegeneration (PLAN) and Parkinson’s disease (PD), and quantitatively examined Lewy bodies (LBs) and neurons. The expression level of αSyn was elevated in PLA2G6-knockdown cells and KO mouse neurons. Strong PαSyn expression was observed in neuronal granules in KO mice before onset of motor symptoms. The granules were mitochondrial outer membrane protein (TOM20)-positive. Ultramicroscopy revealed that PαSyn-positive granules were localized to mitochondria with degenerated inner membranes. After symptom onset, TOM20-positive granules were frequently found in ubiquitinated spheroids, where PαSyn expression was low. Axons were atrophic, but the neuronal loss was not evident in KO mice. In PLAN neurons, small PαSyn-positive inclusions with a TOM20-positive edge were frequently observed and clustered into LBs. The surfaces of most LBs were TOM20-positive in PLAN and TOM20-negative in PD brains. The high proportion of LB-bearing neurons and the preserved neuronal number in PLAN suggested long-term survival of LB-bearing neurons. Elevated expression of αSyn/PαSyn in mitochondria appears to be the early response to PLA2G6-deficiency in neurons. The strong affinity of αSyn for damaged mitochondrial membranes may promote membrane stabilization of mitochondria and neuronal survival in neurons.

Background: Mast cells express cPLA 2 ␣ and iPLA 2 ␤. Results: Knock-out of cPLA 2 ␣, not iPLA 2 ␤, hampers arachidonic acid mobilization in mast cells and adjacent fibroblasts. Conclusion: Mast cell cPLA 2 ␣ is coupled with stromal synthesis of anti-allergic PGE 2 , whereas iPLA 2 ␤ is dispensable for mast cell function. Significance: The cPLA 2 ␣-dependent transcellular PGE 2 synthesis opens new insight into the lipid biochemistry and mast cell biology fields.

The β-catenin mutation is frequently observed in hepatoblastoma (HB), but the underlying mechanism by which Wnt/β-catenin signaling induces HB tumor formation is unknown. Here we show that expression of growth regulation by estrogen in breast cancer 1 (GREB1) depends on Wnt/β-catenin signaling in HB patients. GREB1 is localized to the nucleus where it binds Smad2/3 in a competitive manner with p300 and inhibits TGFβ signaling, thereby promoting HepG2 HB cell proliferation. Forced expression of β-catenin, YAP, and c-Met induces HB-like mouse liver tumor (BYM mice), with an increase in GREB1 expression and HB markers. Depletion of GREB1 strongly suppresses marker gene expression and HB-like liver tumorigenesis, and instead enhances TGFβ signaling in BYM mice. Furthermore, antisense oligonucleotides for GREB1 suppress the formation of HepG2 cell-induced tumors and HB-like tumors in vivo. We propose that GREB1 is a target molecule of Wnt/β-catenin signaling and required for HB progression.

The olfactory epithelial layer contains multipotent horizontal basal cells (HBCs) that differentiate into olfactory sensory neurons. Here, we show that rat HBCs express oligodendrocyte progenitor cell (OPC) and astrocyte markers. We generated olfactory sphere (OS) cells in cultures that were derived from adult rat olfactory mucosa. Fluorescence-activated cell sorting and immunofluorescence analyses showed that OS cells also express OPC and astrocyte markers. Interestingly, OS cells underwent oligodendrocyte differentiation in vitro. To study oligodendrocyte differentiation in vivo, OS cells were transplanted into injured rat spinal cords. The transplanted cells integrated into host tissue and differentiated into oligodendrocytes. When transected saphenous nerve ends were encased in collagen-containing silicone tubes with or without OS cells, the transplanted OS cells differentiated into Schwann cells. Our data provide new insights into of the stemness of OS cells.