PLA2 activity is required for nuclear shrinkage in caspase-independent cell death

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

Isolation of human adult olfactory sphere cells as a cell source of neural progenitors

Olfactory stem cells are generated from olfactory mucosa. Various culture conditions generate olfactory stem cells that differ according to species and developmental stage and have different progenitor or stem cell characteristics. Olfactory spheres (OSs) are clusters of progenitor or stem cells generated from olfactory mucosa in suspension culture. In this study, adult human OSs were generated and their characteristics analyzed. Human OSs were adequately produced from olfactory mucosa with area over 40 mm2. Immunocytochemistry (ICC) and fluorescence-activated cell sorting showed that human OSs were AN2 and A2B5-positive. Immunofluorescence analysis of cell type-specific ICC indicated that the number of Tuj1-positive OS cells was significantly elevated. Tuj1-positive cells displayed typical neuronal soma and dendritic morphology. Human OS cells were also immunopositive for MAP2. By contrast, few RIP-, O4-, and GFAP-positive cells were present. These RIP, O4, and GFAP-positive cells did not resemble bona fide oligodendrocytes and astrocytes morphologically. In culture to induce differentiation of oligodendrocytes, human OS cells also expressed neuronal markers, but neither oligodendrocyte or astrocyte markers. These findings suggest that human OS cells autonomously differentiate into neurons in our culture condition and have potential to be used as a cell source of neural progenitors for their own regenerative grafts, avoiding the need for immunosuppression and ethical controversies

Immunohistochemical analyses of VMAT2 in the striatum of iPLA₂β-KO mice at 100 weeks.

<p>(A, B): Abnormal structures strongly positive for VMAT2 were also seen in KO mice at 100 weeks (large arrows in A and B); these structures contact striatal neurons (N) (small arrows in insets in A and B). In serial sections, an abnormal structure positive for VMAT2 (an arrow in C) is also immunopositive for TH (arrow in D). Scale bar in (A) represents 25 μm in all panels (A-D).</p

Double immunohistochemistry to detect ubiquitin and TH in the striatum of iPLA₂β-KO mice.

<p>(A, B): Double immunostaining of TH (brown) and ubiquitin (blue) in the striatum of iPLA₂β-KO mice at 15 weeks (A) and 100 weeks (B). (A): At 15 weeks, a few round, TH-positive structures were seen (brown, an arrow), while blue staining was not found in this view (immunohistochemistry for ubiquitin is negative). (B): At 100 weeks, several ubiquitin-positive structures were found (blue staining, arrowheads), but these were distinct from the round, TH-positive structures (brown staining, arrows). Scale bar in (A) represents 25 μm in (A) and (B).</p

Immunohistochemical analysis for TH in the striatum of WT control mice and iPLA₂β-KO mice.

<p>Representative photographs of the striatum immunostained with TH in WT mice at 15 weeks (A, B), 56 weeks (C, D), and 100 weeks (E, F) and iPLA₂β-KO mice at 15 weeks (G, H), 56 weeks (I, J), and 100 weeks (K, L). (A), (C), (E), (G), (I), and (K) are low power fields (LPF) and (B), (D), (F), (H), (J), (L) are high power fields (HPF). (A-F): In the low power fields, neuropils of the striatum are diffusely stained with TH in WT mice at 15 weeks (A), 56 weeks (C), and 100 weeks (E). In the high power fields, many nerve fibers strongly immunopositive for TH were observed in neuropils of WT mice at 15 weeks (B), 56 weeks (D), and 100 weeks (F). The insets in (B), (D), (F), and (H) are high magnifications of the dotted square from their respective panel. (G-L): In the low power fields, neuropils of the striatum are diffusely stained with TH in iPLA₂β-KO mice at 15 weeks (G), 56 weeks (I), and 100 weeks (K). In the high power fields, many fibers positive for TH were observed in neuropils of the striatum in iPLA₂β-KO mice at 15 weeks (H), which are almost equal in number to those of WT mice at 15 weeks (B). In iPLA₂β-KO mice at 56 weeks, focal loss of TH-positive fibers is seen in some areas (dotted circle in J-3), while the density of TH-positive fibers (arrows in J-3) is preserved in other areas (J-2, 3). In iPLA₂β-KO mice at 100 weeks, the density of TH-positive fibers (arrows in L-2) are lower than that of WT mice at 100 weeks (F) and iPLA₂β-KO mice at 56 weeks (J-2, 3). Panels (J-2), (J-3), and (L-2) are high magnifications of the dotted squares in (J) and (L), respectively. Scale bar in (A) represents 100 μm in (A), (C), (E), (G), (I), and (K), and 25 μm in (B), (D), (F), (H), (J), and (L).</p

Quantitative analysis of optical densities of TH and DAT in the striatum.

<p>Histograms show quantitative analysis of optical densities of TH (A) and DAT (B) immunostaining in the striatum. WT mice, gray bars; KO mice, black bars. Data are presented as the mean ± standard deviation. The number (n) of animals examined is indicated in each histogram. Vertical axis in both (A) and (B) shows percent density relative to WT mice at 15 weeks. (A) Symbols indicate statistically significant differences; *<i>p</i> < 0.05 vs. age-matched WT mice and iPLA₂β-KO mice at 15 weeks (Wilcoxon’s rank sum test). (B) Symbols indicate statistically significant differences; *<i>p</i> < 0.05 vs. age-matched WT mice and iPLA₂β-KO mice at 15 weeks (Wilcoxon’s rank sum test) and **<i>p</i> < 0.05 vs. iPLA₂β-KO mice at 56 weeks (Wilcoxon’s rank sum test).</p

Immunohistochemical analysis of TH in the amygdala of WT control mice and iPLA₂β-KO mice.

<p>Representative photographs of the amygdala immunostained for TH in WT mice at 15 weeks (A), 56 weeks (B), and 100 weeks (C) and iPLA₂β-KO mice at 15 weeks (D), 56 weeks (E, G), and 100 weeks (F). A section immunostained with TH in the striatum of iPLA₂β-KO mice at 56 weeks is also shown (H). (B), (E), (G), and (H) are frozen sections. The inset in (E) is a high magnification of the dotted square. (A–C): Distal regions of the axons of dopaminergic neurons are positive for TH in the amygdala of WT mice. (D): In iPLA₂β-KO mice at 15 weeks, TH-positive nerve fibers similar to those of WT mice were observed (A-C). (E, G): In iPLA₂β-KO mice at 56 weeks, round structures positive for TH are seen. Some are adjacent to TH-positive nerve fibers (small arrows in E). The round, TH-positive structures lie in a row like a string of beads (G). (F): Many round, TH-positive structures were seen in iPLA₂β-KO mice at 100 weeks (arrows in F). (H): Beaded, round TH-positive structures were also seen in the striatum of iPLA₂β-KO mice at 56 weeks. Scale bar in (A) represents 25 μm in all panels (A-H).</p

Deficiency of Calcium-Independent Phospholipase A2 Beta Induces Brain Iron Accumulation through Upregulation of Divalent Metal Transporter 1

<div><p>Mutations in <i>PLA2G6</i> have been proposed to be the cause of neurodegeneration with brain iron accumulation type 2. The present study aimed to clarify the mechanism underlying brain iron accumulation during the deficiency of calcium-independent phospholipase A2 beta (iPLA₂β), which is encoded by the <i>PLA2G6</i> gene. Perl’s staining with diaminobenzidine enhancement was used to visualize brain iron accumulation. Western blotting was used to investigate the expression of molecules involved in iron homeostasis, including divalent metal transporter 1 (DMT1) and iron regulatory proteins (IRP1 and 2), in the brains of iPLA₂β-knockout (KO) mice as well as in <i>PLA2G6</i>-knockdown (KD) SH-SY5Y human neuroblastoma cells. Furthermore, mitochondrial functions such as ATP production were examined. We have discovered for the first time that marked iron deposition was observed in the brains of iPLA₂β-KO mice since the early clinical stages. DMT1 and IRP2 were markedly upregulated in all examined brain regions of aged iPLA₂β-KO mice compared to age-matched wild-type control mice. Moreover, peroxidized lipids were increased in the brains of iPLA₂β-KO mice. DMT1 and IRPs were significantly upregulated in <i>PLA2G6</i>-KD cells compared with cells treated with negative control siRNA. Degeneration of the mitochondrial inner membrane and decrease of ATP production were observed in <i>PLA2G6</i>-KD cells. These results suggest that the genetic ablation of iPLA₂β increased iron uptake in the brain through the activation of IRP2 and upregulation of DMT1, which may be associated with mitochondrial dysfunction.</p></div