Article Figures & Data
Figures
- Figure 1.
In situ hybridization of iPLA2β mRNA in the nervous system. A–H, The pattern of iPLA2β mRNA expression in the cerebral cortex (A, B), the cerebellum (C, D), the spinal cord (E, F), and the dorsal root ganglion (DRG) (G, H) of 7-week-old female mice. The antisense probe detected signals in all cortical layers, except for layer I, of the cingulate (cg), the motor (mo) cortex, and the somatosensory (ss) cortex, in the Purkinje cell layer (pl) and the granular layer (gl), but not the molecular layer (ml) of the cerebellum, in the dorsal (do) and ventral (ve) horns of the spinal cord, and the soma-rich region of the DRG (so). No signals were detected in the corpus callosum (cc) or in the axon-rich region (ax) that contains mainly axons and oligodendrocytes (cc) or Schwann cells (ax). The sense probe was used as a negative control (B, D, F, H). Scale bars: A–F, 500 μm; G, H, 180 μm.
- Figure 2.
Targeting the iPLA2β gene. A, Diagram of the wild-type iPLA2β gene, the targeting construct, and the disrupted iPLA2β allele. B, C, Southern blot analysis of BglII-digested genomic DNA from ES cell clones (B) and from the tails of iPLA2β+/+, iPLA2β+/−, and iPLA2β−/− mice (C). The DNA probes used for Southern blot analysis were a probe A and B containing exon 4 and neomycin resistance gene (neo), respectively. The iPLA2β+/+ wild-type allele generated a band of 10.9 kbp, whereas the disrupted allele yielded a 15.5 kbp fragment. D, Western blotting of testicular lysates with an anti-iPLA2β antibody (α-tubulin was used as the loading control).
- Figure 3.
Lower body weight and shorter lifespan of iPLA2β−/− mice. A, Body weight of female iPLA2β+/+ mice (n = 9), iPLA2β+/− mice (n = 17), and iPLA2β−/− mice (n = 14) at 91–100 weeks of age. Bars indicate the mean values. p values were calculated using the Student's t test. B, Survival of female iPLA2β+/+ mice (n = 9), iPLA2β+/− mice (n = 20), and iPLA2β−/− mice (n = 19). p values were calculated using the log-rank test. Results obtained with male mice were virtually identical.
- Figure 4.
Motor dysfunction in iPLA2β−/− mice. A, Representative photographs of an iPLA2β+/+ mouse (a–c) and an iPLA2β−/− mouse (d–f) at 95 weeks of age. The iPLA2β−/− mouse adopts a feet-clasping posture when suspended by the tail (d, e), whereas the iPLA2β+/+ mouse holds its hindlimbs outward to steady itself (a, b). The iPLA2β−/− mouse has a hunched posture (f). B, Representative footprint patterns of iPLA2β+/− and iPLA2β−/− mice at 90 and 87 weeks of age, respectively. Forepaws and hindpaws were painted with blue and red ink, respectively. The iPLA2β−/− mouse displays an irregular stride and dragging of the hindlimbs. C, D, Hanging wire grip test. C, Female iPLA2β+/+ mice (n = 9), iPLA2β+/− mice (n = 17), and iPLA2β−/− mice (n = 13) (91–100 weeks old) were tested as described in Materials and Methods, and the total time for three trials was plotted against body weight. D, Female iPLA2β+/+ mice (n = 23), iPLA2β+/− mice (n = 25), and iPLA2β−/− mice (n = 42) (15–55 weeks old) were tested. The total time for three trials was plotted against age.
- Figure 5.
Neuropathological changes in iPLA2β−/− mice. A, B, Cuneate nucleus of an iPLA2β+/+ mouse (A) and an iPLA2β−/− mouse (B) at 2 years (95–103 weeks old). C, Dorsal horn of iPLA2β−/− mouse at 2 years. D–F, Ventral horn of an iPLA2β+/+ mouse at 56 weeks (D) and an iPLA2β−/− mouse at 15 weeks (E) and 2 years (F). G, H, Cerebellum of an iPLA2β+/+ mouse (G) and an iPLA2β−/− mouse (H) at 2 years. I, J, Molecular layer (I) and dentate nucleus (J) of the cerebellum of an iPLA2β−/− mouse at 15 weeks. K, L, Cerebral cortex of an iPLA2β+/+ mouse (K) and an iPLA2β−/− mouse (L) at 2 years. A, B, G–L, H&E stain. C–F, PAS stain. A, No spheroids and vacuoles are detected in the iPLA2β+/+ mouse. B, Many spheroids (arrows) and large vacuoles (large arrows) can be observed. C, Irregular PAS-positive spheroids (small arrows) contain fissures, vacuoles, and PAS-positive granules. The extent of PAS staining is variable. The core of the huge vacuole contains PAS-positive granules (large arrow and open arrow; also shown at a higher magnification). D, No PAS-positive granules are observed in the iPLA2β+/+ mouse. E, Strongly PAS-positive granules can be seen in normal-looking axons or the neuropil. F, PAS-positive granules (arrows) in swollen axons. G, H, The molecular layer (ml) of the cerebellum from the iPLA2β−/− mouse (H) is thinner than that from the iPLA2β+/+ mouse (G). I, Numerous tiny vacuoles (arrows) can be seen in the molecular layer. J, There are only a few spheroids (arrows). K, There are no detectable vacuoles in the iPLA2β+/+ mouse. L, Numerous small vacuoles (arrows) can be observed, but there are no spheroids. Scale bars: A–C, 50 μm; D–F, I–L, 16 μm; G, H, 100 μm. v, Vessels.
- Figure 6.
Pathological changes of the PNS. A–C, Dorsal root of an iPLA2β+/− mouse (A) and an iPLA2β−/− mouse (B, C) at 2 years. D, Subcutaneous nerve fascicle of an iPLA2β−/− mouse at 2 years. A, B, and D are PAS stained. C, Bielschowsky stain. Scale bar, 16 μm. A, Axons of the iPLA2β+/− mouse are PAS negative, although the myelin sheath is weakly stained by PAS. B, Swollen axons (arrows) are heterogeneously stained by PAS and contain strongly PAS-positive granules. C, A single swollen axon contains both spheroids (open arrows) and a vacuole (arrow). The central parts of the spheroids (open arrows) are rarefied. Staining of the axonal structures (arrowheads) between the spheroids is weak. D, PAS-positive granules can be seen in the nerve fascicle.
- Figure 7.
Immunohistochemical analysis of spheroids and vacuoles in iPLA2β−/− mice at 2 years. A, Ventral horn was stained with anti-phosphorylated neurofilament H (SMI31). B, C, Vestibular nucleus (B) and cerebral cortex (C) were stained with anti-ubiquitin antibody. v, Vessels. Scale bars: A, 20 μm; B, 10 μm; C, 16 μm. A, Spheroids (arrows) are positive for SMI31, whereas the axonal portion between the spheroids (arrowhead) is negative. B, Two spheroids (arrows) have positive cores. The other spheroid (asterisk) contains vacuoles that are weakly stained or unstained for ubiquitin. C, Numerous vacuoles (arrows) and tiny circles (arrowheads) that are ubiquitin positive in the neuropil of the cerebral cortex.
- Figure 8.
Electron microscopic features of spheroids. A, B, Dorsal funiculus of a iPLA2β−/− mouse at 56 weeks. B shows a higher magnification of the boxed region in A. Tubulovesicular structures can be seen in the spheroid, as well as degenerated vesicles and vacuoles. Scale bars: A, 2 μm; B, 0.5 μm.
Tables
Region Spheroids Vacuoles Cuneate nucleus +++ ++ Gracile nucleus +++ ++ Trigeminal nucleus +++ ++ Facial nucleus ++ + Cochlear nucleus +++ ++ Vestibular nucleus +++ ++ Hypoglossal nucleus + + Spinal cord dorsal horn +++ ++ ventral horn ++ ++ dorsal funiculus ++ ++ ventral funiculus ++ ++ Striatum + ++ Thalamus + ++ Hypothalamus + ++ Hippocampus + ++ (small)a Amygdala + ++ Cerebellum molecular layer + ++ (small)a granular layer + ++ dentate nucleus ++ ++ Cerebral cortex + / − +++ (small)a -
Distribution of spheroids and vacuoles in the CNS were assessed and graded as follows: +/−, very few; +, mild degree; ++, moderate degree; +++, marked degree.
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↵aVacuoles of small size were predominantly observed. Mice aged 95–103 weeks were examined.
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Density of myelinated fibers (per mm2) Total area of nerve fascicles (mm2) Number of myelinated fibers (density × area) +/+ (n = 4) 20156 ± 4007 0.189 ± 0.037 3735 ± 554 −/− (n = 7) 22922 ± 1695 0.136 ± 0.117 3115 ± 234 (p < 0.05) (p < 0.05) -
The number of myelinated fibers per unit area was counted in sciatic nerves. Mice aged 95–103 weeks old were examined. p values were calculated using the Student's t test.
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Supplemental Data
Files in this Data Supplement:
- supplemental material - Supplemental Legend
- supplemental material - Supplemental Figure 1
- supplemental material - Supplemental Figure 2
