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. 2005 Dec;167(6):1713-28.
doi: 10.1016/S0002-9440(10)61253-9.

Participation of autophagy in storage of lysosomes in neurons from mouse models of neuronal ceroid-lipofuscinoses (Batten disease)

Masato Koike  1 , Masahiro Shibata, Satoshi Waguri, Kentaro Yoshimura, Isei Tanida, Eiki Kominami, Takahiro Gotow, Christoph Peters, Kurt von Figura, Noboru Mizushima, Paul Saftig, Yasuo Uchiyama
Affiliations

Participation of autophagy in storage of lysosomes in neurons from mouse models of neuronal ceroid-lipofuscinoses (Batten disease)

Masato Koike et al. Am J Pathol. 2005 Dec.

Abstract

In cathepsin D-deficient (CD-/-) and cathepsins B and L double-deficient (CB-/-CL-/-) mice, abnormal vacuolar structures accumulate in neurons of the brains. Many of these structures resemble autophagosomes in which part of the cytoplasm is retained but their precise nature and biogenesis remain unknown. We show here how autophagy contributes to the accumulation of these vacuolar structures in neurons deficient in cathepsin D or both cathepsins B and L by demonstrating an increased conversion of the molecular form of MAP1-LC3 for autophagosome formation from the cytosolic form (LC3-I) to the membrane-bound form (LC3-II). In both CD-/- and CB-/-CL-/- mouse brains, the membrane-bound LC3-II form predominated whereas MAP1-LC3 signals accumulated in granular structures located in neuronal perikarya and axons of these mutant brains and were localized to the membranes of autophagosomes, evidenced by immunofluorescence microscopy and freeze-fracture-replica immunoelectron microscopy. Moreover, as in CD-/- neurons, autofluorescence and subunit c of mitochondrial ATP synthase accumulated in CB-/-CL-/- neurons. This suggests that not only CD-/- but also CB-/-CL-/- mice could be useful animal models for neuronal ceroid-lipofuscinosis/Batten disease. These data strongly argue for a major involvement of autophagy in the pathogenesis of Batten disease/lysosomal storage disorders.

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Figures

Figure 1
Figure 1
Immunostaining for subunit c of mitochondrial ATP synthase in the cerebral cortex obtained from CD+/− (A, C) and CD−/− (B, D) mice at P13 (A, B) and P23 (C, D) and from CB+/−CL+/− (E) and CB−/−CL−/− (F) mice at P13. A–D: Positive staining for subunit c is intense in neurons (arrowheads) and microglial cells (arrows) of the CD−/− mouse brain (B, D), but not in those of the control littermate brain (A, C). The immunoreactivity is weaker in neurons of CD−/− at P13 (C, arrowheads). E and F: Similar localization patterns of subunit c to those in A to D are observed in the cerebral cortex of CB+/−CL+/− (E) and CB−/−CL−/− (F) mice, although the immunoreactivity is weak in neurons (arrowheads). G–I: Double immunostaining of subunit c and F4/80 in the cerebral cortex of a CB−/−CL−/− mouse at P13. Positive signals for subunit c (red) are large and intense and co-localized in a F4/80-positive microglial cell (arrows), whereas they are fine granular and localized in a F4/80-negative neuron that is marked by arrowheads. Scale bars, 20 μm.
Figure 2
Figure 2
Electron micrographs of neuronal cells from brains of a CD−/− mouse at P23 (A) and a CB−/−CL−/− mouse at P12 (B–E). A–C: In the perikarya of cerebellar Purkinje cells (A, C) and a cerebral cortical neuron (B), numerous dense bodies, which resemble granular osmiophilic deposit-like inclusions (GROD-l) (arrows), can be seen, and vacuolar/autophagosome-like structures (arrowheads) encircled by ER-like membrane saccules and containing cytoplasmic organelles together with part of the cytoplasm are clearly visible. These autophagosome-like structures and GROD-like inclusions are often encircled by ER-like membrane saccules or multilamellated structures. D and E: In axons located in the corpus callosum of the same mouse as in B and C, accumulated autophagosome-like structures can be seen (D), while these structures accumulated in a myelinated axon are distinctly encircled by ER-like membrane saccules (E, small arrows). Moreover, a microglia-like cell (MG) appears in this field and possesses electron-dense heterophagosomes (asterisk) containing degenerating axons with numerous autophagosome-like structures (inset in D). The cytoplasm around the heterophagosome in the cell appears intact. Scale bars, 1 μm.
Figure 3
Figure 3
Morphometric analysis of various lysosomal compartments in the perikarya of CD−/− and CD+/− mouse neurons. A–E: Representative examples of early (AVi; A, D) and late (AVd; B) AVs, granular osmiophilic deposit-like inclusions (GROD-l; E), and a fingerprint profile (FP; F) from CD−/− neurons obtained at P23, and a normal dense body (C) from a CD+/− neuron obtained at P23 are shown. In AVi, a mitochondrion (m) together with part of the cytoplasm is clearly visible (A), while in AVd, a mitochondrion with partially degraded cytoplasmic materials is also identifiable. D: AVi that contains GROD-l is detectable. Small arrows show double membranes surrounding GROD-l (asterisks). To show the fingerprint pattern clearly, a boxed area in F is enlarged in an inset. G: The volume densities (%) of AVi, AVd, dense body, GROD-l, and fingerprint profiles (FPs) in the perikarya of neurons in the layer V of the cerebral cortex obtained from CD+/− and CD−/− mice at P8, P15, and P23 and CB−/−CL−/− and CB+/−CL+/− littermates at P13 are shown as a stack bar chart. Scale bars: 0.1 μm (A–F); 0.2 μm (inset in F).
Figure 4
Figure 4
Molecular forms of LC3 in nervous tissues of CD−/− and CB−/−CL−/− mice and their littermate controls. A and B: Immunoblotting of LC3 in brains of CD−/− mice and their littermate controls (CD+/−) obtained at P8, P15, and P22 (A), and ratios of densities of LC3-II to LC3-I protein bands (B). C and D: Immunoblotting of LC3 in retinae of CD−/− mice and their littermate controls (CD+/−) obtained at P24 (C), and ratios of densities of LC3-II to LC3-I protein bands (D). E and F: Immunoblotting of LC3 in brains from CB−/−CL−/− and CB+/−CL+/− mice at P13 (E), and the ratios of densities of LC3-II to LC3-I protein bands (F). G and H: Expression of LC-3 mRNA in brain tissues of CD−/− and CB−/−CL−/− mice and their littermate controls. A RT-PCR study. Expression levels of LC-3 mRNA do not differ between CD−/− and CD+/− mice at all stages examined (G, top), and between CB−/−CL−/− and CB+/−CL+/− mice (H, top). Bottom panels show β-actin mRNA, used as internal controls.
Figure 5
Figure 5
Immunohistochemical staining of LC3 in brain tissues of CD−/− and CB−/−CL−/− mice and their littermate controls. A and B: Cerebral cortical regions of CD+/− (A) and CD−/− (B) mouse brains at P24. Positive staining of LC3 appears fibrillary in dendrites of pyramidal neurons and diffuse in their perikarya (A, inset). Its staining is also fibrillary in dendrites of the neurons but appears granular in their perikarya (B, inset). C and D: Purkinje cells in the cerebellum of CB+/−CL+/− (C) and CB−/−CL−/− (D) mice at P12. Fibrillary staining of LC3 is demonstrated in dendrites of Purkinje cells from both control and mutant mice (C, D), whereas its staining is relatively weak and diffuse in the perikarya of CB+/−CL+/− control neurons (C, inset) but intense and granular in those of CB−/−CL−/− neurons (D, inset). E and F: Nerve fibers in the corpus callosum of CB+/−CL+/− (E) and CB−/−CL−/− (F) mice at P12. No clear-cut staining for LC3 can be seen in the control axons (E), whereas intensely stained dots are accumulated in the mutant axons. Scale bars: 50 μm (A–F); 20 μm (insets in A–D).
Figure 6
Figure 6
Double-immunofluorescent staining of LC3 (A, D, G, J; red color) with lamp1 (B, E, H; green color) in cerebral cortical neurons (A–C) of CD−/− mice at P23, cerebellar Purkinje cells (D–F), and the corpus callosum (G–I) of the CB−/−CL−/− mice at P13 or with F4/80 (J, green color) in the corpus callosum (J–L) of the CB−/−CL−/− mice at P13. A–F: A considerable number of LC3-positive granules are also stained for lamp1, but certain numbers of LC3-positive granules (arrows), which are large in size and stain intensely, are distinct from lamp1 (C and F, overlay). G–I: LC3-positive staining in the corpus callosum (arrows) is primarily distinct from lamp1 staining (I, overlay). J–L: F4/80-positive microglial cells possess LC3-positive staining (arrows) (L, overlay). Scale bars, 15 μm.
Figure 7
Figure 7
Immunocytochemical freeze-fracture replica images of an anterior spinal neuron (A) and cerebellar Purkinje cells (B, C) of CD−/− mice at P20 incubated with an anti-LC3 antibody. A: Immunogold labeling is observed on the membrane-bound organelles accumulated in the cytoplasm of the anterior neuron (arrowheads). B and C: In the perikarya of Purkinje cells, positive signals are well localized on membranes of two granules that are further encircled by membrane structures (thin arrows) (B), while they are also detected on multilamellar membranes (thick arrows). Scale bars, 0.25 μm.
Figure 8
Figure 8
Expression of GFP-LC3 in brains of GFP-LC3/CD−/− mice. A: Molecular forms of GFP-LC3 in brains of GFP-LC3/CD+/+ and GFP-LC3/CD−/− mice at P20. GFP-LC3-I is a major form in both GFP-LC3/CD+/+ and GFP-LC3/CD−/− brains, whereas GFP-LC3-II appears faintly in GFP-LC3/CD+/+ brains and distinctly in GFP-LC3/CD−/− brains when immunostained with either anti-GFP or anti-LC3. Moreover, the quantitative relationship of LC3-II to LC3-I in brains of CD+/+ and CD−/− mice does not differ depending on GFP-LC3 expression. B–E: Immunohistochemical images of GFP-LC3 and LC3 (B and C), or GFP-LC3 (D and E) in cerebellar Purkinje cells of GFP-LC3/CD+/+ (B and D) and GFP-LC3/CD−/− (C and E) mice at P20. Immunoreactivity for LC3-GFP and LC3, that was immunoreacted with anti-LC3, is more distinct and intense in dendrites and bodies of Purkinje cells than that for GFP-LC3 that was immunostained with anti-GFP. Diffuse or fibrillary staining for GFP-LC3 and LC3, or GFP-LC3 is clear in dendrites and bodies of Purkinje cells from a GFP-LC3/CD+/+ mouse, whereas granular staining for them (spheroids, arrows) is observed not only in Purkinje cell bodies but also in their dendrites from a GFP-LC3/CD−/− mouse. F–H: Double-fluorescence images of lamp1 (red color, F) and GFP-LC3 (green, G) in cerebral cortical neurons of GFP-LC3/CD−/− brains at P20. Lamp1 immunoreactivity is often localized in GFP-LC3-positive granules, but a considerable number of GFP-LC3-positive granules are distinct from lamp1 immunoreactivity (H). Scale bars: 50 μm (A–E); 10 μm (F–H).
Figure 9
Figure 9
Expression of LC3 in peripheral tissues of CD−/− (A–E) and CB−/−CL−/− (F, G) mice and their littermate controls (CD+/−, CB+/−CL+/−). A and B: Molecular forms of LC3 in the liver of CD+/− and CD−/− mice at P15 and P22 (A), and densities of LC3-II (B). C and D: Molecular forms of LC3 in the heart of CD+/− and CD−/− mice at P15 and P22 (C), and densities of LC3-II (D). E and F: Molecular forms of LC3 in the liver and heart of CB+/−CL+/− and CB−/−CL−/− mice at P13 (E), and densities of LC3-II (F).
Figure 10
Figure 10
Immunostaining of LC3 in hepatic (A, B) and cardiac (C, D) tissues of CD+/− (A, C) and CD−/− (B, D) mice at P23. Punctate staining for LC3 is distinct in hepatocytes and cardiac muscular cells of CD−/− mice. Scale bar, 20 μm.
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