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. 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. Autophagy is a highly regulated process involving the bulk degradation of cytoplasmic macromolecules and organelles in eukaryotic cells via the lysosomal/vacuolar system.1Klionsky DJ Emr SD Autophagy as a regulated pathway of cellular degradation.Science. 2000; 290: 1717-1721Crossref PubMed Scopus (2901) Google Scholar Although it is induced under starvation, differentiation, and normal growth control,2Vittorini S Paradiso C Donati A Cavallini G Masini M Gori Z Pollera M Bergamini E The age-related accumulation of protein carbonyl in rat liver correlates with the age-related decline in liver proteolytic activities.J Gerontol A Biol Sci Med Sci. 1999; 54: B318-B323Crossref PubMed Scopus (94) Google Scholar the participation of autophagy has also been demonstrated in various neurodegenerative disorders.3Larsen KE Sulzer D Autophagy in neurons: a review.Histol Histopathol. 2002; 17: 897-908PubMed Google Scholar Moreover, it has been shown that autophagy can trigger a form of cell death distinct from apoptosis in neurons.4Uchiyama Y Autophagic cell death and its execution by lysosomal cathepsins.Arch Histol Cytol. 2001; 64: 233-246Crossref PubMed Scopus (209) Google Scholar, 5Yuan J Lipinski M Degterev A Diversity in the mechanisms of neuronal cell death.Neuron. 2003; 40: 401-413Abstract Full Text Full Text PDF PubMed Scopus (402) Google Scholar, 6Ohsawa Y Isahara K Kanamori S Shibata M Kametaka S Gotow T Watanabe T Kominami E Uchiyama Y An ultrastructural and immunohistochemical study of PC12 cells during apoptosis induced by serum deprivation with special reference to autophagy and lysosomal cathepsins.Arch Histol Cytol. 1998; 61: 395-403Crossref PubMed Scopus (61) Google Scholar Thus autophagy appears to be involved in neurodegenerative disorders. The most common inherited neurodegenerative disease in childhood is neuronal ceroid-lipofuscinosis (NCL, or Batten disease), which is categorized as a lysosomal storage disorder and pathologically characterized by the accumulation of proteolipids, such as subunit c of mitochondrial ATP synthase and sphingolipid activator proteins in the lysosomes of neurons.7Hall NA Lake BD Dewji NN Patrick AD Lysosomal storage of subunit c of mitochondrial ATP synthase in Batten's disease (ceroid-lipofuscinosis).Biochem J. 1991; 275: 269-272Crossref PubMed Scopus (141) Google Scholar, 8Kominami E Ezaki J Muno D Ishido K Ueno T Wolfe LS Specific storage of subunit c of mitochondrial ATP synthase in lysosomes of neuronal ceroid lipofuscinosis (Batten's disease).J Biochem (Tokyo). 1992; 111: 278-282Crossref PubMed Scopus (110) Google Scholar, 9Palmer DN Martinus RD Cooper SM Midwinter GG Reid JC Jolly RD Ovine ceroid lipofuscinosis. The major lipopigment protein and the lipid-binding subunit of mitochondrial ATP synthase have the same NH2-terminal sequence.J Biol Chem. 1989; 264: 5736-5740Abstract Full Text PDF PubMed Google Scholar, 10Fearnley IM Walker JE Martinus RD Jolly RD Kirkland KB Shaw GJ Palmer DN The sequence of the major protein stored in ovine ceroid lipofuscinosis is identical with that of the dicyclohexylcarbodiimide-reactive proteolipid of mitochondrial ATP synthase.Biochem J. 1990; 268: 751-758Crossref PubMed Scopus (140) Google Scholar We have previously demonstrated that the central nervous system (CNS) neurons in cathepsin D-deficient (CD−/−) mice show a new form of lysosomal accumulation disease with a phenotype resembling NCLs and subunit c of mitochondrial ATP synthase accumulates in the lysosomes of the affected neurons.11Koike M Nakanishi H Saftig P Ezaki J Isahara K Ohsawa Y Schulz-Schaeffer W Watanabe T Waguri S Kametaka S Shibata M Yamamoto K Kominami E Peters C von Figura K Uchiyama Y Cathepsin D deficiency induces lysosomal storage with ceroid lipofuscin in mouse CNS neurons.J Neurosci. 2000; 20: 6898-6906Crossref PubMed Google Scholar, 12Nakanishi H Zhang J Koike M Nishioku T Okamoto Y Kominami E von Figura K Peters C Yamamoto K Saftig P Uchiyama Y Involvement of nitric oxide released from microglia-macrophages in pathological changes of cathepsin D-deficient mice.J Neurosci. 2001; 21: 7526-7533Crossref PubMed Google Scholar Double membrane-bound vacuoles containing part of the cytoplasm are frequently detected in CNS neurons of CD−/− mouse brains near the terminal stage. It has also been demonstrated that the phenotypes of mice deficient in both cathepsins B and L (CB−/−CL−/− mice) resemble NCLs, but the accumulated substances show no autofluorescence and are immunonegative for subunit c and saposins.13Felbor U Kessler B Mothes W Goebel HH Ploegh HL Bronson RT Olsen BR Neuronal loss and brain atrophy in mice lacking cathepsins B and L.Proc Natl Acad Sci USA. 2002; 99: 7883-7888Crossref PubMed Scopus (262) Google Scholar However, in neurons of these CD−/− and CB−/−CL−/− mouse brains, the exact nature and biogenesis of the accumulated lysosomal structures remain unknown. It has recently been found that LC3, light chain 3 of neuronal microtubule-associated protein 1A/B, is a mammalian homolog of yeast Atg8p that is required for autophagosome formation.14Kabeya Y Mizushima N Ueno T Yamamoto A Kirisako T Noda T Kominami E Ohsumi Y Yoshimori T LC3, a mammalian homologue of yeast Apg8p, is localized in autophagosome membranes after processing.EMBO J. 2000; 19: 5720-5728Crossref PubMed Scopus (5306) Google Scholar Immediately after the synthesis of LC3, the protein is converted to the cytoplasmic form, LC3-I, by cleavage at the C-terminal region,14Kabeya Y Mizushima N Ueno T Yamamoto A Kirisako T Noda T Kominami E Ohsumi Y Yoshimori T LC3, a mammalian homologue of yeast Apg8p, is localized in autophagosome membranes after processing.EMBO J. 2000; 19: 5720-5728Crossref PubMed Scopus (5306) Google Scholar which is further converted into a membrane-bound form, LC3-II, by Atg7p and Atg3p, respectively, when autophagy is induced.15Ohsumi Y Molecular dissection of autophagy: two ubiquitin-like systems.Nat Rev Mol Cell Biol. 2001; 2: 211-216Crossref PubMed Scopus (1028) Google Scholar, 16Tanida I Tanida-Miyake E Ueno T Kominami E The human homolog of Saccharomyces cerevisiae Apg7p is a protein-activating enzyme for multiple substrates including human Apg12p, GATE-16, GABARAP, and MAP-LC3.J Biol Chem. 2001; 276: 1701-1706Crossref PubMed Scopus (271) Google Scholar, 17Tanida I Tanida-Miyake E Komatsu M Ueno T Kominami E Human Apg3p/Aut1p homologue is an authentic E2 enzyme for multiple substrates, GATE-16, GABARAP, and MAP-LC3, and facilitates the conjugation of hApg12p to hApg5p.J Biol Chem. 2002; 277: 13739-13744Crossref PubMed Scopus (219) Google Scholar Moreover, LC3-II is localized mainly in the membranes of autophagosomes and, to a lesser extent, within autophagolysosomes.14Kabeya Y Mizushima N Ueno T Yamamoto A Kirisako T Noda T Kominami E Ohsumi Y Yoshimori T LC3, a mammalian homologue of yeast Apg8p, is localized in autophagosome membranes after processing.EMBO J. 2000; 19: 5720-5728Crossref PubMed Scopus (5306) Google Scholar We therefore investigated whether autophagy participates in the accumulation of lysosomal structures in the brain and peripheral tissues of mice deficient in cathepsin D and those doubly deficient in cathepsins B and L by characterizing molecular forms of LC3. Our data showing that the conversion of LC3-I to LC3-II in CD−/− and CB−/−CL−/− mouse brains coincides with the accumulation of lysosomal structures or LC3-positive granules in the neurons, suggest that autophagy is involved in the abnormal storage accumulation in CNS neurons of NCL/lysosomal storage disorders. The experiments described here were performed in compliance with the regulations of Osaka University Medical School Guideline for the Care and Use of Laboratory Animals. For cathepsin D-, B-, or L-deficient mice,18Deussing J Roth W Saftig P Peters C Ploegh HL Villadangos JA Cathepsins B and D are dispensable for major histocompatibility complex class II-mediated antigen presentation.Proc Natl Acad Sci USA. 1998; 95: 4516-4521Crossref PubMed Scopus (226) Google Scholar, 19Saftig P Hetman M Schmahl W Weber K Heine L Mossmann H Koster A Hess B Evers M von Figura K Mice deficient for the lysosomal proteinase cathepsin D exhibit progressive atrophy of the intestinal mucosa and profound destruction of lymphoid cells.EMBO J. 1995; 14: 3599-3608Crossref PubMed Scopus (356) Google Scholar, 20Nakagawa T Roth W Wong P Nelson A Farr A Deussing J Villadangos JA Ploegh H Peters C Rudensky AY Cathepsin L: critical role in Ii degradation and CD4 T cell selection in the thymus.Science. 1998; 280: 450-453Crossref PubMed Scopus (580) Google Scholar heterozygous (+/−) mice were transferred to the Institute of Experimental Animal Sciences, Osaka University Graduate School of Medicine, and kept in conventional or specific pathogen-free facilities on a 12-hour light/dark cycle. To obtain homozygous mice (CD−/−, CB−/−, and CL−/−) mice, heterozygous mice (CD+/−, CB+/−, and CL+/−, respectively) were intercrossed. Selection of homozygous mice from littermates obtained by heterozygous coupling was performed by genomic polymerase chain reaction (PCR) as described previously.21Koike M Shibata M Ohsawa Y Nakanishi H Koga T Kametaka S Waguri S Momoi T Kominami E Peters C Figura K Saftig P Uchiyama Y Involvement of two different cell death pathways in retinal atrophy of cathepsin D-deficient mice.Mol Cell Neurosci. 2003; 22: 146-161Crossref PubMed Scopus (122) Google Scholar, 22Saftig P Peters C von Figura K Craessaerts K Van Leuven F De Strooper B Amyloidogenic processing of human amyloid precursor protein in hippocampal neurons devoid of cathepsin D.J Biol Chem. 1996; 271: 27241-27244Crossref PubMed Scopus (80) Google Scholar Briefly, for the selection of CD−/− mice, the template genomic DNA isolated from tail biopsies was examined by CD-exon 4-specific PCR with primers having the following sequences: MCD14 (5′-AGACTAACAGGCCTGT-TCCC-3′) and MCD15 (5′-TCAGCTGTAGTTGCTCA-CATG-3′). The selection of CB−/− mice from littermates obtained by heterozygous coupling was performed by examining the template genomic DNA isolated from tail biopsies, using CB-exon 4-specific PCR with primers of MCB11 (5′-GGTTGCGTTCGGTGAGG-3′) and MCBGT (5′-AACAAGAGCCGCAGGAGC-3′). The selection of CL−/− mice from littermates obtained by heterozygous coupling was performed by the triple PCR method; the template genomic DNA isolated from tail biopsies was examined using primers of MCL5 (5′-GGAGGAGAGCGATATGGG-3′), MCL9 (5′-AGCCATTCACCACCTGC-C-3′), and neo756-777 (5′-CGCAGAACCTGCGTGCA-ATCC-3′). For the generation of CB and CL double-knockout (CB−/−CL−/−) mice, CB−/− mice were bred to CL−/− mice to create progeny that were heterozygous for both cathepsins B and L deletions (CB+/−CL+/− mice). These double heterozygotes were intercrossed to produce mice doubly deficient in both cathepsins B and L (CB−/−CL−/− mice). Most died at approximately P14, as previously reported.13Felbor U Kessler B Mothes W Goebel HH Ploegh HL Bronson RT Olsen BR Neuronal loss and brain atrophy in mice lacking cathepsins B and L.Proc Natl Acad Sci USA. 2002; 99: 7883-7888Crossref PubMed Scopus (262) Google Scholar The heterozygous mice (CD+/− and CB+/−CL+/− mice) were used as control animals in the present study and showed no pathological phenotypes when examined by histological, immunocytochemical, and biochemical methods. For the generation of CD−/− mice expressing GFP-LC3 (GFP-LC3/CD−/− mice), six GFP-LC3 transgenic mice (line no. 53)23Mizushima N Yamamoto A Matsui M Yoshimori T Ohsumi Y In vivo analysis of autophagy in response to nutrient starvation using transgenic mice expressing a fluorescent autophagosome marker.Mol Biol Cell. 2004; 15: 1101-1111Crossref PubMed Scopus (1867) Google Scholar were obtained from Riken BioResource Center (Tsukaba, Japan), crossed with C57BL/6 mice, and maintained as heterozygotes for the GFP-LC3 transgene in the Institute of Experimental Animal Sciences. As mentioned above, the incorporation of the transgene was confirmed by PCR using primers GFP1 (5′-TCCTGCTGGAGTTCGTGACCG-3′) and LC3* (5′-TTGCGAATTCTCAGCCGTCTTCATCTCTCTCGC-3′). Another primer set mLC3ex3GT (5′-TGAGCGAGCTCATCAAGATAATCAGGT-3′) and mLC3ex4AG (5′-GTTA-GCATTGAGCTGCAAGCGCCGTCT-3′) amplifying the third intron of the LC3 genome as an internal control was used. The positive mice were also crossed with CD+/− mice and CD+/− mice heterozygous for the GFP-LC3 transgene (GFP-LC3/CD+/− mice) were generated. GFP-LC3/CD+/− mice were further crossed with CD+/− mice and CD−/− mice heterozygous for the GFP-LC3 transgene (GFP-LC3/CD−/− mice) were obtained. Rabbit antibodies against rat LC3 and rat subunit c of mitochondrial F1F0ATPase were produced and purified by affinity chromatography, as reported previously.8Kominami E Ezaki J Muno D Ishido K Ueno T Wolfe LS Specific storage of subunit c of mitochondrial ATP synthase in lysosomes of neuronal ceroid lipofuscinosis (Batten's disease).J Biochem (Tokyo). 1992; 111: 278-282Crossref PubMed Scopus (110) Google Scholar, 24Lu Z Dono K Gotoh K Shibata M Koike M Marubashi S Miyamoto A Takeda Y Nagano H Umeshita K Uchiyama Y Monden M Participation of autophagy in the degeneration process of rat hepatocytes after transplantation following prolonged cold preservation.Arch Histol Cytol. 2005; 68: 71-80Crossref PubMed Scopus (41) Google Scholar Monoclonal antibodies against mouse lamp1 (the Developmental Studies Hybridoma Bank, Iowa City, IA) and mouse microglial cells (F4/80) (Serotec, Oxford, UK), respectively, and rabbit polyclonal antibodies against GFP (Abcam, Cambridge, UK) were commercially obtained. CD−/− and CD+/− littermates obtained at postnatal day 8 (P8) to P24 (n = 3 for each stage and each genotype), CB−/−CL−/− and their littermates at P13 (n = 3 each), and GFP-LC3/CD+/+ and GFP-LC3/CD−/− mice at P20 (n = 3 each) were deeply anesthetized with pentobarbital (25 mg/kg i.p.) and fixed by cardiac perfusion with 4% paraformaldehyde buffered with 0.1 mol/L phosphate buffer (pH 7.2), containing 4% sucrose for immunohistochemistry, detection of autofluorescence, and periodic acid-Schiff staining, with 1% paraformaldehyde buffered with 0.1 mol/L phosphate buffer (pH 7.2), containing 4% sucrose for freeze-fracture replica immunogold labeling,25Rash JE Yasumura T Direct immunogold labeling of connexins and aquaporin-4 in freeze-fracture replicas of liver, brain, and spinal cord: factors limiting quantitative analysis.Cell Tissue Res. 1999; 296: 307-321Crossref PubMed Scopus (102) Google Scholar and with 2% paraformaldehyde-2% glutaraldehyde buffered with 0.1 mol/L phosphate buffer for ordinary electron microscopy.11Koike M Nakanishi H Saftig P Ezaki J Isahara K Ohsawa Y Schulz-Schaeffer W Watanabe T Waguri S Kametaka S Shibata M Yamamoto K Kominami E Peters C von Figura K Uchiyama Y Cathepsin D deficiency induces lysosomal storage with ceroid lipofuscin in mouse CNS neurons.J Neurosci. 2000; 20: 6898-6906Crossref PubMed Google Scholar Immediately after perfusion fixation, brains and peripheral tissues were excised from the mice and processed for immunohistochemistry/cytochemistry and ordinary electron microscopy.11Koike M Nakanishi H Saftig P Ezaki J Isahara K Ohsawa Y Schulz-Schaeffer W Watanabe T Waguri S Kametaka S Shibata M Yamamoto K Kominami E Peters C von Figura K Uchiyama Y Cathepsin D deficiency induces lysosomal storage with ceroid lipofuscin in mouse CNS neurons.J Neurosci. 2000; 20: 6898-6906Crossref PubMed Google Scholar, 26Nitatori T Sato N Waguri S Karasawa Y Araki H Shibanai K Kominami E Uchiyama Y Delayed neuronal death in the CA1 pyramidal cell layer of the gerbil hippocampus following transient ischemia is apoptosis.J Neurosci. 1995; 15: 1001-1011Crossref PubMed Google Scholar For light and electron microscopy, brain tissues were quickly removed from the mice and further immersed in the same fixatives for 2 hours. Samples processed for paraffin embedding were cut at 5 μm with a microtome, and those for cryosections were cut at 10 μm with a cryostat (CM3050; Leika, Nussloch, Germany). These sections were placed on silane-coated glass slides and stored at −80°C until used. Samples for electron microscopy were postfixed with 2% OsO4, dehydrated with a graded series of alcohol, and embedded in Epon 812. Ultrathin sections were cut with an ultramicrotome (Ultracut N; Reichert-Nissei, Toyko, Japan), stained with uranyl acetate and lead citrate, and observed with a Hitachi H7100 electron microscope (Hitachi, Tokyo, Japan). For freeze-fracture replica immunogold labeling, the brain tissue was quickly excised from the mice, while cerebral cortical and cerebellar regions were separated from each brain and cut at 100 μm with a vibratome (VT1000S; Leica) at 4°C. Tissue slices were infiltrated with 30% glycerol buffered with 0.1 mol/L phosphate buffer (pH 7.2) until used. Morphometric analyses were performed according to the method of Uchiyama and Watanabe.27Uchiyama Y Watanabe M A morphometric study of developing pancreatic acinar cells of rats during prenatal life.Cell Tissue Res. 1984; 237: 117-122PubMed Google Scholar In samples obtained from CD−/− and CD+/− littermates (n = 4 each) at P8, P15, and P23 and CB−/−CL−/− and CB+/−CL+/− littermates (n = 4 each) at P13, electron micrographs (40 per sample) of the cerebral cortex (layer 5) were taken with systematic random sampling at each corner of 100-mesh grids with a primary magnification of ×7000. Three to five Epon blocks from each mouse were used. After printing at 2.6× the original magnification on projection papers, we estimated the cytoplasmic (perikaryal) volume fraction of lysosomal structures including various types of autophagic vacuoles (AVs), dense bodies, granular osmiophilic deposit (GROD)-like inclusions, and fingerprint profiles by point counting, using a double-lattice test system of 1.5-cm spacing. AVs were classified as early AVs (AVi), which contain the morphologically intact cytoplasm (see Figure 2A), and late AVs (AVd), which contain partially degraded but identifiable cytoplasmic materials (see Figure 2B).28Liou W Geuze HJ Geelen MJ Slot JW The autophagic and endocytic pathways converge at the nascent autophagic vacuoles.J Cell Biol. 1997; 136: 61-70Crossref PubMed Scopus (220) Google Scholar, 29Dunn Jr, WA Studies on the mechanisms of autophagy: formation of the autophagic vacuole.J Cell Biol. 1990; 110: 1923-1933Crossref PubMed Scopus (510) Google Scholar, 30Tanaka Y Guhde G Suter A Eskelinen EL Hartmann D Lullmann-Rauch R Janssen PM Blanz J von Figura K Saftig P Accumulation of autophagic vacuoles and cardiomyopathy in LAMP-2-deficient mice.Nature. 2000; 406: 902-906Crossref PubMed Scopus (710) Google Scholar In CD−/− and CB−/−CL−/− brains, inclusions that, by electron microscopy, were dense granular and amorphous in neurons were quite similar to the so-called GRODs, a typical hallmark in infantile NCL;31Haltia M The neuronal ceroid-lipofuscinoses.J Neuropathol Exp Neurol. 2003; 62: 1-13Crossref PubMed Scopus (213) Google Scholar these granules were defined as GROD-like inclusions. In CD−/− neurons, distinct points falling on GROD-like inclusions or those surrounded by double-layered membranes resembling AVi were counted (see Figure 2D). Moreover, the multilayered lamellar structures that were counted as AVi always contained morphologically intact cytoplasm, and were different from fingerprint profiles that did not contain part of the cytoplasm and possessed tightly and concentrically arrayed membranes as in the structures observed in juvenile NCL.31Haltia M The neuronal ceroid-lipofuscinoses.J Neuropathol Exp Neurol. 2003; 62: 1-13Crossref PubMed Scopus (213) Google Scholar The volume density (Vv) of each lysosomal structure was expressed as the percent volume: Vv = (Pi/Pt) × 100 (%), where Pi is the number of points falling on each lysosomal structure and Pt is the number of points falling on the perikarya of large pyramidal neurons located in the layer V of the cerebral cortex. For the detection of LC3 and subunit c, deparaffinized or frozen sections were used and immunostained according to the method described previously.32Koike M Shibata M Ohsawa Y Kametaka S Waguri S Kominami E Uchiyama Y The expression of tripeptidyl peptidase I in various tissues of rats and mice.Arch Histol Cytol. 2002; 65: 219-232Crossref PubMed Scopus (31) Google Scholar The samples were incubated at 4°C with anti-LC3 (20 μg/ml), anti-subunit c (5 μg/ml), or anti-GFP (4 μg/ml) for 1 to 3 days and further processed for visualization. For double-immunofluorescent staining, deparaffinized or frozen sections of brain tissues from CD−/− and CB−/−CL−/− mice and those from GFP-LC3/CD+/+ and GFP-LC3/CD−/− mice were incubated with anti-LC3 and monoclonal anti-lamp1 or F4/80 for 24 hours at 4°C and further with goat anti-rabbit IgG coupled with Alexa 594 (Molecular Probes, Eugene, OR) and goat anti-rat IgG coupled with fluorescein isothiocyanate (Cappel, Durham, NC), or coupled with Alexa 594 for 1 hour at room temperature. The stained tissues were viewed with a confocal laser-scanning microscope (LSM510; Carl Zeiss, Jena, Germany). To determine the subcellular localization of LC3, the sodium dodecyl sulfate (SDS)-digested freeze-fracture replica labeling (FRL) technique33Fujimoto K Freeze-fracture replica electron microscopy combined with SDS digestion for cytochemical labeling of integral membrane proteins. Application to the immunogold labeling of intercellular junctional complexes.J Cell Sci. 1995; 108: 3443-3449Crossref PubMed Google Scholar was applied to chemically fixed brain tissues.25Rash JE Yasumura T Direct immunogold labeling of connexins and aquaporin-4 in freeze-fracture replicas of liver, brain, and spinal cord: factors limiting quantitative analysis.Cell Tissue Res. 1999; 296: 307-321Crossref PubMed Scopus (102) Google Scholar Samples were mounted between two gold specimen disks in a specimen-mounting medium, polyvinyl alcohol. The disks were quickly frozen in Freon 22 cooled to its freezing point, mounted in a Balzers complementary freeze-fracture apparatus (BAF 400D; Balzers, Furstentum, Lichtenstein) and immediately shadowed unidirectionally by evaporating platinum-carbon at 45° to the vertical carbon support. The thickness of platinum (2.5 nm) was controlled with a quartz crystal monitor. To release the replicas from the specimen carrier, the carrier was immersed gently in phosphate-buffered saline (PBS). After floating off, the pieces of the replicas were transferred to a detergent solution (2.5% SDS, 10 mmol/L Tris-HCl, pH 9). SDS digestion was performed for 24 hours at room temperature with vigorous constant stirring. The replicas were moved to the same detergent solution, washed for at least one additional 1 hour, rinsed four times in PBS (5 minutes each), and placed on drops of 1 to 10% bovine serum albumin (BSA) in PBS (BSA-PBS) for 30 minutes at room temperature. For immunostaining, rabbit polyclonal anti-LC3 antibody and 10-nm colloidal gold conjugated goat anti-rabbit IgG were used. The replicas, picked up on grids, were observed with a Hitachi H7100 electron microscope. Anesthetized mice were killed by decapitation and each excised tissue was independently homogenized in 2 ml of 0.05 mol/L Tris-buffered 0.15 mol/L saline containing 1% Triton X-100 and a protease inhibitor cocktail (Boehringer Mannheim, Mannheim, Germany) by a Polytron homogenizer (Kinematica, Littau, Switzerland) at 80% maximal speed. After centrifuging twice at 10,500 × g for 10 minutes at 4°C, the protein concentration in the supernatants were determined using the BCA protein assay system (Pierce, Rockford, IL). The samples were analyzed by immunoblotting according to the method of Towbin and colleagues.34Towbin H Staehelin T Gordon J Electrophoretic transfer of proteins from polyacrylamide gels to nitrocellulose sheets: procedure and some applications.Proc Natl Acad Sci USA. 1979; 76: 4350-4354Crossref PubMed Scopus (44644) Google Scholar After immunostaining with anti-LC3 (1 μm/ml) or polyclonal anti-GFP (0.4 μm/ml), immunodetection was performed with a chemiluminescent ECL kit (Amersham, Arlington Heights, IL). The density of each protein band in the blotting was measured using a densitometer (Molecular Dynamics, Sunnyvale, CA) and the ratio of LC3-I to LC3-II molecules was calculated for brain tissues. In the case of peripheral tissues, the density of LC3-II was measured and presented as a mean ± SD from three independent experiments. RT-PCR analysis of LC3 was performed according to the method described previously.35Ohsawa Y Zhang G Kametaka S Shibata M Koike M Waguri S Uchiyama Y Purification, cDNA cloning, and secretory properties of FLRG protein from PC12 cells and the distribution of FLRG mRNA and protein in rat tissues.Arch Histol Cytol. 2003; 66: 367-381Crossref PubMed Scopus (2) Google Scholar Total RNAs were extracted from neuronal tissues of CD +/− and −/− mice at P8, P15, and P22 and CB+/−CL+/− and CB−/−CL−/− mice at P13, using a Polytron homogenizer for 1 minute in Isogen (Nippon Gene, Tokyo, Japan). To avoid contamination with genomic DNA, 40 to 50 μg of total RNAs were treated with 0.2 U/ml DNase I (Nippon Gene) for 20 minutes at 37°C. Then, 2.0 μg/ml of the total RNAs were used for cDNA synthesis using AMV reverse transcriptase (Takara, Tokyo, Japan) and amplification of the LC-3 and β-actin cDNAs were performed by PCR using EX taq HS (Takara). The PCR amplification was performed at 95°C for 5 minutes, followed by 30 cycles of 95°C for 30 seconds, 55°C (for LC-3) or 60°C (for β-actin) for 30 minutes, and 72°C for 30 minutes using the primer sets for LC-3 (GenBank accession number NM_026160) (forward: 5′-GTGGGCCGCTCTAGGCACCAA-3′, reverse: 5′-CTCTTTGATGTCACGCACGATTTC-3′; 540 bp) and β-actin (GenBank accession number M12481) (forward: 5′-GTGGGCCGCTCTAGGCACCAA-3′, reverse: 5′-CTC-TTTGATGTCACGCACGATTTC-3′; 540 bp). As described previously,11Koike M Nakanishi H Saftig P Ezaki J Isahara K Ohsawa Y Schulz-Schaeffer W Watanabe T Waguri S Kametaka S Shibata M Yamamoto K Kominami E Peters C von Figura K Uchiyama Y Cathepsin D deficiency induces lysosomal storage with ceroid lipofuscin in mouse CNS neurons.J Neurosci. 2000; 20: 6898-6906Crossref PubMed Google Scholar massive autofluorescent granules, which are immunopositive for subunit c of mitochondrial ATP synthase and lysosomal cathepsin B, had accumulated in the neuronal cell perikarya of CD−/− mouse brains near the terminal stage (data not shown). Indeed, immunoreactivity for subunit c was intensely detected in neurons and microglial cells of CD−/− mouse brains at P13 and P23, but not in those in the control littermate mice (Figure 1, A–D). It has been shown that the phenotype of CB−/−CL−/− is highly reminiscent of NCLs, both of which demonstrate the early and severe brain atrophy; however, the former does not show immunoreactivity for subunit c in brain tissue and ultrastructural characteristics of accumulated lysosomal compartments in CB−/−CL−/− neurons are different from those in NCLs.13Felbor U Kessler B Mothes W Goebel HH Ploegh HL Bronson RT Olsen BR Neuronal loss and brain atrophy in mice lacking c