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• W2034130805 abstract "Ei24 is a DNA damage response gene involved in growth suppression and apoptosis. The physiological function of Ei24, however, is poorly understood. Here we generated conditional knock-out mice of Ei24 and demonstrated that EI24 is an essential component of the basal autophagy pathway. Mice with neural-specific Ei24 deficiency develop age-dependent neurological abnormalities caused by massive axon degeneration and extensive neuron loss in brain and spinal cord. Notably, ablation of Ei24 leads to vacuolated oligodendroglial cells and demyelination of axons. Liver-specific depletion of Ei24 causes severe hepatomegaly with hepatocyte hypertrophy. Ei24 deficiency impairs autophagic flux, leading to accumulation of LC3, p62 aggregates, and ubiquitin-positive inclusions. Our study indicates that Ei24 is an essential autophagy gene and plays an important role in clearance of aggregate-prone proteins in neurons and hepatocytes. Ei24 is a DNA damage response gene involved in growth suppression and apoptosis. The physiological function of Ei24, however, is poorly understood. Here we generated conditional knock-out mice of Ei24 and demonstrated that EI24 is an essential component of the basal autophagy pathway. Mice with neural-specific Ei24 deficiency develop age-dependent neurological abnormalities caused by massive axon degeneration and extensive neuron loss in brain and spinal cord. Notably, ablation of Ei24 leads to vacuolated oligodendroglial cells and demyelination of axons. Liver-specific depletion of Ei24 causes severe hepatomegaly with hepatocyte hypertrophy. Ei24 deficiency impairs autophagic flux, leading to accumulation of LC3, p62 aggregates, and ubiquitin-positive inclusions. Our study indicates that Ei24 is an essential autophagy gene and plays an important role in clearance of aggregate-prone proteins in neurons and hepatocytes. Ei24 (etoposide-induced 2.4 kb transcript), 2The abbreviations used are: Ei24etoposide-induced 2.4 kb transcriptSQSTMsequestosomeAMCaminomethylcoumarinMBPmyelin basic proteinH&Ehematoxylin and eosinDCNdeep cerebellar nuclei. also known as PIG8 (p53-induced gene 8), encodes an ER-localized six transmembrane protein, expression of which is highly induced by the tumor suppressor protein p53 (1.Lehar S.M. Nacht M. Jacks T. Vater C.A. Chittenden T. Guild B.C. Identification and cloning of EI24, a gene induced by p53 in etoposide-treated cells.Oncogene. 1996; 12: 1181-1187PubMed Google Scholar, 2.Gu Z. Flemington C. Chittenden T. Zambetti G.P. ei24, a p53 response gene involved in growth suppression and apoptosis.Mol. Cell. Biol. 2000; 20: 233-241Crossref PubMed Scopus (100) Google Scholar, 3.Zhao X. Ayer R.E. Davis S.L. Ames S.J. Florence B. Torchinsky C. Liou J.S. Shen L. Spanjaard R.A. Apoptosis factor EI24/PIG8 is a novel endoplasmic reticulum-localized Bcl-2-binding protein which is associated with suppression of breast cancer invasiveness.Cancer Res. 2005; 65: 2125-2129Crossref PubMed Scopus (51) Google Scholar). Overexpression of Ei24 suppresses cell growth and induces apoptosis, while depletion of Ei24 results in suppression of apoptosis in response to pro-apoptotic treatment (2.Gu Z. Flemington C. Chittenden T. Zambetti G.P. ei24, a p53 response gene involved in growth suppression and apoptosis.Mol. Cell. Biol. 2000; 20: 233-241Crossref PubMed Scopus (100) Google Scholar, 3.Zhao X. Ayer R.E. Davis S.L. Ames S.J. Florence B. Torchinsky C. Liou J.S. Shen L. Spanjaard R.A. Apoptosis factor EI24/PIG8 is a novel endoplasmic reticulum-localized Bcl-2-binding protein which is associated with suppression of breast cancer invasiveness.Cancer Res. 2005; 65: 2125-2129Crossref PubMed Scopus (51) Google Scholar, 4.Mork C.N. Faller D.V. Spanjaard R.A. Loss of putative tumor suppressor EI24/PIG8 confers resistance to etoposide.FEBS Letters. 2007; 2581: 5440-5444Crossref Scopus (29) Google Scholar). The human EI24 gene is located on chromosome 11q23, a region frequently displaying loss of heterozygosity in several malignancies, including invasive cervical cancers, breast carcinomas and malignant melanoma (5.Gentile M. Ahnström M. Schön F. Wingren S. Candidate tumour suppressor genes at 11q23-q24 in breast cancer: evidence of alterations in PIG8, a gene involved in p53-induced apoptosis.Oncogene. 2001; 20: 7753-7760Crossref PubMed Scopus (50) Google Scholar). The tumor suppression function of Ei24 is further substantiated by reduced expression in invasive breast cancers (3.Zhao X. Ayer R.E. Davis S.L. Ames S.J. Florence B. Torchinsky C. Liou J.S. Shen L. Spanjaard R.A. Apoptosis factor EI24/PIG8 is a novel endoplasmic reticulum-localized Bcl-2-binding protein which is associated with suppression of breast cancer invasiveness.Cancer Res. 2005; 65: 2125-2129Crossref PubMed Scopus (51) Google Scholar, 5.Gentile M. Ahnström M. Schön F. Wingren S. Candidate tumour suppressor genes at 11q23-q24 in breast cancer: evidence of alterations in PIG8, a gene involved in p53-induced apoptosis.Oncogene. 2001; 20: 7753-7760Crossref PubMed Scopus (50) Google Scholar). However, the physiological function of Ei24 is still poorly understood. etoposide-induced 2.4 kb transcript sequestosome aminomethylcoumarin myelin basic protein hematoxylin and eosin deep cerebellar nuclei. Autophagy is an evolutionarily conserved intracellular catabolic system, involving the formation of a closed double-membrane autophagosome and its subsequent delivery to the vacuole/lysosome for degradation (6.Xie Z. Klionsky D.J. Autophagosome formation: core machinery and adaptations.Nat. Cell Biol. 2007; 9: 1102-1109Crossref PubMed Scopus (1711) Google Scholar, 7.Nakatogawa H. Suzuki K. Kamada Y. Ohsumi Y. Dynamics and diversity in autophagy mechanisms: lessons from yeast.Nat. Rev. Mol. Cell Biol. 2009; 10: 458-467Crossref PubMed Scopus (1271) Google Scholar). Under normal physiological conditions, autophagy occurs at a basal constitutive level, removing misfolded, or aggregate-prone proteins and damaged organelles (8.Levine B. Kroemer G. Autophagy in the pathogenesis of disease.Cell. 2008; 132: 27-42Abstract Full Text Full Text PDF PubMed Scopus (5540) Google Scholar). The homeostatic function of basal autophagy plays an important role in neuronal protection and tumor suppression in mammals (8.Levine B. Kroemer G. Autophagy in the pathogenesis of disease.Cell. 2008; 132: 27-42Abstract Full Text Full Text PDF PubMed Scopus (5540) Google Scholar, 9.Ichimura Y. Komatsu M. Pathophysiological role of autophagy: lesson from autophagy-deficient mouse models.Exp. Anim. 2011; 60: 329-345Crossref PubMed Scopus (37) Google Scholar). Neural-specific knock-out of Atg7 or Atg5 (genes essential for autophagosome formation) causes dramatic accumulation of autophagy substrates such as p62 (also known as sequestosome 1, SQSTM1) and ubiquitin-positive aggregates in neurons, accompanied by massive neuron degeneration in various brain regions (10.Hara T. Nakamura K. Matsui M. Yamamoto A. Nakahara Y. Suzuki-Migishima R. Yokoyama M. Mishima K. Saito I. Okano H. Mizushima N. Suppression of basal autophagy in neural cells causes neurodegenerative disease in mice.Nature. 2006; 441: 885-889Crossref PubMed Scopus (3108) Google Scholar, 11.Komatsu M. Waguri S. Chiba T. Murata S. Iwata J. Tanida I. Ueno T. Koike M. Uchiyama Y. Kominami E. Tanaka K. Loss of autophagy in the central nervous system causes neurodegeneration in mice.Nature. 2006; 441: 880-884Crossref PubMed Scopus (2829) Google Scholar, 12.Komatsu M. Waguri S. Koike M. Sou Y.S. Ueno T. Hara T. Mizushima N. Iwata J. Ezaki J. Murata S. Hamazaki J. Nishito Y. Iemura S. Natsume T. Yanagawa T. Uwayama J. Warabi E. Yoshida H. Ishii T. Kobayashi A. Yamamoto M. Yue Z. Uchiyama Y. Kominami E. Tanaka K. Homeostatic levels of p62 control cytoplasmic inclusion body formation in autophagy-deficient mice.Cell. 2007; 131: 1149-1163Abstract Full Text Full Text PDF PubMed Scopus (1709) Google Scholar). The autophagy gene beclin 1 is a haploinsufficient tumor suppressor (8.Levine B. Kroemer G. Autophagy in the pathogenesis of disease.Cell. 2008; 132: 27-42Abstract Full Text Full Text PDF PubMed Scopus (5540) Google Scholar). Atg7 and Atg5 deficiency in liver cause hepatomegaly and subsequently multiple liver adenomas (13.Komatsu M. Waguri S. Ueno T. Iwata J. Murata S. Tanida I. Ezaki J. Mizushima N. Ohsumi Y. Uchiyama Y. Kominami E. Tanaka K. Chiba T. Impairment of starvation-induced and constitutive autophagy in Atg7-deficient mice.J. Cell Biol. 2005; 169: 425-434Crossref PubMed Scopus (1911) Google Scholar, 14.Takamura A. Komatsu M. Hara T. Sakamoto A. Kishi C. Waguri S. Eishi Y. Hino O. Tanaka K. Mizushima N. Autophagy-deficient mice develop multiple liver tumors.Genes Dev. 2011; 25: 795-800Crossref PubMed Scopus (969) Google Scholar). The tumor suppression function of autophagy is at least partially attributed to its elimination of p62, accumulation of which leads to persistent activation of the Nrf2 stress response and dysregulation of NF-κB signaling (14.Takamura A. Komatsu M. Hara T. Sakamoto A. Kishi C. Waguri S. Eishi Y. Hino O. Tanaka K. Mizushima N. Autophagy-deficient mice develop multiple liver tumors.Genes Dev. 2011; 25: 795-800Crossref PubMed Scopus (969) Google Scholar, 15.Mathew R. Karp C.M. Beaudoin B. Vuong N. Chen G. Chen H.Y. Bray K. Reddy A. Bhanot G. Gelinas C. Dipaola R.S. Karantza-Wadsworth V. White E. Autophagy suppresses tumorigenesis through elimination of p62.Cell. 2009; 137: 1062-1075Abstract Full Text Full Text PDF PubMed Scopus (1364) Google Scholar, 16.Inami Y. Waguri S. Sakamoto A. Kouno T. Nakada K. Hino O. Watanabe S. Ando J. Iwadate M. Yamamoto M. Lee M.S. Tanaka K. Komatsu M. Persistent activation of Nrf2 through p62 in hepatocellular carcinoma cells.J. Cell Biol. 2011; 193: 275-284Crossref PubMed Scopus (452) Google Scholar). A group of evolutionarily conserved Atg proteins has been identified from yeast genetic studies that act at distinct steps of autophagosome formation (6.Xie Z. Klionsky D.J. Autophagosome formation: core machinery and adaptations.Nat. Cell Biol. 2007; 9: 1102-1109Crossref PubMed Scopus (1711) Google Scholar, 7.Nakatogawa H. Suzuki K. Kamada Y. Ohsumi Y. Dynamics and diversity in autophagy mechanisms: lessons from yeast.Nat. Rev. Mol. Cell Biol. 2009; 10: 458-467Crossref PubMed Scopus (1271) Google Scholar). In mammalian cells, however, the autophagy process involves more complex membrane dynamics (17.Longatti A. Tooze S.A. Vesicular trafficking and autophagosome formation.Cell Death Differ. 2009; 16: 956-965Crossref PubMed Scopus (175) Google Scholar). The endoplasmic reticulum (ER), Golgi apparatus, recycling endosomes, and plasma membrane all contribute to autophagosomal membranes in mammalian cells (18.Tooze S.A. Yoshimori T. The origin of the autophagosomal membrane.Nat. Cell Biol. 2010; 12: 831-835Crossref PubMed Scopus (434) Google Scholar, 19.Ravikumar B. Moreau K. Jahreiss L. Puri C. Rubinsztein D.C. Plasma membrane contributes to the formation of pre-autophagosomal structures.Nat. Cell Biol. 2010; 12: 747-757Crossref PubMed Scopus (664) Google Scholar). Among them, PI(3)P-enriched subdomains of the ER, called omegasomes, provide a platform for recruiting Atg proteins and subsequent autophagosome formation (18.Tooze S.A. Yoshimori T. The origin of the autophagosomal membrane.Nat. Cell Biol. 2010; 12: 831-835Crossref PubMed Scopus (434) Google Scholar). The more elaborate autophagic machinery in higher eukaryotes requires Atg proteins and also metazoan-specific autophagy components. Genetic screens in Caenorhabditis elegans identified epg-4, the Ei24 homolog, as an essential autophagy gene, loss of function of which causes defective autophagic degradation of a variety of protein aggregates during embryogenesis (20.Tian Y. Li Z.P. Hu W.Q. Ren H.Y. Tian E. Zhao Y. Lu Q. Huang X.X. Yang P.G. Li X. Wang X. Kovács A.L. Yu L. Zhang H. C. elegans screen identifies autophagy genes specific to multicellular organisms.Cell. 2010; 141: 1042-1055Abstract Full Text Full Text PDF PubMed Scopus (315) Google Scholar). epg-4 regulates progression of omegasomes to autophagosomes (20.Tian Y. Li Z.P. Hu W.Q. Ren H.Y. Tian E. Zhao Y. Lu Q. Huang X.X. Yang P.G. Li X. Wang X. Kovács A.L. Yu L. Zhang H. C. elegans screen identifies autophagy genes specific to multicellular organisms.Cell. 2010; 141: 1042-1055Abstract Full Text Full Text PDF PubMed Scopus (315) Google Scholar). Ei24 is also essential for starvation-induced autophagy (20.Tian Y. Li Z.P. Hu W.Q. Ren H.Y. Tian E. Zhao Y. Lu Q. Huang X.X. Yang P.G. Li X. Wang X. Kovács A.L. Yu L. Zhang H. C. elegans screen identifies autophagy genes specific to multicellular organisms.Cell. 2010; 141: 1042-1055Abstract Full Text Full Text PDF PubMed Scopus (315) Google Scholar). Distinct from the role of epg-4 at the early step of autophagosome formation in C. elegans, knockdown of Ei24 by siRNA results in accumulation of degradation-defective autolysosomes (20.Tian Y. Li Z.P. Hu W.Q. Ren H.Y. Tian E. Zhao Y. Lu Q. Huang X.X. Yang P.G. Li X. Wang X. Kovács A.L. Yu L. Zhang H. C. elegans screen identifies autophagy genes specific to multicellular organisms.Cell. 2010; 141: 1042-1055Abstract Full Text Full Text PDF PubMed Scopus (315) Google Scholar). Here we generated mice with tissue-specific deficiency of Ei24. Neural-specific Ei24-deficient mice exhibit massive axonal degeneration and neuronal cell loss in various brain and spinal cord regions. Ei24 deficiency causes vacuolation of oligodendroglial cells. Liver-specific Ei24-deficient mice have hepatocellular enlargement and hepatomegaly. Ei24 deficiency impairs autophagic flux, leading to accumulation of LC3, p62 aggregates, and ubiquitin-positive inclusions. Our study demonstrates that Ei24 is an essential component of basal autophagy in removing aggregate-prone proteins. To construct the Ei24 targeting allele, exon 3 of Ei24 was flanked by two loxP sequences and a neomycin phosphotransferase expression cassette. The targeting vector was electroporated into 129 R1 embryonic stem (ES) cells. Three homologous recombinants were identified by Southern blotting with probes 5′ and 3′ of the genomic sequence present in the targeting vector. Heterozygous Ei24fl/wt ES cell clones were microinjected into C57BL/6N blastocysts. Chimeric offspring were backcrossed to C57BL/6N mice. Heterozygous mutant mice were outbred with C57BL/6 mice and interbred to obtain homozygous mutant mice. Cre-mediated deletion of exon 3 leads to a frameshift, producing a small truncated peptide. The following primers were used to detect wild type Ei24 and Ei24flox alleles: 5′-TAAAGTTCTTAGGACACCTCCTG-3′ (F) and 5′-AATGGAGAACTTTAGAATCTCC-3′ (R). The expected sizes are 273 bp and 377 bp, respectively. To generate neural- or liver-specific Ei24-deficient mice, Ei24flox/flox mice were crossed with nestin-Cre or albumin-Cre mice (Jackson Laboratory), respectively. Ei24+/− mice were obtained from Ei24flox/wt; Zp3-Cre mice (Jackson Laboratory). All mice were raised under specific pathogen-free conditions in the animal facility at the National Institute of Biological Sciences, Beijing. All animal experiments were approved by the institutional committee of the National Institute of Biological Sciences, Beijing. The following antibodies were used: rabbit anti-p62 (PM045, MBL), mouse anti-p62 (ab56416, Abcam), mouse anti-ubiquitin (3936, Cell Signaling), rabbit anti-GFAP (bs-0199R, Bioss), rabbit anti-LC3 (PM046, MBL) for Western, rabbit anti-LC3 (2775, Cell Signaling) for staining, mouse anti-NeuN (MAB377, Millipore), mouse anti-β tubulin III (ab7751, Abcam), mouse anti-calbindin (C9848, Sigma), mouse anti-CNPase (2′,3′-cyclic-nucleotide 3′-phosphodiesterase) (ab6319, Abcam), rabbit anti-MBP (myelin basic protein) (ab40390, Abcam), and rabbit anti-GFP (ab290, Abcam). An accelerating rotarod (YLS-4C, Beijing Zhongshidichuang Science and Technology Development Co., Ltd) was used to measure motor coordination. After 2 consecutive days training, mice were put on the rolling rod with auto-acceleration ranging from 5–20 rpm within 60 s. The time when the mice fell from the rod was recorded, with a maximum observation time of 2 min. Mice were perfused with 10% neutral buffered formalin (Sigma). Tissues were post-fixed, embedded in paraffin, and sectioned at 5 μm. Sections were stained with hematoxylin and eosin for histological examination or 0.1% cresyl violet for Nissl staining and examined by light microscopy (Imager A1, Zeiss). For immunohistochemistry, sections were deparaffinized in xylene three times, then rehydrated in an ethanol series (100% × 3, 95%, and 75%). After heat-activated antigen retrieval (0.1 m citrate buffer), sections were blocked with normal goat serum. Sections were then incubated with primary antibodies at 4 °C overnight in a humidity chamber, washed in PBS three times, and incubated with fluorescently-labeled secondary antibodies in a humidified chamber for 1 h at room temperature. Finally, sections were counterstained with DAPI, mounted and examined under an epifluorescence microscope or a confocal microscope (Zeiss LSM 510 Meta plus Zeiss Axiovert zoom). Fluorescence intensity and dot numbers were analyzed by Image J software. To score the number of spinal cord motor neurons, fixed spinal cords were sequentially sectioned at 5 μm. Every 6th section was stained with 0.1% cresyl violet. Neurons with a distinct nucleus and a diameter of at least 25 μm located in the anterior horn ventral to the line tangential to the ventral tip of the central canal were counted as motor neurons. To examine cortical neurons, large pyramidal cells located in the fifth layer of the motor and sensory cortices were quantified. The number of Purkinje cells in lobules III, IV, and V were quantified and divided by the total length of the lobules. The thickness of the molecular layer was calculated by dividing the distance between lobules III and IV, or lobules V and VI, by 2. Mouse tissues were homogenized in 10 volumes of lysis buffer (50 mm Tris-HCl, pH 7.4, 150 mm NaCl, 1 mm EDTA, 0.1% SDS, 1% Nonidet P-40) supplemented with 1 mm PMSF and protease inhibitor mixture (Roche). After incubation on ice for 30 min, homogenates were centrifuged at 15,000 rpm for 15 min at 4 °C. Supernatants were transferred to new tubes and protein concentrations were determined by Bradford protein assay (Genstar). For preparation of detergent-soluble and -insoluble fractions, mouse tissues were homogenized in five volumes of sucrose buffer (0.25 m sucrose, 50 mm Tris-HCl, pH 7.4, and 1 mm EDTA) supplemented with 1 mm PMSF and protease inhibitors (Roche). After centrifuging at 2500 rpm for 10 min at 4 °C, the supernatants were collected, and the protein concentration was adjusted to 4 μg/μl. An aliquot of 100-μl supernatant was mixed with 100 μl of sucrose buffer containing 1% Triton X-100, then centrifuged at 15,000 rpm for 15 min at 4 °C to separate supernatants (detergent-soluble fraction) and pellets. Pellets were dissolved in 200 μl of 1% SDS in PBS (detergent-insoluble fraction). Equal amounts (30∼40 μg) of proteins were subjected to SDS-PAGE electrophoresis and then transferred onto a PVDF membrane. After blocking with 5% nonfat milk for 1 h at room temperature, membranes were incubated with primary antibodies at 4 °C overnight, washed with PBST three times and incubated with HRP-labeled secondary antibodies for 1 h at room temperature. Immunoreactivity was detected using an Enhanced Chemiluminescent (ECL) kit (Pierce). RNA was extracted using TRIzol (Invitrogen) and cDNA was synthesized by Super Script® III First-Strand Kit (Invitrogen). Quantitative PCR was performed on an Eppendorf Mastercycler® ep Realplex 4 using SYBR® Premix Ex TaqTM (TaKaRa). The following primers were used: 5′-ACTTCCCTCTCGCTGTATTTGAT-3′ (F-Ei24) and 5′-TTGCTTCCGCTCTACACTCTG-3′ (R-Ei24); 5′-GCTGCCCTATACCCACATCT-3′ (F-p62) and 5′-CGCCTTCATCCGAGAAAC-3′ (R-p62); 5′-CTACCTTGCCCGAAAGCAC-3′ (F-Gstm1) and 5′-ATGTCTGCACGGATCCTCTC-3′ (R-Gstm1); 5′-ACCAAGGACACCAAGTTTCG-3′ (F-Cyp2a5) and 5′-AGAGCCCAGCATAGGAAACA-3′ (R-Cyp2a5); 5′-AGCGTTCGGTATTACGATCC-3′ (F-Nqo1) and 5′-AGTACAATCAGGGCTCTTCTCG-3′ (R-Nqo1); 5′-CTGGCTCCTAGCACCATGAAGAT-3′ (F-actin) and 5′-GGTGGACAGTGAGGCCAGGAT-3′ (R-actin). Proteasome activity was determined using aminomethylcoumarin (AMC)-linked synthetic peptide substrates: Ac-Gly-Pro-Leu-Asp-AMC, Suc-Leu-Leu-Val-Tyr-AMC and Ac-Arg-Leu-Arg-AMC (and Boc-Leu-Arg-Arg-AMC) for caspase-like, chymotrypsin-like, or trypsin-like activity, respectively (Proteasome Substrate Pack, Enzo Life Sciences). Mice tissues were homogenized in lysis buffer (50 mm HEPES pH 7.5, 5 mm EDTA, 150 mm NaCl, 1% Triton X-100, and 2 mm ATP). 250 μl of lysate containing equal amounts of protein (2–4 μg) were incubated for 30 min at 37 °C in the dark with 2.5 μl of each substrate (final concentration: 50 nm). The reaction was stopped by adding 252.5 μl pre-cooled 96% ethanol solution. Proteasome activity was measured by detecting fluorescence from AMC hydrolysis (380 nm excitation and 460 nm emission). Mice were transcardially perfused with 2% PFA/2% glutaraldehyde buffered with 0.1 m pH 7.4 phosphate buffer (PB). Tissues were separated, fixed in 2.5% glutaraldehyde, post-fixed with 1% OsO4 in PB buffer for 2 h, dehydrated with graded ethanol solutions, and embedded in Embed812. Ultrathin sections (80 nm) were stained with 2% uranyl acetate for 30 min and lead citrate for 10 min and examined using transmission electron microscopy (TecnaiTM Spirit, FEI). Data from at least three sets of samples were used for statistical analysis. Statistical significance was calculated by Student's t test. A p value less than 0.05 was considered significant. We generated Ei24 conditional knock-out mice by flanking exon 3 of Ei24 with two loxP sequences (Fig. 1A). When we crossed Ei24+/− adults (obtained from Ei24flox/wt; Zp3-Cre mice), no Ei24−/− offspring survived to the neonatal stage, suggesting an essential role of Ei24 during embryogenesis. The function of Ei24 was therefore investigated by crossing Ei24flox/flox mice with mice expressing tissue-specific Cre recombinase. Ei24flox/flox mice were crossed with nestin-Cre mice to produce animals deficient for Ei24 in the central nervous system (referred to as Ei24flox/flox; nestin-Cre) from embryonic stages (Fig. 1B). Nestin is highly expressed in neuroepithelial precursors, which give rise to neurons, astrocytes and oligodendrocytes (21.Lendahl U. Zimmerman L.B. McKay R.D. CNS stem cells express a new class of intermediate filament protein.Cell. 1990; 60: 585-595Abstract Full Text PDF PubMed Scopus (2775) Google Scholar). Ei24flox/flox; nestin-Cre mice appeared normal at birth, but started to show growth retardation at 2 weeks of age and had ∼50% reduction in body weight compared with Ei24flox/+; nestin-Cre littermates (referred to as control littermates in this study) by 3 months (Fig. 1C). The survival rate was also dramatically reduced; 50% of mutant mice died by ∼16 weeks after birth and all mice died by 19 weeks independent of sex (Fig. 1D). Ei24flox/flox; nestin-Cre mice exhibited motor and behavioral deficits at 6 weeks of age and became progressively worse. 8 out of 12 mutant mice at 6 weeks of age and 12 out of 12 of mutant mice at 8 weeks of age exhibited abnormal limb-clasping reflexes (Fig. 1E). All 3-month-old mutants showed poor motor coordination in a rotarod test (Fig. 1F). Mutant mice exhibited marked tremor at 8 weeks and ataxic gait at 12 weeks. At 14 weeks, the hindlimbs of resting mutant mice were flexed, like control littermates, but became hyperextended posteriorly during movement (Fig. 1G; supplemental movie). Thus, neural-specific Ei24 deficiency causes behavioral and motor defects. To investigate the neural defect in Ei24flox/flox; nestin-Cre mice, histological analyses were performed by hematoxylin and eosin (H&E) staining and Nissl staining of sections from cerebrum, cerebellum, and spinal cord of control and mutant mice at 4 months of age. Ei24flox/flox; nestin-Cre brains had enlarged lateral ventricles (Fig. 2A). Mutant mice showed atrophy of the cerebral cortex (Fig. 2A); the ratio of cortical to dorsoventral thickness in Ei24flox/+; nestin-Cre and Ei24flox/flox; nestin-Cre mice was 0.18 ± 0.02 and 0.13 ± 0.01, respectively (p < 0.05). The white matter between the cortex and hippocampal pyramidal cells was thinner in mutants (Fig. 2A). The number of neurons in cortical layers 1–4 was greatly reduced compared with controls, as was the number of pyramidal neurons in the 5th layer of the motor and sensory cortices (Fig. 2B; supplemental Fig. S1, B and C). The hippocampal pyramidal cell layer was much thinner with reduced neuron numbers (Fig. 2C). H&E and axon-specific β-tubulin III staining revealed irregularly arranged nerve fibers in the alveus of the hippocampus (supplemental Fig. S1, D and E). Eosinophilic spheroids, representing axon swellings (10.Hara T. Nakamura K. Matsui M. Yamamoto A. Nakahara Y. Suzuki-Migishima R. Yokoyama M. Mishima K. Saito I. Okano H. Mizushima N. Suppression of basal autophagy in neural cells causes neurodegenerative disease in mice.Nature. 2006; 441: 885-889Crossref PubMed Scopus (3108) Google Scholar), were observed in the cortex (supplemental Fig. S1F). Vacuolated cells were observed in the cingulate cortex and to a lesser extent in other cortices, hippocampus, thalamus, and hypothalamus (Fig. 2D). Numerous vacuolated cells were found in white matter regions including the corpus callosum, the alveus of the hippocampus and the internal capsule (Fig. 2D, supplemental Fig. S1D, data not shown). The vacuolated cells were stained by the oligodendroglial marker anti-2′,3′-cyclic-nucleotide 3′-phosphodiesterase (CNPase) (Fig. 2E). Oligodendrocytes support axonal integrity and myelinate the axons of the CNS (22.Emery B. Regulation of oligodendrocyte differentiation and myelination.Science. 2010; 330: 779-782Crossref PubMed Scopus (457) Google Scholar). Staining with anti-myelin basic protein (MBP) (which labels myelin) or anti-CNPase (which labels cell bodies and myelin) revealed fragmented myelin structures in various cerebral regions in mutant mice (Fig. 2F; supplemental Fig. S1G). The cerebella of Ei24flox/flox; nestin-Cre mice were less foliated and fissured than controls (Fig. 2G). The molecular cell layer was thinner (Fig. 2H). Purkinje cell numbers were reduced by 40% and the remaining cells appeared shrunken (Fig. 2I). The deep cerebellar nuclei (DCN), to which Purkinje cells project axons, contained numerous large eosinophilic spheroid structures (supplemental Fig. S1H), indicative of axonal swellings (10.Hara T. Nakamura K. Matsui M. Yamamoto A. Nakahara Y. Suzuki-Migishima R. Yokoyama M. Mishima K. Saito I. Okano H. Mizushima N. Suppression of basal autophagy in neural cells causes neurodegenerative disease in mice.Nature. 2006; 441: 885-889Crossref PubMed Scopus (3108) Google Scholar). We further stained the DCN region with anti-calbindin, which specifically labels Purkinje axons, and found accumulation of huge bulging calbindin-positive structures in the DCN region of Ei24flox/flox; nestin-Cre mice, indicating abnormal swelling and dystrophy of Purkinje cell axon terminals (Fig. 2J). MBP staining showed that some of the dilated calbindin-positive termini were enwrapped by myelin, confirming the degeneration of Purkinje cell axons (Fig. 2J; supplemental Fig. S1, I–J). Ultrastructurally, axon numbers in the DCN region were dramatically reduced and the remaining axons showed thinned, split myelin sheaths and various signs of degeneration (Fig. 2, K–M; supplemental Fig. S1, K–N). In the anterior horn of the lumbar spinal cord, interneuron numbers were dramatically reduced in Ei24flox/flox; nestin-Cre mice but the number of large motor neurons was not significantly different (Fig. 2N; supplemental Fig. S1, O and P). Numerous vacuolated oligodendrocytes accumulated in spinal cord gray and white matter (Fig. 2O; supplemental Fig. S1Q). Expression of the glial marker GFAP (glial fibrillary acidic protein) dramatically increased in the cerebrum, cerebellum and spinal cord of mutant mice (Fig. 2P), indicating that neurodegeneration was accompanied by reactive astrogliosis. To investigate whether the neural abnormalities in Ei24flox/flox; nestin-Cre mice increase progressively, brain and spinal cord sections from Ei24flox/flox; nestin-Cre mice and controls at 3 weeks, one month, two months, and four months were analyzed. The number of pyramidal neurons in the 5th layer of the cortex in Ei24flox/flox; nestin-Cre mice at 3 weeks of age was comparable to control littermates, but gradually decreased as the mice grew older (Fig. 3A). The number of Purkinje cells and thickness of the molecular layer progressively decreased (Fig. 3, B and C). Vacuolated oligodendrocytes in the cerebrum, cerebellum, and spinal cord were small in size and few in number in mutant mice at 3 weeks and one month of age but became larger and more numerous at 2 and 4 months of age (Fig. 3A; supplemental Fig. S2, A and B). The mutant mice also showed progressively more irregularly arranged nerve fibers in the alveus of the hippocampus, more severe axonal swelling in the DCN of the cerebellum and deterioration of axonal myelin sheaths in various regions of the brain and spinal cord (Fig. 3, D and E; supplemental Fig. 2C). Thus, neural-specific Ei24 deficiency results in progressive axonal degeneration and neuronal loss. We next investigated autophagic flux in mutant mice by examining levels and distribution of LC3. Brain and spinal cord extracts from Ei24flox/flox; nestin-Cre mice at 4 months showed accumulation of LC3-II, a lipidated form of LC3 that normally associates with autophagosomal membranes (Fig. 4A). Immunostaining with anti-LC3 revealed that LC3 diffusely accumulated in various regions of the brain and spinal cord of Ei24flox/f" @default.
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