Systemic Analysis of Atg5-Null Mice Rescued from Neonatal Lethality by Transgenic ATG5 Expression in Neurons

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Systemic Analysis of Atg5-Null Mice Rescued from Neonatal Lethality by Transgenic ATG5 Expression in Neurons Graphical Abstract

Authors Saori R. Yoshii, Akiko Kuma, Takumi Akashi, ..., Hiroshi Shitara, Yoshinobu Eishi, Noboru Mizushima

Correspondence [email protected]

In Brief Loss of the key autophagy factor Atg5 causes neonatal lethality. Yoshii, Kuma et al. show that neuron-specific transgenic expression of ATG5 can rescue lethality, suggesting that neuronal dysfunction is the primary cause of lethality. Rescued mice develop multiple abnormalities and provide a resource for dissection of physiological roles of autophagy.

Highlights d

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Atg5-null neonates can survive if autophagy is restored in neurons The rescued Atg5-null mice develop multiple organ abnormalities

d

These mice show reduced pituitary gland hormones and hypogonadism

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These mice develop hypochromic anemia due to iron malabsorption

Yoshii et al., 2016, Developmental Cell 39, 1–15 October 10, 2016 ª 2016 Elsevier Inc. http://dx.doi.org/10.1016/j.devcel.2016.09.001

Please cite this article in press as: Yoshii et al., Systemic Analysis of Atg5-Null Mice Rescued from Neonatal Lethality by Transgenic ATG5 Expression in Neurons, Developmental Cell (2016), http://dx.doi.org/10.1016/j.devcel.2016.09.001

Developmental Cell

Resource Systemic Analysis of Atg5-Null Mice Rescued from Neonatal Lethality by Transgenic ATG5 Expression in Neurons Saori R. Yoshii,1,2,8 Akiko Kuma,1,2,3,8 Takumi Akashi,4 Taichi Hara,1,9 Atsushi Yamamoto,1,2,5 Yoshitaka Kurikawa,2 Eisuke Itakura,1,10 Satoshi Tsukamoto,6 Hiroshi Shitara,7 Yoshinobu Eishi,4 and Noboru Mizushima1,2,11,* 1Department of Physiology and Cell Biology, Graduate School of Medical and Dental Sciences, Tokyo Medical and Dental University, Tokyo 113-8519, Japan 2Department of Biochemistry and Molecular Biology, Graduate School and Faculty of Medicine, The University of Tokyo, Tokyo 113-0033, Japan 3Japan Science and Technology Agency, PRESTO, Saitama 332-0012, Japan 4Department of Human Pathology 5Comprehensive Reproductive Medicine Graduate School of Medical and Dental Sciences, Tokyo Medical and Dental University, Tokyo 113-8519, Japan 6Laboratory Animal and Genome Sciences Section, National Institute of Radiological Sciences, Chiba 263-8555, Japan 7Center for Basic Technology Research, Tokyo Metropolitan Institute of Medical Science, Tokyo 156-8506, Japan 8Co-first author 9Present address: Laboratory of Molecular Traffic, Institute for Molecular and Cellular Regulation, Gunma University, Gunma 371-8512, Japan 10Present address: Department of Nanobiology, Graduate School of Advanced Integration Science, Chiba University, Chiba 263-8522, Japan 11Lead Contact *Correspondence: [email protected] http://dx.doi.org/10.1016/j.devcel.2016.09.001

SUMMARY

Autophagy is a cytoplasmic degradation system that is important for starvation adaptation and cellular quality control. Previously, we reported that Atg5null mice are neonatal lethal; however, the exact cause of their death remains unknown. Here, we show that restoration of ATG5 in the brain is sufficient to rescue Atg5-null mice from neonatal lethality. This suggests that neuronal dysfunction, including suckling failure, is the primary cause of the death of Atg5-null neonates, which would further be accelerated by nutrient insufficiency due to a systemic failure in autophagy. The rescued Atg5-null mouse model, as a resource, allows us to investigate the physiological roles of autophagy in the whole body after the neonatal period. These rescued mice demonstrate previously unappreciated abnormalities such as hypogonadism and iron-deficiency anemia. These observations provide new insights into the physiological roles of the autophagy factor ATG5.

INTRODUCTION Autophagy is an intracellular degradation system conserved throughout most eukaryotes. When autophagy is initiated, an isolation membrane encloses a portion of the cytoplasm to form an autophagosome, and the autophagosome fuses with lysosomes to degrade its contents; the resulting degradation products are recycled as nutrients (Mizushima and Komatsu,

2011). Autophagy can be induced by various stimuli including starvation to overcome stress, while basal autophagy is important for cellular quality control (Levine and Kroemer, 2008; Mizushima and Komatsu, 2011). Previously, we reported that mice with systemic deletion of Atg5, one of the core autophagy-related (Atg) genes, die at approximately 12 hr after birth (Kuma et al., 2004). Similar neonatal lethality is also observed in Atg3
/
, Atg7
/
, Atg12
/
, and Atg16L1
/
mice (Komatsu et al., 2005; Sou et al., 2008; Saitoh et al., 2008; Malhotra et al., 2015). Autophagy is massively induced immediately after birth as an adaptive response to starvation due to the sudden termination of the transplacental nutrient supply (Kuma et al., 2004). Accordingly, amino acid levels are decreased in the plasma and tissues of autophagy-deficient neonates (Kuma et al., 2004; Komatsu et al., 2005; Sou et al., 2008). Despite the apparent importance of autophagy for the maintenance of the amino acid pool, the exact cause of neonatal lethality in Atg5-null mice remains unclear. Although Atg5-null mice lack obvious anatomical abnormalities at birth, they have a suckling defect, which could be responsible for the neonatal lethality (Kuma et al., 2004). The suckling defect of Atg5-null mice could be due to neuronal dysfunction, because autophagy is important for the proper function and survival of neuronal cells based on the observations of neuron-specific Atg5, Atg7, or FIP200 knockout mice, in which the accumulation of ubiquitinpositive aggregates and neuronal cell death are observed (Komatsu et al., 2006; Hara et al., 2006; Liang et al., 2010). Protein aggregates can already be detected at birth. Indeed, artificial milk feeding extended the survival of Atg5-null neonates, at least partially (Kuma et al., 2004). However, suckling failure alone cannot explain their accelerated death because Atg5-null and Atg7-null neonates die earlier than wild-type neonates, even

Developmental Cell 39, 1–15, October 10, 2016 ª 2016 Elsevier Inc. 1

Please cite this article in press as: Yoshii et al., Systemic Analysis of Atg5-Null Mice Rescued from Neonatal Lethality by Transgenic ATG5 Expression in Neurons, Developmental Cell (2016), http://dx.doi.org/10.1016/j.devcel.2016.09.001

under non-suckling conditions (Kuma et al., 2004; Komatsu et al., 2005). Furthermore, subsequent studies reported that Atg5-null mice have defects in the clearance of apoptotic corpses (Qu et al., 2007) and development of the heart (Lee et al., 2014) and lung (Cheong et al., 2014). Therefore, the neonatal lethality might be a consequence of developmental defects. To address this question, we generated transgenic mice that express GFP-ATG5 under the control of a neuron-specific promoter. Re-expression of GFP-ATG5 in the brain was sufficient to rescue Atg5-null mice from neonatal lethality, suggesting that neuronal dysfunction, including suckling failure, is the primary cause of neonatal lethality. Further analysis of this novel mouse model revealed previously unappreciated roles for the autophagy gene Atg5 in multiple processes including regulation of the hypothalamic-pituitary-gonadal axis and iron absorption in the intestine. These observations provide a valuable resource for understanding the physiological roles of autophagy in the whole body. RESULTS Generation of NSE-GFP-ATG5 Transgenic Mouse Lines To address the hypothesis that the suckling defect may be due to neuronal dysfunction, which causes neonatal lethality, we attempted to rescue Atg5-deficient mice by re-expressing ATG5 in the brain. We generated four lines of transgenic mice expressing GFP-fused mouse ATG5 under the control of the rat neuronspecific enolase (NSE) promoter (Figure 1A) (Sakimura et al., 1985; Forss-Petter et al., 1990). We chose one NSE-GFPATG5 transgenic mouse line because its expression level of exogenous GFP-ATG5 was comparable with that of endogenous ATG5 (Figure 1B). The expression of GFP-ATG5 was confirmed to be highly specific to the brain, although some potential leakiness of GFP-ATG5 expression was observed in the thymus in adult mice and several organs in neonates (Figures 1C and S1A). Re-expression of ATG5 in the Brain Rescues Atg5-Null Mice from Neonatal Lethality We intercrossed NSE-GFP-ATG5 transgenic mice with Atg5+/
mice to generate Atg5
/
;NSE-Atg5 mice, thus expressing exogenous ATG5 in the brain in the context of Atg5-null mice. Next, we tested whether autophagic activity was restored by re-expressing GFP-ATG5 in the brain. Brain homogenates of Atg5-null newborns showed the accumulation of p62 (also known as SQSTM1), a typical selective substrate of autophagy (Figure 1B). This p62 accumulation was effectively reduced to a level comparable with that of wild-type mice by expressing exogenous GFP-ATG5 (Figure 1B). The conversion of LC3-I to LC3-II, which is dependent on ATG5, was also restored (Mizushima et al., 2001). These data suggest that autophagic activity was recovered in the brain of Atg5
/
;NSE-Atg5 mice. Whereas Atg5
/
mice are neonatal lethal, Atg5
/
;NSE-Atg5 mice were able to suckle and survive to adulthood (Figure 1D). If mice were denied milk feeding for 10 hr after cesarean delivery, the plasma amino acid levels were reduced both in Atg5
/
and Atg5
/
;NSE-Atg5 neonates but not in control neonates, suggesting that restoration of Atg5 in the brain (and in some peripheral tissues, if any, as shown in Figure S1A) does not improve the nutritional status (Figure S1B). On the other hand, the plasma 2 Developmental Cell 39, 1–15, October 10, 2016

amino acid levels were comparable between Atg5
/
;NSEAtg5 and control neonates under breast-fed conditions (postnatal day 0.5), suggesting that natural feeding overcomes the nutritional insufficiency in autophagy-deficient neonates (Figure 1E). In adult Atg5
/
;NSE-Atg5 mice, the accumulation of p62 aggregates in the brain was considerably less than in agematched Atg5flox/flox;Nestin-Cre mice (neural cell-specific Atg5 knockout mice), although p62 aggregates were still observed in some neurons but not glial cells in restricted areas within the medulla, midbrain, and hypothalamus in Atg5
/
;NSE-Atg5 mice (Figure 1F). Conversion of LC3 and the accumulation of p62 were not restored in all observed organs except for the brain in both adult mice and neonates, although a weak restoration of LC3 conversion was observed in the thymus in adult mice (Figures 1C and S1A). Thus, autophagy was rescued almost exclusively in the brain in Atg5
/
;NSE-Atg5 mice. To determine the survival rate, we counted the number of all genotypes. However, the number of pups was small for an unknown reason when we generated mutant mice on the C57BL/ 6 genetic background. We therefore generated mutant mice on a mixed genetic background (C57BL/6J 3 ICR) for further analysis because of the prolificacy of ICR mice. Intercross of Atg5+/
and Atg5+/
;NSE-Atg5 mice produced no viable Atg5
/
mice at weaning age, as observed previously (Figure 1G) (Kuma et al., 2004). In contrast, Atg5
/
;NSE-Atg5 mice appeared at close to the predicted Mendelian frequency at weaning age. Atg5
/
;NSE-Atg5 mice were smaller in size and weighed less than their littermates (Figure 1H). The life span of Atg5
/
;NSEAtg5 mice varied greatly among individuals; the majority of them survived for longer than 8 weeks and for 8 months at longest (data not shown). Thus, restoration of autophagy in neurons was sufficient to fully rescue Atg5-null mice from the neonatal lethality and allowed them to survive beyond weaning age. These data suggest that the primary cause of neonatal lethality in Atg5-null mice is neuronal dysfunction, which leads to suckling failure as one of the fatal abnormalities, and that defects in other peripheral organs are not fatal during the neonatal period. Gross and Histological Characterization of Atg5–/–;NSE-Atg5 Mice The rescue of Atg5-null mice from neonatal lethality allowed us to investigate the physiological roles of autophagy in the whole body of adult mice. We performed anatomical and histological analyses of the whole body of Atg5
/
;NSE-Atg5 mice (Figures 2 and 3; Table 1). The liver, spleen, and lymph nodes were significantly enlarged in Atg5
/
;NSE-Atg5 mice, whereas a severe reduction of weight was observed in the skeletal muscle, adipose tissue, pituitary gland, submandibular gland, and reproductive organs (Figures 2 and S2). Several of the previously observed tissue abnormalities were reproduced in Atg5
/
;NSE-Atg5 mice. The liver and pancreas showed tissue damage with cell swelling and vacuolation (Figure 3A) (Komatsu et al., 2005; Karsli-Uzunbas et al., 2014; Diakopoulos et al., 2015; Antonucci et al., 2015). While the granules in the Paneth cells in the small intestine were relatively uniform in control mice, their size and amount varied greatly in Atg5
/
;NSE-Atg5 mice (Figure 3B) (Cadwell et al., 2008, 2009; Wittkopf et al., 2012). Muscle fibers were significantly smaller, and centrally nucleated muscle fibers were observed

Please cite this article in press as: Yoshii et al., Systemic Analysis of Atg5-Null Mice Rescued from Neonatal Lethality by Transgenic ATG5 Expression in Neurons, Developmental Cell (2016), http://dx.doi.org/10.1016/j.devcel.2016.09.001

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H

Figure 1. Transgenic Rescue of Atg5–/– Mice (A) The NSE-GFP-Atg5 transgene is illustrated schematically. pA, SV40 poly(A). (B) Immunoblotting of ATG5, LC3, and p62 showing the effect of exogenous expression of GFP-Atg5 on autophagic activity in the brain from newborn mice. (C) Immunoblotting (IB) of GFP, ATG5, LC3, and p62 in postnuclear supernatant of indicated organs. Atg5+/+;NSE-Atg5 and Atg5
/
;NSE-Atg5 mice at 8–9 weeks of age were analyzed. (D) Representative picture of Atg5+/+ and Atg5
/
;NSE-Atg5 mice (female) at 10 weeks of age. Scale bar, 1 cm. (E) Total plasma amino acid concentration (Asp, Thr, Ser, Asn, Glu, Gln, Pro, Gly, Ala, Val, Cys, Met, Ile, Leu, Phe, Tyr, Lys, His, and Arg) was measured at postnatal day 0.5 under breast-fed conditions (C57BL/6J 3 ICR mixed background). Data represent mean ± SE; n.s., not significant (Student’s t test), n R 5. (F) Representative images of immunostaining of p62, H&E staining, and Nissl staining of brain sections from Atg5+/+, Atg5
/
;NSE-Atg5, and Atg5flox/flox;NestinCre mice at 10 weeks of age (n = 3). Scale bars, 20 mm. (G) Survival rate of each genotype at 4 weeks old. (H) Body weight of male and female Atg5
/
;NSE-Atg5 mice (black) and littermate controls (white, see Experimental Procedures) fed on a chow diet. They were weighed from P7 to week 8 every 3 days. Each point includes data from at least nine individuals. Data represent mean ± SD; *p < 0.05, **p < 0.01 (Student’s t test). (B), (C), (D), and (F) show data obtained from mice on the pure C57BL/6J background, and (E), (G), and (H) show data obtained from mice on the C57BL/6J 3 ICR mixed background. See also Figure S1.

occasionally in Atg5
/
;NSE-Atg5 mice (Figure S3A) (Masiero et al., 2009; Karsli-Uzunbas et al., 2014). Atg5
/
;NSE-Atg5 mice exhibited a varying degree of systemic inflammatory

changes, with increased numbers of the germinal centers in the spleen, and plasma cells and neutrophils in the lymph nodes (Figure 3C) (Deretic et al., 2013; Munz, 2014; Kimmey et al., 2015). Developmental Cell 39, 1–15, October 10, 2016 3

Please cite this article in press as: Yoshii et al., Systemic Analysis of Atg5-Null Mice Rescued from Neonatal Lethality by Transgenic ATG5 Expression in Neurons, Developmental Cell (2016), http://dx.doi.org/10.1016/j.devcel.2016.09.001

(legend on next page)

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Please cite this article in press as: Yoshii et al., Systemic Analysis of Atg5-Null Mice Rescued from Neonatal Lethality by Transgenic ATG5 Expression in Neurons, Developmental Cell (2016), http://dx.doi.org/10.1016/j.devcel.2016.09.001

One of the most stark abnormalities of Atg5
/
;NSE-Atg5 mice was that they have significantly smaller reproductive organs (Figure 2). The ovary of Atg5
/
;NSE-Atg5 mice had morphologically normal primary, secondary, and tertiary vesicular follicles, contrary to previous reports (Figure 3D) (Gawriluk et al., 2011; Song et al., 2015). However, Atg5-deficient ovaries lacked a corpus luteum but had a large number of atretic follicles, indicating that they fail to ovulate, while control mice contained multiple corpora lutea (Figure 3D). Atg5
/
;NSE-Atg5 female mice had thread-like uteri with loss of endometrial gland development, resembling ones before puberty (Figure 3D). In Atg5
/
;NSE-Atg5 male mice, the seminiferous tubules contained fewer mature spermatids while containing normalappearing spermatogonia (Figure 3D). The Leydig cells in the interstitial space were often smaller and indistinct in Atg5
/
;NSE-Atg5 mice, indicating that their maturation was impaired (Figure 3D). A markedly reduced number of mature sperm and a large number of abnormal non-spermatogenic cells were observed in the epididymis (Figure 3D). These data indicate that male Atg5
/
;NSE-Atg5 mice show defective spermatogenesis. Atg5
/
;NSE-Atg5 mice showed additional previously uncharacterized abnormalities. In addition to the Paneth cell abnormalities, the small intestine showed morphological changes with shorter and wider villi and deeper crypts (Figure 3B). The kidney showed mesangial proliferative glomerulonephritis with increased mesangial cells, mesangial matrix, and endocapillary inflammatory cells (Figure 3E). The lung displayed strong interstitial pneumonia with inflammatory cell infiltration of the alveolar wall and collapsed alveoli, likely due to the intense inflammation (Figure 3E). The number of nuclei was increased in muscle fibers of Atg5
/
;NSE-Atg5 mice (Figure 3E), suggesting that these muscles may have repeatedly regenerated (Wang and Rudnicki, 2012). Although submandibular glands are known to show sexual dimorphism (Jayasinghe et al., 1990), they were feminized in male Atg5
/
;NSE-Atg5 mice with markedly reduced eosinophilic granules in the convoluted ducts (Figure 3A). Accumulation of Ubiquitinated Proteins and p62 in Tissues of Atg5–/–;NSE-Atg5 Mice We compared various organs for the accumulation of ubiquitinated proteins and p62 (Figures 3F and S3B; Table 1). Clear increases of ubiquitinated proteins were observed in the heart, liver, pancreas, and muscle, suggesting that autophagy-dependent protein quality control is important in these tissues (Figure 3F). The levels of p62 proteins, but not of its mRNA, increased in all tissues, but especially in the liver, muscle, and testis (Figures 3F and 3G). Overall, the accumulation of ubiquitinated proteins and p62 showed a correlation, except for the testis, where ubiquitinated proteins massively accumulated also in control mice (Figure 3F). p62 slightly accumulated in neurons at this age (8 weeks old), which may be of glial origin or because rescue may be less efficient in adult mice compared

with neonates (Figures 1B and 3F). Accumulation of phosphorylated p62 (Ser351) is known to cause hyperactivation of the transcription factor nuclear factor erythroid 2-related factor 2 (NRF2) (Komatsu et al., 2010; Ichimura et al., 2013). Phosphorylated p62 greatly accumulated in the liver and muscle (Figure 3F). Consistently, a marked increase of NRF2 and NQO1, which is regulated by NRF2 (Komatsu et al., 2007), was observed in these tissues (Figure 3F). At the cellular level, immunohistochemistry revealed that extensive formation of p62 aggregates was observed in exocrine cells, including the liver, pancreas, and gastric gland, endocrine cells (including Leydig cells in the testis), pituitary gland, and adrenal gland, as well as in the muscle and retina (Table 1 and Figure S3B). Some of these tissues did not show apparent histological abnormalities, yet severe accumulation of p62 aggregates may imply the high dependence of these tissues on autophagy for quality control. Atg5–/–;NSE-Atg5 Mice Have Partial Spermatogenetic Failure with Decreased Testosterone and Gonadotropins Anatomical and histological analysis showed that Atg5
/
;NSEAtg5 mice exhibited stark abnormalities in the gonads and accessory sex organs. Consistent with the morphological observations, the number of mature sperm in the cauda epididymis was significantly smaller in Atg5
/
;NSE-Atg5 male mice than in control mice (Figure 4A). Atg5
/
;NSE-Atg5 female mice failed to develop normal estrous cyclicity (data not shown). In addition, the feminization of the submandibular gland of Atg5
/
;NSEAtg5 male mice mentioned above suggests loss of the response to androgen. Collectively, these data suggest an alteration of circulating sex hormone levels in Atg5
/
;NSE-Atg5 mice. In agreement with these observations, testosterone levels were significantly lower in Atg5
/
;NSE-Atg5 male mice than in control mice (Figure 4B). We measured the gene expression of several major enzymes required for testosterone synthesis and found that the mRNA levels of CYP17, CYP11a, and 17b-hydroxysteroid dehydrogenase were significantly decreased and those of steroidogenic acute regulatory protein (StAR) tended to decrease in the testes of Atg5
/
;NSE-Atg5 mice (Figure 4C). The decrease in CYP11A protein was confirmed by immunoblotting (Figure 4D). These data suggest that testosterone synthesis was suppressed in Atg5
/
;NSE-Atg5 mice. Gonadal activities including testosterone production are regulated by the pituitary-derived gonadotropins luteinizing hormone (LH) and follicle-stimulating hormone (FSH). Both serum levels and pituitary mRNA levels of LH and FSH were significantly reduced in Atg5
/
;NSE-Atg5 male mice (Figures 4E and 4F). The pituitary mRNA levels of growth hormone and the serum level of insulin-like growth factor 1 (IGF-1), a downstream effector of growth hormone, were also lower in Atg5
/
;NSE-Atg5 male mice, which may account for their growth retardation (Figures 4E and 4F). On the other hand, thyroid-stimulating hormone

Figure 2. Gross Anatomy of the Organs of Atg5–/–;NSE-Atg5 Mice Representative pictures of various organs from Atg5+/+ and Atg5
/
;NSE-Atg5 female mice (submandibular gland, testis, and seminal vesicle are from male mice) at 11–14 weeks of age. The graphs show wet tissue weight and body weight (male, n R 6; female, n R 5). Data represent mean ± SD; *p < 0.05, **p < 0.01 (Student’s t test). Scale bars, 5 mm. See also Figure S2.

Developmental Cell 39, 1–15, October 10, 2016 5

Please cite this article in press as: Yoshii et al., Systemic Analysis of Atg5-Null Mice Rescued from Neonatal Lethality by Transgenic ATG5 Expression in Neurons, Developmental Cell (2016), http://dx.doi.org/10.1016/j.devcel.2016.09.001

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Figure 3. Histological Analysis of Atg5–/–;NSE-Atg5 Mice (A–E) Representative images of H&E staining of the indicated tissues from Atg5+/+ and Atg5
/
;NSE-Atg5 mice (8–13 weeks of age, n R 3). The exocrine system (A), digestive system (B), lymphatic system (C), reproductive system (D), and others (E) are shown. In (D) the insets are enlarged images of the boxed regions. The arrowheads in (C) indicate irregularly shaped germinal centers (spleen). The dotted lines in (D) indicate corpora lutea (CL, ovary) and the Leydig cell area in the testis (Leydig). (F) Immunoblotting of ubiquitin (Ub), p62, phospho-p62 (Ser351), NRF2, and NQO1 in whole-cell lysates from the indicated organs (8 weeks of age). C, control Atg5+/+;NSE-Atg5 mouse; K, Atg5
/
;NSE-Atg5 mouse. (G) The mRNA levels of p62 (relative to GAPDH) in the indicated organs (n = 4, 8–14 weeks of age). Data represent mean ± SE; *p < 0.05, **p < 0.01 (Student’s t test). See also Figure S3.

6 Developmental Cell 39, 1–15, October 10, 2016

Group

Morphological Changes

p62 Aggregates

Inflammatory Changes

Previous Related Reports

hepatomegaly, hepatitis, and vacuolation

++

++

Komatsu et al., 2005

++

+/


Jung et al., 2008; Ebato et al., 2008; KarsliUzunbas et al., 2014; Antonucci et al., 2015; Diakopoulos et al., 2015


(acinar cells) +/
(ductal epithelium)

+

Morgan-Bathke et al., 2013

+

+




+/


Cadwell et al., 2008; Cadwell et al., 2009; Wittkopf et al., 2012

no obvious change







Tsuboi et al., 2015

proliferative glomerulonephritis

+ (renal tubules)

+

Hartleben et al., 2010; Liu et al., 2012; Martinez et al., 2016

+/





Nakai et al., 2007; Taneike et al., 2010; Lee et al., 2014

+/
(alveolar and bronchiolar epithelium)

++

Inoue et al., 2011; Guo et al., 2013; Cheong et al., 2014; Abdel Fattah et al., 2015; Lu et al., 2016

+/
(osteocyte)




Liu et al., 2013; Nollet et al., 2014

++




Masiero et al., 2009; Karsli-Uzunbas et al., 2014

Exocrine Tissues Liver

disorganized hepatic cell cord swelling of hepatocytes and occasional cell death Pancreas

swelling and vacuolation of acinar cells vacuolation of islet cells degeneration and acinar-to-ductal metaplasia

Submaxillary gland

loss of eosinophilic granules in the convoluted ducts in male swelling and vacuolation of acinar cells

Digestive System Stomach

swelling and vacuolation of chief and parietal cells occasional globular hyaline body

Small intestine

shorter and wider villi, deeper crypt increased proliferation and cell death granule abnormalities in the Paneth cells

Large intestine Urinary System Kidney

swelling of renal tubule epithelial cells Cardiopulmonary System

Developmental Cell 39, 1–15, October 10, 2016 7

Heart

dilatation especially of the right ventricle no obvious histological change

Lung

interstitial pneumonia with inflammatory cell infiltration in the alveolar wall decreased number of dilated alveoli, alveolar and bronchiolar dilation swelling of bronchiolar epithelial cells

Musculoskeletal System Skull

thin calvaria less-clear lamellar structures

Muscle

small size, small muscle fibers vacuolation and occasional centrally nucleated muscle fibers increased number of nuclei

(Continued on next page)

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Table 1. Histological Characterization of Atg5–/–;NSE-Atg5 Mice

Continued

Group

Morphological Changes

p62 Aggregates

Inflammatory Changes

Previous Related Reports

small size


(follicles) ++ (medulla)


+

lack of a corpus luteum

Gawriluk et al., 2011; Gawriluk et al., 2014; Song et al., 2015

thread-like uterus

+/





++ (Leydig cells)
(germline cells)




Karsli-Uzunbas et al., 2014; Wang et al., 2014

+/





Wang et al., 2014

+


+

Reproductive System Ovary Uterus

loss of development of endometrial glands Testis

small size deficient spermatogenesis immature Leydig cells

Epididymis

fewer spermatids in the lumen increased number of non-spermatogenic cells

Endocrine System Pituitary gland

small size no obvious histological change

Thyroid gland

no obvious change







Adrenal gland

vacuolation and eosinophilic change of cortical cells

++ (cortical cells) +/
(medullary cells)


+







Nedjic et al., 2008; Sukseree et al., 2012

+/


+

Mortensen et al., 2010; Karsli-Uzunbas et al., 2014; Rozman et al., 2015

+

++

Rozman et al., 2015




+

Singh et al., 2009; Zhang et al., 2009b; Kim et al., 2013b; Martinez-Lopez et al., 2013

swelling of medullary cells Lymphoid Tissues Thymus

occasionally extremely small no obvious histological change

Spleen

splenomegaly and increased number of megakaryocytes increased number of germinal centers less-clear structures of red and white pulps

Lymph nodes

large size filled with plasma cells and neutrophils, occasional multinuclear giant cells

Others Adipose tissue

small size reduced amount of lipid droplets

Skin

hyperplasia of appendages







Yoshihara et al., 2015

Retina

no obvious change

++ (ganglion cell layer, inner plexiform layer)




Kim et al., 2013a; Zhou et al., 2015

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8 Developmental Cell 39, 1–15, October 10, 2016

Table 1.

Please cite this article in press as: Yoshii et al., Systemic Analysis of Atg5-Null Mice Rescued from Neonatal Lethality by Transgenic ATG5 Expression in Neurons, Developmental Cell (2016), http://dx.doi.org/10.1016/j.devcel.2016.09.001

A

D

B

C

E

F

Figure 4. Impaired Spermatogenesis in Atg5–/–;NSE-Atg5 Male Mice (A) The number of epididymal sperm at 8–12 weeks of age. KO, knockout. Data represent mean ± SE (n = 3); **p < 0.01 (Student’s t test). (B) Serum testosterone levels in control and Atg5
/
;NSE-Atg5 male mice at 10–12 weeks of age. Data represent mean ± SE; n = 4 and n = 6 for the control and Atg5
/
;NSE-Atg5 male mice, respectively; **p < 0.01 (Student’s t test). (C) mRNA levels of StAR, Cyp17, Cyp11a, Hsd17, and Hsd3b1 in testes. The data are presented as the ratio relative to control samples. Data represent mean ± SE (n = 6); *p < 0.05, **p < 0.01 (Student’s t test). (D) Immunoblot analysis of CYP11A in testes at 10–12 weeks of age. (E) mRNA levels of the anterior pituitary hormones in mice at 8–11weeks of age. Data are presented as the ratio relative to control samples. Pro-opiomelanocortin (POMC) is a precursor polypeptide containing ACTH. Data represent mean ± SE; n = 4 and n = 3 for the control and Atg5
/
;NSE-Atg5 male mice, respectively; *p < 0.05, **p < 0.01 (Student’s t test). (F) The serum concentration of anterior pituitary hormones (at 8–14 weeks of age), and IGF-1 and leptin (at 4 weeks of age). Data represent mean ± SE (n R 4); *p < 0.05, **p < 0.01 (Student’s t test).

and adrenocorticotropic hormone (ACTH) were not reduced in Atg5
/
;NSE-Atg5 male mice, suggesting that the pituitary gland functions are not generally impaired (Figures 4E and 4F). In addition, the serum level of leptin, an adipose-derived hormone known to affect the hypothalamic-pituitary-gonadal axis (Caprio et al., 2001), was significantly lower in Atg5
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;NSE-Atg5 mice, consistent with their loss of adipose tissues (Figures 2 and 4F). Taken together, these results suggest that Atg5
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;NSE-Atg5 male mice have defects in the development of reproductive organs and spermatogenesis due to the reduction of circulating testosterone and gonadotropins. Atg5–/–;NSE-Atg5 Mice Develop Iron Deficiency Another notable abnormality observed in Atg5
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;NSE-Atg5 mice was anemia (Figure 5A). Although the reduction of the red blood cell count was consistent with previous reports (Zhang et al., 2009a; Liu et al., 2010; Mortensen et al., 2010), we noticed that the red blood cells of Atg5
/
;NSE-Atg5 mice were thinner and paler than those of control mice, and some of them had a

target cell-like shape (Figure 5B). This morphology was reminiscent of that observed in iron-deficiency anemia. Indeed, the serum iron level of Atg5
/
;NSE-Atg5 mice was significantly lower and the unbound iron-binding capacity, indicating unsaturated transferrin, was significantly higher than in control mice (Figure 5C). Supplementation of iron restored the reduced hemoglobin level and mean corpuscular hemoglobin (average hemoglobin amount per red blood cell) in Atg5
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;NSE-Atg5 mice (Figure 5D). These data suggest that iron deficiency in Atg5
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;NSE-Atg5 mice contributes strongly to their hypochromic anemia. We did not observe obvious external bleeding or fecal occult blood, indicating that Atg5
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;NSE-Atg5 mice do not lose iron by bleeding (data not shown). We then asked whether iron accumulated abnormally in some tissues in Atg5
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;NSE-Atg5 mice and was not able to be remobilized for use. However, iron storage in the liver and spleen as well as iron content in the bone marrow were markedly reduced in Atg5
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;NSE-Atg5 mice, and other organs did not accumulate excessive iron Developmental Cell 39, 1–15, October 10, 2016 9

Please cite this article in press as: Yoshii et al., Systemic Analysis of Atg5-Null Mice Rescued from Neonatal Lethality by Transgenic ATG5 Expression in Neurons, Developmental Cell (2016), http://dx.doi.org/10.1016/j.devcel.2016.09.001

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(Figures 5E and 5F). These data ruled out the possibility that Atg5
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;NSE-Atg5 mice have sufficient iron but fail to utilize it. Expression of Iron-Related Genes Is Insufficient in Atg5–/–;NSE-Atg5 Mice As we did not detect any abnormal bleeding or iron accumulation, we suspected that Atg5
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;NSE-Atg5 mice have malabsorption of iron. Dietary ferric iron (Fe3+) is reduced to ferrous iron (Fe2+) by duodenal cytochrome b (DCYTB), and then is imported into intestinal epithelial cells via divalent metal transporter 1 (DMT1). Incorporated intracellular iron is exported through the sole basolateral iron transporter ferroportin (FPN) to enter the body (Gulec et al., 2014). While Atg5
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;NSE-Atg5 mice expressed DMT1 at a level comparable with that of control mice, the expression levels of DCYTB and FPN were lower, suggesting that Atg5
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;NSE-Atg5 mice show malabsorption of iron due to the reduced expression of proteins involved in iron absorption (Figure 6A). 10 Developmental Cell 39, 1–15, October 10, 2016

Figure 5. Iron-Deficiency Anemia in Atg5–/–; NSE-Atg5 Mice (A) Red blood cell count of mice at 10–12 weeks of age. n = 8 and n = 7 for control and Atg5
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;NSEAtg5 mice, respectively. Data represent mean ± SE; **p < 0.01 (Student’s t test). (B) May-Giemsa staining of peripheral blood smears of 14-week-old mice. Enlarged images of the boxed regions are shown in the lower panels. Scale bars, 10 mm. (C) Serum iron levels and unbound iron-binding capacity (UIBC) of mice at 11–15 weeks of age (n = 4). Data represent mean ± SE; **p < 0.01 (Student’s t test). (D) Rescue of anemia by iron injection. The mice were injected subcutaneously with PBS (iron
) or 500 mg/g body weight of iron-dextran (iron +), and blood was analyzed at 4 weeks after the injection (n R 4 in each group). Red blood cell count, hemoglobin concentration, and mean corpuscular hemoglobin were measured in 10-week-old mice. Data represent mean ± SE; *p < 0.05, **p < 0.01 (one-way ANOVA and Tukey-Kramer). (E) Tissue iron levels in mice at 10–11 week of age (n = 3). Data represent mean ± SE; *p < 0.05, **p < 0.01 (Student’s t test). (F) Perls’/DAB enhanced iron staining of bone marrow of 14-week-old mice. Brown signals indicate the existence of ferric iron. Scale bars, 20 mm.

Hepcidin, a hepatic hormone, is known to bind to FPN and causes its internalization and degradation by endocytosis, thus inhibiting excessive iron absorption (Nemeth et al., 2004). Therefore, we measured hepcidin mRNA levels in the liver and serum hepcidin, but both were significantly reduced in Atg5
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;NSEAtg5 mice, reflecting a normal response to iron deficiency and anemia (Figure 6B). The iron-responsive element (IRE) and iron-regulatory protein (IRP) system is an important mechanism for the regulation of cellular iron homeostasis. IRPs bind to IREs in their target mRNAs and increase the mRNA stability (e.g., transferrin receptor 1 [TfR1]) or decrease their translation (e.g., ferritin) (Wang and Pantopoulos, 2011). The TfR1 mRNA tended to be higher in the intestine of Atg5
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;NSE-Atg5 mice than in control mice (Figure 6C). Ferritin protein levels were lower both in the liver and intestine in Atg5
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;NSE-Atg5 mice than in control mice (Figure 6D). These data suggest that the IRE-IRP system properly responds to iron deficiency in Atg5
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;NSE-Atg5 mice. The expression of proteins involved in iron absorption can be induced in response to iron deficiency and anemia at the transcriptional level (Gulec et al., 2014). To induce transcription of these genes, we fed mice with iron-deficient food. Although control mice markedly increased the mRNA expression of Dcytb, DMT1, and Fpn under iron-deficient conditions, the increase in their expression was significantly lower in Atg5
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;NSE-Atg5 mice, suggesting that they fail to properly upregulate iron-related genes (Figure 6E). It should also be noted that the mRNA levels of

Please cite this article in press as: Yoshii et al., Systemic Analysis of Atg5-Null Mice Rescued from Neonatal Lethality by Transgenic ATG5 Expression in Neurons, Developmental Cell (2016), http://dx.doi.org/10.1016/j.devcel.2016.09.001

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Figure 6. Insufficient Expression of Iron-Related Genes in Atg5–/–;NSE-Atg5 Mice (A) Immunoblotting of DCYTB, DMT1, and FPN in the small intestine (duodenum and upper jejunum) of 4-week-old mice. The graphs show quantification of the immunoblot data. The data are presented as the ratio relative to control samples. Data represent mean ± SE; *p < 0.05, **p < 0.01 (Student’s t test). (B) Hepcidin mRNA levels (relative to GAPDH) in the liver (n = 4, 7–8 weeks old) and serum hepcidin protein levels (n = 6, 7–14 weeks old). Data represent mean ± SE; **p < 0.01 (Student’s t test). (C) The mRNA levels (relative to GAPDH) of transferrin receptor 1 (TfR1) in the small intestine of mice (n R 8 in each group, 10–11 week old). Data represent mean ± SE; n.s., not significant (Student’s t test). (D) Immunoblotting of ferritin in the postnuclear supernatant of the small intestine and liver of mice at 6 weeks of age. (E) The mRNA levels (relative to GAPDH) of Dcytb, DMT1, and Fpn in the small intestine of mice fed with a regular or iron-deficient diet for 5–6 weeks (n R 8 in each group, 10–11 weeks old). Data represent mean ± SE; *p < 0.05, **p < 0.01 (one-way ANOVA and Tukey-Kramer). (F) Immunoblotting of DCYTB, DMT1, and FPN in the small intestine (duodenum and upper jejunum) of mice treated as in (E). The graphs show quantification of the immunoblot data. The data are presented as the ratio relative to those of control mice on a regular diet. Data represent mean ± SE; **p < 0.01 (one-way ANOVA and Tukey-Kramer). (G) Immunostaining of the duodenum and upper jejunum of mice treated as in (E). Frozen sections were stained with an anti-FPN antibody. Enlarged images of the boxed regions are shown in the lower panel. Arrowheads indicate the FPN-positive basolateral membranes. Scale bars, 20 mm. See also Figures S4 and S5.

Developmental Cell 39, 1–15, October 10, 2016 11

Please cite this article in press as: Yoshii et al., Systemic Analysis of Atg5-Null Mice Rescued from Neonatal Lethality by Transgenic ATG5 Expression in Neurons, Developmental Cell (2016), http://dx.doi.org/10.1016/j.devcel.2016.09.001

these factors in Atg5
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;NSE-Atg5 mice were comparable (Dcytb and DMT1) or decreased (Fpn) relative to those in control mice on a regular diet, even though iron was already deficient in Atg5
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;NSE-Atg5 mice (Figure 6E). On the other hand, the mRNA levels of most of other nutrient transporters were not decreased except for GLUT2 and SLC6A20A, suggesting that malabsorption of nutrients is relatively specific to iron (Figure S4). The protein levels of DCYTB and FPN, but not DMT1, also tended to be lower in Atg5
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;NSE-Atg5 mouse intestine under iron-deficient conditions (note that the expression levels of these factors on a regular diet become lower after 4 weeks of age in mice) (Figure 6F). Furthermore, FPN was detected on the basolateral membrane of intestinal mucosa cells in control mice, but not in Atg5
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;NSE-Atg5 mice, under iron-deficient conditions (Figure 6G). These results suggest that Atg5
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;NSE-Atg5 mice develop iron deficiency due to insufficient transcriptional upregulation of proteins involved in iron absorption. Finally, we attempted to determine whether the observed iron malabsorption is due to a cell-autonomous defect using intestinal epithelium-specific Atg5-deficient (Atg5flox/flox; Villin-Cre) mice. ATG5 expression and LC3 conversion slightly remained in the intestine of these mice, which may reflect non-epithelial cell origins (Figure S5A). These mice demonstrated significant reductions in transcriptional upregulation of iron-related genes (Dcytb, DMT1, and Fpn) (Figure S5B). Thus, the iron malabsorption of Atg5
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;NSE-Atg5 mice may be caused partially by cell-autonomous mechanisms in the intestinal epithelium. However, the reduction of these transcripts in Atg5flox/flox;Villin-Cre mice was milder than in Atg5
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;NSEAtg5 mice, and the protein levels of these factors (Figure S5A) and the red blood cell count (Figure S5C) were not significantly reduced, suggesting that cell-non-autonomous mechanisms also contribute to the phenotype. Elucidation of the causative organ(s) needs further investigation. DISCUSSION In this study, we showed that Atg5-null neonates are almost completely rescued from neonatal lethality by re-expressing ATG5 only in neurons on the C57BL/6J 3 ICR background (Figure 1G). Postweaning survivors were also observed on the pure C57BL/6J background (Figures 1C, 1D, and 1F), but we could not evaluate the precise rescue efficiency due to the small number of C57BL/6J pups. These data suggest that the neuronal dysfunction caused by autophagy deficiency, most likely suckling failure, is the primary cause of the neonatal lethality of Atg5-null mice. However, the suckling defect alone cannot explain the observation that Atg5-null neonates survive for only approximately 12 hr after birth, because all wild-type and heterozygous neonates survive longer than 12 hr (20 hr), even without milk feeding (Kuma et al., 2004). Thus, our previous assumption is still valid; lack of a nutrient supply from the autophagic degradation of cytoplasmic macromolecules during the neonatal starvation period exaggerates their malnutrition and shortens their life span under non-suckling conditions, although this function itself is not critical for survival if neonates drink milk. Taken together, our observations highlight two crucial roles for autophagy in neonates: prevention of neuronal dysfunction and adaptation to starvation. Our data also suggest that dysfunction 12 Developmental Cell 39, 1–15, October 10, 2016

of other organs is not fatal, at least for several weeks, in Atg5-null mice. During the past decade, the importance of autophagy and/or Atg genes has been examined in various organs by using mice with spatiotemporal, conditional knockout of Atg genes. Many of the abnormalities reported in these knockout mice were also observed in our Atg5
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;NSE-Atg5 mice (Table 1). In addition, we observed some novel findings such as defects in reproductive organs with low levels of testosterone and gonadotropins and iron deficiency due to the insufficient expression of ironrelated proteins, as well as newly observed histological changes in several organs (Table 1). These abnormalities might not have been observed in other studies due to the lack of appropriate Cre-transgenic mouse lines or incomplete knockout efficiency. Alternatively, the combined dysfunction of more than two cell lineages may produce these abnormalities. Therefore, the information obtained from Atg5
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;NSE-Atg5 mice is valuable for understanding the systemic roles of autophagy. Of course, some of the abnormal findings could be secondary effects rather than direct consequences of autophagy suppression. For example, it appeared that Atg5
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;NSE-Atg5 mice suffered from systemic inflammation, although its severity varied greatly among individuals, highlighting the importance of autophagy for regulation of the immune system. We do not rule out the possibility that some of the abnormalities are due to a disturbance of the autophagy-independent roles of ATG5, such as its recently reported functions in the immune system preventing an unnecessarily severe immune response or in LC3-associated phagocytosis (Kimmey et al., 2015; Martinez et al., 2016). In the present study, we particularly focused on two novel findings: hypogonadism and systemic iron deficiency. The reproductive organs both in male and female Atg5
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;NSE-Atg5 mice show severe defects. Testosterone and gonadotropins (LH and FSH) were decreased in Atg5
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;NSE-Atg5 mice. The reduction of testosterone in Atg5
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;NSE-Atg5 males seems to be sufficient to cause a defect in spermatogenesis, because mice with similar levels of testosterone reduction reportedly display small testes and spermatogenic failure (Pakarainen et al., 2005). As the hypothalamic-pituitary-gonadal axis plays a central role in the regulation of gonadal maturation and function, the low hormone levels in Atg5
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;NSE-Atg5 mice might be due to insufficient rescue of hypothalamic function. However, this is unlikely because Atg5flox/flox;Nestin-Cre mice (neural cellspecific Atg5 knockout mice) do not show such genital abnormalities (data not shown). Thus, the low testosterone levels might be due to defects in the pituitary gland, the adipose tissue, and the testis itself. Recent studies using germ cell-specific Atg7 knockout mice showed a role for autophagy in acrosome biogenesis (Wang et al., 2014). However, spermatogenic failure in germ cell-specific Atg7 knockout mice is milder than in Atg5
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;NSE-Atg5 mice, and folliculogenesis and ovulation were observed in female germ cell-specific Atg7 knockout mice (Song et al., 2015). Thus, multiple factors should contribute to the dysfunction of reproductive organs in Atg5
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;NSE-Atg5 mice. Atg5
/
;NSE-Atg5 mice showed anemia and iron deficiency; however, their anemia is not solely derived from iron deficiency, because we observed that neonates and up to 2-week-old mice did not show iron deficiency, but already exhibited anemia (data

Please cite this article in press as: Yoshii et al., Systemic Analysis of Atg5-Null Mice Rescued from Neonatal Lethality by Transgenic ATG5 Expression in Neurons, Developmental Cell (2016), http://dx.doi.org/10.1016/j.devcel.2016.09.001

not shown). These data are consistent with the roles of autophagy in hematopoiesis; autophagy is required for efficient mitophagy in reticulocytes and for the maintenance of hematopoietic stem cells (Liu et al., 2010; Mortensen et al., 2010, 2011). In this study, we observed a close correlation between the decrease in the mRNA levels of iron-related genes, protein expression, and iron deficiency. As Dcytb, DMT1, and Fpn are all regulated by a common transcription factor, hypoxia-inducible factor 2a (HIF-2a) (Shah et al., 2009; Taylor et al., 2011), one possibility is that HIF-2a is dysfunctional. However, we observed that HIF-2a translocates properly to the nucleus in Atg5
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;NSE-Atg5 mice in response to their iron deficiency and anemia (data not shown). Several reports have connected autophagy and abnormal transcription. Defective autophagy in the liver causes the accumulation of p62, which in turn inactivates KEAP1, resulting in hyperactivation of NRF2 (Komatsu et al., 2010). Autophagy deficiency and defective oxidative phosphorylation in the muscle or liver cause FGF21 production, which is dependent on the transcription factor ATF4 (Kim et al., 2013b). These reports suggest that autophagy deficiency leads to the accumulation of autophagy substrates, which affects transcription positively or negatively. The transcriptional repression of iron-related genes observed in Atg5
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;NSE-Atg5 mice may also be caused by similar mechanisms. How autophagy or Atg5 deficiency causes transcriptional repression of these ironrelated genes will need further investigation.

Antibodies Rabbit polyclonal antibodies against rat LC3B were reported previously (Quy et al., 2013). Antibodies against p62 (GP62-C; Progen), phospho-p62 Ser351 (PM074; MBL), Nqo1 (ab34173; Abcam), Atg5 (A0731; Sigma-Aldrich), GFP (598; MBL), Dcytb (DCYTB11-A; Alpha Diagnostic), DMT1 (NRAMP24-A; Alpha Diagnostic), ferroportin (MTP11-A; Alpha Diagnostic), ubiquitin (z0458; Dako), Cyp11a1 (ab175408; Abcam), ferritin (F6013; Sigma), HSP90 (610419; BD Transduction Laboratories), and b-actin (A2228, Sigma-Aldrich) were purchased. Horseradish peroxidase-conjugated anti-mouse and anti-rabbit immunoglobulin G antibodies were purchased from Jackson ImmunoResearch. Tissue Sample Preparation for Immunoblotting To obtain postnuclear supernatants, we clarified tissue homogenates by centrifugation at 500 3 g for 10 min, and the supernatants were mixed with 63 sample buffer and boiled for 5 min (except for the following). For the detection of intestinal iron-related factors (DCYTB, DMT1, FPN, and b-actin), tissue homogenates containing 1% Triton X-100 were clarified by centrifugation at 500 3 g for 10 min, and the supernatants were mixed with 63 sample buffer and incubated at room temperature. Tissue homogenates in RIPA buffer were sonicated, mixed with 63 sample buffer, and heated at 95 C for 5 min to obtain whole-cell lysates including the nuclear fraction. Statistical Analysis Body and tissue weights are presented as mean ± SD. Other data are presented as mean ± SE. Two groups of data were evaluated by unpaired twotailed Student’s t test. Multiple comparisons were performed by one-way ANOVA followed by the Tukey-Kramer method. p Values of <0.05 were considered significant. SUPPLEMENTAL INFORMATION

EXPERIMENTAL PROCEDURES Mice All animal experiments were approved by the Institutional Animal Care and Use Committee of Tokyo Metropolitan Institute of Medical Sciences, Tokyo Medical and Dental University, and the University of Tokyo. The NSE-GFP-Atg5 transgene was engineered as follows. A 4-kbp DNA fragment encoding the rat NSE promoter and exon 1 was introduced at the 50 site of the GFP-Atg5 plasmid containing mouse Atg5 cDNA in a pEGFP vector (Sakimura et al., 1985). The transgene construct was linearized and the resulting 5.8-kbp fragment was microinjected into C57BL/6J fertilized eggs. NSE-GFP-Atg5 transgenic mice were intercrossed with Atg5+/
mice to yield Atg5
/
;NSE-Atg5 mice. The animals were maintained on C57BL/6J background or a mixedstrain background (C57BL/6J 3 ICR). For genotyping of the NSE-GFP-Atg5 transgene by PCR, the following primer sets were used to amplify an approximately 500-bp fragment in the transgene: 50 -TCCTGCTGGAGTTC GTGACCG-30 and 50 -ATCAGCTTCTTTCATACACGAC-30 . Atg5+/
mice and Atg5flox/flox;Nestin-Cre mice were described previously (Kuma et al., 2004; Hara et al., 2006). Villin-Cre mice (from the Jackson Laboratory) were crossed with Atg5flox/flox mice to obtain Atg5flox/flox;Villin-Cre mice. The mice were maintained on a 12-hr light/12-hr dark cycle and provided with food and water ad libitum. Atg5+/+, Atg5+/+;NSE-Atg5, Atg5+/
, and Atg5+/
;NSE-Atg5 mice were used as control mice. Measurement of Amino Acid Concentration Blood was collected from neonates at postnatal day 0.5 (at 15:00–17:00), when the neonates had already started suckling. Plasma was deproteinized with 3% sulfosalicylic acid. After centrifugation at 20,000 3 g for 5 min at 4 C, free amino acids in the supernatants were measured using an automated L-8900FF amino acid analyzer (Hitachi). Iron Supplementation At 6 weeks of age the mice were injected subcutaneously with either PBS or 500 mg/g body weight of iron-dextran (Sigma) (Nicolas et al., 2002). At 4 weeks after iron injection, the mice were euthanized and blood was obtained by cardiac puncture. Blood counts were performed by Oriental Yeast.

Supplemental Information includes Supplemental Experimental Procedures and five figures and can be found with this article online at http://dx.doi.org/ 10.1016/j.devcel.2016.09.001. AUTHOR CONTRIBUTIONS S.R.Y., A.K., T.H., and N.M. designed the experiments. T.H., H.S., and S.T. generated NSE-GFP-Atg5 transgenic mice. S.R.Y., A.K., Y.K., and E.I. performed general anatomical studies. A.K., A.Y., Y.K., and S.T. analyzed reproductive organs. S.R.Y. and A.K. analyzed the anemia phenotype. S.R.Y., A.K., T.A., and Y.E. performed histological analysis. S.R.Y., A.K., and N.M. wrote the manuscript. All authors commented on the manuscript, data, and conclusions. ACKNOWLEDGMENTS We thank Dr. Kenji Sakimura for the NSE promoter and Keiko Igarashi for technical assistance in histological studies. We thank Ms. Yuka Hiraoka and Drs. Yoshiki Miura and Takashi Ueno of the Laboratory of Proteomics and BioMolecular Science at Juntendo University Graduate School of Medicine for amino acid analysis. This work was supported by JST PRESTO (to A.K.), a Grant-inAid for JSPS Fellows (grant number 25*7082) (to S.R.Y), Funding Program for Next Generation World-Leading Researchers (LS043), and a JSPS KAKENHI Grant-in-Aid for Scientific Research on Innovative Areas (grant number 25111005) (to N.M.). Received: January 22, 2016 Revised: June 23, 2016 Accepted: August 31, 2016 Published: September 29, 2016 REFERENCES Abdel Fattah, E., Bhattacharya, A., Herron, A., Safdar, Z., and Eissa, N.T. (2015). Critical role for IL-18 in spontaneous lung inflammation caused by autophagy deficiency. J. Immunol. 194, 5407–5416.

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