© 2006 Nature Publishing Group Loss of autophagy in the central nervous systemcauses neurodegeneration in miceMasaaki Komatsu1,2*, Satoshi Waguri3*†, Tomoki Chiba1, Shigeo Murata1, Jun-ichi Iwata1,2, Isei Tanida2,Takashi Ueno2, Masato Koike3, Yasuo Uchiyama3, Eiki Kominami2& Keiji Tanaka1Protein quality-control, especially the removal of proteins withaberrant structures, has an important role in maintainingthe homeostasis of non-dividing neural cells1. In addition to theubiquitin–proteasome system, emerging evidence points to theimportance of autophagy—the bulk protein degradation pathwayinvolved in starvation-induced and constitutive protein turn-over—in the protein quality-control process2,3. However, little isknown about the precise roles of autophagy in neurons. Here wereport that loss of Atg7 (autophagy-related 7), a gene essential forautophagy, leads to neurodegeneration. We found that micelacking Atg7 specifically in the central nervous system showedbehavioural defects, including abnormal limb-clasping reflexesand a reduction in coordinated movement, and died within 28weeks of birth. Atg7 deficiency caused massive neuronal loss in thecerebral and cerebellar cortices. Notably, polyubiquitinated pro-teins accumulated in autophagy-deficient neurons as inclusionbodies, which increased in size and number with ageing. Therewas, however, no obvious alteration in proteasome function. Ourresults indicate that autophagy is essential for the survival ofneural cells, and that impairment of autophagy is implicated in thepathogenesis of neurodegenerative disorders involving ubiquitin-containing inclusion bodies.Macroautophagy (hereafter referred to as autophagy) is an evolu-tionarily conserved pathway in which the cytoplasm and organellesare engulfed within double-membraned vesicles, known as autopha-gosomes, in preparation for the turnover and recycling of thesecellular constituents4. Genetic studies using various model organismshave highlighted the importance of autophagy in physiological andpathological events5. The principal role of autophagy is in the supplyof nutrients for survival, as shown in yeast6and early neonatal mice7,8.Autophagy also has a role in cellular remodelling during differen-tiation and the development of multicellular organisms, such asdauer formation in Caenorhabditis elegans9and metamorphosis inDrosophila melanogaster10. Moreover, constitutive autophagy, whichoccurs independently of nutrient stress, contributes to mouse liverhomeostasis8, major histocompatibility class (MHC) II antigenpresentation11, and cellular defence against invading streptococci12and Mycobacterium tuberculosis13. However, the physiological func-tions of autophagy, particularly in neurons, are still largely unknown.To examine the relationship between neuronal pathology andautophagy deficiency in vivo, we crossed Atg7-conditional knockoutmice (Atg7flox/flox) (ref. 8) with transgenic mice expressing Crerecombinase under the control of the nestin promoter (nestin-Cre)(ref. 14), to produce mice deficient for Atg7 specifically in the centralnervous system (Atg7flox/flox; nestin-Cre). Atg7 is an E1-like enzymefor both the Atg12- and Atg8-conjugation systems15, and is essentialLETTERSFigure 1 | Behavioural disorder in Atg7flox/flox; nestin-Cre mice.a, Impairment of two ATG-conjugation systems (Atg12 and LC3) in theAtg7-deficient brain. Brain homogenates from P28 mice wereimmunoblotted with antibodies against Atg7, Atg5 and LC3. Actin was usedas a loading control. Data shown are representative of three separateexperiments. b, Kaplan–Meier survival curves of Atg7flox/þ; nestin-Cre(n ¼ 41) and Atg7flox/flox; nestin-Cre (n ¼ 26) mice over 28 weeks.c, Abnormal limb-clasping reflexes in Atg7flox/flox; nestin-Cre mice at P28.When lifted by the tail, Atg7flox/þ; nestin-Cre mice behave normally,extending their hind limbs and bodies. In contrast, Atg7flox/flox; nestin-Cremice bend their legs towards their trunk or tighten their back limbs to theirbodies and anterior limbs. d, Movement ataxia in Atg7flox/flox; nestin-Cremice at P28. Motor coordination was tested using a rotarod assay. Atg7flox/þ;nestin-Cre (n ¼ 5) and Atg7flox/flox; nestin-Cre (n ¼ 5) mice were placed on arod rotating at 20 r.p.m., and the time spent on the rod was counted. Datashow mean ^ s.d. *, P , 0.01 (Student’s t-test). There was no significant sexdifference in survival rate and onset-stage of abnormal limb-clasping andtremor in Atg7flox/flox; nestin-Cre mice.1Laboratory of Frontier Science, Tokyo Metropolitan Institute of Medical Science, Bunkyo-ku, Tokyo 113-8613, Japan.2Department of Biochemistry, Juntendo University School ofMedicine, Bunkyo-ku, Tokyo 113-8421, Japan.3Department of Cell Biology and Neurosciences, Osaka University Graduate School of Medicine, Osaka 565-0871, Japan. †Presentaddress: Department of Anatomy and Histology, Fukushima Medical University School of Medicine, 1 Hikarigaoka, Fukushima 960-1295, Japan.*These authors contributed equally to this work.Vol 441|15 June 2006|doi:10.1038/nature04723880© 2006 Nature Publishing Group for autophagy8. Atg7 protein was absent at postnatal day (P)28 inbrain from Atg7flox/flox; nestin-Cre but not control (Atg7flox/þ; nestin-Cre) mice (Fig. 1a). The level of Atg7 protein in other tissues such asliver, lung, heart and muscle was comparable between Atg7flox/flox;nestin-Cre and control mice (data not shown). Atg12–Atg5 conjugatewas detected only in the brains of control mice by immunoblottingwith an anti-Atg5 antibody (Fig. 1a). In contrast, free Atg5, whichwas faintly observed in the control mouse brain, was clearly increasedin the mutant brain (Fig. 1a). The mammalian homologue of yeastAtg8, microtubule-associated protein 1 light-chain 3 (LC3), exists intwo forms (LC3-I and LC3-II)16. Both forms were detected in brainsfrom control mice, but only the LC3-I form was detected in Atg7flox/flox;nestin-Cre brain (Fig. 1a). The loss of both Atg7 and LC3-II proteinswas observed from P0 in the brain of Atg7flox/flox; nestin-Cre mice(Supplementary Fig. S1). These results indicate complete impair-ment of autophagy in the central nervous system of Atg7flox/flox;nestin-Cre mice after birth.Atg7flox/flox; nestin-Cre mice were viable at birth and indistinguish-able in appearance from their littermates. However, the survival rateof the mutant mice diminished markedly by four weeks after birth,and all Atg7flox/flox; nestin-Cre mice were dead within 28 weeks(Fig.