© 2006 Nature Publishing Group Suppression of basal autophagy in neural cellscauses neurodegenerative disease in miceTaichi Hara1, Kenji Nakamura2, Makoto Matsui1,3,4, Akitsugu Yamamoto5, Yohko Nakahara2,Rika Suzuki-Migishima2, Minesuke Yokoyama6, Kenji Mishima7, Ichiro Saito7, Hideyuki Okano8,9& Noboru Mizushima1,10Autophagy is an intracellular bulk degradation process throughwhich a portion of the cytoplasm is delivered to lysosomes to bedegraded1–4. Although the primary role of autophagy in manyorganisms is in adaptation to starvation, autophagy is also thoughtto be important for normal turnover of cytoplasmic contents,particularly in quiescent cells such as neurons. Autophagy mayhave a protective role against the development of a number ofneurodegenerative diseases5–8. Here we report that loss of autophagycauses neurodegeneration even in the absence of any disease-associated mutant proteins. Mice deficient for Atg5 (autophagy-related 5) specifically in neural cells develop progressive deficits inmotor function that are accompanied by the accumulation ofcytoplasmic inclusion bodies in neurons. In Atg52/2cells, diffuse,abnormal intracellular proteins accumulate, and then form aggre-gates and inclusions. These results suggest that the continuousclearance of diffuse cytosolic proteins through basal autophagy isimportant for preventing the accumulation of abnormal proteins,which can disrupt neural function and ultimately lead toneurodegeneration.Every eukaryotic cell has two main systems for the degradation ofintracellular components: the ubiquitin–proteasome system andautophagy. Autophagy is a generic term for the degradation ofcellular components in lysosomes1–4. Macroautophagy (hereafterreferred to as autophagy) is believed to be the main pathway amongseveral subtypes of autophagy. During the process of autophagy,small portions of cytoplasm are sequestered by autophagosomesand then degraded on fusion with lysosomes. In contrast to theubiquitin–proteasome system, which accounts for most of theselective intracellular protein degradation, autophagy is less selective.Autophagy induced by starvation is a mechanism for producingamino acids within cells. In yeast, autophagy-defective cells aresusceptible to starvation. In comparison, mice deficient for Atg5and Atg7, which are essential for autophagosome formation9, sufferfrom severe nutrient- and energy-insufficiency soon after birth10,11.Thus, adaptation to starvation is an evolutionarily conserved role ofautophagy.In addition to induced autophagy, a low level of constitutiveautophagy is important for intracellular clearance under normalconditions. Mice bearing a liver-specific conditional knockout alleleof Atg7 show hepatic dysfunction and intracellular ubiquitin-positiveinclusion bodies11. We have also observed the accumulation ofubiquitin-positive inclusion bodies in hepatocytes and a subset ofneurons in Atg5-knockout (Atg52/2) neonates (SupplementaryFig. S1); however, conventional histological analysis revealed nosignificant abnormality10. These data suggest that intracellular pro-tein quality-control by autophagy is particularly important in neuralcells. Indeed, several studies have suggested that impairment ofautophagy could worsen the accumulation of abnormal proteins inneurodegenerative disease models in vitro and in vivo5–8. However,direct evidence demonstrating that autophagy contributes to theprevention of neurodegeneration has been lacking, in part becauseAtg52/2and Atg72/2mice die soon after birth10,11.To determine the role of autophagy in neural cells, we generatedneural-cell-specific Atg52/2mice (Supplementary Fig. S2). Micebearing an Atg5floxallele, in which exon 3 of the Atg5 gene isflanked by two loxP sequences, were crossed with a transgenic lineexpressing Cre recombinase under the control of the nestin promoter(nestin-Cre)12. In these mice, Cre recombinase is expressed in neuralprecursor cells after embryonic day (E)10.5, causing deletion of theloxP-flanked exon 3 (Supplementary Fig. S3). Recombination wassuccessful in over 90% of all brain cells from Atg5flox/flox; nestin-Cremice. The expression of Atg5 (detected as an Atg12–Atg5conjugate13) and the Atg5-dependent conversion of microtubule-associated protein 1 light chain 3 (LC3)-I to LC3-II (LC3–phospha-tidylethanolamine (LC3–PE) conjugate)13,14were almost completelysuppressed in the brains of Atg5flox/flox; nestin-Cre mice after E15.5(Fig. 1a and Supplementary Fig. S3). These data suggest thatautophagosome formation is impaired in the brains of these mutantmice.Atg5flox/flox; nestin-Cre mice were born normally and survivedneonatal starvation. They did not show the suckling defects observedin Atg52/2and Atg72/2neonates10,11, suggesting that an undetect-able, but sufficient, level of Atg5 remains in the neurons controllingthe suckling response at this stage, or that non-neural cells maymediate the suckling deficit in the non-conditional mutants. How-ever, the Atg5flox/flox; nestin-Cre mice showed growth retardation:their mean body weight was about 1.5-times lower than that of control(Atg5flox/þ;nestin-Cre) mice (Fig. 1b). Atg5flox/flox;nestin-Cre micedeveloped progressive motor and behavioural deficits after threeweeks of age, and footprint analysis revealed an ataxic walkingpattern (Fig. 1c). Mean stride lengths corrected for paw base widthswere significantly decreased compared with control (Atg5flox/þ;nestin-Cre) mice. The Atg5flox/flox; nestin-Cre mice showed limb-clasping reflexes when they were suspended by their tails, whereascontrol mice extended their limbs (Fig. 1d). This abnormal reflex isoften observed in mouse models of neurodegenerative disease15,16.LETTERS1Department of Bioregulation and Metabolism, Tokyo Metropolitan Institute of Medical Science, Tokyo 113-8613, Japan.2Mouse Genome Technology Laboratory, MitsubishiKagaku Institute of Life Sciences, Tokyo 194-8511, Japan.3Department of Basic Biology, School of Life Science, the Graduate University for Advanced Studies, Okazaki 444-8585,Japan.4Department of Cell Biology, National Institute for Basic Biology, Okazaki 444-8585, Japan.5Department of Bio-Science, Nagahama Institute of Bio-Science andTechnology, Nagahama 526-0829, Japan.6Brain Research Institute, Niigata University, Niigata 951-8510, Japan.7Department of Pathology, Tsurumi University School of DentalMedicine, Yokohama 230-8501, Japan.8Department of Physiology, Keio University School of Medicine, Tokyo 160-8582,