Human Molecular Genetics, 2007, Vol. 16, No. 12 1495–1503 doi:10.1093/hmg/ddm100 Advance Access published on April 27, 2007 Autophagy in Niemann–Pick C disease is dependent upon Beclin-1 and responsive to lipid trafficking defects Chris D. Pacheco1, Robin Kunkel2 and Andrew P. Lieberman1,2,* 1 2 Neuroscience Program and Department of Pathology, The University of Michigan Medical School, Downloaded from https://academic.oup.com/hmg/article/16/12/1495/2356123 by guest on 27 September 2021 Ann Arbor, MI 48109, USA Received February 7, 2007; Revised April 6, 2007; Accepted April 11, 2007 Niemann–Pick C (NPC) disease is an autosomal recessive lipid storage disorder characterized by a disrup- tion of sphingolipid and cholesterol trafficking that produces cognitive impairment, ataxia and death, often in childhood. Most cases are caused by loss of function mutations in the Npc1 gene, which encodes a protein that localizes to late endosomes and functions in lipid sorting and vesicle trafficking. Here, we demonstrate that NPC1-deficient primary human fibroblasts, like npc12/2 mice fibroblasts, showed increased autophagy as evidenced by elevated LC3-II levels, numerous autophagic vacuoles and enhanced degradation of long- lived proteins. Autophagy because of NPC1 deficiency was associated with increased expression of Beclin-1 rather than activation of the Akt-mTOR-p70 S6K signaling pathway, and siRNA knockdown of Beclin-1 decreased long-lived protein degradation. Induction of cholesterol trafficking defects in wild-type fibroblasts by treatment with U18666A increased Beclin-1 and LC3-II expression, whereas treatment of NPC1-deficient fibroblasts with sphingolipid-lowering compound NB-DGJ failed to alter the expression of either Beclin-1 or LC3-II. Primary fibroblasts from patients with two other sphingolipid storage diseases, NPC2 deficiency and Sandhoff disease, characterized by sphingolipid trafficking defects also showed elevation in Beclin-1 and LC3-II levels. In contrast, Gaucher disease fibroblasts, which traffic sphingolipids normally, showed wild-type levels of Beclin-1 and LC3-II. Our data define a critical role for Beclin-1 in the acti- vation of autophagy because of NPC1 deficiency, and reveal an unexpected role for lipid trafficking in the regulation of this pathway in patients with several sphingolipid storage diseases. INTRODUCTION protein contains a sterol-sensing domain (4) and functions in late endosomes to promote lipid sorting and vesicular traf- The sphingolipid storage diseases encompass a group of ficking (5–8) through mechanisms that are incompletely 40 genetically distinct disorders that result from inherited understood. deficiencies of lysosomal hydrolytic activities or lipid trans- Mice deficient in NPC1, which reproduce the pathology and port. These disorders occur with a collective frequency of lipid trafficking defects of NPC disease, arose from a spon- 1 in 8000 live births (1), and are often associated with devas- taneous mutation in the Npc1 gene (npc12/2 mice) (9). tating neurodegeneration. Among this group is Niemann–Pick Similar defects occur in chimeric mice that lack functional C (NPC) disease, an autosomal recessive disorder of lipid traf- NPC1 in only some cells (10). In both cases, NPC1 deficiency ficking that produces cognitive impairment, ataxia and death, leads to the activation of macroautophagy (hereafter referred most often in childhood (2). NPC disease is characterized by to as autophagy) in the cerebellum, a process by which cyto- the accumulation of unesterified cholesterol and sphingolipids plasmic proteins and organelles are sequestered within autop- in late endosomes and lysosomes. Nine-five percent of NPC hagosomes and are targeted for degradation by lysosomes disease patients have loss of function mutations in the (11). This regulated and evolutionarily conserved pathway Npc1 gene (3). The encoded multipass transmembrane enables recycling of limited or damaged cellular constituents *To whom correspondence should be addressed at: Department of Pathology University of Michigan Medical School, 3510 MSRB1, 1150 W. Medical Center Dr., Ann Arbor, MI 48109, USA. Tel: þ1 7346474624; Fax: þ1 7346153441; Email: [email protected] # The Author 2007. Published by Oxford University Press. All rights reserved. For Permissions, please email: [email protected] 1496 Human Molecular Genetics, 2007, Vol. 16, No. 12 to promote cell survival. However, in other instances, robust E64d and pepstatin A similarly increased LC3-II levels, con- activation of autophagy leads to cell death. sistent with the notion that fusion of autophagosomes to lyso- Here, we have used npc12/2 mice and primary human somes was intact in mutant cells (Fig. 2A). This conclusion fibroblasts deficient in NPC1 to explore the mechanism by was supported by the additive effect of concurrent induction which autophagy is induced in NPC disease. Our data demon- of autophagy by rapamycin and inhibition of lysosomal strate that enhanced basal autophagy in NPC1 deficiency is proteases by E64d and pepstatin A. mediated by increased expression of Beclin-1 rather than by As an independent confirmation that the autophagic activation of the Akt-mTOR-p70 S6K pathway. We further pathway was intact in NPC1-deficient cells, we measured demonstrate that lipid trafficking defects caused by pharma- the degradation of long-lived proteins (18). This assay pro- cologic treatment or by human disease gene mutations occur- vides a functional readout since autophagy is the major ring in other sphingolipid storage diseases also up-regulate pathway through which many of these proteins are degraded Beclin-1 and result in an increased autophagy. Our findings (19,20). Wild-type and NPC1-deficient fibroblasts were Downloaded from https://academic.oup.com/hmg/article/16/12/1495/2356123 by guest on 27 September 2021 establish Beclin-1 as a critical regulator of autophagy in labeled with 3H–leucine for 48 h, then washed and re-fed, several sphingolipid storage diseases. and trichloroacetic acid (TCA) soluble radioactive counts were measured in the medium after 6, 18 and 24 h (Fig. 2B). Significantly higher levels of proteolysis were RESULTS detected by NPC1-deficient fibroblasts at all points, demon- strating increased protein turnover in cells that also exhibited NPC1 deficiency increases basal autophagy enhanced autophagy. We first sought to determine whether elevated levels of auto- phagy occur specifically in the central nervous system of NPC1-deficient (npc12/2) mice or also occur in other organs that exhibit pathology. To accomplish this, we used Beclin-1 mediates increased levels of autophagy the microtubule-associated protein 1 light chain 3 (LC3) as a in NPC1 deficiency marker of autophagy. This protein is modified from its LC3-I cytosolic form to a more rapidly migrating, lipid- The induction of autophagy is achieved through either the conjugated LC3-II form associated with autophagosome dephosphorylation of mTOR or the activation of the membranes when autophagy is induced (12,13). Cerebellar Beclin-1 pathway (11). To determine which of these was pre- and liver lysates from 6-week-old npc12/2 mice had ferentially activated because of NPC1 deficiency, protein elevated levels of LC3-II compared with wild-type littermates lysates from mutant mice and fibroblasts were examined by (Fig. 1A), demonstrating that NPC1 deficiency increased western blot (Fig. 3). Beclin-1, an evolutionarily conserved autophagy in both organs. Similarly, primary human fibro- protein that is part of the Class III PI3K complex that partici- blasts deficient in NPC1 expressed higher total LC3 and pates in autophagosome formation (21), was expressed at LC3-II levels than control fibroblasts (Fig. 1B). This differ- mildly increased levels in both cerebellum and liver ence was observed in untreated cells and following starvation of npc12/2 mice compared with wild-type littermates or rapamycin treatment, indicating that basal levels of auto- (Fig. 3A). Increased Beclin-1 expression was also observed phagy were increased by NPC1 deficiency and that pathways in NPC1-deficient fibroblasts (Fig. 3B), demonstrating that leading to its further activation were intact in mutant cells. up-regulation of Beclin-1 occurred in response to NPC1 High levels of basal autophagy in NPC1-deficient fibro- deficiency in mice and cells. blasts were confirmed by transmission electron microscopy In contrast, our analyses did not reveal activation of the (Fig. 1C). This analysis demonstrated frequent autophagic mTOR pathway as a consequence of NPC1 deficiency. No vacuoles containing rough ER and other cytoplasmic contents alteration in the phosphorylation of mTOR, its regulator in mutant, but not in wild-type fibroblasts. Similarly, staining Akt or its target p70 S6K was detected in npc12/2 mice with monodansylcadaverine (MDC), a dye that preferentially or NPC1-deficient fibroblasts (Fig. 3C and D). We did, incorporates into autophagic vacuoles (14,15), was signifi- however, observe stimulation of the mTOR pathway in cantly higher in NPC1-deficient fibroblasts than that in NPC1-deficient fibroblasts as evidenced by starvation-induced control fibroblasts (Fig. 1D). To determine whether exogenous Akt dephosphorylation or rapamycin-induced p70 S6K depho- NPC1 could decrease autophagy in mutant fibroblasts, we sphorylation, confirming that this pathway was intact in stably expressed NPC1 protein in null cells. We observed mutant cells. These data demonstrated that NPC1 deficiency decreased total LC3 and LC3-II levels in pooled, NPC1- did not activate the Akt-mTOR-p70