Macroautophagy Is Not Directly Involved in the Metabolism of Amyloid Precursor Protein

Barry Boland,David A Smith,Declan Mooney,Sonia S Jung,Dominic M Walsh,Frances M Platt

doi:10.1074/jbc.m110.186411

Abstract

Alterations in the metabolism of amyloid precursor protein (APP) are believed to play a central role in Alzheimer disease pathogenesis. Burgeoning data indicate that APP is proteolytically processed in endosomal-autophagic-lysosomal compartments. In this study, we used both in vivo and in vitro paradigms to determine whether alterations in macroautophagy affect APP metabolism. Three mouse models of glycosphingolipid storage diseases, namely Niemann-Pick type C1, GM1 gangliosidosis, and Sandhoff disease, had mTOR-independent increases in the autophagic vacuole (AV)-associated protein, LC3-II, indicative of impaired lysosomal flux. APP C-terminal fragments (APP-CTFs) were also increased in brains of the three mouse models; however, discrepancies between LC3-II and APP-CTFs were seen between primary (GM1 gangliosidosis and Sandhoff disease) and secondary (Niemann-Pick type C1) lysosomal storage models. APP-CTFs were proportionately higher than LC3-II in cerebellar regions of GM1 gangliosidosis and Sandhoff disease, although LC3-II increased before APP-CTFs in brains of NPC1 mice. Endogenous murine Aβ40 from RIPA-soluble extracts was increased in brains of all three mice. The in vivo relationship between AV and APP-CTF accumulation was also seen in cultured neurons treated with agents that impair primary (chloroquine and leupeptin + pepstatin) and secondary (U18666A and vinblastine) lysosomal flux. However, Aβ secretion was unaffected by agents that induced autophagy (rapamycin) or impaired AV clearance, and LC3-II-positive AVs predominantly co-localized with degradative LAMP-1-positive lysosomes. These data suggest that neuronal macroautophagy does not directly regulate APP metabolism but highlights the important anti-amyloidogenic role of lysosomal proteolysis in post-secretase APP-CTF catabolism.

Highlights

Neurodegenerative proteinopathies such as Alzheimer disease (AD),4 Parkinson disease, and Huntington disease are defined by progressive accumulation of protein aggregates
These findings indicate that macroautophagy does not play a direct role in the metabolism of amyloid precursor protein (APP), it highlights the requirement of efficient lysosomal function in preventing the accumulation of autophagic vacuole” (AV) and amyloidogenic APP metabolites
Central to the pathogenesis of AD is the metabolism of APP, which undergoes proteolytic processing and degradation in endosomal-lysosomal compartments (54 –56)

Summary

EXPERIMENTAL PROCEDURES

Reagents and Primary Antibodies—Unless otherwise stated, all chemical reagents were supplied by Sigma. Preparation of Mouse Brain Extracts and Conditioned Media from Primary Neurons for A␤ ELISA—Brain extracts were prepared for A␤ ELISA in accordance with protocols described previously [31,32,33], with some modifications. Whole mouse brain without the brainstem was Dounce-homogenized (10 strokes) at 40% w/v (wet weight) in Tissue Homogenization Buffer (THB, 250 mM sucrose, 20 mM Tris base) containing protease inhibitors (5 mM EDTA, 1 mM EGTA, 5 ␮g/ml leupeptin, 5 ␮g/ml aprotinin, 2 ␮g/ml pepstatin, 120 ␮g/ml Pefabloc, 2 mM 1,10-phenanthroline). Three wells were pooled for each experimental point, and cell suspensions were probe-sonicated (23 kHz, 5 ␮m amplitude, 5 s) using a Soniprep 150 (MSE Instruments, Sussex, UK) This suspension was layered onto 3 ml of an 8% (w/v) Histodenz/ Sucrose Buffer and centrifuged at 7,000 ϫ g at 4 °C for 30 min. Statistical significance is annotated in figures : *, p Ͻ 0.05; **, p Ͻ 0.01; ***, p Ͻ 0.001

Autophagic Vacuole Accumulation Is a Common Feature of GSL

Nascent Autophagosomes Do Not

DISCUSSION