Autophagic dysfunction in a lysosomal storage disorder due to impaired proteolysis

Abstract

Alterations in macroautophagy (hereafter referred to as “autophagy”) are a common feature of lysosomal storage disorders, and have been hypothesized to play a major role in the pathogenesis of these diseases. We have recently reported multiple defects in autophagy contributing to the lysosomal storage disorder Niemann-Pick type C (NPC). These include increased formation of autophagosomes, slowed turnover of autophagosomes secondary to impaired lysosomal proteolysis, and delivery of stored lipids to the lysosome via autophagy. The study summarized here describes novel methods for the interrogation of individual stages of the autophagic pathway, and suggests mechanisms by which lipid storage may result in broader lysosomal dysfunction.

Autophagy is a multistep process that accomplishes the degradation and recycling of proteins, lipids, carbohydrates and organelles. Any individual step, or several steps simultaneously, could be altered in disease states and may contribute to the overall “autophagic dysfunction” often reported in neurodegenerative diseases. Illustrative of this concept is the lysosomal lipid storage disorder NPC disease. It is caused by mutations in the NPC1 or NPC2 genes, leading to the storage of unesterified cholesterol and glycosphingolipids. Cells from NPC patients have modestly increased autophagic flux, indicating an induction of autophagy, but also demonstrate markedly elevated numbers of autophagosomes and accumulation of autophagic substrates, such as ubiquitinated proteins and SQSTM1/p62. This led us to hypothesize the existence of a second defect in the autophagic pathway interfering with the completion of autophagy. Imaging studies using the autophagosome marker mCherry-GFP-LC3, which loses its GFP fluorescence following fusion with lysosomes to form the acidified autolysosome, confirmed normal autophagosome-to-lysosome fusion in NPC cells. Suspecting that defective completion of autophagy was due to impaired clearance of autolysosomes, we performed live time-lapse imaging of mCherry-GFP-LC3⁺ vesicles. We identified autophagosome-to-lysosome fusion events by noting the transition of vesicles from mCherry⁺/GFP⁺ (yellow) to mCherry⁺/GFP^– (red), and then measured the time until the disappearance of the red vesicle, thus yielding autolysosome lifetime. Autolysosome lifetime is markedly increased in NPC cells.

We hypothesized that prolonged autolysosome lifetimes were due to defective protein degradation. To confirm this proteolysis defect, we used Magic Red substrates, commercially available compounds consisting of cresyl violet fused to peptide sequences that target the molecule for cleavage by a specific cathepsin. Prior to cleavage, the peptide quenches the fluorescence of cresyl violet, and also allows the molecule to be membrane permeable. Following diffusion into the lysosome, Magic Red is cleaved, dequenched and trapped in the lysosome. We used live cell-time lapse imaging to measure the rate of accumulation of the fluorescent marker, thereby providing an estimate of in situ cathepsin activity. In NPC cells, the activity of lysosomal CTSB and CTSK are each markedly reduced. This defect is not attributable to abnormal processing and trafficking of cathepsins or altered lysosomal pH. Instead, removal of lysosomal lipid storage material via treatment with cyclodextrin restores normal cathepsin activity. Lysosomal protease dysfunction therefore most likely results from inhibition of cathepsin activity by lipid storage material.

We also considered whether autophagy plays a direct role in lipid storage in NPC disease. Using pharmacological and genetic manipulations of autophagosome formation, we found increased levels of cholesterol storage when autophagy is induced and decreased cholesterol storage when autophagy is inhibited. This observation suggests that autophagy is an important source of stored cholesterol in the NPC lysosome, and that the observed induction of autophagy in NPC disease actually plays a detrimental role in disease pathogenesis by increasing lipid storage. In support of this conclusion, pharmacological inhibition of autophagy in NPC cells not only decreases cholesterol storage, but also rescues lysosomal cathepsin activity.

Critical to the findings in this study was the development of methods to study specific steps of the autophagic pathway in situ and in real-time. We have demonstrated proof of principle that the mCherry-GFP-LC3 marker is not only able to differentiate autophagosomes from lysosomes in static images, but is actually a useful tool to track these organelles throughout the process of formation and maturation, and to draw meaningful conclusions about their functional properties. The present study required manual curation of images to calculate the lifetime of autolysosomes. However, we suspect the method may be amenable to automated computational analysis, allowing higher throughput investigation of autolysosome lifetime, and perhaps also autophagosome formation, trafficking and latency to fusion. Similarly important, the adaptation of Magic Red to quantitative time-lapse imaging allowed in situ estimation of cathepsin activity in the environment of the lipid-loaded lysosome. Significantly, most cathepsin activity assays involve the measurement of enzyme activity in whole cell lysates, thus separating the enzyme from its environment, and obscuring the sort of observation made in this study. We hope that these tools will prove useful in basic studies of autophagy, and in interrogating the role of autophagy in additional diseases.

Taken together, these data paint a complex picture of autophagy in Niemann-Pick type C disease. On the one hand, lipid storage leads to lysosomal dysfunction through inhibition of lysosomal protease activity. On the other, autophagy is induced through a BECN1- and lipid storage-dependent mechanism. The end result of this autophagy induction, in the absence of efficient lysosome function, is only mildly increased autophagic flux at the expense of the accumulation of autophagic intermediates. Furthermore, autophagy induction increases the delivery of cholesterol to the lysosome, creating a positive feedback loop through which autophagy promotes disease pathogenesis. We suspect that a detailed evaluation of the autophagic pathway in other lysosomal disorders would yield similar findings, and may point to a common final pathway of lysosomal dysfunction. Our findings lead to the conclusion that inhibition of autophagy may be of therapeutic benefit in NPC disease. However, the autophagy inhibitors currently available for in vivo use, such as chloroquine, act by disrupting lysosomal proteolysis. This would be expected to exacerbate, rather than relieve, the defect present in NPC disease. We think that our study provides further motivation for the rational design of drugs targeting additional stages of the autophagic pathway.

Glossary

Abbreviations:

NPC

Niemann-Pick type C

GFP

green fluorescent protein

LC3

microtubule-associated protein 1 light chain 3

Elrick MJ, Yu T, Chung C, Lieberman AP. Impaired proteolysis underlies autophagic dysfunction in Niemann-Pick type C disease. Hum Mol Genet. 2012;21:4876–87. doi: 10.1093/hmg/dds324.

Disclosure of Potential Conflicts of Interest

No potential conflicts of interest were disclosed.