Autophagy in lysosomal storage disorders

Lysosomes are ubiquitous intracellular organelles that have an acidic internal pH, and play crucial roles in cellular clearance. Numerous functions depend on normal lysosomes, including the turnover of cellular constituents, cholesterol homeostasis, downregulation of surface receptors, inactivation of pathogenic organisms, repair of the plasma membrane and bone remodeling. Lysosomal storage disorders (LSDs) are characterized by progressive accumulation of undigested macromolecules within the cell due to lysosomal dysfunction. As a consequence, many tissues and organ systems are affected, including brain, viscera, bone and cartilage. The progressive nature of phenotype development is one of the hallmarks of LSDs. In recent years biochemical and cell biology studies of LSDs have revealed an ample spectrum of abnormalities in a variety of cellular functions. These include defects in signaling pathways, calcium homeostasis, lipid biosynthesis and degradation and intracellular trafficking. Lysosomes also play a fundamental role in the autophagic pathway by fusing with autophagosomes and digesting their content. Considering the highly integrated function of lysosomes and autophagosomes it was reasonable to expect that lysosomal storage in LSDs would have an impact upon autophagy. The goal of this review is to provide readers with an overview of recent findings that have been obtained through analysis of the autophagic pathway in several types of LSDs, supporting the idea that LSDs could be seen primarily as "autophagy disorders."     

Autophagy in astrocytes: A novel culprit in lysosomal storage disorders

Neurodegeneration is a prominent feature of lysosomal storage disorders (LSDs). Emerging data identify autophagy dysfunction in neurons as a major component of the phenotype. However, the autophagy pathway in the CNS has been studied predominantly in neurons, whereas in other cell types it has been largely unexplored. We studied the lysosome-autophagic pathway in astrocytes from a murine model of multiple sulfatase deficiency (MSD), a severe form of LSD. Similar to what was observed in neurons, we found that lysosomal storage in astrocytes impairs autophagosome maturation and this, in turn, has an impact upon the survival of cortical neurons and accounts for some of the neurological features found in MSD. Thus, our data indicate that lysosomal/autophagic dysfunction in astrocytes is an important component of neurodegeneration in LSDs.     

Lysosomal fusion and SNARE function are impaired by cholesterol accumulation in lysosomal storage disorders

The function of lysosomes relies on the ability of the lysosomal membrane to fuse with several target membranes in the cell. It is known that in lysosomal storage disorders (LSDs), lysosomal accumulation of several types of substrates is associated with lysosomal dysfunction and impairment of endocytic membrane traffic. By analysing cells from two severe neurodegenerative LSDs, we observed that cholesterol abnormally accumulates in the endolysosomal membrane of LSD cells, thereby reducing the ability of lysosomes to efficiently fuse with endocytic and autophagic vesicles. Furthermore, we discovered that soluble N‐ethylmaleimide‐sensitive factor attachment protein (SNAP) receptors (SNAREs), which are key components of the cellular membrane fusion machinery are aberrantly sequestered in cholesterol‐enriched regions of LSD endolysosomal membranes. This abnormal spatial organization locks SNAREs in complexes and impairs their sorting and recycling. Importantly, reducing membrane cholesterol levels in LSD cells restores normal SNARE function and efficient lysosomal fusion. Our results support a model by which cholesterol abnormalities determine lysosomal dysfunction and endocytic traffic jam in LSDs by impairing the membrane fusion machinery, thus suggesting new therapeutic targets for the treatment of these disorders.     

Autophagy contributes to lysosomal storage disorders

Degradation in the lysosome/vacuole is not the final step of autophagy. In particular, for starvation-induced autophagy it is necessary to release the breakdown products back into the cytosol. However, some researchers ignore this last step and simply refer to the endpoint of autophagy as degradation, or perhaps even cargo delivery. In many cases this is not a serious issue; however, the analysis of autophagy's role in certain diseases makes clear that this can be a significant error.     

Autophagy contributes to lysosomal storage disorders

Degradation in the lysosome/vacuole is not the final step of autophagy. In particular, for starvation-induced autophagy it is necessary to release the breakdown products back into the cytosol. However, some researchers ignore this last step and simply refer to the endpoint of autophagy as degradation, or perhaps even cargo delivery. In many cases this is not a serious issue; however, the analysis of autophagy’s role in certain diseases makes clear that this can be a significant error.     

Autophagy in astrocytes

Neurodegeneration is a prominent feature of lysosomal storage disorders (LSDs). Emerging data identify autophagy dysfunction in neurons as a major component of the phenotype. However, the autophagy pathway in the CNS has been studied predominantly in neurons, whereas in other cell types it has been largely unexplored. We studied the lysosome-autophagic pathway in astrocytes from a murine model of multiple sulfatase deficiency (MSD), a severe form of LSD. Similar to what was observed in neurons, we found that lysosomal storage in astrocytes impairs autophagosome maturation and this, in turn, has an impact upon the survival of cortical neurons and accounts for some of the neurological features found in MSD. Thus, our data indicate that lysosomal/autophagic dysfunction in astrocytes is an important component of neurodegeneration in LSDs.     

Lysosomal Ca(2+) homeostasis: role in pathogenesis of lysosomal storage diseases

Disrupted cellular Ca²⁺ signaling is believed to play a role in a number of human diseases including lysosomal storage diseases (LSD). LSDs are a group of ∼50 diseases caused predominantly by mutations in lysosomal proteins that result in accumulation of macromolecules within the lysosome. We recently reported that Niemann-Pick type C (NPC) is the first human disease to be associated with defective lysosomal Ca²⁺ uptake and defective NAADP-mediated lysosomal Ca²⁺ release. These defects in NPC cells leads to the disruption in endocytosis and subsequent lipid storage that is a feature of this disease. In contrast, Chediak-Higashi Syndrome cells have been reported to have enhanced lysosomal Ca²⁺ uptake whilst the TRPML1 protein defective in mucolipidosis type IV is believed to function as a Ca²⁺ channel. In this review we provide a summary of the current knowledge on the role of lysosomal Ca²⁺ signaling in the pathogenesis of this group of diseases.     

Culture conditions found to minimize false positive diagnosis of lysosomal storage disorders

Summary The effect of culture conditions on the ultrastructure and enzyme activities of cultured skin fibroblast cells relevant to the diagnosis of lysosomal storage disorders are reported. The parameters examined were: pH of the culture media, type of media, increasing cell passage, and day of harvest. Ultrastructural changes were defined in terms of the number of lysosome-like inclusion bodies per cell according to a method devised in our laboratory and proven reliable in the detection of affected individuals. Our biochemical results included determination of enzyme activities of β-hexosaminidase, α-mannosidase, β-glucuronidase-lysosmal enzymes, arylsulfatase C, a microsomal marker, and 5' nucleotidase, a plasma membrane marker. Our results indicate that the cellular ultrastructure is more sensitive than enzyme activity to changes in culture conditions. The resulting ultrastructural “artifacts” observed under certain conditions were severe enough to result in a mistaken diagnosis. Due to certain difficulties we had previously encountered in heterozygote cultures (for lysosmal storage disorders) of amniotic cells, we decided to examine heterozygote cultures of skin fibroblasts. From these (preliminary) studies it seems that an evaluation in the pH over the pysiologic levels in the culture media may help to define between normal individuals and affected heterozygotes. On the basis of our results, we recommend that to minimize false positive ultrastructural results for the diagnosis of lysosomal storage disorders, cultures be grown in minimal essential medium, the pH of the medium carefully monitored to remain below 7.4, examining the cultures not later than cell Passage 8 and no later than Day 10 after subculture. This work was part of the requirement for the fulfillment of a Ph.D. thesis (J. A.) submitted to the Hebrew University. This work was supported in part by the Richard Meyer Fund for teratological research.     

Autophagy sequesters damaged lysosomes to control lysosomal biogenesis and kidney injury

Diverse causes, including pathogenic invasion or the uptake of mineral crystals such as silica and monosodium urate (MSU), threaten cells with lysosomal rupture, which can lead to oxidative stress, inflammation, and apoptosis or necrosis. Here, we demonstrate that lysosomes are selectively sequestered by autophagy, when damaged by MSU, silica, or the lysosomotropic reagent L ‐Leucyl‐L‐leucine methyl ester (LLOMe). Autophagic machinery is recruited only on damaged lysosomes, which are then engulfed by autophagosomes. In an autophagy‐dependent manner, low pH and degradation capacity of damaged lysosomes are recovered. Under conditions of lysosomal damage, loss of autophagy causes inhibition of lysosomal biogenesis in vitro and deterioration of acute kidney injury in vivo. Thus, we propose that sequestration of damaged lysosomes by autophagy is indispensable for cellular and tissue homeostasis.     

Effects of suramin,a polyanionic drug inducing lysosomal storage disorders on tooth germs in vitro

Summary— Suramin, a potent inhibitor of lysosomal enzymes, is commonly employed as a tool for inducing experimental mucopolysaccharidosis and lipidosis. The effects of the drug on embryonic mouse molars were analysed. Presecretory ameloblasts and odontoblasts were loaded with lysosome-like vacuoles. Staining with MC22-33F, an antibody to choline phospholipids and sphingomyelin, was completely reversed in the suramin-treated germs, in that it stained only presecretory ameloblasts (versus odontoblasts and some pulpal cells in the control group), according to a developmentally regulated pattern. The suramin-induced cytoplasmic changes were reminiscent of the features of mucopolysaccharidoses and lipidoses. The basement membrane, separating the enamel organ from the dental papilla, displayed suramin-induced patches, and in predentin collagen fibrillogenesis was found to be disturbed. Furthermore, autoradiography was employed to reveal uptake and distribution of [³H] suramin in the cells and predentin. Finally, a suramin-induced disturbance of the metabolism of sulphated macromolecules was found. The results imply that suramin effects in vitro on tooth germs can be used as a useful experimental model with to study both the action of the drug as well as cell and extracellular matrix perturbations in a mucopolysaccharidosis-like condition.     

Autophagy impairment with lysosomal and mitochondrial dysfunction is an important characteristic of oxidative stress-induced senescence

Macroautophagy/autophagy has profound implications for aging. However, the true features of autophagy in the progression of aging remain to be clarified. In the present study, we explored the status of autophagic flux during the development of cell senescence induced by oxidative stress. In this system, although autophagic structures increased, the degradation of SQSTM1/p62 protein, the yellow puncta of mRFP-GFP-LC3 fluorescence and the activity of lysosomal proteolytic enzymes all decreased in senescent cells, indicating impaired autophagic flux with lysosomal dysfunction. The influence of autophagy activity on senescence development was confirmed by both positive and negative autophagy modulators; and MTOR-dependent autophagy activators, rapamycin and PP242, efficiently suppressed cellular senescence through a mechanism relevant to restoring autophagic flux. By time-phased treatment of cells with the antioxidant N-acetylcysteine (NAC), the mitochondria uncoupler carbonyl cyanide m-chlorophenyl hydrazone (CCCP) and ambroxol, a reagent with the effect of enhancing lysosomal enzyme maturation, we found that mitochondrial dysfunction plays an initiating role, while lysosomal dysfunction is more directly responsible for autophagy impairment and senescence. Interestingly, the effect of rapamycin on autophagy flux is linked to its role in functional revitalization of both mitochondrial and lysosomal functions. Together, this study demonstrates that autophagy impairment is crucial for oxidative stress-induced cell senescence, thus restoring autophagy activity could be a promising way to retard senescence.     

Gene therapy for lysosomal storage diseases: the lessons and promise of animal models

There are more than 40 different forms of inherited lysosomal storage diseases (LSDs) known to occur in humans and the aggregate incidence has been estimated to approach 1 in 7000 live births. Most LSDs are associated with high morbidity and mortality and represent a significant burden on patients, their families, and health care providers. Except for symptomatic therapies, many LSDs remain untreatable, and gene therapy is among the only viable treatment options potentially available. Therapies for some LSDs do exist, or are under evaluation, including heterologous bone marrow transplantation (BMT), enzyme replacement therapy (ERT), and substrate reduction therapy (SRT), but these treatment options are associated with significant concerns, including high morbidity and mortality (BMT), limited positive outcomes (BMT), incomplete response to therapy (BMT, ERT, and SRT), life-long therapy (ERT, SRT), and cost (BMT, ERT, SRT). Gene therapy represents a potential alternative therapy, albeit a therapy with its own attendant concerns. Animal models of LSDs play a critical role in evaluating the efficacy and safety of therapy for many of these conditions. Naturally occurring animal homologs of LSDs have been described in the mouse, rat, dog, cat, guinea pig, emu, quail, goat, cattle, sheep, and pig. In this review we discuss those animal models that have been used in gene therapy experiments and those with promise for future evaluations.     

Adriamycin-induced leakage of lysosomal enzymes in vitro

Chronic treatment of rats with adriamycin has been shown to affect myocardial lysosomes as well as enzyme activities in the serum fraction. In this study, we examined in vitro effects of adriamycin (10^–6 to 10^–3 M) on the lysosomal fraction isolated from rat ventricular tissue. Morphological examination revealed that the isolated fraction was mainly vesicular in nature. Higher concentrations of adriamycin (10^–3 M) caused a significant loss of acid phosphatase and N-acetyl-B-d-glucosaminidase activity from the lyosomal vesicles. The enzyme leakage was not accompanied by any intravesicular localization of lanthanum, an extravesicular electron dense tracer. Preincubation of lysosomal vesicles with 10 g/ml superoxide dismutase did not protect against adriamycin-induced loss of lysosomal enzymes. The study shows that adriamycin induces loss of lysosomal enzymes in vitro and the superoxide radical may not be involved in this change.