Sphingolipid metabolism diseases

Human diseases caused by alterations in the metabolism of sphingolipids or glycosphingolipids are mainly disorders of the degradation of these compounds. The sphingolipidoses are a group of monogenic inherited diseases caused by defects in the system of lysosomal sphingolipid degradation, with subsequent accumulation of non-degradable storage material in one or more organs. Most sphingolipidoses are associated with high mortality. Both, the ratio of substrate influx into the lysosomes and the reduced degradative capacity can be addressed by therapeutic approaches. In addition to symptomatic treatments, the current strategies for restoration of the reduced substrate degradation within the lysosome are enzyme replacement therapy (ERT), cell-mediated therapy (CMT) including bone marrow transplantation (BMT) and cell-mediated “cross correction”, gene therapy, and enzyme-enhancement therapy with chemical chaperones. The reduction of substrate influx into the lysosomes can be achieved by substrate reduction therapy. Patients suffering from the attenuated form (type 1) of Gaucher disease and from Fabry disease have been successfully treated with ERT.     

Substrate reduction augments the efficacy of enzyme therapy in a mouse model of Fabry disease

Fabry disease is an X-linked glycosphingolipid storage disorder caused by a deficiency in the activity of the lysosomal hydrolase α-galactosidase A (α-gal). This deficiency results in accumulation of the glycosphingolipid globotriaosylceramide (GL-3) in lysosomes. Endothelial cell storage of GL-3 frequently leads to kidney dysfunction, cardiac and cerebrovascular disease. The current treatment for Fabry disease is through infusions of recombinant α-gal (enzyme-replacement therapy; ERT). Although ERT can markedly reduce the lysosomal burden of GL-3 in endothelial cells, variability is seen in the clearance from several other cell types. This suggests that alternative and adjuvant therapies may be desirable. Use of glucosylceramide synthase inhibitors to abate the biosynthesis of glycosphingolipids (substrate reduction therapy, SRT) has been shown to be effective at reducing substrate levels in the related glycosphingolipidosis, Gaucher disease. Here, we show that such an inhibitor (eliglustat tartrate, Genz-112638) was effective at lowering GL-3 accumulation in a mouse model of Fabry disease. Relative efficacy of SRT and ERT at reducing GL-3 levels in Fabry mouse tissues differed with SRT being more effective in the kidney, and ERT more efficacious in the heart and liver. Combination therapy with ERT and SRT provided the most complete clearance of GL-3 from all the tissues. Furthermore, treatment normalized urine volume and uromodulin levels and significantly delayed the loss of a nociceptive response. The differential efficacies of SRT and ERT in the different tissues indicate that the combination approach is both additive and complementary suggesting the possibility of an improved therapeutic paradigm in the management of Fabry disease.     

Substrate deprivation therapy: a new hope for patients suffering from neuronopathic forms of inherited lysosomal storage diseases

Lysosomal storage diseases are a group of disorders caused by defects in enzymes responsible for degradation of particular compounds in lysosomes. In most cases, these diseases are fatal, and until recently no treatment was available. Introduction of enzyme replacement therapy was a breakthrough in the treatment of some of the diseases. However, while this therapy is effective in reduction of many somatic symptoms, its efficacy in the treatment of the central nervous system is negligible, if any, mainly because of problems with crossing the blood-brain-barrier by intravenously administered enzyme molecules. On the other hand, there are many lysosomal storage diseases in which the central nervous system is affected. Results of very recent studies indicate that in at least some cases, another type of therapy, called substrate deprivation therapy (or substrate reduction therapy) may be effective in the treatment of neuronopathic forms of lysosomal storage diseases. This therapy, based on inhibition of synthesis of the compounds that cannot be degraded in cells of the patients, has been shown to be effective in several animal models of various diseases, and recent reports demonstrate its efficacy in the treatment of patients suffering from Niemann-Pick C disease and Sanfilippo disease.     

Biochemistry of glycosphingolipid storage disorders: implications for therapeutic intervention

The physiological importance of the degradative processes in lysosomes is revealed by the existence of at least 40 distinct inherited diseases, the so-called lysosomal storage disorders. Most of these diseases are caused by a deficiency in a single lysosomal enzyme, or essential cofactor, and result in the lysosomal accumulation of one, or sometimes several, natural compounds. The most prevalent subgroup of the lysosomal storage disorders is formed by the sphingolipidoses, inherited disorders that are characterized by excessive accumulation of one or multiple (glyco)sphingolipids. The biology of glycosphingolipids has been extensively discussed in other contributions during this symposium. This review will therefore focus in depth on (type 1) Gaucher disease, a prototypical glycosphingolipidosis. The elucidation of the primary genetic defect, being a deficiency in the lysosomal glucocerebrosidase, is described. Characterization of glucocerebrosidase at protein and gene level has subsequently opened avenues for therapeutic intervention. The development of successful enzyme replacement therapy for type 1 Gaucher disease is discussed. Attention is also paid to the alternative approach of substrate modulation using orally administered inhibitors of glucosylceramide synthesis. Novel developments about the monitoring of age of onset, progression and correction of disease are described. The remaining challenges about pathophysiology of glycosphingolipidoses are discussed in view of further improvements in therapy for these debilitating disorders.     

Suppression of autophagy permits successful enzyme replacement therapy in a lysosomal storage disorder—murine Pompe disease

Autophagy, an intracellular system for delivering portions of cytoplasm and damaged organelles to lysosomes for degradation/recycling, plays a role in many physiological processes and is disturbed in many diseases. We recently provided evidence for the role of autophagy in Pompe disease, a lysosomal storage disorder in which acid alphaglucosidase, the enzyme involved in the breakdown of glycogen, is deficient or absent. Clinically the disease manifests as a cardiac and skeletal muscle myopathy. The current enzyme replacement therapy (ERT) clears lysosomal glycogen effectively from the heart but less so from skeletal muscle. In our Pompe model, the poor muscle response to therapy is associated with the presence of pools of autophagic debris. To clear the fibers of the autophagic debris, we have generated a Pompe model in which an autophagy gene, Atg7, is inactivated in muscle. Suppression of autophagy alone reduced the glycogen level by 50–60%. Following ERT, muscle glycogen was reduced to normal levels, an outcome not observed in Pompe mice with genetically intact autophagy. The suppression of autophagy, which has proven successful in the Pompe model, is a novel therapeutic approach that may be useful in other diseases with disturbed autophagy.     

Suppression of autophagy permits successful enzyme replacement therapy in a lysosomal storage disorder—murine Pompe disease

Autophagy, an intracellular system for delivering portions of cytoplasm and damaged organelles to lysosomes for degradation/recycling, plays a role in many physiological processes and is disturbed in many diseases. We recently provided evidence for the role of autophagy in Pompe disease, a lysosomal storage disorder in which acid alpha-glucosidase, the enzyme involved in the breakdown of glycogen, is deficient or absent. Clinically the disease manifests as a cardiac and skeletal muscle myopathy. The current enzyme replacement therapy (ERT) clears lysosomal glycogen effectively from the heart but less so from skeletal muscle. In our Pompe model, the poor muscle response to therapy is associated with the presence of pools of autophagic debris. To clear the fibers of the autophagic debris, we have generated a Pompe model in which an autophagy gene, Atg7, is inactivated in muscle. Suppression of autophagy alone reduced the glycogen level by 50–60%. Following ERT, muscle glycogen was reduced to normal levels, an outcome not observed in Pompe mice with genetically intact autophagy. The suppression of autophagy, which has proven successful in the Pompe model, is a novel therapeutic approach that may be useful in other diseases with disturbed autophagy.Key words: Pompe disease, lysosomal glycogen storage, myopathy, Atg7, enzyme replacement therapy     

Glucosylceramide modulates membrane traffic along the endocytic pathway

Glycosphingolipids are endocytosed and targeted to the Golgi apparatus, but are mistargeted to lysosomes in numerous sphingolipidoses. Substrate reduction therapy utilizes imino sugars to inhibit glucosylceramide synthase and potentially abrogate the effects of storage. Gaucher disease is a hereditary deficiency in glucocerebrosidase leading to glucosylceramide accumulation; however, Gaucher fibroblasts exhibited normal Golgi transport of lactosylceramide. To better understand the effects of glycosphingolipid accumulation on intracellular trafficking and the use of imino sugar inhibitors, we studied sphingolipid endocytosis in fibroblast and macrophage models for Gaucher disease. Treatment of fibroblasts or RAW macrophages with conduritol B epoxide, an inhibitor of lysosomal glucocerebrosidase, resulted in a change in the endocytic targeting of lactosylceramide from the Golgi to the lysosomes. Co-treatment of macrophages with conduritol B-epoxide and 12-25 microM N-butyldeoxygalactonojirimycin, an inhibitor of glycosphingolipid biosynthesis, prevented the mistargeting of lactosylceramide to the lysosomes and restored trafficking to the Golgi. Surprisingly, higher doses (>25 microM) of NB-DGJ induced targeting of lactosylceramide to the lysosomes, even in the absence of conduritol B-epoxide. These data demonstrate that both increases and decreases in glucosylceramide levels can dramatically alter the endocytic targeting of lactosylceramide and suggest a role for glucosylceramide in regulation of membrane transport.     

A historical perspective of the glycosphingolipids and sphingolipidoses

Glycosphingolipids are a polysaccharide chain between 1 and 40 carbohydrate residues long glycosidically linked to ceramide (a long-chain aliphatic amino-alcohol or sphingoid) that is embedded in the cell plasma membrane with the carbohydrate moiety on the outside. The sphingoid imparts rigidity to the membrane and the carbohydrate tails protect the cell surface and have functions in relation to cell adhesion, growth, regulation, differentiation, cell interaction, recognition and signalling. They provide adhesion sites for pathogens and change during oncogenic transformation. Ceramide is also a component of sphingomyelin. Glycosphingolipids are degraded by lysosomal hydrolysis. The sphingolipidoses are a series of diseases in which mutations affecting the enzymes catalysing the last 11 steps of this process causing abnormal compounds proximal to the metabolic block to accumulate intralysosomally. Thus, they are a sub-group of the lysosomal storage diseases. The degradation of sphingolipids containing three or less carbohydrate residues requires a sphingolipid activator protein and mutations affecting these proteins also cause abnormal glycosphingolipid storage. With one exception (Fabry disease, which is X linked) the sphingolipidoses are inherited autosomally. The phenotypic manifestations of the individual sphingolipidoses are variable although the more severe variants are usually the better known. They have generally been regarded as untreatable but notable therapeutic advances are being made by enzyme replacement therapy and regulating the rate of glycosphingolipid synthesis by inhibiting UDP-glucose-N-acylsphingosine D-glucosyl transferase (CerGlcT), which is the first reaction on the pathway of glycosphingolipid synthesis. The compounds used are N-alkylated iminosugars whose glucose and galactose stereochemistries inhibit CerGlcT. Prenatal and carrier state diagnosis, genetic counselling and the abortion of affected foetuses are reducing the incidence of some of the most severe sphingolipidoses in certain high-incidence populations.     

Lysosomal leukocyte beta-D-glucuronidase during enzyme replacement therapy in Fabry disease

OBJECTIVE: Fabry disease results from a deficiency in the activity of alpha-d-galactosidase A and subsequent accumulation of neutral glycosphingolipids in lysosomes. This study investigated whether lysosomal enzymes can indicate biochemical changes in the lysosomal apparatus induced by enzyme replacement therapy (ERT). DESIGN AND METHODS: Eight patients were monitored by clinical and biochemical tests and several lysosomal glycohydrolases were measured in plasma and leucocytes. RESULTS: Before starting ERT, beta-d-glucuronidase in leukocytes was markedly increased. After 1 month of therapy, enzyme levels dropped in all patients. In the patients who regularly followed the therapy, the enzyme levels remained stable for the next 20 months. In one patient who interrupted therapy for 2 months, the enzyme levels rose again. CONCLUSIONS: Lysosomal enzymes can be useful for monitoring biochemical changes in patients with Fabry disease receiving ERT. Though these findings refer to only a small number of patients, the correlation between beta-d-glucuronidase levels and ERT is interesting and might serve as a basis for further studies to define the potential of this enzyme in monitoring the effects of ERT in lysosomal storage disorders.     

From sheep to mice to cells: Tools for the study of the sphingolipidoses

The sphingolipidoses are a group of inherited lysosomal storage diseases in which sphingolipids accumulate due to the defective activity of one or other enzymes involved in their degradation. For most of the sphingolipidoses, little is known about the molecular mechanisms that lead to disease, which has negatively impacted attempts to develop therapies for these devastating human diseases. Use of both genetically-modified animals, ranging from mice to larger mammals, and of novel cell culture systems, is of utmost importance in delineating the molecular mechanisms that cause pathophysiology, and in providing tools that enable testing the efficacy of new therapies. In this review, we discuss eight sphingolipidoses, namely Gaucher disease, Fabry disease, metachromatic leukodystrophy, Krabbe disease, Niemann–Pick diseases A and B, Farber disease, GM1 gangliosidoses, and GM2 gangliosidoses, and describe the tools that are currently available for their study. This article is part of a Special Issue entitled Tools to study lipid functions.     

Biosynthesis and degradation of mammalian glycosphingolipids

Glycolipids are a large and heterogeneous family of sphingolipids that form complex patterns on eukaryotic cell surfaces. This molecular diversity is generated by only a few enzymes and is a paradigm of naturally occurring combinatorial synthesis. We report on the biosynthetic principles leading to this large molecular diversity and focus on sialic acid-containing glycolipids of the ganglio-series. These glycolipids are particularly concentrated in the plasma membrane of neuronal cells. Their de novo synthesis starts with the formation of the membrane anchor, ceramide, at the endoplasmic reticulum (ER) and is continued by glycosyltransferases of the Golgi complex. Recent findings from genetically engineered mice are discussed. The constitutive degradation of glycosphingolipids (GSLs) occurs in the acidic compartments, the endosomes and the lysosomes. Here, water-soluble glycosidases sequentially cleave off the terminal carbohydrate residues from glycolipids. For glycolipid substrates with short oligosaccharide chains, the additional presence of membrane-active sphingolipid activator proteins (SAPs) is required. A considerable part of our current knowledge about glycolipid degradation is derived from a class of human diseases, the sphingolipidoses, which are caused by inherited defects within this pathway. A new post-translational modification is the attachment of glycolipids to proteins of the human skin.     

Substrate deprivation: a new therapeutic approach for the glycosphingolipid lysosomal storage diseases

The glycosphingolipid (GSL) lysosomal storage diseases are a family of human metabolic diseases that, in their severest forms, cause death in early infancy, as a result of progressive neurodegeneration. They are caused by mutations in the genes encoding the glycohydrolases or the activator proteins that catabolise GSLs within lysosomes. In these diseases the GSL substrate of the defective enzyme accumulates in the lysosome, where it is stored and leads to cellular dysfunction and disease. The therapeutic options for treating these diseases are relatively limited; in fact, there are currently no available therapies for most of these disorders. The problem is further compounded by difficulties in delivering therapeutic agents to the central nervous system, which is where the pathology is frequently manifested. To date, research effort has mainly focused on strategies for augmenting enzyme concentrations to compensate for the underlying defect. These strategies include bone-marrow transplantation, enzyme-replacement therapy and gene therapy. Our group has been exploring the alternative strategy of substrate deprivation. This approach aims to balance the rate of GSL synthesis with the impaired rate of GSL breakdown. Studies using an asymptomatic mouse model of Tay-Sachs disease have shown that substrate deprivation prevents GSL storage. In a severe neurodegenerative mouse model of Sandhoff disease, substrate deprivation delayed the onset of symptoms and disease progression, and significantly increased life expectancy. The implications of this research for human therapy have been discussed.     

Ultrastructural and Immunocytochemical Characterization of Autophagic Vacuoles in Isolated Hepatocytes: Effects of Vinblastine and Asparagine on Vacuole Distributions

The interactions between the autophagic and the endocytic degradation pathways were investigated by means of immunogold labeling of autophagic vacuoles (AVs) in ultrathin frozen sections from isolated rat hepatocytes. AVs were identified by their autophagocytosed contents of the degradation-resistant cytosolic enzyme CuZn-superoxide dismutase (SOD). Another cytosolic enzyme, carbonic anhydrase (CAIII), was rapidly degraded in the lysosomes, making the vacuolar CAIII/SOD ratio useful as a rough indicator of the progress of autophagic-lysosomal degradation. Lysosomes could be recognized by the presence of the lysosomal membrane glycoprotein lgp120, which was absent from hepatocytic endosomes. Endocytic inputs into the AVs were detected by the presence of gold-conjugated bovine serum albumin (BSA-gold), taken up by fluid-phase endocytosis. All vacuoles recognized morphologically as AVs were SOD-positive, as were essentially all of the lysosomes (96%). The majority (72%) of the lysosomes also labeled positively for BSA within 2 h of endocytosis. The data are thus compatible with the notion that all lysosomes can engage in both autophagic and endocytic degradation. Lgp120 appeared to distinguish well between lysosomes and nonlysosomal AVs: the lgp120-negative AVs (nonlysosomes) had a CAIII/SOD ratio identical to that of the cytosol, indicating that no degradation had occurred, In the lgp120-positive AVs (lysosomes), the ratio was only 43% of the cytosolic value, consistent with substantial CAIII degradation. Among the nonlysosomal AVs (about one-third of all AVs), one-half were BSA-positive, suggesting that early AVs (autophagasomes) and intermediary AVs (amphisomes) that had fused with endosomes were equally abundant. These morphological data thus support previous biochemical evidence for a prelysosomal meeting of the autophagic and endocytic pathways. The microtubule inhibitor vinblastine inhibited the autophagic influx to the lysosomes, causing an accumulation of autophagosomes and a reduction in average lysosomal size. Vinblastine also inhibited the endocytic flux, thereby precluding the formation of amphisomes and of BSA-positive lysosomes. High concentrations (20 mM) of asparagine induced swelling of amphisomes and of BSA-positive lysosomes, probably reflecting an acidotropic effect of ammonia generated by asparagine deamination. Asparagine also caused an accumulation of autophagosomes, amphisomes, and BSA-negative lysosomes, presumably as a result of impaired fusion with the swollen BSA-positive lysosomes. The two agents thus appear to perturb the autophagic-endocytic-lysosomal vacuole dynamics by different mechanisms, making them useful in the further study of these complex organelle interactions.     

Genetic defects in the sphingolipid degradation pathway and their effects on microglia in neurodegenerative disease

Sphingolipids, which function as plasma membrane lipids and signaling molecules, are highly enriched in neuronal and myelin membranes in the nervous system. They are degraded in lysosomes by a defined sequence of enzymatic steps. In the related group of disorders, the sphingolipidoses, mutations in the genes that encode the individual degradative enzymes cause lysosomal accumulation of sphingolipids and often result in severe neurodegenerative disease. Here we review the information indicating that microglia, which actively clear sphingolipid-rich membranes in the brain during development and homeostasis, are directly affected by these mutations and promote neurodegeneration in the sphingolipidoses. We also identify parallels between the sphingolipidoses and more common forms of neurodegeneration, which both exhibit evidence of defective sphingolipid clearance in the nervous system.     

Production of recombinant beta-hexosaminidase A, a potential enzyme for replacement therapy for Tay-Sachs and Sandhoff diseases, in the methylotrophic yeast Ogataea minuta

Human beta-hexosaminidase A (HexA) is a heterodimeric glycoprotein composed of alpha- and beta-subunits that degrades GM2 gangliosides in lysosomes. GM2 gangliosidosis is a lysosomal storage disease in which an inherited deficiency of HexA causes the accumulation of GM2 gangliosides. In order to prepare a large amount of HexA for a treatment based on enzyme replacement therapy (ERT), recombinant HexA was produced in the methylotrophic yeast Ogataea minuta instead of in mammalian cells, which are commonly used to produce recombinant enzymes for ERT. The problem of antigenicity due to differences in N-glycan structures between mammalian and yeast glycoproteins was potentially resolved by using alpha-1,6-mannosyltransferase-deficient (och1Delta) yeast as the host. Genes encoding the alpha- and beta-subunits of HexA were integrated into the yeast cell, and the heterodimer was expressed together with its isozymes HexS (alphaalpha) and HexB (betabeta). A total of 57 mg of beta-hexosaminidase isozymes, of which 13 mg was HexA (alphabeta), was produced per liter of medium. HexA was purified with immobilized metal affinity column for the His tag attached to the beta-subunit. The purified HexA was treated with alpha-mannosidase to expose mannose-6-phosphate (M6P) residues on the N-glycans. The specific activities of HexA and M6P-exposed HexA (M6PHexA) for the artificial substrate 4MU-GlcNAc were 1.2 +/- 0.1 and 1.7 +/- 0.3 mmol/h/mg, respectively. The sodium dodecyl sulfate-polyacrylamide gel electrophoresis pattern suggested a C-terminal truncation in the beta-subunit of the recombinant protein. M6PHexA was incorporated dose dependently into GM2 gangliosidosis patient-derived fibroblasts via M6P receptors on the cell surface, and degradation of accumulated GM2 ganglioside was observed.     

β2-Microglobulin Amyloid Fibrils Are Nanoparticles That Disrupt Lysosomal Membrane Protein Trafficking and Inhibit Protein Degradation by Lysosomes

Fragmentation of amyloid fibrils produces fibrils that are reduced in length but have an otherwise unchanged molecular architecture. The resultant nanoscale fibril particles inhibit the cellular reduction of the tetrazolium dye 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT), a substrate commonly used to measure cell viability, to a greater extent than unfragmented fibrils. Here we show that the internalization of β₂-microglobulin (β₂m) amyloid fibrils is dependent on fibril length, with fragmented fibrils being more efficiently internalized by cells. Correspondingly, inhibiting the internalization of fragmented β₂m fibrils rescued cellular MTT reduction. Incubation of cells with fragmented β₂m fibrils did not, however, cause cell death. Instead, fragmented β₂m fibrils accumulate in lysosomes, alter the trafficking of lysosomal membrane proteins, and inhibit the degradation of a model protein substrate by lysosomes. These findings suggest that nanoscale fibrils formed early during amyloid assembly reactions or by the fragmentation of longer fibrils could play a role in amyloid disease by disrupting protein degradation by lysosomes and trafficking in the endolysosomal pathway.     

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.     

Cystic fibrosis transmembrane conductance regulator contributes to reacidification of alkalinized lysosomes in RPE cells

The role of the cystic fibrosis transmembrane conductance regulator (CFTR) in lysosomal acidification has been difficult to determine. We demonstrate here that CFTR contributes more to the reacidification of lysosomes from an elevated pH than to baseline pH maintenance. Lysosomal alkalinization is increasingly recognized as a factor in diseases of accumulation, and we previously showed that cAMP reacidified alkalinized lysosomes in retinal pigmented epithelial (RPE) cells. As the influx of anions to electrically balance proton accumulation may enhance lysosomal acidification, the contribution of the cAMP-activated anion channel CFTR to lysosomal reacidification was probed. The antagonist CFTR(inh)-172 had little effect on baseline levels of lysosomal pH in cultured human RPE cells but substantially reduced the reacidification of compromised lysosomes by cAMP. Likewise, CFTR activators had a bigger impact on cells whose lysosomes had been alkalinized. Knockdown of CFTR with small interfering RNA had a larger effect on alkalinized lysosomes than on baseline levels. Inhibition of CFTR in isolated lysosomes altered pH. While CFTR and Lamp1 were colocalized, treatment with cAMP did not increase targeting of CFTR to the lysosome. The inhibition of CFTR slowed lysosomal degradation of photoreceptor outer segments while activation of CFTR enhanced their clearance from compromised lysosomes. Activation of CFTR acidified RPE lysosomes from the ABCA4(-/-) mouse model of recessive Stargardt's disease, whose lysosomes are considerably alkalinized. In summary, CFTR contributes more to reducing lysosomal pH from alkalinized levels than to maintaining baseline pH. Treatment to activate CFTR may thus be of benefit in disorders of accumulation associated with lysosomal alkalinization.