Lysosomal storage diseases (LSDs) are a family of disorders that result from inherited gene mutations that perturb lysosomal homeostasis. LSDs mainly stem from deficiencies in lysosomal enzymes, but also in some non-enzymatic lysosomal proteins, which lead to abnormal storage of macromolecular substrates. Valuable insights into lysosome functions have emerged from research into these diseases. In addition to primary lysosomal dysfunction, cellular pathways associated with other membrane-bound organelles are perturbed in these disorders. Through selective examples, we illustrate why the term “cellular storage disorders” may be a more appropriate description of these diseases and discuss therapies that can alleviate storage and restore normal cellular function.

β-Glucosidase 2 (GBA2) is an enzyme that cleaves the membrane lipid glucosylceramide into glucose and ceramide. The GBA2 gene is mutated in genetic neurological diseases (hereditary spastic paraplegia and cerebellar ataxia). Pharmacologically, GBA2 is reversibly inhibited by alkylated imino sugars that are in clinical use or are being developed for this purpose. We have addressed the ambiguity surrounding one of the defining characteristics of GBA2, which is its sensitivity to inhibition by conduritol B epoxide (CBE). We found that CBE inhibited GBA2, in vitro and in live cells, in a time-dependent fashion, which is typical for mechanism-based enzyme inactivators. Compared with the well characterized impact of CBE on the lysosomal glucosylceramide-degrading enzyme (glucocerebrosidase, GBA), CBE inactivated GBA2 less efficiently, due to a lower affinity for this enzyme (higher K_I) and a lower rate of enzyme inactivation (k_inact). In contrast to CBE, N-butyldeoxygalactonojirimycin exclusively inhibited GBA2. Accordingly, we propose to redefine GBA2 activity as the β-glucosidase that is sensitive to inhibition by N-butyldeoxygalactonojirimycin. Revised as such, GBA2 activity 1) was optimal at pH 5.5–6.0; 2) accounted for a much higher proportion of detergent-independent membrane-associated β-glucosidase activity; 3) was more variable among mouse tissues and neuroblastoma and monocyte cell lines; and 4) was more sensitive to inhibition by N-butyldeoxynojirimycin (miglustat, Zavesca®), in comparison with earlier studies. Our evaluation of GBA2 makes it possible to assess its activity more accurately, which will be helpful in analyzing its physiological roles and involvement in disease and in the pharmacological profiling of monosaccharide mimetics.

During the last 30 years, an increasing number of cellular functions have been reported to be regulated by the lipid ceramide. The diversity in the ceramide structure, leading to tens of ceramide species and the discrete distribution based on subcellular topology, could explain the wide variety of functions attributed to this bioactive lipid. One of these pools of ceramide resides in the plasma membrane, and several works have suggested that an increase in plasma membrane ceramide (PMCer) in response to stimulation leads to cell death and modulates cell adhesion and migration. However, there is a limitation in studying PMCer content in this location primarily due to the inability to quantify its mass. Our group recently developed a method to specifically quantitate PMCer. In this work, we interrogate what sphingolipid metabolizing enzymes are responsible for modulating the basal levels of plasma membrane ceramide. An in-silico prediction and experimental confirmation found an almost perfect correlation between the endogenous expression levels of neutral sphingomyelinase (nSMase2) and the amount of plasma membrane ceramide in unstimulated cells. Manipulating the expression levels of nSMase2, but not other candidate enzymes of ceramide metabolism, profoundly affected PMCer. Moreover, a physiologic induction of nSMase2 during cell confluence resulted in a nSMase2-dependent dramatic increase in PMCer. Together, these results identify nSMase2 as the primary enzyme to regulate plasma membrane ceramide.

The small-molecular compound miglustat (N-butyldeoxynojirimycin, Zavesca^®) has been approved for clinical use in type 1 Gaucher disease and Niemann–Pick type C disease, which are disorders caused by dysfunction of the endosomal-autophagic-lysosomal system. Miglustat inhibits a number of enzymes involved in glycoconjugate and glycan metabolism, including β-glucosidase 2 (GBA2), which is exceptionally sensitive to inhibition by miglustat. GBA2 is a glucosylceramide-degrading enzyme that is located on the plasma membrane/endoplasmic reticulum, and is distinct from the lysosomal enzyme glucocerebrosidase (GBA). Various strands of evidence suggest that inhibition of GBA2 contributes to the therapeutic benefits of miglustat. To further explore the pharmacology and biology of GBA2, we investigated whether the zebrafish homolog of GBA2 has similar enzymatic properties and pharmacological sensitivities to its human counterpart. We established that zebrafish has endogenous β-glucosidase activity toward lipid- and water-soluble GBA2 substrates, which can be inhibited by miglustat, N-butyldeoxygalactonojirimycin, and conduritol B epoxide. β-Glucosidase activities with highly similar characteristics were expressed in cells transfected with the zebrafish gba2 cDNA and in cells transfected with the human GBA2 cDNA. These results provide a foundation for the use of zebrafish in screening GBA2-targeting molecules, and for wider studies investigating GBA2 biology.

The enzyme β-glucosidase 2 (GBA2) is clinically relevant because it is targeted by the drug miglustat (Zavesca^®) and because it is involved in inherited diseases. Mutations in the GBA2 gene are associated with two neurological diseases on the ataxia-spasticity spectrum, hereditary spastic paraplegia 46 (SPG46) and Marinesco-Sjögren-like syndrome (MSS). To establish how GBA2 mutations give rise to neurological pathology, we have begun to investigate mutant forms of GBA2 encoded by disease-associated GBA2 alleles. Previously, we found that five GBA2 missense mutants and five C-terminally truncated mutants lacked enzyme activity. Here we have examined the cellular locations of wild-type (WT) and mutant forms of GBA2 by confocal and electron microscopy, using transfected cells. Similar to GBA2-WT, the D594H and M510Vfs*17 GBA2 mutants were located at the plasma membrane, whereas the C-terminally truncated mutants terminating after amino acids 233 and 339 (GBA2-233 and -339) were present in the mitochondrial matrix, induced mitochondrial fragmentation and loss of mitochondrial transmembrane potential. Deletional mutagenesis indicated that residues 161–200 are critical for the mitochondrial fragmentation of GBA2-233 and -339. Considering that the mitochondrial fragmentation induced by GBA2-233 and -339 is consistently accompanied by their localization to the mitochondrial matrix, our deletional analysis raises the possibility that that GBA2 residues 161–200 harbor an internal targeting sequence for transport to the mitochondrial matrix. Altogether, our work provides new insights into the behaviour of GBA2-WT and disease-associated forms of GBA2.