Cellular and biochemical response to chaperone versus substrate reduction therapies in neuropathic Gaucher disease

Gaucher disease (GD) is caused by deficiency of the lysosomal membrane enzyme glucocerebrosidase (GCase) and the subsequent accumulation of its substrate, glucosylceramide (GC). Mostly missense mutations of the glucocerebrosidase gene (GBA) cause GCase misfolding and inhibition of proper lysosomal trafficking. The accumulated GC leads to lysosomal dysfunction and impairs the autophagy pathway. GD types 2 and 3 (GD2-3), or the neuronopathic forms, affect not only the Central Nervous System (CNS) but also have severe systemic involvement and progressive bone disease. Enzyme replacement therapy (ERT) successfully treats the hematologic manifestations; however, due to the lack of equal distribution of the recombinant enzyme in different organs, it has no direct impact on the nervous system and has minimal effect on bone involvement. Small molecules have the potential for better tissue distribution. Ambroxol (AMB) is a pharmacologic chaperone that partially recovers the mutated GCase activity and crosses the blood-brain barrier. Eliglustat (EGT) works by inhibiting UDP-glucosylceramide synthase, an enzyme that catalyzes GC biosynthesis, reducing GC influx load into the lysosome. Substrate reduction therapy (SRT) using EGT is associated with improvement in GD bone marrow burden score and bone mineral density parallel with the improvement in hematological parameters. We assessed the effects of EGT and AMB on GCase activity and autophagy-lysosomal pathway (ALP) in primary cell lines derived from patients with GD2-3 and compared to cell lines from healthy controls. We found that EGT, same as AMB, enhanced GCase activity in control cells and that an individualized response, that varied with GBA mutations, was observed in cells from patients with GD2-3. EGT and AMB enhanced the formation of lysosomal/late endosomal compartments and improved autophagy, independent of GBA mutations. Both AMB and EGT increased mitochondrial mass and density in GD2-3 fibroblasts, suggesting enhancement of mitochondrial function by activating the mitochondrial membrane potential. These results demonstrate that EGT and AMB, with different molecular mechanisms of action, enhance GCase activity and improve autophagy-lysosome dynamics and mitochondrial functions.

Pulmonary Involvement Responsive to Enzyme Replacement Therapy in an Elderly Patient with Gaucher Disease

Type 1 Gaucher disease (GD) is a rare autosomal recessive lysosomal storage disorder caused by deficient activity of beta-glucocerebrosidase, leading to accumulation of its substrate (glucosylceramide) in macrophages of the reticuloendothelial system, which are then referred to as Gaucher cells. The most frequent symptoms are asthenia, spleen and liver enlargement, bone abnormalities and cytopenia due to bone marrow infiltration. Lung involvement in GD is a rare finding, and it is unclear whether it may regress under enzyme replacement therapy (ERT) or substrate reduction therapy (SRT). Here we report a case of type 1 GD recently diagnosed in an elderly patient complicated by infiltrative lung disease, which responded to ERT.

Substrate Reduction Therapy Reverses Mitochondrial, mTOR, and Autophagy Alterations in a Cell Model of Gaucher Disease

Substrate reduction therapy (SRT) in clinic adequately manages the visceral manifestations in Gaucher disease (GD) but has no direct effect on brain disease. To understand the molecular basis of SRT in GD treatment, we evaluated the efficacy and underlying mechanism of SRT in an immortalized neuronal cell line derived from a Gba knockout (Gba-/-) mouse model. Gba-/- neurons accumulated substrates, glucosylceramide, and glucosylsphingosine. Reduced cell proliferation was associated with altered lysosomes and autophagy, decreased mitochondrial function, and activation of the mTORC1 pathway. Treatment of the Gba-/- neurons with venglustat analogue GZ452, a central nervous system-accessible SRT, normalized glucosylceramide levels in these neurons and their isolated mitochondria. Enlarged lysosomes were reduced in the treated Gba-/- neurons, accompanied by decreased autophagic vacuoles. GZ452 treatment improved mitochondrial membrane potential and oxygen consumption rate. Furthermore, GZ452 diminished hyperactivity of selected proteins in the mTORC1 pathway and improved cell proliferation of Gba-/- neurons. These findings reinforce the detrimental effects of substrate accumulation on mitochondria, autophagy, and mTOR in neurons. A novel rescuing mechanism of SRT was revealed on the function of mitochondrial and autophagy–lysosomal pathways in GD. These results point to mitochondria and the mTORC1 complex as potential therapeutic targets for treatment of GD.

Miglustat Therapy for SCARB2-Associated Action Myoclonus–Renal Failure Syndrome

ObjectiveWe evaluated whether substrate reduction therapy with miglustat could alter the course of action myoclonus–renal failure syndrome (AMRF), a rare, progressive myoclonic epilepsy with early mortality caused by scavenger receptor class B member 2 (SCARB2) gene mutations.MethodsWe identified an AMRF patient with a biallelic combination of SCARB2 mutations determined by whole exome sequencing. SCARB2 encodes a protein that traffics β-glucocerebrosidase to the lysosomal membrane. Mutations lead to a complex pattern of glucosylceramide accumulation and neurologic symptoms including progressive action myoclonus, seizures, and ataxia. We then evaluated the effect of inhibiting glucosylceramide synthesis, as is used in Gaucher disease. The patient was treated for 3 years with miglustat after several years of steady worsening.ResultsProgression of myoclonus halted, dysphagia resolved, some skills were reacquired, and seizures remained well controlled.ConclusionsThe response suggests that neurologic symptoms of SCARB2-associated AMRF could be ameliorated, at least partly, by targeting glycosphingolipid metabolism with available medications.

Impact of Long-Term Enzyme Replacement Therapy on Glucosylsphingosine (Lyso-Gb1) Values in Patients with Type 1 Gaucher Disease: Statistical Models for Comparing Three Enzymatic Formulations

For three decades, enzyme replacement therapy (ERT), and more recently, substrate reduction therapy, have been the standard-of-care for type I Gaucher disease (GD1). Since 2012, three different ERTs have been available. No clinical trial or academic study has ever compared these ERTs beyond one year. Herein we compare the impact of the ERTs on repeated measurements of glucosylsphingosine (lyso-Gb1; the most sensitive and GD-specific biomarker). A total of 135 adult patients (77 (57%) female) with GD1, followed from July 2014 to March 2020 and treated with a single ERT (imiglucerase (n = 41, 30.4%), taliglucerase alfa (n = 21, 15.6%) and velaglucerase alfa (n = 73, 54.1%)), were included. Disease severity was defined by genotypes (mild: N370S (c.1226A>G) homozygous and N370S/R496H (c.1604G) compound heterozygous; severe: all other genotypes) and by the severity score index (SSI; mild: <7; severe: ≥7). Lyso-Gb1 testing was performed at Centogene™ on dry blood spot samples collected during routine visits. Patients treated with imiglucerase had higher lyso-Gb1 levels at different time points. A huge variation in lyso-Gb1 levels was noticeable both inter-individually and intra-individually for all three ERTs. A steeper and faster decrease of lyso-Gb1 levels was shown in velaglucerase alfa. Nevertheless, the differences between medications were not very large, and bigger numbers and more pretreatment data are required for more powerful conclusions.

Substrate reduction therapy for Krabbe disease and metachromatic leukodystrophy using a novel ceramide galactosyltransferase inhibitor

Krabbe disease (KD) and metachromatic leukodystrophy (MLD) are caused by accumulation of the glycolipids galactosylceramide (GalCer) and sulfatide and their toxic metabolites psychosine and lysosulfatide, respectively. We discovered a potent and selective small molecule inhibitor (S202) of ceramide galactosyltransferase (CGT), the key enzyme for GalCer biosynthesis, and characterized its use as substrate reduction therapy (SRT). Treating a KD mouse model with S202 dose-dependently reduced GalCer and psychosine in the central (CNS) and peripheral (PNS) nervous systems and significantly increased lifespan. Similarly, treating an MLD mouse model decreased sulfatides and lysosulfatide levels. Interestingly, lower doses of S202 partially inhibited CGT and selectively reduced synthesis of non-hydroxylated forms of GalCer and sulfatide, which appear to be the primary source of psychosine and lysosulfatide. Higher doses of S202 more completely inhibited CGT and reduced the levels of both non-hydroxylated and hydroxylated forms of GalCer and sulfatide. Despite the significant benefits observed in murine models of KD and MLD, chronic CGT inhibition negatively impacted both the CNS and PNS of wild-type mice. Therefore, further studies are necessary to elucidate the full therapeutic potential of CGT inhibition.

Human α-Galactosidase A Mutants: Priceless Tools to Develop Novel Therapies for Fabry Disease

Fabry disease (FD) is a lysosomal storage disease caused by mutations in the gene for the α-galactosidase A (GLA) enzyme. The absence of the enzyme or its activity results in the accumulation of glycosphingolipids, mainly globotriaosylceramide (Gb3), in different tissues, leading to a wide range of clinical manifestations. More than 1000 natural variants have been described in the GLA gene, most of them affecting proper protein folding and enzymatic activity. Currently, FD is treated by enzyme replacement therapy (ERT) or pharmacological chaperone therapy (PCT). However, as both approaches show specific drawbacks, new strategies (such as new forms of ERT, organ/cell transplant, substrate reduction therapy, or gene therapy) are under extensive study. In this review, we summarize GLA mutants described so far and discuss their putative application for the development of novel drugs for the treatment of FD. Unfavorable mutants with lower activities and stabilities than wild-type enzymes could serve as tools for the development of new pharmacological chaperones. On the other hand, GLA mutants showing improved enzymatic activity have been identified and produced in vitro. Such mutants could overcome several complications associated with current ERT, as lower-dose infusions of these mutants could achieve a therapeutic effect equivalent to that of the wild-type enzyme.

Fabry Disease: Molecular Basis, Pathophysiology, Diagnostics and Potential Therapeutic Directions

Fabry disease (FD) is a lysosomal storage disorder (LSD) characterized by the deficiency of α-galactosidase A (α-GalA) and the consequent accumulation of toxic metabolites such as globotriaosylceramide (Gb3) and globotriaosylsphingosine (lysoGb3). Early diagnosis and appropriate timely treatment of FD patients are crucial to prevent tissue damage and organ failure which no treatment can reverse. LSDs might profit from four main therapeutic strategies, but hitherto there is no cure. Among the therapeutic possibilities are intravenous administered enzyme replacement therapy (ERT), oral pharmacological chaperone therapy (PCT) or enzyme stabilizers, substrate reduction therapy (SRT) and the more recent gene/RNA therapy. Unfortunately, FD patients can only benefit from ERT and, since 2016, PCT, both always combined with supportive adjunctive and preventive therapies to clinically manage FD-related chronic renal, cardiac and neurological complications. Gene therapy for FD is currently studied and further strategies such as substrate reduction therapy (SRT) and novel PCTs are under investigation. In this review, we discuss the molecular basis of FD, the pathophysiology and diagnostic procedures, together with the current treatments and potential therapeutic avenues that FD patients could benefit from in the future.

Cellular and biochemical response to chaperone versus substrate reduction therapies in neuropathic Gaucher disease

Gaucher disease (GD) is caused by the deficiency of the lysosomal membrane enzyme glucocerebrosidase (GCase), and the subsequent accumulation of its substrate, glucosylceramide substrate (GC). Mostly missense mutations of the glucocerebrosidase gene (GBA) lead to GCase misfolding and inhibiting the lysosome’s proper trafficking. The accumulated GC leads to lysosomal dysfunction and impairs the autophagy pathway.GD types 2 and 3 (GD2-3), or the neuronopathic forms, affect not only the Central Nervous System (CNS) but also have severe systemic involvement and progressive bone disease. Enzyme replacement therapy (ERT) successfully treats the hematologic manifestations; however, due to the lack of equal distribution of the recombinant enzyme in different organs, it has no impact on the nervous system and has minimal effect on bone involvement. Small molecules have the potential for better tissue distribution. Ambroxol (AMB) is a pharmacologic chaperone that partially recovers the mutated GCase activity and crosses the blood-brain barrier. Eliglustat (EGT) works by inhibiting UDP-glucosylceramide synthase, an enzyme that catalyzes the GC biosynthesis, reducing a GC influx load into the lysosome. Substrate reduction therapy (SRT) using EGT is associated with improvement in GD bone marrow burden score and bone mineral density.The effects of EGT and ABX on GCase activity and autophagy-lysosomal pathway (ALP) were assessed in primary cell lines derived from patients with GD2-3 and compared to cell lines from healthy controls. While both compounds enhanced GCase activity in control cells, an individualized response was observed in cells from patients with GD2-3 that varied with GBA mutations. EGT and AMB enhanced the formation of lysosomal/ late endosomal compartments and autophagy, and this effect was independent of GBA mutations. Both AMB and EGT increased mitochondrial mass and density in GD2-3 fibroblasts, suggesting enhancement of the mitochondrial function by activating the mitochondrial membrane potential.These results suggest that EGT and ABX may have different molecular mechanisms of action, but both enhance GCase activity, improve autophagy-lysosome dynamics and mitochondrial functions.