Dietary Supplementation with n-3 Polyunsaturated Fatty Acids Delays the Phenotypic Manifestation of Krabbe Disease and Partially Restores Lipid Mediator Production in the Brain-Study in a Mouse Model of the Disease.

Lipid mediators from fatty acid oxidation have been shown to be associated with the severity of Krabbe disease (KD), a disorder linked to mutations in the galactosylceramidase (GALC) gene. This study aims to investigate the effects of n-3 polyunsaturated fatty acid (PUFA) supplementation on KD traits and fatty acid metabolism using Twitcher (Tw) animals as a natural model for KD. Wild-type (Wt), heterozygous (Ht), and affected Tw animals were treated orally with 36 mg n-3 PUFAs/kg body weight/day from 10 to 35 days of life. The end product of PUFA peroxidation (8-isoprostane), the lipid mediator involved in the resolution of inflammatory exudates (resolvin D1), and the total amount of n-3 PUFAs were analyzed in the brains of mice. In Tw mice, supplementation with n-3 PUFAs delayed the manifestation of disease symptoms (p < 0.0001), and in the bran, decreased 8-isoprostane amounts (p < 0.0001), increased resolvin D1 levels (p < 0.005) and increased quantity of total n-3 PUFAs (p < 0.05). Furthermore, total brain n-3 PUFA levels were associated with disease severity (r = -0.562, p = 0.0001), resolvin D1 (r = 0.712, p < 0.0001), and 8-isoprostane brain levels (r = -0.690, p < 0.0001). For the first time in a natural model of KD, brain levels of n-3 PUFAs are shown to determine disease severity and to be involved in the peroxidation of brain PUFAs as well as in the production of pro-resolving lipid mediators. It is also shown that dietary supplementation with n-3 PUFAs leads to a slowing of the phenotypic presentation of the disease and restoration of lipid mediator production.

Promyelinating drugs ameliorate oligodendrocyte pathologies in a mouse model of Krabbe disease.

Krabbe disease (KD) is a rare inherited demyelinating disorder caused by a deficiency in the lysosomal enzyme galactosylceramide (GalCer) ß-galactosidase. Most patients with KD exhibit fatal cerebral demyelination with apoptotic oligodendrocyte (OL) death and die before the age of 2-4 years. We have previously reported that primary OLs isolated from the brains of twitcher (twi) mice, an authentic mouse model of KD, have cell-autonomous developmental defects and undergo apoptotic death accompanied by abnormal accumulation of psychosine, an endogenous cytotoxic lyso-derivative of GalCer. In this study, we aimed to investigate the effects of the preclinical promyelinating drugs clemastine and Sob-AM2 on KD OL pathologies using primary OLs isolated from the brains of twi mice. Both agents specifically prevented the apoptotic death observed in twi OLs. However, while Sob-AM2 showed higher efficacy in restoring the impaired differentiation and maturation of twi OLs, clemastine more potently reduced the endogenous psychosine levels. These results present the first preclinical in vitro data, suggesting that clemastine and Sob-AM2 can act directly and distinctly on OLs in KD and ameliorate their cellular pathologies associated with myelin degeneration.

Chronic Rapamycin administration via drinking water mitigates the pathological phenotype in a Krabbe disease mouse model through autophagy activation.

Krabbe disease (KD) is a rare disorder arising from the deficiency of the lysosomal enzyme galactosylceramidase (GALC), leading to the accumulation of the cytotoxic metabolite psychosine (PSY) in the nervous system. This accumulation triggers demyelination and neurodegeneration, and despite ongoing research, the underlying pathogenic mechanisms remain incompletely understood, with no cure currently available. Previous studies from our lab revealed the involvement of autophagy dysfunctions in KD pathogenesis, showcasing p62-tagged protein aggregates in the brains of KD mice and heightened p62 levels in the KD sciatic nerve. We also demonstrated that the autophagy inducer Rapamycin (RAPA) can partially reinstate the wild type (WT) phenotype in KD primary cells by decreasing the number of p62 aggregates. In this study, we tested RAPA in the Twitcher (TWI) mouse, a spontaneous KD mouse model. We administered the drug ad libitum via drinking water (15 mg/L) starting from post-natal day (PND) 21-23. We longitudinally monitored the mouse motor performance through grip strength and rotarod tests, and a set of biochemical parameters related to the KD pathogenesis (i.e. autophagy markers expression, PSY accumulation, astrogliosis and myelination). Our findings demonstrate that RAPA significantly enhances motor functions at specific treatment time points and reduces astrogliosis in TWI brain, spinal cord, and sciatic nerves. Utilizing western blot and immunohistochemistry, we observed a decrease in p62 aggregates in TWI nervous tissues, corroborating our earlier in-vitro results. Moreover, RAPA treatment partially removes PSY in the spinal cord. In conclusion, our results advocate for considering RAPA as a supportive therapy for KD. Notably, as RAPA is already available in pharmaceutical formulations for clinical use, its potential for KD treatment can be rapidly evaluated in clinical trials.

Nanomedicines to treat rare neurological disorders: The case of Krabbe disease.

The brain remains one of the most challenging therapeutic targets due to the low and selective permeability of the blood-brain barrier and complex architecture of the brain tissue. Nanomedicines, despite their relatively large size compared to small molecules and nucleic acids, are being heavily investigated as vehicles to delivery therapeutics into the brain. Here we elaborate on how nanomedicines may be used to treat rare neurodevelopmental disorders, using Krabbe disease (globoid cell leukodystrophy) to frame the discussion. As a monogenetic disorder and lysosomal storage disease affecting the nervous system, the lessons learned from examining nanoparticle delivery to the brain in the context of Krabbe disease can have a broader impact on the treatment of various other neurodevelopmental and neurodegenerative disorders. In this review, we introduce the epidemiology and genetic basis of Krabbe disease, discuss current in vitro and in vivo models of the disease, as well as current therapeutic approaches either approved or at different stage of clinical developments. We then elaborate on challenges in particle delivery to the brain, with a specific emphasis on methods to transport nanomedicines across the blood-brain barrier. We highlight nanoparticles for delivering therapeutics for the treatment of lysosomal storage diseases, classified by the therapeutic payload, including gene therapy, enzyme replacement therapy, and small molecule delivery. Finally, we provide some useful hints on the design of nanomedicines for the treatment of rare neurological disorders.

Rapamycin Alleviates Protein Aggregates, Reduces Neuroinflammation, and Rescues Demyelination in Globoid Cell Leukodystrophy.

We have shown in vivo and in vitro previously that psychosine causes dysfunction of autophagy and the ubiquitin-proteasome system underlying the pathogenesis of globoid cell leukodystrophy (GLD), a devastating lysosomal storage disease complicated by global demyelination. Here, we investigated the therapeutic efficacy of the mTOR inhibitor rapamycin in twitcher mice, a murine model of infantile GLD, in biochemical, histochemical, and clinical aspects. Administration of rapamycin to twitcher mice inhibited mTOR signaling in the brains, and significantly reduced the accumulation of insoluble ubiquitinated protein and the formation of ubiquitin aggregates. The astrocytes and microglia reactivity were attenuated in that reactive astrocytes, ameboid microglia, and globoid cells were reduced in the brains of rapamycin-treated twitcher mice. Furthermore, rapamycin improved the cortical myelination, neurite density, and rescued the network complexity in the cortex of twitcher mice. The therapeutic action of rapamycin on the pathology of the twitcher mice's brains prolonged the longevity of treated twitcher mice. Overall, these findings validate the therapeutic efficacy of rapamycin and highlight enhancing degradation of aggregates as a therapeutic strategy to modulate neuroinflammation, demyelination, and disease progression of GLD and other leukodystrophies associated with intracellular aggregates.

A novel brain-penetrant oral UGT8 inhibitor decreases in vivo galactosphingolipid biosynthesis in murine Krabbe disease.

Krabbe disease is a rare, inherited neurodegenerative disease due to impaired lysosomal ß-galactosylceramidase (GALC) activity and formation of neurotoxic ß-galactosylsphingosine ('psychosine'). We investigated substrate reduction therapy with a novel brain-penetrant inhibitor of galactosylceramide biosynthesis, RA 5557, in twitcher mice that lack GALC activity and model Krabbe disease. This thienopyridine derivative selectively inhibits uridine diphosphate-galactose glycosyltransferase 8 (UGT8), the final step in the generation of galactosylceramides which are precursors of sulphatide and, in the pathological lysosome, the immediate source of psychosine. Administration of RA 5557, reduced pathologically elevated psychosine concentrations (72-86%) in the midbrain and cerebral cortex in twitcher mice: the inhibitor decreased galactosylceramides by about 70% in midbrain and cerebral cortex in mutant and wild type animals. Exposure to the inhibitor significantly decreased several characteristic inflammatory response markers without causing apparent toxicity to myelin-producing cells in wild type and mutant mice; transcript abundance of oligodendrocyte markers MBP (myelin basic protein) and murine UGT8 was unchanged. Administration of the inhibitor before conception and during several breeding cycles to mice did not impair fertility and gave rise to healthy offspring. Nevertheless, given the unchanged lifespan, it appears that GALC has critical functions in the nervous system beyond the hydrolysis of galactosylceramide and galactosylsphingosine. Our findings support further therapeutic exploration of orally active UGT8 inhibitors in Krabbe disease and related galactosphingolipid disorders. The potent thienopyridine derivative with effective target engagement here studied appears to have an acceptable safety profile in vivo; judicious dose optimization will be needed to ensure efficacious clinical translation.

Hybrid Nanoparticles as a Novel Tool for Regulating Psychosine-Induced Neuroinflammation and Demyelination In Vitro and Ex vivo.

Polymeric nanoparticles are being extensively investigated as an approach for brain delivery of drugs, especially for their controlled release and targeting capacity. Nose-to-brain administration of nanoparticles, bypassing the blood brain barrier, offers a promising strategy to deliver drugs to the central nervous system. Here, we investigated the potential of hybrid nanoparticles as a therapeutic approach for demyelinating diseases, more specifically for Krabbe's disease. This rare leukodystrophy is characterized by the lack of enzyme galactosylceramidase, leading to the accumulation of toxic psychosine in glial cells causing neuroinflammation, extensive demyelination and death. We present evidence that lecithin/chitosan nanoparticles prevent damage associated with psychosine by sequestering the neurotoxic sphingolipid via physicochemical hydrophobic interactions. We showed how nanoparticles prevented the cytotoxicity caused by psychosine in cultured human astrocytes in vitro, and how the nanoparticle size and PDI augmented while the electrostatic charges of the surface decreased, suggesting a direct interaction between psychosine and the nanoparticles. Moreover, we studied the effects of nanoparticles ex vivo using mouse cerebellar organotypic cultures, observing that nanoparticles prevented the demyelination and axonal damage caused by psychosine, as well as a moderate prevention of the astrocytic death. Taken together, these results suggest that lecithin-chitosan nanoparticles are a potential novel delivery system for drugs for certain demyelinating conditions such as Krabbe's disease, due to their dual effect: not only are they an efficient platform for drug delivery, but they exert a protective effect themselves in tampering the levels of psychosine accumulation.

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.

Adult Krabbe Disease That Was Successfully Treated with Intravenous Immunoglobulin.

Krabbe disease involves the accumulation of neurotoxic metabolites due to lysosomal galactocerebrosidase enzyme deficiency, which results in widespread demyelination of central and peripheral nerves. Generally, Krabbe disease presents as spastic paraplegia with a slow progressive course; however, some cases may show clinical symptoms similar to those of chronic inflammatory demyelinating polyneuropathy (CIDP). No previously reported studies have investigated the efficacy of intravenous immunoglobulin (IVIg) for treating Krabbe disease, and reporting a case involving IVIg treatment may be informative in the clinical setting. A 14-year-old girl who developed Guillain-Barré syndrome-like limb weakness was administered IVIg, and her limb weakness improved. At 16 years old, she developed abnormal sensory perception and weakness of both upper limbs. A nerve conduction study revealed demyelination, which led us to suspect CIDP. IVIg was administered, and her symptoms gradually improved. A nerve biopsy, enzyme activity, and genetic test results indicated adult Krabbe disease. In some cases, IVIg may be an effective treatment for Krabbe disease.

Fingolimod Rescues Demyelination in a Mouse Model of Krabbe's Disease.

Krabbe's disease is an infantile neurodegenerative disease, which is affected by mutations in the lysosomal enzyme galactocerebrosidase, leading to the accumulation of its metabolite psychosine. We have shown previously that the S1P receptor agonist fingolimod (FTY720) attenuates psychosine-induced glial cell death and demyelination both in vitro and ex vivo models. These data, together with a lack of therapies for Krabbe's disease, prompted the current preclinical study examining the effects of fingolimod in twitcher mice, a murine model of Krabbe's disease. Twitcher mice, both male and female, carrying a natural mutation in the galc gene were given fingolimod via drinking water (1 mg/kg/d). The direct impact of fingolimod administration was assessed via histochemical and biochemical analysis using markers of myelin, astrocytes, microglia, neurons, globoid cells, and immune cells. The effects of fingolimod on twitching behavior and life span were also demonstrated. Our results show that treatment of twitcher mice with fingolimod significantly rescued myelin levels compared with vehicle-treated animals and also regulated astrocyte and microglial reactivity. Furthermore, nonphosphorylated neurofilament levels were decreased, indicating neuroprotective and neurorestorative processes. These protective effects of fingolimod on twitcher mice brain pathology was reflected by an increased life span of fingolimod-treated twitcher mice. These in vivo findings corroborate initial in vitro studies and highlight the potential use of S1P receptors as drug targets for treatment of Krabbe's disease.SIGNIFICANCE STATEMENT This study demonstrates that the administration of the therapy known as fingolimod in a mouse model of Krabbe's disease (namely, the twitcher mouse model) significantly rescues myelin levels. Further, the drug fingolimod also regulates the reactivity of glial cells, astrocytes and microglia, in this mouse model. These protective effects of fingolimod result in an increased life span of twitcher mice.

Genetic ablation of acid ceramidase in Krabbe disease confirms the psychosine hypothesis and identifies a new therapeutic target.

Infantile globoid cell leukodystrophy (GLD, Krabbe disease) is a fatal demyelinating disorder caused by a deficiency in the lysosomal enzyme galactosylceramidase (GALC). GALC deficiency leads to the accumulation of the cytotoxic glycolipid, galactosylsphingosine (psychosine). Complementary evidence suggested that psychosine is synthesized via an anabolic pathway. Here, we show instead that psychosine is generated catabolically through the deacylation of galactosylceramide by acid ceramidase (ACDase). This reaction uncouples GALC deficiency from psychosine accumulation, allowing us to test the long-standing "psychosine hypothesis." We demonstrate that genetic loss of ACDase activity (Farber disease) in the GALC-deficient mouse model of human GLD (twitcher) eliminates psychosine accumulation and cures GLD. These data suggest that ACDase could be a target for substrate reduction therapy (SRT) in Krabbe patients. We show that pharmacological inhibition of ACDase activity with carmofur significantly decreases psychosine accumulation in cells from a Krabbe patient and prolongs the life span of the twitcher (Twi) mouse. Previous SRT experiments in the Twi mouse utilized l-cycloserine, which inhibits an enzyme several steps upstream of psychosine synthesis, thus altering the balance of other important lipids. Drugs that directly inhibit ACDase may have a more acceptable safety profile due to their mechanistic proximity to psychosine biogenesis. In total, these data clarify our understanding of psychosine synthesis, confirm the long-held psychosine hypothesis, and provide the impetus to discover safe and effective inhibitors of ACDase to treat Krabbe disease.

Psychosine, a marker of Krabbe phenotype and treatment effect.

Newborn screening (NBS) for Krabbe disease, a rare neurodegenerative disorder caused by deficient galactocerebrosidase (GALC) enzyme activity, has recently been implemented in a number of US states. However, the spectrum of phenotypic manifestations associated with deficient GALC activity complicates the management of screen-positive newborns and underscores the need to identify clinically relevant biomarkers. Earlier studies with a small number of patients identified psychosine, a substrate of the GALC enzyme, as a potential biomarker for Krabbe disease. In this study, we provide, for the first time, longitudinal data on dried blood spot (DBS) psychosine concentrations in different Krabbe disease phenotypes for both untreated patients and those treated with hematopoietic stem cell transplantation (HSCT). Our cohort included patients previously identified by NBS to be at high risk to develop Krabbe disease. Substantially elevated DBS psychosine concentration during the newborn period was found to be a highly specific marker for infantile Krabbe disease. This finding supports the use of DBS psychosine concentration as a second-tier NBS test to aid in the identification of patients who require urgent evaluation for HSCT. In addition, longitudinal assessments showed that both natural disease progression and treatment with HSCT were associated with decreases in DBS psychosine concentrations. Based on these findings we provide recommendations for the interpretation of psychosine concentrations in DBS specimens collected during the first year of life. Future studies should aim to better delineate the relationship between DBS psychosine concentration and disease onset in patients with later-onset forms of Krabbe disease.

Hematopoietic Stem cell transplantation and lentiviral vector-based gene therapy for Krabbe's disease: Present convictions and future prospects.

Currently, presymtomatic hematopoietic stem and progenitor cell transplantation (HSPCT) is the only therapeutic modality that alleviates Krabbe's disease (KD)-induced central nervous system damage. However, all HSPCT-treated patients exhibit severe deterioration in peripheral nervous system function characterized by major motor and expressive language pathologies. We hypothesize that a combination of several mechanisms contribute to this phenomenon, including 1) nonoptimal conditioning protocols with consequent inefficient engraftment and biodistribution of donor-derived cells and 2) insufficient uptake of donor cell-secreted galactocerebrosidease (GALC) secondary to a naturally low expression level of the cation-independent mannose 6-phosphate-receptor (CI-MPR). We have characterized the effects of a busulfan (Bu) based conditioning regimen on the efficacy of HSPCT in prolonging twi mouse average life span. There was no correlation between the efficiency of bone marrow engraftment of donor cells and twi mouse average life span. HSPCT prolonged the average life span of twi mice, which directly correlated with the aggressiveness of the Bu-mediated conditioning protocols. HSPC transduced with lentiviral vectors carrying the GALC cDNA under control of cell-specific promoters were efficiently engrafted in twi mouse bone marrow. To facilitate HSPCT-mediated correction of GALC deficiency in target cells expressing low levels of CI-MPR, a novel GALC fusion protein including the ApoE1 receptor was developed. Efficient cellular uptake of the novel fusion protein was mediated by a mannose-6-phosphate-independent mechanism. The novel findings described here elucidate some of the cellular mechanisms that impede the cure of KD patients by HSPCT and concomitantly open new directions to enhance the therapeutic efficacy of HSPCT protocols for KD. © 2016 The Authors. Journal of Neuroscience Research Published by Wiley Periodicals, Inc.

Chaperones as potential therapeutics for Krabbe disease.

Krabbe's disease (KD) is an autosomal recessive, neurodegenerative disorder. It is classified among the lysosomal storage diseases (LSDs). It was first described in , but the genetic defect for the galactocerebrosidase (GALC) gene was not discovered until the beginning of the 1970s, 20 years before the GALC cloning. Recently, in 2011, the crystal structures of the GALC enzyme and the GALC-product complex were obtained. For this, compared with other LSDs, the research on possible therapeutic interventions is much more recent. Thus, it is not surprising that some treatment options are still under preclinical investigation, whereas their relevance for other pathologies of the same group has already been tested in clinical studies. This is specifically the case for pharmacological chaperone therapy (PCT), a promising strategy for selectively correcting defective protein folding and trafficking and for enhancing enzyme activity by small molecules. These compounds bind directly to a partially folded biosynthetic intermediate, stabilize the protein, and allow completion of the folding process to yield a functional protein. Here, we review the chaperones that have demonstrated potential therapeutics during preclinical studies for KD, underscoring the requirement to invigorate research for KD-addressed PCT that will benefit from recent insights into the molecular understanding of GALC structure, drug design, and development in cellular models. © 2016 Wiley Periodicals, Inc.

New therapeutic approaches for Krabbe disease: The potential of pharmacological chaperones.

Missense mutations in the lysosomal hydrolase ß-galactocerebrosidase (GALC) account for at least 40% of known cases of Krabbe disease (KD). Most of these missense mutations are predicted to disrupt the fold of the enzyme, preventing GALC in sufficient amounts from reaching its site of action in the lysosome. The predominant central nervous system (CNS) pathology and the absence of accumulated primary substrate within the lysosome mean that strategies used to treat other lysosomal storage disorders (LSDs) are insufficient in KD, highlighting the still unmet clinical requirement for successful KD therapeutics. Pharmacological chaperone therapy (PCT) is one strategy being explored to overcome defects in GALC caused by missense mutations. In recent studies, several small-molecule inhibitors have been identified as promising chaperone candidates for GALC. This Review discusses new insights gained from these studies and highlights the importance of characterizing both the chaperone interaction and the underlying mutation to define properly a responsive population and to improve the translation of existing lead molecules into successful KD therapeutics. We also highlight the importance of using multiple complementary methods to monitor PCT effectiveness. Finally, we explore the exciting potential of using combination therapy to ameliorate disease through the use of PCT with existing therapies or with more generalized therapeutics, such as proteasomal inhibition, that have been shown to have synergistic effects in other LSDs. This, alongside advances in CNS delivery of recombinant enzyme and targeted rational drug design, provides a promising outlook for the development of KD therapeutics. © 2016 The Authors. Journal of Neuroscience Research Published by Wiley Periodicals, Inc.

Cell-based high-throughput screening identifies galactocerebrosidase enhancers as potential small-molecule therapies for Krabbe's disease.

Krabbe's disease, also known as globoid cell leukodystrophy (GLD), is a lysosomal storage disease caused by the deficiency of the lysosomal enzyme ß-galactocerebrosidase (GALC), resulting in severe neurological manifestations related to demyelination secondary to elevated galactosylsphingosine (psychosine) with its subsequent cytotoxicity. The only available treatment is hematopoietic stem cell transplantation, which delays disease onset but does not prevent long-term neurological manifestations. This article describes the identification of small molecules that enhance mutant GALC activity, identified by quantitative cell-based high-throughput screening (qHTS). Using a specific neurologically relevant murine cell line (145M-Twi) modified to express common human hGALC-G270D mutant, we were able to detect GALC activity in a 1,536-well microplate format. The qHTS of approximately 46,000 compounds identified three small molecules that showed significant enhancements of residual mutant GALC activity in primary cell lines from GLD patients. These compounds were shown to increase the levels of GALC-G270D mutant in the lysosomal compartment. In kinetic assessments, these small molecules failed to disturb the GALC kinetic profile under acidic conditions, which is highly desirable for folding-assisting molecules operating in the endoplasmic reticulum and not affecting GALC catalytic properties in the lysosomal compartment. In addition, these small molecules rescued the decreased GALC activity at neutral pH and partially stabilized GALC under heat-denaturating conditions. These drug-like compounds can be used as the starting point to develop novel small-molecule agents to treat the progressive neurodegenerative course of GLD. © 2016 Wiley Periodicals, Inc.

Synthesis and evaluation of N-alkylated analogues of aza-galacto-fagomine - potential pharmacological chaperones for Krabbe disease.

Seven novel alkylated or acylated analogues of hexahydropyridazine aza-galacto-fagomine (AGF) was prepared and studied as glycosidase inhibitors with the aim of increasing inhibitory potency and selectivity. The enzyme galactocerebrosidase, implicated in Krabbe disease, was found to be potently inhibited by n-butyl N2-alkylated AGF.

Galactosylsphingosine (psychosine)-induced demyelination is attenuated by sphingosine 1-phosphate signalling.

Globoid cell leukodystrophy (Krabbe disease) is a rare infantile neurodegenerative disorder. Krabbe disease is caused by deficiency in the lysosomal enzyme galactocerebrosidase (GALC) resulting in accumulation, in the micromolar range, of the toxic metabolite galactosylsphingosine (psychosine) in the brain. Here we find that psychosine induces human astrocyte cell death probably via an apoptotic process in a concentration- and time-dependent manner (EC50 ∼ 15 µM at 4 h). We show these effects of psychosine are attenuated by pre-treatment with the sphingosine 1-phosphate receptor agonist pFTY720 (fingolimod) (IC50 ∼ 100 nM). Psychosine (1 µM, 10 µM) also enhances LPS-induced (EC50 ∼ 100 ng/ml) production of pro-inflammatory cytokines in mouse astrocytes, which is also attenuated by pFTY720 (1 µM). Most notably, for the first time, we show that psychosine, at a concentration found in the brains of patients with Krabbe disease (EC50 ∼ 100 nM), directly induces demyelination in mouse organotypic cerebellar slices in a manner that is independent of pro-inflammatory cytokine response and that pFTY720 (0.1 nM) significantly inhibits. These results support the idea that psychosine is a pathogenic agent in Krabbe disease and suggest that sphingosine 1-phosphate signalling could be a potential drug target for this disorder.

Chaperone therapy for Krabbe disease: potential for late-onset GALC mutations.

Krabbe disease is an autosomal recessive leukodystrophy caused by a deficiency of the galactocerebrosidase (GALC) enzyme. Hematopoietic stem cells transplantation is the only available treatment option for pre-symptomatic patients. We have previously reported the chaperone effect of N-octyl-4-epi-ß-valienamine (NOEV) on mutant GM1 ß-galactosidase proteins, and in a murine GM1-gangliosidosis model. In this study, we examined its chaperone effect on mutant GALC proteins. We found that NOEV strongly inhibited GALC activity in cell lysates of GALC-transfected COS1 cells. In vitro NOEV treatment stabilized GALC activity under heat denaturation conditions. We also examined the effect of NOEV on cultured COS1 cells expressing mutant GALC activity and human skin fibroblasts from Krabbe disease patients: NOEV significantly increased the enzyme activity of mutants of late-onset forms. Moreover, we confirmed that NOEV could enhance the maturation of GALC precursor to its mature active form. Model structural analysis showed NOEV binds to the active site of human GALC protein. These results, for the first time, provide clear evidence that NOEV is a chaperone with promising potential for patients with Krabbe disease resulting from the late-onset mutations.