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.

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.

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.

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.

Mechanism-based combination treatment dramatically increases therapeutic efficacy in murine globoid cell leukodystrophy.

Globoid cell leukodystrophy (GLD, Krabbe disease) is a lysosomal storage disease (LSD) caused by a deficiency in galactocerebrosidase (GALC) activity. In the absence of GALC activity, the cytotoxic lipid, galactosylsphingosine (psychosine), accumulates in the CNS and peripheral nervous system. Oligodendrocytes and Schwann cells are particularly sensitive to psychosine, thus leading to a demyelinating phenotype. Although hematopoietic stem-cell transplantation provides modest benefit in both presymptomatic children and the murine model (Twitcher), there is no cure for GLD. In addition, GLD has been relatively refractory to virtually every experimental therapy attempted. Here, Twitcher mice were simultaneously treated with CNS-directed gene therapy, substrate reduction therapy, and bone marrow transplantation to target the primary pathogenic mechanism (GALC deficiency) and two secondary consequences of GALC deficiency (psychosine accumulation and neuroinflammation). Simultaneously treating multiple pathogenic targets resulted in an unprecedented increase in life span with improved motor function, persistent GALC expression, nearly normal psychosine levels, and decreased neuroinflammation. Treating the primary pathogenic mechanism and secondary targets will likely improve therapeutic efficacy for other LSDs with complex pathological and clinical presentations.

Pharmacological chaperones increase residual ß-galactocerebrosidase activity in fibroblasts from Krabbe patients.

Krabbe disease or globoid cell leukodystrophy is a degenerative, lysosomal storage disease resulting from the deficiency of ß-galactocerebrosidase activity. This enzyme catalyzes the lysosomal hydrolysis of galactocerebroside and psychosine. Krabbe disease is inherited as an autosomal recessive trait, and many of the 70 disease-causing mutations identified in the GALC gene are associated with protein misfolding. Recent studies have shown that enzyme inhibitors can sometimes translocate misfolded polypeptides to their appropriate target organelle bypassing the normal cellular quality control machinery and resulting in enhanced activity. In search for pharmacological chaperones that could rescue the ß-galactocerebrosidase activity, we investigated the effect of α-Lobeline or 3',4',7-trihydroxyisoflavone on several patient-derived fibroblast cell lines carrying missense mutations, rather than on transduced cell lines. Incubation of these cell lines with α-lobeline or 3',4',7-trihydroxyisoflavone leads to an increase of ß-galacocerebrosidase activity in p.G553R + p.G553R, in p.E130K + p.N295T and in p.G57S + p.G57S mutant forms over the critical threshold. The low but sustained expression of ß-galactocerebrosidase induced by these compounds is a promising result; in fact, it is known that residual enzyme activity of only 15-20% is sufficient for clinical efficacy. The molecular interaction of the two chaperones with ß-galactocerebrosidase is also supported by in silico analysis. Collectively, our combined in silico-in vitro approach indicate α-lobeline and 3',4',7-trihydroxyisoflavone as two potential pharmacological chaperones for the treatment or improvement of quality of life in selected Krabbe disease patients.

Oxidative stress as a therapeutic target in globoid cell leukodystrophy.

Globoid cell leukodystrophy (GLD, Krabbe Disease) is a lysosomal storage disease, resulting from the genetic deficiency of galactosylceramidase (GALC). This disease is marked by accumulation of the cytotoxic lipid psychosine (Psy). Psychosine is known to induce oxidative stress in cultured cells, and this stress can be ameliorated through co-treatment with the antioxidant N-acetyl cysteine (NAC). Oxidative stress has also been observed in vivo in the mouse model of GLD, the Twitcher mouse (Twi). We hypothesized that treating oxidative stress with NAC; either alone or in combination with bone marrow transplant (BMT) would improve the course of disease. All breeding cages were maintained on water containing NAC. Once born, the pups received IP boluses of NAC three times per week, and were maintained on NAC-containing water. A separate cohort of animals received the same regimen of NAC in addition to a BMT on post-natal days 2-3. Although NAC lowers the level of oxidized proteins in the brains of Twi mice, and dramatically improves immunohistochemical markers of disease, neither treatment results in any clinical improvements in the Twi mouse. Our data suggest that oxidative stress may be sufficiently down-stream in the pathogenic cascade initiated by Psy accumulation as to be difficult or impossible to treat with standard pharmacologic agents. It is possible that NAC may synergize with other therapies or combinations of therapies. A better understanding of the initiating effects of Psy toxicity and oxidative damage may uncover treatable therapeutic targets.

Molecular characterization of mutations that cause globoid cell leukodystrophy and pharmacological rescue using small molecule chemical chaperones.

Globoid cell leukodystrophy (GLD) (Krabbe disease) is an autosomal recessive, degenerative, lysosomal storage disease caused by a severe loss of galactocerebrosidase (GALC) enzymatic activity. Of the >70 disease-causing mutations in the GALC gene, most are located outside of the catalytic domain of the enzyme. To determine how GALC mutations impair enzymatic activity, we investigated the impact of multiple disease-causing mutations on GALC processing, localization, and enzymatic activity. Studies in mammalian cells revealed dramatic decreases in GALC activity and a lack of appropriate protein processing into an N-terminal GALC fragment for each of the mutants examined. Consistent with this, we observed significantly less GALC localized to the lysosome and impairment in either the secretion or reuptake of mutant GALC. Notably, the D528N mutation was found to induce hyperglycosylation and protein misfolding. Reversal of these conditions resulted in an increase in proper processing and GALC activity, suggesting that glycosylation may play a critical role in the disease process in patients with this mutation. Recent studies have shown that enzyme inhibitors can sometimes "chaperone" misfolded polypeptides to their appropriate target organelle, bypassing the normal cellular quality control machinery and resulting in enhanced activity. To determine whether this may also work for GLD, we examined the effect of alpha-lobeline, an inhibitor of GALC, on D528N mutant cells. After treatment, GALC activity was significantly increased. This study suggests that mutations in GALC can cause GLD by impairing protein processing and/or folding and that pharmacological chaperones may be potential therapeutic agents for patients carrying certain mutations.

Effects of treatments on inflammatory and apoptotic markers in the CNS of mice with globoid cell leukodystrophy.

Globoid cell leukodystrophy (GLD) or Krabbe disease is a neurodegenerative disorder caused by the deficiency of the lysosomal enzyme galactocerebrosidase (GALC). GALC deficiency results in a progressive demyelination of the central and peripheral nervous systems. Inflammatory cells and increased levels of cytokines and chemokines are present in the CNS of GLD mice and may play a significant role in the pathogenesis of the disease. In this study we evaluate the effect of non-steroidal anti-inflammatory drugs, such as indomethacin and ibuprofen, and minocycline, a tetracycline analog with neuroprotective and anti-apoptotic properties, on the progression of the disease using a transgenic mouse model of GLD. Real-time quantitative PCR was used to analyze the expression of several markers of the immune/inflammatory response. IL-6, TNF-alpha, MIP-1beta, MCP-1, iNOS/NOS2, CD11b, CD68, CD4 and CD8 mRNA levels were measured in cortex, cerebellum and spinal cord of untreated and treated affected mice at different ages. In addition, the pharmacological treatments were compared to bone marrow transplantation (BMT). The pharmacological treatments significantly extended the life-span of the treated mice and reduced the levels of several of the immuno-related factors studied. However, BMT produced the most dramatic improvements. In BMT-treated mice, factors in the spinal cord were normalized faster than the cerebellum, with the exception of CD68. There was a decrease in the number of apoptotic cells in the cerebellum of mice receiving anti-inflammatory drugs and BMT. These studies indicate a possible role for combined therapy in the treatment of GLD.

Insulin-like growth factor-1 provides protection against psychosine-induced apoptosis in cultured mouse oligodendrocyte progenitor cells using primarily the PI3K/Akt pathway.

Psychosine (galactosylsphingosine) is a toxic metabolite that accumulates in globoid cell leukodystrophy (GLD) due to the deficiency of galactocerebrosidase (GALC) activity. This results in subsequent programmed cell death of oligodendrocytes and demyelination in human patients and animal models. We investigated the potential role of insulin-like growth factor-1 (IGF-1) in modifying the apoptotic effect of psychosine in cultured mouse oligodendrocyte progenitor cells (OLP-II). We show that psychosine inhibits the phosphorylation of Akt and Erk1/Erk2 (Erk1/2), which are the main anti-apoptotic pathways of the IGF-1 receptor (IGF-1R). Although IGF-1 sustained phosphorylation of both of these pathways, it provided maximum protection to OLP-II cells from psychosine-induced cell death in a PI3K/Akt-dependent manner. The effects of IGF-1 were dose-dependent and resulted in increased IGF-1R autophosphorylation levels. Although relatively high concentrations of IGF-1 also resulted in the activation of the insulin receptor (IR), its effect was more significant on the IGF-1R.

Hematopoietic cell transplantation after administration of high-dose busulfan in murine globoid cell leukodystrophy (the twitcher mouse).

We evaluated the effectiveness of administration of high-dose busulfan (BU), an alkylating agent with substantial myeloablative but negligible immunosuppressive properties, on the engraftment of congenic normal bone marrow and spleen cells (hematopoietic cell transplantation; HCT) in murine galactosylceramidase deficiency (the twitcher mouse), a model of human sphingolipid storage disease. Untreated mice died with extensive demyelination and failure to thrive at a median age of 40 d (range, 28-47; n = 51). The life-span of twitcher mice given HCT at age 10 d after 100 mg/kg BU was significantly prolonged (median, 94 d; range, 55-140; n = 17); these animals did not develop the hindlimb paralysis seen in untreated twitchers. Histologic examination of twitcher sciatic nerves after HCT showed remyelinated areas and decreased globoid cell inclusions. Animals given HCT after conditioning with BU had both lymphoid and hematopoietic repopulation with donor cells by 90 d after HCT, as documented by presence of donor glucose phosphate isomerase-1A activity in blood, bone marrow, spleen, and lymph nodes. After BU and HCT, galactosylceramidase activity in livers and spleens of twitcher mice reached 45 and 80% of control, respectively; only modest elevations were observed in kidneys and lymph nodes. Hydrolase activity rose to 20% in the brains and exceeded control values in the sciatic nerves of transplanted twitcher mice, indicating entry of at least some donor-derived cells and/or hydrolase across the blood-brain and blood-nerve barriers after BU and HCT.(ABSTRACT TRUNCATED AT 250 WORDS)