Globoid Cell Research Articles

Krabbe\s disease in a 28 year old adult - A rare case report

Krabbe”s disease (KD) or Globoid cell leukodystrophy (GLD) is a disorder involving the white matter of both the central and peripheral nervous systems. It is caused by congenital deficiency of a lysosomal enzyme, galactosylceramidase I, which is responsible for hydrolyzing the galactolipids in myelin. The disease is extremely rare occurring only one in 100,000 to one in 200,000 live births. The disease is classically of infantile origin but it can also occur in older children as well as adults. Adult variant is the rarest type. We hereby report a 34 year old male patient with progressively increasing ataxia, dysarthria. Typical MRI brain changes and diminished leucocyte galactocerebrosidase (GALC) enzyme levels clinched the diagnosis of Krabbe”s disease . There are very few cases of KD reported from India. This case report is to stress upon the fact that this rare entity should not be missed in an appropriate clinical setting.

Genetic Test and Gene Therapy for Krabbe Disease: An Update

Globoid cell leukodystrophy, also known as Krabbe disease, is an inherited metabolic neurodegenerative disease, due to genetic mutation of β-galactocerebrosidase gene. Here we reviewed how the technological advances in gene analysis have enhanced the enrichment of mutation database. Moreover, we focus on the possibility to develop genetic treatments, hoping that the updating of genetic, clinical and biochemical data will improve for an early and efficient diagnosis, until a definitive treatment option will be identified.

Editorial: 2013—A Year of Clinical Success and Great Scientific Innovation in the Stem Cell Field

The year 2013 has been an extremely excitingone for the field of stem cell research andtherapy. Our “sister journals,” STEM CELLS andSTEM CELLS TRANSLATIONAL MEDICINE, havedone a great job of capturing the excitingadvances in the field, thanks to the outstand-ing manuscripts submitted by our authors.STEM CELLS manuscripts focus primarily onthe functional and mechanistic aspects of stemcell biology and the potential of different typesof stem cells for therapeutic applications, andreports key advances in stem cell clinical trials.Manuscripts must have definitive conclusionsto be considered for potential publication inSTEM CELLS. The manuscripts in STEM CELLSTRANSLATIONAL MEDICINE (SCTM) primarilycover technical advances in delivering cell ther-apy, advances in animal models, and new find-ings that help advance particular stem celltherapies closer to the clinic. SCTM providesan excellent forum for translational, clinical,and technical advances for stem cell therapydevelopment. After only 1 year in press, wewere thrilled to announce that SCTM becamefully indexed in PubMed. This is a wonderfultestimony to the huge amount of excitementin the field and to the truly excellent manu-scripts submitted to both sister journals.During the past year, STEM CELLS has beenproud to publish exciting clinical advances intreating steroid-refractory Graft versus Hostdisease [1], Cerebral Palsy [2], Crohn’s fistula[3], and treating alveolar bone defects with aninjectable tissue engineered stem cell product,avoiding the donor site morbidity associatedwith bone autograft [4].We were also excited to publish an excel-lent review on the current clinical status ofmesenchymal stem/stromal cell (MSC) thera-pies for inflammatory diseases [5], a review ofengineering myocardial tissue [6], and concisereviews on clinical programs of stem celltherapies for liver and pancreas [7], stem cellsfor treating lung disease [8], MSCs for ischemickidney disease [9], and salivary gland stemcells for treating xerostomia/hyposalivation[10].These clinical reports and reviews of com-pleted and ongoing stem cell clinical trialswere accompanied by a dazzling assortment ofbasic and translational research toward betterunderstanding and treating a plethora of dis-eases and injuries. Manuscripts covered manyadvances in understanding the fundamentalmechanisms that control stem and progenitorcell fate, and advances in working towardtreating all tissues, from the lower extremitiesto the brain. Exciting basic and translationalresearch was reported in using stem cells totreat osteoporosis [11], spinal cord injuries[12, 13], radiation-induced fibrosis [14], glau-coma [15], globoid cell leukodystrophy [16],stroke [17], and Huntington’s disease [18], toname just a few. It has truly been an excitingyear, as we watch the stem cell field explode.In order to help readers keep up with theirareas of interest, STEM CELLS provides virtualissues in specific areas, which are collectionsof the most exciting papers over the past 2years in a given field. These can be accessedon the STEM CELLS website. Some of the mostrecent collections are: “Neural Stem CellsPapers,” “Induced Pluripotency—Making StemCells for All Reasons,” and “MSCs for WhatAils You.”The STEM CELLS Portal provides updateson exciting news in the field and links toadvances that are reported in both sister jour-nals. There is a link on the portal to an audiosection called “Innovators and Issues,” whichconnects listeners with leaders in the field ofstem cell research and translational medicine.The website also has a link to the younginvestigator awards. Dr. Jacco van Rheenen’smanuscript, “Intravital Imaging of Cancer StemCell Plasticity in Mammary Tumors,” publishedin the March 2013 issue of the Journal, wasselected as the

Innate Immune Activation in the Pathogenesis of a Murine Model of Globoid Cell Leukodystrophy

Globoid cell leukodystrophy is a lysosomal storage disease characterized by the loss of galactocerebrosidase. Galactocerebrosidase loss leads to the accumulation of psychosine and subsequent oligodendrocyte cell death, demyelination, macrophage recruitment, and astroglial activation and proliferation. To date, no studies have elucidated the mechanism of glial cell activation and cytokine and chemokine up-regulation and release. We explored a novel explanation for the development of the pathological changes in the early stages of globoid cell leukodystrophy associated with toll-like receptor (TLR) 2 up-regulation in the hindbrain and cerebellum as a response to dying oligodendrocytes. TLR2 up-regulation on microglia/macrophages coincided with morphological changes consistent with activation at 2 and 3 weeks of age. TLR2 up-regulation on activated microglia/macrophages resulted in astrocyte activation and marked up-regulation of cytokines/chemokines. Because oligodendrocyte cell death is an important feature of globoid cell leukodystrophy, we tested the ability of TLR2 reporter cells to respond to oligodendrocyte cell death. These reporter cells responded invitro to medium conditioned by psychosine-treated oligodendrocytes, indicating the likelihood that oligodendrocytes release a TLR2 ligand during apoptosis. TLRs are a member of the innate immune system and initiate immune and inflammatory events; therefore, the identification of TLR2 as a potential driver in the activation of central nervous system glial activity in globoid cell leukodystrophy may provide important insight into its pathogenesis.

Psychosine, the cytotoxic sphingolipid that accumulates in globoid cell leukodystrophy, alters membrane architecture

Globoid cell leukodystrophy (GLD) is a neurological disease caused by deficiency of the lysosomal enzyme galactosylceramidase (GALC). In the absence of GALC, the cytotoxic glycosphingolipid, psychosine (psy), accumulates in the nervous system. Psychosine accumulation preferentially affects oligodendrocytes, leading to progressive demyelination and infiltration of activated monocytes/macrophages into the CNS. GLD is characterized by motor defects, cognitive deficits, seizures, and death by 2-5 years of age. It has been hypothesized that psychosine accumulation, primarily within lipid rafts, results in the pathogenic cascade in GLD. However, the mechanism of psychosine toxicity has yet to be elucidated. Therefore, we synthesized the enantiomer of psychosine (ent-psy) to use as a probe to distinguish between protein-psy (stereo-specific enantioselective) or membrane-psy (stereo-insensitive nonenantioselective) interactions. The enantiomer of psychosine has equal or greater toxicity compared with psy, suggesting that psy exerts its toxicity through a nonenantioselective mechanism. Finally, in this study we demonstrate that psy and ent-psy localize to lipid rafts, perturb natural and artificial membrane integrity, and inhibit protein Kinase C translocation to the plasma membrane. Although other mechanisms may play a role in disease, these data strongly suggest that psy exerts its effects primarily through membrane perturbation rather than through specific protein-psy interactions.

Methemoglobinemia In Rhesus Monkeys Due To M-Hemoglobinopathy

M-hemoglobinopathies in humans are usually due to amino acid changes, which tend to stabilize the heme iron in ferric state and inaccessible to methemoglobin reductase. Consequently, blood methemoglobin concentrations are elevated despite normal methemoglobin reductase activity. No naturally-occurring animal models for M-hemoglobinopathies have been described. In contrast to humans where thousands of hereditary disorders and genetic predispositions to disease have been described, inborn errors of metabolism in non-human primates have been very rarely documented (e.g. globoid cell leukodystrophy). As part of an NIH-supported National Referral Center for Animal Models of Human Genetic Disease, which focuses on homologous hereditary diseases in dogs and cats, we present here the discovery and characterization of an M-hemogobinopathy in rhesus monkeys (Macaca mulatta). Adult rhesus monkeys of Chinese origin with persistent cyanotic mucous membranes and healthy controls were studied from two institutions. Despite the apparent cyanosis, these primates did clinically not show any other overt signs. Routine complete blood cell counts, chemistry screens and urinalyses as well as cardiopulmonary examinations did not reveal any abnormalities. Fresh EDTA blood samples were analyzed to determine the degree and cause of methemoglobinemia and results were compared to healthy rhesus monkeys from the same institutions. Blood methemoglobin concentrations were persistently increased to 15-30% in affected monkeys, while control animals had <1% methemoglobin levels. Methemoglobin (cytochrome b5) reductase activities in erythrocytes were repeatedly measured in vitro and found to be normal compared to controls. Based upon the hemoglobin-oxygen dissociation curves and p50 values (14.8 mmHg; compared to 14.5 and 14.6 mmHg for controls) the oxygen transport function was normal. Both cation-exchange as well as reversed-phase and high performance liquid chromatography analyses revealed normal elution patterns of hemoglobin isotype and globin chains. DNA studies identified a disease-associated polymorphism in the alpha globin gene (CAC → CAA) in the affected animals. This change in the alpha globin protein was confirmed by mass spectrometry (α78 His → Gln). Our findings indicate an electrophoretically silent α-globin polymorphism is associated with methemoglobinemia in these rhesus monkeys. This is the first description of methemoglobinemia in a non-human primate and represents the first animal model of an M-hemoglobinopathy. While clinical signs were relatively mild in these rhesus monkeys, our findings represent one of the first inborn errors of metabolism described in non-human primates, thus emphasizing how unusual disorders caused by “experiments of nature” provide rare opportunities to gain a better understanding of both medicine and biology. Rhesus monkeys represent unique, valuable animal models for developing and testing new treatment options for hemoglobinopathies. Disclosures: No relevant conflicts of interest to declare.

Outcomes Of Allogeneic Cord Blood Transplantation For Leukodystrophies; A Joint Study of Eurocord and “Inborn Errors WP-EBMT”

BackgroundThis study aims to describe transplant outcomes and disease-specific clinical outcomes of allogeneic cord blood transplantation (CBT) in leukodystrophies. Leukodystrophies are rare inborn errors of metabolism (IEM) in which the development and maintenance of brain myelin is primarily affected. They are characterized by rapid neurological deterioration. Hematopoietic stem cell transplantation (HSCT) has been shown to halt disease progression for selected leukodystrophies, including adrenoleukodystrophy (ALD), Krabbe disease (globoid cell leukodystrophy) and metachromatic leukodystrophy (MLD). The fast availability of unrelated cord blood (UCB) makes HSCT feasible in leukodystrophy patients who lack an HLA-matched sibling. Patients and methodsAll patients transplanted for leukodystrophies with related and unrelated umbilical cord blood in EBMT centers between 1996 and 2012 were included. HSCT data were collected from the EUROCORD database. An additional questionnaire on disease-specific clinical outcomes was sent to participating centers. Included in the questionnaire were enzyme- and (for ALD) fatty acid levels, general disease status, performance score, MRI/MRS status, peripheral neuropathy, school attendance and performance, mental development testing, adrenal insufficiency (for ALD only), vision and hearing. Kaplan-Meier estimates were used to calculate overall survival (OS) and cumulative incidence methods for secondary outcomes, including neutrophil and platelet engraftment, incidence of graft-versus-host disease (GvHD), donor chimerism and disease-specific characteristics at most recent follow-up. The two-sided log-rank test was used for univariate comparisons. ResultsSeventy patients (31 ALD, 5 Krabbe, 34 MLD) were available for analysis, with a median age at transplant of 6.5 years (range 0-43 years). Median follow-up for survivors was 46 months. Overall survival at 4 years was 60% (±6%). Cumulative incidences of neutrophil and platelet engraftment were 88% (±4%) at day 60, and 73% (±6%) at day 180, respectively. Ten patients experienced graft failure and 5 had autologous recovery; 4 received a 2nd HSCT (2 CBT, 1 Haplo, 1 BM) and of those 3 were alive in a median of 60 months after HSCT. Acute-GvHD (grade II-IV) occurred in 16 patients (22% ±5%) at day 100 and chronic GvHD in 9 patients (13% ±5%). Out of the 24 patients who died, 18 (67%) died of transplant-related causes and 6 (33%) died of disease progression. Higher survival was seen in patients who had no or 1 HLA mismatch (OS 81%), compared to patients who received UCBT with 2 HLA mismatches (OS 47%, p 0.02).On 37 patients (53%), information on disease-specific characteristics was available. OS among these patients was 68% (±8), which was not significantly different from patients without disease-specific information (p=0,307). Of the 37 patients, 19 (51%) were asymptomatic at transplant, 14 (38%) had mild disease and 4 (11%) were severely affected. Overall survival was dramatically worse in patients with severe disease at transplant; all 4 severely affected patients died, while 79% of patients with mild disease and 76% of asymptomatic patients survived. At most recent follow-up, disease status was stable in 15 (57.7%), had improved in 2 (7.7%) and worsened in 9 (34.6%) surviving patients. The majority of patients showed abnormalities on MRI-scanning pre-HSCT (n=25, 68%). In patients with normal MRI-scans pre-transplant OS was 69%; in patients with abnormal MRI-scans 75%. At most recent follow-up, MRI-scans were stable in 12 (43.9%), improved in 5 (17.8%) and worsened in 11 (39.3%) patients. Eighteen patients (50%) showed peripheral neuropathy pre-HSCT, of which 13 were mildly affected and 5 severely affected. Of patients who had no signs of peripheral neuropathy pre-HSCT, 70% survived, versus 77% of patients who were mildly affected and 40% of those severely affected. At most recent follow-up, peripheral neuropathy was stable in 14 (56%), had improved in 3 (12%) and worsened in 8 (32%) patients. ConclusionWe found an overall survival of 60% at 4 years after CBT for leukodystrophies, with relatively low rates of GvHD. Use of a mismatched donor (>1 HLA mismatch) negatively impacts survival. Data on clinical characteristics suggest that overall survival is strongly influenced by disease status at transplant. Disclosures:No relevant conflicts of interest to declare.

Inhibition of angiogenesis by β-galactosylceramidase deficiency in globoid cell leukodystrophy

Globoid cell leukodystrophy (Krabbe disease) is a neurological disorder of infants caused by genetic deficiency of the lysosomal enzyme β-galactosylceramidase leading to accumulation of the neurotoxic metabolite 1-β-d-galactosylsphingosine (psychosine) in the central nervous system. Angiogenesis plays a pivotal role in the physiology and pathology of the brain. Here, we demonstrate that psychosine has anti-angiogenic properties by causing the disassembling of endothelial cell actin structures at micromolar concentrations as found in the brain of patients with globoid cell leukodystrophy. Accordingly, significant alterations of microvascular endothelium were observed in the post-natal brain of twitcher mice, an authentic model of globoid cell leukodystrophy. Also, twitcher endothelium showed a progressively reduced capacity to respond to pro-angiogenic factors, defect that was corrected after transduction with a lentiviral vector harbouring the murine β-galactosylceramidase complementary DNA. Finally, RNA interference-mediated β-galactosylceramidase gene silencing causes psychosine accumulation in human endothelial cells and hampers their mitogenic and motogenic response to vascular endothelial growth factor. Accordingly, significant alterations were observed in human microvasculature from brain biopsy of a globoid cell leukodystrophy case. Together these data demonstrate that β-galactosylceramidase deficiency induces significant alterations in endothelial neovascular responses that may contribute to central nervous system and systemic damages that occur in globoid cell leukodystrophy.

Multipotent Stromal Cells Alleviate Inflammation, Neuropathology, and Symptoms Associated with Globoid Cell Leukodystrophy in the Twitcher Mouse

Globoid cell leukodystrophy (GLD) is a common neurodegenerative lysosomal storage disorder caused by a deficiency in galactocerebrosidase (GALC), an enzyme that cleaves galactocerebroside during myelination. Bone marrow transplantation has shown promise when administered to late-onset GLD patients. However, the side effects (e.g., graft vs. host disease), harsh conditioning regimens (e.g., myelosuppression), and variable therapeutic effects make this an unsuitable option for infantile GLD patients. We previously reported modest improvements in the twitcher mouse model of GLD after intracerebroventricular (ICV) injections of a low-dose of multipotent stromal cells (MSCs). Goals of this study were to improve bone marrow-derived MSC (BMSC) therapy for GLD by increasing the cell dosage and comparing cell type (e.g., transduced vs. native), treatment timing (e.g., single vs. weekly), and administration route (e.g., ICV vs. intraperitoneal [IP]). Neonatal twitcher mice received (a) 2 × 10(5) BMSCs by ICV injection, (b) 1 × 10(6) BMSCs by IP injection, (c) weekly IP injections of 1 × 10(6) BMSCs, or (d) 1 × 10(6) lentiviral-transduced BMSCs overexpressing GALC (GALC-BMSC) by IP injection. All treated mice lived longer than untreated mice. However, the mice receiving peripheral MSC therapy had improved motor function (e.g., hind limb strength and rearing ability), twitching symptoms, and weight compared to both the untreated and ICV-treated mice. Inflammatory cell, globoid cell, and apoptotic cell levels in the sciatic nerves were significantly decreased as a result of the GALC-BMSC or weekly IP injections. The results of this study indicate a promising future for peripheral MSC therapy as a noninvasive, adjunct therapy for patients affected with GLD.

Region- and Age-Dependent Alterations of Glial-Neuronal Metabolic Interactions Correlate with CNS Pathology in a Mouse Model of Globoid Cell Leukodystrophy

Globoid cell leukodystrophy (GLD) or Krabbe disease is a lysosomal storage disorder caused by genetic defects in the expression and activity of galactosylceramidase, a key enzyme in the catabolism of myelin-enriched sphingolipids. While there are several histologic, biochemical, and functional studies on GLD, correlations between morphologic and biochemical alterations in central nervous system (CNS) tissues during disease progression are lacking. Here, we combined immunohistochemistry and metabolic analysis using (1)H and (13)C magnetic resonance (MR) spectra of spinal cord, cerebellum, and forebrain to investigate glial-neuronal metabolic interactions and dysfunction in a GLD murine model that recapitulates the human pathology. In order to assess the temporal- and region-dependent disease progression and the potential metabolic correlates, we investigated CNS tissues at mildly symptomatic and fully symptomatic stages of the disease. When compared with age-matched controls, GLD mice showed glucose hypometabolism, alterations in neurotransmitter content, N-acetylaspartate, N-acetylaspartylglutamate, and osmolytes levels. Notably, age- and region-dependent patterns of metabolic disturbances were in close agreement with the progression of astrogliosis, microglia activation, apoptosis, and neurodegeneration. We suggest that MR spectroscopy could be used in vivo to monitor disease progression, as well as ex vivo and in vivo to provide criteria for the outcome of experimental therapies.

Characterization of adipose-derived stromal/stem cells from the twitcher mouse model of krabbe disease

BackgroundKrabbe disease, also known as globoid cell leukodystrophy, is an autosomal recessive neurodegenerative disease caused by the genetic deficiency of galactocerebrosidase (GALC), a lysosomal enzyme responsible for the degradation of several glycosphingolipids like psychosine and galactosylceramide. In order to investigate whether GALC deficiency in Krabbe disease affects adipose-derived stromal/stem cell (ASC) properties and if the ASCs could be used as a source of autologous stem cell therapy for patients with Krabbe disease, ASCs isolated from subcutaneous adipose tissue of Twitcher mice (a murine model of Krabbe disease) and their normal wild type littermates were cultured, expanded, and characterized for their cell morphology, surface antigen expression, osteogenic and adipogenic differentiation, colony forming units, growth kinetics, and immune regulatory capacities in vitro.ResultsASCs from Twitcher mice (TwiASCs), when compared to ASCs from normal mice (WtASCs), have a reduced osteogenic differentiation potential, have less self-replicating and proliferative capacity, although they have the same fibroblast morphologies and cell sizes. However, surprisingly, the TwiASCs demonstrated similar immune-suppressive capacities as their counterparts WtASCs did when they were transwell co-cultured with macrophages in vitro.ConclusionThis study reveals that Twitcher ASCs exhibit differences in the biologic potential when compared to their counterparts from normal mice. The changes in Twitcher ASCs may be influenced by the GALC deficiency in Twitcher mice. Nevertheless, none of the changes preclude the use of the TwiASCs for autologous applications.

Quantification of psychosine in the serum of twitcher mouse by LC–ESI-tandem-MS analysis

Globoid cell leukodystrophy or Krabbe disease is an inherited autosomal recessive disorder caused by mutations in the galactosylceramidase (GALC) gene. Deficiency of GALC results in the accumulation of a highly cytotoxic metabolite galactosylsphingosine (psychosine). In the present study, we describe the development and validation of a sensitive and specific LC–ESI-tandem-MS method for the determination of psychosine in the serum of twitcher mice, the naturally occurring animal model of this disease. The method was validated in terms of accuracy, precision, specificity, linearity and sensitivity. Calibration plots were linear over the concentration range of 2.5–50ng/mL. Recovery of psychosine from serum was in the range 94.20–98.02%. The results of this study show that in the affected mice the concentration of psychosine (ranging from 2.53 to 33.27ng/mL) increased significantly with the progression of the disease. The maximum level (33.27ng/mL) was detected in the serum of one of the twitcher mice sacrificed at 40PND. Psychosine was not detected at significant levels in wild type mice. These results clearly demonstrate that this noninvasive, rapid, and highly sensitive LC–ESI-tandem-MS method is a very useful approach for the detection of psychosine in serum.

Diagnosis of mitochondrial neurogastrointestinal encephalopathy disease in gastrointestinal biopsies

A 14-year-old boy with mitochondrial neurogastrointestinal encephalopathy (MNGIE) disease had a lifelong history of failure to thrive and gastrointestinal symptoms including vomiting, pain, and diarrhea, leading to progressive cachexia. At the age of 9 years, after an extensive workup, the diagnosis of Crohn disease was strongly suspected, and he underwent colonoscopy with multiple biopsies. At 11 years of age, vision change and poor balance lead to a diagnosis of leukodystrophy by magnetic resonance imaging. Investigations for metachromatic leukodystrophy, adrenal leukodystrophy, and globoid cell leukodystrophy were all negative. A diagnosis of MNGIE disease was suspected when he continued deteriorating with gastrointestinal symptoms, multiple neurologic deficits, and encephalopathy. Markedly diminished thymidine phosphorylase activity and increased thymidine plasma levels confirmed the diagnosis of MNGIE. At autopsy, megamitochondria were observed by light microscopy in submucosal and myenteric ganglion cells and in smooth muscle cells of muscularis mucosae and muscularis propria, along the entire gastrointestinal tract from the esophagus to the rectum. Megamitochondria in ganglion cells were also observed in a retrospective review of the endoscopic intestinal biopsies taken at age 9 and 13 years and in the appendectomy specimen obtained 1 month before his demise. This study corroborates the presence of megamitochondria in gastrointestinal ganglion cells in MNGIE disease, better illustrates their detailed morphology, and describes for the first time similar structures in the cytoplasm of gastrointestinal smooth muscle cells. Pathologists should be able to recognize these structures by light microscopy and be aware of their association with primary mitochondriopathies.

On the role of natural killer cells in neurodegenerative diseases.

Natural killer (NK) cells exert important immunoregulatory functions by releasing several inflammatory molecules, such as IFN-γ and members of chemokines, which include CCL3/MIP-1α and CCL4/MIP-1β. These cells also express heptahelical receptors, which are coupled to heterotrimeric G proteins that guide them into inflamed and injured tissues. NK cells have been shown to recognize and destroy transformed cells and virally-infected cells, but their roles in neurodegenerative diseases have not been examined in detail. In this review, I will summarize the effects of NK cells in two neurodegenerative diseases, namely multiple sclerosis and globoid cell leukodystrophy. It is hoped that the knowledge obtained from these diseases may facilitate building rational protocols for treating these and other neurodegenerative or autoimmune diseases using NK cells and drugs that activate them as therapeutic tools.

MMP‐3 mediates psychosine‐induced globoid cell formation: Implications for leukodystrophy pathology

Globoid cell leukodystrophy (GLD) or Krabbe disease, is a fatal demyelinating disease attributed to mutations in the galactocerebrosidase (GALC) gene. Loss of function mutations in GALC result in accumulation of the glycolipid intermediate, galactosylsphingosine (psychosine). Due to the cytotoxicity of psychosine, it has been hypothesized that accumulated psychosine underlie the pathophysiology of GLD. However, the cellular mechanisms of GLD pathophysiology remain unclear. Globoid cells, multinucleated microglia/macrophages in the central nervous system (CNS), are a defining characteristic of GLD. Here we report that exposure of primary glial cultures to psychosine induces the expression and the production of matrix metalloproteinase (MMP)-3 that mediated a morphological transformation of microglia into a multinucleated globoid cell type. Additionally, psychosine-induced globoid cell formation from microglia was prevented by either genetic ablation or chemical inhibition of MMP-3. These effects are microglia-specific as peripheral macrophages exposed to psychosine did not become activated or express increased levels of MMP-3. In the brain from twitcher mice, a murine model of human GLD, elevated MMP-3 expression relative to wild-type littermates was contemporaneous with disease onset and further increased with disease progression. Further, bone marrow transplantation (BMT), currently the only therapeutically beneficial treatment for GLD, did not mitigate the elevated expression of MMP-3 in twitcher mice. Hence, elevated expression of MMP-3 in GLD may promote microglial responses to psychosine that may represent an important pathophysiological process in this disease and its treatment.

Psychosine, the cytotoxic sphingolipid that accumulates in globoid cell leukodystrophy, alters membrane architecture

Globoid cell leukodystrophy (GLD) is a neurological disease caused by deficiency of the lysosomal enzyme galactosylceramidase (GALC). In the absence of GALC, the cytotoxic glycosphingolipid, psychosine (psy), accumulates in the nervous system. Psychosine accumulation preferentially affects oligodendrocytes, leading to progressive demyelination and infiltration of activated monocytes/macrophages into the CNS. GLD is characterized by motor defects, cognitive deficits, seizures, and death by 2–5 years of age. It has been hypothesized that psychosine accumulation, primarily within lipid rafts, results in the pathogenic cascade in GLD. However, the mechanism of psychosine toxicity has yet to be elucidated. Therefore, we synthesized the enantiomer of psychosine (ent-psy) to use as a probe to distinguish between protein-psy (stereo-specific enantioselective) or membrane-psy (stereo-insensitive nonenantioselective) interactions. The enantiomer of psychosine has equal or greater toxicity compared with psy, suggesting that psy exerts its toxicity through a nonenantioselective mechanism. Finally, in this study we demonstrate that psy and ent-psy localize to lipid rafts, perturb natural and artificial membrane integrity, and inhibit protein Kinase C translocation to the plasma membrane. Although other mechanisms may play a role in disease, these data strongly suggest that psy exerts its effects primarily through membrane perturbation rather than through specific protein-psy interactions.

Crosstalk in human brain between globoid cell leucodystrophy and zinc-alpha-2-glycoprotein (ZAG), a biomarker of lipid catabolism

Zinc-alpha-2-glycoprotein (ZAG) is a protein identified as a lipid-mobilizing factor participating in a lipid catabolism. In spite of intensive studies conducted during last five decades, the role of this protein in processes of neurodegeneration remains unclear. The aim of our study was to examine the presence of ZAG protein in the brain of patients with Krabbe's disease, which is considered as a psychosine lipidosis caused by a mutation of a known gene. We found intracellular and extracellular localization of ZAG in the brain of Krabbe's disease patients but in the brain of control age-matched patients, ZAG was not detected. Distribution of ZAG in the brain suggests that the influx of ZAG into the brain involved a blood-brain barrier mechanism and adenoreceptors localized on astrocytes and some neurons.

A high-throughput screening assay using Krabbe disease patient cells

Globoid cell leukodystrophy (GLD) or Krabbe disease is a lysosomal disease caused by β-galactocerebrosidase (GALC) deficiency resulting in a rapidly progressive neurodegenerative disorder. Unfortunately, the only available treatment is hematopoietic bone marrow transplantation, which prevents its fulminant manifestation but without treating further neurological manifestations. Here, we describe the development of a cellular high-throughput screening (HTS) assay using GLD patient fibroblasts to screen for small molecules that enhance the residual mutant GALC enzymatic activity. Small molecules have substantial therapeutic potential in GLD because they are more prone to cross the blood–brain barrier, reaching the neuronal affected cells. The transformation of primary skin fibroblasts with SV40 large T antigen has been shown to maintain the biochemical characteristics of the GLD cells and generates sufficient cells for the HTS. Using a specific fluorescent substrate, residual GALC activity from an SV40-transformed GLD patient fibroblast was measurable in high-density microplates. The pilot quantitative HTS against a small compound collection showed robust statistics. The small molecules that showed active concentration–response curves were further studied in primary GLD fibroblasts. This cell-based HTS assay demonstrates the feasibility of employing live GLD patient cells to identify therapeutic agents that can potentially be used for the treatment of this progressive neurodegenerative disease.

Extended Normal Life After AAVrh10-mediated Gene Therapy in the Mouse Model of Krabbe Disease

Globoid cell leukodystrophy (GLD) or Krabbe disease is a neurodegenerative disorder caused by the deficiency of the lysosomal enzyme galactocerebrosidase (GALC). This deficiency results in accumulation of certain galactolipids including psychosine which is cytotoxic for myelin-producing cells. Treatment of human patients at this time is limited to hematopoietic stem cell transplantation (HSCT) that appears to slow the progression of the disease when performed in presymptomatic patients. In this study, adeno-associated virus (AAV) serotype rh10-(AAVrh10) expressing mouse GALC was used in treating twitcher (twi) mice, the mouse model of GLD. The combination of intracerebroventricular, intracerebellar, and intravenous (iv) injection of viral particles in neonate twi mice resulted in high GALC activity in brain and cerebellum and moderate to high GALC activity in spinal cord, sciatic nerve, and some peripheral organs. Successfully treated mice maintained their weight with no or very little twitching, living up to 8 months. The physical activities of the long-lived treated mice were comparable to wild type for most of their lives. Treated mice showed normal abilities to mate, to deliver pups, to nurse and to care for the newborns. This strategy alone or in combination with other therapeutic options may be applicable to treatment of human patients.

Multi-Institutional Assessments of Transplantation for Metabolic Disorders

INTRODUCTION more expediently, potentially increasing availability The rational for allogeneic transplantation as therapy for inherited metabolic disease was provided by the fundamentally important work done by Neufeld’s group over 40 years ago, confirming that normal cells could “cross-correct” cells with storage diseases such as Hurler syndrome. Initial reports in the early 1980s confirmed that these patients could be transplanted, resulting in improvement in visceral disease in patients with mucopolysaccharide accumulation. Later investigations suggested that transplantation arrested neurologic deterioration in some disorders, such as Hurler syndrome (mucopolysaccharidosis type IH [MPS IH]), but is less effective in achieving stabilization of cognitive function in MPS II (Hunter) and MPS III (Sanfilippo). It is thought that the engraftment of donor-derived microglia results in the delivery of enzyme in the central nervous system, but the reasons transplantation is more successful for MPS IH than MPS II or MPS III remain elusive. Transplantation was also tested as therapy for lysosomal leukodystrophies, including Krabbe (globoid cell leukodystrophy, or GLD) and metachromatic leukodystrophy (MLD). ALthough isolated reports of success in disease stabilization for GLD and MLD exist, due to heterogeneity of disease phenotypes and degree of disease advancement at the time of transplantation, analysis of these outcomes are difficult, and there are no large multicenter reports of outcomes. Reports of success in the use of transplantation for adrenoleukodystrophy (ALD) were first published in the 1990s. Because ALD is a peroxisomal disorder, the biology by which transplantation exerts its beneficial effects is distinct from that of GLD and MLD. Other rare inherited metabolic diseases, including osteopetrosis, mannosidosis, MaroteauxLamy syndrome (MPS VI), and Wolman disease, for instance, have also been reported to respond to transplantation. New developments have affected approaches to transplantation for these disorders. The availability of enzyme replacement therapy (ERT) has provided the opportunity to explore combination therapy in disorders such as Hurler syndrome, whereas in other diseases, such as MaroteauxLamy syndrome, ERT has essentially replaced transplantation as standard therapy. In addition, cord blood (CB) transplantation has allowed patients to move to transplant