Search

Objective: To provide updated evidence and consensus-based recommendations for the classification of individuals who screen positive for Krabbe Disease (KD) and recommendations for long-term follow-up for those who are at risk for late onset Krabbe Disease (LOKD)., Methods: KD experts (KD NBS Council) met between July 2017 and June 2020 to develop consensus-based classification and follow-up recommendations. The resulting newly proposed recommendations were assessed in a historical cohort of 47 newborns from New York State who were originally classified at moderate or high risk for LOKD., Results: Infants identified by newborn screening with possible KD should enter one of three clinical follow-up pathways (Early infantile KD, at-risk for LOKD, or unaffected), based on galactocerebrosidase (GALC) activity, psychosine concentration, and GALC genotype. Patients considered at-risk for LOKD based on low GALC activity and an intermediate psychosine concentration are further split into a high-risk or low-risk follow-up pathway based on genotype. Review of the historical New York State cohort found that the updated follow-up recommendations would reduce follow up testing by 88%., Conclusion: The KD NBS Council has presented updated consensus recommendations for efficient and effective classification and follow-up of NBS positive patients with a focus on long-term follow-up of those at-risk for LOKD., Competing Interests: Declaration of competing interest Robert Thompson Stone, MD: None. Margie A. Ream, MD: A member of the Evidence Review Group for the Advisory Committee on Heritable Disorders in Newborns and Children. The views expressed herein are solely those of the authors and do not necessarily reflect the views of the Advisory Committee on Heritable Disorders in Newborns and Children, or the members of the Evidence Review Group. Michael Gelb, PhD: Consultant for PerkinElmer Inc. Dietrich Matern, MD, PhD: None. Joseph J. Orsini, PhD: None. Paul A. Levy, MD: None. Jennifer P. Rubin, MD: None. David A. Wenger, PhD: None. Barbara K. Burton, MD: Has received consulting fees and/or honoraria from Biomarin, Shire (Takeda), Sanofi Genzyme, Horizon, Alexion, Moderna, Denali, JCR Pharma, Aeglea, Inventiva and Ultragenyx. She has conducted clinical trials funded by Biomarin, Shire (Takeda), Ultragenyx, Sangamo and Homology Medicines. Maria L. Escolar, MD: Advisory Boards Orphazyme, Shire/Takeda. Consulting Fees, Sanofi, AvroBio, JCR. Contracted Research Abeona, Prevail, Denali, Shire/Takeda, Regenxbio. Salary/Ownership Interest greater than 5%, Forge Biologics, Chief Medical Officer. Speaker's Bureau and Travel Expenses Shire/Takeda, Sanofi. Joanne Kurtzberg, MD: None., (Copyright © 2021 The Authors. Published by Elsevier Inc. All rights reserved.)

Krabbe disease is an autosomal recessive leukodystrophy caused by pathogenic variants in the galactocerebrosidase (GALC) gene. GALC activity is needed for the lysosomal hydrolysis of galactosylceramide, an important component of myelin. While most patients are infants, older patients are also diagnosed. Starting in 1970, a diagnosis could be made by measuring GALC activity in leukocytes and cultured cells. After the purification of GALC in 1993, the cDNA and genes were cloned. Over 260 disease-causing variants as well as activity lowering benign variants have been identified. While some pathogenic variants can be considered "severe," others can be considered "mild." The combination of alleles determines the type of Krabbe disease a person will have. To identify patients earlier, newborn screening (NBS) has been implemented in several states. Low GALC activity in this screening test may indicate a diagnosis of Krabbe disease. Second tier testing as well as neuro-diagnostic studies may be required to identify those individuals needing immediate treatment. Treatment of pre-symptomatic or mildly symptomatic patients at this time is limited to hematopoietic stem cell transplantation. Treatment studies using the mouse and dog models have shown that combining bone marrow transplantation with intra-venous gene therapy provides the best outcomes in terms of survival, behavior, and preservation of normal myelination in the central and peripheral nervous systems. With earlier diagnosis of patients through newborn screening and advances in treatment, it is hoped that more patients will have a much better quality of life.

Introduction: Krabbe disease (KD) is an autosomal recessive disorder caused by mutations in the galactocerebrosidase (GALC) gene resulting in neuro-inflammation and defective myelination in the central and peripheral nervous systems. Most infantile patients present with clinical features before six months of age and die before two years of age. The only treatment available for pre-symptomatic or mildly affected individuals is hematopoietic stem cell transplantation (HSCT). In the animal models, combining bone marrow transplantation (BMT) with gene therapy has shown the best results in disease outcome. In this study, we examine the outcome of gene therapy alone. Methods: Twitcher (twi) mice used in the study, have a W339X mutation in the GALC gene. Genotype identification of the mice was performed shortly after birth or post-natal day 1 (PND1), using polymerase chain reaction on the toe clips followed by restriction enzyme digestion and electrophoresis. Eight or nine-day-old affected mice were used for gene therapy treatment alone or combined with BMT. While iv injection of 4 × 10 13 gc/kg of body weight of viral vector was used originally, different viral titers were also used without BMT to evaluate their outcomes. Results: When the standard viral dose was increased four- and ten-fold (4X and 10X) without BMT, the lifespans were increased significantly. Without BMT the affected mice were fertile, had the same weight and appearance as wild type mice and had normal strength and gait. The brains showed no staining for CD68, a marker for activated microglia/macrophages, and less astrogliosis than untreated twi mice. Conclusion: Our results demonstrate that, it may be possible to treat human KD patients with high dose AAVrh10 without blood stem cell transplantation which would eliminate the side effects of HSCT., (© 2021 The Author(s).)

A transplanted stem cell's engagement with a pathologic niche is the first step in its restoring homeostasis to that site. Inflammatory chemokines are constitutively produced in such a niche; their binding to receptors on the stem cell helps direct that cell's "pathotropism." Neural stem cells (NSCs), which express CXCR4, migrate to sites of CNS injury or degeneration in part because astrocytes and vasculature produce the inflammatory chemokine CXCL12. Binding of CXCL12 to CXCR4 (a G protein-coupled receptor, GPCR) triggers repair processes within the NSC. Although a tool directing NSCs to where needed has been long-sought, one would not inject this chemokine in vivo because undesirable inflammation also follows CXCL12-CXCR4 coupling. Alternatively, we chemically "mutated" CXCL12, creating a CXCR4 agonist that contained a strong pure binding motif linked to a signaling motif devoid of sequences responsible for synthetic functions. This synthetic dual-moity CXCR4 agonist not only elicited more extensive and persistent human NSC migration and distribution than did native CXCL 12, but induced no host inflammation (or other adverse effects); rather, there was predominantly reparative gene expression. When co-administered with transplanted human induced pluripotent stem cell-derived hNSCs in a mouse model of a prototypical neurodegenerative disease, the agonist enhanced migration, dissemination, and integration of donor-derived cells into the diseased cerebral cortex (including as electrophysiologically-active cortical neurons) where their secreted cross-corrective enzyme mediated a therapeutic impact unachieved by cells alone. Such a "designer" cytokine receptor-agonist peptide illustrates that treatments can be controlled and optimized by exploiting fundamental stem cell properties (e.g., "inflammo-attraction")., Competing Interests: Competing interest statement: J.R.S. was one of 17 coauthors with R.G. and E.Y.S. on a 2016 research article. He did not collaborate directly with them., (Copyright © 2020 the Author(s). Published by PNAS.)

Objectives: Nonimmune hydrops fetalis (NIHF) accounts for 90% of hydrops fetalis cases. About 15% to 29% of unexplained NIHF cases are caused by lysosomal storage diseases (LSD). We review the spectrum of LSD and associated clinical findings in NIHF in a cohort of patients referred to our institution., Methods: We present a retrospective case-control study of cases with NIHF referred for LSD biochemical testing at a single center. Cases diagnosed with LSD were matched to controls with NIHF and negative LSD testing and analyzed according to the STROBE criteria to the extent the retrospective nature of this study allowed., Results: Between January 2006 and December 2018, 28 patients with NIHF were diagnosed with a LSD. Eight types of LSD were diagnosed: galactosialidosis 8/28 (28.6%), sialic acid storage disease (SASD) 5/28 (17.9%), mucopolysaccharidosis VII 5/28 (17.9%), Gaucher 4/28 (14.3%), sialidosis 2/28 (7.1%), GM1 gangliosidosis 2/28 (7.1%), Niemann-Pick disease type C 1/28 (3.6%), and mucolipidosis II/III 1/28 (3.6%). Associated clinical features were hepatomegaly 16/21 (76.2%) vs 22/65 (33.8%), P < .05, splenomegaly 12/20 (60.0%) vs 14/58 (24.1%), P < .05, and hepatosplenomegaly 10/20 (50.0%) vs 13/58 (22.4%) P < .05., Conclusion: The most common LSD in NIHF were galactosialidosis, SASD, mucopolysaccharidosis VII, and Gaucher disease. LSD should be considered in unexplained NIHF cases, particularly if hepatomegaly, splenomegaly, or hepatosplenomegaly is visualized on prenatal ultrasound., (© 2020 John Wiley & Sons, Ltd.)

Lysosomal disorders are diseases that involve mutations in genes responsible for the coding of lysosomal enzymes, transport proteins, activator proteins and protein processing enzymes. These defects lead to the storage of specific metabolites within lysosomes resulting in a great variety of clinical features depending on the tissues with the storage, the storage products and the extent of the storage. The methods for rapidly diagnosing patients started in the late 1960's when the enzyme defects were identified eliminating the need for tissue biopsies. The first requests for diagnostic help in this laboratory came in 1973. In that year, patients with Krabbe disease and Niemann-Pick type A were diagnosed. Since that time samples from about 62 000 individuals have been received for diagnostic studies, and 4900 diagnoses have been made. The largest number of diagnosed individuals had metachromatic leukodystrophy and Krabbe disease because of our research interest in leukodystrophies. A number of new disorders were identified and the primary defects in other disorders were clarified. With new methods for diagnosis, including newborn screening, molecular analysis, microarrays, there is still a need for biochemical confirmation before treatment is considered. With new treatments, including gene therapy, stem cell transplantation, enzyme replacement used alone or in combination becoming more available, the need for rapid, accurate diagnosis is critical., Competing Interests: The authors declare no potential conflict of interest., (© 2020 The Authors. Journal of Inherited Metabolic Disease published by John Wiley & Sons Ltd on behalf of SSIEM.)

Saposin A is a post-translation product of the prosaposin (PSAP) gene that serves as an activator protein of the galactocerebrosidase (GALC) enzyme, and is necessary for the degradation of certain glycosphingolipids. Deficiency of saposin A leads to a clinical picture identical to that of early-infantile Krabbe disease caused by GALC enzyme deficiency. Galactosylsphingosine, also known as psychosine, is a substrate of the GALC enzyme that is known to be elevated in classic Krabbe disease. We present the case of an 18-month-old male with clinical and radiological findings concerning for Krabbe disease who had preserved GALC enzyme activity and negative GALC gene sequencing, but was found to have a homozygous variant, c.257 T > A (p.I86N), in the saposin A peptide of PSAP. Psychosine determination on dried blood spot at 18 months of age was elevated to 12 nmol/L (normal <3 nmol/L). We present this case to add to the literature on the rare diagnosis of atypical Krabbe disease due to saposin A deficiency, to report a novel presumed pathogenic variant within PSAP, and to suggest that individuals with saposin A deficiency may have elevated levels of psychosine, similar to children with classic Krabbe disease due to GALC deficiency., (Copyright © 2019 Elsevier Inc. All rights reserved.)

Introduction: Krabbe disease (KD) is an autosomal recessive lysosomal disorder caused by mutations in the galactocerebrosidase (GALC) gene. This results in defective myelination in the peripheral and central nervous systems due to low GALC activity. Treatment at this time is limited to hematopoietic stem cell transplantation (HSCT) in pre-symptomatic individuals. While this treatment extends the lives of treated individuals, most have difficulty walking by the end of the first decade due to peripheral neuropathy. Studies in the murine model of KD, twitcher (twi) combining bone marrow transplantation (BMT) with AAVrh10-mGALC showed a great extension of life from 40 days to about 400 days, with some living a full life time. Methods: In order to find the optimum conditions for dosing and timing of this combined treatment, twi mice were injected with five doses of AAVrh10-mGALC at different times after BMT. Survival, as well as GALC expression were monitored along with studies of sciatic nerve myelination and possible liver pathology. Results: Dosing had a pronounced effect on survival and measured GALC activity. There was window of time after BMT to inject the viral vector and see similar results, however delaying both the BMT and the viral injection shortened the lifespans of the treated mice. Lowering the viral dose too much decreased the correction of the sciatic nerve myelination. There was no evidence for hepatic neoplasia. Conclusion: These studies provide the conditions optimum for successfully treating the murine model of KD. There is some flexibility in dosing and timing to obtain a satisfactory outcome. These studies are critical to the planning of a human trial combining the "standard of care", HSCT, with a single iv injection of AAVrh10-GALC., (© 2020 The Author(s).)

Background: Deficiency in the enzyme β-mannosidase was described over three decades ago. Although rare in occurrence, the presentation of childhood-onset β-mannosidase deficiency consists of hypotonia in the newborn period followed by global development delay, behavior problems, and intellectual disability. No effective pharmacologic treatments have been available., Methods: We report 2-year outcomes following the first umbilical cord blood transplant in a 4-year-old boy with early childhood-onset disease., Results: We show restoration of leukocyte β-mannosidase activity which remained normal at 2 years posttransplant, and a simultaneous increase in plasma β-mannosidase activity and dramatic decrease in urine-free oligosaccharides were also observed. MRI of the brain remained stable. Neurocognitive evaluation revealed test point gains, although the magnitude of improvement was less than expected for age, causing lower IQ scores that represent a wider developmental gap between the patient and unaffected peers., Conclusion: Our findings suggest that hematopoietic cell transplant can correct the biochemical defect in β-mannosidosis, although preservation of the neurocognitive trajectory may be a challenge., (© 2019 The Authors. Molecular Genetics & Genomic Medicine published by Wiley Periodicals, Inc.)

Metachromatic leukodystrophy (MLD) is an autosomal recessive lysosomal storage disease mainly caused by a deficiency of arylsulfatase A activity. The typical clinical course of patients with the late infantile form includes a regression in motor skills with progression to dysphagia, seizures, hypotonia and death. We present a case of a 4-year-old female with rapidly progressive developmental regression with loss of motor milestones, spasticity and dysphagia. MRI showed volume loss and markedly abnormal deep white matter. Enzymatic testing in one laboratory showed arylsulfatase A activity in their normal range. However, extraction of urine showed a large increase in sulfatide excretion in a second laboratory. Measurement of arylsulfatase A in that laboratory showed a partial decrease in arylsulfatase A activity measured under typical conditions (about 37% of the normal mean). When the concentration of substrate in the assay was lowered to one quarter of that normally used, this individual had activity <10% of controls. The patient was found to be homozygous for an unusual missense mutation in the arylsulfatase A gene confirming the diagnosis of MLD. This case illustrates the importance of careful biochemical and molecular testing for MLD if there is suspicion of this diagnosis.

Background: Krabbe disease is a rare neurological disorder caused by a deficiency in the lysosomal enzyme, β-galactocerebrosidase, resulting in demyelination of the central and peripheral nervous systems. If left without treatment, Krabbe disease results in progressive neurodegeneration with reduced quality of life and early death. The purpose of this prospective study was to describe the natural progression of early onset Krabbe disease in a large cohort of patients., Methods: Patients with early onset Krabbe disease were prospectively evaluated between 1999 and 2018. Data sources included diagnostic testing, parent questionnaires, standardized multidisciplinary neurodevelopmental assessments, and neuroradiological and neurophysiological tests., Results: We evaluated 88 children with onset between 0 and 5 months. Median age of symptom onset was 4 months; median time to diagnosis after onset was 3 months. The most common initial symptoms were irritability, feeding difficulties, appendicular spasticity, and developmental delay. Other prevalent symptoms included axial hypotonia, abnormal deep tendon reflexes, constipation, abnormal pupillary response, scoliosis, loss of head control, and dysautonomia. Results of nerve conduction studies showed that 100% of patients developed peripheral neuropathy by 6 months of age. Median galactocerebrosidase enzyme activity was 0.05 nmol/h/mg protein. The median survival was 2 years., Conclusions: This is the largest prospective natural history study of Krabbe disease. It provides a comprehensive description of the disease during the first 2 years of life. With recent inclusion of state mandated newborn screening programs and promising therapeutic interventions, enhancing our understanding of disease progression in early onset Krabbe disease will be critical for developing treatments, designing clinical trials, and evaluating outcomes.

Globoid cell leukodystrophy (GLD), or Krabbe disease, is an inherited, neurologic disorder that results from deficiency of a lysosomal enzyme, galactosylceramidase. Most commonly, deficits of galactosylceramidase result in widespread central and peripheral nervous system demyelination and death in affected infants typically by 2 years of age. Hematopoietic stem-cell transplantation is the current standard of care in children diagnosed prior to symptom onset. However, disease correction is incomplete. Herein, the first adeno-associated virus (AAV) gene therapy experiments are presented in a naturally occurring canine model of GLD that closely recapitulates the clinical disease progression, neuropathological alterations, and biochemical abnormalities observed in human patients. Adapted from studies in twitcher mice, GLD dogs were treated by combination intravenous and intracerebroventricular injections of AAVrh10 to target both the peripheral and central nervous systems. Combination of intravenous and intracerebroventricular AAV gene therapy had a clear dose response and resulted in delayed onset of clinical signs, extended life-span, correction of biochemical defects, and attenuation of neuropathology. For the first time, therapeutic effect has been established in the canine model of GLD by targeting both peripheral and central nervous system impairments with potential clinical implications for GLD patients.

Background: Krabbe disease is a rare neurodegenerative genetic disorder caused by deficiency of galactocerebrosidase. Patients with the infantile form of Krabbe disease can be treated at a presymptomatic stage with human stem cell transplantation which improves survival and clinical outcomes. However, without a family history, most cases of infantile Krabbe disease present after onset of symptoms and are ineligible for transplantation. In 2006, New York began screening newborns for Krabbe disease to identify presymptomatic cases. To ensure that those identified with infantile disease received timely treatment, New York public health and medical systems took steps to accurately diagnose and rapidly refer infants for human stem cell transplantation within the first few weeks of life. After 11 years of active screening in New York and the introduction of Krabbe disease newborn screening in other states, new information has been gained which can inform the design of newborn screening programs to improve infantile Krabbe disease outcomes., Findings: Recent information relevant to Krabbe disease screening, diagnosis, and treatment were assessed by a diverse group of public health, medical, and advocacy professionals. Outcomes after newborn screening may improve if treatment for infantile disease is initiated before 30 days of life. Newer laboratory screening and diagnostic tools can improve the speed and specificity of diagnosis and help facilitate this early referral. Given the rarity of Krabbe disease, most recommendations were based on case series or expert opinion., Conclusion: This report updates recommendations for Krabbe disease newborn screening to improve the timeliness of diagnosis and treatment of infantile Krabbe disease. In the United States, several states have begun or are considering Krabbe disease newborn screening. These recommendations can guide public health laboratories on methodologies for screening and inform clinicians about the need to promptly diagnose and treat infantile Krabbe disease. The timing of the initial referral after newborn screening, the speed of diagnostic confirmation of infantile disease, and the transplantation center's experience and ability to rapidly respond to a suspected patient with newly diagnosed infantile Krabbe disease are critical for optimal outcomes.

Consistent with a tentative diagnosis of neuronal ceroid lipofuscinosis (NCL), autofluorescent cytoplasmic storage bodies were found in neurons from the brains of 2 related Shiba Inu dogs with a young-adult onset, progressive neurodegenerative disease. Unexpectedly, no potentially causal NCL-related variants were identified in a whole-genome sequence generated with DNA from 1 of the affected dogs. Instead, the whole-genome sequence contained a homozygous 3 base pair (bp) deletion in a coding region of HEXB. The other affected dog also was homozygous for this 3-bp deletion. Mutations in the human HEXB ortholog cause Sandhoff disease, a type of GM2 gangliosidosis. Thin-layer chromatography confirmed that GM2 ganglioside had accumulated in an affected Shiba Inu brain. Enzymatic analysis confirmed that the GM2 gangliosidosis resulted from a deficiency in the HEXB encoded protein and not from a deficiency in products from HEXA or GM2A, which are known alternative causes of GM2 gangliosidosis. We conclude that the homozygous 3-bp deletion in HEXB is the likely cause of the Shiba Inu neurodegenerative disease and that whole-genome sequencing can lead to the early identification of potentially disease-causing DNA variants thereby refocusing subsequent diagnostic analyses toward confirming or refuting candidate variant causality., (Copyright © 2017 The Authors. Journal of Veterinary Internal Medicine published by Wiley Periodicals, Inc. on behalf of the American College of Veterinary Internal Medicine.)

Background: Early infantile Krabbe disease is rapidly fatal, but hematopoietic stem cell transplantation (HSCT) may improve outcomes if performed soon after birth. New York State began screening all newborns for Krabbe disease in 2006., Methods: Infants with abnormal newborn screen results for Krabbe disease were referred to specialty-care centers. Newborns found to be at high risk for Krabbe disease underwent a neurodiagnostic battery to determine the need for emergent HSCT., Results: Almost 2 million infants were screened. Five infants were diagnosed with early infantile Krabbe disease. Three died, two from HSCT-related complications and one from untreated disease. Two children who received HSCT have moderate to severe developmental delays. Forty-six currently asymptomatic children are considered to be at moderate or high risk for development of later-onset Krabbe disease., Conclusions: These results show significant HSCT-associated morbidity and mortality in early infantile Krabbe disease and raise questions about its efficacy when performed in newborns diagnosed through newborn screening. The unanticipated identification of "at risk" children introduces unique ethical and medicolegal issues. New York's experience raises questions about the risks, benefits, and practicality of screening newborns for Krabbe disease. It is imperative that objective assessments be made on an ongoing basis as additional states begin screening for this disorder.Genet Med 18 12, 1235-1243.

Newborn screening (NBS) for Krabbe's disease (KD) has been instituted in several states, and New York State has had the longest experience. After an initial screening of dried blood spots, samples from individuals with galactocerebrosidase (GALC) values below a given cutoff level were subjected to additional testing, including sequencing of the GALC gene. This resulted in the identification of mutations that had previously been found in confirmed KD patients and of variants that had never previously been reported. Some individuals had variants considered to be polymorphisms, alone or on the same allele as another mutation. To help with counseling of families on the risk for a newborn to develop KD, expression studies were conducted with these variants identified by NBS. GALC activity was measured in COS1 cells for 140 constructs and compared with mutations that had previously been seen in confirmed cases of KD. When a polymorphism was present on the same allele as the variant, expressed activity was measured with and without the polymorphism. In some cases the presence of the polymorphism greatly lowered the measured GALC activity, possibly making it disease causing. Although it is not possible to predict conclusively whether a variant is severe and will result in infantile KD if two such variants are present or whether a variant is mild and will result in late-onset disease, some variants clearly are not disease causing. This is the largest expression study of GALC variants/mutations found in NBS and confirmed KD cases. This work will be helpful for counseling families of screen-positive newborns found to have low GALC activity. © 2016 Wiley Periodicals, Inc., (© 2016 Wiley Periodicals, Inc.)

This Review summarizes the progress in understanding the pathogenesis and treatment of Krabbe disease from the description of five patients in by Knud Krabbe until 2016. To determine the cause of this genetic disease, pathological and chemical analyses of tissues from the nervous systems of patients were performed. It was determined that these patients had a pathological feature known as globoid cell in the brain and that this consisted partially of galactosylceramide, a major sphingolipid component of myelin. The finding that these patients had a deficiency of galactocerebrosidase (GALC) activity opened the way to relatively simple diagnostic testing with easily obtainable tissue samples, studies leading to the purification of GALC, and cloning of the GALC cDNA and gene. The availability of the gene sequence led to the identification of mutations in patients and to the current studies involving the use of viral vectors containing the GALC cDNA to treat experimentally naturally occurring animal models, such as twitcher mice. Currently, treatment of presymptomatic human patients is limited to hematopoietic stem cell transplantation (HSCT). With recent studies showing successful treatment of animal models with a combination of HSCT and viral gene therapy, it is hoped that more effective treatments will soon be available for human patients. For this Review, it is not possible to reference all of the articles contributing to our current state of knowledge about this disease; however, we have chosen those that have influenced our studies by suggesting research paths to pursue. © 2016 Wiley Periodicals, Inc., (© 2016 Wiley Periodicals, Inc.)

Background: Multiple sulfatase deficiency is an autosomal recessive lysosomal storage disorder characterized by the absence of several sulfatases and resulting from mutations in the gene encoding the human C (alpha)-formylglycine-generating enzyme. There have been a variety of biochemical and clinical presentations reported in this disorder., Patient Description: We present a 4-year-old girl with clinical findings of microcephaly, spondylolisthesis and neurological regression without ichthyosis, coarse facies, and organomegaly., Results: The child's magnetic resonance imaging demonstrated confluent white matter abnormalities involving the periventricular and deep cerebral white matter with the U-fibers relatively spared. Biochemical testing showing low arylsulfatase A levels were initially thought to be consistent with a diagnosis of metachromatic leukodystrophy. The diagnosis of multiple sulfatase deficiency was pursued when genetic testing for metachromatic leukodystrophy was negative., Conclusion: This child illustrates the clinical heterogeneity of multiple sulfatase deficiency and that this disorder can occur without the classic clinical features., (Copyright © 2016 Elsevier Inc. All rights reserved.)

Purpose: Krabbe disease (KD) results from galactocerebrosidase (GALC) deficiency. Infantile KD symptoms include irritability, progressive stiffness, developmental delay, and death. The only potential treatment is hematopoietic stem cell transplantation. New York State (NYS) implemented newborn screening for KD in 2006., Methods: Dried blood spots from newborns were assayed for GALC enzyme activity using mass spectrometry, followed by molecular analysis for those with low activity (≤12% of the daily mean). Infants with low enzyme activity and one or more mutations were referred for follow-up diagnostic testing and neurological examination., Results: Of >1.9 million screened, 620 infants were subjected to molecular analysis and 348 were referred for diagnostic testing. Five had enzyme activities and mutations consistent with infantile KD and manifested clinical/neurodiagnostic abnormalities. Four underwent transplantation, two are surviving with moderate to severe handicaps, and two died from transplant-related complications. The significance of many sequence variants identified is unknown. Forty-six asymptomatic infants were found to be at moderate to high risk for disease., Conclusions: The positive predictive value of KD screening in NYS is 1.4% (5/346) considering confirmed infantile cases. The incidence of infantile KD in NYS is approximately 1 in 394,000, but it may be higher for later-onset forms.

Krabbe disease is an autosomal recessive disorder resulting from defects in the lysosomal enzyme galactocerebrosidase (GALC). GALC deficiency leads to severe neurological features. The only treatment for presymptomatic infantile patients and later-onset patients is hematopoietic stem cell transplantation (HSCT). This treatment is less than ideal with most patients eventually developing problems with gait and expressive language. Several naturally occurring animal models are available, including twitcher (twi) mice, which have been used for many treatment trials. Previous studies demonstrated that multiple injections of AAVrh10-GALC into the central nervous system (CNS) of neonatal twi mice resulted in significant improvements. Recently we showed that one i.v. injection of AAVrh10-GALC on PND10 resulted in normal GALC activity in the CNS and high activity in the peripheral nervous system (PNS). In the present study, a single i.v. injection of AAVrh10-GALC was given 1 day after bone marrow transplantation (BMT) on PND10. The mice show greatly extended lifespan and normal behavior with improved CNS and PNS findings. Since HSCT is the standard of care in human patients, adding this single i.v. injection of viral vector may greatly improve the treatment outcome.

An inherited deficiency of β-galactosylceramidase (GALC) causes the lysosomal storage disease globoid cell leukodystrophy (GLD). The disease is characterized by the accumulation of the cytotoxic metabolite psychosine (galactosylsphingosine), causing rapid degeneration of myelinating cells. Most patients suffer from the infantile form of GLD with onset of disease between 3 and 6 months after birth and death by 2 years of age. The most widely used animal model of GLD, the twitcher mouse, presents with an even more rapid course of disease and death around 40 days of age. We have generated a novel "humanized" mouse model of GLD by inserting a human GALC cDNA containing an adult-onset patient mutation into the murine GALC gene. Humanized GALC mice exhibit pathological hallmarks of GLD including psychosine accumulation, neuroinflammation, CNS infiltration of macrophages, astrogliosis and demyelination. Residual GALC activities in mouse tissues are low and the mice display a median lifespan of 46 days. Due to the expression of the human transgene, the mice do not develop an immune response against rhGALC, rendering the animal model suitable for therapies based on human enzyme. Intravenously injected rhGALC was able to surmount the blood-brain barrier and was targeted to lysosomes of brain macrophages, astrocytes and neurons. High-dose enzyme replacement therapy started at postnatal day 21 reduced the elevated psychosine levels in the peripheral and central nervous system by 14-16%, but did not ameliorate neuroinflammation, demyelination and lifespan. These results may indicate that treatment must be started earlier before pathology occurs., (Copyright © 2015 Elsevier Inc. All rights reserved.)

Background Aims: Cord blood (CB) transplantation slows neurodegeneration during certain inherited metabolic diseases. However, the number of donor cells in the brain of patients does not appear to be sufficient to provide benefit until several months after transplant. We developed the cell product DUOC-01 to provide therapeutic effects in the early post-transplant period., Methods: DUOC-01 cultures initiated from banked CB units were characterized by use of time-lapse photomicroscopy during the 21-day manufacturing process. Antigen expression was measured by means of flow cytometry and immunocytochemistry; transcripts for cytokines and enzymes by quantitative real-time polymerase chain reaction; activities of lysosomal enzymes by direct biochemical analysis; alloreactivity of DUOC-01 and of peripheral blood (PB) mononuclear cells (MNC) to DUOC-01 by mixed lymphocyte culture methods; and cytokine secretion by Bioplex assays., Results: DUOC-01 cultures contained highly active, attached, motile, slowly proliferating cells that expressed common (cluster of differentiation [CD]11b, CD14 and Iba1), M1 type (CD16, inducible nitric oxide synthase), and M2-type (CD163, CD206) macrophage or microglia markers. Activities of 11 disease-relevant lysosomal enzymes in DUOC-01 products were similar to those of normal PB cells. All DUOC-01 products secreted interleukin (IL)-6 and IL-10. Accumulation of transforming growth factor-β, IL-1β, interferon-γ and TNF-α in supernatants was variable. IL-12, IL-2, IL-4, IL-5 and IL-13 were not detected at significant concentrations. Galactocerebrosidase, transforming growth factor-β and IL-10 transcripts were specifically enriched in DUOC-01 relative to CB cells. PB MNCs proliferated and released cytokines in response to DUOC-01. DUOC-01 did not proliferate in response to mismatched MNC., Conclusions: DUOC-01 has potential as an adjunctive cell therapy to myeloablative CB transplant for treatment of inherited metabolic diseases., (Copyright © 2015. Published by Elsevier Inc.)

Background Aims: Cord blood (CB) transplantation slows neurodegeneration during certain inherited metabolic diseases. However, the number of donor cells in the brain of patients does not appear to be sufficient to provide benefit until several months after transplant. We developed the cell product DUOC-01 to provide therapeutic effects in the early post-transplant period., Methods: DUOC-01 cultures initiated from banked CB units were characterized by use of time-lapse photomicroscopy during the 21-day manufacturing process. Antigen expression was measured by means of flow cytometry and immunocytochemistry; transcripts for cytokines and enzymes by quantitative real-time polymerase chain reaction; activities of lysosomal enzymes by direct biochemical analysis; alloreactivity of DUOC-01 and of peripheral blood (PB) mononuclear cells (MNC) to DUOC-01 by mixed lymphocyte culture methods; and cytokine secretion by Bioplex assays., Results: DUOC-01 cultures contained highly active, attached, motile, slowly proliferating cells that expressed common (cluster of differentiation [CD]11b, CD14 and Iba1), M1 type (CD16, inducible nitric oxide synthase), and M2-type (CD163, CD206) macrophage or microglia markers. Activities of 11 disease-relevant lysosomal enzymes in DUOC-01 products were similar to those of normal PB cells. All DUOC-01 products secreted interleukin (IL)-6 and IL-10. Accumulation of transforming growth factor-β, IL-1β, interferon-γ and TNF-α in supernatants was variable. IL-12, IL-2, IL-4, IL-5 and IL-13 were not detected at significant concentrations. Galactocerebrosidase, transforming growth factor-β and IL-10 transcripts were specifically enriched in DUOC-01 relative to CB cells. PB MNCs proliferated and released cytokines in response to DUOC-01. DUOC-01 did not proliferate in response to mismatched MNC., Conclusions: DUOC-01 has potential as an adjunctive cell therapy to myeloablative CB transplant for treatment of inherited metabolic diseases., (Copyright © 2015 International Society for Cellular Therapy. Published by Elsevier Inc. All rights reserved.)

Globoid cell leukodystrophy (GLD) or Krabbe disease is an autosomal recessive disorder resulting from the defective lysosomal enzyme galactocerebrosidase (GALC). The lack of GALC enzyme leads to severe neurological symptoms. While most human patients are infants who do not survive beyond 2 years of age, older patients are also diagnosed. In addition to human patients, several naturally occurring animal models, including dog, mouse, and monkey, have also been identified. The mouse model of Krabbe disease, twitcher (twi) mouse has been used for many treatment trials including gene therapy. Using the combination of intracerebroventricular, intracerebellar, and intravenous (iv) injection of the adeno-associated virus serotype rh10 (AAVrh10) expressing mouse GALC in neonate twi mice we previously have demonstrated a significantly extended normal life and exhibition of normal behavior in treated mice. In spite of the prolonged healthy life of these treated mice and improved myelination, it is unlikely that using multiple injection sites for viral administration will be approved for treatment of human patients. In this study, we have explored the outcome of the single iv injection of viral vector at post-natal day 10 (PND10). This has resulted in increased GALC activity in the central nervous system (CNS) and high GALC activity in the peripheral nervous system (PNS). As we have shown previously, an iv injection of AAVrh10 at PND2 results in a small extension of life beyond the typical lifespan of the untreated twi mice (~40 days). In this study, we report that mice receiving a single iv injection at PND10 had no tremor and continued to gain weight until a few weeks before they died. On average, they lived 20-25 days longer than untreated mice. We anticipate that this strategy in combination with other therapeutic options may be beneficial and applicable to treatment of human patients., (Copyright © 2014 Elsevier Inc. All rights reserved.)

Several families of iminosugar-based galactoside mimics were designed, synthesized, and evaluated as galactocerebrosidase (GALC) inhibitors. They were also tested as inhibitors of lysosomal β- and α-galactosidases in order to find new potent and selective pharmacological chaperones for treatment of the lysosomal storage disorder, Krabbe disease. Whereas 1-C-alkyl imino-L-arabinitols are totally inactive toward the three enzymes, 1-C-alkyl imino-D-galactitols were found to be active only toward α-galactosidase A. Finally, 1-N-iminosugars provided the best results, as 4-epi-isofagomine was found to be a good inhibitor of both lysosomal β-galactosidase and GALC. Further elaboration of this structure is required to achieve selectivity between these two galactosidases., (© 2014 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.)

Metachromatic leukodystrophy (MLD) is a lysosomal storage disorder caused mainly by mutations in the arylsulfatase A (ARSA) gene. In this manuscript we report sixteen novel mutations identified in the ARSA gene of fifteen unrelated patients affected with MLD. Of these 16 mutations nine were missense mutations (p.L11Q, p.S44P, p.L81P, p.R84L, p.V177D, p.P284S, p.R288S, p.G301R, p.P425S), three were nonsense mutations (p.Q51X, p.Y149X, p.C156X), three were frame shift mutations (c.28delG, c.105C>A+106&#95;124dup, c.189delC) and one was a splice-site mutation (c.1102-2A>G). In addition, three previously reported mutations were identified on an allelic background different from the one in the original reports. Two mutations, p.G309S and p.E312D, were identified on the background of the so-called pseudodeficiency (Pd) allele while previously they were reported alone. On the other hand, mutation p.R311X was identified in two unrelated patients not in cis with the Pd mutations, as previously reported., (© 2013 Elsevier B.V. All rights reserved.)

A wild caught juvenile male raccoon with neurological disease was humanely destroyed due to poor prognosis. Necropsy examination revealed hepatomegaly, splenomegaly and multicentric lymphadenomegaly with diffuse hepatic pallor and pulmonary consolidation with pinpoint pale subpleural foci. Microscopically, there was marked pale cytoplasmic swelling of the central and peripheral neurons as well as the glial cells in the brain, accompanied by multiorgan infiltration by abundant foamy macrophages. Ultrastructural investigation revealed accumulation of concentrically arranged lamellar material within lysosomes of the affected neurons, macrophages and endothelial cells. Biochemical enzymatic analysis detected sphingomyelinase deficiency and lysosomal storage disease consistent with sphingomyelin lipidosis (Niemann-Pick disease [NPD]) was diagnosed. This is the first report of NPD in a raccoon., (Copyright © 2013 Elsevier Ltd. All rights reserved.)

Background: New York State has screened over 1.2 million newborns for Krabbe disease, and we identified 4 newborns with infantile Krabbe disease. In addition, 6 other newborns were identified with very low galactosylcerebrosidase (GALC) activity. Because these patients remain asymptomatic, we investigated whether psychosine levels could be a useful marker for disease., Methods: HPLC-MS/MS methodology was used to determine the psychosine concentrations in dried blood spots (DBS) collected from the following cohorts: known Krabbe patients, screened babies that were determined to have infantile Krabbe disease, asymptomatic infants with low GALC activity, and normal controls., Results: The psychosine concentrations from the known Krabbe patients ranged from 7 to 50 ng/ml. Newborns identified by screening who were confirmed with infantile Krabbe disease ranged from 23 to 73 ng/ml. Asymptomatic individuals with low GALC activity had concentrations ranging from 1.7 to 5.7 ng/ml. Concentrations in newborns with normal GALC activity were all <3 ng/ml., Conclusions: The psychosine concentrations in DBS from confirmed infantile patients are at least four times higher than the asymptomatic newborns and nearly an order of magnitude greater than normal newborns. Further studies are needed to determine if psychosine can be used as a predictor of disease status/progression in screen positive newborns., (Copyright © 2013 Elsevier B.V. All rights reserved.)

The name of lysosomal storage diseases stems from the fact that in this category of disorders specific undegraded materials are stored in the lysosomes. This is usually caused by a lysosomal enzyme deficiency and leads to a cascade of pathological outcomes. Apart from deficiency of lysosomal enzymes, lysosomal storage diseases also include deficiencies in proteins necessary for enzyme functioning, proteins needed for post-translational modification of these enzymes and proteins required for export of certain compounds from the lysosomes.

GM2 gangliosidosis is a fatal lysosomal storage disease caused by a deficiency of β-hexosaminidase (EC 3.2.1.52). There are two major isoforms of the enzyme: hexosaminidase A composed of an α and a β subunit (encoded by HEXA and HEXB genes, respectively); and, hexosaminidase B composed of two β subunits. Hexosaminidase A requires an activator protein encoded by GM2A to catabolize GM2 ganglioside, but even in the absence of the activator protein, it can hydrolyze the synthetic substrates commonly used to assess enzyme activity. GM2 gangliosidosis has been reported in Japanese Chin dogs, and we identified the disease in two related Japanese Chin dogs based on clinical signs, histopathology and elevated brain GM2 gangliosides. As in previous reports, we found normal or elevated hexosaminidase activity when measured with the synthetic substrates. This suggested that the canine disease is analogous to human AB variant of G(M2) gangliosidosis, which results from mutations in GM2A. However, only common neutral single nucleotide polymorphisms were found upon sequence analysis of the canine ortholog of GM2A from the affected Japanese Chins. When the same DNA samples were used to sequence HEXA, we identified a homozygous HEXA:c967G>A transition which predicts a p.E323K substitution. The glutamyl moiety at 323 is known to make an essential contribution to the active site of hexosaminidase A, and none of the 128 normal Japanese Chins and 92 normal dogs of other breeds that we tested was homozygous for HEXA:c967A. Thus it appears that the HEXA:c967G>A transition is responsible for the GM2 gangliosidosis in Japanese Chins., (Copyright © 2012 Elsevier Inc. All rights reserved.)

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.

Metachromatic leukodystrophy is a rare disorder with great clinical variability. We report the first case of triplets with the late infantile form of the disease and their systematic progression of symptoms. We reviewed the literature and identified all human studies that reported new cases since 1921. We analyzed survival by decade to assess the impact of historical changes in the management of care. Mean age at death and the 5-year survival from onset of symptoms for late infantile, juvenile, and adult phenotypes were 4.2 years and 24.9%, 17.4 years and 70.3%, and 43.1 years and 88.6%, respectively. The 5-year survival of cases reported after 1990 was significantly better than cases reported before 1970 in all subtypes of metachromatic leukodystrophy (late infantile: 52% vs 14%, juvenile: 100% vs 46%, adult: 95% vs 67%). Survival in the late infantile subtype was worse than that in other subtypes. Survival significantly improved over time in all subtypes.

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.

Krabbe disease or globoid cell leukodystrophy is an autosomal recessive disorder resulting from mutations in the galactocerebrosidase (GALC) gene. These mutations lead to deficient GALC activity, storage of substrates of the enzyme, including psychosine, death to oligodendrocytes, decreased myelination, production of globoid cells and eventually death to the individual. While most affected individuals are infants, late-onset forms are also recognized. In addition to human patients, several animal models have been well characterized, including the twitcher mouse. A spontaneously transformed progenitor cell line was isolated from an astrocyte-enriched fraction of normal mice, partially characterized and transduced with a retrovirus-containing mouse GALC cDNA to produce increased GALC activity (20-30-fold above baseline). These cells, called MAR-52, were injected into the brains of newborn affected twitcher mice. While there was only a modest increase in lifespan and body weight, there was clear evidence for the correction of the astrocytic gliosis, normal appearing oligodendrocytes and evidence for remyelination. We demonstrate that the exogenously supplied neural progenitor cells can donate GALC enzyme to oligodendrocytes in the brains of affected mice resulting in normal myelination in the area of donor cells. At this time, hematopoietic stem cell transplantation provides the best outcome in affected mice and is the only treatment available for human patients, but it does not result in a cure even when performed in asymptomatic newborns. Complete correction probably will require a combined approach to effectively treat patients with Krabbe disease. With developments in the isolation and characterization of stem cells, this approach may improve the outcome for individuals diagnosed in the future.

Krabbe disease is a rare inherited neurologic disorder affecting the central and peripheral nervous systems. The disease has four phenotypes: early infantile, later onset, adolescent, and adult. The only known treatment is hematopoietic stem cell transplantation, which is, in the early infantile form of the disease, most beneficial if performed before onset of clinical symptoms. In August 2006, New York State began screening all newborns for Krabbe disease. A rapid and accurate technique for assessing galactocerebrosidase activity and performing DNA mutation analysis had been developed. Interpreting these results was limited, however, because neither enzyme activity nor genetic mutation reliably predicts phenotype. A series of initiatives were therefore developed by a multidisciplinary group of neurologists, geneticists, metabolic pediatricians, neurodevelopmental pediatricians, and transplant physicians (the Krabbe Consortium of New York State) to enhance the effectiveness of the newborn screening program. A standardized clinical evaluation protocol was designed based on the available literature, criteria for transplantation for the early infantile phenotype were formulated, a clinical database and registry was developed, and a study of developmental and functional outcomes was instituted. This multidisciplinary standardized approach to evaluating infants who have positive results on newborn screening may serve as a model for other states as they begin the process of screening for Krabbe disease and other lysosomal storage disorders.

A 5-month-old Hereford calf with neurologic disease was euthanatized, and a necropsy was done. No gross lesions were seen in the brain. Microscopically, neurons throughout the brain and spinal cord had distended, foamy vacuolated cytoplasm. Ultrastructure showed clear vacuoles filling the neuronal cytoplasm. A lysosomal storage disease was suspected. Sphingomyelinase deficiency was confirmed by biochemical analysis of liver and brain.

The authors report the clinical, neuroradiologic, and neuromuscular pathological findings in a patient with GM1 gangliosidosis. The proton magnetic resonance spectroscopy, previously reported in a single patient with GM1 gangliosidosis, detected a mild reduction of N-acetylaspartate, consistent with relative paucity of axons and neurons and increased levels of myoinositol suggestive of gliotic white matter changes along with the accumulation of an additional compound that could represent either guanidinoacetate or Gal beta 1-6Gal beta 1-4)GlcNAc, an oligosaccharide previously isolated from the urine of GM1 gangliosidosis patients. Although these findings will have to be further confirmed in more patients with GM1 gangliosidosis, they suggest that proton magnetic resonance spectroscopy may provide useful end points to assess the efficacy of novel treatments that could soon become clinically available. Histologically, no significant alterations were found in axons, but there was evidence of redundant and inappropriately folded myelin, which is a feature attributed to disturbed axon-glial interactions.

While transplanted neural stem cells (NSCs) have been shown to hold promise for cell replacement in models of a number of neurological disorders, these examples have typically been under conditions where the host cells become dysfunctional due to a cell autonomous etiology, i.e. a 'sick' cell within a relatively supportive environment. It has long been held that cell replacement in a toxic milieu would not likely be possible; donor cells would succumb in much the same way as endogenous cells had. Many metabolic diseases are characterized by this situation, suggesting that they would be poor targets for cell replacement therapies. On the other hand, models of such diseases could prove ideal for testing the capacity for cell replacement under such challenging conditions. In the twitcher (twi ) mouse -- as in patients with Krabbe or globoid cell leukodystrophy (GLD), for which it serves as an authentic model -- loss of galactocerebrosidase (GalC) activity results in the accumulation of psychosine, a toxic glycolipid. Twi mice, like children with GLD, exhibit inexorable neurological deterioration presumably as a result of dysfunctional and ultimately degenerated oligodendrocytes with loss of myelin. It is believed that GLD pathophysiology is related to a psychosine-filled environment that kills not only host oligodendrocytes but theoretically any new cells placed into that milieu. Through the implantation of NSCs into the brains of both neonatal and juvenile/young adult twi mice, we have determined that widespread oligodendrocyte replacement and remyelination is feasible. NSCs appear to be intrinsically resistant to psychosine -- more so in their undifferentiated state than when directed ex vivo to become oligodendrocytes. This resistance can be enhanced by engineering the NSCs to over-express GalC. Some twi mice grafted with such engineered NSCs had thicker white tracts and lived 2-3 times longer than expected. While their brains had detectable levels of GalC, it was probably more significant that their psychosine levels were lower than in twi mice that died at a younger age. This concept of resistance based on differentiation state extended to human NSCs which could similarly survive within the twi brain. Taken together, these results suggest a number of points regarding cellular therapies against degenerative diseases with a prominent cell non-autonomous component: Cell replacement is possible if cells resistant to the toxic environment are employed. Furthermore, an important aspect of successful treatment will likely be not only cell replacement but also cross-correction of host cells to provide them with enzyme activity and hence resistance. While oligodendrocyte replacement alone was not a sufficient treatment for GLD (even when extensive), the replacement of both cells and molecules -- e.g. with NSCs that could both become oligodendrocytes and 'pumps' for GalC -- emerges as a promising basis for a multidisciplinary strategy. Most neurological disease are complex in this way and will likely require multifaceted approaches, perhaps with NSCs serving as the 'glue'.

Globoid cell leukodystrophy (GLD) is a lysosomal storage disease caused by genetic deficiency of galactocerebrosidase (GALC) activity. Failure in catalyzing the degradation of its major substrate, galactocerebroside, in oligodendrocytes (OLs) and Schwann cells leads to death of these myelinating cells, progressive demyelination, and early demise of GLD patients. Transplantation of bone marrow cells and umbilical cord blood have been attempted as a means of enzyme replacement and have shown limited success. It remains unknown whether or how these therapies support survival of GALC-deficient OLs and myelin maintenance. We report that, upon transplantation, GALC-deficient OLs from the twitcher mouse, a model of GLD, achieved widespread myelination in the brain and spinal cord of the myelin-deficient shiverer mouse, which was preserved for the life of the host. GALC immunohistochemistry showed direct evidence for GALC transfer from the shiverer environment to the engrafted mutant OLs in vivo. These findings suggest that the mutant OLs can internalize exogenous GALC and maintain stable myelin, demonstrating that exogenous enzyme replacement will be a key strategy in the therapy of GLD.

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.

Mucopolysaccharidosis (MPS) type VI, also known as Maroteaux-Lamy disease, is an inherited disorder of glycosaminoglycan catabolism caused by deficient activity of the lysosomal hydrolase, N-acetylgalactosamine 4-sulphatase (4S). A variety of prominent visceral and skeletal defects are characteristic, but primary neurological involvement has generally been considered absent. We report here that the feline model of MPS VI exhibits abnormal lysosomal storage in occasional neurones and glia distributed throughout the cerebral cortex. Abnormal lysosomal inclusions were pleiomorphic with some resembling zebra bodies and dense core inclusions typical of other MPS diseases or the membranous storage bodies characteristic of the gangliosidoses. Pyramidal neurones were shown to contain abnormal amounts of GM2 and GM3 gangliosides by immunocytochemical staining and unesterified cholesterol by histochemical (filipin) staining. Further, Golgi staining of pyramidal neurones revealed that some possessed ectopic axon hillock neurites and meganeurites similar to those described in Tay-Sachs and other neuronal storage diseases with ganglioside storage. Some animals evaluated in this study also received allogeneic bone marrow transplants, but no significant differences in neuronal storage were noted between treated and untreated individuals. These studies demonstrate that deficiency of 4S activity can lead to metabolic abnormalities in the neurones of central nervous system in cats, and that these changes may not be readily amenable to correction by bone marrow transplantation. Given the close pathological and biochemical similarities between feline and human MPS VI, it is conceivable that children with this disease have similar neuronal involvement.

Globoid-cell leukodystrophy (GLD) is an autosomal recessive lysosomal storage disorder caused by mutations in the galactosylceramidase (GALC) gene. Infantile GLD has a lethal course with severe cerebral demyelination that progresses to death by 2 years of age. In the current study twitcher mice, an authentic murine model of infantile GLD, were given intracranial injections of either recombinant adeno-associated virus serotype 2 encoding the murine Galc cDNA (AAV2-GALC) or the same genome pseudotyped with AAV5 capsid proteins (AAV2/5-GALC) on day 3 of age. The group injected intracranially with AAV2/5-GALC had approximately 25-fold greater than normal Galc levels in the brain, while AAV2-GALC-injected animals had 28% normal levels. The average life expectancy of twitcher mice ( approximately 38 days) was significantly (P < 0.0001) increased to 48 and 52 days for the AAV2-GALC- and AAV2/5-GALC-treated groups, respectively. The AAV2/5-GALC group performed significantly better in a battery of behavioral tests compared to untreated, AAV2-GFP-treated, or AAV2-treated twitcher animals. This longitudinal study demonstrated that AAV2/5-GALC-mediated gene therapy resulted in higher levels of Galc expression and slowed the neurologic deterioration more completely than AAV2-GALC in the murine model of globoid-cell leukodystrophy. However, the clinical improvements, as assessed by behavioral tests and life span, were only modest.