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On February 23rd 1936, a boy-child (“Kn”) died in an asylum near Munich after years of severe congenital dis-ease, which had profoundly impaired his development leading to inability to walk, talk and see as well as to severe epilepsy. While a diagnosis of “Little’s disease” was made during life, his postmortem brain investiga-tion at Munich neuropathology (“Deutsche Forschungsanstalt für Psychiatrie”) revealed the diagnosis of “amaurotic idiocy” (AI). AI, as exemplified by Tay-Sachs-Disease (TSD), back then was not yet understood as a specific inborn error of metabolism encompassing several disease entities. Many neuropathological studies were performed on AI, but the underlying processes could only be revealed by new scientific techniques such as biochemical analysis of nervous tissue, deciphering AI as nervous system lipid storage diseases, e.g. GM2-gangliosidosis. In 1963, Sandhoff & Jatzkewitz published an article on a “biochemically special form of AI” reporting striking differences when comparing their biochemical observations of hallmark features of TSD to tissue composition in a single case: the boy Kn. This was the first description of “GM1-Gangliosidosis”, later understood as resulting from genetically determined deficiency in beta-galactosidase. Here we present illus-trative materials from this historic patient, including selected diagnostic slides from the case “Kn” in virtual microscopy, original records and other illustrative material available. Finally, we present results from genetic analysis performed on archived tissue proving beta-galactosidase-gene mutation, verifying the 1963 interpre-tation as correct. This synopsis shall give a first-hand impression of this milestone finding in neuropathology.

GM1-gangliosidosis is a progressive neurodegenerative glycosphingolipidosis resulting from a GLB1 gene mutation causing a deficiency of the lysosomal enzyme β-galactosidase, which leads to the abnormal accumulation of GM1 ganglioside in the central nervous system. In the most severe early infantile phenotype, excessive ganglioside accumulation results in a rapid decline in neurological and psychomotor functions, and death occurs within 2 years of age. Currently, there is no effective therapy for GM1-gangliosidosis. In this study, we evaluated the therapeutic efficacy of ex vivo gene therapy targeting hematopoietic stem cells using a lentiviral vector to increase enzyme activity, reduce substrate accumulation, and improve astrocytosis and motor function. Transplanting GLB1-transduced hematopoietic stem cells in mice increased β-galactosidase enzyme activity in the central nervous system and visceral organs. Specifically, this gene therapy significantly decreased GM1 ganglioside levels in the brain, especially in the cerebrum. More important, this gene therapy rectified astrocytosis in the cerebrum and improved motor function deficits. Furthermore, the elevation of serum β-galactosidase activity in secondary-transplanted mice suggested the ability of transduced hematopoietic stem cells to repopulate long term. These data indicate that ex vivo gene therapy with lentiviral vectors is a promising approach for the treatment of brain deficits in GM1 gangliosidosis.

GM1-gangliosidosis (GM1) is a lysosomal storage disorder caused by mutations in the galactosidase beta 1 gene ( GLB1 ) that leads to reduced β-galactosidase (β-gal) activity. This enzyme deficiency results in neuronal degeneration, developmental delay, and early death. A sensitive assay for the measurement of β-gal enzyme activity is required for the development of disease-modifying therapies. We have optimized fluorometric assays for quantitative analysis of β-gal activity in human cerebrospinal fluid (CSF) and serum for the development of a GLB1 gene replacement therapy. Assay analytical performance was characterized by assessing sensitivity, precision, accuracy, parallelism, specificity, and sample stability. Sensitivity of the CSF and serum β-gal activity assays were 0.05 and 0.20 nmol/mL/3 h, respectively. Assay precision represented by inter-assay percent coefficient of variation of the human CSF and serum was <15% and <20%, respectively. The effect of pre-analytical factors on β-gal activity was examined, and rapid processing and freezing of samples post-collection was critical to preserve enzyme activity. These assays enabled measurement of CSF and serum β-gal activities in both healthy individuals and patients with GM1-gangliosidosis. This CSF β-gal activity assay is the first of its kind with sufficient sensitivity to quantitatively measure β-gal enzyme activity in CSF samples from GM1 patients., Competing Interests: At the time this project was conducted, K.J.Q., D.A.W., S.A.A.-Z., S.E.B., and Y.G.N. were full-time salaried employees of Passage Bio, Inc. and received stock options. C.V., C.D., and C.W. were full-time salaried employees of Thermo Fisher Scientific and have no conflicts of interest to disclose. J.M.W. is a paid advisor to and holds equity in iECURE, Passage Bio, and the Center for Breakthrough Medicines (CBM). He also holds equity in the former G2 Bio asset companies and Ceva Santé Animale. He has sponsored research agreements with Amicus Therapeutics, CBM, Ceva Santé Animale, Elaaj Bio, FA212, Foundation for Angelman Syndrome Therapeutics, former G2 Bio asset companies, iECURE, and Passage Bio, which are licensees of Penn technology. C.J.H. holds equity in a former G2 Bio asset-associated company. J.M.W. and C.J.H. are inventors on patents that have been licensed to various biopharmaceutical companies and for which they may receive payments., (© 2024 The Author(s).)

Satellite glial cells (SGCs) of dorsal root ganglia (DRG) react in response to various injuries in the nervous system. This study investigates reactive changes within SGCs in a murine model for GM1‐gangliosidosis (GM1). DRG of homozygous β‐galactosidase‐knockout mice and homozygous C57BL/6 wild‐type mice were investigated performing immunostaining on formalin‐fixed, paraffin‐embedded tissue. A marked upregulation of glial fibrillary acidic protein (GFAP), the progenitor marker nestin and Ki67 within SGCs of diseased mice, starting after 4 months at the earliest GFAP, along with intracytoplasmic accumulation of ganglioside within neurons and deterioration of clinical signs was identified. Interestingly, nestin‐positive SGCs were detected after 8 months only. No changes regarding inwardly rectifying potassium channel 4.1, 2, 3‐cyclic nucleotide 3‐phosphodiesterase, Sox2, doublecortin, periaxin and caspase3 were observed in SGCs. Iba1 was only detected in close vicinity of SGCs indicating infiltrating or tissue‐resident macrophages. These results indicate that SGCs of DRG show phenotypical changes during the course of GM1, characterized by GFAP upregulation, proliferation and expression of a neural progenitor marker at a late time point. This points towards an important role of SGCs during neurodegenerative disorders and supports that SGCs represent a multipotent glial precursor cell line with high plasticity and functionality. [ABSTRACT FROM AUTHOR]

Elevated serum chitotriosidase (CHITO) is an indication of macrophage activation, and its capacity have been explored as a marker of inflammation in a number of disease states. For over a decade, CHITO plasma levels have been used by clinicians as a biomarker of inflammation in the lysosomal disease, Gaucher disease, including monitoring response to therapies in patients with Gaucher disease type I. Although it is becoming increasingly recognized that inflammation is a prominent component of many lysosomal diseases, the relation of CHITO levels to disease burden has not been well-characterized in the large majority of lysosomal diseases. Moreover, the role of CHITO in lysosomal diseases that affect the central nervous system (CNS) has not been systematically studied. In this study, one hundred and thirty-four specimens of CSF and serum were collected from 34 patients with lysosomal diseases affecting the CNS. This study included patients with GM1-gangliosidosis, GM2-gangliosidosis, mucopolysaccharidoses (MPS), multiple sulfatase deficiency and Gaucher disease. CHITO levels in the CSF were significantly higher in patients with more rapidly progressing severe neurological impairment: GM1-gangliosidosis vs MPS (p

Endoplasmic reticulum-plasma membrane (ER-PM) junctions mediate Ca2+ flux across neuronal membranes. The properties of these membrane contact sites are defined by their lipid content, but little attention has been given to glycosphingolipids (GSLs). Here, we show that GM1-ganglioside, an abundant GSL in neuronal membranes, is integral to ER-PM junctions; it interacts with synaptic proteins/receptors and regulates Ca2+ signaling. In a model of the neurodegenerative lysosomal storage disease, GM1-gangliosidosis, pathogenic accumulation of GM1 at ER-PM junctions due to β-galactosidase deficiency drastically alters neuronal Ca2+ homeostasis. Mechanistically, we show that GM1 interacts with the phosphorylated N-methyl D-aspartate receptor (NMDAR) Ca2+ channel, thereby increasing Ca2+ flux, activating extracellular signal-regulated kinase (ERK) signaling, and increasing the number of synaptic spines without increasing synaptic connectivity. Thus, GM1 clustering at ER-PM junctions alters synaptic plasticity and worsens the generalized neuronal cell death characteristic of GM1-gangliosidosis. [Display omitted] • Neuronal accumulation of GM1 due to β-GAL deficiency increases ER-PM junction formation • GM1 binds pNMDAR at the ER-PM junctions, altering intracellular Ca2+ concentration • pNMDAR-mediated Ca2+ signaling and increased BDNF levels activate the ERK pathway • Altered levels of GM1 increase synaptic spine formation without increasing connectivity Weesner et al. find that, in GM1-gangliosidosis mice, neuronal accumulation of GM1 at the ER-PM junctions alters NMDAR-mediated Ca2+ signaling and activates the synaptogenesis pathway via ERK signaling. This leads to increased synaptic spine density without an increase in synaptic connectivity, contributing to the neurodegeneration characteristic of the disease. [ABSTRACT FROM AUTHOR]

GM1-gangliosidosis is a lysosomal disease resulting from a deficiency in the hydrolase β-galactosidase (β-gal) and subsequent accumulation of gangliosides, primarily in neuronal tissue, leading to progressive neurological deterioration and eventually early death. Lysosomal diseases with neurological involvement have limited non-invasive therapies due to the inability of lysosomal enzymes to cross the blood-brain barrier (BBB). A novel fusion enzyme, labeled mTfR-GLB1, was designed to act as a ferry across the BBB by fusing β-gal to the mouse monoclonal antibody against the mouse transferrin receptor and tested in a murine model of GM1-gangliosidosis (β-gal−/−). Twelve hours following a single intravenous dose of mTfR-GLB1 (5.0 mg/kg) into adult β-gal−/− mice showed clearance of enzyme activity in the plasma and an increase in β-gal enzyme activity in the liver and spleen. Long-term efficacy of mTfR-GLB1 was assessed by treating β-gal−/− mice intravenously twice a week with a low (2.5 mg/kg) or high (5.0 mg/kg) dose of mTfR-GLB1 for 17 weeks. Long-term studies showed high dose mice gained weight normally compared to vehicle-treated β-gal−/− mice, which are significantly heavier than heterozygous controls. Behavioral assessment at six months of age using the pole test showed β-gal−/− mice treated with mTfR-GLB1 had improved motor function. Biochemical analysis showed an increase in β-gal enzyme activity in the high dose group from negligible levels to 20% and 11% of heterozygous levels in the liver and spleen, respectively. Together, these data show that mTfR-GLB1 is a catalytically active β-gal fusion enzyme in vivo that is readily taken up into tissues.Despite these indications of bioactivity, behavior tests other than the pole test, including the Barnes maze, inverted screen, and accelerating rotarod, showed limited or no improvement of treated mice compared to β-gal−/− mice receiving vehicle only. Further, administration of mTfR-GLB1 was insufficient to create measurable increases in β-gal enzyme activity in the brain or reduce ganglioside content (biochemically and morphologically).

Endoplasmic reticulum-plasma membrane (ER-PM) junctions mediate Ca 2+ flux across neuronal membranes. The properties of these membrane contact sites are defined by their lipid content, but little attention has been given to glycosphingolipids (GSLs). Here, we show that GM1-ganglioside, an abundant GSL in neuronal membranes, is integral to ER-PM junctions; it interacts with synaptic proteins/receptors and regulates Ca 2+ signaling. In a model of the neurodegenerative lysosomal storage disease, GM1-gangliosidosis, pathogenic accumulation of GM1 at ER-PM junctions due to β-galactosidase deficiency drastically alters neuronal Ca 2+ homeostasis. Mechanistically, we show that GM1 interacts with the phosphorylated N-methyl D-aspartate receptor (NMDAR) Ca 2+ channel, thereby increasing Ca 2+ flux, activating extracellular signal-regulated kinase (ERK) signaling, and increasing the number of synaptic spines without increasing synaptic connectivity. Thus, GM1 clustering at ER-PM junctions alters synaptic plasticity and worsens the generalized neuronal cell death characteristic of GM1-gangliosidosis., Competing Interests: Declaration of interests The authors declare no competing interests., (Copyright © 2024 The Authors. Published by Elsevier Inc. All rights reserved.)

Background: The gangliosidoses are rare inherited diseases that result in pathologic accumulation of gangliosides in the central nervous system and other tissues, leading to severe and progressive neurological impairment and early death in the childhood forms. No treatments are currently approved for the gangliosidoses, and development of treatments is impaired by limited understanding of the natural history of these diseases. Objective: The objective of this study is to improve understanding of the juvenile gangliosidoses phenotypes and the late-infantile phenotypic subtype. Methods: Through a prospective natural history study of subjects with juvenile GM1- and GM2-gangliosidosis, a timeline of clinical changes was developed for the classic juvenile phenotypes and the late-infantile phenotypes and results of serial neurodevelopmental testing was analyzed. Results: Several candidate ‘outcome measures’ were identified: changes in ambulation and verbalization skills, the communication domain from neurodevelopmental testing and the caregiver-reported socialization domain. Conclusions: The most common symptoms leading caregivers to seek a genetic diagnosis were changes in ambulation and verbalization.

In lysosomal diseases, enzyme deficiency is caused by misfolding of mutant enzyme protein with abnormal steric structure that is expressed by gene mutation. Chaperone therapy is a new molecular therapeutic approach primarily for lysosomal diseases. The misfolded mutant enzyme is digested rapidly or aggregated to induce endoplasmic reticulum stress. As a result, the catalytic activity is lost. The following sequence of events results in chaperone therapy to achieve correction of molecular pathology. An orally administered low molecular competitive inhibitor (chaperone) is absorbed into the bloodstream and reaches the target cells and tissues. The mutant enzyme is stabilized by the chaperone and subjected to normal enzyme protein folding (proteostasis). The first chaperone drug was developed for Fabry disease and is currently available in medical practice. At present three types of chaperones are available: competitive chaperone with enzyme inhibitory bioactivity (exogenous), non-competitive (or allosteric) chaperone without inhibitory bioactivity (exogenous), and molecular chaperone (heat shock protein; endogenous). The third endogenous chaperone would be directed to overexpression or activated by an exogenous low-molecular inducer. This new molecular therapeutic approach, utilizing the three types of chaperone, is expected to apply to a variety of diseases, genetic or non-genetic, and neurological or non-neurological, in addition to lysosomal diseases. [ABSTRACT FROM AUTHOR]

GM1‐gangliosidosis is a rare autosomal recessive lysosomal storage disease caused by deficiency of β‐galactosidase (GLB1). Newborn screening (NBS) may be warranted in the near future given the initiation of a number of gene therapy clinical trials. Here, we report a tandem mass spectrometry (MS/MS) enzymatic assay of GLB1 using dried blood spots (DBS), and the demonstration that GLB1 activities in newborn DBS from seven GM1‐gangliosidosis patients are well below those measured in random newborn DBS. MS/MS analysis of two glycan biomarkers, dp5 and A2G2, shows high elevation in newborn DBS from GM1‐gangliosidosis compared to the levels in the nonaffected reference range. [ABSTRACT FROM AUTHOR]

Over 90% of GM1-gangliosidosis type III cases present with progressive dystonia, which is usually moderate-to-severe and generalized.2,5 In our report, the affected brother (II-2) presented with minimal dystonia if any but parkinsonism over the disease course of 19 years. Keywords: GM1-gangliosidosis; GLB1; parkinsonism; dystonia; genetics EN GM1-gangliosidosis GLB1 parkinsonism dystonia genetics S21 S23 3 09/06/21 20210802 NES 210802 I GLB1 i encodes beta-galactosidase-1, a lysosomal hydrolase that cleaves the terminal beta-galactose from ganglioside substrates. No impaired saccades and limited vertical gaze, seen in our cases, have previously been reported in GM1-gangliosidosis type III.2,4 The presented MRI finding is typical of GM1-gangliosidosis type III and Glutaric aciduria type 1.6 The discussed features seem to expand the clinico-genetic phenotype of the disease. [Extracted from the article]

Gangliosidoses (GM1 and GM2 gangliosidosis) are rare, autosomal recessive progressive neurodegenerative lysosomal storage disorders caused by defects in the degradation of glycosphingolipids. We aimed to investigate clinical, biochemical and molecular genetic spectrum of Turkish patients with infantile gangliosidoses and examined the potential role of serum aspartate transaminase levels as a biomarker. We confirmed the diagnosis of GM1 and GM2 gangliosidosis based on clinical findings with specific enzyme and/or molecular analyses. We retrospectively reviewed serum aspartate transaminase levels of patients with other biochemical parameters. Serum aspartate transaminase level was elevated in all GM1 and GM2 gangliosidosis patients in whom the test was performed, along with normal alanine transaminase. Aspartate transaminase can be a biochemical diagnostic clue for infantile gangliosidoses. It might be a simple but important biomarker for diagnosis, follow up, prognosis and monitoring of the response for the future therapies in these patients. [ABSTRACT FROM AUTHOR]

GM1-gangliosidosis (GM1G) is caused by a deficiency of β-galactosidase, resulting in the excessive accumulation of GM1-ganglioside (GM1) in lysosomes of cells, particularly in the nerve cells (neurons). There is no treatment available for patients with GM1G. Meanwhile, cyclodextrins (CyDs) are cyclic oligosaccharides, which are widely used in the pharmaceutical field. We previously reported that 2, 6-di-O-methyl-α-CyD (DM-α-CyD) extracted phospholipids from lipid rafts, which are abundant with sphingolipids including GM1. Therefore, in the present study, we investigated the effects of α-CyDs on GM1 levels in GM1G model cells and in brain of GM1G model mice. The interaction of DM-α-CyD with GM1 was stronger than that of 2-hydroxypropyl-α-CyD. Additionally, DM-α-CyD significantly reduced GM1 levels in GM1G model cells at 1 mM for 24 h. Furthermore, DM-α-CyD decreased GM1 levels in brain after an intraventricular administration to GM1G model mice without any significant side effects. These results strongly suggest that DM-α-CyD decreased the accumulation of GM1 in not only GM1G model cells but also GM1G model mice. Collectively, DM-α-CyD may have the potential as a therapeutic drug for GM1G. [ABSTRACT FROM AUTHOR]

Background GM1-gangliosidosis and GM2-gangliosidosis (Tay-Sachs disease and Sandhoff disease) are unrelenting heritable neurodegenerative conditions of lysosomal ganglioside accumulation. Although progressive brain atrophy is characteristic, longitudinal quantification of specific brain structures has not been systematically studied. Objectives The goal of this longitudinal study has been to quantify and track brain MRI volume changes, including specific structure volume changes, at different times in disease progression of childhood gangliosidoses, and to explore quantitative brain MRI volumetry (qMRI) as a non-invasive marker of disease progression for future treatment trials. Methods Brain qMRI studies were performed in 14 patients with gangliosidoses (9 infantile, 5 juvenile) yearly. Cerebellar cortex and white matter, caudate, putamen, corpus callosum, ventricles, total brain, and intracranial volumes were measured, as well as total brain volume. Age-matched controls were available for the patients with the juvenile phenotype. Results The infantile phenotype of all gangliosidoses showed a consistent pattern of macrocephaly and rapidly increasing intracranial MRI volume with both (a) brain tissue volume (cerebral cortex and other smaller structures) and (b) ventricular volume (P < 0.01 for all). In contrast to apparent enlargement of the total brain volume, and chiefly the enlarged cerebral cortex, a subset of smaller brain substructures generally decreased in size: the corpus callosum, caudate and putamen became smaller with time. The volume of cerebellar cortex also decreased in patients with infantile GM1-gangliosidosis and juvenile GM1- and GM2-gangliosidosis; however, infantile GM2-gangliosidosis cerebellar cortex was the exception, increasing in size. Elevated intracranial pressure (estimated by lumbar spinal pressure) was a common finding in infantile disease and showed continued increases as the disease progressed, yet lacked MRI signs of hydrocephalus except for increasing ventricular size. Notably, in patients with juvenile gangliosidosis, macrocephaly and elevated intracranial pressure were absent and total brain volume decreased with time compared to controls (P = 0.004). Conclusions The disease course of infantile versus juvenile gangliosidoses is clearly distinguished by the rate of brain disease progression as characterized by qMRI. Assessments by qMRI represent a robust non-invasive method for monitoring CNS changes in the clinical course of gangliosidoses and is ideally suited to monitor effects of novel CNS-directed therapies in future clinical trials. [ABSTRACT FROM AUTHOR]

Juvenile and adult GM1-gangliosidosis are invariably characterized by progressive neurological deterioration. To date only symptomatic therapies are available. We report for the first time the positive results of Miglustat (OGT 918, N-butyl-deoxynojirimycin) treatment on three Italian GM1-gangliosidosis patients. The first two patients had a juvenile form (enzyme activity ≤5%, GLB1 genotype p.R201H/c.1068 + 1G > T; p.R201H/p.I51N), while the third patient had an adult form (enzyme activity about 7%, p.T329A/p.R442Q). Treatment with Miglustat at the dose of 600 mg/day was started at the age of 10, 17 and 28 years; age at last evaluation was 21, 20 and 38 respectively. Response to treatment was evaluated using neurological examinations in all three patients every 4-6 months, the assessment of Movement Disorder-Childhood Rating Scale (MD-CRS) in the second patient, and the 6-Minute Walking Test (6-MWT) in the third patient. The baseline neurological status was severely impaired, with loss of autonomous ambulation and speech in the first two patients, and gait and language difficulties in the third patient. All three patients showed gradual improvement while being treated; both juvenile patients regained the ability to walk without assistance for few meters, and increased alertness and vocalization. The MD-CRS class score in the second patient decreased from 4 to 2. The third patient improved in movement and speech control, the distance covered during the 6-MWT increased from 338 to 475 m. These results suggest that Miglustat may help slow down or reverse the disease progression in juvenile/adult GM1-gangliosidosis. [ABSTRACT FROM AUTHOR]

Mutation analysis of gene GLB1 in 26 patients from different regions of Ukraine diagnosed with GM1-gangliosidosis identified 15 pathogenic mutations. The most common was missense mutation p.His281Tyr (c.841C>T) in exon 8, which was found in at least one allele in 16 of 26 patients (61.5%), accounting for 20 of 52 (38.5%) mutant alleles. Mutation p.His281Tyr (c.841C>T) was described in rare cases in patients from Germany, Italy, and Portugal, while no alleles with such a replacement were found in other countries. This may indicate a much higher frequency of this mutation in Ukraine compared to other countries. Analysis of the information about the spatial distribution of the families with mutation p.His281Tyr (c.841C>T) revealed that most of the alleles with this mutation (14 of 20 or 70%) were found in patients from the western regions of the country, which may be due to the founder effect. [ABSTRACT FROM AUTHOR]

On February 23 rd 1936, a boy-child ("Kn") died in an asylum near Munich after years of severe congenital disease, which had profoundly impaired his development leading to inability to walk, talk and see as well as to severe epilepsy. While a diagnosis of "Little's disease" was made during life, his postmortem brain investigation at Munich neuropathology ("Deutsche Forschungsanstalt für Psychiatrie") revealed the diagnosis of "amaurotic idiocy" (AI). AI, as exemplified by Tay-Sachs-Disease (TSD), back then was not yet understood as a specific inborn error of metabolism encompassing several disease entities. Many neuropathological studies were performed on AI, but the underlying processes could only be revealed by new scientific techniques such as biochemical analysis of nervous tissue, deciphering AI as nervous system lipid storage diseases, e.g. GM2-gangliosidosis. In 1963, Sandhoff & Jatzkewitz published an article on a "biochemically special form of AI" reporting striking differences when comparing their biochemical observations of hallmark features of TSD to tissue composition in a single case: the boy Kn. This was the first description of "GM1-Gangliosidosis", later understood as resulting from genetically determined deficiency in beta-galactosidase. Here we present illustrative materials from this historic patient, including selected diagnostic slides from the case "Kn" in virtual microscopy, original records and other illustrative material available. Finally, we present results from genetic analysis performed on archived tissue proving beta-galactosidase-gene mutation, verifying the 1963 interpretation as correct. This synopsis shall give a first-hand impression of this milestone finding in neuropathology. Original paper: On a biochemically special form of infantile amaurotic idiocy. Jatzkewitz H., Sandhoff K., Biochim. Biophys. Acta 1963; 70; 354-356. See supplement 1.

This report is about the identification, synthesis and initial biological characterization of derivatives of 4- epi -isofagomine as pharmacological chaperones (PC) for human lysosomal β-galactosidase. The two epimers of 4- epi -isofagomine carrying a pentyl group at C-5a, namely (5a R )- and (5a S )-5a- C -pentyl-4- epi -isofagomine, were prepared by an innovative procedure involving in the key step the addition of nitrohexane to a keto-pentopyranoside. Both epimers were evaluated as inhibitors of the human β-galactosidase: the (5a R )-stereoisomer (compound 1 ) was found to be a very potent inhibitor of the enzyme (IC 50 = 8 nM, 30× more potent than 4- epi -isofagomine at pH 7.3) with a high selectivity for this glycosidase whereas the (5a S ) epimer was a much weaker inhibitor. In addition, compound 1 showed a remarkable activity as a PC. It significantly enhanced the residual activity of mutant β-galactosidase in 15 patient cell lines out of 23, with enhancement factors greater than 3.5 in 10 cell lines and activity restoration up to 91% of normal. Altogether, these results indicated that (5a R )-5a- C -pentyl-4- epi -isofagomine constitutes a promising PC-based drug candidate for the treatment of GM1-gangliosidosis and Morquio disease type B. [ABSTRACT FROM AUTHOR]

New enzyme delivery technologies are required for treatment of lysosomal storage disorders with significant pathologies associated with the so-called “hard-to-treat” tissues and organs. Genetic deficiencies in the GLB1 gene encoding acid β-galactosidase lead to GM1-gangliosidosis or Morquio B, lysosomal diseases with predominant disease manifestation associated with the central nervous system or skeletal system, respectively. Current lysosomal ERTs are delivered into cells based on receptor-mediated endocytosis and do not effectively address several hard-to-treat organs including those critical for GM1-gangliosidosis patients. Lectins provide alternative cell-uptake mechanisms based on adsorptive-mediated endocytosis and thus may provide unique biodistribution for lysosomal disease therapeutics. In the current study, genetic fusions of the plant galactose/galactosamine-binding lectin, RTB, and the human acid β-galactosidase enzyme were produced using a plant-based bioproduction platform. β-gal:RTB and RTB:β-gal fusion products retained both lectin activity and β-galactosidase activity. Purified proteins representing both fusion orientations were efficiently taken up into GM1 patient fibroblasts and mediated the reduction of GM1 ganglioside substrate with activities matching mammalian cell-derived β-galactosidase. In contrast, plant-derived β-gal alone was enzymatically active but did not mediate uptake or correction indicating the need for either lectin-based (plant product) or mannose-6-phosphate-based (mammalian product) delivery. Native β-galactosidase undergoes catalytic activation (cleavage within the C-terminal region) in lysosomes and is stabilized by association with protective protein/cathepsin A. Enzymatic activity and lysosomal protein processing of the RTB fusions were assessed following internalization into GM1 fibroblasts. Within 1–4 h, both β-gal:RTB and RTB:β-gal were processed to the ~ 64 kDa “activated” β-gal form; the RTB lectin was cleaved and rapidly degraded. The activated β-gal was still detected at 48 h suggesting interactions with protective protein/cathepsin A. Uptake-saturation analyses indicated that the RTB adsorptive-mediated mechanisms of β-gal:RTB supported significantly greater accumulation of β-galactose activity in fibroblasts compared to the receptor-mediated mechanisms of the mammalian cell-derived β-gal. These data demonstrate that plant-made β-gal:RTB functions as an effective replacement enzyme for GM1-gangliosidosis — delivering enzyme into cells, enabling essential lysosomal processing, and mediating disease substrate clearance at the cellular level. RTB provides novel uptake behaviors and thus may provide new receptor-independent strategies that could broadly impact lysosomal disease treatments. [ABSTRACT FROM AUTHOR]

Objectives GM1-gangliosidosis is an inherited disorder characterized by the accumulation of GM1-gangliosides in many tissues and organs, particularly in the brain. Currently, there is no treatment available for patients with ganglioside storage diseases. Therefore, we investigated the effects of cyclodextrins ( Cy Ds) on the GM1-ganglioside level in EA1 cells, fibroblasts from patients with GM1-gangliosidosis. Methods The concentrations of cholesterol and phospholipids in supernatants were determined by Cholesterol E-test Wako and Phospholipid C-test Wako, respectively. The effects of Cy Ds on GM1-ganglioside levels in EA1 cells using fluorescence-labelled cholera toxin B-subunit, which can bind to GM1-gangliosides specifically, were investigated by flow cytometry and confocal laser scanning microscopy. Key findings The treatment with methylated Cy Ds, hydroxypropylated Cy Ds and branched Cy Ds decreased GM1-ganglioside levels in EA1 cells at 1 m m for 24 h. Unexpectedly, there was no significant change in the efflux of cholesterol or phospholipids from the cells after treatment with Cy Ds under the same experimental conditions, indicating that the efflux of membrane components is not associated with down-regulation of GM1-ganglioside levels in EA1 cells upon Cy Ds treatment. Conclusions Cy Ds may have the potential as drugs for GM1-gangliosidosis, although the mechanism should be thereafter clarified. [ABSTRACT FROM AUTHOR]

Background The gangliosidoses (Tay–Sachs disease, Sandhoff disease, and GM1-gangliosidosis) are progressive neurodegenerative diseases caused by lysosomal enzyme activity deficiencies and consequent accumulation of gangliosides in the central nervous system (CNS). The infantile forms are distinguished from the juvenile forms by age of onset, rate of disease progression, and age of death. There are no approved treatments for the gangliosidoses. In search of potential biomarkers of disease, we quantified 188 analytes in CSF and serum from living human patients with longitudinal (serial) measurements. Notably, several associated with inflammation were elevated in the CSF of infantile gangliosidosis patients, and less so in more slowly progressing forms of juvenile gangliosidosis, but not in MPS disease. Thirteen CSF and two serum biomarker candidates were identified. Five candidate biomarkers were distinguished by persistent elevation in the CSF of patients with the severe infantile phenotype: ENA-78, MCP-1, MIP-1α, MIP-1β, and TNFR2. Correspondence of abnormal elevation with other variables of disease—i.e., severity of clinical phenotype, differentiation from changes in serum, and lack of abnormality in other neurodegenerative lysosomal diseases—identifies these analytes as biomarkers of neuropathology specific to the gangliosidosis diseases. [ABSTRACT FROM AUTHOR]

Abstract: GM1-gangliosidosis is a rare progressive neurodegenerative disorder due to an autosomal recessively inherited deficiency of lysosomal β-galactosidase. We have identified seven American black bears (Ursus americanus) found in the Northeast United States suffering from GM1-gangliosidosis. This report describes the clinical features, brain MRI, and morphologic, biochemical and molecular genetic findings in the affected bears. Brain lipids were compared with those in the brain of a GM1-mouse. The bears presented at ages 10–14months in poor clinical condition, lethargic, tremulous and ataxic. They continued to decline and were humanely euthanized. The T2-weighted MR images of the brain of one bear disclosed white matter hyperintensity. Morphological studies of the brain from five of the bears revealed enlarged neurons with foamy cytoplasm containing granules. Axonal spheroids were present in white matter. Electron microscopic examination revealed lamellated membrane structures within neurons. Cytoplasmic vacuoles were found in the liver, kidneys and chondrocytes and foamy macrophages within the lungs. Acid β-galactosidase activity in cultured skin fibroblasts was only 1–2% of control values. In the brain, ganglioside-bound sialic acid was increased more than 2-fold with GM1-ganglioside predominating. GA1 content was also increased whereas cerebrosides and sulfatides were markedly decreased. The distribution of gangliosides was similar to that in the GM1-mouse brain, but the loss of myelin lipids was greater in the brain of the affected bear than in the brain of the GM1 mouse. Isolated full-length cDNA of the black bear GLB1 gene revealed 86% homology to its human counterpart in nucleotide sequence and 82% in amino acid sequence. GLB1 cDNA from liver tissue of an affected bear contained a homozygous recessive T1042 to C transition inducing a Tyr348 to His mutation (Y348H) within a highly conserved region of the GLB1 gene. The coincidence of several black bears with GM1-gangliosidosis in the same geographic area suggests increased frequency of a founder mutation in this animal population. [Copyright &y& Elsevier]

Abstract: Chaperone therapy is a newly developed molecular therapeutic approach to lysosomal diseases, a group of human genetic diseases causing severe brain damage. Based on early molecular studies during the last decade of the 20th century and early years of the 21st century, mainly on Fabry disease and GM1-gangliosidosis, we found some mutant enzyme proteins were unstable in the cell, and unable to express catalytic activities. Subsequently galactose and other active-site binding substrate analogs were found stabilized and enhance the mutant enzyme activity in culture cells. We concluded that the mutant misfolding enzyme protein and substrate analog competitive inhibitor (chemical chaperone) form a stable complex to be transported to the lysosome, to restore the catalytic activity of mutant enzyme after spontaneous dissociation under the acidic condition. This gene mutation-specific molecular interaction is a paradoxical phenomenon that an enzyme inhibitor in vitro serves as an enzyme stabilizer in situ. First we developed a commercially available compound 1-deoxygalactonojirimycin (DGJ) for Fabry disease, and confirmed the above molecular phenomenon. Currently DGJ has become a new candidate of oral medicine for Fabry disease, generalized vasculopathy involving the kidneys, heart and central nervous system in the middle age. This drug development has reached the phase 3 of human clinical study. Then we found two valienamine derivatives, N-octyl-4-epi-β-valienamine (NOEV) and N-octyl-β-valienamine (NOV), as promising therapeutic agents for human β-galactosidase deficiency disorders (GM1-gangliosidosis and Morquio B disease) and β-glucosidase deficiency disorders (phenotypic variations of Gaucher disease), respectively. Originally NOEV and NOV had been discovered as competitive inhibitors, and then their paradoxical bioactivities as chaperones were confirmed in cultured fibroblasts from patients with these disorders. Subsequently GM1-gangliosidosis model mice have been used for confirmation of clinical effectiveness, adverse effects and pharmacokinetic studies. Orally administered NOEV entered the brain through the blood–brain barrier, enhanced β-galactosidase activity, reduced substrate storage, and improved neurological deterioration clinically. Computational analysis revealed pH-dependent enzyme-chaperone interactions. Our recent study indicated chaperone activity of a new DGJ derivative, MTD118, for β-galactosidase complementary to NOEV. NOV also showed the chaperone effect toward several β-glucosidase gene mutants in Gaucher disease. Furthermore a commercial expectorant drug ambroxol was found to be a chaperone for β-glucosidase. A few Gaucher patients responded to this drug with remarkable improvement of oculomotor dysfunction and myoclonus. We hope chaperone therapy will become available for some patients with Fabry disease, GM1-gangliosidosis, Gaucher disease, and other lysosomal storage diseases particularly with central nervous system involvement. [Copyright &y& Elsevier]

Abstract: Therapeutic chaperone effect of a valienamine derivative N-octyl 4-epi-β-valienamine (NOEV) was studied in GM1-gangliosidosis model mice. Phamacokinetic analysis revealed rapid intestinal absorption and renal excretion after oral administration. Intracellular accumulation was not observed after continuous treatment. NOEV was delivered to the central nervous system through the blood–brain barrier to induce high expression of the apparently deficient β-galactosidase activity. NOEV treatment starting at the early stage of disease resulted in remarkable arrest of neurological progression within a few months. Survival time was significantly prolonged. This result suggests that NOEV chaperone therapy will be clinically effective for prevention of neuronal damage if started early in life hopefully also in human patients with GM1-gangliosidosis. [Copyright &y& Elsevier]

Abstract: GM1 gangliosidosis, a neurodegenerative disorder, and Morquio B disease, a skeletal disorder, are lysosomal storage disorders caused by inherited defects in the enzyme β-galactosidase (GLB1; EC 3.1.2.23; MIM #611458). Enzyme replacement therapy (ERT), a standard of care for a number of non-neuronopathic lysosomal storage disorders, is not yet available for GLB1 deficiency. Although functionally active recombinant human and feline GLB1 precursors have been purified, ERT has not yet been demonstrated in GM1 gangliosidosis or Morquio B disease models. A major obstacle to developing effective therapy may be the stability of human GLB1. We show here that mouse GLB1 has greater stability when compared to human GLB1, and that human GLB1 activity is temperature and protective-dependent on protein cathepsin A, while that of mouse GLB1 is not. These findings may impact on the eventual development of ERT for GLB1 deficiency. Despite our attempts to improve the extracellular stability of human GLB1 through sequence modification and the use of chemical chaperone N-butyldeoxygalactonojirimycin, the specific enzyme activity remained well below that of mGLB1. [Copyright &y& Elsevier]

Abstract: Two simple and reliably accessible intermediates, N-carboxypentyl- and N-aminohexyl-1-deoxy-d-galactonojirimycin were employed for the synthesis of a set of terminally N-dansyl substituted derivatives. Reaction of the terminal carboxylic acid of N-carboxypentyl-1-deoxy-d-galactonojirimycin with N-dansyl-1,6-diaminohexane provided the chain-extended fluorescent derivative. Employing bis(6-dansylaminohexyl)amine, the corresponding branched di-N-dansyl compound was obtained. Partially protected N-aminohexyl-1-deoxy-d-galactonojirimycin served as intermediate for two additional chain-extended fluorescent 1-deoxy-d-galactonojirimycin (1-DGJ) derivatives featuring terminal dansyl groups in the N-alkyl substituent. These new compounds are strong inhibitors of d-galactosidases and may serve as leads en route to pharmacological chaperones for GM1-gangliosidosis. [Copyright &y& Elsevier]

GM1-gangliosidosis is a fatal neurodegenerative disorder caused by deficiency of lysosomal acid β-galactosidase (β-gal). Accumulation of its substrate ganglioside GM1 (GM1) in lysosomes and other parts of the cell leads to progressive neurodegeneration, but underlying mechanisms remain unclear. Previous studies demonstrated an essential role for interaction of GM1 with tropomyosin receptor kinase (Trk) receptors in neuronal growth, survival and differentiation. In this study we demonstrate accumulation of GM1 in the cellsurface rafts and lysosomes of the β-gal knockout (β-gal)/)) mouse brain association with accumulation of Trk receptors and enhancement of its downstream signaling. Immunofluorescence and subcellular fractionation analysis revealed accumulation of Trk receptors in the late endosomes/lysosomes of the β-gal)/) mouse brain and their association with ubiquitin and p62. Administration of a chemical chaperone to β-gal)/) mouse expressing human mutant R201C protein resulted in a marked reduction of intracellular storage of GM1 and phosphorylated Trk. These findings indicate that GM1 accumulation in rafts causes activation of Trk signaling, which may participate in the pathogenesis of GM1-gangliosidosis. [ABSTRACT FROM AUTHOR]

Abstract: β-Galactosidosis is a group of disorder based on heterogeneous mutations of GLB1 gene coding for the lysosomal acid β-galactosidase (β-gal). A decrease of the β-gal enzyme activity results in progressive accumulation of substrates in somatic cells, particularly in neurons, leading to severe neuronal dysfunction. We have previously reported that N-octyl-4-epi-β-valienamine (NOEV), a chemical chaperone compound, stabilized various mutant human β-gal proteins and increased residual enzyme activities in cultured fibroblasts from human patients. These data proved a potential therapeutic benefit of chemical chaperone therapy for patients with missense β-gal. This effect is mutation specific. In this study, we have established a sensitive luciferase-based assay for measuring chaperone effect on mutant human β-gal. A dinoflagellate luciferase (Dluc) cDNA was introduced to the C-terminus of human β-gal. When COS7 cells expressing the Dluc-tagged human R201C β-gal was treated with NOEV, there happened a remarkable increase of the mutant β-gal activity. In the presence of NH4Cl, luciferase level in the medium increased in parallel with the enzyme activity in cell lysates. We also found that proteasome inhibitors enhance chaperone effect of NOEV. These results demonstrate that the luciferase-based assay is a reliable and convenient method for screening and evaluation of chaperone effects on human β-gal mutants, and that it will be a useful tool for finding novel chaperone compounds in the future study. [ABSTRACT FROM AUTHOR]

Abstract: GM1-gangliosidosis (GM1) and Morquio B disease (MBD) are rare lysosomal storage disorders caused by mutations in the gene GLB1. Its main gene product, human acid β-galactosidase (β-Gal) degrades two functionally important molecules, GM1-ganglioside and keratan sulfate in brain and connective tissues, respectively. While GM1 is a severe, phenotypically heterogenous neurodegenerative disorder, MBD is a systemic bone disease without effects on the central nervous system. A MBD-specific mutation, p.W273L, was shown to produce stable β-Gal precursors, normally transported and processed to mature, intralysosomal β-Gal. In accordance with the MBD phenotype, elevated residual activity against GM1-ganglioside, but strongly reduced affinity towards keratan sulfate was found. Most GM1 alleles, in contrast, were shown to affect precursor stability and intracellular transport. Specific alleles, p.R201C and p.R201H result in misfolded, unstable precursor proteins rapidly degraded by endoplasmic reticulum-associated protein degradation (ERAD). They may therefore be sensitive to stabilization by small molecules which bind at the active site and provide proper conformation. Thus the stabilized protein may escape from ERAD processes, and reach the lysosomes in an active state, as proposed for enzyme enhancement therapy (EET). This paper demonstrates that a novel iminosugar, DLHex-DGJ, has potent effects as competitive inhibitor of human acid β-galactosidase in vitro, and describes its effects on activity, protein expression, maturation and intracellular transport in vivo in 13 fibroblasts lines with GLB1 mutations. Beside p.R201C and p.R201H, two further alleles, p.C230R and p.G438E, displayed significant sensitivity against DLHex-DGJ, with an increase of catalytic activity, and a normalization of transport and lysosomal processing of β-Gal precursors. [Copyright &y& Elsevier]

Abstract: We performed a cell transplantation study to treat the brain involvement in lysosomal storage diseases. We used acid β-galactosidase knock-out mice (BKO) from C57BL/6 as recipients. To minimize immune responses, we used cells derived from transgenic mice of C57BL/6 overexpressing the normal human β-galactosidase. Fetal brain cells (FBC), bone marrow-derived mesenchymal stem cells (MSC), and mixed FBC and MSC cells were prepared and injected into the ventricle of newborn BKO mouse brain. The mice were examined at 1, 2, 4, and 8 weeks and 6 months after injection. In each experiment, the injected cells migrated into the whole brain effectively and survived for at least 8 weeks. Decrease in ganglioside GM1 level was also observed. FBC could survive for 6 months in recipient brain. However, the number of transplanted FBC decreased. In the brains of MSC- or mixed cell-treated mice, no grafted cells could be found at 6 months. To achieve sufficient long-term effects on the brain, a method of steering the immune response away from cytotoxic responses or of inducing tolerance to the products of therapeutic genes must be developed. [Copyright &y& Elsevier]

Abstract: β-galactosidase (GLB1) forms a functional lysosomal multienzyme complex with lysosomal protective protein (PPCA) and neuraminidase 1 (NEU1) which is important for its intracellular processing and activity. Mutations in the β-galactosidase gene cause the lysosomal storage disease GM1-gangliosidosis. In order to identify additional molecular changes associated with the presence of β-galactosidase mutations, the expression of canine lysosomal multienzyme complex components in GLB1 +/+, GLB1 +/− and GLB1 −/− fibroblasts was investigated by quantitative RT-PCR, Western blot and enzymatic assays. Quantitative RT-PCR revealed differential regulation of total β-galactosidase, β-galactosidase variants and protective protein for β-galactosidase gene (PPGB) in GLB1 +/− and GLB1 −/− compared to GLB1 +/+ fibroblasts. Furthermore, it was shown that PPGB levels gradually increased with the number of mutant β-galactosidase alleles while no change in the NEU1 expression was observed. This is the first study that simultaneously examine the effect of GLB1 +/+, GLB1 +/− and GLB1 −/− genotypes on the expression of lysosomal multienzyme complex components. The findings reveal a possible adaptive process in GLB1 homozygous mutant and heterozygous individuals that could facilitate the design of efficient therapeutic strategies. [Copyright &y& Elsevier]

Abstract: Background: The glycosphingolipid storage disorder GM1-gangliosidosis is a severe neurodegenerative condition for which no therapy is currently available. Protein misfolding in lysosomal defects may have the potential to be corrected by chemical chaperones: in vitro and clinical approaches are being investigated. Aims: We investigated the in vitro effect of galactose on some lysosomal hydrolases, and its in vitro efficacy as a chemical chaperone in GM1-gangliosidosis. Methods: Galactose was added to the culture medium of fibroblasts from patients, controls and transfected COS-1 cells. Enzyme assays of lysosomal hydrolases, beta galactosidase in particular, were performed. Results: Our data show that galactose alters selectively alpha and beta galactosidases. A significant increase (2,5 fold) in beta galactosidase activity occurred when galactose was added to the cultured fibroblasts of an adult patient. Chemical chaperone therapy requires the presence of residual enzyme activity. The adult patient here reported is heterozygous for the p.T329A mutation that showed no beta galactosidase activity, and for the p.R442Q mutation with residual enzyme activity. The p.R442Q mutation was therefore selected as a potential target for the galactose chaperone; after the addition of galactose, COS-1 cells transfected with this mutation showed an increase in beta galactosidase activity from 6.9% to 12% of control values. Conclusions: These results suggest that galactose or its derivatives with potential chaperone properties could be used in the development of non-invasive therapies for GM1-gangliosidosis. [Copyright &y& Elsevier]

GM1-gangliosidosis is a lysosomal storage disease (LSD) caused by an autosomal recessive deficiency of lysosomal acid β-galactosidase (βgal). This leads to accumulation of GM1-ganglioside and its asialo derivative GA1 in the central nervous system (CNS), and progressive neurodegeneration. Therapeutic AAV-mediated gene delivery to the brain for LSDs has proven very successful in several animal models. GM1-gangliosidosis is also a prime candidate for AAV-mediated gene therapy in the CNS. As global neuropathology characterizes the most severe forms of this disease, therapeutic interventions need to achieve distribution of βgal throughout the entire CNS. Therefore, careful consideration of routes of administration and target structures from where metabolically active enzyme can be produced, released and distributed throughout the CNS, is necessary. The goal of this study was to investigate the pattern and mechanism of distribution of βgal in the adult GM1-gangliosidosis mouse brain upon hippocampal injection of an AAV vector-encoding βgal. We found evidence that three different mechanisms contribute to its distribution in the brain: (1) diffusion; (2) axonal transport within neurons from the site of production; (3) CSF flow in the perivascular space of Virchow–Robin. In addition, we found evidence of axonal transport of vector-encoded mRNA.Gene Therapy (2009) 16, 303–308; doi:10.1038/gt.2008.149; published online 25 September 2008 [ABSTRACT FROM AUTHOR]

Chaperone therapy is a newly developed molecular approach to lysosomal diseases, a group of human genetic diseases causing severe brain damage. We found two valienamine derivatives, N-octyl-4-epi-β-valienamine (NOEV) and N-octyl-β-valienamine (NOV), as promising therapeutic agents for human β-galactosidase deficiency disorders (mainly GM1-gangliosidosis) and β-glucosidase deficiency disorders (Gaucher disease), respectively. We briefly reviewed the historical background of research in carbasugar glycosidase inhibitors. Originally NOEV and NOV had been discovered as competitive inhibitors, and then their paradoxical bioactivities as chaperones were confirmed in cultured fibroblasts from patients with these disorders. Subsequently GM1-gangliosidosis model mice were developed and useful for experimental studies. Orally administered NOEV entered the brain through the blood-brain barrier, enhanced β-galactosidase activity, reduced substrate storage, and improved neurological deterioration clinically. Furthermore, we executed computational analysis for prediction of molecular interactions between β-galactosidase and NOEV. Some preliminary results of computational analysis of molecular interaction mechanism are presented in this article. NOV also showed the chaperone effect toward several β-glucosidase gene mutations in Gaucher disease. We hope chaperone therapy will become available for some patients with GM1-gangliosidosis, Gaucher disease, and potentially other lysosomal storage diseases with central nervous system involvement. [ABSTRACT FROM AUTHOR]

GM1-gangliosidosis is a lysosomal storage disorder caused by a deficiency of β-galactosidase activity. Human GM1-gangliosidosis has been classified into three forms according to the age of clinical onset and specific biochemical parameters. In the present study, a canine model for type II late infantile human GM1-gangliosidosis was investigated 'in vitro' in detail. For a better understanding of the molecular pathogenesis underlying GM1-gangliosidosis the study focused on the analysis of the molecular events and subsequent intracellular protein trafficking of β-galactosidase. In the canine model the genetic defect results in exclusion or inclusion of exon 15 in the mRNA transcripts and to translation of two mutant precursor proteins. Intracellular localization, processing and enzymatic activity of these mutant proteins were investigated. The obtained results suggested that the β-galactosidase C-terminus encoded by exons 15 and 16 is necessary for correct C-terminal proteolytic processing and enzyme activity but does not affect the correct routing to the lysosomes. Both mutant protein precursors are enzymatically inactive, but are transported to the lysosomes clearly indicating that the amino acid sequences encoded by exons 15 and 16 are necessary for correct folding and association with protective protein/cathepsin A, whereas the routing to the lysosomes is not influenced. Thus, the investigated canine model is an appropriate animal model for the human late infantile form and represents a versatile system to test gene therapeutic approaches for human and canine GM1-gangliosidosis. [ABSTRACT FROM AUTHOR]

Abstract: GM1-gangliosidosis is an autosomal recessive lysosomal lipid storage disorder, caused by mutations of the lysosomal β-galactosidase (β-gal) and results in the accumulation of GM1. The underlying mechanisms of neurodegeneration are poorly understood. Here we demonstrate increased autophagy in β-gal-deficient (β-gal−/−) mouse brains as evidenced by elevation of LC3-II and beclin-1 levels. Activation of autophagy in the β-gal−/− brain was found to be accompanied with enhanced Akt–mTOR and Erk signaling. In addition, the mitochondrial cytochrome c oxidase activity was significantly decreased in brains and cultured astrocytes from β-gal−/− mouse. Mitochondria isolated from β-gal−/− astrocytes were morphologically abnormal and had a decreased membrane potential. These cells were more sensitive to oxidative stress than wild type cells and this sensitivity was suppressed by ATP, an autophagy inhibitor 3-methyladenine and a pan-caspase inhibitor z-VAD-fmk. These results suggest activation of autophagy leading to mitochondrial dysfunction in the brain of GM1-gangliosidosis. [Copyright &y& Elsevier]

Abstract: A large number of genetic disease model mice have been produced by genetic engineering. However, phenotypic analysis is not sufficient, particularly for brain dysfunction in neurogenetic diseases. We tried to develop a new assessment system mainly for motor and reflex functions in GM1-gangliosidosis model mice. Two genetically engineered model mouse strains were used for this study: the β-galactosidase-deficient knockout mouse representing infantile GM1-gangliosidosis (severe form), and transgenic mouse representing juvenile GM1-gangliosidosis (mild form). We modified human child neurology techniques, and selected eleven tests for motor assessment and reflex testing. The test results were scored in four grades: 0 (normal), 1 (slightly abnormal), 2 (moderately abnormal), and 3 (severely abnormal). Both disease model mouse strains showed high scores even at the apparently pre-symptomatic stage of the disease, particularly with abnormal tail and hind limb postures. Individual and total test scores were well correlated with the progression of the disease. This method is simple, quick, and reproducible. The testing is sensitive enough to detect early neurological abnormalities, and will be useful for monitoring the natural clinical course and effect of therapeutic experiments in various neurogenetic disease model mice, such as chemical chaperone therapy for GM1-gangliosidosis model mice. [Copyright &y& Elsevier]

Abstract: Objectives: : The aim of the present work was to establish the range of chitotriosidase (CT) activity in normal individuals (controls), patients with Gaucher disease (GD), GM1-gangliosidosis (GM1), Krabbe disease (KD) and heterozygotes for Gaucher disease (HG). The kinetics of the enzyme in the five groups was also investigated. Design and methods: : Plasma CT activity, as well as K m, V max, optimum pH and thermal stability of the enzyme was determined in plasma of controls, GM1, KD, GD and HG subjects. Results: : CT activity in GD, GM1 and KD patients was, respectively, around 600-fold, 15-fold and 12-fold greater than in normal individuals. There was no significant difference between CT activity in the HG and the control group. We also demonstrated that all CT kinetic parameters evaluated (optimum pH, K m, V max, thermal stability) in plasma of GD, KD and GM1 patients were significantly different from those of normal individuals. Regarding to thermal stability, our results show that CT activity in the control group was more stable than in the other groups. Conclusions: : Based on the differences found in the biochemical parameters studied, we presume that the parameters analyzed may be useful in the diagnosis of the Lysosomal Storage Diseases. [Copyright &y& Elsevier]

Abstract: We performed screening of β-galactosidase-deficient fibroblasts for possible chemical chaperone therapy using N-octyl-4-epi-β-valienamine (NOEV) in patients with GM1-gangliosidosis and Morquio B disease (β-galactosidosis). Fibroblasts were cultured with NOEV for 4 days and β-galactosidase activity was measured. Mutation analysis was performed simultaneously. Two separate criteria were set for evaluation of the chaperone effect: a relative increase of enzyme activity (more than 3-fold), and an increase up to more than 10% normal enzyme activity. Among the 50 fibroblast strains tested, more than 3-fold increase was achieved in 17 cell strains (34%), and more than 10% normal activity in 10 (20%). Both criteria were satisfied in 6 (12%), and either of them in 21 (42%). Juvenile GM1-gangliosidosis was most responsive, and then infantile GM1-gangliosidosis. This enhancement was mutation-specific. We estimate that the NOEV chaperone therapy will be effective in 20–40% of the patients, mainly in juvenile and infantile GM1-gangliosidosis patients. A molecular design may produce mutation-specific chaperone compounds for the other disease phenotypes. This cellular screening will be useful for identification of human patients with β-galactosidase deficiency for chaperone therapy to be started in the near future. [Copyright &y& Elsevier]

Abstract: A capillary-scale high-pH anion-exchange chromatography (HPAEC) system for the analysis of carbohydrates was developed, in combination with two parallel on-line detection methods of sub-picomolar sensitivity: (1) pulsed amperometric detection (PAD); (2) capillary-scale desalting followed by electrospray ion-trap (IT) mass spectrometry (MS). The capillary chromatographic system combined the superb selectivity of HPAEC that allows routine separation of isomeric oligosaccharides with the information on monosaccharide sequence and linkage positions obtained by MS/MS fragmentation using the IT-MS. The applicability of the system in biomedical research was demonstrated by its use for the analysis of a urine sample of a GM1-gangliosidosis patient. Isomeric glycans in the sample could be resolved by HPAEC and assigned on the basis of the monosaccharide linkage information revealed by on-line IT-MS/MS. [Copyright &y& Elsevier]

Abstract: Background: Gangliosides are building blocks of cell membranes and their biosynthesis and degradation have been extensively studied in the past. Regulation of the metabolism of these glycolipids controls fundamental cell functions. GM1-gangliosidosis, a neurodegenerative glycosphingolipid storage disease, is caused by deficiency of lysosomal β-galactosidase with consequent disruption of the normal degradative pathway of GM1-ganglioside. We studied the impact of GM1-ganglioside accumulation on its biosynthetic enzyme in cells and tissues from human patients and from the GM1-gangliosidosis mouse model. Methods: We tested the qualitative and quantitative pattern of gangliosides by thin layer chromatography and N-acetylneuraminic acid dosage, respectively. Regulation of GM1-ganglioside biosynthesis was evaluated by GM1 synthase assay in human and murine samples. Results: GM1-ganglioside accumulation has an inhibitory effect on the human but not on the mouse GM1 synthase. We present evidence that GM1 synthase activity in human and murine cells are regulated by different mechanisms. Conclusions: Alternative pathways in the mouse may account for these results and possibly explain some of the phenotypical differences between the human and mouse forms of this disorder. [Copyright &y& Elsevier]