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GM1 gangliosidosis (GM1) is a rare autosomal recessive neurogenerative lysosomal storage disease characterized by deficiency of beta-galactosidase (β-gal) and intralysosomal accumulation of GM1 ganglioside and other glycoconjugates. Resources for GM1 disease modelling are limited, and access to relevant cell lines from human patients is not possible. Generation of iPSC lines from GM1 patient-derived dermal fibroblasts allows for disease modelling and therapeutic testing in 2D and 3D cell culture models relevant to CNS disorders, including various neuronal subtypes and cerebral organoids. The iPSC line described here will be critical to therapeutic development and set the foundation for translational gene therapy work.

GM1 gangliosidosis (GM1) is a rare autosomal recessive neurogenerative lysosomal storage disease characterized by deficiency of beta-galactosidase (β-gal) and intralysosomal accumulation of GM1 ganglioside and other glycoconjugates. Resources for GM1 disease modelling are limited, and access to relevant cell lines from human patients is not possible. Generation of iPSC lines from GM1 patient-derived dermal fibroblasts allows for disease modelling and therapeutic testing in 2D and 3D cell culture models relevant to CNS disorders, including various neuronal subtypes and cerebral organoids. The iPSC line described here will be critical to therapeutic development and set the foundation for translational gene therapy work., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024 The Authors. Published by Elsevier B.V. All rights reserved.)

Abstract GM1 gangliosidosis is a lysosomal storage disorder caused by β‐galactosidase deficiency. To date, prospective studies for GM1 gangliosidosis are not available, and only a few have focused on the adult form. This retrospective cross‐sectional study focused on clinical findings in Brazilian patients with the adult form of GM1 gangliosidosis collected over 2 years. Ten subjects were included in the study. Eight were males and two females, with median age at diagnosis of 11.5 years (IQR, 4‐34 years). Short stature and weight below normal were seen in five out of the six patients with data available. Radiological findings revealed that the most frequent skeletal abnormalities were beaked vertebrae, followed by hip dysplasia, and platyspondyly. Neurological examination revealed that dystonia and swallowing problems were the most frequently reported. None of the patients presented hyperkinesia, truncal hypertonia, Parkinsonism, or spinal cord compression. Clinical evaluation revealed impairment in activities of cognitive/intellectual development and behavioral/psychiatric disorders in all nine subjects with data available. Language/speech impairment (dysarthria) was found in 8/9 patients, fine motor and gross motor impairments were reported in 7/9 and 5/9 patients, respectively. Impairment of cognition and daily life activities were seen in 7/9 individuals. Our findings failed to clearly identify typical early or late alterations presented in GM1 gangliosidosis patients, which confirms that it is a very heterogeneous condition with wide phenotypic variability. This should be taken into account in the evaluation of future therapies for this challenging condition.

Rationale: Monosialotetrahexosylganglioside (GM1) gangliosidosis is a rare lysosomal storage disorder caused by the deficiency of ß-galactosidase. Because clinical symptoms of GM1 gangliosidosis overlap with other neurodevelopmental disorders, the diagnosis of this disease is not easy, specifically in late infantile GM1 gangliosidosis. This report described a case of late-infantile GM1 gangliosidosis mistaken for juvenile idiopathic arthritis., Patient Concerns: A 16-year-old girl was referred to our hospital due to persistent multiple joint deformities and mental retardation, which could not be explained by juvenile idiopathic arthritis., Diagnosis: We made a diagnosis of late infantile GM1 gangliosidosis through enzyme assays and genetic testing after a skeletal survey., Interventions: The patient underwent cervical domeplasty and laminectomy for cord compression and received rehabilitation treatment., Outcomes: The patient is receiving multidisciplinary care at a tertiary center for variable skeletal disease and conditions associated with GM1 gangliosidosis., Lessons: Late infantile GM1 gangliosidosis should be considered in the differential diagnosis of progressive neurologic decline and skeletal dysostosis., Competing Interests: The authors have no conflicts of interests to disclose., (Copyright © 2022 the Author(s). Published by Wolters Kluwer Health, Inc.)

GM1 gangliosidosis is a lysosomal storage disorder caused by mutations in GLB1 encoding a lysosomal β-galactosidase. This disease is a continuum from the severe infantile form with rapid neurological decline to the chronic adult form, which is not life-limiting. The intermediate or type 2 form can be further classified into late infantile and juvenile forms. The frequency and severity of skeletal outcomes in late infantile and juvenile patients have not been characterized. Our goals are to describe the radiological skeletal abnormalities, bone mineral density (BMD), and frequency of fractures in patients with intermediate GM1 gangliosidosis. We evaluated 13 late infantile and 21 juvenile patients as part of an ongoing natural history study. Average time from onset of symptoms to diagnosis was 1.9 and 6.3 years for late infantile and juvenile patients, respectively. All late infantile patients had odontoid hypoplasia and pear-shaped vertebral bodies, the frequency of which was significantly different than in patients with juvenile disease (none and 14%, respectively). Juvenile patients had irregular endplates of the vertebral bodies (15/21), central indentation of endplates (10/21), and squared and flat vertebral bodies (10/21); all allowed radiographic differentiation from late infantile patients. Lumbar spine, femoral neck, and total hip BMD were significantly decreased (−2.1, −2.2, and −1.8 Z -scores respectively). Lumbar spine BMD peaked at 19 years, while distal forearm BMD peaked at 30 years. Despite low BMD, no patients exhibited fractures. We have demonstrated that all late infantile patients have some degree of odontoid hypoplasia suggesting the need for cervical spine evaluation particularly prior to anesthesia, whereas juvenile patients had variable skeletal involvement often affecting activities of daily living. Type 2 GM1 gangliosidosis patients have skeletal abnormalities that are both an early indication of their diagnosis, and require monitoring and management to ensure the highest possible quality of life. • We describe the radiographic and densitometric progression of disease in patients with intermediate GM1 gangliosidosis. • Radiographic changes allow differentiation between late infantile and juvenile patients. • Lumbar spine, femoral neck, and total hip bone mineral density were significantly decreased across the cohort. • Despite low bone mineral density, no patients exhibited fractures. • Odontoid hypoplasia in all late infantile patients suggests the need for cervical spine evaluation prior to anesthesia. [ABSTRACT FROM AUTHOR]

A patient with adrenoleukodystrophy and his mother, a carrier, showed an elevated ratio of very long-chain fatty acids to long-chain fatty acids and decreased beta-galactosidase activity. Other lysosomal enzyme activities were normal except for the borderline level of arylsulfatase-A activity. However, the father and other patients with variant forms of adrenoleukodystrophy showed normal beta-galactosidase and other lysosomal enzyme activities. [ABSTRACT FROM AUTHOR]

GM1 gangliosidosis is a lysosomal storage disorder caused by β-galactosidase deficiency. To date, prospective studies for GM1 gangliosidosis are not available, and only a few have focused on the adult form. This retrospective cross-sectional study focused on clinical findings in Brazilian patients with the adult form of GM1 gangliosidosis collected over 2 years. Ten subjects were included in the study. Eight were males and two females, with median age at diagnosis of 11.5 years (IQR, 4-34 years). Short stature and weight below normal were seen in five out of the six patients with data available. Radiological findings revealed that the most frequent skeletal abnormalities were beaked vertebrae, followed by hip dysplasia, and platyspondyly. Neurological examination revealed that dystonia and swallowing problems were the most frequently reported. None of the patients presented hyperkinesia, truncal hypertonia, Parkinsonism, or spinal cord compression. Clinical evaluation revealed impairment in activities of cognitive/intellectual development and behavioral/psychiatric disorders in all nine subjects with data available. Language/speech impairment (dysarthria) was found in 8/9 patients, fine motor and gross motor impairments were reported in 7/9 and 5/9 patients, respectively. Impairment of cognition and daily life activities were seen in 7/9 individuals. Our findings failed to clearly identify typical early or late alterations presented in GM1 gangliosidosis patients, which confirms that it is a very heterogeneous condition with wide phenotypic variability. This should be taken into account in the evaluation of future therapies for this challenging condition., Competing Interests: Luciana Giugliani, Carlos Eduardo Steiner, Chong Ae Kim, Charles Marques Lourenço, Mara Lucia Schmitz Ferreira Santos, Carolina Fischinger Moura de Souza, Ana Carolina Brusius‐Facchin, Guilherme Baldo, Mariluce Riege, Roberto Giugliani declare that they have no conflict of interest.

Streptococcus pneumoniae is an opportunistic pathogen that colonizes the nasopharynx. Herein we show that carbon availability is distinct between the nasopharynx and bloodstream of adult humans: glucose is absent from the nasopharynx, whereas galactose is abundant. We demonstrate that pneumococcal neuraminidase A (NanA), which cleaves terminal sialic acid residues from host glycoproteins, exposed galactose on the surface of septal epithelial cells, thereby increasing its availability during colonization. We observed that S. pneumoniae mutants deficient in NanA and β-galactosidase A (BgaA) failed to form biofilms in vivo despite normal biofilm-forming abilities in vitro Subsequently, we observed that glucose, sucrose, and fructose were inhibitory for biofilm formation, whereas galactose, lactose, and low concentrations of sialic acid were permissive. Together these findings suggested that the genes involved in biofilm formation were under some form of carbon catabolite repression (CCR), a regulatory network in which genes involved in the uptake and metabolism of less-preferred sugars are silenced during growth with preferred sugars. Supporting this notion, we observed that a mutant deficient in pyruvate oxidase, which converts pyruvate to acetyl-phosphate under non-CCR-inducing growth conditions, was unable to form biofilms. Subsequent comparative transcriptome sequencing (RNA-seq) analyses of planktonic and biofilm-grown pneumococci showed that metabolic pathways involving the conversion of pyruvate to acetyl-phosphate and subsequently leading to fatty acid biosynthesis were consistently upregulated during diverse biofilm growth conditions. We conclude that carbon availability in the nasopharynx impacts pneumococcal biofilm formation in vivo Additionally, biofilm formation involves metabolic pathways not previously appreciated to play an important role., (Copyright © 2016, American Society for Microbiology. All Rights Reserved.)

Background GM1 gangliosidosis is a lysosomal storage disorder caused by mutations in GLB1, encoding β-galactosidase. The range of severity is from type I infantile disease, lethal in early childhood, to type III adult onset, resulting in gradually progressive neurological symptoms in adulthood. The intermediate group of patients has been recently classified as having type II late infantile subtype with onset of symptoms at one to three years of age or type II juvenile subtype with symptom onset at 2-10 years. To characterize disease severity and progression, six Late infantile and nine juvenile patients were evaluated using magnetic resonance imaging (MRI), and MR spectroscopy (MRS). Since difficulties with ambulation (gross motor function) and speech (expressive language) are often the first reported symptoms in type II GM1, patients were also scored in these domains. Deterioration of expressive language and ambulation was more rapid in the late infantile patients. Fourteen MRI scans in six Late infantile patients identified progressive atrophy in the cerebrum and cerebellum. Twenty-six MRI scans in nine juvenile patients revealed greater variability in extent and progression of atrophy. Quantitative MRS demonstrated a deficit of N-acetylaspartate in both the late infantile and juvenile patients with greater in the late infantile patients. This correlates with clinical measures of ambulation and expressive language. The two subtypes of type II GM1 gangliosidosis have different clinical trajectories. MRI scoring, quantitative MRS and brain volume correlate with clinical disease progression and may serve as important minimally-invasive outcome measures for clinical trials., (© 2015 Wiley Periodicals, Inc.)

GM1-gangliosidosis is a glycosphingolipid lysosomal storage disease involving accumulation of GM1 and its asialo form (GA1) primarily in the brain. Thin-layer chromatography and X-ray diffraction were used to analyze the lipid content/composition and the myelin structure of the optic and sciatic nerves from 7- and 10-month old β-galactosidase (β-gal) +/? and β-gal -/- mice, a model of GM1gangliosidosis. Optic nerve weight was lower in the β-gal -/- mice than in unaffected β-gal +/? mice, but no difference was seen in sciatic nerve weight. The levels of GM1 and GA1 were significantly increased in both the optic nerve and sciatic nerve of the β-gal -/- mice. The content of myelin-enriched cerebrosides, sulfatides, and plasmalogen ethanolamines was significantly lower in optic nerve of β-gal -/- mice than in β-gal +/? mice; however, cholesteryl esters were enriched in the β-gal -/- mice. No major abnormalities in these lipids were detected in the sciatic nerve of the β-gal -/- mice. The abnormalities in GM1 and myelin lipids in optic nerve of β-gal -/- mice correlated with a reduction in the relative amount of myelin and periodicity in fresh nerve. By contrast, the relative amount of myelin and periodicity in the sciatic nerves from control and β-gal -/- mice were indistinguishable, suggesting minimal pathological involvement in sciatic nerve. Our results indicate that the greater neurochemical pathology observed in the optic nerve than in the sciatic nerve of β-gal -/- mice is likely due to the greater glycolipid storage in optic nerve., (© The Author(s) 2015.)

G(M1)-gangliosidosis and Morquio B are rare lysosomal storage diseases associated with a neurodegenerative disorder or dwarfism and skeletal abnormalities, respectively. These diseases are caused by deficiencies in the lysosomal enzyme human β-D-galactosidase (h-β-GAL), which lead to accumulations of the h-β-GAL substrates, G(M1) ganglioside and keratan sulfate due to mutations in the h-β-GAL gene. H-β-GAL is an exoglycosidase that catalyzes the hydrolysis of terminal β-linked galactose residues. Here, we present the crystal structures of h-β-GAL in complex with its catalytic product galactose or with its inhibitor 1-deoxygalactonojirimycin. H-β-GAL showed a novel homodimer structure; each monomer was comprised of a catalytic TIM barrel domain followed by β-domain 1 and β-domain 2. The long loop region connecting the TIM barrel domain with β-domain 1 was responsible for the dimerization. To gain structural insight into the molecular defects of h-β-GAL in the above diseases, the disease-causing mutations were mapped onto the three-dimensional structure. Finally, the possible causes of the diseases are discussed.

Although β-galactosidases are physiologically a very important enzyme and have may therapeutics applications, very little is known about the stability and the folding aspects of the enzyme. We have used β-galactosidase from Pisum sativum (PsBGAL) as model system to investigate stability, folding, and function relationship of β-galactosidases. PsBGAL is a vacuolar protein which has a tendency to multimerize at acidic pH with protein concentration ≥100 μg mL⁻¹ and dissociates into its subunits above neutral pH. It exhibits maximum activity as well as stability under acidic conditions. Further, it has different conformational orientations and core secondary structures at different pH. Substantial predominance of β-content and interfacial interactions through Trp residues play crucial role in pH-dependent multimerization of enzyme. Equilibrium unfolding of PsBGAL at acidic pH follows four-state model when monitored by changes in the secondary structure with two intermediates: one resembling to molten globule-like state while unfolding seen from activity and tertiary structure of PsBGAL fits to two-state model. Unfolding of PsBGAL at higher pH always follows two-state model. Furthermore, unfolding of PsBGAL reveals that it has at least two domains: α/β barrel containing catalytic site and the other is rich in β-content responsible for enzyme multimerization.

Urine, amniotic fluid and ascitic fluid samples of galactosialidosis patients were analyzed and structurally characterized for free oligosaccharides using capillary high-performance anion-exchange chromatography with pulsed amperometric detection and online mass spectrometry. In addition to the expected endo-beta-N-acetylglucosaminidase-cleaved products of complex-type sialylated N-glycans, O-sulfated oligosaccharide moieties were detected. Moreover, novel carbohydrate moieties with reducing-end hexose residues were detected. On the basis of structural features such as a hexose-N-acetylhexosamine-hexose-hexose consensus sequence and di-sialic acid units, these oligosaccharides are thought to represent, at least in part, glycan moieties of glycosphingolipids. In addition, C(1)-oxidized, aldohexonic acid-containing versions of most of these oligosaccharides were observed. These observations suggest an alternative catabolism of glycosphingolipids in galactosialidosis patients: oligosaccharide moieties from glycosphingolipids would be released by a hitherto unknown ceramide glycanase activity. The results show the potential and versatility of the analytical approach for structural characterization of oligosaccharides in various body fluids.

beta-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 beta-galactosidase gene cause the lysosomal storage disease G(M1)-gangliosidosis. In order to identify additional molecular changes associated with the presence of beta-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 beta-galactosidase, beta-galactosidase variants and protective protein for beta-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 beta-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.

GM1-gangliosidosis is a lysosomal storage disease (LSD) caused by an autosomal recessive deficiency of lysosomal acid beta-galactosidase (betagal). 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 betagal 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 betagal in the adult GM1-gangliosidosis mouse brain upon hippocampal injection of an AAV vector-encoding betagal. 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.

Objective: Insulin resistance is an independent risk factor for the development of cardiovascular atherosclerosis. A key step in the development of atherosclerosis is endothelial dysfunction, manifest by a reduction in bioactivity of nitric oxide (NO). Insulin resistance is associated with endothelial dysfunction; however, the mechanistic relationship between these abnormalities and the role of impaired endothelial insulin signaling versus global insulin resistance remains unclear., Research Design and Methods: To examine the effects of insulin resistance specific to the endothelium, we generated a transgenic mouse with endothelium-targeted overexpression of a dominant-negative mutant human insulin receptor (ESMIRO). This receptor has a mutation (Ala-Thr(1134)) in its tyrosine kinase domain that disrupts insulin signaling. Humans with the Thr(1134) mutation are insulin resistant. We performed metabolic and vascular characterization of this model., Results: ESMIRO mice had preserved glucose homeostasis and were normotensive. They had significant endothelial dysfunction as evidenced by blunted aortic vasorelaxant responses to acetylcholine (ACh) and calcium ionophore. Furthermore, the vascular action of insulin was lost in ESMIRO mice, and insulin-induced endothelial NO synthase (eNOS) phosphorylation was blunted. Despite this phenotype, ESMIRO mice demonstrate similar levels of eNOS mRNA and protein expression to wild type. ACh-induced relaxation was normalized by the superoxide dismutase mimetic, Mn(III)tetrakis(1-methyl-4-pyridyl) porphyrin pentachloride. Endothelial cells of ESMIRO mice showed increased superoxide generation and increased mRNA expression of the NADPH oxidase isoforms Nox2 and Nox4., Conclusions: Selective endothelial insulin resistance is sufficient to induce a reduction in NO bioavailability and endothelial dysfunction that is secondary to increased generation of reactive oxygen species. This arises independent of a significant metabolic phenotype.

Galactosialidosis is a rare lysosomal storage disease caused by a combined deficiency of lysosomal beta-galactosidase and neuraminidase, due to a primary defect in protective protein/cathepsin A. Three subtypes are recognized: the early infantile type, the late infantile type, and the juvenile/adult type. Here, we report a case of early infantile galactosialidosis in a female who was born at 31 weeks of gestation, after detection of fetal ascites at 21 weeks of gestation and development of fetal hydrops. After birth she received intensive treatment that led to improvement of edema and pleural effusion, but ascites slowly developed. She died of renal failure on day 207. An autopsy showed that all organs contained vacuolated cells, compatible with a storage disease. The patient had decreased activity of beta-galactosidase and undetectable neuraminidase activity in fibroblasts. A single A-G base transition at position 146 of exon 1 (Q49R) in protective protein/cathepsin A gene was found. The mutation has been reported previously in a Japanese patient with different phenotypes. However homozygous Q49R mutation detected in our case was severe prognosis.

G(M1)-gangliosidosis is a lysosomal storage disorder caused by a deficiency of ss-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 G(M1)-gangliosidosis the study focused on the analysis of the molecular events and subsequent intracellular protein trafficking of beta-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 beta-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 G(M1)-gangliosidosis.

Mutations in the coding region of the WFS1 gene cause Wolfram syndrome, a rare multisystem neurodegenerative disorder of autosomal recessive inheritance. Patients with Wolfram syndrome display considerable clinical pleiomorphism, and symptoms such as neurological complications and psychiatric disorders are common. In the present study we have characterized Wfs1 expression pattern in the mouse central nervous system by using a combination of immunohistochemistry on wild-type mice and X-Gal staining of Wfs1 knockout mice with targeted insertion of the lacZ reporter. We identified a robust enrichment of Wfs1 protein in the central extended amygdala and ventral striatum. Prominent Wfs1 expression was seen in the hippocampal CA1 region, parasubiculum, superficial part of the second and third layers of the prefrontal cortex and proisocortical areas, hypothalamic magnocellular neurosecretory system, and central auditory pathway. Wfs1 expression was also detected in numerous brainstem nuclei and in laminae VIII and IX of the spinal cord. Wfs1-positive nerve fibers were found in the medial forebrain bundle, reticular part of the substantia nigra, globus pallidus, posterior caudate putamen, lateral lemniscus, alveus, fimbria, dorsal hippocampal commissure, subiculum, and to a lesser extent in the central sublenticular extended amygdala, compact part of substantia nigra, and ventral tegmental area. The neuroanatomical findings suggest that the lack of Wfs1 protein function can be related to several neurological and psychiatric symptoms found in Wolfram syndrome. Enrichment of Wfs1 protein in the central extended amygdala suggests a role in the modulation of anxiety and fear., (Copyright 2008 Wiley-Liss, Inc.)

The patient is a 24-year-old woman who first came for consultation at age 10 years. Based on clinical phenotype and thin-layer chromatography of urinary oligosaccharides, peripheral leukocytes were sent for beta-galactosidase assay. This testing showed a deficiency in enzyme activity, and gene mutation analysis identified a previously reported mutation p.H281Y (875C > T) and a novel mutation p.W273R (817T > C). Unlike previously reported patients, mutant enzymes in this patient's cultured skin fibroblasts did not respond to treatment with a chaperone compound, N-octyl-4-epi-beta-valienamine., ((c) 2008 Wiley-Liss, Inc.)

Galactosialidosis is an autosomal recessive lysosomal disease associated with a deficiency of beta-galactosidase and neuraminidase. Described herein is the case of a young adult who had been diagnosed with galactosialidosis at 8 years of age. At the age of 30 years, proteinuria and hematuria appeared and the patient underwent a renal biopsy 1 year later. Light microscopy of the kidney sections indicated fine granular contents in the cytoplasm of glomerular endothelial and epithelial cells, arteriolar smooth muscles and proximal tubular epithelial cells on periodic acid silver-methenamin (PAM) stain. Electron microscopy of these cells indicated enlarged, smooth endoplasmic reticulum and lysosomes containing 150 nm-wide rods with a fine lattice structure at 66 A periodicity. Moreover, electron-dense deposits were located in the paramesangial area. Immunofluorescence staining indicated diffuse and global anti-human IgA and C3-positive staining as a mesangial pattern. Given these findings this patient was therefore diagnosed with both galactosialidosis and IgA nephropathy. This is the first report to describe light and electron microscopy observations of storage materials in the kidneys in young/adult galactosialidosis.

G(M1)-gangliosidosis is an autosomal recessive lysosomal lipid storage disorder, caused by mutations of the lysosomal beta-galactosidase (beta-gal) and results in the accumulation of G(M1). The underlying mechanisms of neurodegeneration are poorly understood. Here we demonstrate increased autophagy in beta-gal-deficient (beta-gal(-/-)) mouse brains as evidenced by elevation of LC3-II and beclin-1 levels. Activation of autophagy in the beta-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 beta-gal(-/-) mouse. Mitochondria isolated from beta-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 G(M1)-gangliosidosis.

Different types of herbal yogurts were developed by mixing standardized milk with pretreated herbs, namely tulsi leaf (Ocimum sanctum), pudina leaf (Mentha arvensis) and coriander leaf (Coriandrum sativum), with leaves separately and a 1:1 (v/v) mixture of the strains of lactic starter cultures---Lactobacillus acidophilus (NCIM 2903) and Lactobacillus plantarum (NCIM 2083)-followed by incubation at 40 degrees C for 6 h. The beta-galactosidase enzymatic activity of the abovementioned herbal yogurts was determined and interestingly noted to exhibit higher enzymatic activity compared with the control yogurt (without any herbs). Among all herbal yogurts, tulsi yogurt had the maximum beta-galactosidase activity.

We reported four foodborne disease outbreaks in Taiwan caused by sucrose-nonfermenting and by beta-galactosidase-deficient variants of non-O1, non-O139 Vibrio cholerae. The sucrose-nonfermenting vibrios collected from three outbreaks were biochemically identified to be V. mimicus and the beta-galactosidase-deficient vibrios from an outbreak to be V. alginolyticus. However, molecular methods including DNA-DNA hybridization, fatty acid profile analysis, and sequence analysis of 16S rRNA, oriC, pyrH, recA, and rpoA indicated that these vibrios should be V. cholerae. These V. cholerae variants carried two hemolysin genes, hlyA and hlx, but contained neither cholera toxin gene, ctx, V. mimicus hemolysin gene, vmh, nor thermo-directed hemolysin, tdh. The sucrose-nonfermenting variants of V. cholerae shared a high level of genetic relatedness; they could derive from a common clone. In our record from 1995 to date, this was the first time that V. cholerae variants were discovered as etiologic agents for foodborne disease outbreaks in Taiwan.

Certain low-molecular-weight substrate analogs act both as in vitro competitive inhibitors of lysosomal hydrolases and as intracellular enhancers (chemical chaperones) by stabilization of mutant proteins. In this study, we performed oral administration of a chaperone compound N-octyl-4-epi-beta-valienamine to G(M1)-gangliosidosis model mice expressing R201C mutant human beta-galactosidase. A newly developed neurological scoring system was used for clinical assessment. N-Octyl-4-epi-beta-valienamine was delivered rapidly to the brain, increased beta-galactosidase activity, decreased ganglioside G(M1), and prevented neurological deterioration within a few months. No adverse effect was observed during this experiment. N-Octyl-4-epi-beta-valienamine will be useful for chemical chaperone therapy of human G(M1)-gangliosidosis.

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 G(M1)-gangliosidosis model mice. Two genetically engineered model mouse strains were used for this study: the beta-galactosidase-deficient knockout mouse representing infantile G(M1)-gangliosidosis (severe form), and transgenic mouse representing juvenile G(M1)-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 G(M1)-gangliosidosis model mice.

Objective: Transdifferentiation of bone marrow cells (BMC) into insulin-producing cells might provide a new cellular therapy for type I diabetes, but its existence is controversial. Our aim was to determine if those cells could transdifferentiate, even at low frequency, into insulin-producing cells, in testing optimized experimental conditions., Methods: We grafted mice with total BMC, genetically labeled either ubiquitarily, or with a marker conditionally expressed under the control of the insulin beta-cell specific promoter. We treated some of the recipients with an agent toxic to beta-cells (streptozotocin) and with cytokines stem cell factor (SCF) and granulocyte-colony stimulating factor (G-CSF)., Results: The contribution of grafted cells could be detected neither for natural turnover (n=6), nor for beta-cell regeneration after pancreatic lesion (n=7), 90 days post-transplantation. Cytokine mobilization of BMC in the blood stream, reported to favor their transdifferentiation into cardiac and neural cells, had never been tested before for beta-cell generation. Here, we showed that injection of SCF and G-CSF did not lead to a detectable level of transdifferentiation (n=7)., Conclusions: We conclude that BMC cannot spontaneously transdifferentiate into insulin-producing cells in vivo, even after beta-cell lesion and mobilization induced by cytokines. Interestingly, however, treatment by cytokines may have beneficial indirect effects on STZ-induced hyperglycaemia.

GM1-gangliosidosis is a glycosphingolipid (GSL) lysosomal storage disease caused by autosomal recessive deficiency of lysosomal acid beta-galactosidase (betagal), and characterized by accumulation of GM1-ganglioside and GA1 in the brain. Here we examined the effect of neonatal intracerebroventricular (i.c.v.) injection of an adeno-associated virus (AAV) vector encoding mouse betagal on enzyme activity and brain GSL content in GM1-gangliosidosis (betagal(-/-)) mice. Histological analysis of betagal distribution in 3-month-old AAV-treated betagal(-/-) mice showed that enzyme was present at high levels throughout the brain. Biochemical quantification showed that betagal activity in AAV-treated brains was 7- to 65-fold higher than in wild-type controls and that brain GSL levels were normalized. Cerebrosides and sulfatides, which were reduced in untreated betagal(-/-) mice, were restored to normal levels by AAV treatment. In untreated betagal(-/-) brains, cholesterol was present at normal levels but showed abnormal cellular distribution consistent with endosomal/lysosomal localization. This feature was also corrected in AAV-treated mice. The biochemical and histological parameters analyzed in this study showed that normal brain neurochemistry was achieved in AAV-treated betagal(-/-) mice. Therefore we show for the first time that neonatal AAV-mediated gene delivery of lysosomal betagal to the brain may be an effective approach for treatment of GM1-gangliosidosis.

Lipid storage diseases are debilitating inherited metabolic disorders that stem from the absence of specific lysosomal enzymes that degrade selected lipids. Most characteristically, these disorders affect the nervous and the reticulo-endothelial systems, with massive organomegaly resulting from the presence of engorged, lipid-laden macrophages. In this issue of the JCI, Yildiz et al. describe the role of the ER-resident enzyme beta-glucosidase 2 (GBA2) in mice (see the related article beginning on page 2985). Surprisingly, GBA2 deficiency leaves bile acid and cholesterol metabolism intact, instead causing lipid accumulation in the ER of testicular Sertoli cells, round-headed sperm (globozoospermia), and impaired male fertility.

Lipocalin-type prostaglandin (PG) D synthase (L-PGDS) is a dually functional protein, acting both as a PGD2-synthesizing enzyme and as an extracellular transporter of various lipophilic small molecules. L-PGDS is expressed in oligodendrocytes (OLs) in the central nervous system and is up-regulated in OLs of the twitcher mouse, a model of globoid cell leukodystrophy (Krabbe's disease). We investigated whether up-regulation of L-PGDS is either unique to Krabbe's disease or is a more generalized phenomenon in lysosomal storage disorders (LSDs), using LSD mouse models of Tay-Sachs disease, Sandhoff disease, GM1 gangliosidosis and Niemann-Pick type C1 disease. Quantitative RT-PCR revealed that L-PGDS mRNA was up-regulated in the brains of all these mouse models. In addition, strong L-PGDS immunoreactivity was observed in OLs, but not in either astrocytes or microglia in these models. Thus, up-regulation of L-PGDS appears to be a common response of OLs in LSDs. Moreover, surface plasmon resonance analyses revealed that L-PGDS binds GM1 and GM2 gangliosides, accumulated in neurons in the course of LSD, with high affinities (KD = 65 and 210 nm, respectively). This suggests that L-PGDS may play a role in scavenging harmful lipophilic substrates in LSD.

Unlabelled: Between 40 and 50 lysosomal storage disorders are known at present. Fine details of the pathogenic process involved are in general not known. This overview highlights the basic principles of lysosomal pathogenesis and the clinical consequences of defective genes involved in lysosomal functions. The subject is discussed in the context of the possibility of prevention and reversal of cellular and organ damage by enzyme replacement therapy. Also presented is a mechanical model for the muscle pathology observed in Pompe disease. Direct mechanical effects of the non-contractile inclusions - glycogen-loaded lysosomes - seem to be a key factor in the loss of force during both early and late stages of the disease., Conclusion: Each lysosomal storage disorder and each patient with a given lysosomal disorder has unique molecular, pathological and clinical features. But, the order of pathological events is largely the same. Mutations in a gene cause lysosomal dysfunction which, in turn, results in cellular pathology affecting organ structure and function. Clinical symptoms are the ultimate manifestation. The reversibility of symptoms with enzyme replacement therapy will vary according to the disease, as well as the nature and stage of organ pathology.

We propose a new molecular therapeutic approach to lysosomal diseases with severe neurological manifestations. Some low-molecular-weight compounds, acting as competitive inhibitors of a lysosomal enzyme in vitro, were found to stabilize and restore catalytic activities of the enzyme molecule as a molecular chaperone. We started this trial first in Fabry disease (generalized vasculopathy) using galactose and 1-deoxygalactonojirimycin, and then in beta-galactosidase deficiency disorders (beta-galactosidosis) with generalized neurosomatic and/or systemic skeletal manifestations (GM(1)-gangliosidosis and Morquio B disease), using a newly developed chemical compound N-octyl-4-epi-beta-valienamine (NOEV). Administration of this chaperone compound resulted in elevation of intracellular enzyme activity in cultured fibroblasts from patients and genetically engineered model mice. In addition, substrate storage was improved after NOEV had been transported into the brain tissue via the blood-brain barrier. We hope this new approach (chemical chaperone therapy) will be useful for certain patients with beta-galactosidosis and potentially other lysosomal storage diseases with central nervous system involvement.

The human GLB1 gene encodes a lysosomal beta-galactosidase (beta-Gal) and an elastin-binding protein (EBP). Defect of the EBP as a chaperon for tropoelastin and a component of receptor complex among neuraminidase-1 (NEU1) and protective protein/cathepsin A (PPCA) is suggested responsible for impaired elastogenesis in autosomal recessive beta-Gal, PPCA and NEU1 deficiencies. The purpose of this study is to determine effects of GLB1, PPCA and NEU1 gene mutations on elastogenesis in skin fibroblasts. Elastic fiber formation and the EBP mRNA expression were examined by immunofluorescence with an anti-tropoelastin antibody and RT-PCR selective for EBP in skin fibroblasts with these lysosomal enzyme deficiencies. Apparently normal elastogenesis and EBP mRNA expression were observed for fibroblasts from Morquio B disease cases with the GLB1 gene alleles (W273L/W273L, W273L/R482H and W273L/W509C substitutions, respectively), a galactosialidosis case with the PPCA allele (IVS7+3A/IVS7+3A) and a sialidosis case with the NEU1 allele (V217M/G243R) as well as normal subject. In this study, the W273L substitution in the EBP could impossibly cause the proposed defect of elastogenesis, and the typical PPCA splicing mutation and the V217M/G243R substitutions in the NEU1 might hardly have effects on elastic fiber formation in the dermal fibroblasts.

GM1 gangliosidosis is due to beta-galactosidase deficiency. Only patients with type 3 disease survive into adulthood and develop movement disorders. Clinical descriptions of this form are rare, particularly in non-Japanese patients. We describe four new patients and systematically analyze all previous reports found by a literature search and contacts with the authors for additional information. Generalized dystonia remained the predominant feature throughout the disease course and was often associated with akinetic-rigid parkinsonism. GM1 gangliosidosis must be considered as a cause of early-onset generalized dystonia, particularly in patients with short stature and skeletal dysplasia., (Copyright (c) 2005 Movement Disorder Society.)

The Kluyveromyces lactis lac4 mutants, lacking the beta-galactosidase gene, cannot assimilate lactose, but grow normally on many other carbon sources. However, when these carbon sources and lactose were simultaneously present in the growth media, the mutants were unable to grow. The effect of lactose was cytotoxic since the addition of lactose to an exponentially-growing culture resulted in 90% loss of viability of the lac4 cells. An osmotic stabilizing agent prevented cells killing, supporting the hypothesis that the lactose toxicity could be mainly due to intracellular osmotic pressure. Deletion of the lactose permease gene, LAC12, abolished the inhibitory effect of lactose and allowed the cell to assimilate other carbon substrates. The lac4 strains gave rise, with unusually high frequency, to spontaneous mutants tolerant to lactose (lar1 mutation: lactose resistant). These mutants were unable to take up lactose. Indeed, lar1 mutation turned out to be allelic to LAC12. The high mutability of the LAC12 locus may be an advantage for survival of K. lactis whose main habitat is lactose-containing niches.