Lymphocyte Galactocerebrosidase Activity by LC-MS/MS for Post-Newborn Screening Evaluation of Krabbe Disease.

BACKGROUND: Deficiency of the lysosomal enzyme galactosylcerebrosidase (GALC) causes Krabbe disease. Newborn screening for Krabbe disease is ongoing, but improved methods for follow-up analysis of screen-positive babies are needed to better advise families and to optimize treatment. We report a new assay for the enzymatic activity of GALC in lymphocytes. METHODS: T lymphocytes were isolated from venous blood by magnetic bead technology. The assay used a close structural analog of the natural substrate and LC-MS/MS to quantify the amount of product with the aid of a chemically identical internal standard. RESULTS: The analytical range of the assay (ratio of assay response for the QC high standard to that from all non-enzymatic-dependent processes) was 20-fold greater than that for the conventional radiometric GALC assay. The LC-MS/MS could distinguish cells that were null in GALC from those that contained traces of active enzyme (down to 0.3% of normal). There was a good correlation between the level of residual GALC activity in lymphocytes and the severity of Krabbe disease. CONCLUSIONS: The new assay can measure small amounts of residual GALC activity in leukocytes with high accuracy compared to previous assays and can contribute, along with genotyping, biomarker analysis, and neurological imaging, a better plan for post-newborn screening follow-up for Krabbe disease.

Mesenchymal lineage stem cells have pronounced anti-inflammatory effects in the twitcher mouse model of Krabbe's disease.

The twitcher mouse is an animal model of Krabbe's disease (KD), which is a neurodegenerative lysosomal storage disorder resulting from the absence of functional lysosomal enzyme galactocerebrosidase (GALC). This disease affects the central and peripheral nervous systems and in its most severe form results in death before the age of 2 in humans and approximately 30-40 days in mice. This study evaluates the effect of intracerebroventricular administration of mesenchymal stem cells derived from adipose tissue (ASCs) and bone marrow (BMSCs) on the pathology of KD. Subsequent to the intracerebroventricular injection of ASCs or BMSCs on postnatal day (PND) 3-4, body weight, lifespan, and neuromotor function were evaluated longitudinally beginning on PND15. At sacrifice, tissues were harvested for analysis of GALC activity, presence of myelin, infiltration of macrophages, microglial activation, inflammatory markers, and cellular persistence. Survival analysis curves indicate a statistically significant increase in lifespan in stem cell-treated twitcher mice as compared with control twitcher mice. Body weight and motor function were also improved compared with controls. The stem cells may mediate some of these benefits through an anti-inflammatory mechanism because the expression of numerous proinflammatory markers was downregulated at both transcriptional and translational levels. A marked decrease in the levels of macrophage infiltration and microglial activation was also noted. These data indicate that mesenchymal lineage stem cells are potent inhibitors of inflammation associated with KD progression and offer potential benefits as a component of a combination approach for in vivo treatment by reducing the levels of inflammation.

Specific determination of beta-galactocerebrosidase activity via competitive inhibition of beta-galactosidase.

BACKGROUND: The determination of cellular beta-galactocerebrosidase activity is an established procedure to diagnose Krabbe disease and monitor the efficacy of gene/stem cell-based therapeutic approaches aimed at restoring defective enzymatic activity in patients or disease models. Current biochemical assays for beta-galactocerebrosidase show high specificity but generally require large protein amounts from scanty sources such as hematopoietic or neural stem cells. We developed a novel assay based on the hypothesis that specific measurements of beta-galactocerebrosidase activity can be performed following complete inhibition of beta-galactosidase activity. METHODS: We performed the assay using 2-7.5 microg of sample proteins with the artificial fluorogenic substrate 4-methylumbelliferone-beta-galactopyranoside (1.5 mmol/L) resuspended in 0.1/0.2 mol/L citrate/phosphate buffer, pH 4.0, and AgNO(3). Reactions were incubated for 30 min at 37 degrees C. Fluorescence of liberated 4-methylumbelliferone was measured on a spectrofluorometer (lambda(ex) 360 nm, lambda(em) 446 nm). RESULTS: AgNO(3) was a competitive inhibitor of beta-galactosidase [inhibition constant (K(i)) = 0.12 micromol/L] and completely inhibited beta-galactosidase activity when used at a concentration of 11 micromol/L. Under this condition, the beta-galactocerebrosidase activity was preserved and could be specifically and accurately measured. The assay can detect beta-galactocerebrosidase activity in as little as 2 microg cell protein extract or 7.5 microg tissue. Assay validation was performed using (a) brain tissues from wild-type and twitcher mice and (b) murine GALC(-/-) hematopoietic stem cells and neural precursor cells transduced by GALC-lentiviral vectors. CONCLUSIONS: The procedure is straightforward, rapid, and reproducible. Within a clinical context, our method unequivocally discriminated cells from healthy subjects and Krabbe patients and is therefore suitable for diagnostic applications.

Newborn screening for Krabbe disease: the New York State model.

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

Enzyme replacement therapy results in substantial improvements in early clinical phenotype in a mouse model of globoid cell leukodystrophy.

Globoid cell leukodystrophy (GLD) or Krabbe disease is a devastating, degenerative neurological disorder caused by mutations in the galactosylceramidase (GALC) gene that severely affect enzyme activity. Currently, treatment options for this disorder are very limited. Enzyme replacement therapy (ERT) has been shown to be effective in lysosomal storage disorders with predominantly peripheral manifestations such as type I Gaucher's and Fabry's disease. Little however is known about the possible benefit of ERT in GLD, which has a substantial central nervous system component. In this study, we examined the effect of peripheral GALC injections in the twitcher mouse model of the disease. Although we were unable to block the precipitous decline that normally occurs just before death, we did observe significant early improvements in motor performance, a substantial attenuation in the initial failure to thrive, and an increase in life span. Immunohistochemical and activity analyses demonstrated GALC uptake in multiple tissues, including the brain. This was associated with a decrease in the abnormal accumulation of the GALC substrate psychosine, which is thought to play a pivotal role in disease pathology. These results indicate that peripheral ERT is likely to be beneficial in GLD.

Diagnosis of Krabbe disease by use of a natural substrate.

This chapter describes in detail a practical procedure for the preparation of radiolabeled galactocerebroside and its use in the assay of galactocerebrosidase (GalCase), the enzyme deficient in globoid cell leukodystrophy (Krabbe disease). The reference range for leukocytes and fibroblasts is 0.9-4.4 and 8-36 nmoles substrate hydrolyzed per hour per milligram of protein, respectively. Because of its low abundance this enzyme is difficult to assay in certain situations, such as prenatal diagnosis by chorionic villus sampling. To obviate this a modified assay is used where only the radiolabeled substrate is included in the incubation. This provides a clear separation between affected samples and unaffected controls. The methods detailed here should be reproducible in any laboratory.

Use of a fluorescent analogue of galactocerebroside for assay of galactocerebroside beta-galactosidase activity in skin fibroblasts from patients with Krabbe's disease.

In order to diagnose Krabbe's disease (globoid cell leukodystrophy) or its carrier state in patients, galactocerebroside beta-galactosidase (galactosylceramidase) activity in cultured skin fibroblasts was measured with high-performance liquid chromatography using a fluorescent analogue of galactocerebroside, 1-O-galactosyl-2-N-(1-dimethylaminonaphthalene-5-sulfonyl)-sphingosine, as substrate. The enzyme activities in skin fibroblasts of the patients were found to be dramatically reduced when compared with those of controls. The assay can be adopted for diagnostic use in place of assays using radioisotope-labeled natural substrate.

Prenatal diagnosis of Krabbe disease using a fluorescent derivative of galactosylceramide.

A fluorescent substrate 12-(N-methyl-N(7-nitro-2-oxa-1,3-diazol-4-yl) aminododecanoyl sphingosyl beta-D-galactoside ('NBD galactocerebroside') was synthesized and used for the detection of galactocerebrosidase activity. The enzyme determinations using this substrate were found to be extremely sensitive yielding unambiguous results. This substrate was used for the prenatal diagnosis of a fetus affected with Krabbe disease; the diagnosis was later confirmed in the aborted fetus.

Application of a galactosylceramidase microassay method to early prenatal diagnosis of Krabbe's disease.

An enzymatic microassay method was devised to determine galactosylceramidase activities of microsamples prepared from primary cultures of amniotic fluid cells. A piece of freeze-dried cell mass (0.5--1 microgram dry weight) prepared from a colony on the bottom plate of a culture flask, was weighed and incubated in 4.02 microliter of the assay mixture. A small amount of galactose hydrolyzed from galactosylceramide was amplified about 10 000-fold and determined by using an enzymatic amplification reaction, NAD cycling. Microsamples were also prepared from freeze-dried leukocytes and skin fibroblasts. The present method was applied to prenatal diagnosis using amniotic cells as a sample in a case of high-risk pregnancy for Krabbe's disease. The result was confirmed by analysis of fibroblasts and organs from the aborted fetus.

Pathological and biochemical studies of fetal Krabbe disease.

Morphological and biochemical analysis of tissue from a 21-week-old fetus with Krabbe disease was performed. Galactosylceramidase activity was virtually absent in cultured amniotic cells obtained during the pregnancy of this fetus. The prenatal diagnosis was confirmed by enzymatic analysis of fetal cultured skin fibroblasts and by enzyme analysis of fetal brain, kidney and liver. The galactocerebroside content of brain and spinal cord of the affected fetus was essentially identical to that observed in an age-matched control fetus. Accumulation of galactosylsphingosine was found in all tissues examined from the fetus with Krabbe disease. The highest galactosylsphingosine level was detected in spinal cord of the affected fetus: it was 40 times the concentration observed in controls. The occurrence of inclusion bodies were limited to spinal cord of the fetus with Krabbe disease. These data verify that the pathological and biochemical findings of Krabbe disease are present during the second trimester of pregnancy.

[Study of the specificity of human lysosomal glycolipid hydrolases using synthetic fluorogenic substrates]. / Issledovanie spetsifichnosti lizosomnykh glikolipidgidrolaz cheloveka s pomoshch'iu sinteticheskikh fluorogennykh substratov.

On the basis of o-acylamino-4-methylumbelliferon, a number of beta-galactosides and beta-glucosides have been synthesized. The fluorogenic compounds obtained differ by the length of acyl residues. 6- and 8-hexadecanoylamino-4-methylumbelliferyl-beta-D-galactopyranosides (6-HMGal and 8-HMGal) are shown to be substrates for human galactocerebroside-beta-D-galactosidase. 6-HMGal analogues with shorter acyl residues, octanoyl (OMGal) and butanoyl (BMGal), were cleaved by another type of beta-galactosidase, GM1-ganglioside-beta-galactosidase. It has been established that 6-hexadecanoylamino-4-methylumbelliferyl-beta-D-glucopyranoside (HMGlc) is cleaved by human and animal glucocerebrosidase much slower than its chromogenic analogue (HMGlc). OMGlc did not exceed HNGlc either, though it is cleaved by glucocerebrosidase faster than HMGlc.

[Identification of Krabbe disease in 2 brothers from East Germany using a new fluorogenic substrate for galactocerebrosidase]. / Identifikatsiia bolezni Krabbe u dvukh brat'ev iz GDR s pomoshch'iu novogo fluorogennogo substrata dlia galaktotserebrozidazy.

Activity of several lysosomal hydrolases was studied in skin fibroblasts obtained from two brothers living in GDR. Both patients exhibited distinct clinical symptoms of severe neurovisceral disease. Analysis of the lysosomal enzymes activity enabled to exclude possible occurrence in the patients of such glycolipidoses as Gaucher's disease, Sandhoff's disease, GM1-gangliosidosis and metachromatic leukodystrophy. A new fluorogenic galactoside of lipid nature 6-hexadecanoylamine-4-hethylumbellipheryl-beta-D-galactoside used as a substrate of galactocerebrosidase enabled to detect in the patients distinct decrease in this enzymatic activity and to diagnose Krabb's disease. Biochemical diagnosis of Krabb's disease using the fluorogenic substrate was also confirmed by analysis with labelled galactocerebroside as a substrate.

[Krabbe disease (globoid cell leukodystrophy)].

Krabbe disease is an autosomal recessive inherited demyelinating disease, which is deficient in lysosomal enzyme, galactocerebrosidase. Pathophysiological characteristics of this disease are extreme demyelination in white matter and peripheral nerve, existence of globoid cells, absence of accumulation of main substrates, i.e. galactocerebrosidase in tissues and accumulation of psychosine. Molecular basis of this disease including isolation of a cDNA for human and murine galactocerebrosidase and cloning of genome of this gene are reviewed. The trial of gene therapy on twitcher, the mouse model of Krabbe disease, could break through on therapy on this progressive demyelinating disease.

[Biochemical diagnosis of globoid cell leukodystrophy (Krabbe's disease)]. / Biokhimicheskii diagnoz globoidokletochnoi leikodistrofii (bolezn' Krabbe).

beta-Galactosylceramidase activity was deficient in leukocytes of a 5-month old child with neuro-degenerative disease. The activities of beta-galactosidase and arylsulphatase A were within normal limits. The beta-galactosylcerebrosidase activity in the mother's and father's leukocytes was 25% and 68%, respectively of the mean control values. A sharp decrease of beta-galactosylceramidase activity was found in cultured skin fibroblasts of the child. The data obtained indicate that the child suffered from globoid cell leukodystrophy (Krabbe's disease). The diagnosis was confirmed after liver and brain autopsy. The beta-galactosylceramidase was not revealed in these tissues. Typical globoid cells were observed in microscopical examination of the brain.

Design and optimization of lentiviral vectors for transfer of GALC expression in Twitcher brain.

BACKGROUND: Demyelination in globoid cell leukodystrophy (GLD) is due to a deficiency of galactocerebrosidase (GALC) activity. Up to now, in vivo brain viral gene transfer of GALC showed modest impact on disease development in Twitcher mice, an animal model for GLD. Lentiviral vectors, which are highly efficient to transfer the expression of therapeutic genes in neurons and glial cells, have not been evaluated for direct cerebral therapy in GLD mice. METHODS: Lentiviral vectors containing the untagged cDNA or the hemagglutinin (HA)-tagged cDNA for the full-length mouse GALC sequence were generated and validated in vitro. In vivo therapeutic efficacy of these vectors was evaluated by histology, biochemistry and electrophysiology after transduction of ependymal or subependymal layers in young Twitcher pups. RESULTS: Both GALC lentiviral vectors transduced neurons, oligodendrocytes and astrocytes with efficiencies above 75% and conferred high levels of enzyme activity. GALC accumulated in lysosomes of transduced cells and was also secreted to the extracellular medium. Conditioned GALC medium was able to correct the enzyme deficiency when added to non-transduced Twitcher glial cultures. Mice that received intraventricular injections of GALC vector showed accumulation of GALC in ependymal cells but no diffusion of the enzyme from the ependymal ventricular tree into the cerebral parenchyma. Significant expression of GALC-HA was detected in neuroglioblasts when GALC-HA lentiviral vectors were injected in the subventricular zone of Twitcher mice. Life span and motor conduction in both groups of treated Twitcher mice were not significantly ameliorated. CONCLUSIONS: Lentiviral vectors showed to be efficient for reconstitution of the GALC expression in Twitcher neural cells. GALC was able to accumulate in lysosomes as well as to enter the secretory pathway of lysosomal enzymes, two fundamental aspects for gene therapy of lysosomal storage diseases. Our in vivo results, while showing the capacity of lentiviral vectors to transfer expression of therapeutic GALC in the Twitcher brain, did not limit progression of disease in Twitchers and highlight the need to evaluate other routes of administration.

Prenatal diagnosis of globoid cell leukodystrophy (Krabbe's diseases). Third documented case.

A case of globoid cell leukodystrophy (Krabbe's disease) was diagnosed prenatally by demonstrating a profound deficiency of cerebroside beta-galactosidase in cultured amniotic cells. The diagnosis was confirmed in the fetus aborted in the 19th week. In the cell-free amniotic fluid, normal enzyme activity was found. This finding, which had been demonstrated in a previous case, is discussed.

[A new fluorogenic substrate for human galactocerebroside-beta-D-galactosidase]. / Novyi fluorogenny i substrat dlia galaktotserebrozid-beta-D-galaktozidazy cheloveka.

A new fluorogenic compound--6-hexadecanoylamino-4-methyl-umbelliferyl-beta-D-gala cto pyranoside (HMGal), a substrate for human galactocerebroside beta-D-galactosidase (HG), has been synthesized. A method for determining the HG activity based on the use of HMGal as a fluorogenic substrate has been developed. The specificity of HMGal hydrolysis by HG has been demonstrated in experiments with enzyme preparations from human skin fibroblasts and leukocytes in normally and in hereditary glycolipidosis (GM1-gangliosidosis and Krabbe's disease). The use of HMGal permits to markedly increase the sensitivity of the method used for determining the HG activity.

Analysis of galactocerebrosidase activity in the mouse brain by a new histological staining method.

Gene therapy of galactocerebrosidase (GALC) deficient mice (Twitcher mutants) requires a fast and sensitive assay to detect transduced cells in vitro and in vivo. We have developed a new rapid histochemical method that specifically detects GALC activity in situ in neural cells using 5-Br-3Cl-beta-galactopiranoside (X-Gal) in the presence of taurodeoxycholic and oleic acids to enhance suspension of the substrate at low pH. Using this method, we observed robust X-Gal staining in diverse neuronal populations and interfascicular oligodendrocytes in sections from normal mouse brain. In contrast, sections of Twitcher brain did not show a specific staining pattern in neurons or glial cells. The availability of this new sensitive and rapid in situ detection assay is fundamental for the follow-up of Twitcher mice under gene or cellular therapies to correct central GALC deficiency.