h3. Epidemiology
p<>. Globoid cell leukodystrophy (GLD), or Krabbe’s disease, is a severe, inherited in an autosomal recessive pattern , metabolic disorder in which normal myelin formation is blocked, and multinucleate “globoid cells” accumulate in the brain. Krabbe disease occurs in about 1 in 100,000 births. A higher incidence, about 1 in 6,000, has been reported in some Arab communities in Israel. Scandinavian countries have comparatively high rates of the disease, reported to be 1 in 50,000 births.
h3. DESCRIPTION
p<>. Krabbe disease is caused by mutations in the GALC gene located on chromosome 14 (14q31)
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which causes a deficiency of an enzyme called galactocerebrosidase. The build up of unmetabolized lipids affects the growth of the nerve's protective myelin sheath (the covering that insulates many nerves) and causes severe degeneration of motor skills. As part of a group of disorders known as leukodystrophies, Krabbe disease results from the imperfect growth and development of myelin. GALC deficiency also results in a build up of a glycosphingolipid called Psychosine.
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Figure 1
Structures of lipids.
It has been suggested Psychosine causes axonal degeneration in both the CNS and PNS by disrupting lipid rafts
The primary defect is an absence of the lipid-degrading enzyme galactoceramidase, which cleaves the galactose headgroup from galactoceramide. A related lipid metabolite, psychosine, accumulates in the brain . Psychosine is normally broken down by galactoceramidase, and that in its absence psychosine accumulates, causing death of oligodendrocytes. These are the cells that normally synthesize galactoceramide during myelination, so their death would account for the absence of galactoceramide buildup, and also GLD pathology.
The main sites for the degradation of glycosphingolipids are the lysosomes. These are membrane-bound organelles that comprise a limiting external membrane and internal lysosomal vesicles, which contain digestive enzymes that are active at the acidic pH of this organelle. Most of these enzymes are soluble and localized in the lysosomal lumen. All membrane components are actively transported to the lysosomes to be broken down into their various primary components. In the case of glycosphingolipids, this means to fatty acids, sphingoid bases and monosaccharides, which can be recovered for re-use or further degraded. Degradation of oligoglycosylceramides and gangliosides occurs by sequential removal of monosaccharide units via the action of specific exohydrolases from the non-reducing end until a monoglycosylceramide unit is reached. Then glucosylceramide β-glucosidase or an analogous β-galactosidase removes the final carbohydrate moiety to yield ceramides, which are in turn hydrolysed by an acid ceramidase to fatty acids and sphingoid bases. In addition, a non-lysosomal degrading enzyme for glucosylceramide has been found in the endoplasmic reticulum. The process requires the presence of specific activator proteins, which are glycoproteins of low molecular weight. These are not themselves active catalytically but are required as cofactors either by directing the enzyme to the substrate or by activating the enzyme by binding to it in some manner. Five such proteins are known, the GM2-activator protein (specific for gangliosides) and saposins A, B, C and D. The four saposins are derived by proteolytic processing from a single precursor protein, prosaposin, which is synthesised in the endoplasmic reticulum, transported to the Golgi for glycosylation and then to the lysosomes. Saposin A is essential for the degradation of galactosylceramide, saposin B for that of sulfatide and globotriaosylceramide, and saposin C for that of glucosylceramide. Although there are suggestions that it may activate the acid ceramidase, the function of saposin D is less clear.
Psychosine is the trivial name for a monoglycosylsphingolipid, which is the non-acylated or lyso form of a cerebroside, normally galactosylsphingosine. It is a minor intermediate in the catabolism of monoglycosylceramides, and is normally present in tissues at very low concentrations. A deficiency of the enzyme β-galactosylceramidase, responsible for catabolism of galactosylceramide, is usually noted when psychosine accumulates in significant amounts. Deacylation of the galactosylceramide would then lead to formation of psychosine, although addition of galactose to sphingosine cannot be ruled out. For this reason, psychosine accumulates in tissues in the genetic disorder, Krabbe's disease (globoid cell leukodystrophy), and to a certain extent also in Gaucher's disease. In Krabbe disease the concentration of psychosine in the oligodendroglial cells steadily increases, reaches toxic levels, and kills the cells. The selective destruction of the oligodendroglial cells results in an arrest of the myelin formation. As a consequence of the arrest of the myelin formation and the destruction of the oligodendroglial cells, the concentration of cerebrosides and sulfatides of white matter is significantly lower than in age-matched controls. Also two other glycolipids which normally occur in white matter are reduced, namely glucosylcerarnide and lactosylceramide. These lipids closely resemble galactosylceramides in composition of unsubstituted fatty acids, which suggests that they are normally formed from the same fatty acid pool ( in the oligodendroglial cells). Cytochemistry and time-lapse imaging of dividing cells showed that psychosine did not induce cell fusion; rather, it blocked cytokinesis, uncoupling mitosis from cell division. This discovery is the first reported inhibition of cytokinesis by a physiologically occurring small molecule, and a large step forward in understanding the pathogenesis of GLD. Psychosine is a lysolipid, with detergent-like properties, so its target might in principle be some aspect of membrane biophysics rather than a specific protein. The target of psychosine is a GPCR called TDAG8, previously named for its high expression in T cells undergoing apoptosis but otherwise uncharacterized. Discovering the ligands for “orphan” GPCRs and nuclear receptors (receptors identified by sequence for which ligands and function are unknown) is an important endeavor. GPCRs are involved in regulating many aspects of physiology, and GPCR agonists and antagonists constitute one of the largest categories of therapeutic drugs. Orphan GPCRs may thus be the targets for drugs of the future.
GPCRs are notable for the diversity of their ligands, including proteins, small molecules, and even photons. An important class of GPCR ligands is lipid mediators, metabolites of common lipids that play important and diverse roles in signaling between and within cells.
A GPCR receptor for sphingosylphosphorylcholine , named OGR1, was identified .Im et al. (2000, 2001) were also interested in identifying new sphingosine receptors, and they expressed in cells the orphan GPCR, TDAG8, that is 41% identical to OGR1, with the expectation that its ligand might be a lipid related to sphingosylphosphorylcholine.
This lipid is structurally related to psychosine; both are lyso-sphingolipids; that is, sphingolipids that lack the second fatty acid normally attached as an amide to the amino group of sphingosine . Im et al. (2001) found that TDAG8 is activated by psychosine and related lysosphingolipids. TDAG8 and psychosine promotes accumulation of multinucleate cells.
TDAG8 is the receptor for psychosine and a cytokinesis blocker. The relatively low apparent affinity of psychosine for TDAG8 is consistent with TDAG8 being the psychosine receptor in GLD, where the psychosine accumulates to high concentrations. But it argues caution in concluding that psychosine is the normal physiological ligand for TDAG8. A second potential concern is that psychosine might have additional effects that are required in addition to activating TDAG8 for blocking cytokinesis. This ligand–receptor pair may represent a physiological pathway for regulating cytokinesis and multinucleation .The actin clots were associated with clusters of vacuoles near the plasma membrane. The vacuoles were reminiscent of some endocytic compartment, and endosomes are known to associate with actin. These observations argue for effects of psychosine/TDAG8 on interphase cortical dynamics in addition to their effect on cytokinesis. It is even possible that the block to cytokinesis induced by psychosine/TDAG8 is a secondary effect, due to the actin clot interfering with cytokinesis.
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Figure 2
DNA (red) and actin (green) staining of psychosine-treated U937 cells. These cells are now known to express TDAG8 (Im et al., 2001). The images show sequential stages of failed cytokinesis in fixed cells. Note the large “clot” of actin present in all the images.
At the EM level this clot contains many small vesicles.
Symptoms
Infants with Krabbe's disease are normal at birth. Symptoms begin between the ages of 3 and 6 months with irritability, fevers, limb stiffness, seizures, feeding difficulties, vomiting, and slowing of mental and motor development. In the first stages of the disease, doctors often mistake the symptoms for those of cerebral palsy. Other symptoms include muscle weakness, spasticity, deafness, optic atrophy and blindness, paralysis, and difficulty when swallowing. Prolonged weight loss may also occur. There are also juvenile- and adult-onset cases of Krabbe disease, which have similar symptoms but slower progression
diagnosis
The disease may be diagnosed by its characteristic grouping of certain cells (multinucleated globoid cells), nerve demyelination and degeneration, and destruction of brain cells. Special stains for myelin (e.g.; luxol fast blue) may be used to aid diagnosis. In almost all individuals with Krabbe disease, galactocerebrosidase (GALC) enzyme activity is deficient (0%-5% of normal activity) in leukocytes isolated from whole heparinized blood or in cultured skin fibroblasts. Testing is most reliable when conducted in a laboratory with demonstrated experience in this assay. Carrier testing by measurement of GALC enzyme activity in leukocytes or in cultured skin fibroblasts is not reliable because of the wide range of enzymatic activities observed in carriers and non-carriers. GALC molecular genetic testing is available and may be used for carrier detection in at-risk relatives if the disease-causing alleles have been identified in the family. Prenatal diagnosis is possible either by measurement of GALC enzyme activity or by molecular genetic testing if both disease-causing alleles in an affected family member are known. An exam of the retina in the eye may show damage to the optic nerve. There may be signs or deafness and abnormal posturing in the late stages of the disorder.
Tests that may be done include:
• Blood test to look for galactosylceramidase levels in white blood cells
• CSF total protein
• MRI of the head
• Nerve conduction velocity
• Testing for the GALC gene defect
treatment
Although there is no cure for Krabbe's disease, bone marrow transplantation has been shown to benefit cases early in the course of the disease. Generally, treatment for the disorder is symptomatic and supportive. Physical therapy may help maintain or increase muscle tone and circulation. A recent study in the New England Journal of Medicine reports that cord blood transplants have been successful in stopping the disease as long as they are given before overt symptoms appear. The current strategies for restoration of the reduced substrate degradation within the lysosome are enzyme replacement therapy (ERT), cell-mediated therapy (CMT) and cell-mediated “cross correction”, gene therapy, and enzyme-enhancement therapy with chemical chaperones. The reduction of substrate influx into the lysosomes can be achieved by substrate reduction therapy.
