Lysosomal storage diseases represent a group of about 50 genetic disorders caused by deficiencies of lysosomal and non-lysosomal proteins. Patients accumulate compounds which are normally degraded in the lysosome. In many diseases this accumulation affects various organs leading to severe symptoms and premature death. The revelation of the mechanism by which stored compounds affect cellular function is the basis for understanding pathophysiology underlying lysosomal storage diseases. In the past years it has become clear that storage compounds interfere with various processes on the cellular level. The spectrum covers e.g. receptor activation by non-physiologic ligands, modulation of receptor response and intracellular effectors of signal transduction cascades, impairment of autophagy, and others. Importantly, many of these processes are associated with accumulation of storage material in non-lysosomal compartments. Here we summarize current knowledge on the effects that storage material can elicit on the cellular level.

(Dihydro)ceramide synthase 2 (cers2, formerly called lass2) is the most abundantly expressed member of the ceramide synthase gene family, which includes six isoforms in mice. CERS2 activity has been reported to be specific toward very long fatty acid residues (C22-C24). In order to study the biological role of CERS2, we have inactivated its coding region in transgenic mice using gene-trapped embryonic stem cells that express lacZ reporter DNA under control of the cers2 promoter. The resulting mice lack ceramide synthase activity toward C24:1 in the brain as well as the liver and show only very low activity toward C18:0 -C22:0 in liver and reduced activity toward C22:0 residues in the brain. In addition, these mice exhibit strongly reduced levels of ceramide species with very long fatty acid residues (>C22) in the liver, kidney, and brain. From early adulthood on, myelin stainability is progressively lost, biochemically accompanied by about 50% loss of compacted myelin and 80% loss of myelin basic protein. Starting around 9 months, both the medullary tree and the internal granular layer of the cerebellum show significant signs of degeneration associated with the formation of microcysts. Predominantly in the peripheral nervous system, we observed vesiculation and multifocal detachment of the inner myelin lamellae in about 20% of the axons. Beyond 7 months, the CERS2-deficient mice developed hepatocarcinomas with local destruction of tissue architecture and discrete gaps in renal parenchyma. Our results indicate that CERS2 activity supports different biological functions: maintenance of myelin, stabilization of the cerebellar as well as renal histological architecture, and protection against hepatocarcinomas.

Like many lysosomal storage disorders, metachromatic leukodystrophy shows clinical heterogeneity that seems to reflect genetic heterogeneity. One of the known alleles (allele I) is associated with earlier and more severe disease than the other (allele A).

Metachromatic leukodystrophy is a lysosomal sphingolipid storage disorder caused by the deficiency of arylsulfatase A. The disease is characterized by progressive demyelination, causing various neurologic symptoms. Since no naturally occurring animal model of the disease is available, we have generated arylsulfatase A-deficient mice. Deficient animals store the sphingolipid cerebroside-3-sulfate in various neuronal and nonneuronal tissues. The storage pattern is comparable to that of affected humans, but gross defects of white matter were not observed up to the age of 2 years. A reduction of axonal cross-sectional area and an astrogliosis were observed in 1-year-old mice; activation of microglia started at 1 year and was generalized at 2 years. Purkinje cell dendrites show an altered morphology. In the acoustic ganglion numbers of neurons and myelinated fibers are severely decreased, which is accompanied by a loss of brainstem auditory-evoked potentials. Neurologic examination reveals significant impairment of neuromotor coordination.

Sphingolipids containing 2-hydroxylated fatty acids are among the most abundant lipid components of the myelin sheath and therefore are thought to play an important role in formation and function of myelin. To prove this hypothesis, we generated mice lacking a functional fatty acid 2-hydroxylase (FA2H) gene. FA2H-deficient (FA2H Ϫ/Ϫ ) mice lacked 2-hydroxylated sphingolipids in the brain and in peripheral nerves. In contrast, nonhydroxylated galactosylceramide was increased in FA2H Ϫ/Ϫ mice. However, oligodendrocyte differentiation examined by in situ hybridization with cRNA probes for proteolipid protein and PDGF␣ receptor and the time course of myelin formation were not altered in FA2H Ϫ/Ϫ mice compared with wild-type littermates. Nerve conduction velocity measurements of sciatic nerves revealed no significant differences between FA2H Ϫ/Ϫ and wild-type mice. Moreover, myelin of FA2H Ϫ/Ϫ mice up to 5 months of age appeared normal at the ultrastructural level, in the CNS and peripheral nervous system. Myelin thickness and g-ratios were normal in FA2H Ϫ/Ϫ mice. Aged (18-month-old) FA2H Ϫ/Ϫ mice, however, exhibited scattered axonal and myelin sheath degeneration in the spinal cord and an even more pronounced loss of stainability of myelin sheaths in sciatic nerves. These results show that structurally and functionally normal myelin can be formed in the absence of 2-hydroxylated sphingolipids but that its long-term maintenance is strikingly impaired. Because axon degeneration appear to start rather early with respect to myelin degenerations, these lipids might be required for glial support of axon function.

Metachromatic leukodystrophy (MLD) is a rare lysosomal sphingolipid storage disorder, caused by a deficiency of arylsulfatase A (ASA). It is inherited in an autosomal recessive way, among Caucasians three causing alleles are frequent. Demyelination is the hallmark of MLD. Interest in the disease has increased as therapeutic options such as stem cell transplantation, enzyme replacement and gene therapy are topics of current research. A late-infantile (onset before 3 years of age), a juvenile form (onset before 16 years) and an adult form are usually distinguished. Rapid motor decline is typical for the first and also the second forms, the second may be preceded by cognitive and behavioural problems, which mainly characterize the adult form. There is evidence for a genotype-phenotype correlation: patients homozygous for alleles which do not allow the expression of any enzyme activity (null-allele) suffer from the late infantile form; heterozygosity for a null allele and a non-null allele are more associated with the juvenile form and homozygosity for non-null alleles is more frequent in the most attenuated adult onset form.

ABSTRACT:Hereditary spastic paraplegia (HSP) describes a heterogeneous group of inherited neurodegenerative disorders in which the cardinal pathological feature is upper motor neurone degeneration leading to progressive spasticity and weakness of the lower limbs. Using samples from a large Omani family we recently mapped a gene for a novel autosomal recessive form of HSP (SPG35) in which the spastic paraplegia was associated with intellectual disability and seizures. Magnetic resonance imaging of the brain of SPG35 patients showed white matter abnormalities suggestive of a leukodystrophy. Here we report homozygous mutations in the fatty acid 2-hydroxylase gene (FA2H) in the original family used to define the SPG35 locus (p.Arg235Cys) as well as in a previously unreported Pakistani family with a similar phenotype (p.Arg53_Ile58del). Measurement of enzyme activity in vitro revealed significantly reduced enzymatic function of FA2H associated with these mutations. These results demonstrate that mutations in FA2H are associated with SPG35, and that abnormal hydroxylation of myelin galactocerebroside lipid components can lead to a severe progressive phenotype, with a clinical presentation of complicated HSP and radiological features of leukodystrophy. ©2010 Wiley-Liss, Inc.

For study of the time order of glycosylation, formation of complex oligosaccharides and proteolytic maturation as well as the site of proteolytic maturation of cathepsin D, fibroblasts were subjected to pulse-chase labeling, and cathepsin D was isolated from either total cell extracts or subcellular fractions by immune precipitation and analyzed for its molecular forms and sensitivity to endo-#-N-acetylglucosaminidase H. After a 10-min pulse, It is now well established that cathepsin D is synthesized on membrane-bound ribosomes and cotranslationally translocated into the lumen of the endoplasmic reticulum (1-3). Cathepsin D synthesized in human skin fibroblasts contains two asparagine-linked oligosaccharides per polypeptide chain. During passage through the Golgi apparatus, a portion of these oligosaccharides may become phosphorylated or converted into complex-type oligosaccharides. In the presence of NH4CI, ~90% of the newly synthesized cathepsin D is secreted. In the NH4Cl-induced secretions, about half of the cathepsin D molecules have one high-mannose and one complex oligosaccharide. The remaining cathepsin D polypeptides have either two high-mannose or two complex oligosaccharides (4). Of the high-mannose oligosaccharides, about half are phosphorylated (5). Subcellular fractionation experiments (6) and kinetic studies (7) established that phosphorylation represents an early reaction in the Golgi apparatus and precedes the formation of complex oligosaccharides. The segregation of cathepsin D and of other lysosomal enzymes from the secretory pathway is dependent on binding to mannose 6-phosphate-specific receptors and is thought to occur at an