Biochemical and cell culture approach to study the molecular aspects of Metachromatic Leukodystrophy

Abstract

Metachromatic leukodystrophy (MLD) is a lysosomal lipid storage disease caused by arylsulfatase A deficiency. In MLD patients, the sphingolipid sulfatide increasingly accumulates leading to progressive demyelination. In this study, arylsulfatase A (ASA) knockout (KO) mice were analysed and it was shown that accumulation of sulfatide is not restricted to the lysosomal compartment but occurs in myelin itself. Although this sulfatide storage did not affect the overall composition of most myelin proteins, it specifically caused a severe reduction in myelin and lymphocyte protein (MAL). This demonstrates a regulatory link between sulfatide accumulation and MAL expression and indicates the existence of regulatory mechanisms between lipid and myelin protein synthesis in oligodendrocytes. In addition, in cultured renal epithelial cells, sulfatide accumulation diverts MAL to the late endosomal/lysosomal compartment and thus also affects the intracellular distribution of MAL. The specific reduction and mistargeting of MAL protein as a reaction to sulfatide overload may contribute to the pathogenic mechanisms in metachromatic leukodystrophy. To investigate further the cellular pathomechanism of MLD, spontaneously immortalised Schwann cell (SC) lines from the arylsulfatase A knockout (ASA KO) mice were established. Cultures of SC derived from peripheral sciatic nerves of 2-week-old ASA KO mice were maintained for 6 months and those colonies that spontaneously developed were expanded further and characterised. One of the cell lines, designated SC KO, showed distinct SC phenotype and it was passaged once a week and maintained for over 10 months without phenotypic alterations. The SC KO cells were genotyped and characterised. They showed marked sulfatide storage in the late endosomal/lysosomal compartments. This was demonstrated immunologically, ultra structurally and by biochemical analysis. The stored sulfatide responded to ASA treatment and hence could be a suitable model to study this disease at a molecular level. Preliminary molecular analysis of these cells has paved way to investigate in detail for neurotrophins and their receptors in the sciatic nerve. RT-PCR analysis of sciatic nerve has shown that there is an upregulation of the neurotrophic receptors tyrosine kinase A (TrkA) and tyrosine kinase B (TrkB) and a downregulation of neurotrophins namely, nerve growth factor (NGF) and brain derived neurotrophic factor (BDNF), which are necessary for the normal myelination process. The down regulation of neurotrophins might be the reason for the demyelination observed in the peripheral nervous system (PNS) of the two year old mouse. Finally, the method developed to load sulfatide in the sulfatide non-storing SCs (SC KO NS) can be used as a comparative cell culture model to study the disease further for possible therapeutic approaches.