Janet Deane

Lipid processing defects in neurodegenerative disease

Elucidating how disruptions to lipid recycling in the lysosome cause severe neurodegenerative diseases.

Efficient signal transduction in the nervous system requires that neurons be wrapped in a membrane-rich insulating sheath known as myelin. This sheath is highly enriched with a special class of lipids known as glycosphingolipids. If the recycling and turnover of these lipids is defective the myelin sheath becomes damaged and the neurons degenerate causing severe diseases.

Our research focuses on a rare autosomal recessive disorder, Krabbe disease, which primarily affects infant children. It is caused by deficiencies in the enzyme galactocerebrosidase (GALC), which is responsible for the degradation of the myelin lipid galactocerebroside (GalCer, Fig. 1A). GALC is produced in the ER-Golgi complex and then traffics to the lysosome where it is essential for lipid recycling. Defects in GALC lead to the accumulation of cytotoxic metabolites that elicit complex, and still only partially understood, cellular events that result in apoptosis of myelin-forming cells and rapid neurodegeneration. We have identified several molecular mechanisms by which GALC function can be disrupted including loss of catalytic activity, misfolding and mistrafficking of GALC. However, GALC does not function in isolation and requires an additional protein Saposin A (SapA) which is a lipid-binding and lipid-transfer protein (Fig. 1B).

In order to understand how GALC and SapA function together to coordinate the efficient recycling of sphingolipids we determined the structure of the GALC-SapA complex in the presence of lipid mimics (Fig. 1C). A central SapA dimer binds the hydrophobic tails of the lipids, shielding them from the aqueous solvent (Fig. 1D). This arrangement allows the glycosylated headgroup of the lipid to remain exposed in order to bind the active site of GALC to allow efficient cleavage to occur. This structure provides new insights into the mechanism by which these hydrophobic substrates can be cleaved by soluble enzymes: SapA extracts the sphingolipid from the bilayer and presents it to the GALC active site for processing. A number of disease-relevant mutations are buried in this interface, identifying the critical role of this interaction for correct sphingolipid processing.

We are using a range of techniques from protein biochemistry and structural biology to cell-based assays and proteomics to address how these defects in GALC result in changes in the cell that trigger demyelination and neurodegeneration.

Figure 1. Glycosphingolipid processing by GALC and SapA

(A) The cleavage of galactocerebroside (GalCer) by GALC produces galactose and ceramide. (B) Lipid substrates must be extracted from the membrane in order to be processed. (C) Our crystal structure of the GALC-SapA complex reveals that a central dimer of SapA (yellow and orange) is enclosed by two molecules of GALC (cyan and magenta). (D) A cross-section through the structure reveals a continuous channel from the GALC active site into the SapA core, into which hydrophobic lipid substrates can bind.

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