Molecular Mechanisms of Disease Pathogenesis Differ in Krabbe Disease Variants

Abstract

Krabbe disease is a severe, fatal neurodegenerative disorder caused by defects in the lysosomal enzyme galactocerebrosidase (GALC). The correct targeting of GALC to the lysosome is essential for the degradation of glycosphingolipids including the primary lipid component of myelin. Over 100 different mutations have been identified in GALC that cause Krabbe disease but the mechanisms by which they cause disease remain unclear. We have generated monoclonal antibodies against full‐length human GALC and used these to monitor the trafficking and processing of GALC variants in cell‐based assays and by immunofluorescence microscopy. Striking differences in the secretion, processing and endosomal targeting of GALC variants allows the classification of these into distinct categories. A subset of GALC variants are not secreted by cells, not proteolytically processed, and remain trapped in the ER; these are likely to cause disease due to protein misfolding and should be targeted for pharmacological chaperone therapies. Other GALC variants can be correctly secreted by cells and cause disease due to catalytic defects in the enzyme active site, inappropriate post‐translational modification or a potential inability to bind essential cofactors. The classification of disease pathogenesis presented here provides a molecular framework for appropriate targeting of future Krabbe disease therapies.

Endoplasmic Reticulum Protein Quality Control Failure in Myelin Disorders

“…Moreover, the GALC D528N mutant was found to be hyperglycosylated and misfolded and its function can be partially rescued by a treatment with the pharmacological chaperone α-lobeline (Lee et al, 2010). Another study confirmed that many GALC mutants were trapped within the ER instead of reaching the lysosome (Spratley et al, 2016). These data suggest that failure of ERQC is an important determinant in KD pathogenesis.…”

New therapeutic approaches for Krabbe disease: The potential of pharmacological chaperones

“…The G270D mutation, commonly associated with late-onset forms of the disease, is often found alongside the common polymorphism I546T (found in 35-45% of the population); together, they show lower GALC activity than when expressed on their own . Likewise, it has been shown by Shin et al (2016) and Spratley et al (2016) that the combination of certain cispolymorphisms and disease mutations can significantly reduce the activity and trafficking of GALC. The full impact of known polymorphisms on KD prognosis and therapeutic intervention has yet to be elucidated but must be taken into consideration when characterizing and identifying candidate GALC mutations.…”

“…Missense mutations that lie within the active site of GALC, such as the R380W mutation (Fig. 4), directly disrupt the interactions between enzyme and substrate, critically affecting enzyme activity but not folding (Hill et al, 2013;Spratley, et al, 2016). The R380W mutation thus causes a severe, early-onset form of KD because of catalytic inactivity (Wenger et al, 1997).…”

Large‐scale functionalLIPAvariant characterization to improve birth prevalence estimates of lysosomal acid lipase deficiency

“…A limitation of this approach is that it is not able to provide information on other types of mutations known to give rise to enzyme deficiencies. Examples of these include splicing mutations such as the aforementioned E8SJM/c.894G>A pathogenic mutation found in the majority of patients with CA‐LALD (Bernstein et al, ; Scott et al, ) as well as mutations that inhibit trafficking beyond the endoplasmic reticulum and trans‐Golgi network as has been observed in other lysosomal storage diseases (Parenti et al, ; Ron & Horowitz, ; Spratley et al, ; Zhang et al, ). Another potential limitation of our analysis is the use of the artificial 4‐MU oleate substrate rather than naturally occurring substrates such as cholesteryl esters or triglycerides.…”