Visualization of Active Glucocerebrosidase in Rodent Brain with High Spatial Resolution following In Situ Labeling with Fluorescent Activity Based Probes.

Maria C Renner,Wouter W Kallemeijn,Marco Van Eijk,Elly M Hol,Cindy Van Roomen,Rolf G Boot,Hermen S Overkleeft,Willem Kamphuis,Martina Moeton,Andre R A Marques,Roelof Ottenhoff,Daniela Herrera Moro Chao,Ewout Foppen,Johannes M F G Aerts,Andries Kalsbeek,Jan Aten,Marie Orre

doi:10.1371/journal.pone.0138107

Abstract

Gaucher disease is characterized by lysosomal accumulation of glucosylceramide due to deficient activity of lysosomal glucocerebrosidase (GBA). In cells, glucosylceramide is also degraded outside lysosomes by the enzyme glucosylceramidase 2 (GBA2) of which inherited deficiency is associated with ataxias. The interest in GBA and glucosylceramide metabolism in the brain has grown following the notion that mutations in the GBA gene impose a risk factor for motor disorders such as α-synucleinopathies. We earlier developed a β-glucopyranosyl-configured cyclophellitol-epoxide type activity based probe (ABP) allowing in vivo and in vitro visualization of active molecules of GBA with high spatial resolution. Labeling occurs through covalent linkage of the ABP to the catalytic nucleophile residue in the enzyme pocket. Here, we describe a method to visualize active GBA molecules in rat brain slices using in vivo labeling. Brain areas related to motor control, like the basal ganglia and motor related structures in the brainstem, show a high content of active GBA. We also developed a β-glucopyranosyl cyclophellitol-aziridine ABP allowing in situ labeling of GBA2. Labeled GBA2 in brain areas can be identified and quantified upon gel electrophoresis. The distribution of active GBA2 markedly differs from that of GBA, being highest in the cerebellar cortex. The histological findings with ABP labeling were confirmed by biochemical analysis of isolated brain areas. In conclusion, ABPs offer sensitive tools to visualize active GBA and to study the distribution of GBA2 in the brain and thus may find application to establish the role of these enzymes in neurodegenerative disease conditions such as α-synucleinopathies and cerebellar ataxia.

Highlights

Gaucher disease (GD) is caused by a recessively inherited deficiency of the lysosomal hydrolase glucocerebrosidase (GBA) encoded by the GBA gene
To fluorescently label active GBA molecules in the brain, in vivo i.c.v. infusions with cyclophellitol β-epoxide activity based probe (ABP) were performed in living rats
No methods exist for visualizing active GBA molecules in the intact brain

Summary

Introduction

Gaucher disease (GD) is caused by a recessively inherited deficiency of the lysosomal hydrolase glucocerebrosidase (GBA) encoded by the GBA gene. The enzyme deficiency results in lysosomal accumulation of its glycosphingolipid substrate, glucosylceramide (GlcCer) [1]. Brain of LIMP-2– deficient mice with reduced GBA activity shows increased α-synuclein deposits causing neurotoxicity of dopaminergic neurons as well as apoptotic cell death and inflammation [16]. ERT results in improvements in the viscera, neurological manifestations in type 2 and 3 GD patients are not prevented by the intravenous enzyme infusions. This lack of effect is ascribed to the poor passage of ERT enzyme across the blood brain barrier. The presently registered inhibitors for substrate reduction therapy of GD do not prevent neurological manifestations due to poor brain permeability [19]. Development of better brain-permeable inhibitors of GlcCer synthase would be required for this and is actively pursued [20]

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