title: Visualization of Active Glucocerebrosidase in Rodent Brain with High Spatial Resolution following In Situ Labeling with Fluorescent Activity Based Probes.
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.
authors.affiliation: Department of Medical Biochemistry, Academic Medical Center, Amsterdam, The Netherlands; Department of Endocrinology and Metabolism, Academic Medical Center, Amsterdam, The Netherlands.
authors.forename: Daniela
authors.initials: D
authors.lastname: Herrera Moro Chao
authors.affiliation: Department of Medical Biochemistry, Academic Medical Center, Amsterdam, The Netherlands; Department of Biochemistry, Leiden Insitute of Chemistry, Leiden, The Netherlands.
authors.forename: Wouter W
authors.initials: WW
authors.lastname: Kallemeijn
authors.affiliation: Department of Medical Biochemistry, Academic Medical Center, Amsterdam, The Netherlands.
authors.forename: Andre R A
authors.initials: AR
authors.lastname: Marques
authors.affiliation: Netherlands Institute for Neuroscience, Amsterdam, The Netherlands.
authors.forename: Marie
authors.initials: M
authors.lastname: Orre
authors.affiliation: Department of Medical Biochemistry, Academic Medical Center, Amsterdam, The Netherlands.
authors.forename: Roelof
authors.initials: R
authors.lastname: Ottenhoff
authors.affiliation: Department of Medical Biochemistry, Academic Medical Center, Amsterdam, The Netherlands.
authors.forename: Cindy
authors.initials: C
authors.lastname: van Roomen
authors.affiliation: Department of Endocrinology and Metabolism, Academic Medical Center, Amsterdam, The Netherlands.
authors.forename: Ewout
authors.initials: E
authors.lastname: Foppen
authors.affiliation: Netherlands Institute for Neuroscience, Amsterdam, The Netherlands.
authors.forename: Maria C
authors.initials: MC
authors.lastname: Renner
authors.affiliation: Netherlands Institute for Neuroscience, Amsterdam, The Netherlands.
authors.forename: Martina
authors.initials: M
authors.lastname: Moeton
authors.affiliation: Department of Biochemistry, Leiden Insitute of Chemistry, Leiden, The Netherlands.
authors.forename: Marco
authors.initials: M
authors.lastname: van Eijk
authors.affiliation: Department of Biochemistry, Leiden Insitute of Chemistry, Leiden, The Netherlands.
authors.forename: Rolf G
authors.initials: RG
authors.lastname: Boot
authors.affiliation: Netherlands Institute for Neuroscience, Amsterdam, The Netherlands.
authors.forename: Willem
authors.initials: W
authors.lastname: Kamphuis
authors.affiliation: Netherlands Institute for Neuroscience, Amsterdam, The Netherlands; Department of Translational Neuroscience, Brain Center Rudolf Magnus, University Medical Center Utrecht, Utrecht, The Netherlands; Swammerdam Institute for Life Sciences, Center for Neuroscience, University of Amsterdam, Amsterdam, The Netherlands.
authors.forename: Elly M
authors.initials: EM
authors.lastname: Hol
authors.affiliation: Department of Pathology, Academic Medical Center, Amsterdam, The Netherlands.
authors.forename: Jan
authors.initials: J
authors.lastname: Aten
authors.affiliation: Department of Bio-organic Synthesis, Leiden institute of Chemistry, Leiden, The Netherlands.
authors.forename: Hermen S
authors.initials: HS
authors.lastname: Overkleeft
authors.affiliation: Department of Endocrinology and Metabolism, Academic Medical Center, Amsterdam, The Netherlands; Netherlands Institute for Neuroscience, Amsterdam, The Netherlands.
authors.forename: Andries
authors.initials: A
authors.lastname: Kalsbeek
authors.affiliation: Department of Biochemistry, Leiden Insitute of Chemistry, Leiden, The Netherlands.
authors.forename: Johannes M F G
authors.initials: JM
authors.lastname: Aerts
mesh_terms: D000818:Animals; D001253:Astrocytes / Q000201:enzymology / Q000378:metabolism; D001921:Brain / Q000201:enzymology* / Q000378:metabolism; D002478:Cells, Cultured; D002524:Cerebellar Ataxia / Q000235:genetics / Q000473:pathology; D005455:Fluorescent Antibody Technique; D005456:Fluorescent Dyes / Q000737:chemistry; D005776:Gaucher Disease / Q000235:genetics* / Q000473:pathology; D005962:Glucosylceramidase / Q000235:genetics / Q000378:metabolism*; D005963:Glucosylceramides / Q000378:metabolism*; D008297:Male; D051379:Mice; D008810:Mice, Inbred C57BL; D017628:Microglia / Q000201:enzymology / Q000378:metabolism; D018613:Microscopy, Confocal; D019636:Neurodegenerative Diseases / Q000235:genetics* / Q000473:pathology; D011689:Purkinje Cells / Q000378:metabolism; D051381:Rats; D017208:Rats, Wistar
chemical_list: D005456:Fluorescent Dyes; D005963:Glucosylceramides; D005962:Glucosylceramidase