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

Herrera Moro Chao D, Kallemeijn WW, Marques AR, Orre M, Ottenhoff R, van Roomen C, Foppen E, Renner MC, Moeton M, van Eijk M, Boot RG, Kamphuis W, Hol EM, Aten J, Overkleeft HS, Kalsbeek A, Aerts JM - PLoS ONE (2015)

Bottom Line: 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.

View Article: PubMed Central - PubMed

Affiliation: Department of Medical Biochemistry, Academic Medical Center, Amsterdam, The Netherlands; Department of Endocrinology and Metabolism, Academic Medical Center, Amsterdam, The Netherlands.

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.

No MeSH data available.


Related in: MedlinePlus

In vitro labeling of GBA2 in specific brain areas.(A) SDS-PAGE gel of Anybody MDW1044 labeled rat brains areas. (B) Quantification of Anybody MDW1044 fluorescent signal per μg of protein. (C) GBA2 enzymatic activity in different brain areas. (D) GBA2 protein levels in homogenates of rat brain areas assessed by Western blot analysis. (E) Quantification of GBA2 blot in rat brain areas normalized to the same tubulin signal showed in Fig 3e. Note: cerebellum indicates other parts of cerebellum than cerebellar cortex.
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pone.0138107.g007: In vitro labeling of GBA2 in specific brain areas.(A) SDS-PAGE gel of Anybody MDW1044 labeled rat brains areas. (B) Quantification of Anybody MDW1044 fluorescent signal per μg of protein. (C) GBA2 enzymatic activity in different brain areas. (D) GBA2 protein levels in homogenates of rat brain areas assessed by Western blot analysis. (E) Quantification of GBA2 blot in rat brain areas normalized to the same tubulin signal showed in Fig 3e. Note: cerebellum indicates other parts of cerebellum than cerebellar cortex.

Mentions: We next employed the β-aziridine cyclophellitol ABP which broadly target β-glucosidases [23]. It was first examined whether brain GBA2 can be labeled with these Anybody ABPs. For this purpose, we incubated punches of specific brain areas of untreated rats in vitro with 100 nM green fluorescent Anybody MDW1044 (Fig 7). Following gel electrophoresis and fluorescence scanning, three distinct proteins become visible: GBA2 with MW ~100 kDa, GBA with MW ~60 kDa and a protein of about 40 kDa.


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

Herrera Moro Chao D, Kallemeijn WW, Marques AR, Orre M, Ottenhoff R, van Roomen C, Foppen E, Renner MC, Moeton M, van Eijk M, Boot RG, Kamphuis W, Hol EM, Aten J, Overkleeft HS, Kalsbeek A, Aerts JM - PLoS ONE (2015)

In vitro labeling of GBA2 in specific brain areas.(A) SDS-PAGE gel of Anybody MDW1044 labeled rat brains areas. (B) Quantification of Anybody MDW1044 fluorescent signal per μg of protein. (C) GBA2 enzymatic activity in different brain areas. (D) GBA2 protein levels in homogenates of rat brain areas assessed by Western blot analysis. (E) Quantification of GBA2 blot in rat brain areas normalized to the same tubulin signal showed in Fig 3e. Note: cerebellum indicates other parts of cerebellum than cerebellar cortex.
© Copyright Policy
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC4587854&req=5

pone.0138107.g007: In vitro labeling of GBA2 in specific brain areas.(A) SDS-PAGE gel of Anybody MDW1044 labeled rat brains areas. (B) Quantification of Anybody MDW1044 fluorescent signal per μg of protein. (C) GBA2 enzymatic activity in different brain areas. (D) GBA2 protein levels in homogenates of rat brain areas assessed by Western blot analysis. (E) Quantification of GBA2 blot in rat brain areas normalized to the same tubulin signal showed in Fig 3e. Note: cerebellum indicates other parts of cerebellum than cerebellar cortex.
Mentions: We next employed the β-aziridine cyclophellitol ABP which broadly target β-glucosidases [23]. It was first examined whether brain GBA2 can be labeled with these Anybody ABPs. For this purpose, we incubated punches of specific brain areas of untreated rats in vitro with 100 nM green fluorescent Anybody MDW1044 (Fig 7). Following gel electrophoresis and fluorescence scanning, three distinct proteins become visible: GBA2 with MW ~100 kDa, GBA with MW ~60 kDa and a protein of about 40 kDa.

Bottom Line: 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.

View Article: PubMed Central - PubMed

Affiliation: Department of Medical Biochemistry, Academic Medical Center, Amsterdam, The Netherlands; Department of Endocrinology and Metabolism, Academic Medical Center, Amsterdam, The Netherlands.

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.

No MeSH data available.


Related in: MedlinePlus