Imaging of sialidase activity in rat brain sections by a highly sensitive fluorescent histochemical method

doi:10.1016/j.neuroimage.2011.06.017

NeuroImage

Volume 58, Issue 1, 1 September 2011, Pages 34-40

https://doi.org/10.1016/j.neuroimage.2011.06.017 Get rights and content

Abstract

Sialidase (EC 3.2.1.18) removes sialic acid from sialoglycoconjugates. Since sialidase extracellularly applied to the rat hippocampus influences many neural functions, including synaptic plasticity and innervations of glutamatergic neurons, endogenous sialidase activities on the extracellular membrane surface could also affect neural functions. However, the distribution of sialidase activity in the brain remains unknown. To visualize extracellular sialidase activity on the membrane surface in the rat brain, acute brain slices were incubated with 5-bromo-4-chloroindol-3-yl-α-d-N-acetylneuraminic acid (X-Neu5Ac) and Fast Red Violet LB (FRV LB) at pH 7.3. After 1 h, myelin-abundant regions showed intense fluorescence in the rat brain. Although the hippocampus showed weak fluorescence in the brain, mossy fiber terminals in the hippocampus showed relatively intense fluorescence. These fluorescence intensities were attenuated with a sialidase-specific inhibitor, 2,3-dehydro-2-deoxy-N-acetylneuraminic acid (DANA, 1 mM). Additionally, the fluorescence intensities caused by X-Neu5Ac and FRV LB were correlated with the sialidase activity measured with 4-methylumbelliferyl-α-d-N-acetylneuraminic acid (4MU-Neu5Ac), a classical substrate for quantitative measurement of sialidase activity, in each brain region. Therefore, staining with X-Neu5Ac and FRV LB is specific for sialidase and useful for quantitative analysis of sialidase activities. The results suggest that white matter of the rat brain has intense sialidase activity.

Highlights

► White matter showed intense sialidase activities in rat brain. ► In hippocampus, mossy fiber terminals showed intense sialidase activities. ► Imaging of sialidase activity with X-Neu5Ac is specific for sialidase. ► This imaging method is useful for quantitative analysis of sialidase activities.

Introduction

Sialic acid is an acidic monosaccharide and plays crucial roles in various membrane functions in mammalian central nervous systems (Rutishauser, 2008, Schnaar, 2010). Sialidase removes sialic acid residues from sialoglycoconjugates, such as glycoproteins and glycolipids. Several different types of sialidase have been identified in mammalian tissues by their localization and enzymatic properties. Four types of mammalian sialidase (Neu1, Neu2, Neu3 and Neu4) have been cloned. Neu1, Neu2 and Neu3 are located at lysosomes (Bonten et al., 2009), cytosol (Hasegawa et al., 2000) and plasma membranes (Yamaguchi et al., 2006), respectively. Neu4 exists in the lysosomal lumen, mitochondria and intracellular membranes (Seyrantepe et al., 2004, Shiozaki et al., 2009). These four types of sialidase were reported to be expressed in mammalian brains (Shiozaki et al., 2009).

Extracellulary applied sialidase affects myelin–axon interactions, spinal axon outgrowth (Mountney et al., 2010) and cholinergic neurotransmission (Wieraszko and Seifert, 1984). Removal of polysialic acid (PSA), a polymerized structure of sialic acid with a degree of polymerization ranging from 8 to 400 (Kanato et al., 2008), by applying PSA-specific sialidase (EndoN) affects spatial learning (Becker et al., 1996), synaptic plasticity such as long-term potentiation and long-term depression (Kochlamazashvili et al., 2010, Muller et al., 1996), synaptogenesis (Dityatev et al., 2004), migration (Battista and Rutishauser, 2010) and innervations of GABAergic (Di Cristo et al., 2007) and glutamatergic (Seki and Rutishauser, 1998) neurons. Since extracellular sialidase activities are associated with many neural functions, it seems that enzyme activities on cell surface exhibited by endogenous sialidase could also affect neural functions. However, the distribution of sialidase activity on cell surface in brain is poorly understood.

Previously, Saito et al. developed a novel fluorescent cytochemical method to detect sialidase activity of fixed neuroblastoma cells by using X-Neu5Ac as the substrate and an azo dye, FRV LB (Saito et al., 2002). After cleavage of X-Neu5Ac with sialidase, the compound X reacts with FRV LB, producing a water-insoluble fluorescent compound. This fluorescent method was also employed successfully for detection of sialidase activity of intact cultured cells. In the present study, we evaluated this highly sensitive fluorescent method for use in histochemical staining and examined the distribution of sialidase activity in the rat brain.

Section snippets

Chemicals

The following products were purchased from the vendors indicated: X-Neu5Ac (Peptide Institute, Osaka, Japan); FRV LB and 4MU-Neu5Ac (Sigma-Aldrich, St. Louis, MO, USA); ZnAF-2 DA (Sekisui Medical, Tokyo, Japan); monoclonal antibody toward myelin basic protein (MBP, Cat. No. CP32) and sialidase from Arthrobacter ureafacience (Calbiochem, San Diego, CA, USA); DAPI (Invitrogen, Eugene, OR); fluorescein isothiocyanate-conjugated (FITC-conjugated) secondary antibody (Cat. No. 115-095-062, Jackson

Results

We determined the optimal conditions for detection of sialidase activity with X-Neu5Ac and FRV LB. One mM X-Neu5Ac and 0.1 mg/ml FRV LB themselves showed low background fluorescence (Fig. 1). The sialidase from Arthrobacter ureafacience was added to a solution containing X-Neu5Ac and FRV LB, resulting in a remarkable increase in fluorescent intensity (Fig. 1). The fluorescence intensity acquired with a 550 nm excitation wavelength reached maximum at a 696 nm emission wavelength. We also determined

Discussion

We examined the distribution of sialidase activity in the rat brain by using a highly sensitive fluorescent method. As a result of sialidase activity imaging in acute brain slices, myelin-abundant regions showed intense sialidase activity at pH 7.3. It has been reported that an intrinsic sialidase is present in myelin membranes of rat brain (Saito and Yu, 1986, Yohe et al., 1983). The myelin-associated sialidase may play an important role in the formation and maintenance of the myelin sheath

Acknowledgments

We are grateful to Dr. Naoto Oku and Dr. Atsushi Takeda for helpful discussions. This work was supported by Grant-in-Aid for Young Scientists (B) 21790085, The Sasakawa Scientific Research Grant from the Japan Science Society and The Naito Foundation Subsidy for Promotion of Specific Research Projects.

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Imaging of sialidase activity in rat brain sections by a highly sensitive fluorescent histochemical method

Abstract

Highlights

Introduction

Section snippets

Chemicals

Results

Discussion

Acknowledgments

Neurochem. Int.

J. Biol. Chem.

Int. J. Mass Spectrom.

Mol. Cells

J. Biol. Chem.

Biochem. Biophys. Res. Commun.

J. Biol. Chem.

J. Biol. Chem.

J. Biol. Chem.

Neuron

J. Biol. Chem.

FEBS Lett.

J. Biol. Chem.

J. Biol. Chem.

Neurosci. Lett.

Biochem. Biophys. Res. Commun.

J. Biol. Chem.

Removal of polysialic acid triggers dispersion of subventricularly derived neuroblasts into surrounding CNS tissues

J. Neurosci.

The polysialic acid modification of the neural cell adhesion molecule is involved in spatial learning and hippocampal long-term potentiation

J. Neurosci. Res.