Differential Ganglioside and Cholesterol Depletion by Various Cyclodextrin Derivatives and Their Effect on Synaptosomal Glutamate Release

Abstract

Gangliosides are glycosphingolipids of the plasma membrane and are highly enriched in the nervous system where they play a vital role in normal cell functions. Furthermore, several studies suggest their potential involvement in the pathogenesis of neurological conditions. Since cyclodextrins (CDs) can form inclusion complexes with various lipids, methylated beta-CDs are widely used in biomedical research to extract cholesterol from the membrane and study its cellular role. Despite CDs being known to interact with other membrane lipid components, their effect on gangliosides is poorly characterized. The aim of this research was to investigate the effect of dimethyl-beta-cyclodextrin (DIMEB), hydroxypropyl-beta-cyclodextrin (HPBCD), randomly methylated-alpha-cyclodextrin (RAMEA), and hydroxypropyl-alpha-cyclodextrin (HPACD) on ganglioside and cholesterol levels in rat brain synaptosomes. Their effect on membrane integrity and viability was also assessed. We examined the role of lipid depletion by CDs on the release of the major excitatory neurotransmitter, glutamate. Selective concentration range for cholesterol depletion was only found with HPBCD, but not with DIMEB. Selective depletion of gangliosides was achieved by both RAMEA and HPACD. The inhibition of stimulated glutamate release upon ganglioside depletion was found, suggesting their potential role in neurotransmission. Our study highlights the importance of the characterization of the lipid depleting capability of different CDs.

Keywords: cholesterol; cyclodextrines; gangliosides; glutamate release; lipid rafts; membrane lipids; membrane microdomains; synaptic transmission; synaptosomes.

Figure 1

The effect of DIMEB (A), HPBCD (B), RAMEA (C), and HPACD (D) on the ganglioside and cholesterol content of the rat cerebral synaptosomes. Lipid levels were measured after 40 min of exposure of the synaptosomes to cyclodextrins. Values are expressed as the percentage of vehicle-treated synaptosomes. The IC₅₀ values and their 95% confidence intervals (95% CI) are given. Data are represented as the means ± SEM (n = 3). N/A means non applicable.

Figure 2

The effect of DIMEB (A), HPBCD (B), RAMEA (C), and HPACD (D) on the membrane integrity and viability of the rat cerebral synaptosomes. Synaptosomes were analyzed after 40 min exposure to cyclodextrins. For estimating membrane integrity, LDH release from synaptosomes was calculated as percentage of total LDH content determined by Triton X-100 lysis. Viability was measured by resazurin reduction assay and values are expressed as the percentage of vehicle-treated synaptosomes. IC₅₀ values and their 95% confidence intervals (95% CI) are given. Data are represented as means ± SEM (n = 3). N/A means non applicable.

Figure 3

The basal (non-stimulated) and 4-aminopyridine-evoked release of glutamate from rat cerebral synaptosomes upon DIMEB (A), HPBCD (B), RAMEA (C), and HPACD (D) treatment. Glutamate release was measured after 40 min of exposure of the synaptosomes to cyclodextrins. Glutamate release is expressed as the percentage of baseline release from the vehicle-treated synaptosomes. Data are represented as the means ± SEM (n = 8). One-way ANOVA followed by Bonferroni’s post hoc test was used for data evaluation. *** p ≤ 0.001, ns: not significant.