Stereochemistry of Sphingolipids in Ganglioside GM3 Enhances Recovery of Nervous Functionality

Abstract

GM3 is a simple monosialylated ganglioside (NeuAcα(2-3)Galβ(1-4)Glcβ1-1'-ceramide). Its aberrant expression in adipocytes is involved in a variety of physiological and pathological processes in diabetes mellitus and obesity. GM3 is exposed on the outer surface of cell membranes and is strongly associated with type 2 diabetes and insulin resistance. Exogenously added GM3 promotes neurite outgrowth in a variety of different neuroblastoma cell lines. Neurite outgrowth is a key process in the development of functional neuronal circuits and neuro-regeneration following nerve injury. Therefore, regulating GM3 levels in nerve tissues might be a potential treatment method for these disorders. Here, we demonstrate the comprehensive synthesis of stereoisomeric GM3s and compare their physicochemical properties with those of natural GM3 and diastereomers of sphingolipids in GM3 to examine the enhancement of biological activity. l-erythro-GM3 was confirmed to increase neurite outgrowth, providing valuable insights for potential neuro-regenerative treatments.

Figure 1

Common GM3 structure in biological systems and structures of three unnatural stereoisomers of GM3.

Scheme 1. Synthesis of Benzoyl-Protected Ceramide Derivative 5 for Glycosylation Acceptor

Scheme 2. Synthesis of Trichloroacetimidate Trisaccharide Derivative 10 as Glycosylation Donor

Scheme 3. Synthesis of Three Stereoisomers of GM3 through Glycosylation between Stereoisomeric Ceramides 5 and Activated Trisaccharide 10

Figure 2

Viability assays of PC12 cells exposed to three stereoisomers of GM3 and control normal d-erythro-GM3. Cell viabilities were tested after 24 h of treatment with the four compounds at concentration levels of 5–40 μM. Data are presented as the mean ± standard deviation (SD).

Figure 3

PC12 cells exhibit an enhanced neurite outgrowth upon addition of 10 μM of three stereoisomeric GM3s. (a) Neurite outgrowth activity was measured by staining with CBB and fluorescence detection. The length of each neurite was measured from the CBB detection result, and the mean neurite length was calculated as the percentage of neurite outgrowth activity by scale bar, 50 μm. (b) Quantification of the percentage of neurite outgrowth on PC12 in each group. Results are presented as means ± SD, and experiments were repeated at least five times. ***p < 0.001. (c) Distribution of the neurite lengths in cells following differentiation in the presence of each stereoisomeric GM3, NGF, or no treatment. The data represent the means of five individual experiments with standard deviations.

Figure 4

PC12 cells exhibit an enhanced neurite outgrowth promoted by NGF upon addition of 10 μM of three stereoisomeric GM3s. (a) Neurite outgrowth activity was measured by staining with CBB and fluorescence detection. The length of each neurite was measured from CBB detection results, and the mean neurite length was calculated as the percentage of neurite outgrowth activity by scale bar, 50 μm. (b) Quantification of the percentage of neurite outgrowth on PC12 in each group. Results are presented as means ± SD, and experiments were repeated at least five times. ***p < 0.001. (c) Distribution of the neurite lengths in cells following differentiation in the presence of each stereoisomeric GM3, NGF, or no treatment. The data represents the means of five individual experiments with standard deviations. This experiment was performed simultaneously with the neurite outgrowth test using GM3 alone.