GM1 Oligosaccharide Efficacy in Parkinson's Disease: Protection against MPTP

Abstract

Past evidence has shown that the exogenous administration of GM1 ganglioside slowed neuronal death in preclinical models of Parkinson's disease, a neurodegenerative disorder characterized by the progressive loss of dopamine-producing neurons: however, the physical and chemical properties of GM1 (i.e., amphiphilicity) limited its clinical application, as the crossing of the blood-brain barrier is denied. Recently, we demonstrated that the GM1 oligosaccharide head group (GM1-OS) is the GM1 bioactive portion that, interacting with the TrkA-NGF complex at the membrane surface, promotes the activation of a multivariate network of intracellular events regulating neuronal differentiation, protection, and reparation. Here, we evaluated the GM1-OS neuroprotective potential against the Parkinson's disease-linked neurotoxin MPTP, which destroys dopaminergic neurons by affecting mitochondrial bioenergetics and causing ROS overproduction. In dopaminergic and glutamatergic primary cultures, GM1-OS administration significantly increased neuronal survival, preserved neurite network, and reduced mitochondrial ROS production enhancing the mTOR/Akt/GSK3β pathway. These data highlight the neuroprotective efficacy of GM1-OS in parkinsonian models through the implementation of mitochondrial function and reduction in oxidative stress.

Keywords: GM1 ganglioside; GM1 oligosaccharide; MPTP; Parkinson’s disease; neuroprotection; plasma membrane signaling.

Figure 1

Neuroprotective effects of GM1-OS in primary cultures of rat DA neurons injured with MPP⁺. On day 6 of culture, primary DA neurons were pre-incubated with GM1-OS (100 μM) for 1 h, before MPP⁺ exposure. Next, MPP⁺ (4 µM) was added to the culture medium. After 48 h, TH immunofluorescence was performed as described in the methods section. (a) Representative immunofluorescence images of TH-positive neurons (10X magnifications); (b) number of TH-positive neurons expressed as fold change over untreated control (CTRL) cells, as read-out of DA neuron survival; (c) length of TH-positive neurites in µm, expressed as fold change over untreated CTRL cells, for neurite network of DA neurons. All values are expressed as mean ± SEM (n = 6, **** p < 0.0001; *** p < 0.001; ** p < 0.01; One-way ANOVA followed by Tukey’s multiple comparisons test).

Figure 2

GM1-OS protected mouse CGNs injured with MPP⁺. On day 14 of culture, primary CGNs were pre-incubated with GM1-OS (100 μM) or water (CTRL) for 1 h before MPP⁺ exposure. Next, MPP⁺ (50 µM) or water was added to the culture medium for 24 h. (a) Phase contrast images of CGNs (20X magnification). Images are representative of four independent experiments (n = 4); (b) Viability assays: on the left LDH release, on the right MTT assay. All values are expressed as mean ± SEM (n = 4 independent experiments. *** p = 0.0005, **** p < 0.0001, one-way ANOVA followed by Tukey’s multiple comparisons test).

Figure 3

GM1-OS modulation of mTOR/Akt/GSK-3β pathway. WB of factors sustaining cell survival after 6 h and 24 h of MPP⁺ exposure: mTOR^P-Ser2448, AKT^P-Ser473, GSK3β^P-Ser9, and respective total proteins. Top: WB representative images; bottom: semiquantitative analysis of WB bands represented as fold change over CTRL. All values are expressed as mean ± SEM (n = 4 independent experiments. *** p = 0.0002, **** p < 0.0001, one-way ANOVA followed by Tukey’s multiple comparisons test).

Figure 4

GM1-OS counteracted mitochondrial O₂^•− increase induced by MPP⁺. On day 14 of culture, primary CGNs were pre-incubated with GM1-OS (100 μM) or water (CTRL) for 1 h before MPP⁺ exposure. Next, MPP⁺ (50 µM) or water was added to the culture and mitochondrial O₂^•− was evaluated by MitoSOX Red reagent. (a) Representative fluorescence images of CGNs after 1 h (top) or 6 h (bottom) of MPP⁺ administration (40× magnification). For each condition the small quadrants show single channel images with MitoSOX in red and Nuclei in blue and the big quadrant is the overlayed image (Scale bar: 50 µm); (b) quantification of the MitoSOX Red signal over nuclei number. All values are expressed as mean ± SEM (n = 3 independent experiments. * p < 0.01, *** p = 0.0006 **** p < 0.0001, one-way ANOVA followed by Tukey’s multiple comparisons test).