Parkinson’s disease recovery by GM1 oligosaccharide treatment in the B4galnt1+/− mouse model

Given the recent in vitro discovery that the free soluble oligosaccharide of GM1 is the bioactive portion of GM1 for neurotrophic functions, we investigated its therapeutic potential in the B4galnt1+/− mice, a model of sporadic Parkinson’s disease. We found that the GM1 oligosaccharide, systemically administered, reaches the brain and completely rescues the physical symptoms, reduces the abnormal nigral α-synuclein content, restores nigral tyrosine hydroxylase expression and striatal neurotransmitter levels, overlapping the wild-type condition. Thus, this study supports the idea that the Parkinson’s phenotype expressed by the B4galnt1+/− mice is due to a reduced level of neuronal ganglioside content and lack of interactions between the oligosaccharide portion of GM1 with specific membrane proteins. It also points to the therapeutic potential of the GM1 oligosaccharide for treatment of sporadic Parkinson’s disease.


Immunoblotting analysis
Freshly collected brains from PBS perfused mice were employed and the midbrain cut into 250 μm coronal sections with a vibratome sectioning system in cold PBS. The entire substantia nigra region, including compact and reticulata, was isolated with a dissecting microscope. The pooled sections were extracted with 1 mL/100 mg tissue of Cell Lysis Buffer (Cell Signaling, Danvers, MA). Aliquots containing 30 μg protein were denatured with Laemmli sample buffer (final concentration 0.1 M DTT, 63 mM Tris-HCl, 10 % glycerol v/v, 2% SDS w/v, 0.01% blue bromophenol v/v), boiled at 100°C for 5 min, separated on 4-20% polyacrylamide gels, and transferred to PVDF membranes using the Trans-Blot® Turbo™ Transfer System (Bio-Rad). PVDF membranes were blocked with 5% milk (w/v) in TBS-0.1% tween (v/v) at 23 °C for 1 hour under gently shaking. Phosphorylated α-syn (S129) level and calnexin levels were assayed using respectively anti-phospho-α-syn (S129) rabbit antibody and luminol detection. Finally, the phospho-α-syn (S129) signal was normalized to calnexin signal.
The data acquisition and analysis were performed using Alliance Uvitec (Cleaver Scientific, Ltd, UK).
Whole brains were isolated, post-fixed overnight in 4% PFA at 4°C and soaked in cryoprotective solution (30% sucrose in PBS) until tissue sinking. Brains were snap frozen in pre-cooled isopentane upon OCT embedding and stored at -80°C, prior to cryostat sectioning. Tissues were sectioned (16 μm) using cryostat (MC 5050 Semi-automatic Cryostat, Histo-Line Laboratories) compound embedding in dry ice. Sections through the rostro-caudal extent of the substantia nigra were collected and every fifth section from substantia nigra was processed for phosphorylated α-syn (S129) IHC analysis. SNpc was used for the IHC detection of phosphorylated α-syn (S129), as specifically indicated in Fig. S7 of supplementary files. For immunofluorescence, 16 μm slices were treated for 1 h with citrate buffer and heat in a water bath at 80°C. Then the slices were put in a blocking solution (1h, 23°C) containing 10% fetal calf serum and 0.25% Triton X-100 in PBS. After blocking, samples were incubated with the primary antibody diluted with a same solution overnight at 23°C. The slices were then incubated with the secondary antibody (1h, 23°C), followed by nuclei staining with DAPI 1 (1:5000, 15 min, 23°C).

Supplementary Figure 1
Supplementary Fig. 1 Radioactivity distribution into the brain. Five WT mice (n=5, male, 25 g each) were singly IP injected with 1.3x10 7 dpm of [ 3 H]OligoGM1 plus 0.5 mg of cold OligoGM1 corresponding to 20mg/Kg of OligoGM1. After 24 h following injection, animals were euthanized by heart perfusion with saline solution to remove the blood 2 . Immediately the brain, without cerebellum, was collected, weighted (± 350 mg) and homogenized as reported in Methods section.
Brain homogenate was submitted to determination of total radioactivity by liquid scintillation corresponding to volatile and non-volatile radioactivity (total radioactivity) 2 . For each brain homogenate tree replicates were counted (n=3). To establish the specific amount of volatile and non-volatile radioactivity, the brain homogenate (3 samples from each brain, n=3) was dried under flux of nitrogen, resuspended in cold distilled water and counted for radioactivity content as above.
The difference between the value obtained from not-dry homogenate (total radioactivity) and dry homogenate corresponded to non-volatile radioactivity. The data reported (total radioactivity and volatile radioactivity) are the mean value of three counts for each brain homogenate dried or not. between ■ male and ▼ female; Right B4galnt1 +/mice + GM1 oligosaccharide no significant difference between ■ male and ▼ female; b) Irritant Removal Test. Left: B4galnt1 +/mice + BSS no significant difference between ■ male and ▼ female; Right B4galnt1 +/mice + GM1 oligosaccharide no significant difference between ■ male and ▼ female; c) Pole Climbing Test. Left: B4galnt1 +/mice + BSS no significant difference between ■ male and ▼ female; Right B4galnt1 +/mice + GM1 oligosaccharide no significant difference between ■ male and ▼ female.