Journal: Molecular Neurodegeneration
Article Title: Modulation of O-GlcNAc cycling influences α-synuclein amplification, degradation, and associated neuroinflammatory pathology
doi: 10.1186/s13024-025-00904-2
Figure Lengend Snippet: Regulating O-GlcNAcylation affects dimeric α-syn formation. ( A ) schematic illustration of the procedure for detecting O-GlcNAcylated α-syn (Oα-syn) in PFFs-seeded SH-SY5Y cells and mouse brain treated with OSMI-1 or TMG using the click-iT™ O-GlcNAc enzyme labeling system. ( B ) immunoblot analysis of O-GlcNAcylated α-syn (Oα-syn) in PFFs-seeded SH-SY5Y cells. Ponceau S staining serves as a loading control. Arrowhead indicates the Oα-syn bands that shifted from a higher molecular weight (HMW) of ~ 35 kDa (dimeric α-syn) to ~ 55 kDa in the presence of GalT1 (Y289L). ( C ) quantification of α-syn and the proportion (%) of Oα-syn in the dimeric and total α-syn in the cells treated with OSMI-1 or TMG ( n = 6). ( D ) examination of Oα-syn in SH-SY5Y cells transfected with α-syn-eGFP plasmid. ( E ) representative immunoblot images of Oα-syn in the 6 months post-injection of PFFs. ( F ) quantification of the Oα-syn in the brain of the mice 6 months post-injection of PFFs. Oα-syn levels normalized to GAPDH ( n = 3). ( G - H ) immunoblot analysis of immunoprecipitated α-syn in SH-SY5Y cells transfected with α-syn-eGFP plasmids for 36 h to 72 h ( G ) or exposed to OSMI-1 or TMG ( H ) using a pan O-GlcNAc antibody (RL2). ( I ) sWGA pull-down assay of α-syn in the brains of AAV/PFFs mice treated with OSMI-1 or TMG. ( J ) quantification of α-syn from the input and sWGA pull-down fractions. α-syn and Oα-syn were normalized to GAPDH (input) or the densitometric gray value (IP). Data are shown as means ± SEM. * p < 0.05, ** p < 0.01, * p < 0.001, and ** p < 0.0001 by one-way ANOVA with Tukey’s post-hoc test
Article Snippet: The resulting fragment was inserted into the backbone of the pAAV-hsyn- eGFP -WPRE vector (Addgene item no. #50465).
Techniques: Labeling, Western Blot, Staining, Control, Molecular Weight, Transfection, Plasmid Preparation, Injection, Immunoprecipitation, Pull Down Assay
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