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Fig. 7 | BMC Neuroscience

Fig. 7

From: Limited effects of dysfunctional macroautophagy on the accumulation of extracellularly derived α-synuclein in oligodendroglia: implications for MSA pathogenesis

Fig. 7

Genetic LC3B knockdown does not induce GCI-like formation of extracellularly incorporated recombinant sol and fib α-syn species in oligodendroglial cells. Oligodendroglial cells were transfected using the shRNA plasmid against LC3B ligated to GFP to create a constitutive knockdown of LC3B in oligodendroglial cells. Western blot analysis confirmed a highly significant down-regulation of the LC3B protein in the transfected oligodendroglial cultures irrespective of the treatment with sol and fib α-syn. LC3B levels were normalized to actin levels and a control lysate (a). Intracellular amounts of uptaken α-syn monomers and endogenous α-syn were measured in oligodendroglial cells exposed to extracellular sol or fib α-syn combined with LC3B knockdown using Western blot analysis. α-Syn levels were normalized to actin levels. Minor incorporation of sol and fib α-syn was found in oligodendroglial cells upon transfection with the control plasmids (scrambled shRNA). Knockdown of LC3B revealed an increased uptake of sol and fib α-syn compared to untreated cells and to cells transfected with the scrambled shRNA. However, only treatment with fib α-syn induced a significantly increased incorporation of α-syn upon LC3B knockdown compared to untreated cells. No significant difference was detected comparing LC3B knockdown and scrambled shRNA transfected oligodendroglial cells (b, d). No differences between α-syn levels were found regarding any treatment when measuring endogenously expressed α-syn in oligodendroglial cells (c, d). Two-way analysis of variance with post hoc Bonferroni test was applied. Data are presented as mean ± SEM. *p < 0.05; ***p < 0.001. N number equals 4

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