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Fig. 3 | Molecular Neurodegeneration

Fig. 3

From: Microglial inclusions and neurofilament light chain release follow neuronal α-synuclein lesions in long-term brain slice cultures

Fig. 3

Increased Neurofilament light chain (NfL) in the culture medium in response to the induction of αS lesions. (A) Schematic of a well-plate containing HSCs and medium collection scheme. Each well contains 3 cultures of the same treatment. The entire culture medium (1.2 ml) of each well was collected (and immediately replaced with new one) before seeding (baseline), 2 days (acute), and 1, 2, and 3 weeks after seeding. (B) Longitudinal measurement of NfL in culture medium of tg HSCs measured by immunoassay. Cultures were treated with 35 μM αS pff (pink), or left untreated (black, dotted line). As a control, cultures were also treated with EtOH (dark red). While EtOH treatment had an acute toxic effect, αS pff treated cultures released NfL gradually into the medium with an apparent peak at 2 weeks post-seeding. Mean ± SEM is shown; n = 3 wells (each containing 3 cultures) per group; two-way-ANOVA with repeated measurements (treatment F(1, 4) = 79.86, p = 0.0009; time F(3, 12) = 78.57, p < 0.0001; interaction F(3, 12) = 51.69) with Bonferroni’s corrections for multiple comparisons between treatment groups and within time points (***p < 0.0001). The experiment was repeated with a new batch of αS pff and resulted in somewhat higher NfL levels (171.0 ± 136.8, 2453.7 ± 169.7, 3800.0 ± 431.2 and 2713.7 ± 575.7 pg/mL for 2d, 1 wk., 2 wk., 3 wk., respectively) in comparison to the untreated (43.7 ± 6.6, 248.0 ± 89.5, 234.3 ± 16.6 and 158.3 ± 24.3 pg/mL for 2d, 1 wk., 2 wk., 3 wk., respectively; n = 3 wells (each containing 3 cultures) per group. (C-F) αS seed inactivation blocks NfL increase in culture medium. 35 μM αS pff were mixed 1:1 with 35 μM αS- or ctr-antibody before applying onto each tg HSC. After 3 weeks, αS pff / αS antibody-treated HSCs showed very little pS129-positive inclusions compared to αS pff / ctr antibody treated HSCs (C). Scale bar = 500 μm. Quantification of pS129-positive inclusions (D, unpaired two-tailed t-test, t(4) = 9.22, ***p = 0.0008) and ThioS-positive inclusions (E, unpaired two-tailed t-test, t(4) = 4.19, *p = 0.0138). Mean ± SEM is shown with n = 3 wells (each containing 3 cultures and the mean of the three cultures (black dots) was taken as one data point; grey dots are values for each culture). NfL measurements in the culture medium of αS pff / antibody mix and corresponding PBS-treated cultures (i.e. with no induction of αS lesions) is shown at 2 weeks post-treatment time points (F). Mean ± SEM; n = 3 wells, two-way ANOVA for treatment (pff vs PBS) F(1, 8) = 75.64, p < 0.0001; antibody F(1, 8) = 38.16, p = 0.003; interaction F(1, 8) = 57.850, p < 0.0001). Bonferroni’s correction for multiple comparisons revealed ****p = 0.0001 for αS vs ctr antibody

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