Prion acute synaptotoxicity is largely driven by protease-resistant PrPSc species
Fig 7
Crude M1000 brain homogenate acute synaptotoxicity dose-response relationships and estimation of total PrP and PK-resistant PrPSc in preparations used in electrophysiology experiments.
A dilution series of recombinant full-length mouse PrP was western blotted (probed with 8H4 monoclonal antibody) and analysed with densitometry to generate a standard curve with aliquots of preparations utilised in electrophysiology experiments run on the same gel. (A) Quantification of levels of total PrP species (without PK treatment) in 0.5% (w/v), 0.25% (w/v), and 0.1% (w/v) cM1000, as well as in 0.5% dM1000 (PrP immuno-depleted cM1000). (B) Relative levels of at least modestly PK-resistant PrPSc (after treatment with 5μg/mL PK for 1 hour at 37°C) in 0.5% (w/v), 0.25% (w/v), and 0.1% (w/v) cM1000, as well as in 0.5% (w/v) PK+M1000 and 0.5% (w/v) PK+IP-M1000. (C & D) Percentage of LTP and PTP impairments (calculated as described in Methods) following exposure of 12-week old WT hippocampal slices to non-PK treated 0.5% (w/v), 0.25% (w/v), and 0.1% (w/v) cM1000 compared to 0.5% (w/v) cNBH controls (One-way ANOVA with Bonferroni correction for multiple comparisons [LTP: p<0.0001; PTP: p<0.0001]). In 0.5% (w/v) cM1000, LTP and PTP were significantly impaired by 53 ± 9% and 30 ± 6%, respectively. In 0.25% (w/v) cM1000, LTP and PTP were significantly impaired by 30 ± 2% and 26 ± 7%, respectively. In 0.1% (w/v) cM1000, LTP and PTP were not significantly impaired, reduced by only 19 ± 6% and 0.1 ± 9%, respectively. Percentages of LTP and PTP impairment in WT hippocampal slices following exposure to 0.5% dM1000, PK+M1000, and PK+IP-M1000 were also compared to 0.5% (w/v) cNBH controls (One-way ANOVA with Bonferroni correction for multiple comparisons [LTP: p<0.0001; PTP: p<0.0039]). In 0.5% (w/v) dM1000, the LTP was not impaired significantly (reduced by 14 ± 7%) while PTP was significantly impaired by 38 ± 3%. In 0.5% (w/v) PK+M1000, LTP and PTP were significantly impaired by 48 ± 7% and 26 ± 7%, respectively. In 0.5% (w/v) PK+IP-M1000, LTP and PTP were significantly impaired by 59 ± 5% and 36 ± 5%, respectively. (E & F) Percentage LTP and PTP impairments following exposure of 12-week old PrPo/o hippocampal slices to non-PK treated 0.5% (w/v), 0.25% (w/v), and 0.1% (w/v) cM1000 compared to 0.5% (w/v) cNBH controls (One-way ANOVA with Bonferroni correction for multiple comparisons [LTP: p<0.0002; PTP: p<0.0027]). In 0.5% (w/v) cM1000, LTP and PTP were significantly impaired by 58 ± 8% and 32 ± 8%, respectively. In 0.25% (w/v) cM1000, LTP and PTP were significantly impaired by 48 ± 10% and 39 ± 6%, respectively. In 0.1% (w/v) cM1000, LTP and PTP were not significantly impaired (reduced by only 15 ± 7% and 13 ± 5%, respectively). Data are presented as ± SEM. *p<0.05, **p<0.01, ***p<0.001, ****p<0.0001.