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. 2019 Apr 10;15(4):e1007712. doi: 10.1371/journal.ppat.1007712

Fig 3. Acute synaptotoxicity of cM1000 from earlier time points of M1000 prion disease is associated with prion protein species.

Fig 3

(A) Western blotting of 20 μL 1% (w/v) crude M1000 brain homogenates (cM1000) from 30%, 50%, 70%, and 100% of M1000 prion disease progression to terminal stage disease (TSD) following mock immuno-depletion of total PrP species with normal rabbit serum (control) or immuno-depletion of total PrP species with 03R19 prion antibody (Probed with 8H4 antibody; molecular weight markers are provided at left). (B) Quantification of total PrP species in PrP immuno-depleted normal brain homogenate (dNBH) and PrP immuno-depleted cM1000 (dM1000) from across the disease progression (by densitometry analysis) compared to appropriate negative controls by Unpaired Student’s t-test (mock immuno-depleted NBH or mock immuno-depleted cM1000). (C) Upper panel: A representative western blot of PrP levels in 20 μL cM1000 at 100% TSD following a mock immuno-depletion with normal rabbit serum (NRS) and an immuno-depletion with 03R19 antibody, and mild PK digestion. Lower panel: Quantification of total PrP (n = 4) and at least modestly PK-resistant PrP (n = 2) immuno-depleted from cM1000 (from 100% of the TSD) by 03R19 relative to the controls (probed with 8H4 antibody; molecular weight markers are provided at left). (D to G) LTP of WT mouse hippocampal slices following a five minute treatment with 0.5% (w/v in aCSF) dM1000 from 30% (D), 50% (E), 70% (F), and 100% of the TSD (G) compared with the LTP of slices treated with 0.5% (w/v in aCSF) dNBH controls (One-way ANOVA with Dunnett’s correction for multiple comparisons). (H) The average LTPs, (I) the average percentage of PPF ratio decline, and (J) the average PTPs generated by WT mouse hippocampal slices treated with dNBH and dM1000 from across time points of the disease progression were compared by One-way ANOVA with Dunnett’s correction for multiple comparisons when comparing across time points. (D to G) The five minute treatment started after 8-to-10 minutes of stable baseline. The high frequency stimulation (HFS) trains were applied following 30 minutes of baseline recordings. Results are presented as mean ± standard error of mean. (D-G, I & J) Some examples of raw fEPSP traces are provided as insets. *p<0.05, **p<0.01, ***p<0.001, ns = not statistically significant (p>0.05).