Rescue of Transgenic Alzheimer's Pathophysiology by Polymeric Cellular Prion Protein Antagonists

Abstract

Cellular prion protein (PrP^C) binds the scrapie conformation of PrP (PrP^Sc) and oligomeric β-amyloid peptide (Aβo) to mediate transmissible spongiform encephalopathy (TSE) and Alzheimer's disease (AD), respectively. We conducted cellular and biochemical screens for compounds blocking PrP^C interaction with Aβo. A polymeric degradant of an antibiotic targets Aβo binding sites on PrP^C with low nanomolar affinity and prevents Aβo-induced pathophysiology. We then identified a range of negatively charged polymers with specific PrP^C affinity in the low to sub-nanomolar range, from both biological (melanin) and synthetic (poly [4-styrenesulfonic acid-co-maleic acid], PSCMA) origin. Association of PSCMA with PrP^C prevents Aβo/PrP^C-hydrogel formation, blocks Aβo binding to neurons, and abrogates PrP^Sc production by ScN2a cells. We show that oral PSCMA yields effective brain concentrations and rescues APPswe/PS1ΔE9 transgenic mice from AD-related synapse loss and memory deficits. Thus, an orally active PrP^C-directed polymeric agent provides a potential therapeutic approach to address neurodegeneration in AD and TSE.

Keywords: Alzheimer; Alzheimer’s disease; amyloid-beta; antagonist; hydrogel; memory; oligomer; prion; scrapie; synapse loss; β-amyloid.

Figure 1.. Cefixime or Ceftazidime Degradation Produces HMW Inhibitor of Aβo/PrP^C Interaction

(A) Human stably PrP^C-transfected CV1 cells treated with biotinylated Aβo (~20 nM Aβo, 1 μM Aβ monomer equivalent) followed by 555-streptavidin exhibit signal that is inhibited by the 6D11 antibody (1 μg/mL) directed against the PrP^C 90–111 Aβo-binding domain. Scale bar, 5 μm. (B) Quantitation of Aβo-binding in (A), background subtracted. Data are mean ± SEM, n = 3 wells. *p < 0.05, Student’s t test. (C) Aβo/PrP^C interaction-inhibiting activity of five cephalosporin antibiotics applied to stably PrP^C-transfected CV1 cells followed by treatment with biotinylated Aβo (500 nM monomer equivalent). “Fresh” column: 10 μM drug applied immediately upon dissolution. “Aged” column: 10 μM drug applied 6 days post dissolution. Red circles: acid groups common to cephalosporins that acquire activity post-aging. Scale bar, 5 μm. (D) Absorbance trace from size exclusion chromatography (SEC) fractionation of aged ceftazidime activity (compound “Z”). MW standards at top of graph. (E) Aβo/PrP^C interaction-inhibitory activity of SEC fractions from (D) as measured by PLISA biochemical assay. Maximal activity resides in the HMW fractions of aged ceftazidime.

Figure 2.. Purified Compound Z Binds PrP^C Reversibly, Inhibiting Aβo/PrP^C Interaction via Competition for PrP^C Aβo-Binding Domains

(A) Biolayer interferometric association (60–180 s) and dissociation (180–300 s) traces of 10–20 kDa Z with PrP^C-coated sensor in 4-fold dilution steps from 1 μM top concentration, indicating a dissociation constant of 1.7 nM (top graph). Aβo-coated sensor detects soluble full-length PrP^C interaction but not compound Z (middle and bottom graphs, respectively). (B) PLISA measurement of 10–20 kDa Z Aβo/PrP^C inhibitory activity, indicating an IC₅₀ of 910 pM. Data are mean ± SEM, n = 3 replicates per sample. (C) PrP^C immunoblot of non-denaturing gel-shift assay of full-length PrP^C incubated with 10–40 kDa Z. Laddering indicates multiple PrP^C molecules bound per Z molecule. Incubation at 65°C after co-incubation shows reversible association of Z and PrP^C. (D) Biotinylated 10–20 kDa Z binds full-length PrP^C-coated plate concentration-dependently. Data are mean ± SEM, n = 3 replicates per sample. (E) Binding of biotinylated Z to PrP^C-coated plate is inhibited by antibodies directed against either of the two Aβo-binding domains on PrP^C and not by antibodies against other regions of PrP, indicating direct and selective Z interaction with PrP^C Aβo-binding domains. Data are mean ± SEM, n = 3 replicates per sample. *p <0.05, Student’s t test.

Figure 3.. Z Blocks Neuronal Action of Aβo and Propagation of Proteinase K-Resistant PrP^Sc in Cell Culture

(A) Aβo (1 μM monomer equivalent) binding to DIV 19 mouse hippocampal neurons is blocked by 50 nM 10–20 kDa Z. 80% and 87% of neuronal Aβo binding is inhibited relative to the neuronal markers SV2a and actin, respectively. Scale bar, 10 μM. Data are mean ± SEM, n = 3 wells. **p < 0.01; ***p <0.001 Student’s t test. (B) Induction of phospho-SFK (Src Family Kinase) in DIV 21 mouse cortical neurons by 30-min application of Aβo (1 μM) is blocked by 10–20 kDa Z (50 nM). Phospho-SFK is normalized to total Fyn that is the predominant neuronal SFK family member activated by Aβo (Um et al., 2012). Data are mean ± SEM, n = 3 wells. *p < 0.05 by one-way ANOVA with Tukey’s multiple comparisons test. (C) Neurotoxic action of 6 hr Aβo (3 μM) treatment of DIV 21 hippocampal neurons is blocked dose-dependently by 10–20 kDa Z, as indicated by LDH release, with maximal effect reached at 5 nM Z. *p < 0.05 by one-way ANOVA with Tukey’s multiple comparisons test. (D) Induction of DIV 20 hippocampal neuronal dendritic spine loss by 6 hr application of Aβo (500 nM) is blocked by co-incubation with 10–20 kDa Z (100 nM). *p < 0.05; **p < 0.01 by one-way ANOVA with Tukey’s multiple comparisons test. (E) Propagation of proteinase K-resistant PrP^Sc prion in scN2a cell culture is blocked by 6 day application of 10–20 kDa Z (1 μM) as revealed by anti-PrP immunoblot.

Figure 4.. Intracerebroventricular Z, but Not Peripherally Administered Fresh Ceftazidime, Rescues APP/PS1 Transgenic AD Model Mice from Memory Pathology

(A and B) Ceftazidime (as Fortaz) was dissolved at 333 mg/mL in sodium carbonate per manufacturer’s instructions and allowed to stand 14 days at 23°C. Vehicle (veh) or aged Fortaz (Z) containing PrP antagonist activity equal to 2 μM purified 10–20 kDa compound Z was administered intracerebroventricularly (ICV) by minipump to 12- to 14-month-old wild-type (WT) or APP/PS1 (TG) mice at a constant rate of 0.11 μL/hr for 4 weeks before memory assessment by MWM. Assuming the 24 hr dose distributes equally through the brain and is cleared within 24 hr, this yields a 10 nM predicted steady-state Z level. The same dose was continued throughout the behavior testing period via pump exchange. The time to reach the hidden platform during learning of an initial platform location (A) or a subsequent reversed platform location (B) is plotted as a function of swim block. The vehicle-treated APP/PS1 group differed significantly from all other groups by one-way RM-ANOVA over the last eight trials (two blocks) with Tukey’s multiple comparisons test (*p < 0.05), whereas other comparisons were not different (p > 0.05). (C) A 60-s probe trial test was performed 24 hr after completion of platform reversal training in the MWM. Plotted is the time spent in the area where the platform was previously located (target area). Vehicle-treated APP/PS1 transgenic Alzheimer’s model (TG) mice spent significantly less time in the target area than WT mice, reflecting AD memory deficit. TG mice treated with Z underwent normalization of time spent in the target area relative to WT mice treated with Z, reflecting restored memory function. Data are mean ± SEM of 9–12 mice/group. One-way ANOVA with Tukey’s comparisons as indicated. (D and E) 100 mg/kg ceftazidime (as Fortaz freshly solubilized at 333 mg/mL in sodium carbonate) or vehicle (veh) was administered by intraperitoneal (IP) injection twice daily for 6 weeks to 12- to 14-month-old wild-type (WT) or APP/PS1 transgenic Alzheimer’s model (TG) mice. Four weeks post-treatment initiation, spatial memory was assayed by Morris water maze (MWM) and plotted as the latency to locate a hidden platform during either the initial (forward) set of training blocks (D) or the set of training blocks post-platform relocation (reversal) (E). Data are mean ± SEM of 10–12 mice/group. Both APP/PS1 groups differed significantly from WT groups by one-way RM-ANOVA over the last eight trials (two blocks) with Tukey’s multiple comparisons test (*p < 0.05), whereas there were no differences within genotype groups (p > 0.05).

Figure 5.. Specific Anionic Polymers Are Potent PrP^C-Directed Inhibitors of Aβo/PrP^C Interaction

(A) Dilution series of anionic polymer activities were assayed by PLISA. Presence of the hydrophobic phenyl group in polar polystyrene co-maleic acid correlates with the high activity of the polymer. Data are mean ± SEM, n = 3 replicates per sample. (B) Size-dependence of Aβo/PrP^C inhibitory activity was assayed by PLISA, using polystyrene sulfonate polymers of specific average lengths and repeat number. Activity positively correlated with size up to 3.4 kDa, when an IC₅₀ plateau of ~5 nM was reached. Data are mean ± SEM, n = 3 replicates per sample. (C) Aβo/PrP^C inhibitory activity of selected anionic polymers exceed the activity of Z as assayed by PLISA. IC₅₀s = 900 pM, 700 pM and 300 pM for Z, polystyrene co-maleic acid, and poly (4-styrenesulfonic acid-co-maleic acid) (PSCMA), respectively. Data are mean ± SEM, n = 3 replicates per sample. (D) Known PrP^Sc inhibitors, pentosan polysulfate and dextran sulfate, exhibit much weaker PLISA activity than identified polar anionic polymers. Data are mean ± SEM, n = 3 replicates per sample. (E) Table of IC₅₀ and molecular weight (MW) of selected polymers assayed for Aβo/PrP^C inhibitory activity by PLISA. MWs represent the weight average MW of a bell-shaped range of polymer sizes. IC₅₀ represents activity based on concentrations calculated using that weight average MW of each polymer sample. (F) Biolayer interferometric measurement of PSCMA binding to PrP^C-coated sensor tip. Association (100–360 s) and dissociation (360–600 s) traces of 3.4 kDa PSCMA in 4-fold dilution steps from 1 μM top concentration, indicated a dissociation constant of 540 pM. (G) Biolayer interferometric measurement of soluble full-length PrP^C binding to Aβo-coated sensor tip in assay buffer alone (left sensorgram) or in the presence of PSCMA (P) (right sensorgram). Association (200–400 s) and dissociation (400–600 s) traces of PrP^C in 4-fold dilution steps from 500 nM top concentration, are completely inhibited by PSCMA (1 μM). PSCMA exhibits no affinity for Aβo (100–200 s, right sensorgram) indicating specificity for PrP^C.

Figure 6.. Active Polymers Block Aβo Neuronal Action and Terminate Pathological PrP^Sc Propagation at Low nM Concentrations

(A) PrP^C-transfected COS7 cells were treated with 0 or 200 nM PSCMA for 30 min at RT, followed by addition of 1 μM (monomer equivalent) biotinylated Aβo for 1 hr, fixation and staining of PrP^C (green), Aβo (red), DAPI (blue) as indicated. Scale bar, 20 μm. (B) Quantification of Aβo signal for individual PrP^C-transfected cells as in (A), expressed as the ratio of Aβo signal to PrP^C immunoreactivity. The PSCMA IC₅₀ of 3.4 nM is indicated. n = 3 wells per condition, 10 randomly selected cells per well. Data are mean ± SEM. (C) SNAP-tagged PrP^C-transfected COS7 cells were treated with SNAP-Surface Alexa Fluor647 to fluorescently label cell-surface PrP. Cells were treated with vehicle 1 hr,1 μM Aβo for 1 hr, 1 μM PSCMA for 1 hr, or PSCMA for 15 min followed by Aβo for 1 hr. A 50 μm² square of fluorescent PrP was bleached with a burst of laser light, and recovery of PrP into the bleached area though lateral PrP diffusion in the plasma membrane monitored over 250 s. Scale bar, 1 μm. (D) Quantification of recovery in (C). Vehicle and PSCMA treatment fluorescence recovery curves were indistinguishable. Aβo treatment strongly inhibited PrP^C recovery kinetics, indicating arrested lateral mobility of PrP^C in the plasma membrane as a result of Aβo/PrP^C complexation. PSCMA pre-treatment completely prevented Aβo-induced inhibition PrP^C recovery kinetics. (E) 18 DIV mouse hippocampal neurons were treated with 0 or 200 nM polystyrene sulfonate (PSS) for 30 min at RT, followed by addition of 0.5 μM (monomer equivalent) biotinylated Aβo for 1 hr, fixation and staining for the dendritic marker MAP2 (green) and for Aβo (red). Scale bar, 15 μm. (F) Quantification of total Aβo signal per well for cultures similar to those in (E), measuring PSCMA inhibition of binding 21 DIV rat cortical neurons. The PSCMA IC₅₀ of 32 nM is indicated. n = 3 wells per condition. Data are mean ± SEM. (G) 14 DIV mouse hippocampal neurons were treated 4 days with vehicle or Aβo (1 μM) +/[C0] the designated concentrations of polystyrene sulfonate (PSS). Scale bar, 15 μm (top), 5 μm (bottom). Aβo induced a consistent reduction in spine density per mm dendrite length. (H) Quantitation of dendritic spine density from experiments as in (G.) Co-administered PSS blocked Aβo action dose-dependently with an IC₅₀ between 1–10 nM. n = 3 dendrites per neuron, 5–7 neurons per coverslip, 4–8 coverslips per condition. Data are mean ± SEM (***p < 0.001), one-way ANOVA with Dunnett’s multiple comparisons test. (I) Propagation of proteinase K-resistant PrP^Sc prion in scN2a cell culture is blocked by 6 day application of PSCMA, with an EC₅₀ between 10–40 nM, as revealed by PrP^C immunoblot. Data are mean band densitometry ± SEM, n = 3 replicates per condition. **p < 0.01; ***p < 0.001 relative to veh control, one-way ANOVA with Tukey’s corrected pairwise comparisons.

Figure 7.. Oral Administration Yields Active PSCMA in Mouse Brain and Rescues APP/PS1 Memory Deficits and Synaptopathology

(A) WT mice were administered 40 mg/kg (mpk) PCMA by oral gavage twice daily for 10 days, followed by perfusion, brain lysis, extraction, and PLISA assessment of Aβo/PrP^C inhibitory activity. Data are mean ± SEM, n = 6 mice. Standard curve was made by spiking brain lysate from untreated mice with the designated concentrations of PSCMA, prior to identical processing as brains from treated mice. Orally dosed mouse brain contains an average 40 nM PSCMA. Data are mean ± SEM, n = 3 replicates per sample. (B) APP/PS1 transgenic and wild-type mice were orally gavaged with vehicle (n = 13 transgenic, 15 WT) or 3 mpk PSCMA twice daily (n = 14 transgenic, 14 WT) for a total of 30 days prior to behavioral analysis. After behavioral testing on day 42, PFA-perfused brains were sectioned and immunostained for the pre-synaptic marker SV2A. Representative dentate gyrus confocal images are shown. Scale bar, 10 μm. (C) Synaptotoxicity-induced 42% reduction in SV2a immunoreactivity in the hippocampi of AD model mice was significantly improved by PSCMA treatment, which restored the hippocampal area occupied by SV2a, as determined by one-way ANOVA with Tukey’s multiple comparisons test (*p < 0.05). (D) Dentate gyrus sections prepared as in (B) were immunostained for the post-synaptic marker PSD95. Scale bar, 10 μm. (E) A trend toward PSD95 area reduction was observed in the hippocampus of vehicle-treated transgenic mice compared to WT, but it did not reach statistical significance, as determined by one-way ANOVA and Tukey’s multiple comparisons test. PSCMA reversed this trend. (F) PSCMA-treated APP/PS1 mice are rescued from learning and memory deficits. After treating for 4 weeks twice daily by oral gavage with PSCMA, mice were evaluated for memory performance by Morris water maze. Latency to find the hidden platform is plotted as a function of trial block number. Statistical differences between groups are indicated in the figure. Data are mean ± SEM (*p < 0.05; ***p < 0.001). One-way repeated-measures ANOVA comparing each group to the transgenic group treated with PSCMA, with Dunnett’s multiple comparisons test. (G) For the probe trial, percent time spent in the target quadrant after platform removal is indicated. Vehicle-treated transgenic animals spent significantly less time in the target quadrant compared to either drug-treated WT animals or drug-treated transgenic animals. Data are mean ± SEM (*p < 0.05; **p < 0.01). One-way ANOVA with Dunnett’s test comparing to APP/PS1, veh.