You'll have to ask your doctor to decipher this into layperson terms and where to get these cocktails, Yes, these are in mice but unless YOU light a fire under your doctor nothing will be done for when you need this.
Rescue of Transgenic Alzheimer’s Pathophysiology by Polymeric Cellular Prion Protein Antagonists
Highlights
- •Screen for antagonist of PrPC binding to Aβo identifies polymeric antibiotic degradant
- •Class of polymeric nM-potent PrPC antagonists rescue Aβo-induced phenotypes in vitro
- •PrPC antagonists also clear neuroblastoma cells of PrPSc replication
- •Oral PrPC antagonist rescues transgenic AD mouse synapse loss and memory deficits
Summary
Cellular prion protein (PrPC) binds the scrapie conformation of PrP (PrPSc)
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 PrPC interaction with Aβo. A polymeric degradant of an antibiotic targets Aβo binding sites on PrPC
with low nanomolar affinity and prevents Aβo-induced pathophysiology.
We then identified a range of negatively charged polymers with specific
PrPC 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 PrPC prevents Aβo/PrPC-hydrogel formation, blocks Aβo binding to neurons, and abrogates PrPSc
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 PrPC-directed polymeric agent provides a potential therapeutic approach to address neurodegeneration in AD and TSE.
Graphical Abstract
Keywords
Introduction
Extensive evidence points to the oligomeric form of β-amyloid peptide (Aβo) as the trigger to initiate Alzheimer’s pathology (
,
,
,
), but clinical measures to reduce brain Aβ burden have been therapeutically ineffective (
), inspiring exploration for alternate strategies. Discovery that cellular prion protein (PrPC) acts as a high-affinity neuronal receptor required for toxic Aβo signaling (
,
,
,
) has led to the identification of several effectors downstream of Aβo/PrPC interaction, such as mGluR5 (
,
,
,
), Fyn kinase (
,
,
), and Pyk2 kinase (
,
), that can be targeted pharmacologically to rescue the murine brain from AD model pathology. Abrogation of Aβo/PrPC interaction itself in vivo, genetically (
,
) or with antibodies directed against the Aβo-binding domains on PrPC (
,
,
), also reverses synaptic degeneration and restores behavioral performance to impaired AD model mice, even as Aβ load is unaltered. These data indicate the possibility of disease intervention independently of Aβo clearance and identify Aβo/PrPC interaction as an opportune nexus for pharmacological intervention after Aβ accumulation occurs.
,
,
,
), but clinical measures to reduce brain Aβ burden have been therapeutically ineffective (
), inspiring exploration for alternate strategies. Discovery that cellular prion protein (PrPC) acts as a high-affinity neuronal receptor required for toxic Aβo signaling (
,
,
,
) has led to the identification of several effectors downstream of Aβo/PrPC interaction, such as mGluR5 (
,
,
,
), Fyn kinase (
,
,
), and Pyk2 kinase (
,
), that can be targeted pharmacologically to rescue the murine brain from AD model pathology. Abrogation of Aβo/PrPC interaction itself in vivo, genetically (
,
) or with antibodies directed against the Aβo-binding domains on PrPC (
,
,
), also reverses synaptic degeneration and restores behavioral performance to impaired AD model mice, even as Aβ load is unaltered. These data indicate the possibility of disease intervention independently of Aβo clearance and identify Aβo/PrPC interaction as an opportune nexus for pharmacological intervention after Aβ accumulation occurs.
Cell-surface
PrP is a conformationally diverse protein, originally identified as
effecting transmissible spongiform encephalopathy (TSE) via a
template-induced proteinase K-resistant form (
). Conversion of PrPC to infectious scrapie (PrPSc) refolds the C-terminal segment, while the toxic action of PrPSc also requires natively unfolded N terminus (
). Recently, we showed that cell surface PrPC engages in phase state changes between soluble, liquid, and hydrogel (
). The mobile liquid state exists at endogenous PrPC levels present in lipid rafts. The relatively immobile hydrogel phase is induced upon association with multivalent Aβo. Lateral mobility of PrPC in the membrane is restricted upon Aβo association, and mGluR5 is trapped in the hydrogel. Upon Aβo association, the unstructured N-terminal PrP domain adopts an α-helical structure, which coincides with the engagement of mGluR5 and consequent synaptotoxic signaling though Fyn and Pyk2 kinases.
). Conversion of PrPC to infectious scrapie (PrPSc) refolds the C-terminal segment, while the toxic action of PrPSc also requires natively unfolded N terminus (
). Recently, we showed that cell surface PrPC engages in phase state changes between soluble, liquid, and hydrogel (
). The mobile liquid state exists at endogenous PrPC levels present in lipid rafts. The relatively immobile hydrogel phase is induced upon association with multivalent Aβo. Lateral mobility of PrPC in the membrane is restricted upon Aβo association, and mGluR5 is trapped in the hydrogel. Upon Aβo association, the unstructured N-terminal PrP domain adopts an α-helical structure, which coincides with the engagement of mGluR5 and consequent synaptotoxic signaling though Fyn and Pyk2 kinases.
Transgenic
APPswe/PS1ΔE9 mice (APP/PS1) expressing the human mutant amyloid
precursor protein (APP) and presenilin 1 (PS1) proteins that cause early
onset AD exhibit certain pathological characteristics (
). Age-dependent accumulation of Aβo and abundant amyloid plaques, synaptic degeneration, dendritic spine loss, Fyn dysregulation, microglial and astrocytic activation, and multiple memory deficits are among APP/PS1 histopathologies and functional deficits (
). Because later-stage AD symptoms such as tau tangle accumulation and cell loss are not evident in APP/PS1 and similar strains (
), these models may reflect an early AD stage, at which Aβo-directed intervention might have the greatest impact.
). Age-dependent accumulation of Aβo and abundant amyloid plaques, synaptic degeneration, dendritic spine loss, Fyn dysregulation, microglial and astrocytic activation, and multiple memory deficits are among APP/PS1 histopathologies and functional deficits (
). Because later-stage AD symptoms such as tau tangle accumulation and cell loss are not evident in APP/PS1 and similar strains (
), these models may reflect an early AD stage, at which Aβo-directed intervention might have the greatest impact.
Here, we describe competitive antagonists of Aβo/PrPC interaction. These compounds target PrPC Aβo-binding domains, thereby preventing Aβo association with PrPC, Aβo action in vitro, and APP/PS1 phenotypes in vivo. Additionally, these N terminus-directed ligands potently inhibit PrPSc propagation in culture, suggesting efficacy across PrPC-mediated neurodegenerative diseases.
Results
Ceftazidime Degradation Yields a Potent Polymeric Aβo/PrPC Inhibitor Termed Compound “Z”
To search for inhibitors of Aβo/PrPC interaction we engaged in a high throughput cell-based screen using stably PrPC-transfected CV-1 cells. Aβo prepared from biotinylated synthetic Aβ42 peptide associates with these cells in a PrPC-dependent fashion that can be blocked by an antibody (6D11) directed against the Aβo-binding domain at PrPC 90-111 (Figures 1A
and 1B). From a screen of 2,560 known drug and 10,130 diverse small
molecules, the cephalosporin antibiotic cefixime sample was found to be
highly inhibitory. Upon attempted validation, neither fresh cefixime nor
a range of cephalosporins was found to possess inhibitory activity,
suggesting an impurity or degradation product of cefixime was
responsible (compound “X”). To investigate this possibility, five
different cephalosporins were allowed to stand in DMSO at 23C for 6 days
before re-testing. In addition to cefixime, ceftazidime exhibited
activity resulting from prolonged incubation (compound “Z”), while other
cephalosporins (cefdinir, cefotaxime, and ceftriaxone) exhibited zero
activity either freshly diluted or after 6 days in DMSO (Figure 1C). Inhibitory activity developed progressively from ceftazidime incubated in sodium carbonate at 23°C over 9 days (Figure S1).
Figure 1Cefixime or Ceftazidime Degradation Produces HMW Inhibitor of Aβo/PrPC Interaction
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