Objective
Currently no effective disease‐modifying agents exist for the treatment of Alzheimer disease (AD). The Fyn tyrosine kinase is implicated in AD pathology triggered by amyloid‐ß oligomers ...(Aßo) and propagated by Tau. Thus, Fyn inhibition may prevent or delay disease progression. Here, we sought to repurpose the Src family kinase inhibitor oncology compound, AZD0530, for AD.
Methods
The pharmacokinetics and distribution of AZD0530 were evaluated in mice. Inhibition of Aßo signaling to Fyn, Pyk2, and Glu receptors by AZD0530 was tested by brain slice assays. After AZD0530 or vehicle treatment of wild‐type and AD transgenic mice, memory was assessed by Morris water maze and novel object recognition. For these cohorts, amyloid precursor protein (APP) metabolism, synaptic markers (SV2 and PSD‐95), and targets of Fyn (Pyk2 and Tau) were studied by immunohistochemistry and by immunoblotting.
Results
AZD0530 potently inhibits Fyn and prevents both Aßo‐induced Fyn signaling and downstream phosphorylation of the AD risk gene product Pyk2, and of NR2B Glu receptors in brain slices. After 4 weeks of treatment, AZD0530 dosing of APP/PS1 transgenic mice fully rescues spatial memory deficits and synaptic depletion, without altering APP or Aß metabolism. AZD0530 treatment also reduces microglial activation in APP/PS1 mice, and rescues Tau phosphorylation and deposition abnormalities in APP/PS1/Tau transgenic mice. There is no evidence of AZD0530 chronic toxicity.
Interpretation
Targeting Fyn can reverse memory deficits found in AD mouse models, and rescue synapse density loss characteristic of the disease. Thus, AZD0530 is a promising candidate to test as a potential therapy for AD. Ann Neurol 2015;77:953–971
Dozens of genes have been implicated in late onset Alzheimer's disease (AD) risk, but none has a defined mechanism of action in neurons. Here, we show that the risk factor Pyk2 (PTK2B) localizes ...specifically to neurons in adult brain. Absence of Pyk2 has no major effect on synapse formation or the basal parameters of synaptic transmission in the hippocampal Schaffer collateral pathway. However, the induction of synaptic LTD is suppressed in Pyk2-null slices. In contrast, deletion of Pyk2 expression does not alter LTP under control conditions. Of relevance for AD pathophysiology, Pyk2
slices are protected from amyloid-β-oligomer (Aβo)-induced suppression of LTP in hippocampal slices. Acutely, a Pyk2 kinase inhibitor also prevents Aβo-induced suppression of LTP in WT slices. Female and male transgenic AD model mice expressing APPswe/PSEN1ΔE9 require Pyk2 for age-dependent loss of synaptic markers and for impairment of learning and memory. However, absence of Pyk2 does not alter Aβ accumulation or gliosis. Therefore, the Pyk2 risk gene is directly implicated in a neuronal Aβo signaling pathway impairing synaptic anatomy and function.
Genetic variation at the Pyk2 (PTK2B) locus is a risk for late onset Alzheimer's disease (AD), but the pathophysiological role of Pyk2 is not clear. Here, we studied Pyk2 neuronal function in mice lacking expression with and without transgenes generating amyloid-β (Aβ) plaque pathology. Pyk2 is not required for basal synaptic transmission or LTP, but participates in LTD. Hippocampal slices lacking Pyk2 are protected from AD-related Aβ oligomer suppression of synaptic plasticity. In transgenic AD model mice, deletion of Pyk2 rescues synaptic loss and learning/memory deficits. Therefore, Pyk2 plays a central role in AD-related synaptic dysfunction mediating Aβ-triggered dysfunction.
Alzheimer's disease-related phenotypes in mice can be rescued by blockade of either cellular prion protein or metabotropic glutamate receptor 5. We sought genetic and biochemical evidence that these ...proteins function cooperatively as an obligate complex in the brain. We show that cellular prion protein associates via transmembrane metabotropic glutamate receptor 5 with the intracellular protein mediators Homer1b/c, calcium/calmodulin-dependent protein kinase II, and the Alzheimer's disease risk gene product protein tyrosine kinase 2 beta. Coupling of cellular prion protein to these intracellular proteins is modified by soluble amyloid-β oligomers, by mouse brain Alzheimer's disease transgenes or by human Alzheimer's disease pathology. Amyloid-β oligomer-triggered phosphorylation of intracellular protein mediators and impairment of synaptic plasticity in vitro requires Prnp-Grm5 genetic interaction, being absent in transheterozygous loss-of-function, but present in either single heterozygote. Importantly, genetic coupling between Prnp and Grm5 is also responsible for signalling, for survival and for synapse loss in Alzheimer's disease transgenic model mice. Thus, the interaction between metabotropic glutamate receptor 5 and cellular prion protein has a central role in Alzheimer's disease pathogenesis, and the complex is a potential target for disease-modifying intervention.
Soluble Amyloid-β oligomers (Aβo) can trigger Alzheimer disease (AD) pathophysiology by binding to cell surface cellular prion protein (PrPC). PrPC interacts physically with metabotropic glutamate ...receptor 5 (mGluR5), and this interaction controls the transmission of neurotoxic signals to intracellular substrates. Because the interruption of the signal transduction from PrPC to mGluR5 has therapeutic potential for AD, we developed assays to explore the effect of endogenous ligands, agonists/antagonists, and antibodies on the interaction between PrPC and mGluR5 in cell lines and mouse brain. We show that the PrPC segment of amino acids 91–153 mediates the interaction with mGluR5. Agonists of mGluR5 increase the mGluR5-PrPC interaction, whereas mGluR5 antagonists suppress protein association. Synthetic Aβo promotes the protein interaction in mouse brain and transfected HEK-293 cell membrane preparations. The interaction of PrPC and mGluR5 is enhanced dramatically in the brains of familial AD transgenic model mice. In brain homogenates with Aβo, the interaction of PrPC and mGluR5 is reversed by mGluR5-directed antagonists or antibodies directed against the PrPC segment of amino acids 91–153. Silent allosteric modulators of mGluR5 do not alter Glu or basal mGluR5 activity, but they disrupt the Aβo-induced interaction of mGluR5 with PrPC. The assays described here have the potential to identify and develop new compounds that inhibit the interaction of PrPC and mGluR5, which plays a pivotal role in the pathogenesis of Alzheimer disease by transmitting the signal from extracellular Aβo into the cytosol.
Metabotropic glutamate receptor 5 (mGluR5) has been implicated in Alzheimer’s disease (AD) pathology. We sought to understand whether mGluR5’s role in AD requires glutamate signaling. We used a ...potent mGluR5 silent allosteric modulator (SAM, BMS-984923) to separate its well-known physiological role in glutamate signaling from a pathological role in mediating amyloid-β oligomer (Aβo) action. Binding of the SAM to mGluR5 does not change glutamate signaling but strongly reduces mGluR5 interaction with cellular prion protein (PrPC) bound to Aβo. The SAM compound prevents Aβo-induced signal transduction in brain slices and in an AD transgenic mouse model, the APPswe/PS1ΔE9 strain. Critically, 4 weeks of SAM treatment rescues memory deficits and synaptic depletion in the APPswe/PS1ΔE9 transgenic mouse brain. Our data show that mGluR5’s role in Aβo-dependent AD phenotypes is separate from its role in glutamate signaling and silent allosteric modulation of mGluR5 has promise as a disease-modifying AD intervention with a broad therapeutic window.
Display omitted
•A potent mGluR5 SAM blocks Aβo/PrPC but not glutamate signaling•The mGluR5 SAM rescues age-dependent memory loss in an AD mouse model•Synapse density is restored by mGluR5 SAM treatment over 4 weeks•mGluR5 SAM reveals a glutamate-independent AD role with a wide therapeutic index
Haas et al. investigate the role of mGluR5 in Alzheimer’s disease. They describe a compound that blocks the action of amyloid β through this receptor but preserves glutamate signaling. This amyloid-selective blocker rescues transgenic mouse models of Alzheimer’s disease and, by sparing glutamate signaling, avoids the side effects of typical mGluR5 antagonists.
Alzheimer disease (AD) is characterized by amyloid-β accumulation, with soluble oligomers (Aβo) being the most synaptotoxic. However, the multivalent and unstable nature of Aβo limits molecular ...characterization and hinders research reproducibility. Here, we characterized multiple Aβo forms throughout the life span of various AD mice and in post-mortem human brain. Aβo exists in several populations, where prion protein (PrPC)-interacting Aβo is a high molecular weight Aβ assembly present in multiple mice and humans with AD. Levels of PrPC-interacting Aβo match closely with mouse memory and are equal or superior to other Aβ measures in predicting behavioral impairment. However, Aβo metrics vary considerably between mouse strains. Deleting PrPC expression in mice with relatively low PrPC-interacting Aβo (Tg2576) results in partial rescue of cognitive performance as opposed to complete recovery in animals with a high percentage of PrPC-interacting Aβo (APP/PSEN1). These findings highlight the relative contributions and interplay of Aβo forms in AD.
Background: Amyloid-β (Aβ) oligomers are key in Alzheimer disease (AD) but are diverse and poorly characterized.
Results: Multiple Aβ forms were measured across the life span of AD model mice and human AD brain.
Conclusion: Aβ species interacting with prion protein were tightly linked to behavioral impairment.
Significance: An Aβ oligomer subset with defined biochemical properties is present in multiple AD-relevant samples.
Biochemical and genetic evidence implicate soluble oligomeric amyloid-β (Aβo) in triggering Alzheimer's disease (AD) pathophysiology. Moreover, constitutive deletion of the Aβo-binding cellular prion ...protein (PrP
) prevents development of memory deficits in APP
/PS1ΔE9 mice, a model of familial AD. Here, we define the role of PrP
to rescue or halt established AD endophenotypes in a therapeutic disease-modifying time window after symptom onset. Deletion of
at either 12 or 16 months of age fully reverses hippocampal synapse loss and completely rescues preexisting behavioral deficits by 17 months. In contrast, but consistent with a neuronal function for Aβo/PrP
signaling, plaque density, microgliosis, and astrocytosis are not altered. Degeneration of catecholaminergic neurons remains unchanged by PrP
reduction after disease onset. These results define the potential of targeting PrP
as a disease-modifying therapy for certain AD-related phenotypes after disease onset.
The study presented here further elucidates our understanding of the soluble oligomeric amyloid-β-Aβo-binding cellular prion protein (PrP
) signaling pathway in a familial form of Alzheimer's disease (AD) by implicating PrP
as a potential therapeutic target for AD. In particular, genetic deletion of
rescued several familial AD (FAD)-associated phenotypes after disease onset in a mouse model of FAD. This study underscores the therapeutic potential of PrP
deletion given that patients already present symptoms at the time of diagnosis.
Synaptic loss is critical in Alzheimer's disease (AD), but the dynamics of synapse turnover are poorly defined. We imaged dendritic spines in transgenic APPswe/PSen1∆E9 (APP/PS1) cerebral cortex. ...Dendritic spine turnover is increased far from plaque in aged APP/PS1 mice, and in young APP/PS1 mice prior to plaque formation. Dysregulation occurs in the presence of soluble Aβ oligomer and requires cellular prion protein (PrPC). APP/PS1 mice lack responsiveness of spine turnover to sensory stimulation. Critically, enhanced spine turnover is coupled with the loss of persistent spines starting early and continuing with age. To evaluate mechanisms of experience-independent supranormal spine turnover, we analyzed the transcriptome of young APP/PS1 mouse brain when turnover is altered but synapse density and memory are normal, and plaque and inflammation are absent. Early PrPC-dependent expression changes occur in synaptic and lipid-metabolizing genes. Thus, pathologic synaptic dysregulation underlying AD begins at a young age prior to Aβ plaque.
The dysfunction and loss of synapses in Alzheimer disease are central to dementia symptoms. We have recently demonstrated that pathological Amyloid β oligomer (Aβo) regulates the association between ...intracellular protein mediators and the synaptic receptor complex composed of cellular prion protein (PrPC) and metabotropic glutamate receptor 5 (mGluR5). Here we sought to determine whether Aβo alters the physiological signaling of the PrPC-mGluR5 complex upon glutamate activation. We provide evidence that acute exposure to Aβo as well as chronic expression of familial Alzheimer disease mutant transgenes in model mice prevents protein-protein interaction changes of the complex induced by the glutamate analog 3,5-dihydroxyphenylglycine. We further show that 3,5-dihydroxyphenylglycine triggers the phosphorylation and activation of protein-tyrosine kinase 2-β (PTK2B, also referred to as Pyk2) and of calcium/calmodulin-dependent protein kinase II in wild-type brain slices but not in Alzheimer disease transgenic brain slices or wild-type slices incubated with Aβo. This study further distinguishes two separate Aβo-dependent signaling cascades, one dependent on extracellular Ca2+ and Fyn kinase activation and the other dependent on the release of Ca2+ from intracellular stores. Thus, Aβo triggers multiple distinct PrPC-mGluR5-dependent events implicated in neurodegeneration and dementia. We propose that targeting the PrPC-mGluR5 complex will reverse aberrant Aβo-triggered states of the complex to allow physiological fluctuations of glutamate signaling.
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.
Display omitted
•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
Gunther et al. search for antagonists for Aβ oligomer binding to PrPC and identify a class of potent polymeric compounds. These molecules bind PrPC and competitively antagonize Aβo action at synapses, while also clearing PrPSc replication from neuroblastoma cells. An orally available PrPC antagonist rescues transgenic mouse Alzheimer phenotypes.
PDF
