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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.SIGNIFICANCE STATEMENTGenetic 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.

Objective Amyloid‐beta oligomers (Aßo) trigger the development of Alzheimer's disease (AD) pathophysiology. Cellular prion protein (PrPC) initiates synaptic damage as a high affinity receptor for Aßo. Here, we evaluated the preclinical therapeutic efficacy of a fully human monoclonal antibody against PrPC. This AZ59 antibody selectively targets the Aβo binding site in the amino‐terminal unstructured domain of PrPC to avoid any potential risk of direct toxicity. Methods Potency of AZ59 was evaluated by binding to PrPC, blockade of Aβo interaction and interruption of Aβo signaling. AZ59 was administered to mice by weekly intraperitoneal dosing and brain antibody measured. APP/PS1 transgenic mice were treated with AZ59 and assessed by memory tests, by brain biochemistry and by histochemistry for Aß, gliosis and synaptic density. Results AZ59 binds PrPC with 100 pmol/L affinity and blocks human brain Aßo binding to PrPC, as well as prevents synaptotoxic signaling. Weekly i.p. dosing of 20 mg/kg AZ59 in a murine form achieves trough brain antibody levels greater than 10 nmol/L. Aged symptomatic APP/PS1 transgenic mice treated with AZ59 for 5–7 weeks show a full rescue of behavioral and synaptic loss phenotypes. This recovery occurs without clearance of plaque pathology or elimination of gliosis. AZ59 treatment also normalizes synaptic signaling abnormalities in transgenic brain. These benefits are dose‐dependent and persist for at least 1 month after the last dose. Interpretation Preclinical data demonstrate that systemic AZ59 therapy rescues central synapses and memory function from transgenic Alzheimer's disease pathology, supporting a disease‐modifying therapeutic potential.

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 (PrPC) prevents development of memory deficits in APPswe/PS1ΔE9 mice, a model of familial AD. Here, we define the role of PrPC to rescue or halt established AD endophenotypes in a therapeutic disease-modifying time window after symptom onset. Deletion of Prnp 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/PrPC signaling, plaque density, microgliosis, and astrocytosis are not altered. Degeneration of catecholaminergic neurons remains unchanged by PrPC reduction after disease onset. These results define the potential of targeting PrPC as a disease-modifying therapy for certain AD-related phenotypes after disease onset.

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 (PrP(C)) and metabotropic glutamate receptor 5 (mGluR5). Here we sought to determine whether Aβo alters the physiological signaling of the PrP(C)-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 Ca(2+) and Fyn kinase activation and the other dependent on the release of Ca(2+) from intracellular stores. Thus, Aβo triggers multiple distinct PrP(C)-mGluR5-dependent events implicated in neurodegeneration and dementia. We propose that targeting the PrP(C)-mGluR5 complex will reverse aberrant Aβo-triggered states of the complex to allow physiological fluctuations of glutamate signaling.

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.

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.

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.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.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.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.

Soluble Amyloid-β oligomers (Aβo) can trigger Alzheimer disease (AD) pathophysiology by binding to cell surface cellular prion protein (PrP(C)). PrP(C) 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 PrP(C) 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 PrP(C) and mGluR5 in cell lines and mouse brain. We show that the PrP(C) segment of amino acids 91-153 mediates the interaction with mGluR5. Agonists of mGluR5 increase the mGluR5-PrP(C) 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 PrP(C) and mGluR5 is enhanced dramatically in the brains of familial AD transgenic model mice. In brain homogenates with Aβo, the interaction of PrP(C) and mGluR5 is reversed by mGluR5-directed antagonists or antibodies directed against the PrP(C) 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 PrP(C). The assays described here have the potential to identify and develop new compounds that inhibit the interaction of PrP(C) and mGluR5, which plays a pivotal role in the pathogenesis of Alzheimer disease by transmitting the signal from extracellular Aβo into the cytosol.