Metabotropic glutamate receptor 5 is a coreceptor for Alzheimer aβ oligomer bound to cellular prion protein

Abstract

Soluble amyloid-β oligomers (Aβo) trigger Alzheimer's disease (AD) pathophysiology and bind with high affinity to cellular prion protein (PrP(C)). At the postsynaptic density (PSD), extracellular Aβo bound to lipid-anchored PrP(C) activates intracellular Fyn kinase to disrupt synapses. Here, we screened transmembrane PSD proteins heterologously for the ability to couple Aβo-PrP(C) with Fyn. Only coexpression of the metabotropic glutamate receptor, mGluR5, allowed PrP(C)-bound Aβo to activate Fyn. PrP(C) and mGluR5 interact physically, and cytoplasmic Fyn forms a complex with mGluR5. Aβo-PrP(C) generates mGluR5-mediated increases of intracellular calcium in Xenopus oocytes and in neurons, and the latter is also driven by human AD brain extracts. In addition, signaling by Aβo-PrP(C)-mGluR5 complexes mediates eEF2 phosphorylation and dendritic spine loss. For mice expressing familial AD transgenes, mGluR5 antagonism reverses deficits in learning, memory, and synapse density. Thus, Aβo-PrP(C) complexes at the neuronal surface activate mGluR5 to disrupt neuronal function.

Figure 1. Metabotropic GluR5 Links Aβo/PrP^C Complexes to Fyn

A Schematic indicating that Aβo/PrP^C complexes require a co-receptor to activate Fyn in PSDs. B Pie charts show transmembrane proteins (81) among total PSD proteins (651). Transmembrane proteins were subdivided; those that were screened in Aβo/Fyn assays (56, yellow) and those not (25, gray). C Immunoblot of screening for Fyn activation. HEK293T cells were transfected with expression vectors for Fyn, PrP^C or candidate genes. After treatment with 1 μM Aβo for 15 min, lysates were analyzed by anti-phospho-SFK (Tyr 416) or anti-Fyn immunoblot. D The ratio of phospho-SFK to Fyn is plotted from 3 experiments with Aβo. Blue line indicates the mean of all controls. Dark gray is one standard deviation, and light gray is two standard deviations from control. E HEK293T cells were transfected with vectors for Fyn, PrP^C or mGluR5, and treated with 0 or 1 μM Aβo for 15 min. Some cultures were pre-incubated for 30 min with 100 μM MPEP prior to Aβ. Lysates were analyzed by anti-phospho-SFK (Tyr 416), anti-Fyn, anti-mGluR5, or anti-PrP^C immunoblot. Actin is loading control. F HEK293T cells were transfected with vectors for Fyn, PrP^C or Myc-mGluR1 as indicated. After cell treatment with 0 or 1 μM Aβo for 15 min, lysates were analyzed by anti-phospho-SFK (Tyr 416), anti-Fyn, anti-Myc, or anti-PrP^C immunoblot. Actin is loading control. G Quantification of phospho-SFK level in lysates from E, F, normalized to Fyn. Mean±sem, n=4 experiments. **, P<0.01; *, P<0.05; ANOVA, Tukey post-hoc pairwise comparisons. H Cortical neurons from E17 WT mice at 21 DIV were treated with 0 or 1 μM of Aβo for 15 min. Indicated samples were treated with 100 μM MTEP or 10 μM MPMQ for 30 min prior to Aβo. Lysates were subjected to immunoblot with anti-Fyn, or anti-phospho-SFK (Tyr 416). Actin is loading control. I Cortical neurons from WT or Grm5−/− mice after 21 DIV were treated with 0 or 1 μM Aβo for 15 min. Lysates were analyzed by anti-phospho-SFK (Tyr 416), anti-Fyn, anti-mGluR5 immunoblot. Actin is loading control. J Quantification of phospho-SFK level in lysates from H, I, normalized to Fyn. Mean±sem, n=3 experiments. ***, P<0.001; *, P<0.05; ANOVA, Tukey post-hoc pairwise comparisons. See also Fig. S1.

Figure 2. PrP^C Interacts with mGluR5

A Aβo (250 nM) binding to COS7 cells expressing mGluR5, PrP^C, or co-expressing mGluR5 with PrP^C (upper). Protein expression was confirmed by immunofluorescence (bottom). Scale bar, 50 μm. B Dose-response for Aβo binding to COS7 cells from experiments as in A. Mean±sem, n=3 experiments. C HEK293T cells were transfected with vector for either mGluR5 or PrP^C, or co-transfected for mGluR5 and PrP^C. After treatment with 0 or 1 μM Aβo for 1 h, lysates (input) and anti-PrP^C immunoprecipitates were immunoblotted with either anti-mGluR5 or anti-PrP^C. D HEK293T cells were transfected with vector for PrP^C or co-transfected with different Myc-tagged mGluRs and PrP^C, as indicated. Lysates (input) and anti-Myc immunoprecipitates were immunoblotted with either anti-Myc or anti-PrP^C antibodies. E Indicated fractions (20 μg protein) were prepared and analyzed by immunoblot with anti-PrP^C, anti-mGluR5, anti-EphB2, anti-PSD95, anti-synaptophysin, and anti-actin antibodies. F Brain lysates from WT or Prnp −/− mice were cross-linked with the cleavable DTSSP. Whole lysates (5% Input) and anti-PrP^C immunoprecipitates were immunoblotted with anti-mGluR5, anti-mGluR8, anti-NR2B, anti-GluR1 or anti-PrP antibodies. Asterisk, Ig light chain. G His-tagged human PrP^C was incubated with DIV21 WT or Grm5 −/− neurons for 1 h. Neurons were then fixed and stained with human-specific anti-PrP^C antibody and Alexa-568 secondary antibody with rhodamine-phalloidin as counterstain. Scale bar, 10 μm. H Quantification of His-PrP^C bound to WT or Grm5 −/− neurons. His-PrP^C immunofluorescence was measured by ImageJ in segments of primary dendrites 10 μm from the soma and background-subtracted. Mean±sem, n=3 embryos for each genotype.***, P<0.001; Student’s two-tailed t test. See also Fig. S2.

Figure 3. Aβo directly stimulate mGluR5

A Representative traces obtained from oocytes expressing mGluR5 plus PrP^C, or mGluR1 plus PrP^C. Bath application (bars) of Aβo (1.5 μM monomer equivalent, estimated 15 nM oligomer) or Glu (100 μM) elicits an inward current from mGluR5 plus PrP^C oocyte. Exposure to Glu, but not Aβo, elicits a response in oocytes expressing mGluR1 and PrP^C. B, C Quantification of the peak current elicited by glutamate (B) or Aβo (C) in oocytes expressing the indicated proteins, with or without pre-treatment with 5 μg/ml anti-PrP^C antibody, 6D11. Data are Mean±sem, n=8–28 oocytes. **, P<0.01; ANOVA, Tukey post-hoc pairwise comparisons. D Dose response for Aβo-induced current in oocytes expressing mGluR5 and PrP^C. Mean±sem for n = 4–7 oocytes. E Oocytes expressing mGluR5 and PrP^C were either treated for 1 min with 100 μM Glu to maximally stimulate a response 3 min prior to testing for an Aβo response, or not pre-stimulated with Glu. Alternatively, some oocytes were preincubated with 100 μM BAPTA-AM for 60 minutes. After these pretreatments, the peak inward current was measured in response to 1.5 μM Aβo. Mean±sem, n=6–12 oocytes. **, P<0.005; ANOVA, Tukey post-hoc pairwise comparisons. See also Fig. S4.

Figure 4. Aβ species from human AD brains or synthetic Aβo increase intracellular calcium via PrP and mGluR5

A Change of intracellular calcium in E17 cortical neurons from WT, Prnp −/− or Grm5 −/− mice in response to Aβo, vehicle (Veh) or ionomycin (500 nM) was monitored by FLIPR calcium assay. Mean±sem, n=24–40 wells from 3–5 embryos per genotype. B Quantification of calcium response induced by Aβo or vehicle (Veh) from A. *, P<0.05, **, P<0.01 ***, P<0.001; ANOVA, Tukey post-hoc pairwise comparisons. C Quantification of calcium response induced by oligomeric Aβ (Aβo), monomeric Aβ (Aβm) or vehicle (Veh). ***, P<0.001; ANOVA, Tukey post-hoc pairwise comparisons. D Intracellular calcium in E17 cortical neurons in response to human AD brain extracts (AD), n=25, age-matched control brain (Con), n=19 (1.5 μg total protein/ml) or ionomycin (500 nM) monitored by FLIPR calcium assay. Mean±sem, ***, P<0.001 by Repeated Measures ANOVA for 10 seconds window after AD brain extract. E Quantification of calcium response induced by AD or Con extracts from C. Each dot is from a different brain. ***, P<0.001 by Student’s two-tailed t test. F Correlation between AD extract-induced calcium and PrP(23-111)-interacting Aβ species is plotted. Each point is from a different brain sample. Pearson coefficient of linear correlation reported with two-tailed P. G Calcium response induced by AD extracts preabsorbed with Fc (AD Fc) or PrP^C-Fc resin (AD PrP-Fc), or Con extracts preabsorbed with Fc (Con Fc) or PrP^C-Fc resin (Con PrP-Fc). *, P<0.05, **, P<0.01; ANOVA, Tukey post-hoc pairwise comparisons. H Calcium response induced by AD or Con extracts in E17 cortical neurons from WT, Prnp−/− or Grm5−/− mice. Mean±sem, n = 3 independent embryos for each genotype. *, P<0.05, **, P<0.01; ANOVA, Tukey post-hoc comparisons. I Calcium response induced by AD or Con extracts in WT cortical neurons. Neurons were pretreated with 100 μM MPEP, 100 μM MTEP, 10 μM MPMQ, 500 nM saracatinib for 1 h or 100 nM thapsigargin for 24 h prior to AD extract. *, P<0.05, **, P<0.01, ***, P<0.001; ANOVA, Tukey post-hoc pairwise comparisons to AD extract only samples. J WT cortical neurons were treated with 0 or 1 μM Aβo for 15 min. Indicated sample was treated with 100 nM thapsigargin for 24 h prior to Aβo. Lysates were analyzed by anti-phospho-SFK or anti-Fyn immunoblot. K Quantification of phospho-SFK level in the lysate normalized to Fyn from 3 experiments. Mean±sem, *, P<0.05; **, P<0.01; ANOVA, Tukey post-hoc pairwise comparisons. See also Fig. S3.

Figure 5. eEF2 phosphorylation is enhanced by Aβo through PrP and mGluR5

A DIV21 cortical neurons from WT, Prnp−/− or Grm5−/− mice were treated with 0 or 1 μM Aβo for 5 min. Lysates were analyzed by anti-phospho-eEF2, or anti-eEF2 immunoblot. GAPDH is loading control. B Phospho-eEF2 level in the lysate normalized to eEF2. WT, n=3; Prnp−/−, n=3; Grm5−/−, n=3. Mean±sem, **, P<0.01; Student’s two-tailed t test. C Immunohistology of DIV21 WT, Prnp−/− or Grm5−/− cortical neurons stained with anti-phospho-eEF2 and anti-MAP2 antibodies after exposure of 0 or 1 μM Aβo. Scale bar, 10 μm. D Phospoho-eEF2 intensity normalized to MAP2 signal. WT, n=3; Prnp−/−, n=3; Grm5−/−, n=3. 8–10 images were analyzed per experiment. Mean±sem, ***, P<0.001; Student’s two-tailed t test. E Immunohistology of DIV 21 WT neurons stained with anti-Arc antibody after 1 μM Aβo for 5 min. Scale bar, 5 μm. F Arc intensity in dendrites after 1 μM Aβo or 100 μM DHPG for 5 min. For dendritic Arc levels, average pixel intensity was measured in secondary dendrites 10 μm from first branch point. WT, n=6. Mean±sem, **, P<0.01, *, P<0.05; ANOVA, Tukey post-hoc pairwise comparisons. G DIV21 cortical neurons from WT, Grm5−/− or Prnp−/− mice were treated with human control or AD brain extracts (30 μg total protein/ml) for 5 min. Lysates were analyzed by anti-phospho-eEF2 or anti-eEF2 immunoblot. H Phospho-eEF2 level normalized to eEF2 from G. WT, n=6; Grm5−/−, n=4; Prnp−/−, n=3. Mean±sem, *, P<0.05; **, P<0.01; ***, P<0.001; ANOVA, Tukey post-hoc pairwise comparisons. I, J WT cortical neurons were treated with 0 or 1 μM Aβo for 15 min. Indicated samples were treated with 500 nM saracatinib for 1 hour (I) or 100 nM thapsigargin (TG) for 24 hours (J) prior to Aβo. Lysates were analyzed by anti-phospho-SFK, anti-Fyn, anti-phospho-eEF2, or anti-eEF2 immunoblot. GAPDH is loading control. K Phospho-eEF2 level normalized to eEF2 from 3 experiments. Mean±sem, *, P<0.05; ANOVA, Tukey post-hoc pairwise comparisons. See also Fig. S5.

Figure 6. Aβo-induced toxicity is blocked by mGluR5 antagonists

A Hippocampal neurons (21DIV) expressing myristoyl-GFP from WT or Grm5−/− mice were imaged for 6 h with or without MPEP (100 μM). Aβo (500 nM monomer equivalent) or vehicle was added at 1 h. Note loss of several spines after Aβo addition in the WT neurons. Scale bar, 1 μm. B, C Dendritic spine loss over 5 hours is plotted as a function of Aβo and MPEP (B) or Grm5 genotype (C). Mean±sem, n=3–5 cultures from separate mice of each genotype. *, P<0.05; **, P<0.01; ANOVA, Tukey post-hoc pairwise comparisons. D Neurons at DIV21 from WT mice were treated with 0 or 1 μM Aβo for 2 h. Some cultures were treated with 100 μM MTEP for 1 h prior to Aβo. Cell toxicity was determined by LDH release. Mean ± sem, n=3 experiments. ***, P<0.001; ANOVA, Tukey post-hoc pairwise comparisons. E WT or Grm5−/− cortical neurons were treated with 0 or 1 μM Aβo for 2 h. Toxicity was determined by LDH release. Mean±sem, n=4 experiments. **, P<0.01; ANOVA, Tukey post-hoc pairwise comparisons.

Figure 7. mGluR5 Antagonist Reverses Learning and Memory Deficits in AD Mouse Models

A Spatial learning is plotted as the number of errors in finding a hidden platform in a radial arm water maze at age 9 months. Mean±sem for vehicle treated C57BL/6, n=11; MTEP treated C57BL/6, n=12; vehicle treated APP/PS1, n=10; MTEP treated APP/PS1, n=10. Performance differed across the last 15 swims by genotype and treatment (two-way RM-ANOVA APP/PS1, P<0.001; MTEP, P<0.001). There was an interaction between genotype and treatment (two-way RM-ANOVA APP/PS1xMTEP P<0.001). By Tukey post-hoc pairwise comparisons across trials, the vehicle-treated APP/PS1 group differed from each of the other groups (P<0.001), while none of the other groups differed from each other (P>0.05). For indicated trial blocks, the vehicle-treated APP/PS1 group differed from each of the other groups (*P<0.05; **P<0.01; ***P<0.001), while none of the other groups differed from each other (P>0.05). B Time spent with a novel object for the same mice as A: vehicle C57BL/6, n=11; MTEP C57BL/6, n=12; vehicle APP/PS1, n=9; MTEP APP/PS1, n=9. After being acclimated to an object, vehicle APP/PS1 mice showed no preference for a novel object (two tailed Student’s t test P>0.05). The other 3 groups showed preference for a novel object (two tailed Student’s t test P<0.001). C Spatial learning is plotted as latency to find a hidden platform in a Morris water maze at age 9 months in a cohort different from A. Mean±sem for untreated (naïve) C57BL/6, n=32; MTEP treated C57BL/6, n=13; untreated APP/PS1, n=38; MTEP treated APP/PS1, n=17. Performance differed across the last 16 swims by genotype and treatment (two-way RM-ANOVA for APP/PS1, P<0.001; for MTEP, P<0.001). There was an interaction between genotype and treatment (two-way RM-ANOVA, APP/PS1xMTEP P<0.001). By Tukey post-hoc pairwise comparisons across trials, the vehicle-treated APP/PS1 group differed from each of the other groups (***P<0.001), while none of the other groups differed from each other (P>0.05). For specific trial blocks, the untreated APP/PS1 group differed from each of the other groups (*P<0.05; ***P<0.001), while none of the other groups differed from each other (P>0.05). D Performance during a 60 sec probe trial, 24 h after learning, where time spent in the target quadrant was measured. Random chance is 25%. Mean±sem for the groups in C. Target quadrant time differed by genotype and treatment (two-way ANOVA APP/PS1, P<0.001; MTEP, P<0.01). There was an interaction between genotype and treatment (ANOVA, APP/PS1xMTEP P<0.001). By Tukey post-hoc, the untreated APP/PS1 group differed from others (P<0.001), while none of the other groups differed from each other (P>0.05). E WT and 3XTg mice of the same genetic background at 8–9 months of age were tested for novel object recognition. WT mice show preference the novel object (two-tailed Student’s t test P<0.05), but 3XTg mice show no preference (P>0.05). Mean±sem. F The effect of MTEP administration on object recognition is tested in 3XTg mice at age 8 months. Mean±sem for vehicle 3XTg, n=12; MTEP 3XTg, n=8. Mice that received MTEP had a significant preference for the novel object (two tailed Student’s t test P<0.01) while vehicle 3XTg mice showed no preference (two-tailed Student’s t test P >0.05). See also Fig. S6.

Figure 8. Synaptic Markers Recover After 10-Day Treatment With mGluR5 Antagonist

WT and APP/PS1 mice at 10 months of age were treated with 15 mg/kg of MTEP, or saline, by intraperitoneal injection twice a day for 10 days and then sacrificed for histological analysis. A–C The molecular layer of the dentate gyrus of the indicated groups was stained with anti-PSD-95 antibody and imaged with a confocal microscope and a 60X objective lens. Scale bar, 6 μm. D Fractional area of immunoreactive puncta for PSD-95 from images as in A–C. P<0.05, ANOVA with post-hoc pairwise LSD. Mean±sem, n=5 mice per group with 3 images per mouse. E Fractional area of immunoreactive puncta for synaptophysin. P < 0.05, ANOVA with post-hoc pairwise LSD. Mean±sem, for n=5 mice per group with 3 images per animal. F Ultrastructure of the molecular layer of the dentate gyrus from a vehicle APP/PS1 mouse. Arrows point to synapses scored in G. Magnification is 20,500X. Scale bar, 1 μm. G Synapses were counted in a 25 μm² area in drug-treated and saline-treated samples. P<0.05, two-tailed Student’s t test. Mean±sem, n=5 mice per group with 30 images per mouse.