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α-synuclein interacts with PrPC to induce cognitive impairment through mGluR5 and NMDAR2B

Nature Neuroscience volume 20, pages 1569–1579 (2017)Cite this article

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Abstract

Synucleinopathies, such as Parkinson's disease and dementia with Lewy bodies, are neurodegenerative disorders that are characterized by the accumulation of α-synuclein (aSyn) in intracellular inclusions known as Lewy bodies. Prefibrillar soluble aSyn oligomers, rather than larger inclusions, are currently considered to be crucial species underlying synaptic dysfunction. We identified the cellular prion protein (PrPC) as a key mediator in aSyn-induced synaptic impairment. The aSyn-associated impairment of long-term potentiation was blocked in Prnp null mice and rescued following PrPC blockade. We found that extracellular aSyn oligomers formed a complex with PrPC that induced the phosphorylation of Fyn kinase via metabotropic glutamate receptors 5 (mGluR5). aSyn engagement of PrPC and Fyn activated NMDA receptor (NMDAR) and altered calcium homeostasis. Blockade of mGluR5-evoked phosphorylation of NMDAR in aSyn transgenic mice rescued synaptic and cognitive deficits, supporting the hypothesis that a receptor-mediated mechanism, independent of pore formation and membrane leakage, is sufficient to trigger early synaptic damage induced by extracellular aSyn.

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Figure 1: PrPC mediates synaptic impairment induced by extracellular aSyn oligomers.
Figure 2: PrPC dependent-toxic effects of aSyn oligomers are mediated by Src family kinases.
Figure 3: aSyn oligomers, but not monomers, increase intracellular Ca2+ levels in primary neuronal cultures in a PrPC/NMDAR2B-dependent mechanism.
Figure 4: PrPC blockade rescues LTP impairment induced by extracellular aSyn oligomers through a mechanism dependent on mGluR5.
Figure 5: In vivo treatment of Thy1-aSyn (aSyn Tg) mice with KW-6002 rescues aSyn-associated cognitive deficits.
Figure 6: aSyn-associated synaptic and NMDAR dysfunction are rescued by KW-6002 in vivo treatment.

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Acknowledgements

The authors thank L. Gros for figure layout design and C. Fahlbusch (University Medical Center Göttingen), A. Margarida Nascimento and J. Rino (Instituto de Medicina Molecular (iMM) Bioimaging facility), I. Moreira and J. Marques (iMM Rodent facility), and the iMM Histology and Comparative Pathology laboratory for technical assistance. M.T.F., H.V.M. and J.E.C. were supported by individual grants from Fundação para a Ciência e Tecnologia (FCT) (SFRH/BD/52228/2013; SFRH/BPD/109347/2015; SFRH/BPD/87647/2012); L.V.L. and T.F.O. were supported by a grant from the Fritz Thyssen Stiftung (Az. 10.12.2.165), Germany. L.V.L. received an iMM Lisboa internal fund (BIG – Breakthrough Idea Grant) for part of the project. L.V.L. is an Investigator FCT, Portugal. T.F.O. is supported by the DFG Center for Nanoscale Microscopy and Molecular Physiology of the Brain (CNMPB), Germany. LISBOA-01-0145-FEDER-007391, project co-financed by FEDER, POR Lisboa 2020 - Programa Operacional Regional de Lisboa, from PORTUGAL 2020 and by Fundação para a Ciência e a Tecnologia.

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Author notes

  1. Luísa V Lopes and Tiago F Outeiro: These authors jointly directed this work.

Authors and Affiliations

  1. Instituto de Medicina Molecular, Faculdade de Medicina de Lisboa, Universidade de Lisboa, Lisboa, Portugal

    Diana G Ferreira, Mariana Temido-Ferreira, Hugo Vicente Miranda, Vânia L Batalha, Joana E Coelho, Inês Marques-Morgado, Sandra H Vaz & Luísa V Lopes

  2. Department of Experimental Neurodegeneration, Center for Nanoscale Microscopy and Molecular Physiology of the Brain, Center for Biostructural Imaging of Neurodegeneration, University Medical Center Göttingen, Göttingen, Germany

    Diana G Ferreira, Éva M Szegö & Tiago F Outeiro

  3. Department of Pharmacology and Therapeutics, Faculty of Medicine, University of Porto, Porto, Portugal

    Diana G Ferreira

  4. MedInUP - Center for Drug Discovery and Innovative Medicines, University of Porto, Porto, Portugal

    Diana G Ferreira

  5. CEDOC, Chronic Diseases Research Center, NOVA Medical School, Faculdade de Ciências Médicas, Universidade NOVA de Lisboa, Lisboa, Portugal

    Hugo Vicente Miranda & Tiago F Outeiro

  6. Max Planck Institute for Experimental Medicine, Göttingen, Germany

    Jeong Seop Rhee & Tiago F Outeiro

  7. Department of Neurology, University Medical Center Göttingen, and German Center for Neurodegenerative Diseases (DZNE)-site Göttingen, Göttingen, Germany

    Matthias Schmitz & Inga Zerr

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Contributions

D.G.F. performed most of the experimental work, analyzed data and wrote the manuscript. M.T.-F., J.E.C., V.L.B. and S.H.V. assisted with behavior and calcium experiments. E.M.S. assisted with animal experiments. I.M.-M. performed the immunohistochemistry experiments. M.S., J.S.R. and I.Z. provided the Prnp−/− mice and experimental support. H.V.M. produced and characterized aSyn species. L.V.L. and T.F.O. coordinated the study, designed the experiments and wrote the manuscript. All of the authors approved the manuscript.

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Correspondence to Luísa V Lopes or Tiago F Outeiro.

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Integrated supplementary information

Supplementary Figure 1 Characterization of the aSyn species and biological effects.

(a) SDS–PAGE separation of the different aSyn species. Monomers (aSyn mon) migrate with monomeric molecular weight (15 kDa) whereas aSyn oligomers (aSyn olig), and fibrils (aSyn fib) display SDS-resistant high-molecular weight species. (b) Changes in fEPSP slope induced by theta-burst stimulation recorded from WT rat hippocampal slices pre-incubated with extracellular aSyn monomers (aSyn mon, 90 min, 500 nM, n = 4), fibrils (aSyn fib, 90 min, 500 nM, n = 3) or in control conditions (CTR, n = 4). (c) Plot of the LTP magnitude represented in b (change in fEPSP slope at 50–60 min after theta-burst stimulation, compared to baseline) (means ± s.e.m., P < 0.01, one-way ANOVA followed by a Bonferroni’s Multiple Comparison Test). (d) Plot of the LTP magnitude obtained from WT and Prnp-/- hippocampal slices pre-incubated with extracellular aSyn oligomers (aSyn olig, 90 min, 500 nM, n = 10, 6) or in control conditions (CTR, n = 7, 6) (means ± s.e.m., P < 0.001, one-way ANOVA followed by a Bonferroni’s Multiple Comparison Test). (e) Input/Output (I/O) curves corresponding to fEPSP slope evoked by various stimulation intensities (10 – 120 μA) from WT hippocampal slices pre-incubated with or without aSyn oligomers (n = 5, 7; means ± s.e.m., P < 0.001, F-test). (f) I/O curves from Prnp-/- hippocampal slices pre-incubated with (n = 4) or without (n = 4) aSyn oligomers obtained by the same method as in e (means ± s.e.m., P > 0.05, F-test). (g) I/O curves obtained by the same method as in e, from hippocampal slices CTR (n = 6), pre-incubated with aSyn oligomers alone (aSyn olig, 90 min, 500 nM, n = 7) or in the presence of the anti-PrP 6D11 antibody (6D11 + aSyn olig, 110 min, 100 nM, n = 4) (means ± s.e.m., P < 0.01, F-test). (h) Changes in fEPSP slope, obtained by the same methods as in b, from WT hippocampal slices in control conditions (CTR, n = 4) and in the presence of aSyn oligomers alone (aSyn olig, 90 min, 500 nM, n = 6) or together with the anti-PrP antibodies, 8B4 (8B4 + aSyn, 110 min, 10 μg, n = 4) or C-20 (C-20 + aSyn olig, 110 min, 10 μg, n = 4) (means ± s.e.m., P < 0.001, one-way ANOVA followed by a Bonferroni’s Multiple Comparison Test). (i) Paired Pulse Facilitation (PPF) plotted against 200 ms interpulse intervals in WT slices submitted to the same conditions as in g (n = 3-5; means ± s.e.m., P > 0.05, one-way ANOVA followed by a Bonferroni’s Multiple Comparison Test).

Supplementary Figure 2 Src pharmacological blockade prevents aSyn oligomer-induced synaptic impairment.

(a) Plot of the LTP magnitude (change in fEPSP slope at 50–60 min after theta-burst stimulation, compared to baseline) from control WT hippocampal slices (CTR, n = 4), slices pre-incubated with the Src antagonist 1-naphthyl-PP1 (PP1, 110 min, 30 μM, n = 3) and slices pre-incubated with extracellular aSyn oligomers alone (aSyn olig, 90 min, 500 nM, n = 6) or in the presence of PP1 (PP1 + aSyn olig, n = 3) (means ± s.e.m., P < 0.01, one-way ANOVA followed by a Bonferroni’s Multiple Comparison Test). (b) Input/Output (I/O) curves corresponding to fEPSP slope evoked by various stimulation intensities (10 – 120 μA) from control WT hippocampal slices (CTR, n = 5) and slices pre-incubated with extracellular aSyn oligomers alone (aSyn olig, 90 min, 500 nM, n = 5) or in the presence of PP1 (PP1 + aSyn olig, n = 4) (means ± s.e.m., P < 0.001, F-test). (c) Quantification of the effects of the NMDA receptor antagonist APV (50 μM, 30 min) perfusion on basal fEPSP slope from control WT hippocampal slices (CTR, n = 5), and slices pre-incubated with extracellular aSyn oligomers alone (aSyn olig, 90 min, 500 nM, n = 4) or in the presence of PP1 (PP1 + aSyn olig, n = 4) (change in slope between baseline and the last 10 min of APV application) (means ± s.e.m., P < 0.01, one-way ANOVA followed by a Bonferroni’s Multiple Comparison Test). (d) Effect of NMDA receptor antagonist APV (50 μM, 30 min) perfusion on basal fEPSP slope in WT hippocampal slices in control conditions (n = 3) or in the presence of aSyn oligomers (n = 4). (e) Quantification of the APV perfusion (50 μM, 30 min) effects on basal fEPSP slope (change in slope between baseline and the last 10 min of APV application) from WT and Prnp-/-hippocampal control slices (CTR) or slices pre-incubated with aSyn oligomers (n = 3-4; means ± s.e.m., P < 0.001). (f) Representative immunoblot and quantitation of NMDA receptor subunit 2B (NMDAR2B) and NMDA receptor subunit 1 (NMDAR1) levels in hippocampal slices from WT and Prnp-/- mice in the same conditions as in d (n = 4; means ± s.e.m., P < 0.001, one-way ANOVA followed by a Bonferroni’s Multiple Comparison Test). GAPDH was used as a loading control.

Supplementary Figure 3 Extracellular aSyn oligomers induce phosphorylation of SFK kinases and NR2B subunit of NMDA receptors.

(a) Representative image of primary cultures from WT animals at 12 DIV. Mature neurons are labeled with green fluorescence with MAP-2 antibody, astrocytes, in red, are probed with anti-GFAP antibody, and cell nucleus are labeled with Hoechst 33342, in blue fluorescence. Z-stack images were acquired using a confocal microscope at 40x magnification and converted into maximum intensity projections. At the bottom a schematic representation of the aSyn incubation protocol used. (b) Representative immunoblots and quantitation of the aSyn levels in neuronal cultures incubated with extracellular aSyn oligomers over time (n = 4-5; P < 0.01, one-way ANOVA followed by a Dunnett’s Multiple Comparison Test). GAPDH was used as a loading control. (c) Representative immunoblots and quantitation of the Fyn levels in neuronal cultures incubated with extracellular aSyn oligomers over time (n = 4; P > 0.05, one-way ANOVA followed by a Dunnett’s Multiple Comparison Test). GAPDH was used as a loading control. (d) Representative immunoblots and quantitation of the SFK kinases phosphorylation levels, normalized to Fyn immunoreactivity, in primary neuronal cultures incubated with extracellular aSyn oligomers over time (n = 3-10; P < 0.01, one-way ANOVA followed by a Dunnett’s Multiple Comparison Test). (e) Representative immunoblots and quantitation of the PrPC levels in neuronal cultures incubated with extracellular aSyn oligomers over time (n = 6; P > 0.05, one-way ANOVA followed by a Dunnett’s Multiple Comparison Test). GAPDH was used as a loading control. (f) Representative immunoblots and quantitation of the NMDAR2B levels in neuronal cultures incubated with extracellular aSyn oligomers over time (n = 4; P > 0.05, one-way ANOVA followed by a Dunnett’s Multiple Comparison Test). GAPDH was used as a loading control. (g) Representative immunoblots and quantification of NMDA receptors subunit NR2B phosphorylation levels, normalized to NMDA receptor immunoreactivity, in neuronal cultures incubated with extracellular aSyn oligomers over time. (n = 3-7; P < 0.05, one-way ANOVA followed by a Dunnett’s Multiple Comparison Test). (h) Quantitative analysis of IP:aSyn and IP:PrPC (n = 3; means ± s.e.m., P < 0.05, two-sided unpaired t test). (i) Immunohistochemistry in 1.5 μm hippocampal sections from WT and aSyn Tg mice. aSyn is labelled in red and PrPC is labelled in green (scale bar: 25 μm). At the bottom, details from the 63x magnification images are presented (scale bar: 5 μm).

Supplementary Figure 4 mGluR5 mediated aSyn/PrPC long term potentiation impairment.

(a) Plot of the LTP magnitude (change in fEPSP slope at 50–60 min after theta-burst stimulation, compared to baseline) from control WT hippocampal slices (CTR, n = 4), slices pre-incubated with the mGluR5 antagonist MPEP (110 min, 5 μM, n = 3) and slices pre-incubated with extracellular aSyn oligomers alone (aSyn olig, 90 min, 500 nM, n = 6) or in the presence of MPEP (MPEP + aSyn olig, n = 4) (means ± s.e.m., P < 0.05, one-way ANOVA followed by a Bonferroni’s Multiple Comparison Test). (b) Changes in fEPSP slope induced by theta-burst stimulation recorded from WT rat hippocampal slices pre-incubated with extracellular aSyn oligomers alone (aSyn mon, 90 min, 500 nM, n = 6), in the presence of the selective A2AR antagonist SCH-58261 (110 min, 50 nM, SCH + aSyn olig, n = 3) and in the presence of SCH-58261 together with the mGluR5 agonist DHPG (110 min, 10 μM; SCH + DHPG + aSyn olig, n = 4). (c) Plot of the LTP magnitude represented in b and in Fig. 4d (change in fEPSP slope at 50–60 min after theta-burst stimulation, compared to baseline) (means ± s.e.m., P < 0.01, one-way ANOVA followed by a Bonferroni’s Multiple Comparison Test).

Supplementary Figure 5 Characterization of Thy1-aSyn (aSyn Tg) overexpressing mice.

(a) Representative western blot of independent experiments to evaluate aSyn levels in the hippocampus (HIP) and striatum (STR) of WT and aSyn Tg mice. GAPDH was used as a loading control. At the bottom quantification of aSyn immunoreactivity in relation to WT (n = 2-5; means ± s.e.m., P < 0.05, two-sided unpaired t test). (b) Top panels: compositional images of fluorescence immunohistochemistry of WT and aSyn Tg mice hippocampus (scale bar: 500 μm). aSyn is identified in red fluorescence and cell nuclei are stained with Hoechst in blue fluorescence. Bottom panels: maximum intensity projection images of z-stack taken at 63x magnification in the CA1 area of hippocampus (scale bar: 25 μm). At the right, details from the 63x magnification images are presented (scale bar: 5μm). aSyn is identified in red fluorescence and SNAP25 is labeled in green. (c) Representative western blot of independent experiments to evaluate TH levels in the striatum of WT and aSyn Tg mice. GAPDH was used as a loading control. At the bottom the respective quantification of TH immunoreactivity in relation to WT (n = 4; means ± s.e.m., P > 0.05, two-sided unpaired t test). (d) Representative images of TH immunohistochemistry of WT and aSyn Tg mice coronal brain sections. At the bottom the respective quantitation of TH immunoreactivity in relation to WT (n = 3-4; means ± s.e.m., P > 0.05, two-sided unpaired t test). (e) Representative western blot of 3 independent experiments to evaluate PrP levels in the hippocampus of WT, aSyn Tg, and Prnp-/- mice. GAPDH was used as a loading control. At the bottom the respective quantification of PrP immunoreactivity in relation to WT (n = 5-6; means ± s.e.m., P < 0.05, two-sided unpaired t test).

Supplementary Figure 6 Summary diagram of the mechanism by each aSyn oligomers induce an aberrant PrPC-mGluR5-NMDAR2B signaling at the post-synaptic density.

aSyn oligomers physically interact with PrPC (1) to activate mGluR5 (2)50. This leads to the phosphorylation and activation of the Src kinase Fyn (3) followed by the phosphorylation of NMDAR2B (Y1472) (4)27 and robust increase in intracellular Ca2+ (5). Adenosine A2AR are required for the mGluR5-induced NMDAR2B phosphorylation (6)37.

Supplementary Figure 7 Uncropped gels and blots with molecular weight standards complete images

Uncropped versions of representative western blot images from Fig. 2e, 2f, 2g, 2h, 5g, 6g, and 6h and pre-IP lysates corresponding to IPs shown in Fig. 2g and 2h. Membranes were cut prior to antibody staining to allow for simultaneous detection of proteins running at different sizes on the same membrane without reprobing. Representative membranes with the molecular weight (MW) markers used are shown at the bottom right panels.

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aSyn oligomers increase intracellular Ca2+ in neurons

Initial calcium response of FURA-2 AM loaded wildtype neurons to aSyn oligomers (aSyn mon, 500 nM) (AVI 28473 kb)

aSyn oligomers do not increase intracellular Ca2+ in Prnp (-/-) neurons

Initial calcium response of FURA-2 AM loaded Prnp (-/-) neurons to aSyn oligomers (aSyn mon, 500 nM) (AVI 31418 kb)

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Ferreira, D., Temido-Ferreira, M., Vicente Miranda, H. et al. α-synuclein interacts with PrPC to induce cognitive impairment through mGluR5 and NMDAR2B. Nat Neurosci 20, 1569–1579 (2017). https://doi.org/10.1038/nn.4648

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