Skip to main content

Mutation of copper binding sites on cellular prion protein abolishes its inhibitory action on NMDA receptors in mouse hippocampal neurons

Abstract

We have previously reported that cellular prion protein (PrPC) can down-regulate NMDA receptor activity and in a copper dependent manner. Here, we employed AAV9 to introduce murine cellular prion protein into mouse hippocampal neurons in primary cultures from PrP null mice to determine the role of the six copper binding motifs located within the N-terminal domain of PrPC. The results demonstrate that viral expression of wild type PrPC lowers NMDAR activity in PrP null mouse hippocampal neurons by reducing the magnitude of non-desensitizing currents. Elimination of the last two copper binding sites alone, or in combination with the remaining four attenuates this protective effect. Thus our data suggest that copper ion interactions with specific binding sites on PrPC are critical for PrPC dependent modulation of NMDA receptor function.

N-methyl-d-aspartate receptors (NMDARs) are a key class of glutamate receptors that mediates a wide range of central nervous system functions [1, 2]. Excessive NMDAR activity causes calcium toxicity and may result in neurodegeneration in disorders such as stroke and Alzheimer’s disease [3, 4]. Cellular prion protein (PrPC) is widely expressed in the mammalian nervous system and mediates neuroprotective functions. Previous studies revealed that PrPC can physically interact with NMDARs and exert an inhibitory functional regulation [5,6,7]. Specifically, knockout of PrPC leads to slowly desensitizing NMDAR currents across a range of glycine co-agonist concentrations, leading to tonic receptor activity that causes neurotoxicity [5,6,7]. Conversely, we recently explored transgenic PrPC mouse models to demonstrate that overexpression of PrPC downregulates NMDAR activity [8]. PrPC is a known copper binding protein that interacts with these metal ions with up to attomolar affinity, with six putative copper binding sites localized within the N-terminal region of PrPC [9]. Copper interactions with PrPC have been shown to affect its ability to regulate NMDA receptor desensitization. For example, copper chelation by bathocuproine disulfonate (BCS) [10] results in more slowly desensitizing NMDAR currents, thus suggesting that copper ions mediate this effect via PrPC. Here, we tested the hypothesis that copper binding sites on PrPC are required for regulation of NMDAR receptor activity.

Animal experiments were conducted with the approval of the animal care committee of the University of Calgary. Wild-type C57 mice were purchased from Charles River, PrPC knockout (KO) mice and Tga20 mice (overexpressing the murine cellular prion protein) were provided by Dr. Frank Jirik. P0–P1 pups were obtained to prepare hippocampal neurons for primary culture as described by us [8]. We first performed whole cell patch clamp experiments on hippocampal pyramidal neurons from WT, PrPC null and Tga20 mice at 11–13 DIV to measure NMDA currents. The external solution contained 140 mM NaCl, 5 mM KCl, 1 mM CaCl2, 25 mM HEPES, and 33 mM d-Glucose, (pH 7.4, NaOH), and supplemented with 15 µM 2,3-dihydroxy-6-nitro-7-sulfamoyl-benzo[f]quinoxaline, 100 μM picrotoxin, 1 μM tetrodotoxin, 500 nM CuSO4 (to standardize [Cu2+] in the external medium), and different concentrations of the NMDAR co-agonist glycine as indicated. The pipette solution contained 140 mM CsCl, 11 mM EGTA, 1 mM CaCl2, 2 mM MgCl2, 10 mM HEPES, 4 mM K2ATP and 0.6 mM GTP (pH 7.3, CsOH). 500 µM NMDA (Tocris Bioscience) was applied with a microperfusion system (EVH-9, Biologic Science Instruments) to achieve rapid solution exchange and activation of NMDA currents. The holding potential was − 60 mV throughout, and agonist was applied for 7 s before washout.

Figure 1a represents typical NMDA currents in hippocampal pyramidal neurons from mouse lines with different PrPC expression levels in the presence of 1 μM glycine. Consistent with our previous work, NMDA currents in Tga20 neurons exhibited decreased steady state current compared to wild type. On the other hand, PrPC KO leads to increased steady state current as described by us previously. This effect is quantified over a range of glycine concentrations in Fig. 1b. These data reconfirm that higher levels of PrPC reduce non-desensitizing NMDA current activity whereas removal of PrPC has the opposite effect. We attribute these effects primarily to alterations in glycine regulation [5]. However, we previously reported that the absence of PrPC alters NMDAR subunit composition and this might contribute to changes in the decay kinetics (although we note that this subunit switch predominantly affected deactivation rather than desensitization) [7].

Fig. 1
figure1

a NMDAR-mediated currents from hippocampal neuron cultures of PrPc knock-in Tga20 mice, PrPc knock-out PrPc KO mice versus wild-type C57. Neurons were held at − 60 mV throughout and currents were evoked by application of 500 µM NMDA and 1 μM glycine. The dashed lines indicate baseline, steady state current, and peak current, and the arrows indicate the magnitude of the non-desentitizing (steady state) current. b Glycine dose response curve of the percentage of steady-state current (normalized to peak) in wild-type C57, Tga20 and PrPc KO muse neurons (n = 5). Asterisks denote statistical significance for C57 vs Tga20, and number symbols indicate statistical significance between C57 and PrPc KO at the 0.05, 0.1 and 0.001 levels for one, two and three symbols, respectively (one way ANOVA with Bonferroni post hoc test). c Structure of PrPc illustrating the location of copper binding motifs in the unstructured N-terminal region (taken from [11]). d Illustration of constructs of recombinant AAV-GFP-PrPc and the various copper site mutants. e Confocal images of hippocampal neurons from PrPc KO mice transduced with AAV9 expressing eGFP (left), mPrPc (middle), and mPrPc-6HA (right). Green signal reflects eGFP fluorescence and thus PrPc expression. The primary hippocampal neurons were cultured for 3 days before transduction and confocal images were collected 8 days later. The dose was 1 × 1011 GC/ml for all constructs. Scale bar = 50 µm. f Representative traces of NMDAR currents from hippocampal neurons of PrPc KO mice infected with AAV-GFP, AAV-GFP-PrPc, and AAV-GFP-PrPc-6HA. Neurons were held at − 60 mV throughout and currents were evoked by application of 500 µM NMDA and 1 μM glycine g. Glycine dose response curve of the percentage of steady-state current (normalized to peak) in neurons from PrPc KO mice transduced with AAV-GFP, AAV-GFP-PrPc, AAV-GFP-PrPc-6HA, AAV-GFP-PrPc-4HA and AAV-GFP-PrPc-2HA (n = 6 for AAV-GFP and AAV-GFP-PrPc, n = 5 for AAV-GFP-PrPc-6HA, AAV-GFP-PrPc-4HA and AAV-GFP-PrPc-2HA). Asterisks refer to statistical difference relative to AAV-GFP-PrPc (One way ANOVA with Tamhane-Dunnett's Test [13]), with colour of the asterisks corresponding to the colour denoting the various PrPc mutant constructs

The unstructured N-terminus of PrPC contains copper binding sites formed by four histidines within the octapeptide repeats closer to the N-terminus and two histidines outside of the octapeptide repeats closer to the C-terminal end of this region Fig. 1c [11]. To test the roles of these copper sites, we packaged cDNA of murine cellular prion protein, along with the signal peptide (SP) and green fluorescent protein (GFP) into AAV9 vectors to generate AAV-PrPC constructs (Fig. 1d). To monitor putative effects of GFP overexpression, we also created an AAV-GFP construct. Partial or full ablation of copper binding domains was achieved by replacing the two histidines outside the octarepeat, four histidines inside the octarepeat, or all six key histidines with alanines. We then infected PrP null mouse hippocampal cultures with AAV-GFP, wild type GFP-PrPC, or the three different copper mutant AAV constructs. Figure 1e illustrates hippocampal neurons 8 days post infection, with robust GFP expression being evident. We then performed whole cell patch clamp experiments as outlined above, by selectively patching on to the GFP positive neurons. Figure 1f illustrates NMDAR currents in pyramidal neurons from PrPC null mouse neurons transduced with AAV-GFP, AAV-GFP-PrPC, or a mutant in which all six copper binding sites had been abolished (AAV-GFP-PrPC-6HA). With the reintroduction of PrPC, NMDAR current desensitization kinetics were normalized to levels similar to those seen in WT mouse neurons (compare Fig. 1a and f). This rescue effect was abolished when the six copper binding sites were mutated (Fig. 1f). We further tested two mutants in which either the first four or the last two histidines were mutated. Figure 1g summarizes aggregate data for NMDAR desensitization over a range of glycine concentrations for the various constructs, illustrating the differing NMDAR desensitization kinetics. Removal of all six (AAV-GFP-PrPC-6HA), the final two (AAV-GFP-PrPC-2HA) or the first four histidines (AAV-GFP-PrPC-4HA) abolished the rescue effect (i.e., lower desensitization plateau) observed with the AAV-GFP-PrPC construct.

The first four copper binding motifs on PrPC vary in affinity from femto to nanomolar, with the fifth site (His96) being of lower affinity and being modulated by His111 (see Fig. 1c) [12]. Our data indicate that elimination of the two low affinity copper binding sites (and perhaps associated alterations in affinity for the other sites due to disruptions in cooperativity that is known to occur among these sites [12]) is sufficient to compromise the inhibitory function of PrPC on NMDAR currents. By inference, we thus suggest that copper chelation with BCS may mediate its effect on NMDARs in part by stripping copper from these two lower affinity PrPC copper binding motifs. Our data also show that ablation of the four high affinity sites similarly compromises PrPC function. This then suggests that multiple PrPC copper binding sites participate in the ability of PrPC to inhibit NMDAR activity. Further work with individual substitution of the various histidine residues will be needed to further dissect the copper dependent modulation of NMDARs by PrPC, and additional work will be needed to determine if these copper mutants alter NMDAR subunit composition.

Availability of data and materials

All data generated or analysed during this study are included in this published article.

Abbreviations

AAV:

Adeno-associated virus

NMDAR:

N-methyl-d-aspartate receptor

PrPC :

Cellular prion protein

GFP:

Green fluorescent protein

References

  1. 1.

    Traynelis SF, Wollmuth LP, et al. Glutamate receptor ion channels: structure, regulation, and function. Pharmacol Rev. 2010;62:405–96.

    CAS  Article  Google Scholar 

  2. 2.

    Collingridge G. Synaptic plasticity. The role of NMDA receptors in learning and memory. Nature. 1987;330:604–5.

    CAS  Article  Google Scholar 

  3. 3.

    Palop JJ, Mucke L. Amyloid-β-induced neuronal dysfunction in Alzheimer’s disease: from synapses toward neural networks. Nat Neurosci. 2010;13:812–8.

    CAS  Article  Google Scholar 

  4. 4.

    Parsons MP, Raymond LA. Extrasynaptic NMDA receptor involvement in central nervous system disorders. Neuron. 2014;82(2):279–93.

    CAS  Article  Google Scholar 

  5. 5.

    You H, et al. Aβ damages neurons by altering copper-dependent prion protein regulation of NMDA receptors. Proc Natl Acad Sci U S A. 2012;109:1737–42.

    CAS  Article  Google Scholar 

  6. 6.

    Stys PK, You H, Zamponi GW. Copper-dependent regulation of NMDA receptors by cellular prion protein: implications for neurodegenerative disorders. J Physiol. 2012;590(6):1357–68.

    CAS  Article  Google Scholar 

  7. 7.

    Khosravani, et al. Prion protein attenuates excitotoxicity by inhibiting NMDA receptors. J Cell Biol. 2008;181(3):551–65.

    CAS  Article  Google Scholar 

  8. 8.

    Huang S, Chen L, Bladen C, Stys PK, Zamponi GW. Differential modulation of NMDA and AMPA receptors by cellular prion protein and copper ions. Mol Brain. 2018;11(1):62.

    CAS  Article  Google Scholar 

  9. 9.

    Millhauser GL. Copper and the prion protein: methods, structures, function, and disease. Annu Rev Phys Chem. 2007;58:299–320.

    CAS  Article  Google Scholar 

  10. 10.

    Mohindru A, Fisher JM, Rabinovitz M. Bathocuproine sulphonate: a tissue culture-compatible indicator of copper-mediated toxicity. Nature. 1983;303(5912):64–5.

    CAS  Article  Google Scholar 

  11. 11.

    Legname G. Elucidating the function of the prion protein. PLoS Pathog. 2017;13(8):e1006458.

    Article  Google Scholar 

  12. 12.

    Thompsett AR, Abdelraheim SR, Daniels M, Brown DR. High affinity binding between copper and full-length prion protein identified by two different techniques. J Biol Chem. 2005;280(52):42750–8.

    CAS  Article  Google Scholar 

  13. 13.

    R Core Team. R: A language and environment for statistical computing. R Foundation for Statistical Computing. Vienna, Austria. 2019; https://www.R-project.org/.

Download references

Acknowledgements

We thank Dr. Frank Jirik for providing us with access to transgenic mice. We thank Dr. Marco Prado for having provided GFP-PrPC.

Funding

This work was supported by the Alberta Prion Research Institute. GWZ and PKS are Canada Research Chairs. SAB held a Fellowship from the Heart and Stroke Foundation of Canada.

Author information

Affiliations

Authors

Contributions

SH, SAB and JH performed experiments. GWZ and PKS conceived and supervised the study. SH and GWZ wrote the manuscript. All authors read and approved the final manuscript.

Corresponding author

Correspondence to Gerald W. Zamponi.

Ethics declarations

Ethics approval and consent to participate

This study was approved by the University of Calgary’s animal care committee.

Consent for publication

Not applicable.

Competing interests

The authors declare no competing interest.

Additional information

Publisher's Note

Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.

Rights and permissions

Open Access This article is licensed under a Creative Commons Attribution 4.0 International License, which permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons licence, and indicate if changes were made. The images or other third party material in this article are included in the article's Creative Commons licence, unless indicated otherwise in a credit line to the material. If material is not included in the article's Creative Commons licence and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder. To view a copy of this licence, visit http://creativecommons.org/licenses/by/4.0/. The Creative Commons Public Domain Dedication waiver (http://creativecommons.org/publicdomain/zero/1.0/) applies to the data made available in this article, unless otherwise stated in a credit line to the data.

Reprints and Permissions

About this article

Verify currency and authenticity via CrossMark

Cite this article

Huang, S., Black, S.A., Huang, J. et al. Mutation of copper binding sites on cellular prion protein abolishes its inhibitory action on NMDA receptors in mouse hippocampal neurons. Mol Brain 14, 117 (2021). https://doi.org/10.1186/s13041-021-00828-0

Download citation

Keywords

  • NMDA receptor
  • Cellular prion protein
  • AAV system
  • Knock-out mice
  • Hippocampal neurons
  • Whole-cell patch clamp
  • CNS disorders
  • Copper