Regulation of NMDA receptors by cellular prion protein – impact on neuronal survival and pain

1. Regulation of NMDA receptors by cellular prion protein – impact on neuronal survival and pain Gerald W. Zamponi, PhD, FRSC, FCAHS Department of Physiology and Pharmacology University of Calgary

2. Aguzzi and Heikenwalder Nature Reviews Microbiology 4, 765–775 (October 2006) | doi:10.1038/nrmicro1492

3. NMDA receptors and PrPC Localized to synapses (i.e. synaptosome preparations) PrP-null mice exhibit altered LTP (controversial) but have otherwise a mild phenotype PrP-null mice exhibit greater kainate-mediated excitotoxicity; cell death reduced with MK-801 Mice lacking PrP display depressive like behavior (reversed by MK801) MK-801 and memantine block protects against cell death in neuronal cultures infected with PrPSc

4. Pulse 2 Pulse 1 Field Excitatory Post-Synaptic Potential (fEPSP) Caused by dendritic currents during population firing Recording Electrode CA1 Population Spike Proportional to many neurons firing synchronously CA3 DG Stimulation Electrode Paired-Pulse Facilitation Stimulation Artifact Stimulation Artifact

5. Hippocampal brain slices from PrP-null mice exhibit enhanced basal excitability Khosravani et al, J Cell Biol., 2008

6. Hippocampal brain slices from PrP-null mice exhibit enhanced excitability in an in vitro seizure model Khosravani et al, J Cell Biol., 2008

7. PrP-null mice exhibit altered NMDA mEPSCs 8 4 Khosravani et al, J Cell Biol., 2008

8. Evoked NMDA currents Khosravani et al, J Cell Biol., 2008

9. Evoked NMDA currents

10. siRNA phenocopies the knockout data whereas overexpression of PrP rescues the phenotype Khosravani et al, J Cell Biol., 2008

11. NMDA receptor structure and function

12. NMDA receptor subunit-dependent kinetics

13. Neonatal PrP+/+ Neonatal PrP-/- Adult PrP+/+ Adult PrP-/- (kDa) 250 150 75 IB:NR2D Actin NMDA receptor subunits:mRNA transcript and protein levels Khosravani et al, J Cell Biol., 2008

14. (kDa) PrP NR2D Merge IP:NR2D control IP:PrP input 50 37 25 IB:PrP NR2D PrP (kDa) 25 control IP:PrP input 250 15 AIU(x10) 150 5 0 5 10 100 Distance (μm) IB:NR2D PrP- NR2D subunit complexes Khosravani et al, J Cell Biol., 2008

15. in vivo (apoptotic) excitotoxicity assay Khosravani et al, J Cell Biol., 2008

16. What is the physiological role of the PrPC copper binding domain? Bathocuproine disulfonate (BCS) Burns et al., Biochemistry, 2003

17. NMDA Receptor Desensitization From Stys & Lipton, TiPS, 2007 • is a spontaneous shutdown of channel activity in the presence of agonist • prevents toxic calcium buildup during prolonged glutamate exposure • appears to involve multiple molecular mechanisms • is antagonized by high concentrations of glycine or d-serine

18. Copper chelation slows NMDAR desensitization persistent (residual)current

19. Different copper chelators have similar effects onNMDAR desensitization

20. Cleavage of amyloid precursor protein Robbins and Cotran 2009 Pathologic Basis of Disease • Aβ1-42: • is a high affinity copper binding protein (attomolar affinity) • forms copper dependent oligomers

21. Aβ1-42 slows NMDAR desensitization

22. Aβ1-42 slows NMDAR desensitizationby regulating copper

23. Slowed NMDAR desensitization results in cell death (20) (27) (38) % Tunel positive (20) (35) (15) (41) (28) (13) (10)

24. Antibody binding near the octarepeat regionalters NMDA currents 6D11 Burns et al., Biochemistry, 2003

25. Both Aβ1-42 monomers and oligomers affect NMDA currents HFIP - treated

26. PrP KO mouse neurons display altered NMDA current kinetics

27. Neurons from 5XFAD mice show altered NMDA currents • 5XFAD mice (Oakley et al., J,. Neurosci. 2006) • APPmutations: • "Swedish" double mutation (K670N/M671L) • "Florida" mutation (I716V) • "London" mutation (V717I). • PS1 mutations: • L286V and L286V • increased production of Aβ1-42 • plaque deposits at two months • memory deficits at 4 months

28. PrP regulates glycine affinity for NMDA receptors IP PrP kDa PrP KO CuSO4 Beads Lysate Control BCS 150 NR1 100 IB NR1 * 140 120 100 80 band intensity (% of control) 60 n=4 40 20 0 BCS CuSO4 Control

29. Proposed model of NMDAR, Cu, Aβ, and prion protein interaction

30. White Matter (WM) in Alzheimer’s • WM degeneration seen in 60% of AD patients • partial demyelination, axonal degeneration, loss of oligos • WM disorder occurs independently of cortical degeneration • preclinical AD: prominent and selective shrinkage of WM, with normal cortex • may arise in part from compromised vasculature Brun & Englund, 1986; De la Monte 1989

31. Detection of NMDAR subunits in myelin NR1 NR1 120KDa NR1 NR2 180 KDa NR1 NR3 115 kDa Micu et al., Nature, 2006

32. Measuring myelin [Ca] with 2-photon microscopy Micu et al., Nature, 2006; Micu et al., Nat Med, 2007

33. Pharmacological profile of Ca changes in myelin Micu et al., Nature, 2006

34. BCS triggers a calcium rise in myelin Copper rescue 10 mM BCS DF/F0 (%) DCKA+BCS BCS

35. BCS and Aβ effects on myelin depend onNMDA receptors 10 µM BCS 2 µM Aβ1-42 DF/F0 (%) (n=20-40) ifen+NVP 12 µM Cu 4 µM Cu DCKA DCKA

36. BCS induced Ca2+ entry mediates WM damage QD9 AB – 1:1000

37. Aβ-mediated GM and WM degeneration: Interplay between copper, PrP and NMDARs • normal Aβ and copper levels fine tune synaptic transmission • PrP mediates normal copper homeostasis near the NMDAR complex • excessive Aβ results in a copper dysregulated state • abnormally low free copper leads to reduced NMDAR desensitization • this causes abnormally high persistent NMDAR currents • the ensuing calcium overload mediates cell death • applies independently to neurons (GM – NR2A/B) and myelin (WM – NR3)

38. Broader implications for human disease: • Menkes disease involves a dysfunction of a copper ATPaseenzyme and is linked to NMDA receptor mediated excitotoxicity(Schlief et al., PNAS, 2006) – Menkes disease is characterized by neurodegeneration and demyelination • copper deficiency leads to impaired brain development • α-synuclein and huntingtin also bind copper ions • NMDA receptor hyperfunction due to copper dysregulation may result in increased cell death in a wide spectrum of disorders.

39. Periphery CNS (Spinal cord) Pain transmission Pain Brain PAG NMR Spinal Cord

40. Time course of nociceptive threshold Mechanical Threshold Thermal Threshold • Dynamic Plantar Anesthesiometer (Ugo Basile) • Plantar Test apparatus (Hargreaves, Ugo Basile) Gadotti and Zamponi, Mol.Pain, 2011

41. Acute Nociception: Formalin and Glutamate (i.pl.)

42. Acute nociception: NMDA (i.t.) Gadotti and Zamponi, Mol.Pain, 2011

43. Nociception and inflammatory pain: involvement of NMDA receptors Formalin (1.25%) Phase 1 Formalin (1.25%) phase 2 Mechanical Threshold 3 months Gadotti and Zamponi, Mol.Pain, 2011

44. Neuropathic pain: Sciatic nerve ligation (CCI) 3 month old male mice 3 days later Gadotti and Zamponi, Mol.Pain, 2011

45. Neuropathic pain: involvement of NMDA receptors Gadotti and Zamponi, Mol.Pain, 2011

46. Summary • PrPC null mice exhibit a decreased nociceptive threshold thatis reversed by MK-801 delivered intrathecally (i.t.). • This fits with the notion of hyperactivity of NMDA receptors in PrPC null mice – perhaps in a state of chronic central sensitization (i.e., “wind up”) • Suggests yet another protective role of cellular prion protein

47. ACKNOWLEDGMENTS ZAMPONI LAB Dr. Haitao You Dr. Jawed Hamid Dr. Shahid Hameed Lina Chen Chris Bladen Dr. Clint Doering Dr. Vinicius Gadotti Brett Simms Dr. Agustin Caballero Dr. Norbert Weiss Renata Rehak Dr. Stefanie Black Dr. Fang-Xiong Zhang Dr. Ivana Souza Dr. Houman Khosravani COLLABORATORS Dr. Peter Stys Dr. Shigeki Tsutsui Dr. Frank Jirik

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