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Behavioral state-dependent Ras signaling and AMPA-R trafficking J. Julius Zhu

This study explores the role of Ras signaling in AMPA receptor trafficking and synaptic plasticity. By linking molecular signaling pathways to plasticity, this work enhances our understanding of synaptic function. The findings have potential clinical implications for neurological diseases such as neurofibromatosis, schizophrenia, X-linked mental retardation, Alzheimer's, and tuberous sclerosis.

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Behavioral state-dependent Ras signaling and AMPA-R trafficking J. Julius Zhu

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  1. Behavioral state-dependent Ras signaling and AMPA-R trafficking J. Julius Zhu Department of Pharmacology University of Virginia School of Medicine

  2. Silent synapse Mature synapse AMPA Receptors Maturation AMPAR delivery NMDA Receptors Na+ Na+ & Ca2+ Na+ & Ca2+ K+ K+ K+ Glutamate receptors relay excitatory synaptic transmission Nature 375:400; Nature Neurosci 5:513

  3. AMPA receptor subunits AMPA-R GluR1 GluR2 GluR2L GluR3 GluR4

  4. Delivering genes with Sindbis virus GFP Make virus carrying GluR-GFP and prepare slices + Inject virus extracellularly Incubate 15 hrs Biochemistry, Electrophysiology, EM and imaging

  5. +40 mV +40 mV -60 mV -60 mV Detecting delivery of recombinant receptor Endogenous Receptors GluR2/X Recombinant Receptors GluRX/X X= 1, 3, 4

  6. GluR1/4/2L Constitutive Replacement GluR2 Activity dependent Lysosome GluR3 Slot Model for AMPA receptor trafficking at synapses Science 284:1811; Science 287:2262; Nature Neurosci 3:1098; Cell 105:331

  7. NMDAR GluR1/2L Ca2+ GluR2 Ca2+  GluR3 CaMKII p38MAPK p42/44MAPK Slot Rap-GEF Ras-GEF Rap-GDP Rap-GTP Ras-GDP Ras-GTP Rap-GAP Ras-GAP Ras and Rap signal delivery and removal of AMPA-Rs Cell 110:443; Neuron 39:in press

  8. Significance • This work will further our understanding of synaptic plasticity, by linking molecular signaling to plasticity. • Clinical implications: important for understanding and suggesting better treatments for many neurological diseases, such as: 1. von Recklinghausen neurofibromatosis (incidence = ~1/3000 individuals; Cell 63:851; Nature 403:495).2. Schizophrenia (incidence = ~0.7% adults; Psychiatry Res 63:25).3. X-linked mental retardation (incidence = ~1/3000 males; Nature 392:923).4. Alzheimer’s disease (incidence = ~10% over 65-year old; >50% over 85-year old; Ann Neurol 30:572).5. Tuberous sclerosis (incidence = ~0.3% adults; Hum Genet 107:97).

  9. ? Ca2+  PKB P P P P P P P P CaMKII V/ES P V/ES Pi3K LS RasGEF Ras-GTP Ras-GDP V/ES RasGAP MEK ERK1/2 NM-R NM Slot GluR2 GluR3 GluR1 GluR2L NMDA-R Ras-MEK-ERK and Pi3K-PKB signal delivery of GR1 and GR2L

  10. Point mutations (T35S, E37G or Y40C) was made on constitutively active Ras (S12V) background to selectively stimulate Ras-Raf, Ras-Ral and Ras-Pi3K signaling pathways. Scheffzek et al. Science 277:333-338; Boriack-Sjodin et al. Nature 394:337-343 Rodriguez-Viciana et al.Cell 89:457-467; White et al., Cell 80:533-541 Structure of small GTPases

  11. Ras(S35)-GFP+RFP Ras(G37)-GFP+RFP Ras(C40)-GFP+RFP Expression of active Ras mutants in CA1 neurons

  12. 25 pA +40 mV +40 mV +40 mV 20 ms Ras(G37)-GFP Ctrl Ras(S35)-GFP Ras(C40)-GFP -60 mV -60 mV -60 mV Expression of active Ras mutants in CA1 neurons IAMPA (%) INMDA (%)

  13. Anti-phospho-ERK1/2 Anti-phospho-PKB Anti-ERK1/2 Anti-PKB Control Control Ras(S35) Ras(S35) Ras(C40) Ras(C40) Ras(G37) Ras(G37) Activation of Ras pathways increases p-ERK or p-PKB

  14. Ras(G37)-GFP Control Ras(C40)-GFP Ras(S35)-GFP Immunoreactivity (%) Activation of Ras pathways increases p-ERK or p-PKB

  15. ? Ca2+  PKB P P P P P P P P CaMKII V/ES P V/ES Pi3K LS RasGEF Ras-GTP Ras-GDP V/ES RasGAP MEK ERK1/2 NM-R NM Slot GluR2 GluR3 GluR1 GluR2L NMDA-R Ras-MEK-ERK and Pi3K-PKB signal delivery of GR1 and GR2L

  16. GluR1 -/- GluR2 -/- 40 pA +40 mV +40 mV 25 ms Ctrl Ras(C40)-GFP Ras(G37)-GFP -60 mV -60 mV IAMPA (%) INMDA (%) GluR1 -/- GluR2 -/- GluR1 -/- GluR2 -/- Expression of active Ras mutants in knockout mice

  17. Ras-MEK-ERK and PI3K-PKB target S845 & S831 of GR1 Anti-p845-GluR1 Anti-p831-GluR1 Anti-GluR1 Anti-GluR1 Control Control Ras(C40) Ras(C40) Ras(G37) Ras(G37)

  18. Ras(G37)-GFP Control Ras(C40)-GFP Immunoreactivity (%) Immunoreactivity (%) Ras-MEK-ERK and PI3K-PKB target S845 & S831 of GR1

  19. ? Ca2+  PKB P P P P P P P P CaMKII V/ES P V/ES Pi3K LS RasGEF Ras-GTP Ras-GDP V/ES RasGAP MEK ERK1/2 NM-R NM Slot GluR2 GluR3 GluR1 GluR2L NMDA-R Ras-MEK-ERK and Pi3K-PKB signal delivery of GR1 and GR2L

  20. LTP experimental configuration Control pathway Stimulated pathway

  21. Before pairing After pairing Test pathway Control pathway 50 pA 20 ms Normalized IAMPA (%) Expression of Ras(G37)-GFP occludes 50% of LTP

  22. Before pairing After pairing Test pathway Control pathway 50 pA 20 ms Normalized IAMPA (%) Expression of Ras(C40)-GFP occludes 50% of LTP

  23. Before pairing After pairing Ctrl GR1(845D)+PD Ctrl+LY Ras(G37)+Ras(C40) 50 pA 20 ms Normalized IAMPA (%) Blocking Ras-MEK-ERK or Pi3K-PKB blocks 50% of LTP

  24. ? Ca2+  PKB P P P P P P P P CaMKII V/ES P V/ES Pi3K LS RasGEF Ras-GTP Ras-GDP V/ES RasGAP MEK ERK1/2 NM-R NM Slot GluR2 GluR3 GluR1 GluR2L NMDA-R Ras-MEK-ERK and Pi3K-PKB signal delivery of GR1 and GR2L

  25. With Medium With LY With Medium +40 mV +40 mV +40 mV -60 mV -60 mV -60 mV 25 pA 20 ms Ctrl GluR1-GFP GluR1ct-GFP IAMPA (%) INMDA (%) Synaptic delivery of GluR1 by neuromodulator agonists

  26. McN McN Control Control CaMKII CaMKII Histamine Histamine Neuromodulator agonists stimulate Ras-MEK-ERK and Pi3K-PKB Anti-phospho-ERK1/2 Anti-phospho-PKB Anti-ERK1/2 Anti-PKB

  27. Immunoreactivity (%) Neuromodulator agonists stimulate Ras-MEK-ERK and Pi3K-PKB Control CaMKII Histamine McN

  28. ? Ca2+  PKB P P P P P P P P CaMKII V/ES P V/ES Pi3K LS RasGEF Ras-GTP Ras-GDP V/ES RasGAP MEK ERK1/2 NM-R NM Slot GluR2 GluR3 GluR1 GluR2L NMDA-R Ras-MEK-ERK and Pi3K-PKB signal delivery of GR1 and GR2L

  29. Control vs. GluR1-GFP Control vs. GluR1ct-GFP 40 pA +40 mV +40 mV 25 ms Ctrl Awaking animals Sleeping animals -60 mV -60 mV Synaptic delivery of GluR1 at different behavioral states IAMPA (%) INMDA (%) Control vs. GluR1ct-GFP Control vs. GluR2Lct-GFP Control vs. GluR1ct-GFP Control vs. GluR2Lct-GFP

  30. Sleeping Sleeping Awaking Awaking Activation of Ras-MEK-ERK and Pi3K-PKB at different states Anti-phospho-ERK1/2 Anti-phospho-PKB Anti-ERK1/2 Anti-PKB

  31. Sleeping animals Awaking animals Immunoreactivity (%) Activation of Ras-MEK-ERK and Pi3K-PKB at different states

  32. ? Ca2+  PKB P P P P P P P P CaMKII V/ES P V/ES Pi3K LS RasGEF Ras-GTP Ras-GDP V/ES RasGAP MEK ERK1/2 NM-R NM Slot GluR2 GluR3 GluR1 GluR2L NMDA-R Ras-MEK-ERK and Pi3K-PKB signal delivery of GR1 and GR2L

  33. Trafficking & Signaling

  34. Acknowledgements Dept of Pharmacology, UVA Yi Qin, MD, MS (UW-Madison) Joel Baumgart, BA, BS (Missouri) Anders Killend, MS (Oslo) Hailan Hu, PhD (HHMI/Berkeley) Yinghua Zhu, MD, MS (Peking/CSHL) Cold Spring Harbor Laboratory Roberto Malinow Linda van Alest MPI Medical Research Peter Seeburg Pavel Osten

  35. Behavioral state-dependent activation of Ras signaling pathways and synaptic delivery of AMPA-Rs Joel P. Baumgart1, Yi Qin1,2, Yinghua Zhu1, Kenneth Seidenman2, Pavel Osten3, Peter H. Seeburg3, Linda van Aelst2, Roberto Malinow2 and J. Julius Zhu1* 1Department of Pharmacology and Neuroscience Training Program University of Virginia School of Medicine, Charlottesville, VA 22908 2Cold Spring Harbor Laboratory, Cold Spring Harbor, NY 11724 3Department of Molecular Neurobiology, Max Planck Institute for Medical Research, Heidelberg D-69120, Germany

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