Translational Control by MAPK Signaling in Long-Term Synaptic Plasticity and Memory Raymond J. Kelleher III,1,2 Arvind

1. Translational Control by MAPK Signaling in Long-Term Synaptic Plasticity and Memory Raymond J. Kelleher III,1,2 Arvind Govindarajan,1 Hae-Yoon Jung,1 Hyejin Kang,1 and Susumu Tonegawa1,3

2. ERK-MAPK Pathway in Neurons Calcium Influx ERK-MAPK Pathway Synaptic Plasticity Learning and Memory

3. Component of ERK Pathway Activated Normal MEK1 ERK1 P P MEK1 MEK1 + ERK1 ERK2 Kinase Activity Kinase Activity ERK2 Inactive Dominant-negative form of MEK1 ERK1 MEK1 MEK1 + ERK1 ERK2 Kinase Activity Kinase Activity ERK2

4. Does the dnMEK1 mutation have an Effect on memory?

6. Does the dnMEK1 mutation Effect The general function of the synapsis?

8. Does the dnMEK1 mutation effect early-LTP?

10. Does the dnMEK1 mutation effect Late-LTP?

12. Hippocampal L-LTP and long term memory ERK-MapK pathway Early phases of LTP and Memory +

13. Does the dnMEK1 mutation effect transcription or translation?

14. Transcriptional inhibitor Actinomycin-D (Black) Translational inhibitor Anisomycin. (Red)

16. What are the effects of the dnMEK1 mutant on translation resulting from different form of neuronal stimulation?

17. Translation reporter assay Culture Plate Cultured Primary Hippocampal Neuron

18. Translation reporter assay Transfection of cultured neurons with synthetic mRNAs Culture Plate Cultured Primary Hippocampal Neuron

19. They Stimulated the neurons with three different forms of stimulants: • BDNF • The GABA-A receptor inhibitor bicuculline • Membrane depolarization

20. Stimulators

21. Does the dnMEK1 have a neuron wide effect on Translation?

22. Step 1: Inject one or a mixture of substances Step 2: Basal Level translation Step 3: Measure the amount of translated product Step 4: Remove cytoplasm separate proteins via electrophoresis and Step 5: Probe for proteins

23. MAPK-BLOCKER Stimulator Stimulator

24. ERK-MapK pathway Neuron wide Protein synthesis

25. What effect does the dnMEK1 mutant have on the number of activated translation Initiation factors?

26. P P P E-Bp1 S6 4E 4

27. MAPK-BLOCKER Stimulator Stimulator

28. Blocking the ERK-MAPK Pathway Translational induction and translation factor phosphorylation

29. Does the dnMEK1 mutation have an effect on the amount of activated translation initiation factors located within the synapse?

30. ERK-dependence of protein synthesis and translation factor phosphorylation in synapto-neurosomes Synapse Translation initiation factors U0126 pERK ? + pS6 ? + pelF4E ?

31. Translation initiation factors U0126 pERK + pS6 + pelF4E

32. Local protein synthesis ERK-MapK pathway

33. During hippocampal Memory Formation ? Translational Activity

34. During hippocampal Memory Formation ? Translational Activity

35. During hippocampal Memory Formation ? Translational Activity P-ERK, P-elF4E, and P-S6 control and dnMeK1 30 Min P-ERK, P-elF4E, and P-S6 control and dnMeK1 30 Min

36. During hippocampal Memory Formation Translational Activity Conditioned Control Mice pERK, pS6, and pelF4E Conditioned mutant mice pERK, pS6, and pelF4E

37. ERK-MapK Pathway Phosphorylation translation machinery Neuronal activity

38. In conclusion: Hippocampal L-LTP and long term memory ERK-MapK pathway Early phases of LTP and Memory protein synthesis dependent events +

39. In conclusion: Hippocampal L-LTP and long term memory ERK-MapK pathway Early phases of LTP and Memory protein synthesis dependent events + Local protein synthesis ERK-MapK pathway Neuron wide Protein synthesis

40. In conclusion: Hippocampal L-LTP and long term memory ERK-MapK pathway Early phases of LTP and Memory protein synthesis dependent events + Local protein synthesis ERK-MapK pathway Neuron wide Protein synthesis ERK-MapK Pathway Phosphorylation translation machinery Neuronal activity