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Yu Tian Wang Brain Research Centre Faculty of Medicine University of British Columbia

LTP promotes proliferation and neuronal differentiation of neuronal progenitor cells. Yu Tian Wang Brain Research Centre Faculty of Medicine University of British Columbia. Neurodegenerative diseases require cell replacement therapies. Parkinson’s Disease. Huntington Disease.

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Yu Tian Wang Brain Research Centre Faculty of Medicine University of British Columbia

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  1. LTP promotes proliferation and neuronal differentiation of neuronal progenitor cells Yu Tian Wang Brain Research Centre Faculty of Medicine University of British Columbia

  2. Neurodegenerative diseases require cell replacement therapies Parkinson’s Disease Huntington Disease

  3. Neurons in the core region die following stroke insults • Core • Neurons die immediately • Penumbra • Neurons die 1 to 3 days later -Delayed cell death

  4. Stem cell therapies to replace the dead neurons in the brain Neurostem Cells Core Replacement with neuronal progenitor stem cells

  5. Current challenges for stem cell transplantation • Not survive long enough • Not fully differentiate into neurons • Not fully integrate into functional network with host neurons • Improvements in all these fronts will be the key for the successful application of stem cell therapy in replacing injured and/or repairing neuronal circuits in neurodegenerative diseases.

  6. How can we go about it?

  7. How can we go about it? 2007, 25:562–570 J. Neural Eng. 6 (2009) 055001

  8. How can we go about it? 2007, 25:562–570 • Can we create an effective brain stimulation protocol that promotes neurogenesis of stem cell/neural progenitor cells, thereby facilitating brain repair? J. Neural Eng. 6 (2009) 055001

  9. Discovery of High-Frequency-Stimulation to induce LTP in the hippocampus Hippocampus • Bliss and Lomo (1973) J Physiol. 232:331-56 • Bliss and Gardner-Medwin (1973) J Physiol. 232:357-74.

  10. Synaptic transmission in the brain Synapse Synaptic transmission

  11. T840-p NMDAR NMDAR activation stimulates PI3K-Akt, thereby leading to LTP expression Basal LTP Ca2+ CaMKII Ras PI3K Akt AMPAR

  12. PI3K-Akt signaling pathway is critical for cell proliferation and survival PI3-Kinase PTEN Growth Proliferation survival Akt/PKB

  13. Hypothesis:LTP-inducing brain stimulation promotes proliferation/survival and neural differentiation of NPCs • LTP increases neurogenesis of endogenous NPCs • LTP increases neurogenesis of transplanted NPCs • LTP promotes neurogenesis partially via BDNF

  14. Hippocampal DG exhibits LTP in response to HFS, and also contains endogenous neural progenitor cells (NPCs) NPCs shown in green, neurons in red. Photo: Univ. of California-Irvine NPCs are are self-renewing cells between stem cells and neurons, capable of giving rise to three main cell types of the nervous system: neurons, astrocytes and oligodendrocytes.

  15. In Vivo microinjection and electrophysiology setup

  16. HFS reliably induces NMDAR-mediated LTP in rat hippocampal DG in vivo 0.05Hz + 0.9% Saline LTP 5ms, 2mV fEPSP slope (% baseline) fEPSP slope (% baseline) Time (min), n=8 Time (min), n=9 0.05Hz + CPP CPP + HFS fEPSP slope (% baseline) fEPSP slope (% baseline) Time (min), n=9 Time (min), n=8

  17. Induction of LTP promotes proliferation/survival of endogenous DG-NPCs in the rat hippocampus PCNA staining LTP Protocol: Day 0 Day 7 PCNA DAPI PCNA DAPI 300 ** Control DG DG 200 PCNA+/DAPI cells (% control) 100 LTP Control (0.05Hz+Saline) LTP LTP CPP+sTPS 0.05Hz+CPP 0.05Hz+Saline

  18. Can LTP promote adult neurogenesis in hippocampal DG? HFS Retrovirus-GFP Sacrifice - 90min Day 3 Day 10 Day 0 Immunohistochemistry CPP

  19. Induction of LTP promotes proliferation/survival and neuronal differentiation of hippocampal DG endogenous NPCs in the adult rat

  20. Summaries • LTP increases neurogenesis of endogenous NPCs

  21. Summaries • LTP increases neurogenesis of endogenous NPCs Can LTP induction also increase neurogenesis of transplanted NPCs?

  22. Isolation and proliferation of NSCs from the embryonic rat brain in vitro Neurospheres Nestin NSCs from the telencephalon of E14 Wistar rats were isolated and maintained in N2 supplemented with bFGF, EGF, and LIF. Nestin/DAPI MAP2/DAPI Neurons NSCs Nestin Vimentin MAP2 β-actin GFAP

  23. Transplantation of GFP-NPCs into hippocampal CA1 region following LTP induction in the rat GFP-NPCs Immunohistochem. LTP CA1 Day 0 Day 7-14 After 2-6hr CA1 DG CA1 ML GFP-NPCs 100 80 1mV 20ms 60 EPSP slope (% Change) LTP 40 CONT 20 0 -20 -30 -15 0 15 30 45 60 75 90 Time (minutes)

  24. LTP promotes the proliferation/survival and neuronal differentiation of NPCs transplanted into the hippocampal CA1 region in rats Control

  25. Summaries • LTP increases neurogenesis of endogenous NPCs • LTP increases neurogenesis of transplanted NPCs

  26. Summaries • LTP increases neurogenesis of endogenous NPCs • LTP increases neurogenesis of transplanted NPCs How does LTPpromote neurogenesis?

  27. Studying mechanisms underlying LTP-promoted neurogenesis in NPC-Neuron co-cultures Neurospheres PBS or NSCs+Neurons cLTP Immunocytochem. BrdU cLTP Day 11 Day 25 Day 14 Day 3 Day 0 Day 2 Neurons NSCs-GFP LTP in animal models Mechanisms in cultures GFP

  28. LTP promotes neurogenesis in NPC-Neuron co-cultures 200 150 *** 100 50 cLTP MAP2+ GFP/GFP+ (% control) 0 Neurons NSCs-GFP PBS APV cLTP LTP+APV PBS cLTP

  29. Glycine stimulation does not alter neurogenesis in NPC cultures cLTP Pure NSC culture NSCs-GFP NSC MAP2 LTP Control 100 MAP+ on GFP (% Control) 50 23mm 0 LTP Control

  30. How does LTP induction in neurons affect neurogenesis of NPCs in co-cultures? GFP MAP2 Direct contacts (synapses) or diffusible factors?

  31. Glycine-induced LTP promotes NPC neurogenesis in co-cultures via releasing diffusible trophic factors No stim PBS LTP Neurons Neurons Media Media Media NSCs NSCs NSCs NSCs Neurons 0.2 ㎛ filter, centrifugation MAP2 DAPI * 200 150 MAP2+ /DAPI cells (% control) 100 NSCs alone 50 0 PBS No stim. Cond. Med.

  32. 50 50 50 LTP-conditioned medium promotes NPC neurogenesis in pure NPC cultures in part by BDNF-TrkB signaling ** Conditioned medium from LTP stimulated cells 40 40 40 165kDa 30 30 30 Neurotrophins (pg/ml) TfR 96kDa * 20 20 20 * 200 10 10 10 Phospho-TrkB 150 0 0 0 0 10 0 10 0 10 30 60 1 day 30 60 1 day 30 60 1 day Phospho-TrkB/TfR (% control) Time (min) Time (min) Time (min) 100 BDNF NGF NT-3 50 0 Control LTP (C.M.) LTP.+ K252a 40 DAPI MAP2 30 MAP2+ (% DAPI) 20 ** 10 100mm Control Control (C.M.) LTP (C.M.) LTP + K-252a LTP+K252a LTP (C.M.)

  33. Neurogenesis induced by glycine-induced LTP in NPC-Neuron co-cultures was prevented by TrkB inhibitor 40 ** MAP2 GFP 30 MAP2+GFP-cells 20 10 50㎛ 0 LTP Control LTP+K252a Control LTP LTP + K252a

  34. Summaries • LTP increases neurogenesis of endogenous NPCs • LTP increases neurogenesis of transplanted NPCs • LTP promotes neurogenesis partially via BDNF

  35. Clinical Relevance Chronic LTP-inducing Electrical stimulation increases neurogenesis, thereby facilitating recovery following neuronal damage such as stroke. LTP induction with electrical stimulation or glycine may be performed prior to NPC transplantations, thereby promoting their survival and neural differentiation, and ultimately functional integration into hosting neuronal network

  36. Acknowledgements Collaborators: Dr. James G. McLarnon Jak Kyu Ryu Supports: CIHR HHMI HSFC/BC Taesup Cho Dr. Changiz Taghibiglou Dr. Jie Lu Gary Evans Yuping Li Dr. Yuan Ge

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