1. 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

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

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

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

6. 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.

7. How can we go about it?

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

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

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

11. Synaptic transmission in the brain
Synapse
Synaptic
transmission

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

13. PI3K-Akt signaling pathway is critical for cell proliferation and survival
PI3-Kinase
PTEN
Growth
Proliferation
survival
Akt/PKB 1 1

14. 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

15. 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.

16. In Vivo microinjection and electrophysiology setup

17. 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

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

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

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

21. Summaries
LTP increases neurogenesis of endogenous NPCs

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

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

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

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

26. Summaries LTP increases neurogenesis of endogenous NPCs
LTP increases neurogenesis of transplanted NPCs

27. Summaries LTP increases neurogenesis of endogenous NPCs
LTP increases neurogenesis of transplanted NPCs
How does LTP promote neurogenesis?

28. Mechanisms in cultures
Studying mechanisms underlying LTP-promoted neurogenesis in NPC-Neuron co-cultures
LTP in animal models
Mechanisms in cultures
Day 0
Neurospheres
Day 2
Day 3
NSCs+Neurons
BrdU
Day 14
Day 11
cLTP
PBS or
Day 25
Immunocytochem.
Neurons
NSCs-GFP
cLTP
GFP 1 1

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

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

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

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

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

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

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

36. 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

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