Wang Haoa Tian Zhixingb Hao Linac * Wang Jinmingd Yu Zhiminge
Yu Tian Wang Brain Research Centre Faculty of Medicine University of British Columbia
description
Transcript of Yu Tian Wang Brain Research Centre Faculty of Medicine University of British Columbia
Yu Tian WangBrain Research Centre
Faculty of MedicineUniversity of British Columbia
LTP promotes proliferation and neuronal differentiation of neuronal progenitor cells
Huntington Disease Parkinson’s Disease
Neurodegenerative diseases require cell replacement therapies
– Core
Neurons die immediately
– Penumbra
Neurons die 1 to 3 days later -Delayed cell death
Neurons in the core region die following stroke insults
Stem cell therapies to replace the dead neurons in the brain
Replacement with neuronal progenitor stem cells
Core
Neurostem Cells
Current challenges for stem cell transplantation
1. Not survive long enough
2. Not fully differentiate into neurons
3. 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.
How can we go about it?
2007, 25:562–570
J. Neural Eng. 6 (2009) 055001
How can we go about it?
2007, 25:562–570
J. Neural Eng. 6 (2009) 055001
Can we create an effective brain stimulation protocol that promotes neurogenesis of stem cell/neural progenitor cells, thereby facilitating brain repair?
How can we go about it?
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. 232:357-74.
Synaptic transmission in the brain
Synapse
Synaptictransmission
NMDAR activation stimulates PI3K-Akt, thereby leading to LTP expression
Basal LTP
AMPARNMDAR T840-p
Ca2+
CaMKIIRas
PI3KAkt
PI3-Kinase
PTEN
Akt/PKBGrowth
Proliferationsurvival
PI3K-Akt signaling pathway is critical for cell proliferation and survival
• LTP increases neurogenesis of endogenous NPCs
• LTP increases neurogenesis of transplanted NPCs
• LTP promotes neurogenesis partially via BDNF
Hypothesis: LTP-inducing brain stimulation promotes proliferation/survival and neural differentiation of 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.
Hippocampal DG exhibits LTP in response to HFS, and also contains endogenous neural progenitor cells (NPCs)
In Vivo microinjection and electrophysiology setup
HFS reliably induces NMDAR-mediated LTP in rat hippocampal DG in vivo
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fEPS
P sl
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(% b
asel
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Time (min), n=8
fEPS
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LTP
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fEPS
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fEPS
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LTPControl (0.05Hz+Saline)
DAPIPCNA DAPIPCNA
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(% c
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Hz+CPP
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Hz+Sali
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Induction of LTP promotes proliferation/survival of endogenous DG-NPCs in the rat hippocampus
Control
LTP
Day 0
LTP
Day 7
PCNA staining
Protocol:
Day 0 Day 3
Retrovirus-GFP HFS
Day 10
Sacrifice
ImmunohistochemistryCPP
- 90min
Can LTP promote adult neurogenesis in hippocampal DG?
Induction of LTP promotes proliferation/survival and neuronal differentiation of hippocampal DG endogenous NPCs in the adult rat
• LTP increases neurogenesis of endogenous NPCs
Summaries
• LTP increases neurogenesis of endogenous NPCs
Can LTP induction also increase neurogenesis of transplanted NPCs?
Summaries
Isolation and proliferation of NSCs from the embryonic rat brain in vitro
Neur
ons
Nestin
Vimentin
MAP2
NSCs
β-actin
Neurospheres Nestin
Nestin/DAPI
GFAP
MAP2/DAPI
NSCs from the telencephalon of E14 Wistar rats were isolated and maintained in N2 supplemented with bFGF, EGF, and LIF.
Day 0
LTP
After 2-6hr Day 7-14
Immunohistochem.
GFP-NPCs
GFP-NPCs
ML DG CA1 CA1
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Time (minutes)
EPSP
slo
pe (%
Cha
nge)
LTPCONT
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1mV
CA1
Transplantation of GFP-NPCs into hippocampal CA1 region following LTP induction in the rat
Control
LTP promotes the proliferation/survival and neuronal differentiation of NPCs transplanted into the hippocampal CA1 region in rats
• LTP increases neurogenesis of endogenous NPCs
• LTP increases neurogenesis of transplanted NPCs
Summaries
• LTP increases neurogenesis of endogenous NPCs
• LTP increases neurogenesis of transplanted NPCs
Summaries
How does LTP promote neurogenesis?
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
cLTPPBS or
Day 25
Immunocytochem.
GFP
NeuronsNSCs-GFP
cLTP
cLTP
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MAP
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FP+
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ontr
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PBScL
TP
LTP+A
PVAPV
LTP promotes neurogenesis in NPC-Neuron co-cultures
PBS
NeuronsNSCs-GFP
cLTP
Glycine stimulation does not alter neurogenesis in NPC cultures
MAP2NSC LTPControl
LTP
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GFP
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ontr
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Pure NSC cultureNSCs-GFP
cLTP
MAP2GFP
How does LTP induction in neurons affect neurogenesis of NPCs in co-cultures?
Direct contacts (synapses) or diffusible factors?
NSCs
Neurons
LTP
NSCs
Neurons
PBS
NSCs
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Media MediaMedia
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Cond. Med
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Neurons0.2 ㎛ filter,
centrifugation
NSCs
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DAPIMAP2
Glycine-induced LTP promotes NPC neurogenesis in co-cultures via releasing diffusible trophic factors
Conditioned medium from LTP stimulated cells
LTP (C.M.) LTP + K-252aControl (C.M.)
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% D
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LTP (C.M
.)
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Time (min)
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Phospho-TrkB 165kDa
96kDaTfR
Control
LTP (C.M
.)
LTP.+ K252a
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Phos
pho-
TrkB
/TfR
(%
con
trol
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BDNF NT-3NGF
DAPIMAP2
100mm
LTP-conditioned medium promotes NPC neurogenesis in pure NPC cultures in part by BDNF-TrkB signaling
Neurogenesis induced by glycine-induced LTP in NPC-Neuron co-cultures was prevented by TrkB inhibitor
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LTP
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GFPMAP2
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• LTP increases neurogenesis of endogenous NPCs
• LTP increases neurogenesis of transplanted NPCs
• LTP promotes neurogenesis partially via BDNF
Summaries
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
Clinical Relevance
Acknowledgements
Taesup ChoDr. Changiz TaghibiglouDr. Jie LuGary EvansYuping LiDr. Yuan Ge
Collaborators:Dr. James G. McLarnonJak Kyu Ryu
Supports:CIHRHHMIHSFC/BC