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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
Huntington Disease
Parkinson’s Disease
Neurons in the core region die following
stroke insults
– Core
Neurons die immediately
– Penumbra
Neurons die 1 to 3 days later
-Delayed cell death
Stem cell therapies to replace the dead neurons
in the brain
Neurostem Cells
Core
Replacement with neuronal progenitor stem 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?
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?
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
Synaptic
transmission
NMDAR activation stimulates PI3K-Akt, thereby
leading to LTP expression
Basal
LTP
Ca2+
CaMKII
Ras
PI3K
Akt
NMDAR
AMPAR
T840-p
PI3K-Akt signaling pathway is critical for cell
proliferation and survival
PI3-Kinase
PTEN
Growth
Proliferation
survival
Akt/PKB
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
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.
In Vivo microinjection and electrophysiology setup
0.05Hz + 0.9% Saline
180
160
5ms, 2mV
140
120
100
80
60
0
20
40
60
80
100
120
fEPSP slope (% baseline)
fEPSP slope (% baseline)
HFS reliably induces NMDAR-mediated LTP in rat
hippocampal DG in vivo
LTP
180
160
140
120
100
80
60
0
20
180
CPP + HFS
160
140
120
100
80
60
0
20
40
60
80
Time (min), n=9
60
80
100
120
100
120
Time (min), n=9
100
120
fEPSP slope (% baseline)
fEPSP slope (% baseline)
Time (min), n=8
40
180
0.05Hz + CPP
160
140
120
100
80
60
0
20
40
60
Time (min), n=8
80
Induction of LTP promotes proliferation/survival of
endogenous DG-NPCs in the rat hippocampus
PCNA staining
LTP
Protocol:
PCNA DAPI
DG
Day 7
PCNA DAPI
Control
DG
LTP
Control (0.05Hz+Saline)
LTP
PCNA+/DAPI cells (% control)
Day 0
300
200
100
**
Can LTP promote adult neurogenesis in hippocampal DG?
HFS
Sacrifice
Day 3
Day 10
Retrovirus-GFP
- 90min
Day 0
CPP
Immunohistochemistry
Induction of LTP promotes proliferation/survival and neuronal
differentiation of hippocampal DG endogenous NPCs in the adult rat
Summaries
• LTP increases neurogenesis of endogenous NPCs
Summaries
• LTP increases neurogenesis of endogenous NPCs
Can LTP induction also increase neurogenesis of
transplanted NPCs?
Isolation and proliferation of NSCs from the
embryonic rat brain in vitro
NSCs from the
telencephalon of
E14 Wistar rats
were isolated and
maintained in N2
supplemented with
bFGF, EGF, and LIF.
Neurospheres
Nestin/DAPI
MAP2/DAPI
Nestin
Nestin
Vimentin
MAP2
β-actin
GFAP
Transplantation of GFP-NPCs into hippocampal CA1 region
following LTP induction in the rat
GFP-NPCs
LTP
Immunohistochem.
CA1
Day 0 After 2-6hr
Day 7-14
DG
ML
GFP-NPCs
100
80
1mV
EPSP slope (% Change)
CA1
20ms
60
LTP
CONT
40
20
0
-20
-30
-15
0
15
30
45
Time (minutes)
60
75
90
LTP promotes the proliferation/survival and neuronal differentiation
of NPCs transplanted into the hippocampal CA1 region in rats
Control
Summaries
• 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
How does LTP promote neurogenesis?
Studying mechanisms underlying LTP-promoted
neurogenesis in NPC-Neuron co-cultures
LTP in animal models
Mechanisms in cultures
Neurospheres
cLTP
NSCs-GFP
GFP
NSCs+
Neurons
PBS or
cLTP
BrdU
Day
11
Day
14
Immunocytochem.
Neurons
Day Day Day
3
0
2
Day
25
LTP promotes neurogenesis in NPC-Neuron co-cultures
PBS
Neurons
150
(% control)
NSCs-GFP
MAP2+ GFP/GFP+
200
cLTP
100
50
0
cLTP
***
Glycine stimulation does not alter neurogenesis in NPC cultures
cLTP
Pure NSC culture
NSC MAP2
LTP
Control
23mm
MAP+ on GFP (% Control)
NSCs-GFP
100
50
0
How does LTP induction in neurons affect neurogenesis
of NPCs in co-cultures?
GFP MAP2
Direct contacts (synapses) or diffusible factors?
Glycine-induced LTP promotes NPC neurogenesis in
co-cultures via releasing diffusible trophic factors
No
stim
PBS
LTP
Neurons
Media
Media
NSCs
Neurons
NSCs
Media
Neurons
NSCs
200
MAP2+ /DAPI cells
(% control)
NSCs alone
MAP2 DAPI
150
100
50
0
0.2 ㎛ filter,
centrifugation
NSCs
*
LTP-conditioned medium promotes NPC neurogenesis
in pure NPC cultures in part by BDNF-TrkB signaling
Phospho-TrkB
Conditioned medium from LTP stimulated cells
40
30
50
*
*
*
20
10
0
0
10
30
60 1 day
Time (min)
50
NGF
40
40
30
30
20
20
10
10
0
0
10
30
60 1 day
Time (min)
0
NT-3
0
10
30
96kDa
200
(% control)
BDNF
TfR
Phospho-TrkB/TfR
Neurotrophins
(pg/ml)
50
165kDa
*
150
100
50
0
60 1 day
Time (min)
40
MAP2+ (% DAPI)
MAP2 DAPI
100mm
Control (C.M.)
LTP (C.M.)
LTP + K-252a
30
20
10
*
*
Neurogenesis induced by glycine-induced LTP in NPCNeuron co-cultures was prevented by TrkB inhibitor
40
GFP
**
MAP2+ GFP-cells
MAP2
30
20
10
0
50㎛
Control
LTP
LTP + K252a
Summaries
• LTP increases neurogenesis of endogenous NPCs
• LTP increases neurogenesis of transplanted NPCs
• LTP promotes neurogenesis partially via BDNF
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
Acknowledgements
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