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

0 20 40 60 80 100 12060

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180

0 20 40 60 80 100 12060

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100

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180

0 20 40 60 80 100 12060

80

100

120

140

160

180

0.05Hz + 0.9% Saline

fEPS

P sl

ope

(% b

asel

ine)

Time (min), n=8

fEPS

P sl

ope

(% b

asel

ine)

Time (min), n=9

LTP

0 20 40 60 80 100 12060

80

100

120

140

160

180CPP + HFS 0.05Hz + CPP

Time (min), n=9 Time (min), n=8

fEPS

P sl

ope

(% b

asel

ine)

fEPS

P sl

ope

(% b

asel

ine)

5ms, 2mV

LTPControl (0.05Hz+Saline)

DAPIPCNA DAPIPCNA

DG DG

PC

NA

+/D

AP

I ce

lls

(% c

ontr

ol)

**

0.05

Hz+CPP

0.05

Hz+Sali

neLT

P

CPP+sTPS

100

200

300

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

-20

0

20

40

60

80

100

-30 -15 0 15 30 45 60 75 90

Time (minutes)

EPSP

slo

pe (%

Cha

nge)

LTPCONT

20ms

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

***

MAP

2+ G

FP/G

FP+

(% c

ontr

ol)

100

150

0

50

200

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

50

100

MAP

+ on

GFP

(% C

ontr

ol)

Control23mm 0

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

No stim

Media MediaMedia

50

100

200

0

MA

P2+

/DA

PI c

ells

(%

co

ntr

ol)

*

No stim

.

Cond. Med

.

150

PBS

Neurons0.2 ㎛ filter,

centrifugation

NSCs

NS

Cs

alo

ne

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.)

10

20

30

40

MAP

2+ (

% D

API)

LTP+K252a

Control

LTP (C.M

.)

**

Time (min)

Neu

rotr

ophi

ns

(pg/

ml)

0 10 30 60 1 day 0 10 30 60 1 day 0 10 30 60 1 dayTime (min) Time (min)

20

30

40

50

10

20

30

40

50

10

20

30

40

50

10

*

**

0 0 0

Phospho-TrkB 165kDa

96kDaTfR

Control

LTP (C.M

.)

LTP.+ K252a

*

50

100

150

0

200

Phos

pho-

TrkB

/TfR

(%

con

trol

)

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

10

20

30

40

Control

LTP

LTP+K252a

MAP

2+ GFP

-cel

ls

LTP LTP + K252aControl

GFPMAP2

50㎛0

**

• 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