talk 2008-meeting about nad

NAD+ / NADH在神经细胞死亡中的作用

殷卫海教授上海交通大学Med-X 研究院

上海交通大学医学院附属瑞金医院神经病学研究所

NAD+ / NADH

OLD COUPLE

POWERFUL COUPLE

I. Based on the above discussion, it appears that the classical paradigm regarding the biological functions of NAD and NADP is too narrow to generalize the growing functions of these molecules. It is tempting to propose that a novel paradigm about the biological functions of NAD and NADP may be emerging.

From: Ying W. (2008) Antioxidants & Redox Signaling

Two of my major new thoughts about NADTwo of my major new thoughts about NAD

NADPH NADP+ NAD+ NADH

NAADP

AntioxidationOxidative StressReductive biosynthesis

Calcium homeostasis Mitochondrial functionEnergy metabolismOxidative stressCalcium homeostasisGene expression

Mitochondrial functionEnergy metabolismCalcium homeostasisGene expressionCell deathAging

DehydrogenasesPARPsSirtuinsARCsARTs

NADKDehydrogenases/Oxidases

GRx

NADPH oxidase

G6PDH6GPDHIDPMEPTDH

de novo pathway

Salvage pathway

L-Trp NMN/NaMN

ARCsETCOxidases


http://www.nightskyinfo.com/sky_highlights/hunters_moon/full_moon_small.jpg

II. NAD, together with ATP and Ca2+, may be the most fundamental components in life which mediate nearly all of the key biological processes. The close interactions among these components may

constitute a ‘Central Regulatory Network’ in life.


ATP NAD/NADP

Ca2+

Biological processes

1. A brief overview of the biological functions of NAD+ and NADH

2. Roles of NAD+ in PARP-1-mediated cell death

3. Therapeutic potential of NAD+

4. NADH transport across plasma membranes of cells

5. Roles of Ca2+-Mg2+-depenent endonuclease in cell death

OUTLINE OF THIS TALK


NAADP

AntioxidationOxidative StressReductive biosynthesis

Calcium homeostasis Mitochondrial functionEnergy metabolismOxidative stressCalcium homeostasisGene expression

Mitochondrial functionEnergy metabolismCalcium homeostasisGene expressionCell deathAging

DehydrogenasesPARPsSirtuinsARCsARTs

NADKDehydrogenases/Oxidases

GRx

NADPH oxidase

G6PDH6GPDHIDPMEPTDH

de novo pathway

Salvage pathway

L-Trp NMN/NaMN

ARCsETCOxidases


1. Roles of NAD+ and NADH in cellular functions

1.1. NAD+ and NADH in energy metabolism (a) Glycolysis (GAPDH);(b) pyruvate / lactate conversion;(c) TCA cycle; (d) electron transport chain; and(e) energy metabolism affected by NAD-dependentSIR2 / PARPs.

1) NAD+ / NADH ratio is an important regulator of mitochondrial permeability transition (MPT);

2) NADH can directly interact with and inhibit voltage-dependent anion channels (VDAC);

3) Indirectly affecting mitochondria by mediating calcium homeostasis and the activities of PARPs and sirtuins.

1.2. NAD+ and NADH in mitochondrial

functions

1.3. NAD+ and NADH in calcium homeostasisNAD+

PARP/PARG

ARTs

cADPR

NADP+

Sirtuins

O-acetyl-ADPR

ADP-R-P2X7R

ARCs

NADH

RyR

IP3-gated Ca2+ channels RyR

ADP-ribose

TRPM2

NAADP

Ca2+ store

MPT

NADPH

Antioxidation/ROS

Ca2+ pumps, Ca2+ channels

Calcium homeostasis


NAD+ NADH

Sirtuins

Histonedeacylation

Gene silencing

p53

PARP-1 Tankyrases

AP-1, NFkB, p53

Telomerases

Gene Expression

Corepressor CtBP

Clock:BMAL1;

NPAS2:BMAL1

Gene Expression

1.4. NAD+ and NADH in regulation of gene

expression

Ying W. (2007) Antioxidants & Redox Signaling

1.5. NAD+ and NADH in aging


Sirtuins PARP-1 Tankyrases

DNA repairGenomic stability

Telomere

AntioxidationROS

Mitochondria

Reducing potentialROS

Aging process

Nam

Nampt

NADPH oxidase


Summary

NAD+ and NADH have emerged as one of the most influential couples in nearly all of the major biological processes in life, including calcium homeostasis, mitochondrial functions, energy metabolism, gene expression, immunological functions, aging and cell death.

2. Roles of NAD+ in poly(ADP-ribose) polymerase-1 (PARP-1)-mediated cell death

NAD+Dehydrogenases

PARP

Poly(ADP-ribosyl)ated

proteins + Nam

ARTs

cADPR + Nam

NAD+ kinaseNADP+

sirtuins

Deacylated proteins + Nam

+ O-acetyl-ADP-ribose

(ADP-ribosyl)ated proteins + Nam

ADP-ribosyl cyclases

Salvage pathway

Nam / NA

de novo pathway

NaMNL-Trp L-Kyn Qa

Energy metabolism / Mitochondrial functions

NADH

DNA repair

Cell death

Gene expression

Genomic stability

Gene silencing

Aging

Cell death

Calcium homeostasis

Antioxidation

Calcium homeostasis

Signal transduction

Immunological regulation

Ying W. (2006)

Roles of Oxidative Stress in Pathological and Biological Processes

1) Aging;

2) necrosis and apoptosis;

3) ischemic brain and myocardial injury;

4) Alzheimer’s disease;

5) Parkinson’s disease;

6) cancer; and

7) diabetes.

Excessive PARP-1 activation has been indicated to play key roles in:

1) Cell death induced by:

a) oxidative stress;

b) excitotoxicity; and

c) oxygen-glucose deprivation

2) Multiple diseases models:

a) Ischemic brain injury;

b) MPTP-induced parkinsonism;

c) diabetes;

d) inflammation; and

e) hypoglycemic brain injury

Poly(ADP-ribose) Polymerase-1 (PARP-1)

1. An abundant nuclear protein; 113 kDa;

2. a major member of PARP family proteins;

3. three domains: DNA binding domain;

regulatory domain and catalytic domain;

4. rapidly activated by ssDNA damage; catalyzes poly(ADP-ribosyl)ation of proteins by consuming NAD+;

5. biological functions: DNA repair; gene expression; genomic stability; cell cycle;

long term memory; cell death.

From: Weihai Ying. (2006) Frontiers in Bioscience 11:3129-3148.

Ischemia/Reperfusion Oxidative stress MNNG

DNA Damage

PARP-1 Activation

PARG PAR-Protein Protein

ADP-RiboseNAD+ Depletion

Glycolysis

MPT

Mitochondrial Depolarization CyC/AIF Release ATP

Cell Death

From: Weihai Ying. (2006) Frontiers in Bioscience 11:3129-3148.

% A

str

ocy

te D

eath

0

20

40

60

80

100

wt

PARP-1-/-

****

MNNG (M)

0 50 100 200 300

****

OGD (min)

C

Fig. 1. PARP-1 activation mediates neuronal death induced by MNNG and OGD, and astrocyte death induced by MNNG. Pre-treatment with 50 M DPQ decreased MNNG- (A) and OGD-induced (B) neuronal death. The astrocytes prepared from PARP-1 ko mice were also highly resistant to cell death induced by 30-min MNNG exposures.

% As

trocy

te De

ath

0

20

40

60

80

100

wt

PARP-1-/-

****

MNNG (M)

0 50 100 200 300

****

OGD (min)

C

Fig. 1. PARP-1 activation mediates neuronal death induced by MNNG and OGD, and astrocyte death induced by MNNG. Pre-treatment with 50 M DPQ decreased MNNG- (A) and OGD-induced (B) neuronal death. The astrocytes prepared from PARP-1 ko mice were also highly resistant to cell death induced by 30-min MNNG exposures.

PARP-1 mediates MNNG- and chemical OGD-induced

Neuronal and astrocyte death

A

Fig. 2A. MNNG induces PARP activation in astrocytes. MNNG treatment induced formation of PAR (green fluorescence) in the nuclei (red fluorescence), indicting PARPactivation in the nuclei.

MNNG induced increased PAR in the nucleus of neurons

[Me

tab

olite

s]

( nm

ol / m

g p

rote

in)

0

10

20

30

40

50

60

ConMNNGMNNG / 50 M DPQ

** **

** **

Fig. 2C. PARP activation decreased ATP and total adenylate pool (ATP+ADP+AMP). Astrocytes were pre-treated with the PARP inhibitor DPQ, followed by MNNG treatment. Intracellular ATP, ADP and AMP were determined by HPLC assay.

PARP-1 activation causes not only ATP depletion, but also depletion of the total pool of (ATP + ADP + AMP)

Fig. 2B. PARP activation decreased intracellular NAD+ levels in astrocytes. Pre-treatment with 50 mM DPQ prevented MNNG-induced NAD+ depletion.

PARP-1 produces NAD+ depletion in cells

• How PARP-1 activation causes cell death?

• What is the role of NAD+ depletion in PARP-1 cytotoxicity?

• How to test the hypothesis that NAD+ depletion mediates PARP-1 toxicity ?

NA

D+

(nm

ol / m

g p

rote

in)

0

2

4

6

8

10

12

14

**

MNNG

Ying W. et al. (2003) BBRC 308:809-813.

NAD+ treatment can restore the intracellular NAD+ levels in astrocytes treated with the PARP activator MNNG

Post treatment delay (h)

% C

ell D

eath

0

20

40

60

80

100

control100 M MNNG

100 M MNNG / 5 mM NAD+

100 M MNNG / 10 mM NAD+

0 1 2 3

****

****

OGD

Fig. 3. NAD+ post-treatment profoundly decreased neuronal death induced by MNNG or OGD. After neuron-astrocyte co-cultures were exposed to MNNG or OGD, the cells were washed and treated with NAD+ for 24 hrs. Neuronal death was determined by PI staining.

AIF Nuclei Overlay

Control

MNNG + NAD+

AIF Nuclei Overlay

Fig. 14. NAD+ treatment can decrease MNNG-induced AIF translocation of astrocytes. The astrocytes were treated with 100 M MNNG for 30 min. After washout the cells were treated with 10 mM NAD+ for 3 hrs. After three hrs AIF immunostaining was conducted, and the images were photographed under a confocal microscope.

Con

MNNG

MNNG + NAD+

NAD+ treatment blocked MNNG-induced AIF translocation

Gly

co

lyti

c R

ate

(% o

f C

on

tro

l)

0

20

40

60

80

100

**

NAD+ post-treatment for 4 hours reversed MNNG-induced

glycolytic blockade

Glu

tam

ate

Up

tak

e(n

mo

l/m

in/m

g p

rote

in)

0

2

4

6

8

10

12

100 M MNNG

****

NAD+ post-treatment for 4 hours attenuated MNNG-induced glutamate

transport inhibition

* We have further found that NAD+ treatment can abolish MNNG-induced mitochondrial permeability transition and mitochondrial depolarization (Alano, Ying and Swanson JBC (2004).

Other studies that further indicate that NAD+ depletion mediates PARP-1-induced cell death

1) Liposome-based NAD+ delivery can decrease peroxynitrite-induced mitochondrial depolarization in neurons (Du et al.);

2) our colleagues Drs. Alano and Dr. Swanson have recently shown that BioPorter-Based delivery of NADase can induced NAD+ depletion and cell death;

3) NAD+ depletion by an inhibitor of a NAD+-synthesizing enzyme Nampt can induce cell death; and

4) a latest study published in Cell suggests that mitochondrial NAD+ depletion mediates cell survival in certain cell lines with low levels of mitochondria

Other studies have further indicated therapeutic potential of NAD+ for various diseases

1. NAD+ treatment can block transection-induced axonal injury by activating SIRT1 (Science (2004)) or locally enhancing energy metabolism (JCB (2005));

2. NAD+ treatment can block zinc-induced neuronal death (Eur. J. Neurosci. (2006); and

3. NAD+ treatment can decrease oxidative stress-induced myocyte death (JBC (2005)).

Summary

1. Our study provides the first direct evidence that NAD+ depletion mediates PARP-1-induced cell death; and

2. our study also provides the first evidence that NAD+ may be used for treating oxidative stress-mediated diseases

Can NAD+ be used in vivo to decrease brain injury in cerebral ischemia and other PARP-1 related diseases?

We used a rat model of transient focal ischemia to test our hypothesis that NAD+ administration can decrease ischemic brain damage.

3. Therapeutic potential of NAD+

A key problem for treatment of CNS diseases:William M. Pardridge. (2005) The Blood-Brain Barrier: Bottleneck in Brain Drug Development. NeuroRx. 2: 3–14.

A key challenge in establishing effective strategies for neuroprotection: Searching for drug delivery approaches that can overcome the limitations of BBB.

The Nose May Help the Brain --- Intranasal Drug Delivery for Treating Neurological Diseases

Ying W. (Editorial) Future Neurology

Intranasal Pathway (Slow process)

Olfactory bulb

Blood Pathway

Extracellular Pathway (Rapid process)

Drugs in Nasal Cavity

Gaps between the olfactory neurons

Trigeminal nerve

Olfactory bulb

BBB

CNS

Olfactory neurons in olfactory epithelium

Total

Infa

rct

Siz

e (

mm

3 )

0

50

100

150

200

250

300

350

IschemiaIschemia + GT

StriatumCortex

****

**

Total

Infa

rct

Siz

e (

mm

3 )

0

50

100

150

200

250

300

350

IschemiaIschemia + GT (i.v.)

StriatumCortex

Intranasal administration, but not intravenous administration, with the PARG inhibitor gallotannin,

decreased ischemic brain injury

0

50

100

150

200

250

300

NA

D

+ in

Bra

in S

lic

es

(% o

f c

on

tro

l)

*

Fig. 6. Acutely prepared brain slices were incubated in 10 mM NAD+ for 60 min. After 60 min the slices were washed 5 times with artificial CSF. For controls, the brain slices were incubated with NAD+ for 1 min only to control for non-specific binding, followed by 5 washes. NAD+ was determined by the cycling assay.

NAD+ treatment can increase intracellular NAD+

in a brain slice model

Ischemia Ischemia +

10 mg / kg NAD+

Intranasal administration with 10 mg / kg NAD+ at 2 hrs after ischemic onset can profoundly decreased infarct formation.

This treatment did not affect multiple major physiological parameters including temperature, blood pressure, pH etc.

Total Cortex StriatumInfa

rct

Vo

lum

e (m

m3 )

050100150200250300Ischemia+ 5 mg / kg NAD++ 10 mg / kg NAD++ 10 mg / kg NAm

***

**

*

0.5

1.0

1.5

2.0

2.5

3.0

3.5

4.0

Neu

rolo

gical D

eficits

Control

**

10 mg / kg NAD +

Intranasal NAD+ administration significantly decreased neurological deficits in rats subject to ischemia-reperfusion

What are the mechanisms underlying the protective effects of intranasal NAD+ administration against ischemic brain injury?

4 Hrs post-reperfusion

24 Hrs post-reperfusion

Ischemia

Ischemia + GT

AIF Nucleus Merge

Ischemia-reperfusion induced AIF translocation in rat brains

Can NAD+ be used for treating other PARP-1-associated diseases ?

Our latest study: Intranasal NAD+ delivery could decrease traumatic brain injury.

TBI

TBI + NAD+

Conclusions1) Intranasal NAD+ administration can significantly

decrease ischemic brain injury, suggesting that this might become a new strategy for reducing ischemic brain damage;

2) our study provides a useful tool for determining the roles of NAD+ metabolism in ischemic brain damage and other PARP-1-related diseases; and

3) future studies are needed to determine the mechanisms underlying the protective effects of intranasal NAD+ administration against ischemic brain injury, and to determine if this approach can decrease brain damage in other CNS diseases.

4. NADH transport across plasma membranes of cells

% C

ell D

eath

0

20

40

60

80

100

Con

MNNG

**

+ 5 M

NADH**

**

**+ 10

M NADH

+ 500 M

NADH

+ 1 mM

NADH

+ 100 M

NADH

50

75

100

125

150

175

Intracellular NADH (% of control) Control

**10

M NADH

**100

M NADH

1 mM

NADH10 m

M NADH

50

75

100

125

150

175

Intracellular NAD+

(% o

f co

ntr

ol)

Control

**10

M NADH

**100

M NADH

1 mM

NADH10 m

M NADH

****NADH treatment can increase intracellular

NADH levels in astrocytesNADH treatment can increase intracellular

NAD+ levels in astrocytes

020406080

100120140160

Intracellu

lar NA

D

+

(% o

f co

ntr

ol)

Control

**

NADH / 1 mM

PPADS

10 mM

NADH

**

Intrace

llular N

AD

+

(% o

f co

ntr

ol)

80

100

120

140

Control+ 10 mM NADH

P2X7 siRNAScrambled

** **

P2X7

-Actin

RNA silencing study in murine astrocytes

P2X7R

-Actin

-actin

1g plasmidNo plasmid

P2X7 Receptor

P2X7 Receptor

No Plasmids 1 g Plasmids

-actin

Intracellu

lar NA

D

+

(% o

f co

ntr

ol)

80

100

120

140

160

180

Control+ 10 mM NADH

mP2X7 Control

**

Control P2X7R

mP2X7R cDNA transfection studyTransfection of HEK293 cells with P2X7 receptors led

to increased NADH transport

Summary

1) We provided first evidence that NADH can decrease PARP-1 toxicity;

2) we provided the first evidence that NADH can be transported across the plasma membranes of astrocytes

5. Roles of Ca2+-Mg2+ -depenent endonuclease in cell death

PARG inhibition

PAR turnover

NAD+ depletion

PAR degradation

ADP-ribose

TRPM2 receptor opening

Calcium homeostasis

Cell death

PAR- CME

CME inhibition

DNA fragmentation

PAR- PARP-1

PARP-1

PARG inhibition may decrease genotoxic agent-induced cell death by multiple mechanisms

Post-treatment of the astrocytes with the CME inhibitor ATA abolished MNNG-induced chromatin condensation.

ATA post-treatment abolished MNNG-induced DNA fragmentation

ATA post-treatment, but not ATA pre-treatment, decreased MNNG-induced cell necrosis

Control 7.5 mM SIN-1 7.5 mM SIN-1 + 100 M ATA (2 hr)

Fig. 11. ATA post-treatment can decrease peroxynitrite-induced DNA damage. Astrocytes were exposed to 5 mM SIN-1 for 1 h. After washout of the drug, the cells were treated with 100 μM ATA or 25 μM DPQ for 2 h. Subsequently, DNA damage was assessed by Comet assay. The photomicrograph was taken in a randomly selected field (A). Quantification of the comet extend shows that treatment with ATA prevented SIN-1-induced increase in comet extend, while DPQ only minimally affected the DNA damage (B). Bar 1, Control; Bar 2, SIN-1; Bar 3, SIN-1 plus ATA; and Bar 4, SIN-1 plus DPQ. **p< 0.01; n = 3; data are representative of three independent experiments.

Fig. 13. Detection of the mRNA of Ca2+-Mg2+-dependent endonuclease in murine astrocytes and neurons. We conducted RT-PCRs using the primers for detecting the cDNA of Ca2+-Mg2+-dependent endonuclease in astrocytes or neurons. There was a single, distinct PCR product at approximately 250 bp.

Both astrocytes and neurons express CME

Summary

• Post-treatment with the CME inhibitor ATA can abolish genotoxic agent-induced DNA fragmentation and nuclear condensation; and

• CME may be an important target to decrease oxidative stress-induced nuclear alterations in multiple diseases

NAD+

ARTs

NADP+

ADP-R-P2X7R

NADHNADPH

ROS burst in phagocytes

Immunological functions

Treg cell death

NFB

PARP-1

CD38

cADPR

Cell signaling in immune cells

Cytokine release

NADPH oxidase


PARP-1

Nucleosomes,

Histones

Transcriptional factors (AP-1, NFB, p53)

RNA polymerase II

Chromatin compaction and de-condensation

NAD+

Sirtuins

DNA methylationPromoters

Gene expression


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