April 1, 2009Lech Kiedrowski, Ph.D.UICDepartment of Psychiatry

lkiedr@psych.uic.edu

Neuroprotective agents

Objectives

1. Learn about the mechanisms of neurodegeneration caused by brain ischemia (stroke, heart attack).

2. Learn about neuroprotective agents being developed to counteract ischemic brain damage.

Outline

1. High susceptibility of brain to ischemia

2. Mechanisms of ischemic neuronal death and the role of Ca2+ and Zn2+ in triggering these mechanisms

3. Agents developed to protect the brain from ischemic damage

American Heart Association 2009

Ischemic brain damage may occur after:

• Heart attack (global ischemia)

• Stroke (focal ischemia)– ischemic (occlusion of a blood vessel) 87%– hemorrhagic (bleeding in the brain) 13%

Heart attack and brain damage

• Brain damage can start to occur just 4-6 min after the heart stops pumping blood

• Survival rate is only 2% if heart is arrested for more than 12 min

Stroke and brain damage• About 795,000 cases each year• Every 40 seconds someone in the USA has a

stroke and every 3 min someone dies of it• Stroke is the third leading cause of death, after

heart disease and cancer• Stroke is the leading cause of long-term disability

(60% of survivors become handicapped)• The estimated direct and indirect cost of stroke

for 2009 is $68.9 billion.

American Heart Association 2009

Stroke and brain damage

• Lack of effective neuroprotective therapy

• The only FDA-approved therapy for stroke is intravenous injection of t-PA (Tissue Plasminogen Activator, a clot-dissolving agent)

• However, t-PA can only be applied during ischemic stroke and it must be applied during the first 3 hours of stroke

High energy requirements of the brain

The human brain constitutes only 2% of the body’s weight, yet it utilizes approximately 25% of total glucose and almost 20% of oxygen.

What happens when the blood supply to the brain is suddenly

interrupted?

Arch. Neurol. Psych. 50 (1943) 510-528

This was the first (fortunately the last) controlled investigation on the effects of acute arrest of the circulation to the human

brain

126 volunteers !

Arch. Neurol. Psych. 50 (1943) 510-528

The Kabat-Rossen-Anderson apparatus

Dramatic Symptoms

Arch. Neurol. Psych. 50 (1943) 510-528

7 seconds of brain ischemia will make you unconscious, but will not damage your brain

Arch. Neurol. Psych. 50 (1943) 510-528

Hansen, Acta Physiol. Scand. (1978) 102: 324-329.

EEG is flat within 10 sec of global brain ischemia

Ischemic depolarization (high elevation in external K+) takes place about 2 min after the onset of ischemia.

Sagital section through rat brain

Hippocampus

Selective vulnerability of CA1 neurons to ischemia

Yokota et al. Stroke (1995) 26: 1901-1907.

Sham operated

3 days after 10-min ischemia

7 days after 10-min ischemia

CA1 neurons dieCA3 and DG neurons survive

CA = Cornu Ammonis (Ammon’s horn)DG = Dentate Gyrus

DG

CA1

CA3

2 min of ischemia

3 min of ischemia

Kato et al. Brain Res. (1991) 553: 238-242.

Hippocampal CA1 regionin gerbil brain 7 days after ischemia

Ischemia has to last over 2 min to kill CA1 neurons

What kills the CA1 neurons?

The Pulsinelli et al. experiment

Denervation protected the CA1 neurons from ischemic death

Pulsinelli (1985) Prog Brain Res 63: 29-37

?

?

?

Death

10 20 30 10 20 10 20 30 60 120

Baseline Ischemia Reperfusion

Extracellular glutamate during ischemia and reperfusion

Glutamate (µM) sampled from various brain regions of the rat subjected to 20-min ischemia.

Globus et al. (1988) J Neurochem 51:1455-1464

Glutamate is neurotoxic

Olney, J.W., Brain lesions, obesity, and other disturbances in mice treated with monosodium glutamate. Science, 1969. 164: p. 719-721.

A single subcutaneous injection of glutamate (4 mg/g) produces brain lesions and kills 2 – 9 day-old mice within 1 to 48 hours

Glu

tam

ate Receptor

Death

?

In cultured spinal neurons, glutamate deregulates Ca2+ homeostasis in a Ca-dependent manner

Tymianski et al. J. Neurosci. 13 (1993) 2085-2104

Glutamate activates a number of receptors

Glutamate

NMDA AMPA(-Glu-R2)

AMPA(+Glu-R2)

Kainate

mGluRsgroup 1

mGluRsgroup 2 and 3

in

out

Ionotropic receptors

Metabotropic receptors

Some of these receptors are Ca-permeable channels

in

out

Glutamate

NMDA AMPA(-Glu-R2)

AMPA(+Glu-R2)

Kainate

mGluRsgroup 1

mGluRsgroup 2 and 3

IP3 cAMP

Na+ Ca2+ Na+ Na+Na+ Ca2+

K+ K+ K+ K+

NBQX NBQXNBQXMK-801

Some of these receptors are Ca-permeable channels

in

out

Glutamate

NMDA AMPA(-Glu-R2)

AMPA(+Glu-R2)

Kainate

mGluRsgroup 1

mGluRsgroup 2 and 3

IP3 cAMP

Na+ Ca2+ Na+ Na+Na+ Ca2+

K+ K+ K+ K+

NBQX NBQXNBQXMK-801

Tymianski et al. J. Neurosci. 13 (1993) 2085-2104

Ca2+ deregulation

Dead Neurons

Fra

ctio

n d

ere

gul

ate

d/d

ea

d

APV – NMDA receptor inhibitorCNQX – AMPA/kainate receptor inhibitorNIM – voltage-gated Ca channel inhibitor

Blocking NMDA receptors prevents glutamate-induced deregulation of Ca2+ homeostasis and neuronal death

Conclusion: Inhibiting NMDA receptors is sufficient to protect the neurons against glutamate-induced death

Failure of clinical stroke trials with glutamate receptor antagonist

Drugs Mode of action Result

Selfotel competitive NMDA antagonist trial discontinued Aptiganel noncompetitive NMDA antagonist adverse effectsMK-801 noncompetitive NMDA antagonist adverse effectsDextrorfan noncompetitive NMDA antagonist adverse effects

GV150526 glycine site antagonist of NMDA rec. no efficacy

Eliprodil polyamine site antagonist of NMDA rec. no efficacy

NBQX competitive AMPA receptor antagonist trial discontinuedadverse effectsrenal toxicity

Cerebrovasc. Dis. 11, suppl 1 (2001) 60-70

!

Zin

c-sp

ecifi

c flu

ores

cenc

e in

rat

hi

ppoc

ampu

s be

fore

isch

emia

CA

1 re

gion

3 d

ays

afte

r 10

-min

isch

emia

Zinc-specific fluorescence

Fuchsin staining (pink) of degenerating neurons

Koh et al. Science 272 (1996) 1013-1016

CaEDTA but not ZnEDTA protectsCA1 neurons against ischemic death

The role of zinc in ischemic neuronal death

The data indicate that preventing zinc translocation, using CaEDTA,

prevents the ischemic death of CA1 neurons

What are the zinc-induced neurotoxic phenomena?

Activation of Poly(ADP-ribose) polymerase-1 (PARP-1) may lead to neuronal death

Kauppinen and Swanson, Neuroscience 145 (2007) 1267-1272

Zhang et al. Science 263 (1994) 686 - 689

NO – nitric oxidePARS – Poly(ADP-ribose) synthetaseNAm – nicotinamideNMN – nicotinamide mononucleotidePRPP – phosphoribosyl pyrophosphatePPi – inorganic phosphate

PARP-1 (called also PARS)-mediated NAD- and ATP-depletion leads to cell death

Does PARP play a role in ischemic neuronal death?

Endres et al. J Cereb Blood Flow Metab 17 (1997) 1143-1151

PBS = phosphate buffered saline (control)3-AB = 3-aminobenzamide (PARP inhibitor)

PARP-1 knockout PARP-1 inhibition

PARP knockout or PARP inhibition reduce the size of ischemic brain infarct caused by the middle cerebral artery occlusion (MCAO)

PARP plays a role in ischemic brain infarct formation in vivo

Inhibition of PARP with 3-AB prevents the MCAO-induced NAD depletion

Endres et al. J Cereb Blood Flow Metab 17 (1997) 1143-1151

These data implicate that the mechanism of ischemic neuronal death in vivo involves PARP activation

Dark color represents NAD staining. This staining is decreased in the MCAO-affected territory

Is there a link between zinc exposure and PARP activation?

Exposure of cultured cortical neurons to toxic concentrations of zinc (400 µM) for 15 min activates PARP and causes

NAD and ATP depletion

ABAM = 3-aminobenzamide, 3-AB (PARP inhibitor) NAM = nicotinamide (another PARP inhibitor)PAR = Poly(ADP-ribose)

Kim et al. Exp Neurology 177 (2002) 407-418

PARP inhibitors prevent zinc-induced NAD-depletion and neuronal death

Kim et al. Exp Neurology 177 (2002) 407-418

ABAM = 3-aminobenzamide, 3-AB (PARP inhibitor) NAM = nicotinamide (another PARP inhibitor)LDH = lactate dehydrogenase (LDH release from cells is used as an index of cell death)

Kim et al. Exp Neurology 177 (2002) 407-418

The effect of zinc (15 min exposure) on PARP and neuronal death is dose-dependent

What about lower zinc concentrations?

The data presented indicate that a transient exposure of neuronal cell cultures to over 100 µM zinc activates a PARP-dependent mechanism of neuronal death

Prolonged exposure (24 hours) of neuronal cultures to low (<100 µM) concentrations of zinc is neurotoxic and involves

p75NTR and NADE activation

LDH = lactate dehydrogenase (LDH release from cells is used as an index of cell death) p75NTR = a nonselective low-affinity neurotrophin receptor belonging to TRK family; nerve growth factor (NGF) is an agonist of this receptorNADE = a 22 kDa cytosolic protein called p75NTR-associated death executorNeuN = neuron-specific nuclear protein (negative control)

Park et al. J Neurosci 20 (2000) 9096 - 9103

Exposure of neuronal cultures to 25 µM zinc for 24 hours is neurotoxic and involves a capase-dependent mechanism

AS#1 and #2 – NADE antisense #1 and #2 oligonucleotides used to knockdown NADENS – a nonsense oligonucleotide (negative control)

Note that both NADE knockdown and caspase inhibitors protect against zinc toxicity

Park et al. J Neurosci 20 (2000) 9096 - 9103

The data show PARP, caspases, p75NTR, and NADE being involved in zinc-induced toxicity in vitro

Do these mechanisms play a role in ischemic neuronal death in vivo?

p75NTR

Park et al. J Neurosci 20 (2000) 9096 - 9103

NADETUNEL

(staining of apoptotic cells)

Sham operated control

3 days after 15

min ischemia

3 days after 15

min ischemia

+ CaEDTA

Zinc chelation with CaEDTA prevents ischemic activation of p75NTR and NADE, and also prevents neurodegeneration

ApoptosisProgrammed Cell Death

Requires activation of caspases

NecrosisDoes not involve caspases

• Cells shrink • Chromatin becomes pyknotic (condensed)• Cytoplasmic organelles remain intact• Plasma membrane remains intact• Eventually nucleus and cytoplasm break into

apoptotic bodies that are phagocytized by macrophages or adjacent cells

• Cells swell • Only modest condensation of chromatin • Cytoplasmic vacuolization and breakdown of

organelles• Rupture of plasma membrane followed by

leakage of cellular content to the extracellular space

Two types on cell deathLow zinc

concentrationsHigh zinc

concentrations

How could we protect the brain against ischemic damage?

From Lee et al.,J. Neurosci. (2001) RC171

Lee et al. J. Neurosci. 21 (2001) RC171

CA1 region of the hippocampus 3 days after 12 min forebrain ischemia. TUNEL staining of apoptotic cells (green dots) and cresyl violet staining of surviving cells (insets). 30 min AFTER ischemia, the rats were injected (i.p.) with NaCl (A) or sodium pyruvate (500 mg/kg B).

Neuroprotection offered by intraperitoneal injection of pyruvate lasts up to 1 month after ischemia!

Lee et al., J. Neurosci. 21 (2001) RC171

How does pyruvate work? Does it inhibit zinc neurotoxicity?

Pyruvate protects cultured neurons against zinc toxicity better than a PARP inhibitor - niacinamide

Sheline et al. J Neurosci 20 (2000) 3139 - 3146

Neuronal cultures were exposed to 40 µM zinc for 24 h in the presence of the indicated concentrations of pyruvate or niacinamide

In neuronal cultures exposed to 40 µM zinc, pyruvate (4 mM) prevents ATP and NAD depletion

Sheline et al. J Neurosci 20 (2000) 3139 - 3146

NAD+ measure 4 h after the addition of zinc

Could pyruvate be used as a drug for stroke?

Transient MCAO (t-MCAO)

Yi et al. Neurobiology of Disease 26 (2007) 94 - 104

Permanent MCAO (p-MCAO)

Pyruvate applied 30 min after reperfusion Pyruvate applied 30 min after the p-MCAO onset

A delayed application of pyruvate treatment decreases the size of brain infarct after transient or permanent occlusion of

middle cerebral artery (MCAO)

Note that high doses of pyruvate (500 mg/kg) are not protective

Yi et al. Neurobiology of Disease 26 (2007) 94 - 104

Pyruvate (125 mg/kg) is effective even if applied 1h

after p-MCAO

The neuroprotective effect lasts for at least 2 weeks

after p-MCAO

and

Yi et al. Neurobiology of Disease 26 (2007) 94 - 104

Pyruvate protects against MCAO-elicited decline of motor skills

How does pyruvate work?

cytosol

mitochondria

cytosol

mitochondria

Zhang et al. Science 263 (1994) 686 - 689

NO – nitric oxidePARS – Poly(ADP-ribose) synthetaseNAm – nicotinamideNMN – nicotinamide mononucleotidePRPP – phosphoribosyl pyrophosphatePPi – inorganic phosphate

PARP-mediated NAD- and ATP-depletion leads to cell death

NO – nitric oxidePARS – Poly(ADP-ribose) synthetaseNAm – nicotinamideNMN – nicotinamide mononucleotidePRPP – phosphoribosyl pyrophosphatePPi – inorganic phosphate

By promoting ATP production in the mitochondria, pyruvate prevents NAD- and ATP-depletion, and promotes neuronal

survival

ATP Pyruvate

The future of neuroprotective therapy for stroke looks good

Pyruvate is emerging as the most promising neuroprotective agent As a natural metabolite of the glycolytic pathway, pyruvate is

unlikely to have major side effects Pyruvate is effective even if applied 1 h after the onset of

ischemia Unlike t-PA, pyruvate could be applied during ischemic and

hemorrhagic strokes Its application at the early stages of stroke (for example by

paramedics arriving to pick up a stroke victim) could extend the window of opportunity for t-PA application

Conclusion

Readings

De Keyser J., G. Sulter, & P. G. Luiten. (1999) Clinical trials with neuroprotective drugs in acute ischaemic stroke: are we doing the right thing? Trends Neurosci 22: 535-40.

Koh J.-Y., Suh S. W., Gwag B. J., He Y. Y., Hsu C. Y. and Choi D. W. (1996) The role of zinc in selective neuronal death after transient global cerebral ischemia. Science 272: 1013-1016.

Lee, J. Y., Y. H. Kim, & J. Y. Koh. (2001) Protection by pyruvate against transient forebrain ischemia in rats. J Neurosci 21: RC171.

Yi J. S., Kim T. Y., Kyu Kim D. and Koh J. Y. (2007) Systemic pyruvate administration markedly reduces infarcts and motor deficits in rat models of transient and permanent focal cerebral ischemia. Neurobiol Dis 26: 94-104.