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April 1, 2009 L ech Kiedrowski, Ph.D. UIC Department of Psychiatry [email protected] Neuroprotective agents. Objectives. Learn about the mechanisms of neurodegeneration caused by brain ischemia (stroke, heart attack).

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slide1
April 1, 2009

Lech Kiedrowski, Ph.D.

UIC

Department of Psychiatry

[email protected]

Neuroprotective agents

objectives
Objectives
  • Learn about the mechanisms of neurodegeneration caused by brain ischemia (stroke, heart attack).
  • Learn about neuroprotective agents being developed to counteract ischemic brain damage.
outline
Outline
  • High susceptibility of brain to ischemia
  • Mechanisms of ischemic neuronal death and the role of Ca2+ and Zn2+ in triggering these mechanisms
  • Agents developed to protect the brain from ischemic damage
ischemic brain damage may occur after
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%

American Heart Association 2009

heart attack and brain damage
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
Stroke and brain damage
  • About795,000 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 damage1
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
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.

slide10
126 volunteers !

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

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

slide13
Dramatic Symptoms

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

slide14
7 seconds of brain ischemia will make you unconscious,

but will not damage your brain

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

slide15
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.

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

slide17
Selective vulnerability of CA1 neurons to ischemia

CA1

CA = Cornu Ammonis (Ammon’s horn)

DG = Dentate Gyrus

Sham operated

DG

CA3

CA1 neurons die

CA3 and DG neurons survive

3 days after

10-min ischemia

7 days after

10-min ischemia

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

slide18
Ischemia has to last over 2 min to kill CA1 neurons

2 min of ischemia

3 min of ischemia

Hippocampal CA1 region

in gerbil brain 7 days after ischemia

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

slide21
Denervation protected the CA1 neurons from ischemic death

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

slide22
?

?

?

Death

slide23
Extracellular glutamate during ischemia and reperfusion

Baseline Ischemia Reperfusion

10 20 30 10 2010 20 30 60 120

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

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

slide24
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

slide25
Receptor

Glutamate

?

Death

slide26
In cultured spinal neurons, glutamate deregulates Ca2+ homeostasis in a Ca-dependent manner

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

slide27
Ionotropic receptors

Metabotropic receptors

Glutamate activates a number of receptors

Glutamate

AMPA

(-Glu-R2)

AMPA

(+Glu-R2)

NMDA

Kainate

mGluRs

group 1

mGluRs

group 2 and 3

out

in

slide28
MK-801

NBQX

NBQX

NBQX

Some of these receptors are Ca-permeable channels

Glutamate

AMPA

(-Glu-R2)

AMPA

(+Glu-R2)

NMDA

Kainate

mGluRs

group 1

mGluRs

group 2 and 3

Na+ Ca2+

Na+ Ca2+

Na+

Na+

out

in

IP3

K+

K+

K+

K+

cAMP

slide29
MK-801

NBQX

NBQX

NBQX

Some of these receptors are Ca-permeable channels

Glutamate

AMPA

(-Glu-R2)

AMPA

(+Glu-R2)

NMDA

Kainate

mGluRs

group 1

mGluRs

group 2 and 3

Na+ Ca2+

Na+ Ca2+

Na+

Na+

out

in

IP3

K+

K+

K+

K+

cAMP

slide30
Blocking NMDA receptors prevents glutamate-induced deregulation of Ca2+ homeostasis and neuronal death

Ca2+ deregulation

Dead Neurons

Fraction deregulated/dead

APV – NMDA receptor inhibitor

CNQX – AMPA/kainate receptor inhibitor

NIM – voltage-gated Ca channel inhibitor

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

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

slide31
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 effects

MK-801 noncompetitive NMDA antagonist adverse effects

Dextrorfan 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 discontinued

adverse effects

renal toxicity

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

slide33
CaEDTA but not ZnEDTA protects

CA1 neurons against ischemic death

The role of zinc in ischemic neuronal death

Zinc-specific fluorescence in rat hippocampus before ischemia

CA1 region 3 days after 10-min ischemia

Zinc-specific

fluorescence

Fuchsin staining (pink)

of degenerating neurons

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

slide34
The data indicate that preventing zinc translocation,

using CaEDTA,

prevents the ischemic death of CA1 neurons

What are the zinc-induced neurotoxic phenomena?

slide35
Activation of Poly(ADP-ribose) polymerase-1 (PARP-1) may lead to neuronal death

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

slide36
PARP-1 (called also PARS)-mediated NAD- and ATP-depletion leads to cell death

NO – nitric oxide

PARS – Poly(ADP-ribose) synthetase

NAm – nicotinamide

NMN – nicotinamide mononucleotide

PRPP – phosphoribosyl pyrophosphate

PPi – inorganic phosphate

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

slide38
PARP plays a role in ischemic brain infarct formation in vivo

PARP-1 knockout

PARP-1 inhibition

PBS = phosphate buffered saline (control)

3-AB = 3-aminobenzamide (PARP inhibitor)

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

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

slide39
Inhibition of PARP with 3-AB prevents the MCAO-induced NAD depletion

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

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

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

slide41
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

slide42
PARP inhibitors prevent zinc-induced NAD-depletion and neuronal death

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

slide43
The effect of zinc (15 min exposure) on PARP and neuronal death is dose-dependent

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

slide44
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

What about lower zinc concentrations?

slide45
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 receptor

NADE = a 22 kDa cytosolic protein called p75NTR-associated death executor

NeuN = neuron-specific nuclear protein (negative control)

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

slide46
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 NADE

NS – 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

slide47
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?

slide48
Zinc chelation with CaEDTA prevents ischemic activation of p75NTR and NADE,

and also prevents neurodegeneration

TUNEL

(staining of apoptotic cells)

p75NTR

NADE

Sham operated control

3 days after 15 min ischemia

3 days after 15 min ischemia

+ CaEDTA

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

slide49
Two types on cell death

Low zinc

concentrations

High zinc

concentrations

Apoptosis

Programmed Cell Death

Requires activation of caspases

Necrosis

Does not involve caspases

  • 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
  • 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
slide51
From Lee et al.,

J. Neurosci. (2001)

RC171

slide52
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).

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

slide53
Neuroprotection offered by intraperitoneal injection of pyruvate lasts up to 1 month after ischemia!

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

slide54
How does pyruvate work?

Does it inhibit zinc neurotoxicity?

slide55
Pyruvate protects cultured neurons against zinc toxicity better than a PARP inhibitor - niacinamide

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

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

slide56
In neuronal cultures exposed to 40 µM zinc, pyruvate (4 mM) prevents ATP and NAD depletion

NAD+ measure 4 h after the addition of zinc

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

slide59
A delayed application of pyruvate treatment decreases the size of brain infarct after transient or permanent occlusion of middle cerebral artery (MCAO)

Transient MCAO (t-MCAO)

Permanent MCAO (p-MCAO)

Pyruvate applied 30 min after reperfusion

Pyruvate applied 30 min after the p-MCAO onset

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

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

slide60
The neuroprotective effect lasts for at least 2 weeks after p-MCAO

Pyruvate (125 mg/kg) is effective even if applied 1h after p-MCAO

and

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

slide61
Pyruvate protects against MCAO-elicited decline of motor skills

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

slide63
cytosol

mitochondria

a

a

slide64
cytosol

mitochondria

a

a

slide65
PARP-mediated NAD- and ATP-depletion leads to cell death

NO – nitric oxide

PARS – Poly(ADP-ribose) synthetase

NAm – nicotinamide

NMN – nicotinamide mononucleotide

PRPP – phosphoribosyl pyrophosphate

PPi – inorganic phosphate

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

slide66
By promoting ATP production in the mitochondria, pyruvate prevents NAD- and ATP-depletion, and promotes neuronal survival

NO – nitric oxide

PARS – Poly(ADP-ribose) synthetase

NAm – nicotinamide

NMN – nicotinamide mononucleotide

PRPP – phosphoribosyl pyrophosphate

PPi – inorganic phosphate

ATP

Pyruvate

slide67
Conclusion

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
readings
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.

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