april 1, 2009 lech kiedrowski, ph.d. uic department of psychiatry [email protected]...
TRANSCRIPT
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April 1, 2009Lech Kiedrowski, Ph.D.UICDepartment of Psychiatry
Neuroprotective agents
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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.
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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
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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%
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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
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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
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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
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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.
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What happens when the blood supply to the brain is suddenly
interrupted?
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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 !
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Arch. Neurol. Psych. 50 (1943) 510-528
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The Kabat-Rossen-Anderson apparatus
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Dramatic Symptoms
Arch. Neurol. Psych. 50 (1943) 510-528
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7 seconds of brain ischemia will make you unconscious, but will not damage your brain
Arch. Neurol. Psych. 50 (1943) 510-528
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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.
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Sagital section through rat brain
Hippocampus
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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
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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
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What kills the CA1 neurons?
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The Pulsinelli et al. experiment
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Denervation protected the CA1 neurons from ischemic death
Pulsinelli (1985) Prog Brain Res 63: 29-37
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?
?
?
Death
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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
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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
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Glu
tam
ate Receptor
Death
?
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In cultured spinal neurons, glutamate deregulates Ca2+ homeostasis in a Ca-dependent manner
Tymianski et al. J. Neurosci. 13 (1993) 2085-2104
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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
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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
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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
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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
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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
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!
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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
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The data indicate that preventing zinc translocation, using CaEDTA,
prevents the ischemic death of CA1 neurons
What are the zinc-induced neurotoxic phenomena?
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Activation of Poly(ADP-ribose) polymerase-1 (PARP-1) may lead to neuronal death
Kauppinen and Swanson, Neuroscience 145 (2007) 1267-1272
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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
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Does PARP play a role in ischemic neuronal death?
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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
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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
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Is there a link between zinc exposure and PARP activation?
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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
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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)
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Kim et al. Exp Neurology 177 (2002) 407-418
The effect of zinc (15 min exposure) on PARP and neuronal death is dose-dependent
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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
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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
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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
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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?
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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
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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
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How could we protect the brain against ischemic damage?
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From Lee et al.,J. Neurosci. (2001) RC171
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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).
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Neuroprotection offered by intraperitoneal injection of pyruvate lasts up to 1 month after ischemia!
Lee et al., J. Neurosci. 21 (2001) RC171
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How does pyruvate work? Does it inhibit zinc neurotoxicity?
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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
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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
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Could pyruvate be used as a drug for stroke?
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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
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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
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Yi et al. Neurobiology of Disease 26 (2007) 94 - 104
Pyruvate protects against MCAO-elicited decline of motor skills
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How does pyruvate work?
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cytosol
mitochondria
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cytosol
mitochondria
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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
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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
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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
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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.