April 4, 2007 L. Kiedrowski, Ph.D. UIC Department of Psychiatry [email protected]
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April 4, 2007 L. Kiedrowski, Ph.D. UIC Department of Psychiatry [email protected] Neuroprotective agents
description
April 4, 2007 L. Kiedrowski, Ph.D. UIC Department of Psychiatry [email protected]. Neuroprotective agents. Outline. Extreme vulnerability of brain to ischemia Early stages of brain ischemia Delayed neuronal death after ischemia Neuroprotective agents and strategies. - PowerPoint PPT Presentation
Transcript of April 4, 2007 L. Kiedrowski, Ph.D. UIC Department of Psychiatry [email protected]
April 4, 2007L. Kiedrowski, Ph.D.UICDepartment of Psychiatry
Neuroprotective agents
Outline
1. Extreme vulnerability of brain to ischemia2. Early stages of brain ischemia3. Delayed neuronal death after ischemia4. Neuroprotective agents and strategies
American Heart Association 2004
Ischemic brain damage may occur after: • Heart attack (global ischemia)• Stroke (focal ischemia)
– ischemic (occlusion of a blood vessel) 88%– hemorrhagic (bleeding in the brain) 12%
American Heart Association 2004
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• 600,000 new cases each year• Every 45 seconds someone in the USA has a stroke and
every 3 min someone dies of it• Stroke is the third leading cause of death, behind heart
disease and cancer• Stroke is the leading cause of long-term disability (60% of
survivors become handicapped)• In 2004, the overall cost of stroke-induced brain injury was
over $53.6 billion• Lack of effective neuroptotective agents• The only currently available therapy: intravenous injection
of t-PA (Tissue Plasminogen Activator, a clot-dissolving agent)
High energy requirements of the brain
• The human brain constitutes only 2% of the body weight, yet it utilizes approximately 25% of total glucose and almost 20% of oxygen.
Arch. Neurol. Psych. 50 (1943) 510-528
“This is the first controlled investigation on the effects of acute arrest of the circulation to the human brain.”
Arch. Neurol. Psych. 50 (1943) 510-528
Humans become unconscious within 7 seconds of brain ischemia
Where does the consciousness reside?
ILN – intralaminar nuclei of thalamus
MRF – mesencephalic reticular formation
Consciousness requires MRF activity
Permanent post-stroke coma results from bilateral lesions within MRF
from Hansen, (1978) Acta Physiol. Scand.
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 of rat brain
Hippocampus
Selective vulnerability of hippocampal CA1 neurons to ischemia
From 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
Ischemia
2 min
3 min
From Kato et al. Brain Res. (1991) 238-242
Hippocampal CA1 regionin gerbil brain 7 days after ischemia
Ischemia has to last over 2 min to kill CA1 neurons
Denervation protected CA1 neurons from ischemic death.
This indicates that CA1 neurons are damaged indirectly.
from 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.
From 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.
Gluta
mat
e Receptor
Death
?
In cultured spinal neurons, application of glutamate deregulates Ca2+ homeostasis. This deregulation depends
on extracellular Ca2+ concentration
From Tymianski et al. J. Neurosci. 13 (1993) 2085-2104
The data imply that Ca2+ homeostasis is deregulated by glutamate-induced Ca2+ influx
Two classes of glutamate receptors: ionotropic and metabotropic
Glutamate
NMDA AMPA(-Glu-R2)
AMPA(+Glu-R2)
Kainate
mGluRsgroup 1
mGluRsgroup 2 and 3
in
out
Ionotropic receptors
Metabotropic receptors
Some ionotropic glutamate receptors mediate Ca2+ influx
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+
MK-801 and NBQX inhibit NMDA and AMPA/kainate receptors, respectively
in
out
Glutamate
NMDA AMPA(-Glu-R2)
AMPA(+Glu-R2)
Kainate
mGluRsgroup 1
mGluRsgroup 2 and 3
IP3 cAMP
NBQXCNQX
NBQXCNQX
NBQXCNQX
MK-801APV
From Tymianski et al. J. Neurosci. 13 (1993) 2085-2104
Ca2+ deregulationDead Neurons
Frac
tion
dere
gula
ted/
dead
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 trials with glutamate receptor antagonist
Drugs Mode of action ResultSelfotel competitive NMDA antagonist trial discontinued Aptiganel noncompetitive NMDA antagonist adverse effectsMK-801 noncompetitive NMDA antagonist adverse effectsDextrorfan noncompetitive NMDA antagonist adverse effects Racemide noncompetitive NMDA antagonist phase III planed
GV150526 glycine site antagonist of NMDA rec. no efficacyEliprodil 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
!
What methodological problems?
Answer: Many in vitro studies underestimated the impact of early ischemic events on the mechanism of toxic Ca2+ influx.
What early events ?
1. Oxygen depletion2. Drop in pH3. Plasma membrane depolarization by K+ efflux4. Cytosolic [Na+] elevation
These events affect the mechanism by which glutamate kills neurons!
Early Stages of Global Brain IschemiaOxygen Depletion
From Halsey et al. Microvasc. Res. (1977)
In the presence of oxygen and glucose, Ca2+ accumulates in the mitochondria
Glucose
Pyruvate
Krebscycle
4NADH
Glycolysis
e-
2H2O
O2
H+ H+ H+
-180 mV
Ca2+Ca2+
Pi
Ca-phosphate
Glutamate
+
ATPCa-pump
ATP
endoplasmicreticulum
plasmalemma
mitochondrion
Pyruvate
Krebscycle
4NADH
Glycolysis
e-
-180 mV
Ca2+Ca2+
Pi
Ca-phosphate
Glutamate
+
Ca-pump
Lactate
Ca2+
In the absence of oxygen and glucose, Ca2+ accumulates in the cytosol
calpain
+
Mg2+ blocks NMDA channel
in
out NMDA rec.
Na+ Ca2+
GluMg
Na+ influx via AMPA channel depolarizes the plasma membrane and removes the Mg2+ block
AMPA rec.
Na+ Ca2+ Na+
Depolarization
GluGlu
in
out NMDA rec.
Many in vitro studies were performed in the absence of Mg2+.
Glucose
Pyruvate
Krebscycle
4NADH
Glycolysis
e-
2H2O
O2
H+ H+ H+
-180 mV
Ca2+Ca2+
Pi
Ca-phosphate
Glutamate
+
NMDA channel not blocked by Mg2+
Under Mg-free conditions, the Ca2+ influx via NMDA receptors is artificially enhanced.
Early events of brain ischemia
1. Oxygen depletion2. Drop in pH3. Plasma membrane depolarization by K+ efflux4. Cytosolic [Na+] elevation
Early Event of Global Brain Ischemia Low pH inhibits NMDA receptors
From Traynelis and Cull-Candy (1991)
NMDA rec.
AMPA rec.
Kainate rec.
From Siemkowicz and Hansen (1981)
min
Pyruvate Lactate
Mitochondria
Early events of brain ischemia
1. Oxygen depletion2. Drop in pH3. Plasma membrane depolarization by K+ efflux4. Cytosolic [Na+] elevation
Early Stages of Brain IschemiaANOXIC DEPOLARIZATION
100
50
10
3
mM
[K+]o
From Hansen (1978)
Depolarization of the plasma membrane due to [K+]o increase, reduces (by about 50 mV) the electrochemical driving force for Ca2+ influx via NMDA and other Ca-permeable channels.
Early events of brain ischemia
1. Oxygen depletion2. Drop in pH3. Plasma membrane depolarization by K+ efflux4. Cytosolic [Na+] elevation
Early Stages of Brain Ischemia
100
50
10
3
mM
[K+]o
From Hansen (1978)
17010050
5
.1.05
.51
mM
[Na+]o
[Ca2+]om
M
From Hansen and Zeuthen (1981)
How do the early ischemic ionic fluxes affect the plasmalemmal Na/Ca (NCX) and Na/Ca+K (NCKX) exchange operation?
in
out3Na+
K+Ca2+
4Na+
Ca2+
NCX
Forward mode
150 mM Na+ 5 mM K+
5 mM Na+ 150 mM K+
NCKX
Normal conditionsNCX and NCKX are electrogenic exchangers. The charge associated with Na+ transport exceeds that associated with Ca2+ transport.
Therefore, NCX and NCKX generate membrane potential and are affected by the existing membrane potential.
Normally, NCX and NCKX remove Ca2+ from the cytosol (forward mode to the exchange).
-65 mV
100
50
10
3
mM
[K+]o
From Hansen (1978)
17010050
5
.1.05
.51
mM
[Na+]o
[Ca2+]om
M
From Hansen and Zeuthen (1981)
Lets examine NCX and NCKX operation
before ischemiaafter anoxic depolarization
ENCX = 3ENa – 2ECa
Em = - 65 mV
Ion Out InNa+ 150 mM 5 mM Ca2+ 1300 M 0.1 M
ENCX = 3ENa – 2ECa = (3 x 90.9) – (2 x 126.6) = 272.7 – 253.2 = 19.5 mV
Em and ENCX before ischemia
ENa = 2.303 RT/F log ([Na]o/[Na]i) = 2.303 x 26.73 x log (150/5) = 61.56 x 1.477 = 90.9 mV ECa = 2.303 RT/2F log ([Ca]o/[Ca]i) = (2.303 x 26.73)/2 x log (1300/0.1) = 30.78 x 4.114 = 126.6 mV
Em (- 65 mV) < ENCX (19.5 mV) When Em < ENCX, NCX operates in the forward mode
ENCKX = 4ENa – 2ECa – EK
Em = - 65 mV
Ion Out InNa+ 150 mM 5 mM Ca2+ 1300 M 0.1 MK+ 5 mM 150 mM
ENCKX = 4ENa – 2ECa – EK = (4 x 90.9) – (2 x 126.6) – (– 90.9) = 201.3 mV
Em and ENCKX before ischemia
ENa = 2.303 RT/F log ([Na]o/[Na]i) = 2.303 x 26.73 x log (150/5) = 61.56 x 1.477 = 90.9 mV ECa = 2.303 RT/2F log ([Ca]o/[Ca]i) = (2.303 x 26.73)/2 x log (1300/0.1) = 30.78 x 4.114 = 126.6 mVEK = 2.303 RT/F log ([K]o/[K]i) = 2.303 x 26.73 x log (5/150) = 61.65 x -1.477 = - 90.9 mV
Em (- 65 mV) < ENCKX (201.3 mV)When Em < ENCKX, NCKX operates in the forward mode
ENCX = 3ENa – 2ECa
Em = - 15 mV
Ion Out InNa+ 50 mM 45 mM Ca2+ 130 M 1 M
ENCX = 3ENa – 2ECa = (3 x 2.82) – (2 x 65.1) = 8.46 – 130.2 = –121.7 mV
ENa = 2.303 RT/F log ([Na]o/[Na]i) = 2.303 x 26.73 x log (50/45) = 61.56 x 0.0458 = 2.82 mV ECa = 2.303 RT/2F log ([Ca]o/[Ca]i) = (2.303 x 26.73)/2 x log (130/1) = 30.78 x 2.114 = 65.1 mV
Em (–15 mV) > ENCX (–121.7 mV)When ENCX < Em, NCX operates in the reverse mode
Em and ENCX after anoxic depolarization
ENCKX = 4ENa – 2ECa – EK
Em = - 15 mV
Ion Out InNa+ 50 mM 45 mM Ca2+ 130 M 1 MK+ 65 mM 120 mM
ENCKX = 4ENa – 2ECa – EK = (4 x 2.82) – (2 x 65.1) – (– 16.4) = – 102.5 mV
ENa = 2.303 RT/F log ([Na]o/[Na]i) = 2.303 x 26.73 x log (50/45) = 61.56 x 0.0458 = 2.82 mV ECa = 2.303 RT/2F log ([Ca]o/[Ca]i) = (2.303 x 26.73)/2 x log (130/1) = 30.78 x 2.114 = 65.1 mVEK = 2.303 RT/F log ([K]o/[K]i) = 2.303 x 26.73 x log (65/120) = 61.65 x -0.266 = -16.4 mV
Em (–15 mV) > ENCKX (–102.5 mV)When ENCKX < Em, NCKX operates in the reverse mode
Em and ENCKX after anoxic depolarization
NCX and NCKX reverse during anoxic depolarization
in
out3Na+
K+Ca2+
4Na+
Ca2+
NCX
Forward mode
150 mM Na+ 5 mM K+
5 mM Na+ 150 mM K+
NCKX
Normal conditions
3Na+
K+Ca2+
4Na+
Ca2+
NCX
Reverse mode
50 mM Na+ >65 mM K+
45 mM Na+ 120 mM K+
NCKX
in
out
Anoxic depolarization
How high do NCX and NCKX elevate [Ca2+]c?
ENCKX = 4ENa – 2ECa – EK
ENCX = 3ENa – 2ECa
When Em > ENCKX , NCKX reversesWhen Em > ENCX , NCX reverses
ENa = RT/F ln ([Na]o/[Na]i)EK = RT/F ln ([K]o/[K]i) ECa = RT/2F ln ([Ca]o/[Ca]i)
0 10 20 30 40 50 60 70 80
EN
CX o
r EN
CK
X (m
V)
-200
-150
-100
-50
0
[Ca2+]i (µM)
Em = -15 mV
NCKX NCX
Forward
Reverse
Ion Out InNa+ 50 mM 45 mM Ca2+ 130 M x MK+ 65 mM 120 mM
Very shortly after the onset of ischemia, in hippocampal CA1 neurons in vivo, [Ca2+]i elevates to ~50 M
From Silver and Erecinska, J. Gen. Physiol. (1990)
2 min
Krebscycle
4NADHe-
Glutamate
+
SEA0400 is a potently inhibits NCX1 reversal
AMPA/kainate rec.
+
Na+
Ca2+
K+
Ca2+
NCX
NCKX
SEA0400
NMDA rec.
SEA0400
Comparison of effects of SEA0400 versus MK-801 on ischemic brain damage
The drugs were applied i.p. immediately after occlusion of the middle cerebral artery from Matsuda et al. J Pharmacol Exp Therap 298 (2001) 249-256
cerebral cortex striatum
Krebscycle
4NADHe-
-180 mVPi
Ca-phosphate
Glutamate
+
NCKX reversal inhibitors have not yet been developed
NMDA rec.
AMPA/kainate rec.
+
Na+
Ca2+
K+
Ca2+
NCX
NCKX
SEA0400
No inhibitor available
NCKX mRNA expression in the rat brain
Krebscycle
4NADHe-
Glutamate
+
Elevation in cytosolic [Ca2+] activates calpain
AMPA/kainate rec.
+
Na+
Ca2+
K+
Ca2+
NCX
NCKX
NMDA rec.
Calpain
Calpain
What is calpain?1. Calpains are cytosolic Ca-dependent proteases.2. Two calpain isoforms: µ-calpain and m-calpain
are expressed in the brain. 3. µ-calpain and m-calpain are activated by
micromolar and milimolar concentrations of Ca2+, respectively.
Does calpain-mediated proteolysis play a role in ischemic brain damage?
After 10-min of global ischemia, calpain is specifically activated in the hippocampal CA1 region
Sham operated 1 day
15 min
12 hours
3 days
7 days
From Yokota et al. (1995) Stroke, 26: 1901-1907.
Stained by a specific antibody is the degraded spectrin, a product of calpain proteolysis.
Calpain inhibitor (ALLNaI) prevents ischemia-induced calpain activation
Sham operated
Control Ischemia Ischemia + ALLNaI
4 hours
7 days
From Yokota et al. (1999) Brain Res, 819: 8-14.
ALLNaI = N-acetyl-leucyl-leucyl-norleucile amide
ALLNI was entrapped in lyposomes and injected i.v. 30 min before ischemia.
Lyposomes are artificially made phospholipid vesicles. They help ALLNaI to cross the blood-brain barrier.
Note that ALLNaI prevents calpain activation (dark staining shows calpain-dependent degradation of spectrin).
Calpain inhibition protects CA1 neurons from ischemic death
From Yokota et al. (1999) Brain Res, 819: 8-14.
From Lee et al.,J. Neurosci. (2001) RC171
From Lee et al., J. Neurosci. (2001) RC171
30 min after 12 min forebrain ischemia, the rats were injected (i.p.) with NaCl (A) or sodium pyruvate (500 mg/kg (B). Shown is CA1 region of the hippocampus 3 days later. Green staining - dying (apoptotic) cells. In the insets, cresyl violet staining of surviving cells.
From Lee et al., J. Neurosci. (2001) RC171
Neuroprotection offered by intraperitoneal injection of pyruvate lasts up to 1 month after ischemia!
cytosol
mitochondria
How does pyruvate rescue ischemic neurons?
ATP is a necessary substrate for glycolytic reactions catalysed by hexokinase and phosphofructokinase. If cells are deprived of ATP, hexokinase and phosphofructokinase cannot work.
Pyruvate, applied at early stages of reperfusion, facilitates mitochondiral ATP production.
The mitochondrial ATP is then used to re-activate hexokinase and phosphofructokinase, the first two stages of glycolysis.
We may try to: Generate potent inhibitors of NCKX and NCX reversal. Generate potent inhibitors of calpain. Prevent NCKX and NCX reversal. Inhibit calpain activation. Apply pyruvate after ischemia.
What can we do to improve the outcome of brain ischemia?
Despite the failure of all phase III clinical trials conducted so far, the future of neuroprotective therapy is promising.
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.
Lee, J.M., G.J. Zipfel, D.W. Choi, The changing landscape of ischaemic brain injury mechanisms. (1999) Nature, 399: A7-14.
Lee, J. Y., Y. H. Kim, & J. Y. Koh. (2001) Protection by pyruvate against transient forebrain ischemia in rats. J Neurosci 21: RC171.
