Neuroprotective Effects of Memantine

1
Neuroprotective Effects of Memantine
2
Memantine in an In Vitro Model for
Neurodegeneration
Semi-chronic 3-NP toxicity in organotypic
hippocampal cultures
Hippocampal slice cultures
Brown et al., Soc. Neurosci 2003
3
Neuroprotective Effect of MemantineIn Vitro
Semi-chronic 3-NP toxicity in organotypic
hippocampal cultures
300 200 100 0
3-NP for 7 days
GluR1 (arbitary units)
Control
10 µM
1 µM
5 µM Memantine
Vehicle
Brown et al., Soc. Neurosci 2003
4
Neuroprotective Effect of MemantineIn Vitro
Semi-chronic 3-NP toxicity in organotypic
hippocampal cultures
3-NP for 7 days
300 200 100 0
Synapsin II b (arbitary units)
Control
10 µM
1 µM
5 µM Memantine
Vehicle
Brown et al., Soc. Neurosci 2003
5
Neuroprotective Effect of MemantineIn Vitro
Semi-chronic 3-NP toxicity in organotypic
hippocampal cultures
Control
3-NP 14 days
3-NP Memantine
CA1
DG
Brown et al., Soc. Neurosci 2003
6
Memantine in an Animal Model for
Neurodegenerative Dementia
Memantines effect on lesions of the nucleus
basalis magnocellularis (NBM)
Memantine injection 30 min before NMDA
Memantine infusion for 2 weeks
Unilateral injection of NMDA (7.5 nmol) or 3NP
(250 nmol) into the NBM
or
Wenk et al., Eur J Pharmacol 1995, NeuroReport
1996
7
Protection of Cholinergic Neurons by Memantine
Memantine injection (i.p.) attenuated
NMDA-induced lesion of the NBM
8 6 4 2 0
? Memantine (ED50 2.8 mg/kg) ? MK-801 (ED50
0.077 mg/kg)
ChAT activity control-lesioned side (nmol
ACh/hmg proteine)
1
0.01
0.1
10
100
1000
Dose (mg/kg)
Wenk et al., Eur J Pharmacol 1995
8
Degeneration of Cholinergic Neurons was
Attenuated by Memantine
Infusion of memantine attenuated damage to NBM
neurones induced by injection of NMDA or 3-NP
0 -4 -8 -12 -16 -20

Cortical ChAT activity control-lesioned side
(nmol ACh/hmg proteine)
? Memantine (20 mg/day) ? Control

3-NP lesion
NMDA lesion
p lt 0.01 versus control
Wenk et al., NeuroReport 1996
9
Memantine in an Animal Model for Alzheimers
Disease
Memantines effect on ß-amyloid-induced lesion of
the hippocampus
Memantine infusion (20 mg/kg/day)
Miguel-Hidalgo et al., Brain Res 2002
10
Protection by Memantine Against Aß-Induced
Neurodegeneration
Extent of ß-amyloid-induced damage in the CA1
region
1,000 800 600 400 200 0
Vehicle
Extent (µm)

Vehicle
Memantine
Memantine
p lt 0.02 versus placebo
Miguel-Hidalgo et al., Brain Res 2002
11
Protection by Memantine Against A?-Induced
Neurodegeneration
Area of GFAP profiles around the injection site
30 25 20 15 10 5 0

Area ()
Vehicle
Memantine
Vehicle
Memantine
p lt 0.03 versus vehicle
Miguel-Hidalgo et al., Brain Res 2002
12
Effect of Memantine on Inflammation Induced
Neurodegeneration
Memantine effect on lipopolysaccharide
(LPS)-induced brain insult and inflammation
Memantine infusion (s.c. 20 mg/kg/day for 37
days)
Infusion of LPS into the basal forebrain (37 days)
Willard et al., Exp Brain Res 2000
13
Memantine Protected Cholinergic Neurons from
Damage by Inflammation
Effect of LPS infusion into the basal forebrain
on cortical ChAT activity
5 0 -5 -10 -15 -20 -25
Decline in cortical ChAT activity ()


Control
LPS
LPS Memantine
p lt 0.0001 versus control p lt 0.05 versus
LPS
Willard et al., Exp Brain Res 2000
14
Memantine in an Animal Model for
Neurodegenerative Dementia
Effects of memantine on quinolinic acid-induced
neurodegeneration
Misztal et al., Eur J Pharmacol 1996
15
Attenuation of Quinolinic Acid Induced Memory
Loss by Memantine
Infusion of Memantine (20 mg/kg/day) attenuated
T-maze learning deficit induced by chronic i.c.v.
infusion of quinolinic acid (QA)
4 3 2 1 0
? Control ? QA ? QA Memantine





Number of errors
1
5
3
2
4
Day
p lt 0.05 versus control and QA Memantine
Misztal et al., Eur J Pharmacol 1996
16
Attenuation of Quinolinic Acid Induced
Neurodegeneration by Memantine
Memantine (20 mg/kg/day) attenuated the
hippocampal cholinergic deficit induced by
chronic i.c.v. infusion of quinolinic acid (QA)
100 80 60 40 20 0


H3Hemicholinium-3 binding (µmol/mg tissue)
Control
QA
QA Memantine
p lt 0.05 versus quinolinic acid
Misztal et al., Eur J Pharmacol 1996
17
Summary

• Neuroprotective effects of memantine were shown,
• in vivo on
• Excitotoxic induced neurodegeneration
• ß-amyloid induced neuronal damage
• LPS induced inflammation
• in vitro on
• Metabolic disturbances due to mitochondrial
  dysfunction
• Conclusion
• Neuroprotective effects of memantine have been
  shown under various conditions which are
  clinically relevant for Alzheimers disease

18
Memantine Inhibits and Reverses the Alzheimer
Type Abnormal Hyperphosphorylation of tau and
Associated Neurodegeneration

• Li L., Sengupta A., Haque N., Grundke-Iqbal I.
  and Iqbal K.
• FEBS Letters, 2004, 566 (13)261269

19
Tau Hyperphosphorylation in Alzheimers Disease
In vitro model
Alzheimers disease
Hippocampal culture okadaic acid (OA) PP-2A
activity ? CaMKII activity ? PKA activity
? Hyperphosphorylation of tau
Therapeutic approach
Hyperphosphorylation of tau Tangle
formation Neurodegeneration
20
Effects of Memantine on Phosphorylation of
tau-Methods
Okadaic acid for 24 h
Memantine or vehicle for 24 h
Analysis

• Assay for phosphatase- or kinase activity
• Assay for cell death
• Western blots (p-tau)

Hippocampal slices
21
Memantine Counteracted OA-induced PP-2A
Inhibition
PP-2A activity
120 100 80 60 40 20 0

PP-2A activity ( of control)
? 24 h OA 24 h vehicle ? 24 h OA 24 h
Mem ? 24 h Mem
100 100 100 0 0
nM OA
0 1 10 1 10
µM Mem
p lt 0.05 versus OA treated tissue
22
Memantine Restored CaMKII and PKA Activity
CaMKII activity
PKA activity
250 200 150 100 50 0
120 100 80 60 40 20 0
? 24 h OA 24 h vehicle ? 24 h OA 24 h
Mem ? 24 h Mem


Kinase activity ( of control)

100 100 100 0 0
nM OA
100 100 100 0 0
0 1 10 1 10
µM Mem
0 1 10 1 10
p lt 0.05 versus OA treated tissue
23
Memantine Counteracted OA-induced Cell Death
Cell death assay
10 8 6 4 2 0
LDH release (ratio after/before treatment)
? Control ? 24 h OA 24 h vehicle ? 24 h OA 24
h Mem ? 24 h Mem


0 100 100 100 0 0
nM OA
0 0 1 10 1 10
µM Mem
p lt 0.05 versus OA treated tissue
24
Memantine Counteracted CaMKII-induced
Phosphorylation of tau
? Control ? 24 h OA 24 h vehicle ? 24 h OA 24
h Mem (10 µM)
7 6 5 4 3 2 1 0
125I Western blot with Antibody against pS-262
1 2 3
Phosphorylation of tau
68
43
pSer262
pSer422
10 nM OA

10 µM Mem

25
Memantine Counteracted OA-induced Phosphorylation
of tau
Immunocytochemical staining of pSer-262
100 nM OA 10 µM Mem
100 nM OA 10 µM Mem
26
Summary

• In an in vitro model using okadaic acid memantine
  was shown to
• Restore normal PP-2A, CaMKII and PKA activities
• Prevent cell death
• Positively influence phosphorylation/dephosphoryla
  tion imbalance
• Conclusion
• Apart from the symptomatic benefits, memantine
  might also positively influence pathological
  changes in AD