Neuroprotective Effects of Memantine

Neuroprotective Effects of

Memantine

Hippocampal slice cultures

Brown et al., Soc. Neurosci 2003

Semi-chronic 3-NP toxicity in organotypic hippocampal cultures

Parallel incubation of 3-NP and memantine for 7 or 12 days

Cresyl violet staining

Homogenization Immunoblot Incubation with

synaptic markers

Memantine in an In Vitro Model for Neurodegeneration

Semi-chronic 3-NP toxicity in organotypic hippocampal cultures

Brown et al., Soc. Neurosci 2003

3-NP for 7 days300

200

100

0Control 10 µM1 µM 5 µM

MemantineVehicle

Glu

R1

(a

rbit

ary

un

its

)Neuroprotective Effect of Memantine

In Vitro

Semi-chronic 3-NP toxicity in organotypic hippocampal cultures

Brown et al., Soc. Neurosci 2003

3-NP for 7 days300

200

100

0VehicleControl 10 µM1 µM 5 µM

Memantine

Sy

na

ps

in II

b (

arb

itar

y u

nit

s)

Neuroprotective Effect of MemantineIn Vitro

CA1

Control 3-NP 14 days 3-NP + Memantine

DG

Semi-chronic 3-NP toxicity in organotypic hippocampal cultures

Brown et al., Soc. Neurosci 2003

Neuroprotective Effect of MemantineIn Vitro

orMemantine injection 30 min before NMDA

Memantine infusion for 2 weeks

Unilateral injection of NMDA (7.5 nmol) or 3NP (250 nmol) into the NBM

Biochemical assay

Choline acetyltransferase in the frontal cortex

2 weeks

Wenk et al., Eur J Pharmacol 1995, NeuroReport 1996

Memantine‘s effect on lesions of the nucleus basalis magnocellularis (NBM)

Memantine in an Animal Model for Neurodegenerative Dementia

Ch

AT

act

ivit

y c

on

tro

l - le

sio

ne

d s

ide

(n

mo

l AC

h/h

*mg

pro

tein

e)

Wenk et al., Eur J Pharmacol 1995

8

6

4

2

0

10.01 0.1 10 100 1000

Memantine (ED50 = 2.8 mg/kg)

MK-801 (ED50 = 0.077 mg/kg)

Dose (mg/kg)

Memantine injection (i.p.) attenuated NMDA-induced lesion of the NBM

Protection of Cholinergic Neurons by Memantine

Wenk et al., NeuroReport 1996

Co

rtic

al C

hA

T a

cti

vit

y c

on

tro

l - le

sio

ne

d s

ide

(n

mo

l AC

h/h

*mg

pro

tein

e)

* p < 0.01 versus control

3-NPlesion

NMDAlesion

0

-4

-8

-12

-16

-20

*

* Memantine

(20 mg/kg/d)

Control

Infusion of memantine attenuated damage to NBM neurones induced by injection of NMDA or 3-NP

Degeneration of Cholinergic Neurons was Attenuated by Memantine

Memantine infusion(20 mg/kg/day)

7 days Immunohistochemistry in the hippocampus:

neuronal damage

GFAP

Miguel-Hidalgo et al., Brain Res 2002

Injection of ß-amyloid (1-40)into the hippocampus

2 days

Memantine‘s effect on ß-amyloid-induced lesion of the hippocampus

Memantine in an Animal Model for Alzheimer´s Disease

Memantine

Placebo Extent of ß-amyloid-induced damage in the CA1 region

1000

800

600

400

200

0

Ex

ten

t (µ

m)

Vehicle Memantine

*

Miguel-Hidalgo et al., Brain Res 2002

* p < 0.02 versus placebo

Protection by Memantine Against A-Induced Neurodegeneration

Vehicle

Memantine

Miguel-Hidalgo et al., Brain Res 2002

* p < 0.03 versus vehicle

Are

a (

%)

Protection by Memantine Against A-Induced Neurodegeneration

Area of GFAP profilesaround the injection site

30

25

20

15

10

5

0

Vehicle Memantine

*

Vehicle Memantine

Memantine effect on lipopolysaccharide (LPS)-induced brain insult and inflammation

Memantine infusion(s.c. 20 mg/kg/day for 37 days)

Infusion of LPSinto the basal forebrain (37 days)

Willard et al., Exp Brain Res 2000

Biochemical analysis in the frontal cortex:

ChAT activity

10 days

Effect of Memantine on Inflammation Induced Neurodegeneration

* p < 0.0001 versus control; ** p < 0.05 versus LPS

Willard et al., Exp Brain Res 2000

Effect of LPS infusion into the basal forebrain on cortical ChAT activity

5

0

-5

-10

-15

-20

-25

**

Control LPS LPS + Memantine

*De

clin

e in

co

rtic

al C

hA

T

act

ivit

y [%

]Memantine Protected Cholinergic

Neurons from Damage by Inflammation

Effects of memantine on quinolinic acid-induced neurodegeneration

15 sec

10 trials/day5 days

T-maze alternation

Biochemicalanalysis

Removal of minipumps

3 days

Parallel infusion of memantine (s.c. 20 mg/kg/day) and

quinolinic acid (i.c.v.)

2 weeks

Misztal et al., Eur J Pharmacol 1996

Memantine in an Animal Model for Neurodegenerative Dementia

Infusion of Memantine (20 mg/kg/day) attenuated T-maze learning deficit induced by chronic i.c.v. infusion of quinolinic acid (QA)

Day

Misztal et al., Eur J Pharmacol 1996

* p < 0.05 versus control and QA + Memantine

Control QA QA + Memantine

4

3

2

1

01 532 4

*

* **

*

Nu

mb

er

of

erro

rsAttenuation of Quinolinic Acid Induced

Memory Loss by Memantine

Memantine (20 mg/kg/day) attenuated the hippocampal cholinergic deficit induced by chronic i.c.v. infusion of quinolinic acid (QA)

* p < 0.05 versus quinolinic acid

**

100

80

60

40

20

0Control QA QA + Memantine

Misztal et al., Eur J Pharmacol 1996

[H3]H

em

ich

olin

ium

-3

bin

din

g (

µm

ol/m

g t

iss

ue)

Attenuation of Quinolinic Acid Induced Neurodegeneration by Memantine

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 Alzheimer's

disease.

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 (1-3): 261-269

Tau Hyperphosphorylation in Alzheimer‘s Disease

Alzheimer‘s disease In vitro model

Hippocampal culture

+ okadaic acid (OA)

PP-2A activity

CaMKII activity

PKA activity

Hyperphosphorylation

of tau

Therapeutic approach

Hyperphosphorylation

of tau

Tangle formation

Neurodegeneration

Okadaic acid for 24h Memantine or vehicle for 24h

Hippocampal slices

Analysis

Effects of Memantine on Phosphorylation of tau - Methods

Assay for phosphatase- or kinase activity

Western blots (p-tau)

Assay for cell death

Memantine Counteracted OA-induced PP-2A Inhibition

100 100 100 0 0

PP-2A activity

0 1 10 1 10

PP

-2A

ac

tivi

ty (

% o

f c

on

tro

l)

24 h OA + 24 h vehicle 24 h OA + 24 h Mem 24 h Mem

* p < 0.05 versus OA treated tissue

120

100

80

60

40

20

0nM OA

µM Mem

*

CaMKII activity PKA activity

Memantine Restored CaMKII and PKA Activity

Kin

ase

ac

tiv

ity

( %

of

co

ntr

ol)

* p < 0.05 versus OA treated tissue

250

200

150

100

50

0

100 100 100 0 0

120

100

80

60

40

20

0

0 1 10 1 10

nM OA µM Mem

100 100 100 0 0 0 1 10 1 10

24 h OA + 24 h vehicle 24 h OA + 24 h Mem 24 h Mem

*

**

Memantine Counteracted OA-induced Cell Death

10

8

6

4

2

0

Cell death assay

Control 24 h OA + 24 h vehicle 24 h OA + 24 h Mem 24 h Mem

LD

H r

ele

as

e(r

ati

o a

fte

r / b

efo

re t

rea

tme

nt)

nM OA 0 100 100 100 0 0

µM Mem 0 0 1 10 1 10

* *

* p < 0.05 versus OA treated tissue

3 21

68

43

100 nM OA - + + 10 µM Mem - - +

Memantine Counteracted CaMKII-induced Phosphorylation of tau

[125I] Western blot with Antibody against pS-262

Control 24 h OA + 24 h vehicle 24 h OA + 24 h Mem (10 µM)

Ph

osp

ho

ryla

tio

n o

f ta

u

7

6

5

4

3

2

1

pSer262 pSer422

Immunocytochemical staining of pSer-262

100 nM OA + + + 10 µM Mem - - -

100 nM OA - + + 10 µM Mem - - +

Memantine Counteracted OA-induced Phosphorylation of tau

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 / dephosphorylation

imbalance

Conclusion

Apart from the symptomatic benefits, memantine might also

positively influence pathological changes in AD.

Summary