1. Neuroprotective Effects of Memantine

2. 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

3. Semi-chronic 3-NP toxicity in organotypic hippocampal cultures Brown et al., Soc. Neurosci 2003 3-NP for 7 days 300 200 100 0 Control 10 µM1 µM5 µM Memantine Vehicle GluR1 (arbitary units) Neuroprotective Effect of Memantine In Vitro

4. Semi-chronic 3-NP toxicity in organotypic hippocampal cultures Brown et al., Soc. Neurosci 2003 3-NP for 7 days 300 200 100 0 Vehicle Control 10 µM1 µM5 µM Memantine Synapsin II b (arbitary units) Neuroprotective Effect of Memantine In Vitro

5. CA1 Control3-NP 14 days3-NP + Memantine DG Semi-chronic 3-NP toxicity in organotypic hippocampal cultures Brown et al., Soc. Neurosci 2003 Neuroprotective Effect of Memantine In Vitro

6. or 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 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

7. ChAT activity control - lesioned side (nmol ACh/h*mg proteine) Wenk et al., Eur J Pharmacol 1995 8642086420 10.010.1101001000 Memantine (ED 50 = 2.8 mg/kg) MK-801 (ED 50 = 0.077 mg/kg) Dose (mg/kg) Memantine injection (i.p.) attenuated NMDA-induced lesion of the NBM Protection of Cholinergic Neurons by Memantine

8. Wenk et al., NeuroReport 1996 Cortical ChAT activity control - lesioned side (nmol ACh/h*mg proteine) * p < 0.01 versus control 3-NP lesion NMDA lesion 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

9. Memantine infusion (20 mg/kg/day) 7 daysImmunohistochemistry 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

10. Memantine Placebo Extent of ß-amyloid-induced damage in the CA1 region 1000 800 600 400 200 0 Extent (µm) VehicleMemantine * Miguel-Hidalgo et al., Brain Res 2002 * p < 0.02 versus placebo Protection by Memantine Against A  - Induced Neurodegeneration Vehicle Memantine

11. Miguel-Hidalgo et al., Brain Res 2002 * p < 0.03 versus vehicle Area (%) Protection by Memantine Against A  - Induced Neurodegeneration Area of GFAP profiles around the injection site 30 25 20 15 10 5 0 Vehicle Memantine * VehicleMemantine

12. 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 Biochemical analysis in the frontal cortex: ChAT activity 10 days Effect of Memantine on Inflammation Induced Neurodegeneration

13. * 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 ** ControlLPSLPS + Memantine * Decline in cortical ChAT activity [%] Memantine Protected Cholinergic Neurons from Damage by Inflammation

14. Effects of memantine on quinolinic acid-induced neurodegeneration 15 sec 10 trials/day 5 days T-maze alternation Biochemical analysis 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

15. 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 4321043210 15324 * * * * * Number of errors Attenuation of Quinolinic Acid Induced Memory Loss by Memantine

16. 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 0 ControlQAQA + Memantine Misztal et al., Eur J Pharmacol 1996 [H 3 ]Hemicholinium-3 binding (µmol/mg tissue) Attenuation of Quinolinic Acid Induced Neurodegeneration by Memantine

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 Alzheimer's 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 (1-3): 261-269

19. 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

20. Okadaic acid for 24hMemantine 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

21. Memantine Counteracted OA-induced PP-2A Inhibition 100 100 100 0 0 PP-2A activity 0 1 10 1 10 PP-2A activity (% of control) 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 0 nM OA µM Mem *

22. CaMKII activity PKA activity Memantine Restored CaMKII and PKA Activity Kinase activity ( % of control) * 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 * * *

23. 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 LDH release (ratio after / before treatment) nM OA 0 100 100 100 0 0 µM Mem 0 0 1 10 1 10 * * * p < 0.05 versus OA treated tissue

24. 3 2 1 68 43 100 nM OA -++ 10 µM Mem - -+ Memantine Counteracted CaMKII- induced Phosphorylation of tau [ 125 I] Western blot with Antibody against pS-262 Control 24 h OA + 24 h vehicle 24 h OA + 24 h Mem (10 µM) Phosphorylation of tau 76543217654321 pSer262 pSer422

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

26. 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