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 Control10 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 Synapsin II b (arbitary units) Neuroprotective Effect of Memantine In Vitro 300 200 100 0 Control10 µM1 µM5 µM Memantine Vehicle

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. 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 or

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 lesionNMDA lesion 0 -4 -8 -12 -16 -20 * * 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 (20 mg/day) Control

9. Memantine infusion (20 mg/kg/day) 7 daysImmunohistochemistry in the hippocampus: neuronal damage GFAP Miguel-Hidalgo et al., Brain Res 2002 Injection of β-amyloid (1–40) into the hippocampus 2 days Memantine’s effect on β-amyloid-induced lesion of the hippocampus Memantine in an Animal Model for Alzheimer’s Disease

10. Extent of β-amyloid-induced damage in the CA1 region 1,000 800 600 400 200 0 Extent (µm) VehicleMemantine * Miguel-Hidalgo et al., Brain Res 2002 * p < 0.02 versus placebo Protection by Memantine Against Aβ- 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 VehicleMemantine * 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 4321043210 15324 * * * * * Number of errors Attenuation of Quinolinic Acid Induced Memory Loss by Memantine Control QA QA + 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 β-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 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 Alzheimer’s disease

20. Okadaic acid for 24 hMemantine or vehicle for 24 h Hippocampal slices Analysis Assay for phosphatase- or kinase activity Assay for cell death Western blots (p-tau) Effects of Memantine on Phosphorylation of tau-Methods

21. 120 100 80 60 40 20 0 Memantine Counteracted OA-induced PP-2A Inhibition 100100100 00 PP-2A activity 0110110 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 nM OA µM Mem *

22. * * 250 200 150 100 50 0 100100100 00 0110110 nM OA µM Mem CaMKII activityPKA activity Memantine Restored CaMKII and PKA Activity Kinase activity (% of control) * p < 0.05 versus OA treated tissue * 120 100 80 60 40 20 0 100100100 00 0110110 24 h OA + 24 h vehicle 24 h OA + 24 h Mem 24 h Mem

23. ** Memantine Counteracted OA-induced Cell Death Cell death assay LDH release (ratio after/before treatment) * p < 0.05 versus OA treated tissue 10 8 6 4 2 0 0100100100 00 00110110 nM OA µM Mem Control 24 h OA + 24 h vehicle 24 h OA + 24 h Mem 24 h Mem

24. 68 43 [ 125 I] Western blot with Antibody against pS-262 Phosphorylation of tau pSer262pSer422 Memantine Counteracted CaMKII- induced Phosphorylation of tau 7654321076543210 Control 24 h OA + 24 h vehicle 24 h OA + 24 h Mem (10 µM) 10 nM OA 10 µM Mem––+––+ –++–++ 123123

25. Immunocytochemical staining of pSer-262 100 nM OA+++ 10 µM Mem––– Memantine Counteracted OA-induced Phosphorylation of tau 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/dephosphorylation imbalance Conclusion: Apart from the symptomatic benefits, memantine might also positively influence pathological changes in AD