Neuroprotective Effects of Memantine

1. Neuroprotective Effects of Memantine

2. Memantine in an In Vitro Model for Neurodegeneration Parallel incubation of 3-NP and memantine for 7 or 12 days • Cresyl violet staining • Homogenization • Immunoblot • Incubation with synaptic markers 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 Vehicle 1 µM 5 µM Memantine 10 µM 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 Vehicle 1 µM 5 µM Memantine 10 µM 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 2 weeks • Biochemical assay • Choline acetyltransferase in the frontal cortex Memantine’s effect on lesions of the nucleus basalis magnocellularis (NBM) Memantine injection 30 min before NMDA or Memantine infusion for 2 weeks Unilateral injection of NMDA (7.5 nmol) or 3NP (250 nmol) into the NBM 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/h*mg proteine) 0.01 0.1 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/h*mg proteine) Memantine (20 mg/day) Control * NMDA lesion 3-NP lesion * p < 0.01 versus control Wenk et al., NeuroReport 1996

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

10. Protection by Memantine Against Aβ-Induced Neurodegeneration Extent of β-amyloid-induced damage in the CA1 region 1,000 800 600 400 200 0 Vehicle Extent (µm) * Vehicle Memantine Memantine * p < 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 < 0.03 versus vehicle Miguel-Hidalgo et al., Brain Res 2002

12. 10 days Biochemical analysis in the frontal cortex:  ChAT activity 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 < 0.0001 versus control; ** p < 0.05 versus LPS Willard et al., Exp Brain Res 2000

14. Parallel infusion of memantine (s.c. 20 mg/kg/day) and quinolinic acid (i.c.v.) T-maze alternation 3 days Removal of minipumps 15 sec 2 weeks 10 trials/day 5 days Biochemical analysis 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 2 3 4 5 Day * p < 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 * * [H3]Hemicholinium-3 binding (µmol/mg tissue) Control QA QA + Memantine * p < 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 • 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 Alzheimer’s disease In vitro model Therapeutic approach Hippocampal culture + okadaic acid (OA) PP-2A activity  CaMKII activity  PKA activity  Hyperphosphorylation of tau 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 nM OA 100 100 100 0 0 µM Mem 0 1 10 1 10 * p < 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) * nM OA 100 100 100 0 0 100 100 100 0 0 µM Mem 0 1 10 1 10 0 1 10 1 10 * p < 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 * * nM OA 0 100 100 100 0 0 µM Mem 0 0 1 10 1 10 * p < 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/dephosphorylation imbalance Conclusion: • Apart from the symptomatic benefits, memantine might also positively influence pathological changes in AD