1. Astrocytic contribution to deficient Ca 2+ signalling and oxidative stress mediated by TRPV4 channels in Aβ 40 -induced hippocampal cell death Ji-Zhong Bai and Janusz Lipski Department of Physiology, Faculty of Medical and Health Sciences, University of Auckland, New Zealand

2. AD, Aβ, Astrocytes, TRPV4 Brain cell death & amyloid deposition: pathological hallmarks of Alzheimer’s disease (AD) Ca 2+ -permeable TRPV4 channels : expression in rat hippocampal astrocytes oxidative stress-induced cell damage ischemia-evoked Ca 2+ entry in reactive astrocytes. Toxic amyloid β (Aβ) species: primary factor in AD pathogenesis Astrocytes as primary target of toxic Aβ action: Ca 2+ signalling, oxidative states, brain cell death in AD

3. Objective: To investigate the potential role of TRPV4 channels in Aβ-evoked in vitro damage of the hippocampus, a brain region highly vulnerable in AD

4. Methods Model systems: Monolayer co-culture of neurons and astrocytes Organotypic slice culture of rat hippocampus Cell death induction: Exogenous Amyloid β 1-40 peptide (Aβ 40 ) Endogenous increase in ROS evoked by BSO GSHGSSG Catalase GSHPx H2O2H2O2 GSH synthetase H 2 O + O 2 H2OH2O Superoxide dismutase Τ BSO (Buthionine sulfoximine ) Fe 2+, Fenton Reaction O 2 OH NAD + /ADPR

5. Methods (cont.) Detection of TRPV4 expression: RT-PCR, Western blotting, Immunocytochemistry Detection of cell death: Uptake of fluorescent propidium iodide (PI) Effect of Aβ 40 on activation of TRPV4 channels: [Ca 2+ ] i changes in neurons and astrocytes by fluorescence digital imaging (Olympus Live Cell Confocal System)

6. Aβ 40 -evoked hippocampal damage is region-specific 10 μM 20 μM Ctrl5 μM N = 7-16, p < 0.001 vs corresponding controls 24 hr DG CA1-3 Ctrl 16 hr 30 hr 50 µm

7. Aβ 40 -evoked hippocampal damage is region- specific with altered TRPV4 and GFAP expression GFAPMerged TRPV4 20 μm GFAPMerged MAP2 500 μm Ctrl Aβ 40 20 µM Western 1 2 RT-PCR 1 2 3 4 400 200 650 bp 427bp370bp +ve v4 -RT 100 KDa

8. Oxidative stress induces astrocytic damage in organotypic hippocampal cultures Ctrl BSO + Glut 500  m BSO (4 µM) / PI PI / GFAP TRPM2 / GFAP GFAP TRPM2 500  m

9. Cell death induced by Aβ 40 (or oxidative stress) is attenuated by TRPV4 blockers and antioxidants * * * * n = 9 - 30, p < 0.001 vs A β 40 * * * * or BSO * * * * RR /2µM Gd 3+ /500mM Trolox /500µMMAFP /5mM DG CA1-3 Aβ 40 /20 μM 50 µm

10. Aβ 40 evoked mainly neuronal damage in co-cultures of hippocampal neurons and astrocytes n = 3 - 4, p < 0.001 vs relative controls /15 µM 0 hr * ** * * 24 hr 40 µm * ** * *

11. Aβ 40 enhanced-[Ca 2+ ] i in astrocytes is attenuated by RR and in Ca 2+ -free media n = 16 - 26, p < 0.001 relative to the period before Aβ 40 treatment 60 µm 0 min * * * * * * 30 min * * * * * * 100 µm

12. Aβ 40 enhanced the expression of TRPV4 and GFAP proteins in astrocytes n = 18 - 31, p < 0.001 relative to corresponding controls MergedTRPV4GFAP Ctrl Aβ 40 /20 µM 40 µm

13. We propose that the altered astrocytic state affects neuronal survival due to lack of trophic and other support, forming a link between astrocyte dysfunction and neurodegeneration in AD. Summary TRPV4 modulators inhibit A β 40 -evoked: region-specific damage that alters TRPV4 & GFAP expression astrocytic Ca 2+ influx, while Aβ 40 damages more neurons TRPV4-expressing astrocytes protect Hippocampal pyramidal neurons against Aβ 40 and oxidative damage A β 40 primarily activates astrocytic TRPV4 channels, leading to neuronal death with limited astrocyte damage

14. Dr Justin Dean & co-workers Department of Physiology, University of Auckland, New Zealand