Astrocytic Contribution to Aβ40-Induced Hippocampal Cell Death: TRPV4 Channel Mediation

1. Astrocytic contribution to deficient Ca2+ signalling and oxidative stress mediated by TRPV4 channels in Aβ40-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) •Toxic amyloid β (Aβ)species: primary factor in AD pathogenesis •Astrocytesas primary target of toxic Aβ action: Ca2+ signalling,oxidative states, braincell death in AD •Ca2+-permeable TRPV4 channels : expression in rat hippocampal astrocytes oxidative stress-induced cell damage ischemia-evoked Ca2+ entry in reactive astrocytes.

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. Superoxide dismutase Fe2+, Fenton Reaction Methods •Model systems: •Monolayer co-culture of neurons and astrocytes • Organotypic slice culture of rat hippocampus •Cell death induction: • Exogenous Amyloid β1-40 peptide (Aβ40) • Endogenous increase in ROS evoked by BSO H2O + O2 O2• Catalase H2O2 GSHPx OH• H2O GSH synthetase GSH GSSG Τ NAD+/ADPR BSO(Buthioninesulfoximine )

5. Methods (cont.) •Detection of TRPV4 expression: RT-PCR, Western blotting, Immunocytochemistry •Detection of cell death: Uptake of fluorescent propidium iodide (PI) •Effect of Aβ40 on activation of TRPV4 channels: [Ca2+]i changes in neurons and astrocytes by fluorescence digital imaging (Olympus Live Cell Confocal System)

6. Aβ40-evoked hippocampal damage is region-specific Ctrl 16 hr Ctrl 5 μM 50 µm 30 hr 24 hr 20 μM 10 μM CA1-3 DG N = 7-16, p < 0.001 vs corresponding controls

7. Aβ40-evoked hippocampal damage is region-specific with altered TRPV4 and GFAP expression GFAP Merged TRPV4 MAP2 GFAP Merged Ctrl 500 μm Aβ40 20 µM RT-PCR Western 650 427bp 370bp 400 100 KDa 200 bp v4 -RT +ve 1 2 1 2 3 4 20 μm

8. 500 mm Oxidative stress induces astrocytic damagein organotypic hippocampal cultures Ctrl BSO (4 µM) /PI BSO + Glut TRPM2 GFAP TRPM2 /GFAP PI /GFAP 500 mm

9. Cell death induced by Aβ40 (or oxidative stress) is attenuated by TRPV4 blockers and antioxidants Aβ40 /20 μM CA1-3 DG * * * * * * * * RR /2µM Gd3+ /500mM n = 9 - 30, p < 0.001 vs Aβ40 or BSO * Trolox /500µM MAFP /5mM * * * 50 µm

10. Aβ40 evoked mainly neuronal damage in co-cultures of hippocampal neurons and astrocytes * * * * * * * * * * 40 µm 24 hr 0 hr /15 µM n = 3 - 4, p < 0.001 vs relative controls

11. Aβ40 enhanced-[Ca2+ ]i in astrocytes is attenuated by RR and in Ca2+ -free media 0 min * * * * * * * * * * * * 100 µm 30 min 60 µm n = 16 - 26, p < 0.001 relative to the period before Aβ40 treatment

12. Aβ40 enhanced the expression of TRPV4 and GFAP proteins in astrocytes TRPV4 GFAP Merged Ctrl n = 18 - 31, p < 0.001 relative to corresponding controls 40 µm Aβ40 /20 µM

13. Summary •TRPV4 modulators inhibit Aβ40-evoked: • region-specific damage that alters TRPV4 & GFAP expression •astrocytic Ca2+ influx, while Aβ40 damages more neurons • TRPV4-expressing astrocytes protect Hippocampal pyramidal neurons against Aβ40 and oxidative damage •Aβ40 primarily activates astrocytic TRPV4 channels, leading to neuronal death with limited astrocytedamage 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.

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