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

AD, Aβ, Astrocytes, TRPV4

• Brain cell death & amyloid deposition: pathological hallmarks of Alzheimer’s disease (AD)

• Ca2+-permeable TRPV4 channels : expression in rat hippocampal astrocytes oxidative stress-induced cell damage ischemia-evoked Ca2+ entry in reactive astrocytes.

• Toxic amyloid β (Aβ) species: primary factor in AD pathogenesis

• Astrocytes as primary target of toxic Aβ action: Ca2+ signalling, oxidative states, brain cell death in AD

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

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

GSH GSSG

Catalase

GSHPxH2O2

GSH synthetase

H2O + O2

H2O

Superoxide dismutase

ΤBSO (Buthionine sulfoximine )

Fe2+, Fenton

Reaction

O2•

OH•

NAD+/ADPR

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)

Aβ40-evoked hippocampal damage is region-specific

10 μM

20 μM

Ctrl 5 μM

N = 7-16, p < 0.001 vs corresponding controls

24 hr

DG

CA1-3

Ctrl 16 hr

30 hr

50 µm

Aβ40-evoked hippocampal damage is region-specific with altered TRPV4 and

GFAP expression GFAP MergedTRPV4

20 μm

GFAP MergedMAP2

500 μm

Ctrl

Aβ40

20 µM

Western

1 2

RT-PCR

1 2 3 4

400

200

650

bp

42

7b

p

37

0b

p

+ve v4 -RT

100 KDa

Oxidative stress induces astrocytic damage

in organotypic hippocampal culturesCtrl BSO + Glut

500 m

BSO (4 µM) / PI

PI / GFAP

TRPM2 / GFAP GFAPTRPM2

500 m

Cell death induced by Aβ40 (or oxidative stress) is attenuated by TRPV4 blockers and

antioxidants

* * *

*

n = 9 - 30, p < 0.001 vs Aβ40

* * *

*

or BSO

*

*

* *

RR /2µM Gd3+ /500mM

Trolox /500µM MAFP /5mM

DG

CA1-3Aβ40 /20 μM

50 µm

Aβ40 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

* * *

**

Aβ40 enhanced-[Ca2+ ]i in astrocytes is attenuated by RR and in Ca2+ -free media

n = 16 - 26, p < 0.001 relative to the period before Aβ40 treatment

60 µm

0 min

*

*

*

**

*

30 min

*

*

**

*

*

100 µm

Aβ40 enhanced the expression of TRPV4 and GFAP proteins in astrocytes

n = 18 - 31, p < 0.001 relative to corresponding controls

MergedTRPV4 GFAP

Ctrl

Aβ40 /20 µM

40 µm

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 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 astrocyte damage

Dr Justin Dean & co-workers

Department of Physiology,

University of Auckland,

New Zealand