Astrocytic contribution to deficient Ca 2+ signalling and oxidative stress mediated by TRPV4...
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Transcript of Astrocytic contribution to deficient Ca 2+ signalling and oxidative stress mediated by TRPV4...
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