Neurotoxicology. 2014 Mar;41:64-72.

Involvement of TRPV4 channels in 40-induced hippocampal cell death and astrocytic Ca2+ signalling

Ji-Zhong Bai* and Janusz Lipski

Department of Physiology, Faculty of Medical and Health Sciences, University of Auckland, Private bag 92-019, Auckland, New Zealand

 

Abstract

Previous studies suggested that amyloid β (Aβ)-induced disruption of astrocytic Ca2+ signallingand oxidative stressplay a major role in the progression towards neuronal and glial death in Alzheimer’s disease. We have recently demonstrated that Ca2+-permeable TRPV4 channels are highly expressed in rat hippocampal astrocytes and are involved in oxidative stress-induced cell damage. The aim of this study was to test the hypothesis that TRPV4 channels also contribute to hippocampal damage evoked by Aβ. Synthetic Aβ40 evoked cell death in hippocampal slice cultures in a concentration (0-20 mM) and time (12-48 hr) dependent manner, after cultures were preconditioned with sublethal concentration of buthionine sulfoximine (BSO, 1.5 µM) which enhanced endogenous ROS production. As demonstrated by propidium iodide fluorescence, damage was observed in the granule cell layer of the dentate gyrus and to a smaller degree in pyramidal neurons of the CA1-CA3 region, as well as in glia cells mainly at the edge of the slice. Immunocytochemistry revealed an altered pattern of TRPV4 and GFAP protein expression, andreactive astrogliosis surrounding pyramidal CA1-CA3 neurons. Neuronal and astrocytic damage was attenuated by the antioxidant Trolox, TRPV4 channel blockers Gd3+ and ruthenium red (RR), and a specific inhibitor of the redox and Ca2+-sensitive phospholipase A2 enzyme (MAFP). In disassociated co-cultures of hippocampal neurons and astrocytes without BSO preconditioning, Aβ40 evoked pronounced neuronal damage,enhanced the expression of TRPV4 and GFAP proteins (indicative of reactive astrogliosis), and increased intracellular free Ca2+ concentration in astrocytes. The latter effect was attenuated by RR and in Ca2+-free media. These data show that Ab40 can activate astrocytic TRPV4 channels in the hippocampus, leading to neuronal and astrocytic damage in a Ca2+ and oxidative stress-dependent manner.

PMID: 24457011

 

Supplementary information

Amyloid deposition and neuron death are the pathologic hallmarks of Alzheimer’s disease (AD). Accumulation of toxic amyloid β (Aβ) species in the brain is widely accepted as the primary factor in AD pathogenesis. While previous research on the action of Aβ has predominantly focused on neurons, glial cells (mainly astrocytes, the most abundant group of brain cells) have been largely ignored due to their spongiform appearance as ‘passive bystanders’. There is strong evidence that astrocytes supply neurons with metabolic substrates and the precursors of antioxidant glutathione (GSH), as well as play an active role in other aspects of neuronal function. Although reactive astrocytes are known to surround amyloid plaques indicating their role in the deposition and clearance of Aβand activation of the inflammatory response, a role of dysfunctional astrocytes in the pathogenesis of AD remains unanswered.

Previous studies suggested that astrocytes can be the primary target of Aβ, and that Aβ-induced changes in astrocytic [Ca2+]i, ROS generation and GSH depletion play a role in the progression towards neuronal death (e.g., 1).However,the cellular and molecular mechanisms of these effects remain to be determined. We have recently demonstrated that Ca2+-permeable TRPV4 channels are highly expressed in rat hippocampal astrocytes and are involved in oxidative stress-induced cell damage (2). The aim of the current study was to examine the potential role of these channels in Aβ-induced damage of the hippocampus, a brain region highly vulnerable in AD.

Our results show thatin hippocampal slice cultures obtained from rats, Ab40 evokes oxidative stress-dependent and region-specific cell damage associated with a change of TRPV4 immunoreactivity. Aβ40 mainly damaged neurons in the dentate gyrus and the CA1 pyramidal region, similarly to what has been reportedin the hippocampus in AD human brains. Under our experimental conditions, oxidative preconditioning of slice cultures with low concentrations of BSO was essential for Ab40 cytotoxicity.This is consistent with the notion that oxidative stress in brain tissue increases with age, representing a profound risk factor for AD. Most interestingly, immunocytochemistry revealed an altered pattern of TRPV4 and GFAP protein expression, andreactive astrogliosis surrounding pyramidal CA1-CA3 neurons.  Cell damage could be prevented by the antioxidant Trolox, non-selective TRPV4 channel blockers, and inhibition of redox and Ca2+-sensitive PLA2.

fig-p

Figure 1. Fluorescent microscopic image ofdisassociated co-cultures of rat hippocampal neurons and astrocytes labelled for neuronal marker MAP2 (green), astrocyte marker GFAP (red) and DAPI-stained nuclei (blue).

In primary co-cultures of hippocampal neurons and astrocytes, as shown in the figure, Aβ40 exposure damaged mainly neurons. This was associated withenhanced expression of TRPV4 and GFAP proteins, and increased [Ca2+]i in astrocytes. The Aβ40-induced Ca2+ rise was suppressed by TRPV4 channel blocker ruthenium red, and in Ca2+ -free media. These data indicate that TRPV4-expressing astrocytes protect hippocampal pyramidal neurons against oxidative damage, and that Ab40 primarily activates astrocytic TRPV4 channels,leading totheir altered function and secondary death of neurons in a Ca2+ and oxidative stress-dependent manner. In conclusion, we propose that dysfunctional TRPV4 channels in astrocytes contribute to oxidative stress and Ca2+ overload, resulting in their failure to adequately support neurons and eventually neuronal death in AD.

Scientific and clinical significance of this study to neurological research

The onset of AD pathology is a complex, multifactorial process. While previous research into the effects of Aβ in AD has predominantly focused on neurons, this study addressed the potential role of Ca2+-permeable TRPV 4 channels in Aβ-induced disruption of astrocyte Ca2+ signalling and subsequent effects of this process on neuron survival. Our study suggests that activation of TRPV4 channels by Aβ forms a link between astrocytic dysfunction and neuron demise in AD. Further studies into the role of these channels in AD pathology could lead to development of novel therapeutic interventions in this brain disorder.

References

  1. Abramov AY, Canevari L and Duchen MR (2003) Changes in [Ca2+]i and glutathione in astrocytes as the primary mechanism of amyloid neurotoxicity. Journal of Neuroscience 23: 5088–5095.
  2. Bai JZ and Lipski J (2010) Differential expression of TRPM2 and TRPV4 channels and their potential role in oxidative stress-induced cell death in organotypic hippocampal culture. Neurotoxicology 31:204-14.

Acknowledgements: This study was supported by the Neurological Foundation of New Zealand, and by the Performance-Based Research Fund (University of Auckland).

 

Contact:

Ji-Zhong Bai, PhD

Department of Physiology

University of Auckland

Faculty of Medical and Health Science

85 Park Rd

Auckland 1023, New Zealand

j.bai@auckland.ac.nz

 

Multiselect Ultimate Query Plugin by InoPlugs Web Design Vienna | Webdesign Wien and Juwelier SchönmannMultiselect Ultimate Query Plugin by InoPlugs Web Design Vienna | Webdesign Wien and Juwelier Schönmann