Cell-specific roles of GRK2 in onset and severity of hypoxic-ischemic brain damage in neonatal mice

doi:10.1016/j.bbi.2009.11.009

Brain, Behavior, and Immunity

Volume 24, Issue 3, March 2010, Pages 420-426

https://doi.org/10.1016/j.bbi.2009.11.009 Get rights and content

Abstract

The ubiquitously expressed kinase GRK2 protects against cellular overstimulation by desensitizing G protein-coupled receptors and regulating intracellular signaling. Recently, we described that hypoxia-ischemia (HI)-induced brain damage was accelerated and increased in GRK2^+/− neonatal mice. Using Cre-Lox technology we now investigated the role of decreased GRK2 in only microglia/macrophages or forebrain neurons in development of HI brain injury.

Low GRK2 in microglia/macrophages (LysM-GRK2^f/+ mice) was sufficient to accelerate onset of HI damage, without affecting the severity of brain injury at 24 h post-HI as compared to LysM-GRK2^+/+ littermates. Consistently, the ipsilateral hemisphere of GRK2^+/− mice contained microglia with a more rounded phenotype compared to WT mice at 3 h post-HI. Inhibition of microglial/macrophage activity by minocycline treatment prevented the early onset of HI injury in GRK2^+/− mice. In vitro, primary GRK2^+/− microglia stimulated with LPS produced more TNF-α than WT microglia via a p38-dependent pathway. In vivo, HI-induced cerebral p38 activation and TNF-α production were increased in GRK2^+/− mice or in LysM-GRK2^f/+ mice. Our findings indicate that low GRK2 in microglia/macrophages accelerates brain damage via a GRK2/p38/TNF-α-dependent pathway.

Reduced GRK2 only in forebrain neurons (CamKIIα-GRK2^f/+ mice) significantly increased severity of HI brain damage without affecting the onset of brain damage.

In conclusion, our data indicate that low GRK2 in microglia/macrophages facilitates activation of these cells which may contribute to the earlier onset of cerebral HI injury associated with increased p38 phosphorylation and TNF-α production. The level of GRK2 in neurons is crucial for determining the ultimate severity of HI damage in the newborn brain.

Introduction

Perinatal hypoxia-ischemia (HI) often leads to serious and permanent brain injury in the human neonate. The underlying mechanisms leading to cell death in the brain after HI are numerous and complex and only partially understood (Ferriero, 2004).

We have shown previously that the level of G protein-coupled receptor kinase 2 (GRK2) in whole brain homogenates is decreased after neonatal HI (Lombardi et al., 2004, Nijboer et al., 2008). Cerebral GRK2 expression is also reduced in animal models of Alzheimer and Parkinson disease (Burgos-Ramos et al., 2008, Suo et al., 2004, Ahmed et al., 2008). Moreover, GRK2^+/− mice that express 50% of the protein develop both earlier and more pronounced brain injury after exposure to neonatal HI (Nijboer et al., 2008). Homozygous GRK2^−/− mice die in utero (Jaber et al., 1996).

GRKs are key kinases involved in regulation of signaling via G protein-coupled receptors (GPCR) such as chemokine receptors and metabotropic glutamate receptors, via a process called homologous desensitization. More recently, it has been revealed that GRK2 also regulates cellular signaling by direct interaction with several downstream molecules including p38 MAP kinase (Reiter and Lefkowitz, 2006, Ribas et al., 2007). In vitro studies have demonstrated that GRK2 binds and phosphorylates p38 MAP kinase, thereby preventing activation of p38 (Peregrin et al., 2006). GRK2 can thus function as an endogenous p38 inhibitor in vitro. However, the in vivo relevance of changes in GRK2 for p38-dependent processes is unknown. Numerous factors that contribute to brain injury, like reactive oxygen species and cytokines, upregulate the activity of p38 (Cuenda and Rousseau, 2007). In addition, p38 is a major kinase involved in microglial activation and cytokine production in the brain (Koistinaho and Koistinaho, 2002). Activation of cerebral microglia and production of pro-inflammatory cytokines is thought to aggravate brain injury (Silverstein et al., 1997, Hedtjarn et al., 2004). In the literature, the general idea is that microglial activation is induced as a consequence of neuronal injury (Saliba and Henrot, 2001). More recent evidence suggests that in the neonatal HI brain microglia also contribute to an early phase of inflammation by increasing early cytokine expression (Dommergues et al., 2003, Fox et al., 2005, Bye et al., 2007, Nijboer et al., 2009).

The aim of this study was to delineate the role of GRK2 in microglia/macrophages or in forebrain neurons in determining the onset and magnitude of neonatal HI brain damage, p38 activation and cytokine production.

Section snippets

Animals

Experiments were performed in accordance with national guidelines (WOD, 1996) and were approved by the University Medical Center Utrecht experimental animal care committee.

We used heterozygous GRK2⁺/⁻ and wildtype (WT) C57BL/6J littermates (Jaber et al., 1996). Animals with cell-specific reduction of GRK2 were obtained using Cre-Lox technology; LysM-Cre^+/+ males (B6.129P2-Lyzs^tm1(cre)Ifo/J) or CamKIIα-Cre^+/+ males ((Camk2a-cre)T29-1Stl/J; both Jackson Laboratory) were crossed with GRK2^f/+

Role of microglial/macrophage GRK2 in HI brain damage

To determine the contribution of GRK2 in microglia/macrophages to onset and severity of brain damage, we used mice in which GRK2 was only reduced in lysozyme M (LysM)-positive cells (microglia/macrophages/granulocytes). GRK2 protein was ∼50% reduced in isolated LysM-GRK2^f/+ microglia compared to control LysM-GRK2^+/+ microglia, but not in LysM-GRK2^f/+ astrocytes (Fig. 1A). LysM is also expressed in granulocytes and therefore, GRK2 will also be reduced in these cells. We have previously shown

Discussion

Previously, we showed that neonatal HI brain damage is accelerated and aggravated in mice with reduced GRK2 levels in all cells (GRK2^+/- mice) (Nijboer et al., 2008). These findings indicated that the endogenous level of GRK2 is a critical player in HI brain damage. In the present study, we employed Cre-Lox technology to decrease GRK2 in specific cell types. This approach allowed us to identify cell-specific effects of low GRK2 on onset and severity of neonatal HI brain damage.

In LysM-GRK2^f/+

Acknowledgments

This study was supported by the Wilhelmina Children’s Hospital Fund and the European Commission (Sixth Framework Program, contract no. LSHM-CT-2006-036534, NEOBRAIN). The authors thank I. den Hartog and J. Zijlstra for excellent technical assistance.

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In the present study, we used GFAP-GRK2+/− mice to study the role of GRK2 in astrocytes. In contrast to the exacerbated HI brain damage that develops in mice with cell-specific reduction of GRK2 in forebrain neurons (CamKIIα-GRK2+/− mice) (Nijboer et al., 2010), we show here that reduction of GRK2 in astrocytes reduces cerebral infarct size after HI. These data point out that GRK2 has a cell-specific effect on HI-induced brain damage.
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Cell-specific roles of GRK2 in onset and severity of hypoxic-ischemic brain damage in neonatal mice

Abstract

Introduction

Section snippets

Animals

Role of microglial/macrophage GRK2 in HI brain damage

Discussion

Acknowledgments

Neuroscience

Exp. Neurol.

Biochim. Biophys. Acta

J. Biol. Chem.

Neuroscience

Cell Signal.

J. Biol. Chem.

Neuron

Curr. Biol.

Trends Endocrinol. Metab.

Biochim. Biophys. Acta

Neurochem. Int.

Neuropharmacology

Exp. Neurol.

J. Neuroimmunol.

Altered expression and subcellular distribution of GRK subtypes in the dopamine-depleted rat basal ganglia is not normalized by l-DOPA treatment

J. Neurochem.

Minocycline: a neuroprotective agent for hypoxic-ischemic brain injury in the neonate?

J. Neurosci. Res.

Neonatal brain injury

N. Engl. J. Med.

Minocycline confers early but transient protection in the immature brain following focal cerebral ischemia-reperfusion

J. Cereb. Blood Flow Metab.