Neonatal mice lacking functional Fas death receptors are resistant to hypoxic–ischemic brain injury

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Abstract

Neonatal hypoxia–ischemia (HI) upregulates Fas death receptor expression in the brain, and alterations in expression and activity of Fas signaling intermediates occur in neonatal brain injury. B6.MRL-Tnfrsf6lpr mice lacking functional Fas death receptors are protected from HI brain damage in cortex, striatum, and thalamus compared to wild-type mice. Expression of Fas death receptor and active caspases increase in the cortex after HI. In wild-type mice, the hippocampus is most severely injured, and the hippocampus is the only region not protected in the B6.MRL-Tnfrsf6lpr mice. The selective vulnerability of the hippocampus to injury correlates with (1) lower basal expression of [Fas-associated death-domain-like IL-1β-converting enzyme]-inhibitory protein (FLIP), (2) increased degradation of spectrin to its 145 or 150 kDa breakdown product, and (3) a higher percentage of non-apoptotic cell death following neonatal HI. We conclude that Fas signaling via both extrinsic and intrinsic caspase cascades causes brain injury following neonatal HI in a region-dependent manner. Basal levels of endogenous decoy proteins may modulate the response to Fas death receptor signaling and provide a novel approach to understanding mechanisms of neonatal brain injury.

Introduction

Cerebral palsy occurs as a result of neonatal brain injury from a variety of causes including antenatal chorioamnionitis, perinatal hypoxia–ischemia (HI), and stroke with resultant activation of inflammatory cascades Foster-Barber and Ferriero, 2002, Foster-Barber et al., 2001, Grow and Barks, 2002, Hagberg et al., 2002, Shalak et al., 2002, Willoughby and Nelson, 2002. A number of cell surface receptors respond to cytokine (inflammatory) stimulation resulting in activation of cell death signaling programs Schulze-Osthoff et al., 1994, Schneider and Tschopp, 2000, Leist and Jaattela, 2001. Fas death receptor is one of the most extensively studied of this group of cytokine-responsive receptors (Nagata, 1999). Lack of functional Fas death receptor is neuroprotective in adult models of hypoxia–ischemia (HI) Martin-Villalba et al., 1999, Rosenbaum et al., 2000. Hypoxia–ischemia also activates Fas death receptor signaling in the neonatal brain (Northington et al., 2001a). Using an established model of neonatal HI in murine brain (Ditelberg et al., 1996), the functional importance of Fas death receptor upregulation in neonatal brain injury can be investigated.

The intracellular signaling by the Fas death receptor is complex. Depending on the type of stimulus applied, a cell may undergo apoptosis, necrosis, or survival and proliferation Kawahara et al., 1998, Matsumura et al., 2000, Nagata, 1999 in response to activation of the Fas death receptor. In addition to activation of the extrinsic caspase-directed apoptosis cascade in the presence of increased Fas death receptor expression, as we have shown in the developing thalamus following neonatal HI (Northington et al., 2001a), there is abundant evidence that the intrinsic- or mitochondrial-directed caspase-apoptosis cascade is activated following Fas death receptor signaling and functions to amplify Fas-mediated cell death Li et al., 1998, Luo et al., 1998. Fas-mediated apoptosis is also amplified via recruitment of the JNK pathway via DAXX, a Fas death receptor binding protein in some cell types (Yang et al., 1997). How Fas signals for necrotic cell death in the presence of caspase inhibition is less well understood; however, in fetal motoneurons, Fas activation leads to transcription and translation of neuronal nitric oxide synthase and production of the potent oxidant, peroxynitrite (Raoul et al., 2002). The overall response to Fas signaling is further complicated by the presence of [Fas-associated death-domain-like IL-1β-converting enzyme]-inhibitory protein (FLIP) (Ahn et al., 2001), a naturally occurring, dominant negative inhibitor of Fas-mediated apoptosis (Hu et al., 1997). FLIP is expressed in the immature rat cortex (Cheema et al., 1999), but neither the basal regional expression of FLIP nor the expression of FLIP following brain injury has been studied.

Originally, Fas death receptor was detected in fibroblasts, tumor cell lines, and lymphocytes, but no Fas expression was detected in normal adult brain (Park et al., 1998). Subsequently, RT-PCR and immunocytochemical analysis of P14 mice revealed significant basal expression of Fas death receptor in both hippocampal and cortical neurons and upregulation of Fas death receptor following HI (Felderhoff-Mueser et al., 2000). Most recently, increases in Fas death receptor message, expression, and function have been found in the neonatal p7 rat brain following traumatic brain injury (Felderhoff-Mueser et al., 2002).

That Fas death receptor signaling is important in human disease is now obvious. Activation of Fas death receptor pathways in traumatic, hemorrhagic, and stroke brain injury Felderhoff-Mueser et al., 2003, Martin-Villalba et al., 2001, Mehmet, 2001 is well documented. Additionally, asphyxiated infants with pontosubicular neuronal necrosis secondary to severe perinatal asphyxia show marked increase in expression of Fas death receptor protein in degenerating neurons (Van Landeghem et al., 2002). Reports of Fas death receptor expression in the pediatric clinical literature, combined with studies of Fas signaling in animal models, suggest that additional studies be undertaken to understand the importance and mechanisms of Fas death receptor signaling in the immature brain. Because there is prominent activation of Fas signaling in the neonatal thalamus following HI, we hypothesize that neonatal mice lacking functional Fas death receptors will have less thalamic injury than wild-type controls. Neuroprotection may also be found in the forebrain, where necrotic cell death is more prominent following neonatal HI Nakajima et al., 2000, Northington et al., 2001b, in the absence of functional Fas death receptors. Our study is the first to evaluate neuroprotection following neonatal HI in genetically altered neonatal mice lacking functional Fas death receptors. We also investigate the regional specificity of neuroprotection associated with lack of Fas death receptor function, and whether or not cell death phenotype within a brain region in wild-type mice predicts whether that region will exhibit neuroprotection in the absence of functional Fas death receptors. Finally, in the setting of neonatal HI, we investigate expression of downstream Fas cell-signaling proteins including (1) members of both the intrinsic and extrinsic caspase-apoptosis pathways and (2) the caspase 8 decoy protein, FLIP.

Section snippets

Material and methods

These studies received prior approval from the Animal Care and Use Committee of Johns Hopkins University School of Medicine and were performed in accordance with the NIH Guide for the Care and Use of Laboratory Animals, U.S. Department of Health and Human Services 85-23, 1985.

Results

Selective increase in Fas death receptor protein following neonatal hypoxia–ischemia is associated with activation of both extrinsic and intrinsic apoptosis pathways in neonatal cortex In p7 wild-type mice; at baseline (control), there is minimal Fas death receptor expression in the membrane-enriched P2 fraction from cortex; however, by 24 h after hypoxia–ischemia, there is a large increase in Fas death receptor protein expression in the ipsilateral cortex. Increased expression of Fas death

Discussion

The most important findings of the present work are that (1) lack of functional Fas death receptors provides significant neuroprotection in the thalamus, striatum, and cortex in the very immature brain but does not provide neuroprotection in the hippocampus following neonatal HI, (2) basal levels of the endogenous caspase 8 decoy protein, FLIP, are greater in the cortex and thalamus than in the hippocampus, which sustains more injury following HI, and (3) injury in the hippocampus differs

Acknowledgements

These studies were supported by NS 45059, HD 39672 (FJN), United Cerebral Palsy (FJN), U.S. Army Department of Defense DAMD17-99-1-9553, NIH AG16282 (LJM), and NS 35902 (DMF).

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