Elsevier

Neurobiology of Disease

Volume 43, Issue 2, August 2011, Pages 388-396
Neurobiology of Disease

The RNA-binding protein RBM3 is involved in hypothermia induced neuroprotection

https://doi.org/10.1016/j.nbd.2011.04.010Get rights and content

Abstract

Induced hypothermia is the only therapy with proven efficacy to reduce brain damage after perinatal asphyxia. While hypothermia down-regulates global protein synthesis and cell metabolism, low temperature induces a small subset of proteins that includes the RNA-binding protein RBM3 (RNA-binding motif protein 3), which has recently been implicated in cell survival. Here, immunohistochemistry of the developing postnatal murine brain revealed a spatio-temporal neuronal RBM3 expression pattern very similar to that of doublecortin, a marker of neuronal precursor cells. Mild hypothermia (32 °C) profoundly promoted RBM3 expression and rescued neuronal cells from forced apoptosis as studied in primary neurons, PC12 cells, and cortical organotypic slice cultures. Blocking RBM3 expression in neuronal cells by specific siRNAs significantly diminished the neuroprotective effect of hypothermia while vector-driven RBM3 over-expression reduced cleavage of PARP, prevented internucleosomal DNA fragmentation, and LDH release also in the absence of hypothermia. Together, neuronal RBM3 up-regulation in response to hypothermia apparently accounts for a substantial proportion of hypothermia-induced neuroprotection.

Research highlights

► RBM3 is expressed in fetal and early postnatal brain neurons. ► Hypothermia promotes RBM3 expression in neurons. ► Neuroprotection afforded by hypothermia involves RBM3. ► RBM3 rescues neuronal cells from apoptosis.

Introduction

The only available therapy for neonatal asphyxia is cooling the infant's body to about 33 °C (mild hypothermia) for 48–72 h. A series of randomized multicenter trials have established a protective effect of therapeutic hypothermia in reducing long-term sequelae (Edwards et al., 2010, Jacobs et al., 2007, Schulzke et al., 2007, Shah et al., 2007).

Hypoxic-ischemic brain damage following asphyxia occurs in a biphasic manner, where the early phase (first hours) is classified as necrosis and the later phase (by 48 h) display characteristics of neuronal apoptosis (Northington et al., 2001a, Northington et al., 2001b). Thus, the primary target of hypothermia is the mitigation of early cell death and the prevention of delayed neuronal apoptosis (Robertson et al., 2009).

In neurons, hypoxia-ischemia triggers an intrinsic apoptotic pathway (Ferrand-Drake and Wieloch, 1999, Perez-Pinzon et al., 1999, Zhu et al., 2003), characterized by a biochemical cascade leading to activation of caspases, cleavage of poly ADP-ribose polymerase (PARP), internucleosomal DNA fragmentation, and eventually cell death with release of intracellular enzymes, such LDH (Liu et al., 1996, Susin et al., 1999a, Susin et al., 1999b). Hypothermia ultimately appears to stop cell death (Edwards et al., 1995, Fukuda et al., 2001, Han et al., 2002, Zhu et al., 2004) but the precise molecular events of how and where hypothermia retards this biochemical cascade are poorly understood.

The RNA-binding protein RBM3 belongs to a very small group of cold inducible proteins being synthesized in response to either hypothermia or other conditions of mild stress (Lleonart, 2010). RBM3 is evolutionary highly conserved (Derry et al., 1995) and has been suggested to play a role in hibernation (Fedorov et al., 2009, Williams et al., 2005). While a temperature drop from 37 to 32–34 °C is sufficient to induce the expression of RBM3 (Danno et al., 1997), elevated temperature decreases the expression of RBM3 in the setting of therapeutic hyperthermia (Zeng et al., 2009) and in cryptorchid testis, a condition in which the testes do not descend from the 37 °C abdominal compartment to the 33–34 °C scrotal environment (Danno et al., 2000).

Recent studies have attributed RBM3 a role in cell survival. Blocking RBM3 signaling by siRNAs triggers cells mitotic catastrophe in colon carcinoma cells (Sureban et al., 2008) and causes loss of proliferation and ultimately cell death in human embryonic kidney cells (HEK-293) (Wellmann et al., 2010). In contrast, exogenous over-expression of RBM3 inhibits apoptosis in neurons transfected with mutated huntingtin (Kita et al., 2002) and increases protein synthesis in mouse neuroblastoma N2a cells (Dresios et al., 2005, Smart et al., 2007).

These findings on RBM3 prompted us to hypothesize that the cold-inducible RBM3 might serve as a candidate to mediate hypothermia-induced neuroprotection after neonatal asphyxia, via its up-regulation in response to hypothermia. Therefore, we first determined RBM3 expression in the developing brain of mice. Second, we studied RBM3 induction in response to hypothermia in cortical organotypic slice cultures (COSCs), primary neurons and rat pheochromocytoma cells (PC12). Third, we investigated whether in PC12 cells neuroprotection by hypothermia might involve RBM3, employing siRNA knockdown and vector-driven RBM3 over-expression experiments.

Section snippets

Animals, slice preparations, cortical organotypic slice cultures, and primary neurons

All animal experiments were performed with permission of the local animal care committee and in accordance with international guidelines on handling laboratory animals and current Swiss law.

C57BL/6J mice from postnatal days 1, 10, 19, and adult (4 months old) were used. Mice were anesthetized with pentobarbital (Vetanarcol®, 0.04 g/kg ip) and perfused with 4% paraformaldehyde in 0.1 M phosphate buffer. Brains were dissected and immersed in 4% paraformaldehyde overnight. Serial sagittal 40 μm

RBM3 is expressed in the developing mouse brain in a temporally and spatially restricted pattern

During postnatal murine brain development, RBM3 levels were found to be high immediately after birth (P1) and low in adult brain, paralleled by a very similar DCX and p35 mRNA and protein expression dynamic (Figs. 1A and B). DCX is a microtubule associated protein and is expressed in newborn and migrating neurons as well as in neuronal stem cells of the adult brain (for review of DCX and p35 see Feng and Walsh, 2001). The decrease in mRNA expression alongside maturation was stronger for DCX

Discussion

Here, we present several lines of evidence for an involvement of the cold-inducible RNA binding protein RBM3 in mediating hypothermia-induced neuroprotection. First, RBM3 is widely expressed in juvenile neurons of murine brain. Second, mild hypothermia of 32 °C causes significant RBM3 mRNA and protein up-regulation in juvenile as well as in adult neurons. Third, the degree of neuronal apoptosis is inversely related to the level of RBM3 expression. Fourth, blocking RBM3 over-expression that

Acknowledgments

This work was supported by a grant from the Bangerter-Rhyner-Foundation, Bern, Switzerland. SW was supported by a Swiss National Science Foundation Career Award for Medical Scientists (33CM30-124101).

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