Elsevier

Molecular Brain Research

Volume 63, Issue 2, 8 January 1999, Pages 225-232
Molecular Brain Research

Research report
Activation of MYD116 (gadd34) expression following transient forebrain ischemia of rat: implications for a role of disturbances of endoplasmic reticulum calcium homeostasis

https://doi.org/10.1016/S0169-328X(98)00276-9Get rights and content

Abstract

MyD116 is the murine homologue of growth arrest- and DNA damage-inducible genes (gadd34), a gene family implicated in growth arrest and apoptosis induced by endoplasmic reticulum dysfunction. The present study investigated changes in MyD116 mRNA levels induced by transient forebrain ischemia. MyD116 mRNA levels were measured by quantitative PCR. After 2 h of recovery following 30 min forebrain ischemia, MyD116 mRNA levels rose to about 550% of control both in the cortex and hippocampus. In the cortex, MyD116 mRNA levels gradually declined to 290% of control 24 h after ischemia, whereas in the hippocampus they remained high (538% of control after 24 h of recovery). To elucidate the possible mechanism underlying this activation process, MyD116 mRNA levels were also quantified in primary neuronal cell cultures under two different experimental conditions, both leading to a depletion of endoplasmic reticulum (ER) calcium pools. Changes in cytoplasmic calcium activity were assessed by fluorescence microscopy of fura-2-loaded cells, and protein synthesis (PS) was evaluated by measuring the incorporation of l-[4,5-3H]leucine into proteins. The first procedure, exposure to thapsigargin (Tg), an irreversible inhibitor of ER Ca2+-ATPase, produced a parallel increase in cytoplasmic calcium activity and a long-lasting suppression of PS, while the second, immersion in a calcium-free medium supplemented with the calcium chelator EGTA, caused a parallel decrease in cytoplasmic calcium levels and a short-lasting suppression of PS. Exposure of neurons to Tg induced a permanent increase in MyD116 mRNA levels. Exposure of cells to calcium-free medium supplemented with EGTA produced only a transient rise in MyD116 mRNA levels peaking after 6 h of recovery. The results demonstrate that depletion of ER calcium stores without any increase in cytoplasmic calcium activity is sufficient to activate MyD116 expression. A similar mechanism may be responsible for the increase in MyD116 mRNA levels observed after transient forebrain ischemia. It is concluded that those pathological disturbances triggering the activation of MyD116 expression after transient forebrain ischemia are only transient in the cerebral cortex but permanent in the hippocampus.

Introduction

The most prominent stress response elicited by transient cerebral ischemia is a suppression of protein synthesis (PS) 12, 22, 25and an activation of stress gene expression [29]. The mechanisms which trigger this stress response have still not been fully elucidated. It is believed that the activation of stress gene expression is induced by the increase in cytoplasmic calcium activity occurring during ischemia [35]. Recently, evidence has been presented indicating that a depletion of endoplasmic reticulum (ER) calcium stores in the absence of changes in cytoplasmic calcium activity is sufficient to activate the expression in stress genes [17]. Knowledge of the molecular mechanisms underlying the activation of the stress response may help to elucidate the pathological process of ischemic cell injury. In this respect, genes activated under conditions of growth arrest are of particular interest because transient cerebral ischemia causes a suppression of the initiation process of PS 4, 12, 13similar to the state of growth arrest induced by disturbances of ER calcium homeostasis 14, 32.

MyD116 is a myeloid differentiation primary response gene expressed in myeloblastic leukemia cells induced for terminal differentiation by IL6 [27]. MyD116 is the murine homologue of gadd34, a member of the gadd gene family isolated from hamster cells by hybridization substraction from cDNA of cells that have been exposed to DNA damaging conditions [15]. A subset of these genes, which responded to both DNA damaging agents and conditions of growth arrest induced by serum reduction or medium depletion, were named growth arrest- and DNA damage-inducible genes (gadd) [16]. Recently, it has been suggested that gadd genes are more responsive to ER stress than to DNA damage or growth arrest, see Section 4[38].

In the present series of experiments we investigated changes in MyD116 mRNA levels induced by transient forebrain ischemia. In addition, to elucidate the possible mechanisms underlying ischemia-induced changes in MyD116 mRNA levels, we evaluated MyD116 expression in two different experimental paradigms which cause disturbances of ER calcium homeostasis: a transient exposure of neurons to thapsigargin (Tg, an irreversible inhibitor of ER Ca2+-ATPase), and an exposure of cells to calcium-free medium supplemented with the calcium chelator EGTA. In the first paradigm, depletion of ER calcium stores caused an increase in cytoplasmic calcium activity, whereas in the second paradigm the emptying of the ER calcium stores was accompanied by a reduction in cytoplasmic calcium activity. MyD116 mRNA levels were evaluated by quantitative PCR to be able to compare directly the pattern and temporal profile of changes produced by the different experimental paradigms studied, transient cerebral ischemia in vivo and experimental depletion of ER calcium pools in vitro.

Section snippets

Materials and methods

Animal experiments were carried out following the NIH guidelines for the care and use of laboratory animals, and approved by the local authorities. Experiments were performed with Wistar rats weighing 200–250 g. Animals were anesthetized with 1.5% halothane in 70% N2O/30% O2 and transient forebrain ischemia was induced using the four-vessel occlusion technique [33]with modifications [34]. After 30 min forebrain ischemia, brains were spontaneously recirculated for periods of 2, 4, 8 or 24 h. At

Results

The procedure for the quantification of MyD116 mRNA levels of brain and tissue culture samples is illustrated in Fig. 1. PCR reactions were run with different concentration ratios of plasmids containing sample cDNA or internal standard cDNA as inserts. PCR products were separated by gel electrophoresis. Bands were photographed and O.D. of bands was evaluated by image analysis. The O.D. ratio of bands (O.D. internal standard band/O.D. sample band) was correlated to the concentration ratio of

Discussion

MyD116 is a myeloid differentiation primary response gene expressed in myeloblastic leukemia cells induced for terminal differentiation by IL6 [27]. MyD116 is the murine homologue of gadd34, a member of a family of genes which respond to both DNA damaging agents and conditions of growth arrest [16]. Lately, the question has arisen whether the expression of genes of the gadd family is specifically activated by conditions of DNA damage and growth arrest. It has been demonstrated that gadd153,

Acknowledgements

The excellent technical assistance of Cordula Strecker and Änne Pribliczki is gratefully acknowledged. We would also like to thank Dr. Akira Uto for providing the brain tissue of rats subjected to transient cerebral ischemia, and Ilka Mühlhöver and Bertram Huth for preparation of the graphics and figures.

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