Regulation of protein synthesis in lymphoblasts from vanishing white matter patients
Introduction
Leukoencephalopathy with vanishing white matter (VWM), also called childhood ataxia with central nervous system hypomyelination (CACH), was first documented by Eicke in 1962 (Eicke, 1962) and is now known to be one of the most prevalent inherited childhood white matter disorders (van der Knaap et al., 1999). Neurological signs include progressive cerebellar ataxia and spasticity and relatively preserved mental abilities. Magnetic resonance imaging (MRI) of the brain shows diffuse abnormality of the cerebral white matter beginning in the presymptomatic stage (Schiffmann et al., 1994, van der Knaap et al., 1997, van der Knaap et al., 1998). With time, increasing amounts of white matter disappear to be replaced by fluid (van der Knaap et al., 1997, van der Knaap et al., 1998). The mode of inheritance of this disease is autosomal recessive (Leegwater et al., 1999).
Mutations in any of the five genes encoding the subunits of eukaryotic initiation factor 2B (eIF2B) can cause VWM (Leegwater et al., 2001, van der Knaap et al., 2002). eIF2B plays a crucial role in protein synthesis in all eukaryotic cells. Although in severe VWM variants multiple organs can be affected (van der Knaap et al., 2003), the fact that the cerebral white matter is predominantly affected in VWM is still unexplained.
The process of eukaryotic translation initiation involves several essential steps, including the formation of a 48S ribosomal complex consisting of the 40S small ribosomal subunit, the mRNA, several initiation factors, and the initiator Met-tRNAi. Pairing between the anticodon of Met-tRNAi and the AUG start-codon on the mRNA triggers hydrolysis of GTP on the initiation factor eIF2. The released eIF2·GDP complex is inactive and has to be reactivated by eIF2B. Exchange of GDP for GTP is essential for protein synthesis as eIF2-GTP is required for each initiation event (Hinnebusch, 2000).
eIF2B is a major target of translational control by the phosphorylation of eIF2 (Proud, 2001). When phosphorylated on Ser51 in its α-subunit, eIF2 binds eIF2B and acts as a competitive inhibitor of eIF2B (Kaufman et al., 1989). Phosphorylation can be induced by various stress conditions, such as viral infections, amino acid deprivation, and increased temperature (Kimball, 1999, Mathews, 1990, Scheper et al., 1997, Webb and Proud, 1997).
Recent publications indicate a correlation between the eIF2B mutation and the age of onset of VWM (Fogli et al., 2002, Fogli et al., 2004a, van der Knaap et al., 2004). Three reports have addressed the functional consequences of VWM mutations on eIF2B activity and eIF2·eIF2B complex formation (Fogli et al., 2004b, Li et al., 2004, Richardson et al., 2004). VWM mutations affected eIF2B activity in diverse ways in transfected HEK293 cells (Li et al., 2004) and in yeast (Li et al., 2004, Richardson et al., 2004) and, most importantly, caused a decrease in eIF2B activity in patient-derived cells (Fogli et al., 2004b).
VWM patients may experience considerable neurological deterioration following fever. This prompted us to study eIF2B activity, the levels of expression of eIF2B, eIF2·eIF2B complex formation, eIF2α phosphorylation, protein synthesis, and cell survival under normal and heat shock conditions in lymphoblasts derived from VWM patients, which express endogenous levels of the mutated eIF2B subunits.
Section snippets
Cell culture
The lymphoblast cell lines were derived from patients carrying the following homozygous mutations in different subunits; in eIF2Bε: T91A and R113H; in eIF2Bβ: E213G; and in eIF2Bδ: R374C. White blood cells were isolated using a Lymphoprep column (Nycomed) and transformed with B95-8 Epstein–Barr virus supernatant. The cells were cultured in suspension at 5% CO2 in RPMI medium containing 10% fetal calf serum (Gibco BRL). Cells were used at a density of 106 cells/ml. Control cells were derived
eIF2B activity in VWM-derived lymphoblasts
To confirm the reduced eIF2B activity in VWM-patient-derived lymphoblasts, as reported earlier (Fogli et al., 2004b), GDP–GTP exchange on eIF2 was measured in a number of lymphoblast cell lines. eIF2B activity was moderately to severely reduced in VWM lymphoblasts as compared to control cells (Fig. 1).
For many of the subsequent studies, immortalized lymphoblast cell lines were used. To verify that the expression level of the subunits in lymphoblasts was not altered upon viral transformation, as
Discussion
The discovery that mutations in the translation initiation factor eIF2B cause VWM suggests that the regulation of protein synthesis is likely to be affected in VWM patients. Furthermore, the rapid deterioration of VWM patients after fever suggests that the heat shock response could be compromised (van der Knaap et al., 2002, van der Knaap et al., 2003). The heat shock response is a protective reaction of cells to elevated temperatures and includes inhibition of protein synthesis with the
Acknowledgments
We would like to thank all patients and their families for cooperation. Financial support for this work was provided by “Princes Beatrix Fonds” (PBF) (grant MAR01-0201), “Stichting Spieren voor Spieren”, Dr. W.M. Phelps Stichting (grant 00026WO), the Wellcome Trust, and the Optimix Foundation for Scientific Research. We thank Dr. F. Wiegant (Utrecht University) for the HSP72-antibody. We also thank Dr. Maarten Terlou (Utrecht University) for the quantification of the Western blots and Dr. P.D.
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