Abstract
Phenylketonuria (PKU), an autosomal recessive disorder of amino acid metabolism caused by mutations in the phenylalanine hydroxylase (PAH) gene, leads to childhood mental retardation by exposing neurons to cytotoxic levels of phenylalanine (Phe). A recent study showed that the mitochondria-mediated (intrinsic) apoptotic pathway is involved in Phe-induced apoptosis in cultured cortical neurons, but it is not known if the death receptor (extrinsic) apoptotic pathway and endoplasmic reticulum (ER) stress-associated apoptosis also contribute to neurodegeneration in PKU. To answer this question, we used specific inhibitors to block each apoptotic pathway in cortical neurons under neurotoxic levels of Phe. The caspase-8 inhibitor Z-IETD-FMK strongly attenuated apoptosis in Phe-treated neurons (0.9 mM, 18 h), suggesting involvement of the Fas receptor (FasR)-mediated cell death receptor pathway in Phe toxicity. In addition, Phe significantly increased cell surface Fas expression and formation of the Fas/FasL complex. Blocking Fas/FasL signaling using an anti-Fas antibody markedly inhibited apoptosis caused by Phe. In contrast, blocking the ER stress-induced cell death pathway with salubrinal had no effect on apoptosis in Phe-treated cortical neurons. These experiments demonstrate that the Fas death receptor pathway contributes to Phe-induced apoptosis and suggest that inhibition of the death receptor pathway may be a novel target for neuroprotection in PKU patients.
Highlights
Phenylketonuria (PKU), one of the most common inborn errors of amino acid metabolism, leads to progressive mental retardation in children
We pretreated cortical neurons with Sal, an inhibitor of eIF2 dephosphorylation recently shown to counteract endoplasmic reticulum (ER) stress-induced cell death in cultured cells [16] and compared apoptosis to cultures treated with Phe alone
Our previous studies demonstrated that high concentrations of phenylalanine (Phe) can increase apoptosis in cultured primary neurons, suggesting that direct Phe-mediate cytotoxicity contributes to neuronal injury in PKU [21]
Summary
Phenylketonuria (PKU), one of the most common inborn errors of amino acid metabolism, leads to progressive mental retardation in children. A deficiency in PAH activity leads to accumulation of Phe in brain tissue and cerebrospinal fluid, resulting in brain damage [1,2]. Many of the neurodegenerative effects of PKU-associated Phe accumulation are indirect, including decreased creatine kinase activity, deficient myelin production, and reduced dopamine synthesis due to the lack of tyrosine [3]. High concentrations of Phe can trigger neuronal apoptosis directly [4]. There are two major pathways that lead to apoptosis [5]. One is the mitochondria-initiated intrinsic pathway, in which the release of cytochrome c from the mitochondrial matrix following loss of inner mitochondrial membrane integrity triggers formation of the apoptosome composed of Apaf-1, pro-caspase-9, dATP, and cytochrome c. Activated m-calpain cleaves Bcl-xL and proteolytically activates caspase-12 [10], which activates caspase-9 followed by activation of caspase-3 [11]
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