Abstract
Alzheimer's disease (AD) is characterized by an early synaptic loss, which strongly correlates with the severity of dementia. The pathogenesis and causes of characteristic AD symptoms are not fully understood. Defects in various cellular cascades were suggested, including the imbalance in production of reactive oxygen and nitrogen species. Alterations in S-nitrosylation of several proteins were previously demonstrated in various AD animal models and patients. In this work, using combined biotin-switch affinity/nano-LC-MS/MS and bioinformatic approaches we profiled endogenous S-nitrosylation of brain synaptosomal proteins from wild type and transgenic mice overexpressing mutated human Amyloid Precursor Protein (hAPP). Our data suggest involvement of S-nitrosylation in the regulation of 138 synaptic proteins, including MAGUK, CamkII, or synaptotagmins. Thirty-eight proteins were differentially S-nitrosylated in hAPP mice only. Ninety-five S-nitrosylated peptides were identified for the first time (40% of total, including 33 peptides exclusively in hAPP synaptosomes). We verified differential S-nitrosylation of 10 (26% of all identified) synaptosomal proteins from hAPP mice, by Western blotting with specific antibodies. Functional enrichment analysis linked S-nitrosylated proteins to various cellular pathways, including: glycolysis, gluconeogenesis, calcium homeostasis, ion, and vesicle transport, suggesting a basic role of this post-translational modification in the regulation of synapses. The linkage of SNO-proteins to axonal guidance and other processes related to APP metabolism exclusively in the hAPP brain, implicates S-nitrosylation in the pathogenesis of Alzheimer's disease.
Highlights
From the ‡Institute of Biochemistry and Biophysics, Polish Academy of Sciences, Warsaw, Poland; §Institute for Molecular Medicine Finland (FIMM), Helsinki, Finland; ¶Biomedicum Helsinki, Institute of Biomedicine, Biochemistry/Developmental Biology, Meilahti Clinical Proteomics Core Unit, University of Helsinki, Finland; ʈFolkhalsan Institute of Genetics, Helsinki, Finland
Endogenous Protein S-nitrosylation is Increased in human Amyloid Precursor Protein (hAPP) Synaptosomes—To assess whether the increased expression of nitric oxide synthases visible in the hAPP mouse brain results in pattern changes of endogenous protein S-nitrosylation, we used a biotin switch technique (BST) assay in which SNO-proteins are selectively labeled by biotin, followed by Western blot detection with anti-biotin antibodies
Numerous protein bands across a broad mass range were revealed, both in FVB and hAPP derived synaptosomal fractions indicating the presence of endogenously S-nitrosylated proteins (Fig. 2, lane 3 and 4)
Summary
Nitric oxide is a freely diffusible, very reactive radical molecule It readily reacts with various endogenous substrates forming that is, iron and copper adducts in prosthetic groups of proteins [4], peroxynitrite in the reaction with reactive oxygen species, ROS [5], and S-nitrosothiols with endogenous low-molecular weight thiols like cysteine and glutathione [6]. N-methyl-D-aspartate receptor (NMDAR) and caspase enzyme activity can be decreased by S-nitrosylation, thereby facilitating neuroprotection [15] This finding led to development of nitro-memantine, a nitric oxide donor and selective NMDAR interacting drug. It selectively S-nitrosylates the NMDA receptor and prevents its’ hyperactivation, observed in Alzheimer’s disease [16]. The present study utilized one such model, a transgenic mice expressing human APP with London mutation and its wild-type, agedmatched counterparts, for targeted, differential proteomic analysis of S-nitrosylation of proteins located at the synaptic terminals
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