Preparation of human tau exon-2- and -10-specific monoclonal antibodies for the recognition of brain tau proteins in various mammals.

The aggregations of tau protein in brain tissue have been described in a large number of neurodegenerative diseases; however, due to the lack of tau isoform- or exon-specific antibodies, the exact situations under which various brain tau isoforms can be found and their exact contributions during disease progression remain unknown. Therefore, in this study, we prepared tau exon-specific monoclonal antibodies (mAbs) that recognize different mammalian tau isoforms. Briefly, 3 Balb/c mice were separately immunized (3 mice per antigen) with the recombinant GST-fusion proteins, GST-tE2 and GST-tE10. Two hybridoma cell lines, 4A8 and 3E12, secreting antibodies against human tau exon-2 and -10 were established using the hybridoma technique. The sensitivity and specificity of the prepared mAbs were evaluated using indirect ELISA and western blot analysis. The ability of the prepared mAbs, 4A8 and 3E12, to recognize endogenous tau protein in the brain tissues of various mammals was estimated by immunoprecipitation. Based on the results of various verification methods, we found that the prepared mAbs, 4A8 and 3E12, not only specifically reacted with the individual recombinant GST tau exon fusion proteins, but also correctly recognized the recombinant human tau isoforms containing respective exon sequences, as shown by western blot analysis. Furthermore, western blot analysis and immunoprecipitation assays verified that the mAbs, 4A8 and 3E12, recognized endogenous tau proteins in human brain tissue, as well as tau proteins in a series of mammalian tissues, including goat, bovine, rabbit, hamster and mouse. Thus, in the present study, using the hybridoma technique, we successfully prepared the mAbs, 4A8 against tau exon-2 and 3E12 against tau exon-10, which provide useful tools for determining potential alternations of tau isoforms in neurodegenerative diseases.

Analyses of the mitochondrial mutations in the Chinese patients with sporadic Creutzfeldt-Jakob disease.

Pathogenic mitochondrial DNA (mtDNA) mutations leading to mitochondrial dysfunction can cause a variety of chronic diseases in central nervous system (CNS). However, the role of mtDNA mutations in sporadic Creutzfeldt-Jakob disease (sCJD) has still been unknown. In this study, we comparatively analyzed complete mtDNA sequences of 31 Chinese sCJD patients and 32 controls. Using MITOMASTER and PhyloTree, we characterized 520 variants in sCJD patients and 507 variants in control by haplogroup and allele frequencies. We classified the mtDNAs into 40 sub-haplogroups of 5 haplogroups, most of them being Asian-specific haplogroups. Haplogroup U, an European-specific haplogroups mtDNA, was found only in sCJD. The analysis to control region (CR) revealed a 31% increase in the frequency of mtDNA CR mutations in sCJD versus controls. In functional elements of the mtDNA CR, six CR mutations were in conserved sequence blocks I (CSBI) in sCJD, while only one in control (P<0.05). More mutants in transfer ribonucleic acid-Leu (tRNA-Leu) were detected in sCJD. The frequencies of two synonymous amino-acid changes, m.11467A>G, p.(=) in NADH dehydrogenase subunit 4 (ND4) and m.12372G>A, p.(=) in NADH dehydrogenase subunit 5 (ND5), in sCJD patients were higher than that of controls. Our study, for the first time, screened the variations of mtDNA of Chinese sCJD patients and identified some potential disease-related mutations for further investigations.