Analysis of the molecular heterogeneity of the microtubule-associated protein tau by two-dimensional electrophoresis and RT-PCR.
The microtubule-associated protein tau is a member of a group of proteins, promoting assembly and stabilization of microtubules. In several tauopathic neurodegenerative disorders, namely Alzheimer's and Pick's disease and frontotemporal dementia with parkinsonism linked to chromosome 17 (FTDP 17) this protein is converted into fibrilar polymers which form the component of insoluble proteanous deposits such as neurofibrillary tangles. The formation of these fibrils is believed to interrupt the physiological function of neurons resulting in degeneration and cell death. Tau protein exists as a family of heterogeneous isoforms derived by both, differential splicing of tau-mRNA and posttranslational modification of the protein. Since the role of the different isoforms during the process of neurodegeneration is not well understood and as their balance might be altered in some cases of tauopathies (Spillantini et al., Proc. Natl. Acad. Sci. USA 1998;95:7737-7741), the detailed analysis of the molecular heterogeneity gained outstanding interest. The method presented here allows the analysis of both, differential splicing and phosphorylation of tau protein by the application of two-dimensional (2D) electrophoresis and Western blot detection. Tau protein isoforms could be identified from the 2D pattern of dephosphorylated tau in concordance with the results of tau-mRNA analysis by RT-PCR. The protocol presented was successfully applied to analysis of tau isoforms of human brain (Janke et al., FEBS Lett. 1996;379:222-226) and of several species, revealing a phylogenetic correlation of tau protein patterns in mammals (Janke et al., Mol. Brain Res. 1999;68:119-128). The present paper provides a detailed description of the technique and discusses its prospects and limits.
Janke
C
C
Department of Neuroanatomy, University of Leipzig, Paul Flechsig Institute of Brain Research, Jahnallee 59, 04109, Leipzig, Germany.
Beck
M
M
Holzer
M
M
Bigl
V
V
Arendt
T
T
D000818:Animals; D001483:Base Sequence; D015153:Blotting, Western; D003704:Dementia / Q000235:genetics; D015180:Electrophoresis, Gel, Two-Dimensional / Q000379:methods*; D018740:Genetic Heterogeneity; D006801:Humans; D008969:Molecular Sequence Data; D010766:Phosphorylation; D012326:RNA Splicing / Q000235:genetics; D012333:RNA, Messenger / Q000032:analysis; D020133:Reverse Transcriptase Polymerase Chain Reaction / Q000379:methods*; D016875:tau Proteins / Q000032:analysis / Q000235:genetics* / Q000378:metabolism*
D012333:RNA, Messenger; D016875:tau Proteins