Research reportMissense tau mutations identified in FTDP-17 have a small effect on tau–microtubule interactions
Introduction
Formation of filamentous inclusions consisting predominantly of microtubule (MT)-associated protein tau is a cardinal neuropathologic feature of many neurological disorders including Alzheimer's disease (AD), Pick's disease (PD), corticobasal degeneration (CBD), progressive supranuclear palsy (PSP) and frontotemporal dementia with Parkinsonism linked to chromosome 17 (FTDP-17) 7, 9, 28. The morphology and distribution of the lesions and the ultrastructural appearance of filaments vary in these disorders. Depending on their structure, the filaments are referred to as paired helical filaments, twisted ribbons or straight filaments [4]. In affected neurons, the accumulation of tau filaments is thought to be accompanied by depletion of MT [20], raising the possibility that the formation of inclusions may be related to the disruption of the MT system. This is presumably due to abnormal modifications of MT-associated proteins [15]. This view is supported by findings in which tau proteins extracted from PHF exhibit a decreased ability to promote the assembly of purified tubulin [1]and are abnormally modified by phosphorylation, glycosylation, ubiquitination and glycation [35]. Moreover, in vitro phosphorylation or glycation has been documented to reduce the function of tau in MT polymerization [18].
Recent genetic analyses of FTDP-17 revealed that some families have mutations or deletion in the tau gene 13, 14, 27, 29. These mutations were found in exons 9–13 and in the intron at the 5′ splice site of exon 10 (+3, +13, +14 and +16). The missense mutations reside mainly in a portion of tau protein whose normal function involves MT-binding [8]. This region, referred to as the MT-binding domain, is composed of imperfect repeats of 31 or 32 amino acid residues. Based on the numbering of the longest tau isoform (441 amino acids), the six missense mutations and a deletion identified to date include: G272V, N279K, Δ280, P301L, S305N, V337M and R406W 13, 14, 24, 27, 29. Three of these mutations, N279K, P301L, and S305N being within exon 10, are only expressed in four-repeat tau, but the others are found in both three- and four-repeat tau. The MT-binding domain has been established as the core component of Alzheimer paired helical filaments [32], and in vitro assembly studies with recombinant tau fragments revealed that this domain contains all the information needed to form a myriad of structures including paired helical filaments 5, 31. The splice site mutations have been proposed to destabilize the formation of a stemloop structure in tau pre-mRNA which leads to an increase in the splicing of exon 10 and thus to an increase in the ratio of four-repeat to three-repeat tau [13].
The missense mutations have recently been shown to reduce the ability of tau to promote MT assembly 11, 12, raising the possibility that these mutations may cause neurodegeneration in FTDP-17 through the inhibition of tau–MT interactions. This could lead to an increase in the pool of soluble tau available for tau filament assembly. There is disagreement, however, as to whether the P301L or R406W mutation has the most impact on the ability of tau to promote MT assembly in vitro. It is also not clear whether the defect observed in cell-free systems can be detected in cells expressing mutant tau. The present report focused on the ability of the wild type and mutant (P301L, V337M and R406W) four-repeat tau to bind MT, to bundle MT and to promote MT assembly in cell-free assays and in transfected cultured cells. The results indicate that in cell-free assays only recombinant P301L tau has a significant impact on both MT binding and polymerization. Cultured neuronal or non-neuronal cells transfected with mutant tau are qualitatively indistinguishable from wild type transfectants with respect to the distribution of tau-decorated MT networks.
Section snippets
Construction of four-repeat tau vectors
The wild type tau cDNA containing four repeats and two amino-terminal inserts was obtained from a T40 clone [10]encoding the longest isoform of human brain tau (kindly provided by Dr. M. Goedert, Medical Research Council, Laboratory of Molecular Biology, Cambridge, UK) and cloned into pUC19 vector using NdeI and EcoRI. The cDNA was mutated using the Gene Editor site-directed mutagenesis kit (Promega, Madison, WI). The sequence for the bottom strand, selection oligo was:5′
Results
Recombinant tau proteins were isolated from bacteria lysates and tubulin was purified from bovine brains. These preparations were demonstrated by gel electrophoresis to be highly homogeneous (Fig. 1B and C). Each contained a major band with molecular weight consistent with that of tubulin or tau. The electrophoretic mobility of different mutant tau proteins was comparable to that of wild type tau. The results indicate that these mutations do not mimic phosphorylation events in vivo as observed
Discussions
Our studies demonstrate that in a cell-free system, the P301L mutation significantly reduced the ability of tau to bind and polymerize MTs, while the V337M and R406W mutations showed smaller effects on the ability of tau to function as a MT-associated protein. This data is in partial agreement with that recently obtained by Hasegawa et al. [11]. Similar to the present results, Hasegawa et al. found that the P301L mutant exhibits the greatest effect on MT assembly, and the mutation has very
Acknowledgements
We thank Dr. W-L Lin for help with photography and Dr. D.W. Dickson for helpful discussions and comments on the manuscript. This work was supported by NIH grants AG01136 (SHY) and NS37143 (MH).
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