Phosphorylated forms of microtubule-associated protein tau accumulate in neurofibrillary tangles in Alzheimer's disease. To investigate the effects of specific phosphorylated tau residues on its function, wild type or phosphomutant tau was expressed in cells. Elevated tau phosphorylation decreased its microtubule binding and bundling, and increased the number of motile tau particles, without affecting axonal transport kinetics. In contrast, reducing tau phosphorylation enhanced the amount of tau bound to microtubules and inhibited axonal transport of tau. To determine whether differential tau clearance is responsible for the increase in phosphomimic tau, we inhibited autophagy in neurons which resulted in a 3-fold accumulation of phosphomimic tau compared with wild type tau, and endogenous tau was unaffected. In autophagy-deficient mouse embryonic fibroblasts, but not in neurons, proteasomal degradation of phosphomutant tau was also reduced compared with wild type tau. Therefore, autophagic and proteasomal pathways are involved in tau degradation, with autophagy appearing to be the primary route for clearing phosphorylated tau in neurons. Defective autophagy might contribute to the accumulaton of tau in neurodegenerative diseases.
Frontotemporal dementia with parkinsonism linked to chromosome 17 (FTDP-17) is caused by mutations in the gene encoding the microtubule-associated protein, tau. Some FTDP-17 mutations affect exon 10 splicing. To correct aberrant exon 10 splicing while retaining endogenous transcriptional control, we evaluated the feasibility of using spliceosome-mediated RNA trans-splicing (SMaRT) to reprogram tau mRNA. We designed a pre-trans-splicing molecule containing human tau exons 10 to 13 and a binding domain complementary to the 3 end of tau intron 9. A minigene comprising tau exons 9, 10, and 11 and minimal flanking intronic sequences was used as a target. RT-PCR analysis of SH-SY5Y cells or COS cells cotransfected with a minigene and a pre-trans-splicing molecule using primers to opposite sides of the predicted splice junction generated products containing exons 9 to 13. Sequencing of the chimeric products showed that an exact exon 9 -exon 10 junction had been created, thus demonstrating that tau RNA can be reprogrammed by trans-splicing. Furthermore, by using the same paradigm with a minigene containing full-length intronic sequences, we show that cis-splicing exclusion of exon 10 can be by-passed by trans-splicing and that conversion of exon 10 ؊ tau RNA into exon 10 ؉ tau RNA could be achieved with Ϸ34% efficiency. Our results demonstrate that an alternatively spliced exon can be replaced by trans-splicing and open the way to novel therapeutic applications of SMaRT for tauopathies and other disorders linked to aberrant alternative splicing.T au is a microtubule-associated protein predominantly expressed in neurons and specifically localized in axons that promotes microtubule polymerization and stabilization (1, 2). Mutations in the MAPT gene, encoding tau, on chromosome 17, cause frontotemporal dementia with parkinsonism linked to chromosome 17 (FTDP-17) (3-5). FTDP-17 is characterized by intraneuronal aggregates of tau and, as such, belongs to the group of dementias that includes Alzheimer's disease and collectively referred to as tauopathies.The human MAPT gene is a large gene of Ϸ134 kb comprising 16 exons (6). Alternative splicing of exons 2, 3, and 10 in the tau pre-mRNA results in the expression of six isoforms in the brain. Exon 10 encodes the second of four imperfect microtubulebinding repeats in the C-terminal half of the tau protein.Exclusion or inclusion of exon 10 gives rise to tau isoforms with three (tau3R, exon 10 Ϫ ) or four (tau4R, exon 10 ϩ ) microtubulebinding repeats (7). Furthermore, inclusion or exclusion of exons 2 and 3 produces tau isoforms with zero, one, or two inserts near the N terminus. Only tau3R is expressed during embryogenesis whereas tau3R and tau4R are expressed in approximately equal amounts in adult human brain. Exon 10 splicing is regulated by multiple cis-acting elements comprising exonic and intronic silencers and enhancers (for reviews, see refs. 8 and 9). In particular, intron 10 contains a bipartite element comprising a silencer and a modulator downstream to the 5Ј splice...
scite is a Brooklyn-based organization that helps researchers better discover and understand research articles through Smart Citations–citations that display the context of the citation and describe whether the article provides supporting or contrasting evidence. scite is used by students and researchers from around the world and is funded in part by the National Science Foundation and the National Institute on Drug Abuse of the National Institutes of Health.
hi@scite.ai
10624 S. Eastern Ave., Ste. A-614
Henderson, NV 89052, USA
Copyright © 2024 scite LLC. All rights reserved.
Made with 💙 for researchers
Part of the Research Solutions Family.