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. 2022 Sep 13;119(37):e2204179119.
doi: 10.1073/pnas.2204179119. Epub 2022 Sep 6.

Truncated Tau caused by intron retention is enriched in Alzheimer's disease cortex and exhibits altered biochemical properties

Zhen-Kai Ngian  1   2 , Yow-Yong Tan  1   2 , Ching-Thong Choo  1 , Wei-Qi Lin  1 , Chao-Yong Leow  1 , Shan-Jie Mah  1 , Mitchell Kim-Peng Lai  3   4 , Christopher Li-Hsian Chen  3   4 , Chin-Tong Ong  1   2
Affiliations

Truncated Tau caused by intron retention is enriched in Alzheimer's disease cortex and exhibits altered biochemical properties

Zhen-Kai Ngian et al. Proc Natl Acad Sci U S A. .

Abstract

Alzheimer's disease (AD) is characterized by the accumulation of amyloid-β plaques and Tau tangles in brain tissues. Recent studies indicate that aberrant splicing and increased level of intron retention is linked to AD pathogenesis. Bioinformatic analysis revealed increased retention of intron 11 at the Tau gene in AD female dorsal lateral prefrontal cortex as compared to healthy controls, an observation validated by quantitative polymerase chain reaction using different brain tissues. Retention of intron 11 introduces a premature stop codon, resulting in the production of truncated Tau11i protein. Probing with customized antibodies designed against amino acids encoded by intron 11 showed that Tau11i protein is more enriched in AD hippocampus, amygdala, parietal, temporal, and frontal lobe than in healthy controls. This indicates that Tau messenger RNA with the retained intron is translated in vivo instead of being subjected to nonsense-mediated decay. Compared to full-length Tau441 isoform, ectopically expressed Tau11i forms higher molecular weight species, is enriched in Sarkosyl-insoluble fraction, and exhibits greater protein stability in cycloheximide assay. Stably expressed Tau11i also shows weaker colocalization with α-tubulin of microtubule network in human mature cortical neurons as compared to Tau441. Endogenous Tau11i is enriched in Sarkosyl-insoluble fraction in AD hippocampus and forms aggregates that colocalize weakly with Tau4R fibril-like structure in AD temporal lobe. The elevated level of Tau11i protein in AD brain tissues tested, coupled with biochemical properties resembling pathological Tau species suggest that retention of intron 11 of Tau gene might be an early biomarker of AD pathology.

Keywords: Alzheimer disease; Tau; intron retention.

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Conflict of interest statement

The authors declare no competing interest.

Figures

Fig. 1.
Fig. 1.
Increased retention of intron 11 of Tau gene in AD female. (A) Intron 11 contains a premature stop codon (red) with a canonical polyadenylation site that is located 1 kb downstream. Arrows indicate qPCR primers. (B) Integrative genomic viewer of intron 11 from control (blue) and AD (orange) patients. (C, Left) Statistically significant higher intron retention (IR) is observed only in female AD dorsal lateral prefrontal cortex (n = 34) as compared to control (CT, n = 32). Each dot represents individual normalized IR ratio. Right: No differential expression in the Tau gene between CT and AD cohorts. (D) qPCR validation of intron 11 (relative to Tau exon1/exon2) and Tau expression (exon1/exon 2 and exon5/exon6 relative to GAPDH) from three distinct pairs of brain tissues. FL, frontal lobe; TL, temporal lobe. Data presented as mean of triplicates ± SD, P values calculated by two-tailed t test.
Fig. 2.
Fig. 2.
Truncated Tau11i oligomerizes and is found in Sarkosyl-insoluble fraction. (A) Schematic of Flag-tag full-length and truncated Tau proteins with specific antibody (red) recognition sites. Amino acids encoded by intron 11 are colored orange. pT181, phosphor-Tau (T181); Tau4R, 275–291; Tau46, 404–421; AT8, phosphor-Tau (S202/T205). (B and C) Immunoblot and quantification of Tau11i HMW species in (B) 293T transfected with Tau441 or Tau11i, and (C) NSC expressing Tau441 or Tau11i proteins. Data presented as mean ± SEM. (D) Immunoblot and quantification showed elevated Sarkosyl-insoluble fraction of Tau11i in 293T cells (n = 7), NPC and day 44 neurons (n = 7) expressing Flag-Tau441 or Flag-Tau11i. The level of insoluble Tau was first normalized to total lysate. The relative level of insoluble fraction from each biological replicate was combined and presented as mean ± SD. All P values were calculated by two-tailed t test.
Fig. 3.
Fig. 3.
Elevated level of Tau11i in different AD brain tissues. (A) Immunoblot of control (blue) and AD (orange) temporal lobe, amygdala (AMYDG.), parietal lobe, frontal lobe, and hippocampus showed enrichment of Tau11i (red arrowhead) in AD samples. Samples were also immunoblotted with AT8 and actin antibodies. Each number depicts unique human subject. Quantification of seven pairs of control and AD samples with data presented as mean ± SD. The level of Tau11i in control for each brain region was set as 1. Sample quantified was underlined. The P value was calculated by paired two-tailed t test. (B) Immunoblots showed that Tau11i is found in Sarkosyl-insoluble fraction (red arrow) in AD hippocampus and temporal lobe. 10% of total lysate (Lys), 15% of soluble fraction (Sol.) and 50% of Sarkosyl-insoluble fraction (Insol.) were loaded. Tech Rep, technical replicate. (C) Immunofluorescence showed weak colocalization of Tau11i aggregates with Tau4R fibrils in AD temporal lobe. (Scale bar: 20 μm.) Each circle represents one field. Data presented as mean ± SD with P values calculated by two-tailed t test.
Fig. 4.
Fig. 4.
Tau11i protein associates poorly with microtubule network and is more stable. (A) Images of Day 21 and 44 neurons expressing Tau441 or Tau11i stained with α-tubulin and Flag antibodies. (Scale bar: 50 μm.) Quantification showed lower colocalization of Tau11i with α-tubulin as compared to Tau441. Each circle represents one field with filled circle from second biological replicate. Data presented as mean ± SD (B) Immunoblot using Flag and actin antibodies in 293T cells harvested on different day “D” after transfection. Quantification of Tau level (D7 relative to D3) is presented as mean ± SEM (n = 5). (C) Immunoblot of Tau level after cycloheximide (CHX) treatment of 293T cells on day 5 after transfection. Rep, replicate. Quantification of Tau level (8 h relative to 0 h) is presented as mean ± SD where each circle represents one replicate (n = 12). All P values were calculated by two-tailed t test.
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