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. 2013 Oct 8;110(41):16562-7.
doi: 10.1073/pnas.1310249110. Epub 2013 Sep 10.

U1 small nuclear ribonucleoprotein complex and RNA splicing alterations in Alzheimer's disease

Bing Bai  1 , Chadwick M Hales, Ping-Chung Chen, Yair Gozal, Eric B Dammer, Jason J Fritz, Xusheng Wang, Qiangwei Xia, Duc M Duong, Craig Street, Gloria Cantero, Dongmei Cheng, Drew R Jones, Zhiping Wu, Yuxin Li, Ian Diner, Craig J Heilman, Howard D Rees, Hao Wu, Li Lin, Keith E Szulwach, Marla Gearing, Elliott J Mufson, David A Bennett, Thomas J Montine, Nicholas T Seyfried, Thomas S Wingo, Yi E Sun, Peng Jin, John Hanfelt, Donna M Willcock, Allan Levey, James J Lah, Junmin Peng
Affiliations

U1 small nuclear ribonucleoprotein complex and RNA splicing alterations in Alzheimer's disease

Bing Bai et al. Proc Natl Acad Sci U S A. .

Abstract

Deposition of insoluble protein aggregates is a hallmark of neurodegenerative diseases. The universal presence of β-amyloid and tau in Alzheimer's disease (AD) has facilitated advancement of the amyloid cascade and tau hypotheses that have dominated AD pathogenesis research and therapeutic development. However, the underlying etiology of the disease remains to be fully elucidated. Here we report a comprehensive study of the human brain-insoluble proteome in AD by mass spectrometry. We identify 4,216 proteins, among which 36 proteins accumulate in the disease, including U1-70K and other U1 small nuclear ribonucleoprotein (U1 snRNP) spliceosome components. Similar accumulations in mild cognitive impairment cases indicate that spliceosome changes occur in early stages of AD. Multiple U1 snRNP subunits form cytoplasmic tangle-like structures in AD but not in other examined neurodegenerative disorders, including Parkinson disease and frontotemporal lobar degeneration. Comparison of RNA from AD and control brains reveals dysregulated RNA processing with accumulation of unspliced RNA species in AD, including myc box-dependent-interacting protein 1, clusterin, and presenilin-1. U1-70K knockdown or antisense oligonucleotide inhibition of U1 snRNP increases the protein level of amyloid precursor protein. Thus, our results demonstrate unique U1 snRNP pathology and implicate abnormal RNA splicing in AD pathogenesis.

Keywords: RNA-seq; U1A; liquid chromatography-tandem mass spectrometry; premature cleavage and polyadenylation; proteomics.

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Conflict of interest statement

The authors declare no conflict of interest.

Figures

Fig. 1.
Fig. 1.
Proteomic comparison reveals that U1-70K and U1A are enriched in the sarkosyl-insoluble proteome of AD. (A) Scheme for profiling the aggregated proteins in AD postmortem brains, with nondemented cases as controls (Ctl). (B) A stained SDS gel showing detergent-insoluble proteins in one set of pooled control and AD cases. (C) Similar proteomics analysis of seven groups of neurodegenerative disease samples. One set of sarkosyl-insoluble fractions was immunoblotted by phosphorylated tau antibodies to confirm tauopathies. (D) Relative level of representative sarkosyl-insoluble proteins across different diseases. The level was estimated by spectral counts of these identified proteins, and normalized to set the maximum to 100. Two replicates were analyzed, and the bars indicate the values of mean ± SEM. (E–I) Western blotting analysis of U1-70K or U1A in biochemical brain extracts from control and neurodegenerative cases, and the strategy for protein sequential extraction. The case numbers are shown. B, blank. The exposure time was longer in I Left than in others. At least one AD sample and one control sample were loaded on every gel for comparison.
Fig. 2.
Fig. 2.
U1-70K and U1A show neurofibrillary tangles in AD pathology. (A–D) Representative immunohistochemistry images with diaminobenzidine staining of selected control and AD brain slides (50-µm sections). (Scale bar, 5 μm.) (E–H) Representative adjacent sections of FTLD-U and FTLD-tau cases demonstrating normal U1-70K distribution despite the presence of TDP-43 and tau pathology, respectively. (I–L) Double-immunofluorescence staining indicates partial colocalization of U1-70K with tau in AD. (Scale bar, 5 μm.)
Fig. 3.
Fig. 3.
RNA splicing impairment in AD, and APP up-regulation upon splicing inhibition. (A) The frequency of summed intron reads is higher in AD than in control. The bars indicate mean ± SEM (P value derived by Student t test). The Emory and UKY samples were processed independently. The batch discrepancy may be due to sample quality difference and experimental variations. (B) The histograms of splicing deficiency scores of all mapped genes show a statistically significant difference between AD and control in both Emory and UKY groups (P < 2.2 × 10−16 for both groups, Kolmogorov–Smirnov test). (C) Evaluation of RNA splicing efficiency by measuring mRNAs and pre-mRNAs of selected genes in control and AD cases. The bars indicate the values of mean ± SEM (AD: n = 15; control: n = 14; asterisks: P < 0.05, Student t test). (D) Poly(A)-containing reads from 5′ to 3′ of every gene were defined and normalized according to the total poly(A) reads of the gene. Every transcript was divided into coding region (0–100, from start to stop codon) and 3′ UTR region (100–200), then into 20 bins. The poly(A) read percentage in each bin was averaged for all genes in every case, and plotted to represent the frequency of PCPA. The PCPA frequency was markedly different between control and AD cases (P < 2.2 × 10−16 for Emory group, P < 6.9 × 10−15 for UKY group, Kolmogorov–Smirnov test). (E) U1-70K knockdown increases APP and Aβ40 levels in HEK293 cells. The cells were transfected for 2 d, then cultured in a low-serum medium and harvested at day 0, 1, and 2 for analysis (asterisks: P < 0.05, Student t test; N.D., not detected). APP and Aβ40 were analyzed by immunoblotting and ELISA, respectively. (F) PCR to examine the specificity of U1 AMO. The reaction was designed to amplify the U1 RNA 5′-end region with the addition of control AMO or U1 AMO as inhibitory competitor. (G) The APP level increases upon AMO inhibition of U1 snRNP.
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