The intellectual disability gene PQBP1 rescues Alzheimer’s disease pathology

Early-phase pathologies of Alzheimer’s disease (AD) are attracting much attention after clinical trials of drugs designed to remove beta-amyloid (Aβ) aggregates failed to recover memory and cognitive function in symptomatic AD patients. Here, we show that phosphorylation of serine/arginine repetitive matrix 2 (SRRM2) at Ser1068, which is observed in the brains of early phase AD mouse models and postmortem end-stage AD patients, prevents its nuclear translocation by inhibiting interaction with T-complex protein subunit α. SRRM2 deficiency in neurons destabilized polyglutamine binding protein 1 (PQBP1), a causative gene for intellectual disability (ID), greatly affecting the splicing patterns of synapse-related genes, as demonstrated in a newly generated PQBP1-conditional knockout model. PQBP1 and SRRM2 were downregulated in cortical neurons of human AD patients and mouse AD models, and the AAV-PQBP1 vector recovered RNA splicing, the synapse phenotype, and the cognitive decline in the two mouse models. Finally, the kinases responsible for the phosphorylation of SRRM2 at Ser1068 were identified as ERK1/2 (MAPK3/1). These results collectively reveal a new aspect of AD pathology in which a phosphorylation signal affecting RNA splicing and synapse integrity precedes the formation of extracellular Aβ aggregates and may progress in parallel with tau phosphorylation.

３ Supplementary Figure 2 SRRM2 phosphorylation at pSer1068 was increased in specific brain regions Immunohistochemistry of 5xFAD and B6/SJL mice at 1, 3, and 6 months of age revealed an increase of pSer1068-SRRM2 in neurons. Supplementary data to Figure 1c are shown. Cytoplasmic staining of neurons was detected in secondary motor cortex (M2), secondary visual cortex (V2), and the other cortex areas. Cytoplasmic staining of neurons at field CA1 in the hippocampus (CA1) did not change considerably at 1 and 3 months, although some cytoplasmic granules of pSer1068-SRRM2 were detected in 5xFAD mice at 6 months. No definite stains of pSer1068-SRRM2 were detected in the cerebellum and brain stem. Quantitative analyses of intensities are shown in right graphs at each time point and in each area. Signal intensities were determined in six cytoplasmic areas of a single cell, and the mean was adjusted to the background intensity. The corrected mean values from 30 cells in each brain area were used to calculate the representative value of a mouse. Statistical analysis was performed with the values of three mice in each area at each time point by Student's ttest.

Supplementary Figure 3
Phosphorylation at Ser1068 shifts SRRM2 to the cytoplasm a. Antigen regions of two anti-SRRM2 antibodies and anti-pSer1068-SRRM2 antibody. b. Anti-pSer1068-SRRM2 antibody stained cytoplasmic granules of pSer1068-SRRM2, while anti-SRRM2 antibody (ab122719) stained nuclear speckles of non-phosphorylated SRRM2. c. High osmolarity increased pSer1068-SRRM2 in cytoplasm. The pSer1068-SRRM2 did not merge with cytoplasmic stress granule (TIA), P-body (GW182) or nuclear stress granule (HSF1). The concept of exon skipping and calculation of the skipping ratio. The exon skipping ratios of whole exons from all genes were compared between wild-type male and Synapsin-Cre PQBP1-cKO male mice, and genes containing more than one exon that changed significantly in Fisher's exact test were selected for further analyses. b. Gene ontology (GO) enrichment analysis of selected genes in a. c. Network analysis of GO-enriched groups indicated neuron-specific PQBP1-deficiency affects mainly synapse function, cytoskeleton and RNA metabolism. d. A functional network of genes showing pathologically altered splicing under conditions of PQBP1 deficiency in mature neurons (Synapsin-Cre PQBP1-cKO) was generated based on the integrated protein-protein interaction database (http://genomenetwork.nig.ac.jp/index_e.html). The high quality figure is posted at http://suppl.atgc.info/021/. e. Core genes were selected based on the betweenness score in the pathological network under conditions of PQBP1 deficiency in mature neurons. The top three core genes with the highest betweenness scores were GAPDH, Ywhae (14-3-3 e), and hnRNPK. APP was ranked at the 5 th position.

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Supplementary Figure 7 PQBP1 rescues synapse and cognitive function of female 5xFAD mice a. AAV-PQBP1 mediated rescue of synapse pathology in 5xFAD female mice. 5xFAD female mice received a single injection of AAV-PQBP1 into RSD at 5.5 months and were evaluated 2 weeks later at 6 months of age. The protocol was exactly similar to that for male 5xFAD mice. b. Two-photon microscopic images of dendritic spines in the first layer of RSD in 5xFAD or B6/SJL female mice after injection of AAV-EGFP or AAV-PQBP1. The right graph shows the quantitative analysis of spine number. c. Two-photon microscopic images of contact of axon terminals and dendritic spines in the first layer of RSD in 5xFAD female mice after injection of AAV-Vamp-Cherry with AAV-EGFP or AAV-PQBP1. The right graph shows the quantitative analysis of the axon terminals merged on the spine. d. Alteration ratios in the Y-maze test of 5xFAD female mice after injection of AAV-EGFP or AAV-PQBP1 are shown.

Supplementary Figure 8 AAV-PQBP1 rescues RNA splicing of synapse-related genes in 5xFAD mice
We compared genes changed in splicing (p <0.05 in Fisher's exact test, q-value is also sown) between PQBP1-Syn-Cre mice and 5xFAD mice. 47 genes and their 103 exons were commonly changed in two mouse models. The lower Ben graph shows recovery of the abnormal RNA splicing by AAV-PQBP1. 41 in 103 exons and 11 in 47 genes were recovered with a statistical significance. Corresponding Supplementary Table and Sheet Numbers are indicated.

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Supplementary Figure 9 The Erk1 pathway was not significantly activated at later AD stages Pathway analysis of downstream target proteins of Erk1/2 and Clk1. Proteins shown in red indicate increased phosphorylation at 3, 6, and 12 month of age in whole cerebral cortex tissues of 5xFAD. The high quality figures are posted at http://suppl.atgc.info/021/.