Proteogenomics Reveals Orthologous Alternatively Spliced Proteoforms in the Same Human and Mouse Brain Regions with Differential Abundance in an Alzheimer's Disease Mouse Model

Abstract

Alternative splicing (AS) may increase the number of proteoforms produced by a gene. Alzheimer's disease (AD) is a neurodegenerative disease with well-characterized AS proteoforms. In this study, we used a proteogenomics strategy to build a customized protein sequence database and identify orthologous AS proteoforms between humans and mice on publicly available shotgun proteomics (MS/MS) data of the corpus callosum (CC) and olfactory bulb (OB). Identical proteotypic peptides of six orthologous AS proteoforms were found in both species: PKM1 (gene PKM/Pkm), STXBP1a (gene STXBP1/Stxbp1), Isoform 3 (gene HNRNPK/Hnrnpk), LCRMP-1 (gene CRMP1/Crmp1), SP3 (gene CADM1/Cadm1), and PKCβII (gene PRKCB/Prkcb). These AS variants were also detected at the transcript level by publicly available RNA-Seq data and experimentally validated by RT-qPCR. Additionally, PKM1 and STXBP1a were detected at higher abundances in a publicly available MS/MS dataset of the AD mouse model APP/PS1 than its wild type. These data corroborate other reports, which suggest that PKM1 and STXBP1a AS proteoforms might play a role in amyloid-like aggregate formation. To the best of our knowledge, this report is the first to describe PKM1 and STXBP1a overexpression in the OB of an AD mouse model. We hope that our strategy may be of use in future human neurodegenerative studies using mouse models.

Keywords: Alzheimer’s disease; mRNA splicing; neurodegeneration; proteogenomics.

Figure 1

Flowchart for ortholog assessment.

Figure 2

Distribution of genes and peptides across the corpus callosum and olfactory bulb in humans and mice. (A) Genes of canonical proteoforms with orthologous relationships between humans and mice or those shared across brain tissues are represented at intersections; (B) genes of noncanonical proteoforms with orthologous relationships between humans and mice or those shared across brain tissues are represented at intersections; (C) peptides of canonical proteoforms with identical sequences across human and mouse and brain regions are represented at intersections; (D) peptides of noncanonical proteoforms with identical sequences across human and mouse and brain tissues are represented at intersections.

Figure 3

Exons and shared peptides of the orthologous alternatively spliced proteoforms. (A) Table with general information about the human (Hs) and mouse (Mm) proteoforms: gene symbol, proteoform name, identity, splicing pattern, identified peptides, and their spectral count in each brain region. (B) Schematic representation of the spliced transcripts of the genes CADM1/Cadm1; STXBP1/Stxbp1; CRMP1/Crmp1; HNRNPK/Hnrnpk; PKM/Pkm; PRKCB/Prkcb and the exon location of their identified peptides. Human proteoforms are represented in blue and mouse proteoforms are represented in green. Exons are represented by full (coding sequences) and empty (UTRs) boxes, while introns are represented by lines. Each peptide is represented with orange boxes. The two slashes in the intronic region indicate a fragment of mRNA structure not represented in the schematic.

Figure 4

Log₂ ratio of the normalized intensities of the AS proteoform peptides (genes Pkm, Stxbp1 and Crmp1). Samples more abundant in the wild-type (log₂ ratio < 0) are represented as red dots and samples more abundant in the AD model APP/PS1 (log₂ ratio > 0) are shown as blue dots. One-sample t-test; not significant (ns), p-value < 0.05 (*) and p-value < 0.01 (**).

Figure 5

RNA-Seq expression levels for the orthologous alternatively spliced proteoforms selected for mRNA experimental validation. (A) Comparative analysis of transcript expression levels in transcripts per million (TPM) across the corpus callosum and olfactory bulb in mice and humans. The Y-axis shows the expression of transcripts in TPM, and the X-axis shows the different transcripts. (B) Transcript expression levels (2^−∆∆Ct) of the AS variants of the genes Cadm1, Crmpd1, Hnrnpk, Pkm, Prkcb, and Stxbp in the mouse CC and OB. Two-sample t-test; p-value ≤ 0.05 (*) and p-value ≤ 0.01 (**).