Increased APOE abundance in the brain in Down syndrome - Alzheimer’s disease compared with early onset-Alzheimer's disease is associated with elevated APP and C- terminal fragment abundance

Trisomy of chromosome 21, the cause of Down syndrome (DS), is the most commonly occurring genetic cause of Alzheimer’s disease (AD). Here we compare the proteome of the frontal cortex of people who have Down syndrome-Alzheimer’s disease (DSAD) with demographically matched cases of early-onset AD and healthy ageing controls from the general population. We find the abundance of several chromosome 21 encoded proteins are increased in DSAD compared to both comparative groups. Moreover, wider dysregulation of the proteome occurs beyond proteins encoded by chromosome 21, including an increase in the abundance of the key AD protein APOE, in people with DSAD compared to matched cases of early-onset AD. To understand the cell-types which may contribute to these changes in protein abundance we undertook a single-nuclei RNA-sequencing study on the same cases. This demonstrated that trisomy of chromosome 21 altered transcription of these proteins in a range of cell-types. Specifically, APOE expression was raised in subtypes of astrocytes, endothelial cells and pericytes. In our case series, we also found that the abundance of APOE correlates with the abundance of full-length APP and C-terminal-fragments-α but not with amyloid-β, the key constituent of AD plaque pathology. Having increased abundance of APOE may impact the development of, or response to, AD pathology in the brain of people who have DSAD, altering the disease mechanisms, with clinical implications. Overall, these data highlight that trisomy 21 alters both the transcriptome and proteome of people who have DS in the context of AD, and that these differences should be considered when selecting therapeutic strategies for this important group of individuals who are at high risk of early-onset dementia. Nuclei were isolated from frozen frontal cortex grey matter from discovery cohort cases (8 DS-AD, 4 EOAD, and 4 healthy controls). Tissue was transferred directly onto ice-cold sucrose buffer and homogenised by hand. The homogenate was layered on top of a sucrose cushion and nuclei were recovered after centrifugation. Nuclei were resuspended in PBS containing BSA (to prevent clumping) and RNase inhibitors, counted and diluted to 1,000 nuclei/μl. Sequencing libraries were prepared using the “single cell 3’ library kit v3” with the Chromium instrument (10X Genomics). Aiming to obtain libraries for ~5,000 independent nuclei per sample. Libraries were sequenced on an Illumina NovaSeq6000 instrument (UCL Genomics), to an average of at least 100,000 reads/nucleus. Raw sequencing data were processed through the software CellRanger (10X Genomics) to assign each transcript read to its respective nucleus, we performed alignment to the transcriptome, counting reads, and processing of the unique molecular identifier tag to reduce PCR bias. The resulting gene count table was analysed using the Seurat package (version 4.4). Quality control was performed to remove low-quality cells based on the number of detected genes and unique molecular identifiers (UMIs). Cells with an unusually high number of UMIs, suggesting potential doublet events, were also excluded using scDblFinder. Using SCTransform, gene expression data were normalised to adjust for differences in sequencing depth between cells and scaled to correct for cell-to-cell variation in total UMI count. Data were also normalised to remove confounding sources of variation such as mitochondrial mapping percentage, post-mortem delay, age of death, and batch number.