07 Dec 2024

Strapped with an extra APP, people with Down’s syndrome are all but destined to develop Alzheimer’s dementia if they live past middle age. Compared with sporadic forms of the disease, DSAD starts younger and progresses faster. Yet few studies have compared the molecular signatures of DSAD to those of sporadic or familial AD. In a first in the field, researchers led by Vivek Swarup of the University of California, Irvine, merged single-cell and spatial transcriptomics to map the molecular signatures of DSAD and late-onset sporadic AD. On November 22 in Nature Genetics, they reported that the two forms of AD had much in common, but because DSAD is more aggressive, its signatures were sharper.

In pathology-ridden layers of the cortex, synaptic genes faded while inflammation flared. In women, inflammatory and vascular pathways stood out, while neuron and oligodendrocyte disease signatures dominated in men.

In DSAD, the scientists picked up signals of toxic communication between astrocytes and neurons; this may have hastened the demise of neurons in regions burdened with neuropathology. Finally, they detected packs of genes that changed expression around amyloid plaques. These overlapped with plaque-associated genes in a mouse model of amyloidosis. Together, the data allowed the scientists to identify amyloid-related disease signatures in their human samples.

Because the spatial transcriptomics zeros in on the molecular changes taking place in regions where the brain is most plagued by neuropathology, Swarup thinks it will help identify therapeutic targets.

The scientists also used an old mouse model of amyloidosis—5xFAD—to pinpoint amyloid-related molecular signatures. Mouse models of DS also exist. Making them is a challenge, because DS is caused by an extra copy of chromosome 21, and the genes on it are scattered across multiple chromosomes in mice. Alzforum recently added five DS models into its research model database, where they are described in detail: Dp1Tyb; Dp9Tyb; Dp(16)1Yey/+; TcMAC21; and ts65dn. None develop bona fide amyloid plaques, although some churn out excess Aβ peptides, accumulate hyperphosphorylated tau, and/or show signs of neurodegeneration.

Digging Deeper Into DSAD
Although it is the most common genetic cause of AD, only one single-cell study has ever been conducted on brain samples from people with DSAD, sans spatial transcriptomics (Palmer et al., 2021).

For their study, co-first authors Emily Miyoshi and Samuel Morabito and colleagues ran spatial transcriptomics on sections spanning all six layers of the frontal cortex from 10 controls, 29 people with late-onset AD, and 10 with DSAD. The LOAD samples were split into early stage and late stage based on amyloid plaque and neurofibrillary tangle pathology.

Swarup told Alzforum that across the board, DSAD samples contained a heftier burden of plaques and tangles than did those from people with LOAD. This even though, at an average age of 56, DSAD participants died three decades earlier. To corroborate their spatial transcriptomics data, the scientists used single-nucleus RNA sequencing on frontal and posterior cingulate cortical samples from 27 healthy controls and 21 people with DSAD. In addition, they ran spatial transcriptomics on 5xFAD mouse samples at various stages of amyloidosis to see how well the model tracked changes in AD (see workflow below).

AD in Time and Space. Spatial transcriptomes of frontal cortex samples from controls were compared with those from early stage LOAD, late-stage LOAD, DSAD (top), and from 5xFAD mice at progressive stages of amyloidosis (bottom). [Courtesy of Miyoshi et al., Nature Genetics, 2024.]

These analyses yielded a cornucopia of data, all available for interactive perusal online. A few highlights? The spatial transcriptomics analysis took stock of six neocortical layers and three white-matter layers, each of which had a distinct transcriptional profile (image below). In keeping with their extra copy of chromosome 21, people with DSAD had more APP transcripts relative to controls in most cortical layers, except for cortical L3/L4. Here, APP levels were the same as in controls. This echoes a broader trend of neuronal gene shushing in this region across both types of AD, while glial and vascular gene expression ramped up. L3/L4 of the cortex host dense synaptic connections and are heavily burdened by amyloid plaques. This implies the gene-expression changes observed here may reflect central disease-related mechanisms early in pathology, and are likely to impact cognition, the authors proposed.

Layer by Layer. Spatial transcriptomics identifies nine gene-expression clusters corresponding to the six known neocortical layers and three white-matter layers. Two representative samples from each group are shown. [Courtesy of Miyoshi et al., Nature Genetics, 2024.]

To characterize region-specific changes across the groups, Miyoshi and colleagues turned to gene co-expression analyses of differentially expressed genes, finding 15 cortex-wide “meta-modules.” Each was enriched for genes involved in specific functions, for example myelination, synaptic transmission, glutamate signaling, inflammatory responses, and amyloid fibril formation.

Findings from this analysis included an uptick of the “M6” module in neurons inhabiting the outermost layer of the cortex. M6 includes genes involved in APP metabolism, macroautophagy, and RNA splicing. This occurred in DSAD, as well as in both early and late stages of sporadic LOAD. An inflammatory module—M11—included genes involved in immune responses, neuronal death, the complement pathway, and disease-associated astrocyte genes. It ramped up broadly across cortical layers L1-L5 in DSAD, predominantly in L1 and L3/L4 in late-stage LOAD, and only faintly in early stage AD. In single-nucleus sequencing data, M11 genes were predominantly expressed in microglia, and correlated with AD risk.

Disease Landscape. The dendrogram shows 166 co-expression modules, grouped into 15 meta-modules with expression levels in cortical (L1-L6) and white matter (WM1-3) layers.

In the network plot (center) each dot is a gene colored by meta-module. Heatmap shows differential expression level in early and late-stage LOAD and in DSAD. [Courtesy of Miyoshi et al., Nature Genetics, 2024.]

The scientists identified striking differences between the sexes across the different types of AD, although because each diagnosis group was split by sex, sample sizes were small. In DSAD, more genes were upregulated among the seven women than in the three men across the board (image below). In women with DSAD, glia and the vasculature accounted for most of this, as genes involved in inflammation, oxidative stress, and glucose metabolism were cranked up across brain regions. In men, neurons and oligodendrocytes shouldered the brunt of differentially expressed genes, including those related to alternative splicing, chromatin organization, and cytoskeletal transport.

Sex Differences. More genes are upregulated in women than in men. [Courtesy of Miyoshi et al., Nature Genetics, 2024.]

Many of the elevated genes in women with DSAD, for example C1QB, hailed from the inflammatory M11 module. These genes were upregulated across cortical regions, with the strongest expression in white-matter layers (image below).

The scientists also identified sex differences in sporadic LOAD, where they were able to look at how those evolved from early to late-stage disease. For example, in layer 1 of the neocortex, inflammatory genes rose in the early stage of LOAD in men, but not until late-stage disease in women. Swarup thinks this might help explain the observation that women tend to keep cognitively stable for longer as their neuropathology builds up, but decline faster once symptoms start.

Can You Hear Me Now?
The scientists merged spatial and single nuclei transcriptomics to investigate how AD compromises cellular communication. Using CellChat, an algorithm that detects co-expressed ligand and receptor pairs, they mapped changes in those potential pairings to regional addresses in the brain. The findings hint at “toxic communication” between astrocytes and neurons. For example, in people with late-stage LOAD or DSAD, angiopoietin-like 4 signaling from astrocytes to upper layer neurons intensified. This pathway would be predicted to shut down neuronal survival signals, Swarup said. Meanwhile, neurons broadly hushed signaling through the synapse maintenance protein Nectin in DSAD and late-stage LOAD, possibly reflecting neurodegeneration in response to AD pathology, Swarup proposed.

Finally, the scientists deployed spatial techniques to zero in on gene-expression changes taking place around amyloid—both dense plaques and diffuse fibrillar ones. This turned up 65 and 215 genes associated with the two types of aggregate, respectively, of which 50 were in common. Many were expressed by microglia and astrocytes and in the inflammatory M11 module. Other plaque-induced genes figure in filament assembly, long-term potentiation, and blood-brain border transport, while genes associated with diffuse deposits were also related to synaptic function and hemopoiesis.

In 5xFAD mice, a similar analysis revealed substantial overlap in plaque-associated genes between mouse and human samples. Swarup said the findings bolster previous studies on plaque-induced genes, aka “PiGs” (Jul 2020 news; Feb 2023 news). The agreement across species suggests a common, amyloid-related signature at the heart of different forms of AD, the authors conclude.—Jessica Shugart

Comments

1. • Fanny Elahi
     Icahn School of Medicine at Mount Sinai
   • Posted: 10 Dec 2024

   The work led by Swarup, Miyoshi, and Head is a tour de force and a valuable contribution of incredibly rich datasets to the field. The design is strong with comparisons between early stage AD and late-stage AD and Down’s syndrome to derive shared and specific disease-associated insights. Given that no cell is an island and that we increasingly know that disrupted cell-cell communications and altered extracellular matrix are important disease components, the use of spatial transcriptomics provides the opportunity to investigate multicellular selective vulnerabilities.

   This is a very rich paper that confirms some things that were known, such as the importance of vascular and glial abnormalities, and adds novel molecular and cellular insights, such as spatial pathologies surrounding amyloid plaques. I was especially pleased to see their sex-specific analyses. We have recently reported on the role of angiogenesis in sex-dimorphic trajectories in brain aging (Torres-Espin et al., 2024). Here they found that many of the upregulated genes in women were glial and vascular, as opposed to neuronal or oligodendrocytic in men, and they noted vascular smooth-muscle cell proliferation, vascular development, and angiogenesis, among top dysregulated pathways in both AD and DSAD. 

   References:

   Torres-Espin A, Radabaugh HL, Treiman S, Fitzsimons SS, Harvey D, Chou A, Lindbergh CA, Casaletto KB, Goldberger L, Staffaroni AM, Maillard P, Miller BL, DeCarli C, Hinman JD, Ferguson AR, Kramer JH, Elahi FM. Sexually dimorphic differences in angiogenesis markers are associated with brain aging trajectories in humans. Sci Transl Med. 2024 Nov 27;16(775):eadk3118. Epub 2024 Nov 27 PubMed.

   View all comments by Fanny Elahi

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References

Mutations Citations

1. Trisomy 21

Research Models Citations

1. Dp1Tyb
2. Dp9Tyb
3. Dp(16)1Yey/+
4. TcMAC21
5. Ts65Dn

News Citations

1. Paper Alert: Those PIGs! Spatial Transcriptomics Add Human Data 30 Jul 2020
2. Higher-Resolution Spatial Transcriptomics Maps Mayhem Near Plaques 8 Feb 2023

Paper Citations

1. Palmer CR, Liu CS, Romanow WJ, Lee MH, Chun J. Altered cell and RNA isoform diversity in aging Down syndrome brains. Proc Natl Acad Sci U S A. 2021 Nov 23;118(47) PubMed.

External Citations

1. online

Further Reading

No Available Further Reading