Unweaving the Microscopic Threads of Alzheimer’s Tapestry: Single-cell Dissection of Vascular Changes Across Six Brain Regions

Your brain is powered by 400 miles of blood vessels that provide nutrients, clear out waste products, and form a tight protective barrier – the blood brain barrier – that restricts which molecules can enter or exit. However, it has remained unclear how these brain vascular cells change between brain regions, or in Alzheimer’s disease, at single-cell resolution.

To address this challenge, a team of scientists from MIT’s Computer Science and Artificial Intelligence Laboratory (CSAIL), The Picower Institute for Learning and Memory, and The Broad Institute, recently unveiled a systematic molecular atlas of human brain vasculature and its changes in Alzheimer’s Disease (AD) across six brain regions, in a paper published in Nature Neuroscience.

Alzheimer’s Disease is a leading cause of death, affecting 1 in 9 Americans over 65, and usually gives rise to debilitating and devastating cognitive symptoms. Impaired blood brain barrier (BBB) function has long been associated with Alzheimer’s and other neurodegenerative diseases such as Parkinson’s and multiple sclerosis. However, the molecular and cellular underpinnings of BBB dysregulation remain ill-defined, particularly at a single-cell resolution across multiple brain regions and many donors.

Navigating vascular complexity

Embarking deep into the complexities of our gray matter, the researchers created a molecular atlas of human brain vasculature across 428 donors. In the cohort, 220 had Alzheimer’s Disease and 208 did not, and they profiled six different regions of the brain with varying levels of pathology. They characterized over 22,514 vascular cells, each measured for the expression of thousands of genes. Examining these cells unveiled intriguing changes in gene expression across different brain regions, and stark contrasts between individuals afflicted with AD and those without.

“Alzheimer’s therapy development faces a significant hurdle – brain alterations commence decades before cognitive signs make their debut, at which point, it might already be too late to intervene effectively,” comments MIT CSAIL principal investigator and MIT EECS Professor Manolis Kellis, the study’s senior author. “Our work presents a beacon of hope: by charting the terrain of vascular changes, one of the earliest markers of Alzheimer’s, across multiple brain regions, we now have a map to guide biological and therapeutic investigations earlier in disease progression.”

The little cell that could

The team used gene expression signatures to distinguish 11 types of vascular cells. These included endothelial cells that line the interior surface of blood vessels and control which substances pass through the BBB, pericytes that wrap around small vessels and provide structural support and blood flow control, smooth muscle cell that form the middle layer of large vessels and whose contraction and relaxation regulates blood flow and pressure, fibroblasts that surround blood vessels and hold them in place, and distinguished arteriole, venule, and capillary veins responsible for the different stages of blood oxygen exchange.

Armed with these annotations, the next phase was studying how each of these cell types change in AD, and identifying 2,676 genes whose expression levels change significantly. They found that the expression of certain genes, specific to some vascular cell types, was reduced in AD patients, including growth factor receptors in pericytes, and transporter and energy ATPase in endothelial cells.

“Single-cell RNA sequencing provides an extraordinary microscope to peer into the intricate machinery of life, and ‘see’ millions of RNA molecules bustling with activity within each cell,” says Kellis, who is also a member of the Broad Institute. “This level of detail was inconceivable just a few years ago, and the resulting insights can be transformative to comprehend and combat complex psychiatric and neurodegenerative disease.”

Maestros of the cellular orchestra

Genes do not act in isolation, and they do not act on a whim. Cellular processes are governed by a complex cast of regulators, or transcription factors, that dictate which genes should be turned on or off in different conditions, and in different cell types. These regulators are responsible for interpreting our genome, the ‘book of life’, and turning into the myriad of distinct cell types in our bodies and in our brains. These regulators might be responsible when something goes wrong, and they could also be critical in fixing things and restoring healthy cellular states.

With thousands of genes showing altered expression levels in Alzheimer’s Disease, the researchers then sought to find the potential masterminds behind these changes. They asked if common regulatory control proteins target numerous altered genes, which may provide candidate therapeutic targets to restore the expression levels of large numbers of target genes. Indeed, they found several such ‘master controllers’, involved in regulating endothelial differentiation, inflammatory response and epigenetic state.

Tapping into cellular murmurings

Cells do not function in isolation; rather, they rely on communication with each other to coordinate biological processes. This intercellular communication is especially fascinating within the complex and diverse cellular landscape of the brain. Specifically, the intricate interactions between vascular cells and other resident cells of the brain – neurons, microglia and other glial cells – are of particular importance. These dialogues take on heightened significance during pathological events, such as in Alzheimer’s disease, where dysregulation of this cellular communication can contribute to the progression of the disease.

“The dynamics of multi-cellular interactions in AD call attention to the development of multicellular in vitro systems and provide a specific point of view at a multicellular level to therapy in the future,” says Kellis. “Our work provides a blueprint for understanding the dysregulation of the cerebrovasculature in Alzheimer’s disease. By unraveling how genetic variants influence vascular differential genes, we can begin to decipher the intricate mechanisms underlying BBB disruption.”

Going off script: genetic plot twists

Disease onset in our bodies (and in our brains!) is shaped by a combination of genetic predispositions and environmental exposures. On the genetic level, most complex traits are shaped by hundreds of minuscule sequence alterations, known as single-nucleotide polymorphisms (or SNPs, pronounced snips), most of which act through subtle changes in gene expression levels.

No matter how subtle their effects might be, these genetic changes can reveal causal contributors to disease, which can greatly increase the chance of therapeutic success for genetically-supported target genes, compared to targets lacking genetic support.

To understand how genetic differences associated with Alzheimer’s might act in the vasculature, the researchers then sought to connect genes that showed altered expression in Alzheimer’s with genetic regions associated with increased Alzheimer’s risk through genetic studies of thousands of individuals.

They linked the genetic variants (SNPs) to candidate target genes using three lines of evidence: physical proximity in the three-dimensional folded genome, genetic variants that affect gene expression, and correlated activity between distant regulatory regions and target genes that go on and off together between different conditions.

This resulted in not just one hit, but 125 hits, where Alzheimer’s dysregulated genes were linked to Alzheimer’s associated genetic variants. Some of these hits were direct, where the genetic variant acted directly on a nearby gene. Others were predicted to be indirect, when the genetic variant instead affected the expression of a regulator, which then affected expression of its target genes. And yet others were predicted to be indirect through cell-cell communication networks.

ApoE4: maestro of misfortune

While most changes are subtle, not just in Alzheimer’s, but nearly all complex disorders, exceptions exist. One such exception is FTO in obesity, that increases obesity risk by one standard deviation. Another one is ApoE4 in Alzheimer’s Disease, which increases risk more than 10-fold for carriers of two risk alleles – one ‘unlucky’ copy inherited from each parent.

With such a strong effect size, the researchers then asked if ApoE4 carriers showed specific changes in vascular cells that were not found in ApoE3 carriers. They found that these changes significantly shared with AD-associated changes, providing valuable insights into the cellular and molecular changes involved in APOE4-associated cognitive decline. They also studied differences in vascular cell type abundance and cell-type-specific gene expression levels across six brain regions. This analysis highlighted the regional heterogeneity of the BBB and the importance of single-cell multi-region characterization of the cerebrovasculature. Translating these findings into viable therapeutics will be a journey of exploration, demanding rigorous preclinical and clinical trials. To bring these potential therapies to patients, the scientists need to understand how to target the discovered dysregulated genes safely and effectively, and determine whether modifying their activity can ameliorate or reverse AD symptoms.

“Unearthing these AD-differential genes gives us a glimpse into how they may be implicated in the deterioration or dysfunction of the brain’s protective barrier in Alzheimer’s patients, shedding light on the molecular and cellular roots of the disease’s development,” says Na Sun, an MIT CSAIL and EECS graduate student and first author on the study. “But, our findings do far more than just enrich our knowledge of Alzheimer’s. They subtly point towards new potential avenues for therapy, hinting at a future where these differential genes might be harnessed to devise innovative treatments for Alzheimer’s. The possibility of slowing or even halting the disease’s progression is truly mesmerizing.”

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