The pathophysiology of the brains of Alzheimer’s disease (AD) patients is widely studied. So too are global expression changes revealed by bulk transcriptomic analyses. Now, for the first time, researchers have performed a comprehensive analysis of genes expressed in individual brain cells of patients with AD using single-nucleus RNA-seq (snRNA-seq) profiles, identifying distinctive cellular pathways that are affected in neurons and other types of brain cells.
“This study provides, in my view, the very first map for going after all of the molecular processes that are altered in Alzheimer’s disease in every single cell type that we can now reliably characterize,” said Manolis Kellis, PhD, professor of computer science and a member of the Computer Science and Artificial Intelligence Laboratory at MIT. “It opens up a completely new era for understanding Alzheimer’s.”
The work, published in an article titled, “Single-cell transcriptomic analysis of Alzheimer’s disease” in Nature, showed that axon myelination is significantly disrupted in patients with AD. The researchers also found that the brain cells of men and women vary significantly in how their genes respond to the disease.
The researchers analyzed postmortem brain samples from 24 people who exhibited high levels of AD pathology and 24 people of similar age who did not have these signs of disease. They performed snRNA-seq on about 80,000 cells from these subjects. Previous studies of gene expression in Alzheimer’s patients have measured overall RNA levels from a section of brain tissue, but these studies don’t distinguish between cell types, which can mask changes that occur in less abundant cell types, noted Li-Huei Tsai, PhD, director of MIT’s Picower Institute for Learning and Memory and a senior author on the study.
“We wanted to know if we could distinguish whether each cell type has differential gene expression patterns between healthy and diseased brain tissue,” Tsai said. “This is the power of single-cell-level analysis: You have the resolution to really see the differences among all the different cell types in the brain.”
The researchers were able to analyze not only the most abundant cell types, which include excitatory and inhibitory neurons, but also rarer, non-neuronal brain cells such as oligodendrocytes, astrocytes, and microglia. They found that each of these cell types showed distinct gene expression differences in Alzheimer’s patients.
Some of the most significant changes occurred in genes related to axon regeneration and myelination. Myelin is a fatty sheath that insulates axons, helping them to transmit electrical signals. In people with AD, genes related to myelination were affected in both neurons and oligodendrocytes, the cells that produce myelin.
Most of these cell-type-specific changes in gene expression occurred early in the development of the disease. In later stages, the researchers found that most cell types had very similar patterns of gene expression change. Specifically, most brain cells turned up genes related to stress response, programmed cell death, and the cellular machinery required to maintain protein integrity.
Correlations between gene expression patterns and other measures of Alzheimer’s severity such as the level of amyloid plaques and neurofibrillary tangles, as well as cognitive impairments were noted. This allowed the identification of “modules” of genes that appear to be linked to different aspects of the disease.
“To identify these modules, we devised a novel strategy that involves the use of an artificial neural network and which allowed us to learn the sets of genes that are linked to the different aspects of Alzheimer’s disease in a completely unbiased, data-driven fashion,” Hansruedi Mathys, PhD, postdoctoral associate and co-first author on the paper said. “We anticipate that this strategy will be valuable to also identify gene modules associated with other brain disorders.”
The most surprising finding, the researchers said, was the discovery of a dramatic difference between brain cells from male and female Alzheimer’s patients. They found that excitatory neurons and other brain cells from male patients showed less pronounced gene expression changes in Alzheimer’s than cells from female individuals, even though those patients did show similar symptoms, including amyloid plaques and cognitive impairments. By contrast, brain cells from female patients showed dramatically more severe gene-expression changes in Alzheimer’s disease, and an expanded set of altered pathways.
“That’s when we realized there’s something very interesting going on. We were just shocked,” Tsai said.
So far, it is unclear why this discrepancy exists. The sex difference was particularly stark in oligodendrocytes, which produce myelin, so the researchers performed an analysis of patients’ white matter, which is mainly made up of myelinated axons. Using a set of MRI scans from 500 additional subjects, the researchers found that female subjects with severe memory deficits had much more white matter damage than matched male subjects.
More study is needed to determine why men and women respond so differently to Alzheimer’s disease, the researchers say, and the findings could have implications for developing and choosing treatments.
“There is mounting clinical and preclinical evidence of a sexual dimorphism in Alzheimer’s predisposition, but no underlying mechanisms are known. Our work points to differential cellular processes involving non-neuronal myelinating cells as potentially having a role. It will be key to figure out whether these discrepancies protect or damage the brain cells only in one of the sexes—and how to balance the response in the desired direction on the other,” said Jose Davila-Velderrain, PhD, a postdoctoral associate at the Computer Science and Artificial Intelligence Laboratory (CSAIL) at MIT and co-first author on the paper.
The researchers are now using mouse and human induced pluripotent stem cell models to further study some of the key cellular pathways that they identified as associated with Alzheimer’s in this study, including those involved in myelination. They also plan to perform similar gene expression analyses for other forms of dementia that are related to Alzheimer’s, as well as other brain disorders such as schizophrenia, bipolar disorder, psychosis, and diverse dementias.