The XX Factor: What's the Key to Female Longevity?

Dena Dubal’s interest in sex differences in aging started early. Growing up in rural India, she was surrounded by great-grandmothers, great-aunts and others who far outlived the men in the family. Her family wasn’t unusual. Across the world, women typically outlive men by approximately five years and experience slower rates of age-related cognitive decline.

Now, as a neuroscientist at the University of California, San Francisco, Dubal is starting to uncover why those differences occur. In videos of aging mice wandering through a maze, she points out how some animals appear confused, meandering much like people trying to find their cars in a parking garage. Others take quicker, more focused routes through the maze, as if they remember exactly where they left their vehicle. Dubal recently discovered that the difference between these animals stems, in part, from a protein made by a gene on the X chromosome. Over the last several years, her research has homed in on sex chromosomes as a vital contributor to how our brains age.

Two X chromosomes lead to female hormones and features, while an X and Y lead to male traits. These differences affect glucose metabolism, cellular energy production and much more, according to Dubal, an investigator with the Simons Collaboration on Plasticity and the Aging Brain. “Sex chromosomes and gonadal hormones alter fundamental biology in ways that we are just beginning to understand,” she says. “The X chromosome is 5 percent of our genome, and it has the largest density of brain-related genes compared to any individual autosome. I don’t think it’s coincidence that variations or human mutations in these different X factors affect brain function.”

Understanding these differences will be important for developing new treatments. In 2003, the National Institute on Aging (NIA) launched a program to help assess drugs or dietary interventions that could safely extend the life span of mice — and eventually humans. This Intervention Testing Program supports studies of approximately 10 to 15 treatments each year, and researchers test interventions in both male and female animals. In 2015, researchers reviewed 14 interventions that had been tested over the previous decade. They found that only five appeared to actually extend life span — and four of those worked differently in male and female animals. “That suggests there’s a fundamental difference in the way we age,” said Bérénice Benayoun, a biologist at the University of Southern California, in a 2020 podcast.

Dubal and Benayoun are now probing the mechanisms underlying these observations, examining how steroid hormones and sex chromosomes alter the trajectory of cognitive decline with age in different sexes.* Their studies, part of an SCPAB project on sex differences in aging, will help reveal why women are on average more resilient to cognitive decline, memory loss and other brain functions that erode as we grow old. Identifying these factors could inspire interventions that benefit everyone — improving not just a person’s life span but their quality of life as they age. “The things that help us live longer also tend to be the things that help us live better,” Dubal says.

Dissimilar mechanisms

The link between longevity and ‘better’ aging is apparent in the brain: Women’s longer life spans appear to correlate with slower rates of cognitive decline. In one 2016 study, for instance, researchers tracked people between the ages of 50 and 96 over a period of nine years using tests of memory, verbal learning and other cognitive tasks. Over time, men’s performance declined more rapidly on some of these tests compared to women, hinting that women had more resilience to age-related decline. Researchers seeking the underlying mechanisms have unearthed clues in gene regulation, cellular energy production and how hormones control energy production.

Seeking clues as to why a 70-year-old woman is likely to appear far more youthful than a man of the same age, Steve Horvath, a geneticist at the University of California, Los Angeles, and his colleagues looked for changes in DNA methylation, also known as epigenetic markers, that control gene expression. They examined blood, brain, lung, liver and other tissues across the human life span and found many sex-associated differences. Samples from older adults showed signs of demethylation, particularly at enhancers and regulatory elements in the genome, which control when a particular gene produces protein and how much. Men typically appeared ‘older’ and showed more signs of such loss compared to women of the same age. The contrast was most pronounced in liver and blood, and to a lesser extent in brain tissues.

Methylation hints at the cellular mechanisms underlying female cognitive resilience as well as their longer life spans, Horvath says. “It’s a very consistent finding that women age more slowly than men in several different organs, so on some level methylation reflects that mortality advantage,” he says. “It could easily be part of the explanation for cognitive resilience — but I would never say it explains it all.”

In 2019, another clue emerged from studies of how the brain consumes glucose. Manu Goyal, a neuroradiologist at Washington University in St. Louis, found that younger brains use a mix of two cellular pathways, oxidative and non-oxidative metabolism, to break glucose down and fuel their activity. Some older brains shift mostly to oxidative. When Goyal and his colleagues imaged glucose metabolism in the brains of 205 cognitively normal adults aged 20 to 82, they found that female brains on average appeared younger than those of age-matched males by approximately three years.

Clues from chromosomes

To uncover the source of these differences, Dubal and her team developed a mouse model that allows them to differentiate the effects of sex chromosomes and sex hormones on aging. Researchers moved the gene in mice and humans that determines male sex, called SRY, from the Y chromosome to an autosome. Once unhitched, the gene expressed itself in progeny that bore this autosome, independent of their sex chromosomes. Some were XX and carried the SRY gene and thus formed testes. Others were XY and lacked the gene, forming ovaries instead.

Surprisingly, mice with two X chromosomes — whether they bore ovaries or testes — lived longer than their XY littermates. Dubal and her colleagues were the first to note this effect of the X chromosome on life span in a 2018 study. The XX mice also performed better on tests of memory and spatial learning, such as finding their way to a platform through a water maze. “If estrogen were the only factor responsible for longevity, then all mice with ovaries should show these advantages,” Dubal says. “But only XX mice did, whether they had ovaries or testes.”

Typically, cells with two X chromosomes silence one set of X-linked genes by epigenetic mechanisms such as methylation, to avoid a double dose of X-linked proteins. In principle, a silenced X should be inactive. But at least 15 percent of X-linked genes in humans escape this silencing. “There’s really a handful of factors that escape X inactivation in the brains of both humans and mice,” Dubal says.

In a study published in August 2020, the team homed in on four such genes that are expressed in the brain and appear to slip past their epigenetic off-switches, resulting in increased levels of these proteins in XX cells. Some have been linked to intellectual disability and autism.

At a lab meeting, Dubal and her team took a vote on which of the four they’d pursue; the winner was a gene named lysine demethylase 6a, or KDM6A. “Not much was known about it,” she says. “People with mutations in this gene have syndromes that involve intellectual disability — and it seemed to be important to how brain cells connect at the synapse in mice. That was enough of a clue for us to wonder whether this was involved in cognitive aging and neurodegenerative diseases.”

The KDM6A protein unspools DNA from histones, opening it up to be transcribed and controlling the expression of many other genes. In a series of experiments, Dubal and her team discovered that XX cells, which have higher levels of KDM6A, were more resistant to neurotoxins than XY cells. Decreasing KDM6A levels in XX cells made them more vulnerable to toxins, including β-amyloid, a misfolded peptide linked to Alzheimer’s disease. Boosting levels of the KDM6A protein in XY cells made them more resilient to these exposures and improved XY animals’ spatial memory, enabling them to remember the location of a platform hidden within a maze and move swiftly toward it — much like finding one’s car in a garage.

Dubal’s team has also analyzed databases of genetic variants in Alzheimer’s disease and found an association between the presence of a genetic variant that increases levels of KDM6A in the brain and lower levels of cognitive decline. Such data could eventually lead to new interventions to prevent age-related loss in brain function — and not just in women. “If we can unravel what makes one sex more resilient or vulnerable than the other in a specific measure,” Dubal says, “that could mean novel therapies that could protect both sexes.”

The hold of hormones

Our brains’ resilience or vulnerability to aging is also rooted in the sex hormones testosterone and estrogen. Their effects are so profound — and begin so early in life — that many researchers group the brain with sexually differentiated organs such as gonads when studying the hormones’ effects. Their influence is especially relevant to one hallmark of aging:  an increase in inflammation unrelated to infections. This so-called ‘sterile’ inflammation also follows distinct trajectories in male and female animals and is implicated in age-related cognitive differences. “Aging creates many more changes in female cells than in male cells that could explain a lot of the differences in immune diseases,” Benayoun says.

In a study published in Nature Aging in July, Benayoun and her colleagues studied how immune cells known as neutrophils changed with age in male and female mice. They found that an animal’s sex was a strong predictor of immune responses. Neutrophil activity followed sex-specific trajectories as animals aged: Cells from male mice showed higher levels of inflammatory proteins that can damage surrounding tissues, while those from female animals increased the production of extracellular structures correlated with autoimmune disease. “These findings suggest that sex differences can become amplified with aging, at least for neutrophils,” Benayoun said in a press release.

Benayoun is now developing a mouse model to understand how testosterone and estrogen regulate these processes. Their model allows researchers to externally reverse an adult animal’s gonads. They can reprogram an ovary-bearing mouse, born with two X chromosomes, to convert the ovaries into testes and produce levels of testosterone that are nearly identical to those of their XY-chromosome-bearing littermates. The model will allow them to answer questions such as how a lifelong exposure to testosterone affects neuro-inflammation or microglia in two animals with identical genetic backgrounds. “It’s going to help us identify what’s beneficial or not in terms of these hormones which are known to modify brain physiology,” she says.

To understand how sex hormones shape brain function over a lifetime, Benayoun has turned to the African killifish, a brightly colored tropical fish that’s the shortest-lived vertebrate scientists can breed in labs. Killifish life spans range from 3 to 6 months, because they evolved in seasonal ponds of rainwater that dry up for half a year. Their ‘live fast, die young’ lifestyle makes them ideal subjects for studying aging. “They age five to six times faster than a mouse and almost 10 times as fast as zebra fish,” Benayoun says. “It’s so powerful because we can recapitulate most of what we expect from human aging, including cognitive decline, in that short amount of time.”

Her team studies two strains of the fish — one that evolved in regions with a longer dry season, and another from a region with longer monsoons that allow the strain to live 30 to 40 percent longer than the other. Killifish sex is determined by X and Y chromosomes, so Benayoun aims to use single-cell RNA sequencing and other analyses to seek out sex differences in males and females in both the long-lived and short-lived strains. “There’s not a lot of work in the species yet,” she says. “But we have every reason to believe they will have sex differences like any other vertebrate species.”

*Note: Their projects and this story focus on the biological classification of worms, mice and humans as male and female. They do not capture gender, a person’s innate sense of identity as male, female, a combination, or a different gender.

Reference: Lu RJ, Taylor S, Contrepois K, et al. Multi-omic profiling of primary mouse neutrophils predicts a pattern of sex- and age-related functional regulation. Nat Aging. 2021:1-19. doi: 10.1038/s43587-021-00086-8

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