Of the roughly 6.5 million Americans who currently have Alzheimer’s disease, 4 million are women. The extreme gender divide likely stems from biological and cultural factors, but attempts to decipher the genetic or hormonal risk factors of the neurodegenerative disease have yielded inconclusive results, according to the Alzheimer’s Association. Now, a study published in Science Advances on December 14 offers a new clue: Brain samples taken from women who had Alzheimer’s disease are more likely than men’s to contain a particular post-translationally modified protein that the study links to Alzheimer’s pathology.

The experiment began with a search for S-nitrosylated proteins in samples taken from 40 postmortem brains, including those of men and women who had Alzheimer’s disease when they died as well as healthy controls. Protein S-nitrosylation (SNO) is a protein modification that study coauthor Stuart Lipton, a neurodegenerative medicine researcher at Scripps Research in California and one of the scientists behind the first FDA-approved Alzheimer’s drug (memantine), has been studying in the context of neurodegeneration for decades. The SNO process involves a protein’s cysteine residue binding to a nitrosonium (NO+) cation, according to Lipton’s earlier work, which can result in protein misfolding and aggregation, as is seen in neurodegeneration. But the modification is also employed by healthy cells, he explains, as a part of normal post-translational modification.

Rachel Hendrix, a neuroscientist studying Alzheimer’s at Washington University in St. Louis, says in an email to The Scientist that “Post-translational modifications, such as SNO, are important to consider in proteomics research as they can provide additional information about protein function and regulation.” However, SNO “has been difficult to measure in the past,” adds Hendrix, who didn’t work on the new study.

Lipton notes that “we’ve only more recently developed tools to look at it in real detail.” In this case, the search for SNO proteins in the brain tissue was made possible by a modified version of SNOTRAP, a technique developed by study coauthor and MIT biological engineer Steven Tannenbaum that uses nitrogen-binding chemical probes and nano-liquid chromatography to detect and isolate specific proteins. In doing so, the team identified 1,449 SNO proteins—a finding that Lipton says indicates that SNO is likely important for a variety of biomedical processes—then narrowed down their list to the 10 proteins that were more prevalent in Alzheimer’s brains than healthy controls.

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One of them, a SNO protein called complement component 3 (C3) that plays a role in the autoimmune system, was found in considerably greater quantities in the brains of female Alzheimer’s patients than in the brains of male Alzheimer’s patients. In the brains of males, C3 levels were 5.3 times higher in Alzheimer’s brains than male controls. However, the study identified a 34.2-fold increase in C3 levels among female Alzheimer’s brains than in the female controls, indicating that it’s linked to both sex and the neurodegenerative disease.

Hendrix complimented the study’s rigor, noting that the authors measured ten samples from each group four times, “which is a larger sample size than is often used in mass spectrometry studies” as samples are often pooled to reduce costs. “This increased the reliability of the results and allowed the researchers to identify some protein hits that were highly significant within a subset of individual [Alzheimer's] males and females.”

However, Hendrix also notes that nearly all samples, even controls, displayed some level of neurodegenerative pathology, therefore “further research with larger sample sizes may be needed to fully understand the differences presented in this paper.”

C3 was previously linked to Alzheimer’s disease but wasn’t known to be nitrosylated or distributed unevenly between men and women, according to the study. So, the team performed further experiments to determine what drives that sex difference, looking specifically at hormonal changes in men and women. In follow-up in vitro studies using human induced pluripotent stem cells (hiPSCs) derived from brain microglia, the researchers found that the sex hormone β-estradiol inhibited SNO-C3 formation. Based on that finding, the researchers conclude that the sudden drop in estrogen levels that women experience during menopause—known to result in increased NO in the brain—acts as a trigger that increases nitrosylation and induces widespread SNO-C3 formation.

“The problem I think with the women is [C3] suddenly goes up when the estrogen is removed,” says Lipton, explaining why female brains had a greater jump in C3 than male brains. “Not only is it higher in women, but it’s a sudden event.”

In further in vitro experiments, Lipton and his colleagues found that SNO-C3 activates microglia in the brain and causes them to set their sights on the synapses between neurons, digesting and destroying these connections between brain cells. It also results in the destruction of mitochondria in the brain, leaving neurons without the fuel they need to operate and survive. This, Lipton argues, is clear evidence that the C3-activated microglia are driving Alzheimer’s disease progression.

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“Alzheimer’s and other demented illnesses—yes they have misfolded and other aggregated proteins,” Lipton says, regarding other purported biomarkers for Alzheimer’s such as tau or amyloid plaque aggregation—“but that is not the disease.” Instead, he notes that synapse loss closely correlates with disease progression. “If we have a therapy that can protect synapses, we may really have something,” he says.

Lipton adds that he and his team are now aiming to commercialize the finding for use in new Alzheimer’s medications by developing denitrosylating drugs “that, with surgical precision, aim right at the one protein that’s nitrosylated and either preventing it from being nitrosylated or denitrosylate it.”

“You can’t just reduce NO—that’s not going to work—you have to go after the protein of interest,” he explains.

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Still, he says he hopes that other labs will join in, using SNOTRAP and other techniques to explore how nitrosylated proteins influence Alzheimer’s disease as well as other neurodegenerative diseases and medical conditions—after all, there are another 1,498 SNO proteins identified in this study that could use investigation.

Hendrix was also eager to see how the new work assists in the development of new treatments. “One aspect of this study that is particularly interesting is the potential for personalized medicine in [Alzheimer’s disease] research,” she writes in her email. “Overall, this study brings to light the importance of considering sex and other individual differences in the development of potential therapies for [Alzheimer’s].”