Scientists at Scripps Research and Massachusetts Institute of Technology (MIT) have found a clue to the molecular cause of Alzheimer’s disease (AD), which may also explain why women are at greater risk for the disease than men.

The team found that a particularly harmful, chemically modified form of an inflammatory immune protein called complement C3 was present at much higher levels in the brains of women who had died with AD, compared with the brains of men who had died with the disease. The results also showed that estrogen—levels of which drop during menopause—normally protects against the production of this form of complement C3.

“Our new findings suggest that chemical modification of a component of the complement system helps drive Alzheimer’s, and may explain, at least in part, why the disease predominantly affects women,” said research lead Stuart Lipton, MD, PhD, professor and Step Family Foundation endowed chair in the department of molecular medicine at Scripps Research and a clinical neurologist in La Jolla, CA. Lipton is senior author of the team’s published paper in Science Advances, which is titled, “Mechanistic insight into female predominance in Alzheimer’s disease based on aberrant protein S-nitrosylation of C3.” In their paper the researchers concluded, “Collectively, we demonstrate robust alterations in the S-nitrosoproteome that contribute to AD pathogenesis in a sex-dependent manner.” The study was a collaboration with a team led by Steven Tannenbaum, PhD, post-tenure Underwood-Prescott professor of biological engineering, chemistry, and toxicology at MIT.

AD, the most common form of dementia that occurs with aging, currently afflicts about six million people in the U.S. alone. “AD—characterized by the accumulation of misfolded amyloid-β (Aβ) peptide and neurofibrillary hyperphosphorylated tau tangles in the brain—is arguably the most common neurodegenerative disorder leading to dementia,” the team wrote.

Alzheimer’s disease is always ultimately fatal, usually within a decade of onset, and there is no approved treatment that can halt the disease process, let alone reverse it. The shortcomings of treatments reflect the fact that scientists have never fully understood how Alzheimer’s develops. “The etiology and pathogenesis of AD are incompletely understood, and effective, disease-modifying drug treatments are lacking,” they continued. Scientists also don’t know fully why women account for nearly two-thirds of cases. “Although tremendous strides have been made in AD research over the past decade, additional consideration of sex differences will be important to explain the increased incidence of disease in females.”

Lipton’s lab studies biochemical and molecular events that may underlie neurodegenerative diseases, including the chemical reaction that forms a modified type of complement C3—a process called protein S-nitrosylation (SNO). Lipton and his colleagues previously discovered this chemical reaction, which happens when a nitric oxide (NO)-related molecule binds tightly to a sulfur atom (S) on a particular amino acid building-block of proteins to form a modified SNO-protein.

Protein modifications by small clusters of atoms such as NO are common in cells and typically activate or deactivate a target protein’s functions. For technical reasons, S-nitrosylation has been more difficult to study than other protein modifications, but Lipton suspects that “SNO-storms” of these proteins could be a key contributor to Alzheimer’s and other neurodegenerative disorders. “SNO can influence protein activity, localization, conformation, or interactions with other proteins; aberrant protein SNO may play a key role in the pathogenesis of various neurodegenerative diseases,” the investigators commented in their paper. “Prior work has linked SNO proteins in neurons and glial cells to neurodegenerative diseases, including AD.”

For their newly reported study, the researchers used novel methods for detecting S-nitrosylation to quantify proteins modified in 40 postmortem human brains. Half of the brains were from people who had died of Alzheimer’s, and half were from people who hadn’t—and each group was divided equally between males and females.

In these brains, the scientists found 1,449 different proteins that had been S-nitrosylated. Among the proteins most often modified in this way, there were several that have already been tied to Alzheimer’s, including complement C3. Strikingly, the levels of S-nitrosylated C3 (SNO-C3) were more than six-fold higher in female Alzheimer’s brains compared to male Alzheimer’s brains. “Notably, SNO proteins associated with complement pathways are enriched in both male and female AD brains,” they stated. “However, SNO of C3, representing the point of convergence of the various complement cascades, was detected predominantly in female AD brains … In our SNO protein datasets, we observed a significant increase in SNO-C3 in female AD brains, exhibiting a 34.2-fold increase over female non-AD control brain.”

estrogen, Alzheimer's Disease, Women, S-nitrosylation Complement protein
In postmenopausal women, depletion of estrogen causes excessive elevation of nitric oxide (NO) in the brain and thus generates S-nitrosylated complement factor C3 (SNO-C3). SNO-C3 triggers activated microglial cells, the innate immune cells in the brain, to phagocytose (or “eat”) neuronal synapses—the connections that mediate signaling between nerve cells in the brain. This aberrant chemical biology process results in synapse loss, leading to cognitive decline in Alzheimer’s disease. [Chang-ki Oh and Stuart Lipton, Scripps Research]

The complement system is an evolutionarily older part of the human immune system. It consists of a family of proteins, including C3, that can activate one another to drive inflammation in what is called the “complement cascade.” Scientists have known for more than 30 years that Alzheimer’s brains have higher levels of complement proteins and other markers of inflammation, compared to neurologically normal brains. More recent research has shown specifically that complement proteins can trigger brain-resident immune cells called microglia to destroy synapses—the connection points through which neurons send signals to one another. Many researchers now suspect that this synapse-destroying mechanism at least partly underlies the Alzheimer’s disease process, and loss of synapses has been demonstrated to be a significant correlate of cognitive decline in Alzheimer’s brains.

Why would SNO-C3 be more common in female brains with Alzheimer’s? There has long been evidence that the female hormone estrogen can have brain-protective effects under some conditions; thus, the researchers hypothesized that estrogen specifically protects women’s brains from C3 S-nitrosylation—and this protection is lost when estrogen levels fall sharply with menopause.

“We hypothesized that menopause-associated up-regulation of inflammation in the brain could be causally linked to the aberrant increase in SNO-C3 that we observed,” the authors noted. “Experiments with cultured human brain cells supported this hypothesis, revealing that SNO-C3 increases as estrogen (β-estradiol) levels fall, due to the activation of an enzyme that makes NO in brain cells. This increase in SNO-C3 activates microglial destruction of synapses. “Mechanistically, we show that formation of SNO-C3 is dependent on falling β-estradiol levels, leading to increased synaptic phagocytosis and thus synapse loss and consequent cognitive decline,” they stated. “Thus, dysregulation of the complement system may play a role in the pathogenesis of AD and explain, at least in part, the female predominance of the disease.”

“Why women are more likely to get Alzheimer’s has long been a mystery, but I think our results represent an important piece of the puzzle that mechanistically explains the increased vulnerability of women as they age,” Lipton said. “To our knowledge, this is the first investigation comparing changes in NO-modified protein levels in the brains of male and female humans with AD,” the team stated. “ … our data suggest a unique mechanism by which protein SNO modulates complement (C3) activity in a sex-dependent manner, thereby providing a molecular link between NO signaling and the complement cascade in AD pathogenesis.”

The researchers now hope to conduct further experiments with de-nitrosylating compounds—which remove the SNO modification—to see if they can reduce pathology in animal models of Alzheimer’s and eventually in humans.

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