The most common white blood cells in your body — immune cells called neutrophils — can make a protein nobody knew they were making, Stanford Medicine investigators have discovered. That unexpected sighting joins a growing list of hints tying schizophrenia, a disorder of the brain, to events occurring elsewhere in our bodies.
The findings are summarized in a paper published online May 11 in Proceedings of the National Academy of Science.
The newly noticed neutrophil nexus, as a source of the protein called C4A, links a long list of other observations that are somewhat puzzling when looked at in isolation: For example, large-scale population-genetic studies have identified C4A, already known to be produced mainly in the liver, as a pronounced risk factor in schizophrenia. People with schizophrenia tend to have increased numbers of neutrophils in their blood. And the most effective medication for schizophrenia inhibits neutrophils.
"Connecting such seemingly disparate relationships could give us a better understanding of schizophrenia and, eventually, better treatments and diagnostics for it," said Agnes Kalinowski, MD, PhD, a clinical assistant professor of psychiatry and behavioral sciences.
Kalinowski, a researcher as well as a practicing psychiatrist focusing on schizophrenia, is the PNAS study's lead author. The senior author is Alexander Urban , PhD, associate professor of psychiatry and behavioral sciences and of genetics.
Schizophrenia affects one in every 100 persons globally almost without variation by geography or ethnicity. Its most noticeable symptoms are hallucinations, delusions and fixations. A fundamental feature of the disease is cognitive impairment: inability to think clearly, reduced working memory, disorganized thinking and behavior.
Current treatments for schizophrenia are palliatives, Kalinowski said. They don't stop disease progression or restore motivation or cognitive sharpness. "The best drug we've got now is clozapine," she said. "It reduces manifestations such as hallucinations, fixations and delusions. That can be life-changing. But it has side effects ranging from weight gain to heart attack risk."
Intriguingly, another of clozapine's major side effects — or is it a "side" effect? — is that it depletes the numbers of circulating neutrophils, she noted.
C4A: a neutrophil-brain connection?
You may not have heard much about neutrophils, but you've probably seen them. They account for a good half of a healthy person's circulating white blood cells.
These short-lived kamikaze warriors have a tough life. They're the first immune cells to arrive on the scene of bacterial infections. There they gobble up microbial pathogens; spew out toxic bactericidal substances; and extrude gooey microbe-trapping webs made of long stringy molecules, including their own DNA. Neutrophils routinely die on the job — their life expectancies range from less than a day to a week — only to become the main cellular ingredient of pus.
C4A is one of the 50 or so proteins aligned in a close, complex collaboration in our bloodstreams called the complement system. This ancient evolutionary kernel of our modern immune system, which traces back to the sponges of some 500 million years ago, can rapidly recognize various inflammatory events such as microbial invasions.
In response, a bucket brigade of events ensues. One by one, the component proteins become activated and bind to the next protein in the brigade, activating it. C4A's activation gets tripped off when a small fragment of it, called C4-ana, gets snipped off.
Activated complement, the result of that bucket brigade, engages in tasks such as punching holes in bacterial cells' outer membranes; curiously, it's consistently seen in blood samples from people with schizophrenia.
C4A pops up in more places than just our blood. In the brain, it engages in a very different capacity. It has been implicated in a process called synaptic pruning, in which the brain periodically rids itself of excessive nerve-cell-to-nerve-cell contact points called synapses.
A healthy adult human brain has an estimated 100 trillion to 500 trillion synapses. During early brain development, that number rockets even higher, resulting in many unnecessary, redundant and even obfuscating connections. Like an editor slashing out extraneous verbiage in a potentially great story, synaptic pruning ordinarily enhances cognitive coherence. It occurs in waves during fetal development, early childhood and adolescence.
But this can go too far. The schizophrenic brain's outermost layer, the cerebral cortex, crucial to high-level mental functioning, has been shown to contain some 30% fewer synapses than a healthy brain does, and to be thinner than normal.
Researchers have found a link between brain thickness and circulating neutrophil counts, Kalinowski said.
A major schizophrenia risk factor
Risk factors for schizophrenia include high levels of emotional stress or severe fever in early childhood (before age 5) and daily marijuana use, especially during adolescence.
But these environmental factors are dwarfed by the genetic ones.
"The heritability of schizophrenia is very strong — about 80%," Kalinowski said. The biggest single hereditary risk factor involves C4A.
Genes are recipes for proteins. Our genomes typically carry two copies of each gene – one inherited from Mom, one from Dad. But over the course of evolution, some of our genes have undergone mutations called duplications, resulting in more copies of those genes in some people's genomes.
That's the case for C4A: Some of us have two copies of the gene for it, while others may have several. The number of C4A-gene copies in a person's genome is the strongest common genetic risk factor associated with schizophrenia. Nobody's been able to say why. But C4A's concentration in schizophrenia patients' plasma (blood's contents after its resident cells have been removed) has been found to track with the number of copies of the C4A gene in those patients' genomes.
Schizophrenic patients' symptom intensity correlates with their brain levels of C4A, inferred from its measured levels in their cerebrospinal fluid.
In 2021, Kalinowski and Urban co-authored a paper in Translational Psychiatry showing a strong connection between the number of copies of the gene for C4A in the genomes of people with schizophrenia and the amount, in their plasma, of C4-ana, that small snippet of C4A that gets excised to kick-start C4A's activation.
C4-ana can be thought of as a kind of footprint of activated C4A's presence.
Neutrophils make C4A
In the new PNAS study, Kalinowski, Urban and their colleagues looked at a massive gene-expression database. Gene expression analyses measure, gene by gene, the intensity of their participation in an early step in individual cells' production of proteins.
Our genomes contain roughly 20,000 genes. But only a small, selected set of any single cell's genes are actively engaged in directing the production of proteins, depending on what type of cell it is (nerve, skin, heart, etc.) and what state it's in (healthy or diseased, agitated or at peace, young and frisky or old and cranky). The investigators also tested blood samples from 10 anonymous volunteers.
They saw that neutrophils can and do make C4A.
What's more, neutrophils' C4A protein-production intensity in individuals with schizophrenia correlated with their clinical measures. Was C4A from schizophrenia patients' neutrophils getting into the brain and revving up synaptic pruning in these individuals?
"It turns out neutrophils are able to be little factories for C4A," Kalinowski said. "And neutrophils from schizophrenic patients are initiating far more CA4 production than healthy controls' neutrophils are."
Paradoxically, they're holding on to less of it: There was no difference in the amount of this protein itself in plasma, and there was less of it in neutrophils from schizophrenic patients than in neutrophils from controls. Yet levels of C4-ana — the telltale marker of C4A's activation — were higher in schizophrenic patients' plasma.
This all suggests to Kalinowski that the protein is being made in abundance in neutrophils but is somehow getting consumed.
"Schizophrenia patients' neutrophils appear to be exhausting their initially plentiful C4A supplies," she said. "Something, somewhere, is using it up. We don't know exactly where that's happening, but we're hoping to find out."
The study raises the possibility that neutrophils may play a direct role in schizophrenia. If they do, then maybe blocking their activity could interfere with the disease process.
"Drugs that block neutrophil activation wouldn't need to penetrate the blood-brain barrier, because they'd be working in our peripheral bloodstream," Kalinowski said.
Neutrophil assessments could also be part of a diagnostic, Kalinowski said. "Being able to extract a diagnostic signal from something in blood, in combination with clinical information, would be game-changing."
Might looking for elevated neutrophil counts predict the onset of symptoms?
"We don't have a full explanation of schizophrenia yet, but we're putting together the jigsaw puzzle," Kalinowski said. "Figuring out where each piece goes helps you snap the rest of them into place more quickly."
The study was funded by the Stanford Department of Medicine's Translational Research and Applied Medicine Program and the Stanford Department of Psychiatry and Behavioral Sciences.
