Deep within the recesses of the brain, long before amyloid plaques or tau tangles appeared, there was a spark that led to Alzheimer’s disease. The exact identity of that spark, which ignited the neuroinflammation and cognitive decline associated with Alzheimer’s disease, remains a mystery.

Genetics, the environment, and lifestyle contribute to a person’s risk for Alzheimer’s disease. Some of the most well-studied causes are genetic mutations and alleles that increase the chance of developing the disease. While specific genetic mutations cause early onset familial Alzheimer’s disease, these account for less than 1% of all Alzheimer’s disease cases (1). People carrying certain genetic variants like the apolipoprotein E4 (APOE4) allele have an increased risk of developing Alzheimer’s disease, but many people who have the APOE4 allele never develop the disease.

One of the first indications of Alzheimer’s disease is the accumulation of amyloid beta plaques in the brain. For this reason, scientists and drug companies have focused on developing therapies to reduce amyloid beta with the hope that the drugs will slow or reverse the cognitive decline associated with the disease. While multiple drugs successfully deplete amyloid beta, they have little to no effect on cognitive decline, as exemplified most recently by the controversy surrounding the less than stellar performance of Biogen’s drug, aduhelm. The disappointing results of amyloid beta treatments have prompted some scientists to wonder if the amyloid beta plaques do not cause Alzheimer’s disease, but are simply a consequence of it.

“What triggered the amyloid?” asked Rudolph Tanzi, an Alzheimer’s disease researcher at Harvard Medical School. “If it wasn't just genetics, it could have been a microbial infection.”

Relegated to the fringe for decades, a theory called the infection hypothesis, which proposes that there might be a link between microbial infections and Alzheimer’s disease is gaining traction in the field. While the popularity of this hypothesis has ebbed and flowed since the 1970s, recent advances suggest that both viral and bacterial infections may be involved in the progression of Alzheimer’s disease. With multiple clinical trials now testing the potential for antiviral and antibacterial drugs to slow cognitive decline in patients with Alzheimer’s disease, this controversial hypothesis is heading into the mainstream.

An infection connection

Microbial infections are involved in neurological disorders surprisingly often. When the bacteria Treponema pallidum invade the brain of late-stage syphilis patients, they can cause dementia (2). In a landmark study published earlier this year, scientists reported that becoming infected with the Epstein-Barr virus (EBV) increased a person’s risk for developing multiple sclerosis 32-fold, suggesting that EBV may cause this immuno-neurological disease (3). Now, evidence is emerging that the SARS-CoV-2 virus associates with a reduction in grey matter thickness and overall brain size (4).

When Ruth Itzhaki, now an emeritus Alzheimer’s disease researcher at the University of Manchester and visiting professor at the University of Oxford, first started investigating the infection hypothesis in 1989, she reasoned that because Alzheimer’s disease affects approximately 44 million people worldwide, an infectious cause would need to come from a pretty prolific microbe. Itzhaki figured that a herpes simplex virus 1 (HSV-1) infection was as common as they come.

“More importantly, though, the virus can become latent in the body, and once you're infected, it doesn't leave you,” she said. Additionally, herpes infections can be asymptomatic, so many people may be infected but not realize it. “You can be infected but not affected,” she emphasized.

She wondered if perhaps the reactivation of latent herpes virus in the central nervous system due to stress or the suppression of the immune system over a person’s lifetime could trigger the progression of Alzheimer’s disease.

Early attempts to look for microbes in brains of deceased people led to somewhat variable results due to the lack of sensitive techniques available at the time. But with the advent of PCR just a few years before, Itzhaki used the technique to look for evidence of herpes virus in the brains of people with Alzheimer’s disease and those without.

Because herpes viral infections are so common in the general population, Itzhaki and her team were careful not to accidentally contaminate any of the brain samples with herpes viral particles that they might have shed themselves. To their surprise, they found herpes virus in the brains of Alzheimer’s disease patients and in healthy elderly people. The problem, though, was that nobody believed them.

“Now there's a lot of work suggesting that there's a whole menagerie of viruses and bacteria [in the brain], but in those days, the thought was quite anathema to most people,” Itzhaki explained. “We had the most awful, awful trouble getting it published.” They did eventually publish their findings in the Journal of Medical Virology, but she and her team were still puzzled by the result (5).

A few years later, however, they discovered the key: people carrying the APOE4 risk allele and herpes virus in the brain had an increased risk for developing Alzheimer’s disease compared to people who carried the APOE4 allele or an HSV-1 infection alone (6). Subsequent epidemiological studies confirmed this association (7-9), but researchers in the Alzheimer’s disease community still resisted the idea that the disease could be caused by an infection.

Many of the critics pushed back, conceding that herpes virus might be in the brain, but questioning if it did anything there. Itzhaki and her colleagues rolled up their sleeves and demonstrated that HSV-1 antibodies were present in the cerebral spinal fluid of both healthy people and people with Alzheimer’s disease, suggesting that the virus indeed reactivated in the brain (10).

She and others hypothesized that as the brain’s immune system responded to these periodic reactivations, it triggered the neuroinflammation seen in Alzheimer’s disease. But if that is the case, how does viral reactivation connect to the known hallmarks of Alzheimer’s disease such as amyloid beta plaques and tau tangles?

Amyloid beta, a protector

When Tanzi first heard about the infection hypothesis, he wasn’t a big fan of it.

“People way back then were saying, ‘No, it's an infection that causes inflammation, and the plaques and the tangles are just meaningless biomarkers,” Tanzi said. “I said, ‘No, that can't be because the first four Alzheimer's genes we found in the 80s and 90s all had one thing in common: They led to more amyloid in the brain.'” 

As Tanzi started investigating the infection hypothesis further, in Manchester, Itzhaki made an exciting discovery. When she and her team infected neurons in a dish with HSV-1, to their surprise, they saw that HSV-1 stimulated the production of amyloid beta and phosphorylated tau — the hallmarks of Alzheimer’s disease (11-12). They also reported that HSV-1 DNA co-localized with amyloid plaques and that plaques containing HSV-1 appeared significantly more frequently in brains affected by Alzheimer’s disease than in healthy, age-matched brains (13).

“It suggested, of course, that the plaques sort of encapsulated or encaged the virus,” Itzhaki said.

Back at Harvard Medical School, Tanzi and his colleague Robert Moir demonstrated that amyloid beta acts as an antimicrobial peptide, inhibiting growth of both bacterial and fungal pathogens in vitro (14). They went on to show that amyloid beta protected against bacterial and fungal infections in mouse and worm models of Alzheimer’s disease (15). 

“We argued that amyloid beta protein evolved as a host defense peptide in the brain, and plaques were originally forming in response to infections,” Tanzi said. “Amyloid is the match that triggers. Tangles are the brush fires that spread. But it's not until you get inflammation — the forest fire — that you get dementia.”

Subsequent studies revealed that amyloid beta also protects against HSV-1  infection (16-17), and HSV-1 infection itself triggers amyloid beta deposition in mouse brains (18).

Tanzi sees two ways for amyloid plaques to accumulate in the Alzheimer’s disease brain: genetic susceptibility that leads to a buildup of amyloid beta, or an infection that triggers amyloid beta production to defend against invading microbes. But he argues that these two possibilities are not mutually exclusive.

“The mutations that predispose you to making amyloid more readily or having more amyloid beta protein in your brain would have been conserved every time there was a big epidemic of brain infection or encephalitis,” he explained. “So, you had evolutionary conservation of these mutations that today lead to more amyloid and predispose to Alzheimer's because originally they were protective.”

While the connection between infections and amyloid beta is strong, it is still only an association. To prove that an infection could actually cause Alzheimer’s disease, researchers would need to show that treating a microbial infection slows or reverses the progression of the disease.

Antimicrobials as potential treatments

The most fascinating prospect of a microbial cause for Alzheimer’s disease is that if it proves true, multiple antiviral and antibacterial treatments already exist.

The bacteria Porphyromonas gingivalis, which causes gum disease, associates with Alzheimer’s disease and the development of amyloid beta plaques, tau tangles, neuroinflammation, and cognitive impairment in an Alzheimer’s disease mouse model (19). The company Cortexyme recently tested their drug that inhibits vital P. gingivalis enzymes in patients with mild to moderate Alzheimer’s disease in a clinical trial. While the treatment had no significant effect on the overall group, the team found that in a sub-group of patients with high levels of P. gingivalis in their saliva, the drug slowed cognitive decline by 30-50%. They reported their results at the recent AD/PD 2022 International Conference on Alzheimer’s and Parkinson’s Diseases (20).

“To put this in context, 30 to 50% slowing is, I think, the best that anybody has ever shown in this MMSE [Mini-Mental State Exam] range, even in a subgroup,” said Michael Detke, the chief medical officer at Cortexyme. The team hopes to do a confirmatory trial to replicate this result.

In terms of viral infections, epidemiological studies have reported a strong association between antiviral treatment and a decreased risk for Alzheimer’s disease. In a landmark study of more than 33,000 people in Taiwan, epidemiologists reported that people positive for herpes virus had a 2.5 times greater risk of developing dementia compared to uninfected people (21). However, those who had received antiviral treatment at some point after contracting herpes had a decreased risk of developing dementia compared to their untreated, herpes positive peers. A recent study of more than 68,000 French people followed for eight years revealed a similar association: at least one course of anti-herpes medication associated with a 15% reduction in risk for developing Alzheimer’s disease (22).

While researchers were encouraged to see this association, many were surprised that just a short course of antivirals — in some cases only given once during a person’s life — was enough to reduce the risk of cognitive decline.

“Antiherpetic drugs don't suppress the infection. They just suppress the replication of the virus during the treatment period and maybe a little longer,” said Morgane Linard, a postdoctoral researcher in studying the role of infections in neurodegenerative disease at the University of Bordeaux and lead author of the epidemiological study.

But, she added, “I don't think that one dose can decrease the risk. I think that the association that we found and that others found is either reflecting a more chronic use of antiherpetic drugs before the inclusion in the study… or the association doesn't really exist and is caused by methodological biases that we didn't take into account.”

To probe the potential of antivirals to influence the progression of Alzheimer’s disease, Swedish scientists and clinicians performed a small clinical trial to assess the safety, feasibility, and tolerability of a month-long antiviral treatment in 33 patients with early-stage Alzheimer’s disease (23). These patients were positive for HSV-1 and carried the APOE4 risk allele.

“Most of them didn't even know that they were herpes simplex carriers,” said Bodil Weidung, a geriatrics researcher at Uppsala University and lead author of the study. “It's very common to be an asymptomatic carrier, and most of these patients had never had symptoms.”

The researchers reported that people with early-stage Alzheimer’s disease had no problem taking their antiviral medication every day for a month and showed a significant improvement in cognitive ability after the treatment period. However, because there was no placebo group in this pilot study, Weidung cautioned, “absolutely no conclusions can be made regarding these changes. But still, I'd rather have them increase than decrease.”

Hugo Lövheim, the senior author of the study and a herpes virus and Alzheimer’s disease researcher at Umeå University, hypothesized that the antivirals may improve the outcomes of Alzheimer’s disease by acting on reactivated herpes virus.

“If there is some kind of ongoing process affecting the brain, then we could also see small improvements over a short period of time when we have the viral replication,” he said. 

Similar to Weidung and Lövheim’s study, investigators at Columbia University are currently recruiting patients for a similar clinical trial that will be both placebo-controlled and include more Alzheimer’s disease patients (24). The researchers plan to complete the trial by the end of 2023.

“The potential is enough for me to want to go on investigating this. Imagine that there is already a drug on the market. It’s widely used. It's widely distributed. It's easy for clinicians to prescribe,” said Weidung. “To be able to use this tool and repurpose it for this horrible disease where we have so few options, I think it's really exciting.”

No smoking gun yet

While scientists continue to probe the infection hypothesis, for many researchers in the Alzheimer’s disease field, the inability so far to prove a definitive microbial cause for Alzheimer’s disease leaves them unconvinced.

“The idea is not crazy. It’s perfectly plausible that AD may be facilitated or exacerbated by a microbial pathogen,” said Gary Landreth, an Alzheimer’s disease researcher studying the role of neuroinflammation in the disease at Indiana University. But, he added, “There's nothing solid to hang your hat on here. And it's hard to go through life doing experiments that have small effect sizes, and people are openly skeptical of the concept.”

Landreth explained that many of the associations reported in epidemiological studies have had low numbers of participants, and other studies have been plagued by poor replicability. As a recent example of this, researchers at the University of Maryland School of Medicine reported that amyloid beta does not protect against HSV-1 infection in mice — the opposite result of what others reported (25).

“This is extremely disappointing. It's not what we thought we were going to find, but we have to stay true to the science,” said Ilia Baskakov, a prion and neurodegenerative disease researcher and senior author of the study. “Just on a personal level, it's been extremely frustrating to us because we put a lot of effort into this topic.”

Baskakov’s team observed an infiltration of the immune cells, microglia and macrophages, into mouse brains after infection by HSV-1, which may trigger the neuroinflammation associated with Alzheimer’s disease. Baskakov added that while his team’s results do not invalidate the infection hypothesis, “it requires more tests and more rigorous research.”

Whether or not Baskakov and many other researchers investigating the infection hypothesis will be able to perform additional research depends on their ability to fund their work. Many prominent researchers in the Alzheimer’s disease field who sit on the grant review boards still view the infection hypothesis as fringe, making it difficult to win funding. Baskakov said he has about one year’s worth of funding left on his infection hypothesis line of research, but he would like to do more “because I think that it's very scientifically challenging. It's a very interesting topic. I will do my best, but at a certain point, it's not up to us.”

The progressive nature of Alzheimer’s disease also means that experiments investigating a potential microbial cause are time consuming and thus expensive to perform, which without consistent funding, makes a lot of the needed research impossible.

Researchers at Tufts University, however, may have just the solution to the timing problem of infection hypothesis research. The team, led by David Kaplan a biomedical engineer with Dana Cairns, a postdoctoral fellow in Kaplan’s group, developed a 3D human brain-like tissue model that can be used to study the effect of microbial infections in the brain. By seeding neural precursor cells onto collagen-infused sponges arranged in a doughnut-shaped scaffold, Kaplan’s model mimics the matrix of the brain. The neural precursor cells develop into neurons that grow toward the center of the model and can fire across to each other.

Organoids can suffer from necrosis over long periods of time due to their high cell density, but Kaplan and Cairns’ 3D model enables the use of lower cell densities, so the cells in the model survive longer. “It allows us to do not just acute studies, but chronic studies with the brain systems,” said Kaplan.

Cairns and Kaplan initially developed the model to study the effect of Zika virus on the brain, but when Cairns saw the studies linking herpes virus to Alzheimer’s disease, she thought, why not see what a herpes infection did in their brain model?

“They formed these huge beta amyloid plaques. And then, I said, ‘David, we’ve got to run with this,’” said Cairns. “It was very clear that what we were seeing relative to Alzheimer's-like phenotypes was specific to herpes and not just any virus.” In addition to inducing amyloid beta plaques, the HSV-1 infected brain models also showed evidence of increased inflammation and impaired neuronal function (26).

In new work that has just recently been accepted for publication, Cairns and Kaplan used their 3D brain system to screen approximately 100 compounds to see if they could prevent the herpes virus-induced Alzheimer’s disease pathology in their brain tissue model.

“We're able to tease out a mechanism in some sense to see if maybe it's antiviral or maybe it prevents the formation of these beta amyloid plaques, but either way, it really opens the floodgates for drug discovery,” said Cairns.

Kaplan and Cairns would like to get their 3D brain model into the hands of other researchers who want to investigate the infection hypothesis for Alzheimer’s disease, but like many others have experienced, getting people to accept the infection hypothesis as a possibility is still a challenge.

“Even when submitting papers now, we still get pushback for just the concept,” said Cairns. “It seems like it's gaining a little bit more acceptance, but there are a lot of naysayers who seem to be kind of powerful.”

Overcoming the resistance in the Alzheimer’s disease field to a potential viral cause is especially important now as new research emerges about the role SARS-CoV-2 plays in the brain. Many researchers studying the infection hypothesis worry about the potential role that the SARS-CoV-2 virus might play in Alzheimer’s disease.

“We don't know anything yet about the long-term effects of COVID,” said Kaplan. “If it's going to affect the brain, which we know it does either directly or indirectly, that means it could then catalyze or accelerate Alzheimer's-like symptoms from other pre-infections or for other reasons. So, I think studies on those chronic effects need to start now.”

While there is still no smoking gun to say that microbes cause Alzheimer’s disease, new data from mechanistic studies and clinical trials will add to scientists’ understanding of the potential role they may play in the disease. Future work will tell if early antiviral treatment or even a childhood vaccine against HSV-1 or other microbes might prevent the devastating cognitive decline of Alzheimer’s disease. The infection hypothesis may be on the sidelines for now, but it is gaining traction in the Alzheimer’s disease field, giving hope that new treatments may not be far behind.

“Eventually, it's going to get accepted,” said Cairns. “Hopefully we get to be involved in the wave of discovery.”

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