Vaccine may unravel Alzheimer’s tangled web

 P. Rajendran

Kiran Bhaskar with graduate student Nikki Maphis

Kiran Bhaskar (left) and graduate student Nicole Maphis. They worked on the vaccine to combat Alzheimer’s disease. Courtesy: Kiran Bhaskar

Medicine’s loss was perhaps also medicine’s gain.

Kiran Bhaskar, a professor at the University of New Mexico, could not become a clinical doctor in India, but went on to complete his PhD there and now has come up with a vaccine that wipes out the visible effect of Alzheimer’s disease in mice.

Most people know that Alzheimer’s is a common form of dementia. It is marked by plaques, tangles of otherwise useful proteins, loss of nerve connections and brain inflammation. It is also associated with damage to the hippocampus, a sea horse-shaped part of the brain important to memory and learning.

The first recorded case

Auguste Deter, the first patient diagnosed with Alzheimer’s disease. Courtesy: Wikimedia

Alzheimer’s was identified as a distinct disorder only in the 20th century. The story goes that one Auguste Deter began showing signs of acute confusion, memory loss, delusion and other signs of early dementia while still in her forties. In November 1901, she was taken to the Städtische Anstalt für Irre und Epileptische (Asylum for Lunatics and Epileptics) in Frankfurt. Despite the insensitive name, the institute, managed by Emil Sioli, Franz Nissl and Alois Alzheimer, was being transformed at the time. Instead of restraining the mentally ill and leaving them in filthy conditions, Sioli helped clean up the place and let patients even have warm baths, a luxury measure that was reputedly very popular.

Franz Nissl had made his name in histology, eventually having the site of protein synthesis in a cell named after him. He taught Alzheimer some of the techniques that would make them both famous.

Alzheimer was struck by the symptoms the 51-year-old Deter, who he treated for two years and maintained detailed records of. He moved to to the Royal Psychiatric Hospital in Munich but continued to monitor Deter’s decline from afar. So, when she died in 1906, Sioli passed on her records and brain to Alzheimer for further study.

This made sense because Alzheimer was a follower of Wilhelm Griesinger, who argued that mental diseases were brain diseases. In that case, Alzheimer concluded, there would be visual evidence of Deter’s mental illness, too.

And there it was – the plaques and neurofibrillary tangles always associated with the disease and later named after Alzheimer.

Whatdunnit?

Alois Alzheimer. Courtesy: Wikimedia

It seemed clear in the past at the cleaning out the plaques and tangles could reverse the progression of the disease. What was not as clear was what causes the disease to develop. In those affected, fewer cells produce acetylcholine. So researchers developed drugs that would reduce how much acetylcholine would be broken down by the enzyme acetylcholine esterase. This treatment reduced the symptoms but did not reverse the disease itself. The drop in acetylcholine was gradually perceived as being a result, not the cause of the disease. That view received support when levels of other brain chemicals, such as dopamine and serotonin, were also found to be affected.

There is a longstanding argument that levels of amyloid beta, clumps of certain proteins, are linked to whether the disease shows up since amyloid is part of the plaques. It got support from the fact that those with Down’s syndrome, where there are three copies of chromosome 21 instead of the usual two, have higher levels of amyloid beta.

Earlier, researchers thought amyloid beta levels in those with Alzheimer’s may just not be able to undergo clearance at the same rate that it was produced. But low levels were also seen in Alzheimer’s. They conjectured it could have something to do with the ratio between a long and short forms of amyloid. And so the theories continued to bubble.

All the while there was another protein lurking around in the neurofibrillary tangles that dodged attention – tau.

Amyloid is an earlier marker of Alzheimer’s than tau,” Bhaskar told Truly Curious. “There was less interest in tau because it came up later in the disease progression.”

Now normal tau is not a rogue protein. It is believed to help the work of microtubules, thin internal tubes that act both as a cell’s internal struts, and as bridges for the caravans of chemicals crisscrossing it. Yet, there it is – in the neurofibrillary tangles. Bhaskar thought he could confirm the emerging view that warped tau proteins may not be just a symptom of Alzheimer’s, but its immediate cause.

Bhaskar considered ways to just remove them and do cognitive tests. It helped that he had done his PhD work on the cell’s cytoskeleton, post-doc research on tau proteins and, later, on amyloid.

An artist’s impression of the neurofibrillary tangles of tau protein seen in the brains of those with Alzheimer’s disease. Courtesy: National Institute on Aging

Other researchers had also got the immune system to fight pathological tau proteins (called pTau for brevity – or because it sounds coolly Vulcan). They produced antibodies that fight the erring protein, but the process is costly and has to repeated many times. Besides, the antibodies cannot easily penetrate the brain and spinal cord. In some cases, they generate an immune response themselves.

The UNM team decided to use a virus that attack bacteria to get the immune system to target the tangles. Such viruses, called bacteriophages, have been used since early in the last century to treat human illnesses. But they fell out of favor in the West after the discovery of the antibiotic penicillin.

Guilt by association

Bhaskar relied on earlier work done by researchers in his department, David Peabody, Bryce Chackerian and others. They had created viral vaccines to target problematic protein segments, such as those found in nicotine, or in Plasmodium, the single-celled creature that causes malaria. Since the viruses used attacked only bacteria, it was safe to inject them into a variety of animals, including humans.

Ethics boards do tend to frown on injections of germs or germ-like material into humans. Therefore, the team used fully-grown otherwise normal mice, and rTg4510, a variety of mice that naturally produces a lot of abnormal tau. The rTg4510 mice exhibit early signs of dementia, the effects being most prominent in brain areas behind the eyes and the temples.

Susceptible mice vaccinated with only the virus-like particle (Qß) had no protection from Alzheimer’s. But if the Qß was tagged with a bit of the pathological tau protein (pT181) then the cortex and hippocampus of comparable mice had lower levels of iba1, a protein found in microglia, the brain’s scavenger cells (see a. and b.). Compared to control-vaccinated animals, those injected with Qß-pT181 also had lower levels of CD45, a protein found in generic white blood cells (see c. and d.). This is evidence that there is less damage in the brains of mice vaccinated with Qß bonded with pT181. Courtesy: npj Vaccines, CC BY 4.0

The researchers plugged a phosphate group (phosphorus bonded to four oxygen atoms) to threonine, the 181st amino acid in the problematic tau protein. This pT181 was attached to the virus-like particle to act as bait for the immune system. pT181 also tends to increase in the cerebrospinal fluid of Alzheimer’s patients.

The plan was to give three injections of the virus-like particle ferrying the pT181 a few times into the mouse. When the immune attacked the virus-like particle, it could also expand the attack to the pT181 already in the patient’s tangled brain.

Often, research has this way of going wrong, even when all the bases appeared covered. In that sense, the team led a relatively charmed life, though it had some trouble deciding on the right bit of pTau protein to attach to the virus-like particle.

I think the only problem was with the rTg4510 mice,” Bhaskar said.

While Nicole Maphis was an experienced technician, her subjects were a little, well, demented in that they did exhibit the murine version of early onset Alzheimer’s.

It was hard to handle the mice. They were aggressive, jumpy. They were the only problems we faced,” said Bhaskar. He concluded that the lesson they got from the experience was: “Stay away from these mice.”

After three injections, given over six weeks, all the mice were tested for their ability to recognize things they had seen before, and to find a geographic location.

To test for recognition, the researchers put each mouse in an open area with two identical glass jars for five minutes for two days in a row. The third day they added a novel object, a plastic water bottle. If the rTg4510 mice still had dementia, they would not remember the glass bottles. In that case, they were likely to spend about the same time exploring the glass and plastic bottles. But if they spent more time with the new object that suggested they were already familiar with the glass bottles.

Cognitive confirmation

Gratifyingly for Bhaskar and the others, vaccinated rTg4510 mice, whose immune systems attacked pT181 in the tangles, clearly preferred the novel plastic bottle to the glass ones. Those that had received injections of the virus-like particle with no attached pT181 had trouble remembering the glass bottles they had seen the first two days. Clearly, the immune systems in these control vaccinated mice were not trained to destroy pT181. The “normal” mice, which had no neurofibrillary tangles anyway, were not affected by the treatment they received.

The other test, on the Morris Water Maze, involved putting the mice in a drum of cold milky water with a small platform just beneath the surface that they could swim to. Since the platform was not visible, the mice would have to rely on cues on the room’s walls to swim to it and get some rest. The animals were given four daily trials, each lasting about 25 minutes, for five days. The platform was in a different quadrant for each mouse. On the sixth day, the platform was diabolically removed. If the bewildered mouse swam in the quadrant where it had earlier found the platform, then it clearly knew where to look. Again, the vaccinated rTg4510 did significantly better in this spatial memory task than their control-vaccinated compatriots, and about as well as all the normal mice.

How neurofibrillary tangles form

When tau proteins  change in a brain cell, its scaffolding of microtubules break down. The freed tau proteins form tangled clumps that hamper cellular functioning. Pic courtesy: National Institute on Aging

Brain samples supported the argument for the efficacy of the vaccine. The pT181-virus particle vaccine reduces pTau in the hippocampus, that area involved with memory and spatial learning. It does the same thing in the cortex, which is involved in judgment, and sensory, motor and cognitive skills. The vaccination also prevented the shrinkage of the hippocampus and the corpus callosum, the latter being the bridge between the left and right halves of the brain.

Bhaskar’s team had a vaccine that worked – even if it was only in mice. If the results can be replicated in humans, an Alzheimer’s diagnosis will no longer be the debilitating death sentence it is today.

Clearly, it is a very good thing Bhaskar did not become a clinical doctor.

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