The effects of Covid-19 on people are varied but city doctors have observed that there is a possibility of heart attacks, especially in youngsters.
Some city experts say heart attacks after Covid-19 are caused due to the presence of blood clots in the body. However, others believe there is neither any scientific evidence to prove blood clot-related heart attacks occur due to Covid-19 nor that the virus causes disproportionally more heart attacks than otherwise. The jury is still out but the fact that heart attacks and blood clots and their presence in people who have suffered from Covid-19 is being discussed cannot be ignored.
Mid-day Online spoke to Dr Manish Hinduja, consultant-cardio thoracic and vascular surgery, Fortis Hospital and Dr Pravin Kahale, consultant, cardiology, Kokilaben Dhirubhai Ambani Hospital to understand more about the causes of blood clots and the effects post-Covid. They also shed light on the symptoms one must be aware of and the preventive measures but while taking expert advice.
What causes blood clots in people after they suffer from Covid-19?
Hinduja: In Covid, clots form in blood vessels because once the virus binds to ACE 2 receptors on blood vessel lining cells, it activates the release of clot-forming proteins. It is also sometimes due to hyperactive inflammation caused by the virus in the body.
Kahale: Any infection which damages the wall of the blood vessels increases the chances of clotting in the body and that is not particularly due to Covid, many infections can also lead to blood clots.
Do blood clots cause heart attacks in people who have suffered from Covid-19? What are the other complications that could occur due to these clots besides heart attacks?
Hinduja: Yes, clots if formed in blood vessels of the heart, can lead to heart attack. Clots can also cause stroke, pulmonary embolism, deep vein thrombosis in legs or arms, and kidney but rarely liver damage.
Kahale: Blood clots can occur due to multiple infections. There is no evidence of blood clot-related heart attacks due to Covid-19. Apart from leg veins called deep vein thrombosis, other complications that can occur are paralysis due to clots in the brain, and lung arteries.
Has there been an increase in the number of heart attacks due to blood clots or people coming with clots after suffering from Covid-19?
Hinduja: Definitely. There is an increase in the number of heart attacks after Covid infection (especially in the younger age group).
Kahale: There is no evidence that Covid causes disproportionally more heart attacks.
What are the chances of the blood clots occurring? Do they appear more in any particular age group?
Hinduja: About 20-30 per cent of patients with Covid-19 infection needing ICU treatment, show features of blood clot formation within six months of infection. Although it is more common in elderly hospitalised patients, it is also seen in young patients who have no comorbidities.
Kahale: Blood clots in mild to moderate Covid cases are uncommon. In case of severe Covid, the chances of blood clots occurring are still less. There is no particular age which is more susceptible.
Which part of the body do the blood clots occur the most?
While Hinduja says blood clots occur in the lungs, heart and brain vessels, Kahale adds that they mostly occur in leg veins and lung circulation.
Can people avoid getting blood clots after Covid-19?
Hinduja: Yes, preventive treatment with blood thinners and early diagnosis is the key.
Kahale: A patient who has suffered from severe Covid-19 infection can take a blood thinner based on the need, and guidance of a doctor.
Are there any foods people can eat to prevent getting blood clots eventually causing heart attacks? Do they need to make lifestyle changes?
Hinduja: Staying active, avoiding smoking and reducing weight for obese patients can help in reducing the risk. Common foods like ginger, turmeric and garlic have been shown to have some blood thinning effects. However, their role in preventing Covid 19-related blood clots, is not well-documented.
Kahale: In terms of blood clotting due to Covid-19, there are no specific food or lifestyle changes required. The risk of developing blood clots for a patient suffering from severe Covid-19 is only a potential threat until a patient is Covid positive.
What are the signs or symptoms for people to realise they have a blood clot? Why should they be concerned?
Hinduja: There is sudden chest pain, swelling in arms or legs, drowsiness and weakness in limbs.
Kahale: Blood clots depend upon the area where the patient is affected. If it occurs in the lungs, it can cause breathlessness. If it is in the legs, then it can cause swelling of the legs; heart blood clots lead to a heart attack-like chest pain, and clots in the brain can cause paralysis or stroke.
When Lara Hawthorne, an illustrator in Bristol, UK, began developing strange symptoms after having COVID-19, she hoped that they weren’t due to the virus. Her initial illness had been mild. “I’ve been triple vaccinated. I felt quite protected,” she says. But months later, she was still sick with a variety of often debilitating symptoms: earaches, tinnitus, congestion, headaches, vertigo, heart palpitations, muscle pain and more. On some days, Hawthorne felt so weak that she could not get out of bed. When she finally saw her physician, the diagnosis was what she had been dreading: long COVID.
Unable to find relief, she became increasingly desperate. After reading an opinion piece in The Guardian newspaper about how blood clots might be to blame for long COVID symptoms, Hawthorne contacted a physician in Germany who is treating people with blood thinners and a procedure to filter the blood. She hasn’t heard back yet — rumour has it that people stay on the waiting list for months — but if she has the opportunity to head there for these unproven treatments, she probably will. “I don’t want to wait on my health when I’m feeling so dreadful,” she says.
Researchers are baffled by long COVID: hundreds of studies have tried to unpick its mechanism, without much success. Now some scientists, and an increasing number of people with the condition, have been lining up behind the as-yet-unproven hypothesis that tiny, persistent clots might be constricting blood flow to vital organs, resulting in the bizarre constellation of symptoms that people experience.
Proponents of the idea (#teamclots, as they sometimes refer to themselves on Twitter) include Etheresia Pretorius, a physiologist at Stellenbosch University in South Africa, and Douglas Kell, a systems biologist at the University of Liverpool, UK, who led the first team to visualize micro-clots in the blood of people with long COVID. They say that the evidence implicating micro-clots is undeniable, and they want trials of the kinds of anticoagulant treatment that Hawthorne is considering. Pretorius penned the Guardian article that caught Hawthorne’s attention.
But many haematologists and COVID-19 researchers worry that enthusiasm for the clot hypothesis has outpaced the data. They want to see larger studies and stronger causal evidence. And they are concerned about people seeking out unproven, potentially risky treatments.
When it comes to long COVID, “we’ve now got little scattered of bits of evidence”, says Danny Altmann, an immunologist at Imperial College London. “We’re all scuttling to try and put it together in some kind of consensus. We’re so far away from that. It’s very unsatisfying.”
Cascade of clots
Pretorius and Kell met about a decade ago. Pretorius had been studying the role of iron in clotting and neglected to cite some of Kell’s research. When he reached out, they began chatting. “We had a Skype meeting and then we decided to work together,” Pretorius says. They observed odd, dense clots that resist breaking down for years in people with a variety of diseases. The research led them to develop the theory that some molecules — including iron, proteins or bits of bacterial cell wall — might trigger these abnormal clots.
Blood clotting is a complex process, but one of the key players is a cigar-shaped, soluble protein called fibrinogen, which flows freely in the bloodstream. When an injury occurs, cells release the enzyme thrombin, which cuts fibrinogen into an insoluble protein called fibrin. Strands of fibrin loop and criss-cross, creating a web that helps to form a clot and stop the bleeding.
Under a microscope, this web typically resembles “a nice plate of spaghetti”, Kell says. But the clots that the team has identified in many inflammatory conditions look different. They’re “horrible, gunky, dark”, Kell says, “such as you might get if you half-boiled the spaghetti and let it all stick together.” Research by Kell, Pretorius and their colleagues suggests that the fibrin has misfolded1, creating a gluey, ‘amyloid’ version of itself. It doesn’t take much misfolding to seed disaster, says Kell. “If the first one changes its conformation, all the others have to follow suit”, much like prions, the infectious misfolded proteins that cause conditions such as Creutzfeldt–Jakob disease.
Pretorius first saw these strange, densely matted clots in the blood of people with a clotting disorder2, but she and Kell have since observed the phenomenon in a range of conditions1 — diabetes, Alzheimer’s disease and Parkinson’s disease, to name a few. But the idea never gained much traction, until now.
When the pandemic hit in 2020, Kell and Pretorius applied their methods almost immediately to people who had been infected with SARS-CoV-2. “We thought to look at clotting in COVID, because that is what we do,” Pretorius says. Their assay uses a special dye that fluoresces when it binds to amyloid proteins, including misfolded fibrin. Researchers can then visualize the glow under a microscope. The team compared plasma samples from 13 healthy volunteers, 15 people with COVID-19, 10 people with diabetes and 11 people with long COVID3. For both long COVID and acute COVID-19, Pretorius says, the clotting “was much more than we have previously found in diabetes or any other inflammatory disease”. In another study4, they looked at the blood of 80 people with long COVID and found micro-clots in all of the samples.
So far, Pretorius, Kell and their colleagues are the only group that has published results on micro-clots in people with long COVID.
But in unpublished work, Caroline Dalton, a neuroscientist at Sheffield Hallam University’s Biomolecular Sciences Research Centre, UK, has replicated the results. She and her colleagues used a slightly different method, involving an automated microscopy imaging scanner, to count the number of clots in blood. The team compared 3 groups of about 25 individuals: people who had never knowingly had COVID-19, those who had had COVID-19 and recovered, and people with long COVID. All three groups had micro-clots, but those who had never had COVID-19 tended to have fewer, smaller clots, and people with long COVID had a greater number of larger clots. The previously infected group fell in the middle. The team’s hypothesis is that SARS-CoV-2 infection creates a burst of micro-clots that go away over time. In individuals with long COVID, however, they seem to persist.
Dalton has also found that fatigue scores seem to correlate with micro-clot counts, at least in a few people. That, says Dalton, “increases confidence that we are measuring something that is mechanistically linked to the condition”.
In many ways, long COVID resembles another disease that has defied explanation: chronic fatigue syndrome, also known as myalgic encephalomyelitis (ME/CFS). Maureen Hanson, who directs the US National Institutes of Health (NIH) ME/CFS Collaborative Research Center at Cornell University in Ithaca, New York, says that Pretorius and Kell’s research has renewed interest in a 1980s-era hypothesis about abnormal clots contributing to symptoms. Pretorius, Kell and colleagues found amyloid clots in the blood of people with ME/CFS, but the amount was much lower than what they’ve found in people with long COVID5. So clotting is probably only a partial explanation for ME/CFS, Pretorius says.
Where these micro-clots come from isn’t entirely clear. But Pretorius and Kell think that the spike protein, which SARS-CoV-2 uses to enter cells, might be the trigger in people with long COVID. When they added the spike protein to plasma from healthy volunteers in the laboratory, that alone was enough to prompt formation of these abnormal clots6.
Bits of evidence hint that the protein might be involved. In a preprint7 posted in June, researchers from Harvard University in Boston, Massachusetts, reported finding the spike protein in the blood of people with long COVID. Another paper8 from a Swedish group showed that certain peptides in the spike can form amyloid strands on their own, at least in a test tube. It’s possible that these misfolded strands provide a kind of template, says Sofie Nyström, a protein chemist at Linköping University in Sweden and an author of the paper.
A California-based group found that fibrin can actually bind to the spike. In a 2021 preprint9, it reported that when the two proteins bind, fibrin ramps up inflammation and forms clots that are harder to degrade. But how all these puzzle pieces fit together isn’t yet clear.
If the spike protein is the trigger for abnormal clots, that raises the question of whether COVID-19 vaccines, which contain the spike or instructions for making it, can induce them as well. There’s currently no direct evidence implicating spike from vaccines in forming clots, but Pretorius and Kell have received a grant from the South African Medical Research Council to study the issue. (Rare clotting events associated with the Oxford–AstraZeneca vaccine are thought to happen through a different mechanism (Nature596, 479–481; 2021).)
Raising safety concerns about the vaccines can be uncomfortable, says Per Hammarström, a protein chemist at Linköping University and Nyström’s co-author. “We don’t want to be over-alarmist, but at the same time, if this is a medical issue, at least in certain people, we have to address that.” Gregory Poland, director of the Mayo Clinic’s vaccine research group in Rochester, Minnesota, agrees that it’s an important discussion. “My guess is that spike and the virus will turn out to have a pretty impressive list of pathophysiologies,” he says. “How much of that may or may not be true for the vaccine, I don’t know.”
Dearth of data
Many researchers find it plausible and intriguing that micro-clots could be contributing to long COVID. And the hypothesis does seem to fit with other data that have emerged on clotting. Researchers already know that people with COVID-19, especially severe disease, are more likely to develop clots. The virus can infect cells lining the body’s 100,000 kilometres of blood vessels, causing inflammation and damage that triggers clotting.
Those clots can have physiological effects. Danny Jonigk, a pathologist at Hanover Medical School in Germany, and his colleagues looked at tissue samples from people who died of COVID-19. They found micro-clots and saw that the capillaries had split, forming new branches to try to keep oxygen-rich blood flowing10. The downside was that the branching introduces turbulence into the flow that can give rise to fresh clots.
Several other labs have found signs that, in some people, this tendency towards clotting persists months after the initial infection. James O’Donnell, a haematologist and clotting specialist at Trinity College Dublin, and his colleagues found11 that about 25% of people who are recovering from COVID-19 have signs of increased clotting that are “quite marked and unusual”, he says.
What is less clear is whether this abnormal clotting response is actually to blame for any of the symptoms of long COVID, “or is it just, you know, another unusual phenomenon associated with COVID?” O’Donnell says.
Alex Spyropoulos, a haematologist at the Feinstein Institutes for Medical Research in New York City, says the micro-clot hypothesis presents “a very elegant mechanism”. But he argues that much more work is needed to tie the lab markers to clinical symptoms. “What’s a little bit disturbing is that these authors and others make huge leaps of faith,” Spyropoulos says.
Jeffrey Weitz, a haematologist and clotting specialist at McMaster University in Hamilton, Canada, points out that the method Pretorius’s team is using to identify micro-clots “isn’t a standard technique at all”. He adds: “I’d like to see confirmation from other investigators.” Micro-clots are difficult to detect. Pathologists can spot them in tissue samples, but haematologists tend to look for markers of abnormal clotting rather than the clots themselves.
Other, larger studies of long COVID have failed to find signs of clotting. Michael Sneller, an infectious-disease specialist, and his colleagues at the NIH in Bethesda, Maryland, thoroughly examined 189 people who had been infected with SARS-CoV-2, some with lingering symptoms and some without, and 120 controls12. They did not specifically look for micro-clots. But if micro-clots had been clogging the capillaries, Sneller says, they should have seen some evidence — tissue damage in capillary-rich organs such as the lungs and kidneys, for example. Micro-clots might also damage red blood cells, leading to anaemia. But Sneller and his colleagues found no signs of this in any of the lab tests.
Kell and Pretorius argue that just because this study didn’t find any evidence of micro-clots doesn’t mean they aren’t there. One of the key issues with long COVID is that “every single test comes back within the normal ranges”, Pretorius says. “You have desperately ill patients with no diagnostic method.” She hopes that other researchers will read their papers and attempt to replicate their results. “Then we can have a discussion,” she says. The ultimate causal proof, she adds, would be people with long COVID feeling better after receiving anticoagulant therapies.
There is some limited evidence of this. In an early version of a preprint, posted in December 2021, Kell, Pretorius and other researchers, including physician Gert Jacobus Laubscher at Stellenbosch University, reported that 24 people who had long COVID and were treated with a combination of two antiplatelet therapies and an anticoagulant experienced some relief13. Participants reported that their main symptoms resolved and that they became less fatigued. They also had fewer micro-clots. Pretorius and Kell are working to gather more data before they try to formally publish these results. But other physicians are already using these medications to treat people with long COVID. Some are even offering a dialysis-like procedure that filters fibrinogen and other inflammatory molecules from the blood. To O’Donnell, such treatment feels premature. He accepts that some people with long COVID are prone to clots, but leaping from a single small study to treating a vast number of people is “just not going to wash in 2022 in my book”, he says. Sneller agrees. “Anticoagulating somebody is not a benign thing. You basically are interfering with the blood’s ability to clot,” he says, which could make even minor injuries life-threatening.
Kell says he’s tired of waiting for a consensus on how to treat long COVID. “These people are in terrible pain. They are desperately unwell,” he says. Altmann understands that frustration. He gets e-mails almost daily, asking: “Where are the drug trials? Why does it take so long?” But even in the midst of a pandemic, he argues, researchers have to follow the process. “I’m not rubbishing anybody’s data. I’m just saying we’re not there yet,” he says. “Let’s join up the dots and do this properly.”
Authors: Wes Ely August 25, 2022 The Washington Post
The young man pulled something from behind both ears. “I can’t hear anything without my new hearing aids,” said the 32-year-old husband and father. “My body is broken, Doc.” Once a fireman and emergency medical technician, he’d had covid more than 18 months before and was nearly deaf. He was also newly suffering from incapacitating anxiety, cognitive impairment and depression. Likewise, a 51-year-old woman told me through tears: “It’s almost two years. My old self is gone. I can’t even think clearly enough to keep my finances straight.” These are real people immersed in the global public health catastrophe of long covid, which the medical world is struggling to grasp and society is failing to confront.
As such stories clearly indicate, covid is biologically dangerous long after the initial viral infection. One of the leading hypotheses behind long covid is that the coronavirus is somehow able to establish a reservoir in tissues such as the gastrointestinal tract. I believe the explanation for long covid is more sinister.
The science makes it increasingly clear that covid-19 turns on inflammation and alters the nervous system even when the virus itself seems to be long gone. The virus starts by infecting nasal and respiratory lining cells, and the resulting inflammation sends molecules through the blood that trigger the release of cytokines in the brain. This can happen even in mild covid cases. Through these cell-to-cell conversations, cells in the nervous system called microglia and astrocytes are revved up in ways that continue for months — maybe years. It’s like a rock weighing down on the accelerator of a car, spinning its engine out of control. All of this causes injury to many cells, including neurons. It is past time we recognized this fact and began incorporating it into the ways we care for those who have survived covid.
These studies left most medical professionals mistakenly convinced that the virus was not damaging the brain. Accordingly, we narrowed our focus to the lungs and heart and then scratched our heads in wonder at the coma and delirium found in more than 80 percent of covid ICU patients. A robust study from the Netherlands showed that at least 12.5 percent of covid patients end up with long covid three months afterward, yet because “brain fog” wasn’t identified until later in the pandemic, these investigators didn’t include cognitive problems or mental health disorders in the data they collected. Thus, this otherwise beautifully executed study almost certainly underestimated the rate of long covid.
Since the early days of the pandemic, we’ve learned a great deal about the neurological effects of SARS-CoV-2. Earlier this year, the UK Biobank neuroimaging study showed that even mild covid can lead to an overall reduction in the size of the brain, with notable effects in the frontal cortex and limbic system. These findings help explain the profound anxiety, depression, memory loss and cognitive impairment experienced by so many long-covid patients.
A new study published in the Lancet of more than 2.5 million people matched covid-19 patients with non-covid patients to determine the rate of recovery from mental health complaints and neurological deficits like the depression and brain fog in my own patients. What it revealed is partly encouraging and partly devastating: The anxiety and mood disorders in long covid tend to resolve over months, while serious dementia-like problems, psychosis and seizures persist at two years.
Authors: Frances Stead Sellers Fri, August 19, 2022 Washington Post
A study published this week in the Lancet Psychiatry showed increased risks of some brain disorders two years after infection with the coronavirus, shedding new light on the long-term neurological and psychiatric aspects of the virus.
The analysis, conducted by researchers at the University of Oxford and drawing on health records data from more than 1 million people around the world, found that while the risks of many common psychiatric disorders returned to normal within a couple of months, people remained at increased risk for dementia, epilepsy, psychosis and cognitive deficit (or brain fog) two years after contracting covid. Adults appeared to be at particular risk of lasting brain fog, a common complaint among coronavirus survivors.
The study was a mix of good and bad news findings, said Paul Harrison, a professor of psychiatry at the University of Oxford and the senior author of the study. Among the reassuring aspects was the quick resolution of symptoms such as depression and anxiety.
“I was surprised and relieved by how quickly the psychiatric sequelae subsided,” Harrison said.
David Putrino, director of rehabilitation innovation at Mount Sinai Health System in New York, who has been studying the lasting impacts of the coronavirus since early in the pandemic, said the study revealed some very troubling outcomes.
“It allows us to see without a doubt the emergence of significant neuropsychiatric sequelae in individuals that had covid and far more frequently than those who did not,” he said.
Because it focused only on the neurological and psychiatric effects of the coronavirus, the study authors and others emphasized that it is not strictly long-covid research.
“It would be overstepping and unscientific to make the immediate assumption that everybody in the [study] cohort had long covid,” Putrino said. But the study, he said, “does inform long-covid research.”
Between 7 million and 23 million people in the United States have long covid, according to recent government estimates – a catchall term for a wide range of symptoms including fatigue, breathlessness and anxiety that persist weeks and months after the acute infection has subsided. Those numbers are expected to rise as the coronavirus settles in as an endemic disease.
The study was led by Maxime Taquet, a senior research fellow at the University of Oxford who specializes in using big data to shed light on psychiatric disorders.
The researchers matched almost 1.3 million patients with a diagnosis of covid-19 between Jan. 20, 2020, and April 13, 2022, with an equal number of patients who had other respiratory diseases during the pandemic. The data, provided by electronic health records network TriNetX, came largely from the United States but also included data from Australia, Britain, Spain, Bulgaria, India, Malaysia and Taiwan.
The study group, which included 185,000 children and 242,000 older adults, revealed that risks differed according to age groups, with people age 65 and older at greatest risk of lasting neuropsychiatric affects.
For people between the ages of 18 and 64, a particularly significant increased risk was of persistent brain fog, affecting 6.4 percent of people who had had covid compared with 5.5 percent in the control group.
Six months after infection, children were not found to be at increased risk of mood disorders, although they remained at increased risk of brain fog, insomnia, stroke and epilepsy. None of those affects were permanent for children. With epilepsy, which is extremely rare, the increased risk was larger.
The study found that 4.5 percent of older people developed dementia in the two years after infection, compared with 3.3 percent of the control group. That 1.2-point increase in a diagnosis as damaging as dementia is particularly worrisome, the researchers said.
The study’s reliance on a trove of de-identified electronic health data raised some cautions, particularly during the tumultuous time of the pandemic. Tracking long-term outcomes may be hard when patients may have sought care through many different health systems, including some outside the TriNetX network.
“I personally find it impossible to judge the validity of the data or the conclusions when the data source is shrouded in mystery and the sources of the data are kept secret by legal agreement,” said Harlan Krumholz, a Yale scientist who has developed an online platform where patients can enter their own health data.
Taquet said the researchers used several means of assessing the data, including making sure it reflected what is already known about the pandemic, such as the drop in death rates during the omicron wave.
Also, Taquet said, “the validity of data is not going to be better than validity of diagnosis. If clinicians make mistakes, we will make the same mistakes.”
The study follows earlier research from the same group, which reported last year that a third of covid patients experienced mood disorders, strokes or dementia six months after infection with the coronavirus.
While cautioning that it is impossible to make full comparisons among the effects of recent variants, including omicron and its subvariants, which are currently driving infections, and those that were prevalent a year or more ago, the researchers outlined some initial findings: Even though omicron caused less severe immediate symptoms, the longer-term neurological and psychiatric outcomes appeared similar to the delta waves, indicating that the burden on the world’s health-care systems might continue even with less-severe variants.
Hannah Davis, a co-founder of the Patient-Led Research Collaborative, which studies long covid, said that finding was meaningful. “It goes against the narrative that omicron is more mild for long covid, which is not based on science,” Davis said.
“We see this all the time,” Putrino said. “The general conversation keeps leaving out long covid. The severity of initial infection doesn’t matter when we talk about long-term sequalae.
A team of international researchers may have uncovered the cause of the neurological conditions seen in patients with long-Covid, such as brain fog. The team from Swinburne University of Technology and La Trobe University in Australia and Luxembourg University in Luxembourg revealed that fragments of proteins from the SARS-CoV-2 virus can form amyloid clumps in the brain that look similar to the amyloids found in patients with neurodegenerative diseases such as Alzheimer’s and Parkinson’s.
Further, the study published in the journal Nature Communications showed that these amyloids are highly toxic to brain cells. To understand, the team designed, performed and analysed the biochemical flow cytometry assays used to determine the mechanism of brain cell death triggered by the amyloids and assisted with physical characterisation of the amyloids at the Australian Synchrotron.
“If further studies are able to prove that the formation of these amyloids is causing long-Covid then anti-amyloid drugs developed to treat Alzheimer’s might be used to treat some of the neurological symptoms of long-Covid,” said Dr Mirren Charnley, a postdoctoral researcher at Swinburne.
Long-Covid is marked by neurological symptoms, such as memory loss, sensory confusion, severe headaches, and even stroke.
These neurological symptoms are similar to the early stages of neurodegenerative diseases such as Alzheimer’s and Parkinson’s, which are characterised by the presence of clumps of ordered proteins a” known as amyloids – in the brain.
The long-Covid symptoms can persist for months after the infection is over. While there is evidence that the virus can enter the brain of infected people, the precise mechanisms causing these neurological symptoms are unknown.
COVID-19 is primarily known as a respiratory disease caused by SARS-CoV-2. However, neurological symptoms such as memory loss, sensory confusion, severe headaches, and even stroke are reported in up to 30% of cases and can persist even after the infection is over (long COVID). These neurological symptoms are thought to be produced by the virus infecting the central nervous system, however we don’t understand the molecular mechanisms triggering them. The neurological effects of COVID-19 share similarities to neurodegenerative diseases in which the presence of cytotoxic aggregated amyloid protein or peptides is a common feature. Following the hypothesis that some neurological symptoms of COVID-19 may also follow an amyloid etiology we identified two peptides from the SARS-CoV-2 proteome that self-assemble into amyloid assemblies. Furthermore, these amyloids were shown to be highly toxic to neuronal cells. We suggest that cytotoxic aggregates of SARS-CoV-2 proteins may trigger neurological symptoms in COVID-19.
The disease caused by viral infection with severe acute respiratory syndrome (SARS)-COV-2 is known as COVID-19 and whilst predominantly a respiratory disease affecting the lungs it has a remarkably diverse array of symptoms. These include a range of moderate to severe neurological symptoms reported in as many as 30% of patients, which can persist for up to 6 months after infection1. These symptoms include memory loss, sensory confusion (e.g., previously pleasant smells become fixed as unpleasant), cognitive and psychiatric issues, severe headaches, brain inflammation and haemorrhagic stroke1,2,3,4,5. COVID-19-related anosmia and phantosmia have been shown to correlate with a persistence of virus in the olfactory mucosa and in the olfactory bulb of the brain, and with persistent inflammation; however, negative evidence for continuing viral replication has also been shown for long-term anosmia6. Furthermore, there is evidence that SARS-CoV-2 is neuroinvasive with either the full virus7,8 or viral proteins8 being found in the CNS of mouse models and the post-mortem brain tissue of COVID-19 patients. Whilst the neuroinvasiveness of SARS-CoV-2 is apparent the molecular origin of the associated neurological symptoms is as yet unknown, although they are similar to hallmarks of amyloid-related neurodegenerative diseases such as Alzheimer’s (AD)9,10, and Parkinson’s11. For instance, impaired olfactory identification ability and mild cognitive impairment have also been reported in the early stages of AD and prodromal AD12.
A number of in vitro studies have shown that proteins from SARS-CoV-2 can detrimentally affect a variety of cell types including kidney, liver and immune cells13,14. Furthermore, experiments on brain organoids show that SARS-CoV-2 can infect neuronal cells resulting in cell death15. Combined these papers point to a potential cytotoxic cause of neurological symptoms in COVID-19.
Proteins from the Zika virus16 and also the coronavirus responsible for the SARS outbreak in 2003 (SARS-COV-1)17 have been shown to contain sequences that have a strong tendency to form amyloid assemblies. As the proteome of SARS-CoV-1 and SARS-CoV-2 possess many similarities18, we propose amyloid nanofibrils formed from proteins in SARS-CoV-2 may be implicated in the neurological symptoms in COVID-19. Therefore, amyloid-forming proteins from the SARS-CoV-2 virus in the CNS of COVID-19 infected patients could have similar cytotoxic and inflammatory functions to amyloid assemblies that are the molecular hallmarks of amyloid-related neurodegenerative diseases such as AD (Aβ, Tau) and Parkinson’s (α-synuclein). The worst-case scenario given the present observations is that of the progressive neurological amyloid disease being triggered by COVID-19. To the authors’ knowledge, there has so far been no documented example of this; however, it has been noted that up-regulation of Serum amyloid A protein driven by inflammation in COVID-19 seems like a probable trigger for the systemic (non-neurological) amyloid disease AA amyloidosis19, which is already known to be a concomitant of inflammatory disease in general.
If the proteome of SARS-COV-2 does contain amyloid-forming sequences, this raises the question, what is their function? It is known that viral genomes evolve rapidly and are highly constrained by size; therefore, every component is typically functional either to help the virus replicate or to impede the host immune system. To this end, there are several potential roles for amyloid assemblies in pathogens generally20 and specifically in coronaviruses such as SARS-CoV-2. The simplest is that amyloid is an inflammatory stimulus21, and proinflammatory cytokines can up-regulate the expression of the spike protein receptor ACE-2 such that intercellular transmissibility of SARS-CoV-2 is increased. Alternatively Tayeb-Fligelman et al.22 found that the nucleocapsid protein in SARS-CoV-2, which is responsible for packaging RNA into the virion, contains a number of highly amyloidogenic short peptide sequences within its intrinsically disordered regions22. It has been shown that the self-assembly of these peptides is enhanced in the presence of viral RNA, during liquid–liquid phase separation (LLPS is an important stage in the viral replication cycle)23,24. These findings suggest amyloids may play an important role in RNA binding and packaging during the viral replication cycle. Finally, it is also possible that amyloid assemblies in coronaviruses might have a role in inhibiting the action of the host antiviral response similar to a discussed role for amyloid in other viruses. Pham et al.25 observed that amyloid aggregates from murine cytomegalovirus can interfere with RIPK3 kinase activation and potentially inhibit its antiviral immune signalling capabilities.
In this study, we choose to focus on a selection of proteins from the SARS-CoV-2 proteome known as the open reading frames (ORFs). These ORF proteins were chosen as they have no obvious roles in viral replication26, perhaps freeing them up to have yet uncharacterised roles in disrupting the host antiviral responses. By sequence and length, they appear to be largely unstructured, making them good candidates for amyloid formation in vivo. We performed a bioinformatic screening of the ORF proteins to look for potential amyloidogenic peptide sequences. This analysis was used to select two sub-sequences, one each from ORF6 and ORF10, for synthesis. The synthesised peptides were both found to rapidly self-assemble into amyloid assemblies with a variety of polymorphic morphologies. Cytotoxicity assays on neuronal cell lines showed these peptide assemblies to be highly toxic at concentrations as low as 0.0005% (0.04 mg mL−1).
Since commencing this work, others have found that ORF6 is the most cytotoxic single protein of the SARS-CoV-2 proteome, showing localisation to membranes when overexpressed in human and primate immune cell lines13. In contrast, ORF10 has been reported as an unimportant gene with very low expression and no essential role in virus replication26; however, the functions of immune suppression or inflammation promotion via amyloid formation would be non-essential, if present, and should not necessarily require transcription in large volumes, making ORF10 an intriguing second candidate for the present study. It is also interesting that ORF10 and ORF8 are the only two coded proteins present in SARS-CoV-2 which do not have a homologue in SARS-CoV-127, perhaps suggesting a unique amyloid etiology for COVID-19. While long-term consequences from SARS-CoV-1 infection were severe, including tiredness, depression, and impaired respiration, few or zero unequivocally neurological post-viral symptoms were recorded from the (admittedly quite small) set of documented cases28.
Results and discussion
Amyloid aggregation prediction algorithms identified two short peptides from ORF6 and ORF10 that are likely to form amyloids
Figure 1 shows selected output from bioinformatics tools applied to predict the amyloidogenicity of peptide sequences within larger polypeptides. Application of the ZIPPER tool to ORF6 provides more than ten choices of six-residue windows of the sequence predicted to be highly amyloidogenic (Fig. 1a), while ORF10 shows only three such highly amyloidogenic sequence windows (Fig. 1b). To narrow down our search for candidate peptides we also used the TANGO algorithm, for ORF6 there are two regions that are predicted to be highly aggregation prone, I14LLIIMR and D30YIINLIIKNL. The region I14-R20 overlaps almost perfectly with the hexapeptide I14LLIIM identified by ZIPPER. The region 30–40 also contains multiple hits in ZIPPER, but as this study was limited to two candidate peptides we chose ILLIIM as our first candidate as it closely resembles the sequence ILQINS from Hen Egg White Lysozyme that has previously been seen to be highly amyloidogenic (the mutation TFQINS in human lysozyme is disease-linked)29,30,31. Looking now at the TANGO plots for ORF10 the main aggregation-prone sequence is residues F11TIYSLLLC, although there are no high stability hexapeptides in this sequence predicted by ZIPPER. The octapeptide R24NYIAQVD was chosen due to its zwitterionic residue pair R-D which should strongly enhance interpeptide association, despite being too far apart in the sequence to trigger the highly local bioinformatics algorithms. Encouragingly ZIPPER also predicts the hexapeptide NYIAQV contained within RNYIAQVD to be highly amyloidogenic. Based on the outputs from ZIPPER and TANGO and also on the experience in making and studying amyloid, we selected RNYIAQVD and ILLIIM to be synthesised and their amyloid-forming capability investigated.
Nanoscale imaging reveals both peptide sequences self-assemble into polymorphic amyloid assemblies
Atomic force microscopy (AFM) imaging of the two peptide assemblies revealed that both peptides assembled at 37 °C almost immediately at 1 mg mL−1 (Supplementary Figs. 1 and 2) into a highly polymorphic mixture of nanofibrous and crystalline structures (Supplementary Fig. 3). For both peptides, the dominant polymorph was needle-like crystalline assemblies as seen in Fig. 2. In an attempt to ensure any observed polymorphism was not due to a heterogeneous mixture of insoluble peptide seeds we added warm PBS (90 °C) to the lyophilised peptides, and maintained this elevated temperature for at least 3 h in order dissolve as much of the monomeric peptide as possible. Self-assembly was subsequently initiated by slowly reducing the temperature, using a previously developed protocol32. This method produced less polymorphism resulting in the needle-like crystalline polymorph being overwhelmingly dominant, however a number of twisted fibrillar polymorphs were still present for RNYIAQVD (Fig. 2i and Supplementary Fig. 4a). To facilitate repeatable quantitative analysis of the biochemical and biophysical properties of the assemblies we used the slow cooling assembly method for all further experiments.
AFM and transmission electron microscopy (TEM) imaging of assemblies formed at either 1 or 5 mg mL−1 for 24 h revealed that assemblies from both peptides tend to stack on top of each other forming multi-laminar structures (Fig. 2a–d and Supplementary Figs. 5 and 6). Evidence of lateral assembly of the needles was also observed but this appears to happen more frequently in the ILLIIM assemblies (Fig. 2g) compared to RNYIAQVD. ILLIIM tends to form very large (2–3 μm in width) multi-laminar crystalline assemblies (Fig. 2g), whereas RNYIAQVD predominantly forms long linear needle-like structures. The apparent lower tendency of RNYIAQVD to form large two-dimensional lateral assemblies can be explained by the polymorphism seen in this peptide, which does not promote translational symmetry (i.e., extended crystals). Figure 2i and Supplementary Fig. 4a both clearly show that in addition to the flat needle-like crystals seen elsewhere, RNYIAQVD can also form non-planar partially twisted fibrillar assemblies. This polymorphism observed in RNYIAQVD assemblies may reduce the ability of the crystals to laterally associate and stack into multi-laminar species, simply because of a mismatch in planarity between two adjacent assemblies, the molecular basis for this polymorphism is briefly discussed in the next section of the manuscript. AFM was further used to investigate the height of the individual assemblies of both ILLIIM and RNYIAQVD. Figure 2a, b shows a line section through a multi-laminar RNYIAQVD assembly with two distinct layers with a step height of 5.5 nm between each layer. Similarly for ILLIIM (Fig. 2c and Supplementary Fig. 5), we see multi-laminar stacking with individual step heights that vary between 4 and 12 nm. Turning to RNYIAQVD we see single crystals with step heights varying between 5 and 20 nm (Supplementary Fig. 6). Together this heterogeneous distribution of crystal heights provides further evidence for the polymorphic nature of both the ILLIIM and RNYIAQVD assemblies.
Quantitative analysis of the distribution of assembly heights and contour lengths was performed using the freely available software tool FiberApp33. The analysis of assembly height distribution was taken from the z-axis (height) of the AFM images, both peptide assemblies show a heterogeneous distribution of fibril heights due to the previously observed tendency of both assemblies to form polymorphic multi-laminar stacks (Fig. 2f, h). Analysis of the distribution of the contour length of the two assemblies showed a biphasic distribution of lengths for both fibrils with two broad sub-populations centred around 1 and 3 µm (Fig. 2j, l). The sub-population at 3 µm was seen to be much larger for the RNYIAQVD peptide (Fig. 2j) compared to the ILLIIM (Fig. 2l). This population of longer fibrils correlates with the observation from Fig. 2 that for RNYIAQVD longer, thinner assemblies are favoured (self-assembly via fibril extension) over the wider shorter assemblies more commonly seen for ILLIIM (assembly via lateral association of protofilaments). Analysis of the persistence length of the fibrils (Supplementary Fig. 7) showed that whilst both peptides formed very straight linear assemblies, the persistence length of RNYIAQVD (λ = 41.92 µm) is greater than that of ILLIIM (λ = 31.96 µm).
To further investigate the polymorphic nature of the assembly of these peptides, we investigated the structures formed from 1:1 mixtures of the two peptides. Interestingly when mixed prior to assembly the peptides form a wide range of polymorphic structures exceeding that of either peptide assembled individually. Supplementary Fig. 8 shows a selection of some of the polymorphs formed, especially interesting are the large flat structures with well-defined edges that seem almost to interlock (Supplementary Fig. 8c). Such well-ordered 2D crystals were never observed for either peptide individually, and provide clear evidence of co-crystallisation. At this stage we have no evidence for the biological relevance of this co-crystallisation; however, as the ORF proteins from which these peptides are identified are themselves very small proteins (ORF6 is 61 amino acids in length), it is feasible that these small proteins may undergo similar co-crystallisation during their viral replication cycle facilitating an as yet unknown biological function.
X-ray scattering, spectroscopic characterisation, fluorescent microscopy and molecular modelling confirm the amyloid nature of the assemblies
Figure 3a shows the radially averaged 1D small-angle X-ray scattering (SAXS) plots for ILLIIM and RNYIAQVD at the lower concentration studied (at the higher concentration, sedimentation made recording X-ray scattering spectra impossible). In the central part of the scattering curve, the ILLIIM assemblies produced a slope with a q−2 dependence which is consistent with the form factor of an infinite 2D surface30, and is most likely arising due to the broader lateral dimensions observed by AFM and TEM for ILLIIM compared to RNYIAQVD. RNYIAQVD, however, displays a q−4 dependence in the central part of the scattering curve, appearing more towards the high-q limit. Porod’s law indicates that q−4 scaling (at high q but still less than 0.1 Å−1) is consistent with any aggregates having sharp surfaces but does not otherwise specify shape34. The data from the SAXS plots provide supporting evidence that the laterally associated amyloid assemblies seen by AFM and TEM are not artefacts induced either by the dehydration (AFM), applying vacuum conditions (TEM) or the mica (AFM) or carbon (TEM) substrates used, but a genuine structure observed also in bulk.
Figure 3b shows the CD spectra of mature assemblies, of both peptides. Assemblies of ILLIIM display a quite simple spectrum indicating the dominance of β-sheets, with a minimum between 225 and 230 nm and a strong maximum at 205 nm29. The CD spectra of RNYIAQVD possess a well-defined minimum at 203 nm and a distinct shoulder at around 215 nm.
To further investigate the predicted secondary structure of both peptide assemblies we employed the secondary structure analysis software BeStSel (Supplementary Table 1)35,36. As expected from the classic shape of the spectra the predicted secondary structure of ILLIIM at 5 mg mL−1 is exclusively made up of β-sheets (41.8%) and β-turns (58.2%). At these high concentrations, the composition of these β-sheets is shown to be exclusively left twisted, whilst at lower concentrations (1 mg mL−1) a more complex mixture of right, left and non-twisted (relaxed) β-sheets are predicted. At both concentrations, the CD spectra of RNYIAQVD again suggest the secondary structure is dominated by β-sheets; however, now they appear to be exclusively in the form of higher energy right-twisted β-sheets, similar to that observed in the highly strained structure of other amyloidogenic ultra-short peptides30. This additional strain introduced by β-sheets opposing the left-handed chirality seen in natural amino acids may explain the additional polymorphism and twisted microstructures seen in the AFM and TEM images of the RNYIAQVD assemblies (Fig. 2i and Supplementary Fig. 4a)29. The BeStSel fitting algorithm predicted the remainder of the RNYIAQVD secondary structure is composed of α-helical structure and further backbone conformations that could not be assigned (Supplementary Table 1). Part-helical CD spectra do not necessarily imply helical structure, especially considering that a single octapeptide cannot literally be 19% helix (two residues). Backbone conformation as reported by CD correlates through sheet structure to the assembled tertiary structure but no single level of organisation exclusively dictates any other, this is especially true in the case of coupling the twist of a peptide strand to the overall twist of the aggregate, which can relax to meet surface and shape-driven constraints through intersheet and interchain as well as intrachain degrees of freedom29.
To further investigate the conformation of the amyloid assemblies formed we utilised the conformation-specific antibody A11 and the fluorescent probe thioflavin T (ThT). The former binds specifically to non-fibrillar amyloid oligomers and the latter is a commonly used molecular probe that becomes highly fluorescent when binding to amyloid assemblies37. As expected, when ILLIIM and RNYIAQVD assemblies were stained with ThT both demonstrated clearly visible fluorescent emission at 590 nm, providing further evidence of their amyloid nature (Supplementary Fig. 9a, b). Conversely, A11 exhibited no positive binding; specifically, the level of fluorescence observed was similar to the background staining seen in the negative controls, as confirmed by similar fluorescent intensities for both assemblies (Supplementary Fig. 9e, f) and the negative controls (Supplementary Fig. 9d, h). Higher levels of A11 binding were seen for the positive control that consisted of phenylalanine assemblies known to form oligomeric species38 (Supplementary Fig. 9g), with fluorescent intensities for these assemblies over 4 times greater than for ILLIIM or RNYIAQVD. Combined these data confirm the amyloid nature of the two ORF peptide fragments and suggest that non-fibrillar oligomeric amyloid species are absent.
The amyloid nature of the two assemblies is further confirmed by the wide-angle X-ray scattering (WAXS) spectra (Fig. 3d) of the peptide assemblies which possessed a number of strongly diffracting Bragg peaks. Both peptides have a clear peak at 1.38 Å−1 corresponding to a d-spacing of 4.6 Å which is indicative of an amyloid assembly composed of extended β-sheets39. It is worth noting that the apparent intrastrand spacing of ILLIIM assemblies is very slightly lower than the typically reported values (4.7–4.8 Å). This may be explained by the BeStCell analysis of the ILLIIM assemblies at 1 mg mL−1, which suggests that the β-sheets in these assemblies are composed of a complex mixture of left-handed, strained right-handed and relaxed β-sheets (Supplementary Table 1); therefore, it is perhaps not surprising that the observed average intersheet spacing very slightly differs from that which is commonly reported. Furthermore, Lomont et al.40 report that the observed intrastrand spacing from a range of amyloid crystal structures can vary by as much as 0.45 Å. ILLIIM also has a very strong Bragg reflection at 0.58 Å−1 (11 Å) corresponding to a typical intersheet spacing given moderately bulky hydrophobic sidechains forming a steric zipper. RNYIAQVD has a number of well-defined Bragg peaks between 0.3 and 0.75 Å−1 that are consistent with a mixture of first and second-order reflections corresponding to an amyloid-like 3D symmetry. Typical reflections arising from the combinations of the longer two axes of the unit cell of short peptide amyloid crystals arise in the 0.3–0.75 Å−1 region with a qualitatively similar appearance to the pattern from RNYIAQVD, although in this case the peaks could not be individually assigned.
Discovery of sub-Å resolution structures from solution WAXS is highly challenging; however, given the simple nature of the scattering from the ILLIIM system, it was possible to produce an atomistic model matching the positions of the observed peaks, although not their sharpness. Physically, peak sharpness increases with the ordering length scale, indicating that some structures in the solution were larger than could be managed in the simulation. The sheet-like shape factor and the presence of peaks at roughly 2π/4.6 and 2π/11 Å−1 indicate assembly in solution dominated by the hydrogen bonding axis (with the typical parallel β sheet period of ≈ 4.7 Å) and by a sidechain-sidechain hydrophobic zipper interface. A metastable candidate structure of size 6 × 50 × 1 peptides was constructed following this geometry (see Methods) and found to reproduce the observed WAXS and to fully exclude water at the steric zipper (Fig. 4). The q−2 dependence of ILLIIM scattering at low q in solution (Fig. 3a) is consistent with a 2D sheet-like structure similar to that produced in the modelling. Initial assembly into sheets is also consistent with the eventual formation of multi-laminar structures as shown in the AFM (Fig. 2), as well as with the tendency of ILLIIM in particular to form lateral assemblies of needle microcrystals (Fig. 2g, k vs. e, i). Atomistic details of the interaction of the 2D sheet-like oligomer structure of Fig. 4 with neuronal cell membrane are difficult to predict and would be an interesting subject for further work. However, the juxtaposition of hydrophobic sidechains with polar termini in the ILLIIM fragment (or with titratable residues in the longer fragment E13ILLIIMR, which unfortunately could not be synthesised) has a length of approximately 10 Å, comparable to the polar-hydrophobic-polar length scale of 40 Å for the two leaflets of the eukaryotic cell membrane, indicating a potential for planar aggregates of, in particular, four sheets in thickness (four peptides end-to-end, linked in the middle by salt bridges) to disrupt the cell membrane.
The ThT stain, which becomes highly fluorescent upon binding to β-sheet rich amyloid assemblies was used to assess the assembly kinetics of both ILLIIM and RNYIAQVD (Fig. 3c). Both peptides show rapid kinetics with significant assembly occurring almost instantaneously and reaching a plateau after 30–60 min. Longer amyloidogenic polypeptides typically show a distinct lag phase in their assembly kinetics; however, this was not observed in these sequences. This apparent lack of a lag phase in the assembly kinetics behaviour is typical of amyloidogenic short peptides, which have been previously seen to assemble very rapidly29,41. This is highly likely to be due to a lack of additional non-amyloidogenic amino acid sequences acting as a kinetic barrier to amyloid formation. The ThT signal for ILLIIM at 5 mg mL−1 plateaus at about 300 a.u., this is slightly stronger than the maximum signal generated from mature fibrils of the somewhat homologous peptide ILQINS, which was around 250 a.u29,30,31,42, suggesting that the amyloidogenicity of the two peptides is comparable. RNYIAQVD, whilst showing similar ThT values at low concentrations (1 mg mL−1), generated a ThT signal nearly 3 times as large at 5 mg mL−1 suggesting that the assembly of this peptide is highly concentration dependent. For reasons yet unknown, it seems that RNYIAQVD appears to reach a maximum ThT value and then begin to drop, this can be seen at both concentrations but is most obvious at the higher concentration. This could be due to a reversible self-assembly as seen in other functional amyloids39,41,43,44, or to self-quenching of the amyloid bound aromatic ThT molecules, or simply to a reduction of exposed ThT-binding sites as larger aggregates with a lower surface area to volume ratio come to dominate the solution.
Cytotoxicity of both studied peptides also began to drop slightly (without statistical significance) at the highest concentrations tested (vide infra, Fig. 5), together with the drop in ThT response this supports the existence of a kinetically or thermodynamically available aggregate structure with reduced ‘amyloid activity’. This is reminiscent of strongly amyloid correlated diseases such as AD, where the toxicity of amyloid can vary dramatically, with the relationship between the amount of amyloid deposited to the progress of the disease being idiosyncratic and highly non-linear45. Combined the CD spectroscopy (Fig. 3b), the ThT spectroscopy (Fig. 3c) and confocal microscopy (Supplementary Fig. 9), the presence of the Bragg peaks corresponding to the intra- and inter-β-sheet spacings (Fig. 3d) and the molecular modelling (Fig. 4) confirm beyond doubt the β-sheet rich, amyloid nature of these two fragments.
ILLIIM and RNYIAQVD peptide assemblies are both highly toxic towards the neuroblastoma cell line SH-SY5Y
Given the physical evidence and the discussions referred to in the introduction of various means by which SARS-CoV-2 and other viral infections could enhance their fitness (to the detriment of the host) by the production of amyloidogenic peptides, we hypothesised that the SARS-CoV-2 viral transcript fragments ILLIIM and RNYIAQVD are toxic to human neurons. This is in particular supported by the previously reported neuroinvasive capabilities of SARS-CoV-27,8, the noted similarities of the symptoms to a (hopefully transient form of) AD5 and the previous detection of amyloid assemblies driven by other viruses20. To investigate this further we performed a number of cytotoxicity assays of the two peptide sequences against a human-derived neuroblastoma cell line (SH-SY5Y) often used as a model cell line for studying Parkinson’s and other neurodegenerative diseases46. Using an MTT assay we found that cells grown in the presence of both peptide assemblies possessed much lower viability after 48 h incubation. Concentrations as low as 0.04 and 0.03 mM (for RNYIAQVD and ILLIIM, respectively) were seen to reduce the viability of cultured cells after 48 h to <50% (IC50) compared to the cells cultured without the peptides (Fig. 5a, b). This toxicity in relation to concentration is similar to that reported for Aβ4247 although expression levels and time-scales (sudden for COVID versus chronic for AD) are likely to be very different.
To gain further insight into the mechanism of cell death occurring in the peptide exposed cells, we performed a detailed flow cytometry analysis using the apoptotic stain Annexin V and the viability dye 7-AAD. Figure 5c shows representative flow cytometry plots; cells can be identified as viable (bottom left quadrant), viable but undergoing early apoptosis (bottom right), non-viable and necrotic (top left) or non-viable due to late-stage apoptosis (top right). The percentages of cells in these quadrants are roughly equal for all conditions tested except in the case of late-stage apoptosis where we see a large increase in the cells exposed to the peptide assemblies (a 6.25-fold increase for RNYIAQVD at 2.5 mg mL−1). Quantification over a range of concentrations showed that on average cells exposed to both ILLIIM and RNYIAQVD had a 3–5-fold increase in late-stage apoptosis compared to SH-SY5Y cells cultured in the absence of peptide assemblies (Fig. 5d, e). No evidence of increasing necrosis was seen in any of the samples, suggesting that the amyloid assemblies are triggering programmed cell death via an apoptotic pathway. This triggering of late-stage apoptosis in the cells was more pronounced for ILLIIM than for RNYIAQVD, showing statistically significant increases in apoptotic cells at concentrations as low as 0.04 mg mL−1 for ILLIIM compared to 0.15 mg mL−1 for RNYIAQVD. This increase in apoptosis down to low concentration provides convincing evidence, especially for ILLIIM, that the amyloid aggregates are responsible for this toxicity, as at these low concentrations we would expect very little un-assembled peptide to exist. The mechanisms of cell death in neurodegenerative diseases are complex and can vary between different diseases48, and here we provide evidence that induction of apoptosis may be an important mechanism of neuronal death in COVID-19. Intriguingly, the conserved protein ORF6 from SARS-CoV-1 (not SARS-CoV-2) has previously been shown to induce apoptosis49. Furthermore, we performed a series of cell counting experiments and demonstrated that after 48 h incubation we saw statistically significant decreases in cell number for both peptides at concentrations as low as 0.04 mg mL−1 for ILLIIM and 0.32 mg mL−1 for RNYIAQVD. These results confirm that in addition to the cytotoxic nature of the peptide assemblies, they significantly reduce cell number especially in the case of ILLIIM. The significant increase in apoptosis and reduction in cell number seen for ILLIIM correlates with the work of Lee et al. who have previously shown that the ORF6 protein (that contains the ILLIIM sequence) is the most cytotoxic protein in the proteome of SARS-CoV-213. Combined with our data, this suggests that this toxicity might be due to the amyloidogenic nature of this short protein.
Previous research has shown that the polymorphism, size distribution and the morphology of amyloid aggregates can have a large influence on their cytotoxicity. Marshall et al.50 showed that a range of crystal-forming assemblies formed from short peptide sequences show surprisingly little toxicity to the same neuroblastoma cell line used in this study. Our TEM and AFM images (Fig. 2) confirm that the assemblies formed by the sequences identified from ORF6 and ORF10 look very similar to the assemblies in Marshall et al.50 but the SARS-CoV-2-related peptides are significantly more toxic, suggesting a specific mechanism of toxicity for these assemblies. Xue et al.51 showed that shorter amyloid assemblies from a range of different proteins/peptides have increased the ability to disrupt the bilayer of unilamellar vesicles and provide a greater cytotoxic effect on neuroblastoma cells. Mocanu et al.52 showed a dose-dependent cytotoxic effect in epithelial cells for long-thin lysozyme amyloid fibrils, and a threshold dependent mechanism for the larger laterally associated fibrils. We see similar effects to both Xue et al. and Mocanu et al. suggesting that the observed toxicity of the assemblies may be related to their aspect ratio. We observed that ILLIIM assemblies are both more toxic, wider (Fig. 2h) and shorter than their RNYIAQVD counterparts (Fig. 2k), this is shown schematically in Fig. 6. Similarly to Mocanu et al.52 we see that the long-thin RNYIAQVD fibrils show a clear dose-dependent increase in apoptosis (Fig. 5e), and the laterally associated ILLIIM fibrils show similarly high levels of apoptosis induction at all concentrations above a threshold of 0.04 mg mL−1 (Fig. 5d).
There is a wealth of literature suggesting that in neurodegenerative diseases like Alzheimer’s and Parkinson’s amyloid oligomers are the main toxic culprits and mature amyloid fibrils are a more inert assembly end-point. This is seemingly at odds with our data; however, there have also been multiple studies that show mature assemblies can also display significant toxicity37,53,54. Alternatively, it may be the nature of the amyloids species seen here that differs from amyloids in neurodegenerative diseases; the amyloids seen here appear to be largely crystalline (especially in the case of ILLIIM). Previous work has shown that amyloid crystals are deeper in the free energy landscape compared to twisted protofilaments and amyloid ribbons30,55, representing a global energy minima. AFM and TEM data have shown that these stable amyloid crystals are the dominant polymorph for ILLIIM (Fig. 2c, g, k) and that RNYIAQVD shows examples of higher energy (partially) twisted fibrils (Fig. 2i and Supplementary Figs. 4a and 6). Therefore, we hypothesise that the low energy ILLIIM crystalline assemblies are more slowly metabolised and cells are exposed for longer timeframes to the cytotoxic effect compared to RNYIAQVD assemblies. To date, there have been few investigations into the toxicity of amyloid crystals compared to other more commonly reported amyloid species. For the reasons above, the toxic nature of these amyloid assemblies warrants further investigations into the potential presence of amyloid aggregates from SARS-CoV-2 in the CNS of COVID-19 patients, and the potential role of amyloids in the neurological symptoms observed.
In conclusion, using a bioinformatics approach we identified two strongly amyloidogenic sub-sequences from the ORF6 and ORF10 sections of the SARS-COV-2 proteome. Nanoscale imaging, X-ray scattering, molecular modelling, spectroscopy and kinetic assays revealed that these self-assembled structures are amyloid in nature, and screening against neuronal cells revealed that they are highly toxic (approximately as toxic as the toxic amyloid assemblies in AD) to a cell line frequently used as a neurodegenerative diseases model. The neuroinvasive nature of SARS-COV-2 has been established previously7,8; therefore, it is entirely plausible that amyloid assemblies either from these ORF proteins or other viral proteins could be present in the CNS of COVID-19 patients. The cytotoxicity and protease-resistant structure of these assemblies may result in their persistent presence in the CNS of patients post-infection that could partially explain the lasting neurological symptoms of COVID-19, especially those that are novel in relation to other post-viral syndromes such as that following the original SARS-CoV-1. The outlook in relation to triggering of progressive neurodegenerative disease remains uncertain. Given the typically slow progress of neurodegenerative disease if such a phenomenon exists, it will most probably take some time to become evident epidemiologically.
Amyloid prediction algorithms
The online amyloid prediction algorithms TANGO and ZIPPER were used to predict peptide sequences with a tendency to form β-rich amyloid assemblies. TANGO is an algorithm that predicts aggregation nucleating regions in unfolded polypeptide chains56. It works on the assumption that the aggregating regions are buried in the hydrophobic core of the natively folded protein. ZIPPER is an algorithm that predicts hexapeptides within larger polypeptide sequences that have a strong energetic drive to form the two complementary β-sheets (known as a steric zipper) that give rise to the spine of an amyloid fibril57. Both methods are physically motivated but rely on statistically determined potentials.
Self-assembly of peptides
NH2-ILLIIM-CO2H and Ac-RNYIAQVD-NH2 (>95% pure) were purchased from GL Biochem Ltd (Shanghai, China). Ideally, it would have been preferred to have both peptides capped (N-terminus: Acetyl and C-terminus: Amide), as they would better represent small fragments of a larger peptide sequence. Due to the fact ILLIIM contains no charged sidechains, synthesising capped sequences to high purity would have been very challenging, therefore only the RNYIAQVD sequence remained capped and the ILLIIM sequence had regular carboxyl and amino termini. To ensure that all peptide seeds were fully dissolved before self-assembly was initiated the peptides were solubilised in warmed PBS (90 °C) at either 1 or 5 mg mL−1 The warmed peptide solutions were vortexed vigorously and held at 90 °C for 3 h to ensure maximum dissolution. After the second round of vortexing, the peptide suspensions were cooled slowly. This protocol has been previously used to maximise a homogenous starting population of monomeric peptide32. Alternatively, self-assembly was carried out at a constant temperature of 37 °C without pre-solubilising the peptides in hot PBS.
Atomic force microscopy (AFM)
AFM imaging was performed on a Bruker Multimode 8 AFM and a Nanoscope V controller. Tapping mode imaging was used throughout, with antimony (n)-doped silicon cantilevers having approximate resonant frequencies of 525 or 150 kHz and spring constants of either 200 or 5 Nm−1 (RTESPA-525, Bruker or RTESPA-150, Bruker). No significant differences were observed between cantilevers. 50 µL aliquots of the peptide (either at 1 or 5 mg mL−1) were drop cast onto freshly cleaved muscovite mica disks (10 mm diameters) and incubated for 20 min before gently rinsing in MQ water and drying under a nitrogen stream. All images were flattened using the first order flattening algorithm in the nanoscope analysis software and no other image processing occurred. Statistical analysis of the AFM images was performed using the open-source software FiberApp33 from datasets of no less than 900 fibres.
Transmission electron microscopy (TEM)
Copper TEM grids with a formvar-carbon support film (GSCU300CC-50, ProSciTech, Qld, Australia) were glow discharged for 60 s in an Emitech k950x with k350 attachment. Then, 5 µL drops of sample suspension were pipetted onto each grid, allowed to adsorb for at least 30 s and blotted with filter paper. Two drops of 2% uranyl acetate were used to negatively stain the particles with excess negative stain removed by blotting with filter paper after 10 s each. Grids were then allowed to dry before imaging. Grids were imaged using a Joel JEM-2100 (JEOL (Australasia) Pty Ltd) transmission electron microscope equipped with a Gatan Orius SC 200 CCD camera (Scitek Australia).
Small- and wide-angle X-ray scattering (SAXS/WAXS)
SAXS/WAXS experiments were performed at room temperature on the SAXS/WAXS beamline at the Australian Synchrotron. Peptide assemblies in PBS prepared at both 1 and 5 mg mL−1 were loaded into a 96-well plate held on a robotically controlled x–y stage and transferred to the beamline via a quartz capillary connected to a syringe pump. Data from the 5 mg mL−1 assemblies were discarded due to sedimentation of the assemblies preventing reliable sample transfer into the capillaries. The experiments used a beam wavelength of λ = 1.03320 Å−1 (12.0 keV) with dimensions of 300 µm × 200 µm and a typical flux of 1.2 × 1013 photons per second. 2D diffraction images were collected on a Pilatus 1M detector. SAXS experiments were performed at q ranges between 0.002 and 0.25 Å−1 and WAXS experiments were performed at a q range between 0.1 and 2 Å−1. These overlapping spectra provide a total q range of 0.002–2.2 Å−1. Spectra were recorded under flow (0.15 mL min−1) to prevent X-ray damage from the beam. Multiples of approximately 15 spectra were recorded for each time point (exposure time = 1 s) and averaged spectra are shown after background subtraction against PBS in the same capillary.
Circular dichroism spectroscopy
CD spectroscopy was performed using an AVIV 410-SF CD spectrometer. Spectra were collected between 190 and 260 nm in PBS using 1 mm quartz cuvettes with a step size of 0.5 nm and 2 s averaging time. Data were analysed using the BeStSel (Beta Structure Selection) method of secondary structure determination35.
Atomistic models were constructed using the Nucleic Acid Builder58. Simulations were run in explicit water (TIP3P59) using the ff15ipq forcefield60 and the pmemd time integrator61. In order to hold the unit cell geometry to values consistent with the observed scattering, the alpha carbon of the central residue of each chain was subjected to a restraining force with spring constant 2 kcal mol−1 Å−2. Periodic boundaries were applied to the system such that it formed a truncated octahedron, which was relaxed during equilibration to a volume of 10,310 nm3, giving a density of 0.973 reference with 323,535 water molecules. The system state after 10 ns of equilibration was stripped of water molecules more than 10 Å from any non-hydrogen solute atom, and passed to CRYSOL3 for calculation of orientationally averaged scattering profile given the example state (including the ordered waters from the explicit solvent shell, and also including an approximate treatment of ordered water beyond this shell)42,62.
Thioflavin T amyloid kinetic assays
Peptide assemblies were made up to concentrations of 1 or 5 mg mL−1 suspensions containing 25 µM ThT in PBS. The first fluorescence measurement (t = 0) was recorded immediately after sample preparation. All the samples were then stored at room temperature and fluorescence intensity was recorded at different time points. Measurements were performed in triplicate using a ClarioStar fluorimeter equipped with a 96-well plate reader (excitation wavelength: 440 nm, emission wavelength: 482 nm).
Cell line and cultures
Human-derived neuroblastoma cells (SH-SY5Y, ATCC Product Number: CRL-2266) were cultured in DMEM-F12 (Invitrogen) medium supplemented with 10% (v/v) foetal calf serum (FCS), 100 UmL−1 penicillin and 100 µgmL−1 streptomycin (Invitrogen, Carlsbad, CA). Cells were cultured at 37 °C in a humidified atmosphere containing 5% CO2.
Immunofluorescent and thioflavin T microscopy
ThT staining was performed by incubating the amyloid assemblies with a 25 µM solution of ThT in PBS for 15 min, in an 8-well Labtek II chamber (Nunc). For the antibody stain the same assemblies were incubated in a 1:200 dilution of the A11 polyclonal antibody raised in rabbit (Invitrogen, REF: AHB0052, LOT:VF299837) in 2% BSA in PBS for 1 h. Following this the wells were carefully washed in PBS and a solution of the 2° antibody (Goat-Anti Rabbit IgG-Alexa Fluor 647, Product A21244, Lot #: 1871168, Molecular Probes) at 1:1000 dilution in PBS was incubated with the peptides for 1 h. Finally the assemblies were once again washed in PBS before being imaged via laser scanning confocal microscopy using a FV3000 microscope (Olympus) and 60× objective lens (1.35 NA Oil Plan Apochromat) using the following settings: ThT channel λex = 450 nm, λem = 490 nm, Alexa Fluora 647 channel λex = 650 nm, λem = 665 nm. The same imaging settings were used for all samples and the negative controls (peptide assemblies + 2° antibody only) were used to determine the level of background fluorescence. For positive controls, amyloid assemblies of phenylalanine were used under concentrations known to readily form oligomeric amyloid assemblies38, which were shown here to bind strongly to the A11 antibody (Supplementary Fig. 9e, f).
Cell viability assay
Cells were seeded into 96-well plates at 1 × 105 cells per mL and incubated for 24 h to ensure good attachment to the surface. A stock solution of peptide assemblies (10 mg mL−1) was serially diluted into DMEM-F12 media 2.5–0.02 mg mL−1 or 3.3–0.027 mM for ILLIIM and 2.45–0.02 mM for RNYIAQVD) and seeded onto the SH-SY5Y cells and incubated for 48 h, cell viability was determined using 3-(4,5-dimethylthiazol-2-yl)2,5-diphenyltetrazolium bromide (MTT) (Sigma-Aldrich) as described previously63. Equivalent MTT assays were performed on cells cultured in the same ratios of PBS to media, but in the absence of peptide, assemblies to confirm that the culture conditions were non-toxic (Supplementary Fig. 10). Absorbance readings of untreated control wells in 100% cell culture media were designated as 100% cell viability. Statistical analysis was performed by one-way ANOVA tests with Tukey comparison in the software GraphPad (Prism) ***p < 0.001. Flow cytometry assays to determine cell viability were performed in a similar manner to the MTT assays. Briefly, to determine the effect of the peptides on cellular viability SH-SY5Y cells were cultured in the presence of the peptides for 48 h, harvested and stained with the apoptosis stain Annexin V for 10 min on ice (Cat No. 550474, BD Biosciences, 5 µL in 100 µL of 2% FCS in PBS). Samples were diluted with 300 µL of 2% FCS in PBS and stained with the viability dye 7-AAD (559925 BD Biosciences, 5 µL per sample) and analysed using flow cytometry (FACS Aria III; BD Biosciences). Cell counts were performed manually using a hemocytometer, with tryphan blue to differentiate non-viable cells.
All code used in this study is either free (NAB 1.3, pymol 2, TANGO 2.2 and ZIPPER) or commercially available (pmemd 19, CRYSOL3 3.0.3).
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N.P.R. would like to acknowledge The La Trobe Institute of Molecular Sciences (LIMS) for the receipt of a Nicholas Hoogenraad fellowship, and the CASS foundation for partially funding this work through a philanthropic grant (#10053, ‘Determining the role of protein aggregation in COVID-19’). N.P.R. would also like to acknowledge that Fig. 6 was created using Biorender.com. The authors thank Dr Susi Seibt for assistance on the SAXS/WAXS beamline at the Australian Synchrotron. This research was undertaken, in part, on the SAXS/WAXS beamline at the Australian Synchrotron, part of ANSTO. Molecular dynamics calculations made use of the HPC service of the University of Luxembourg64. The project was part-funded by grant C20/MS/14588607 of the Fonds Nationale de la Recherche, Luxembourg.
Authors: University of Otago July 12, 2022: Science Daily
Summary: Researchers have uncovered how post-viral fatigue syndromes, including Long COVID, become life-changing diseases and why patients suffer frequent relapses.
Arising commonly from a viral infection, Myalgic Encephalomyelitis/Chronic Fatigue Syndrome (ME/CFS), is known to cause brain-centred symptoms of neuroinflammation, loss of homeostasis, brain fog, lack of refreshing sleep, and poor response to even small stresses.
Long-COVID has similar effects on people and is believed to also be caused by neuroinflammation.
Lead author Emeritus Professor Warren Tate, of the University of Otago’s Department of Biochemistry, says how these debilitating brain effects develop is poorly understood.
In a study published in Frontiers in Neurology, he and colleagues from Otago, Victoria University of Wellington and University of Technology Sydney, developed a unifying model to explain how the brain-centred symptoms of these diseases are sustained through a brain-body connection.
They propose that, following an initial viral infection or stressor event, the subsequent systemic pathology moves to the brain vianeurovascular pathways or through a dysfunctional blood-brain barrier. This results in chronic neuroinflammation, leading to a sustained illness with chronic relapse recovery cycles.
The model proposes healing does not occur because a signal continuously cycles from the brain to the body, causing the patient to relapse.
The creation of this model is not only important for the “huge research effort ahead,” but also to provide recognition for ME/CFS and Long COVID sufferers.
“These diseases are very closely related, and it is clear the biological basis of Long COVID is unequivocally connected to the original COVID infection — so there should no longer be any debate and doubt about the fact that post viral fatigue syndromes like ME/CFS are biologically based and involve much disturbed physiology,” Emeritus Professor Tate says.
This work will enable best evidence-based knowledge of these illnesses, and best management practices, to be developed for medical professionals.
“Patients need appropriate affirmation of their biological-based illness and help to mitigate the distressing symptoms of these very difficult life-changing syndromes which are difficult for the patients to manage by themselves.
“This work highlighted that there is a susceptible subset of people who develop such syndromes when exposed to a severe stress, like infection with COVID-19, or the glandular fever virus Epstein Barr, or in some people with vaccination that is interpreted as a severe stress.
“What should be a transient inflammatory/immune response in the body to clear the infection, develop immunity and manage the physiological stress, becomes chronic, and so the disease persists.”
Warren Tate, Max Walker, Eiren Sweetman, Amber Helliwell, Katie Peppercorn, Christina Edgar, Anna Blair, Aniruddha Chatterjee. Molecular Mechanisms of Neuroinflammation in ME/CFS and Long COVID to Sustain Disease and Promote Relapses. Frontiers in Neurology, 2022; 13 DOI: 10.3389/fneur.2022.877772
The COVID-19 pandemic has been unpredictable as more is learned about the varied side effects of the virus. A typical respiratory infection, such as the flu, usually has a specific set of symptoms and potential complications. With COVID-19, the long-term effects range from neurological complications to loss of taste and smell, trouble focusing (“brain fog”), and chronic fatigue. Another surprising finding from several studies is the heightened risk of stroke and heart attack—and not just for older adults. People under the age of 50 appear to be at much higher risk of these complications too.
One study published in JAMA in April 2021 found that the risk of stroke was more than twice as high for COVID-19 patients when compared to people of the same age, sex, and ethnicity in the general population—82.6 cases per 100,000 people compared to 38.2 cases for those without a COVID-19 diagnosis.
In another Swedish study published in the August 14, 2021 issue of The Lancet, researchers found that within a week of a COVID-19 diagnosis, a person’s risk of heart attack was three to eight times higher than normal, and their risk of stroke was three to six times higher. The study revealed these risks remained high for at least a month. The average age of people in the study was only 48 years. The data from those diagnosed was compared with 348,000 Swedish people in a similar age range who did not have the virus.
“We are definitely seeing a huge increase in younger stroke survivors who are post-COVID diagnosis,” Kinzinger says. “We know that vascular complications go along with COVID infections, which can lead to strokes and other cardiovascular issues.”
A group of researchers headed by Mark Ellul, PhD, NIHR Clinical Lecturer in Neurology at the Institute of Infection, Veterinary and Ecological Sciences from the University of Liverpool, first observed this in September 2020. They found that the number of patients admitted to the hospital with a large vessel stroke who also had a COVID-19 diagnosis was seven times higher than normal.
Similar findings have also come out of other countries, where the median age for patients who needed thrombectomy surgery to remove a blood clot was down across the board. In one New York Medical Center, the average age of patients with confirmed stroke and COVID-19 diagnosis was 63 years. The average age of stroke patients who tested negative for COVID was much higher (70 years), even when they controlled for age, sex, and other risk factors.
Researchers are still studying the cause of the increased risk. But doctors know that COVID-19 causes an inflammatory response that thickens a person’s blood. Thicker blood is more likely to clot, and clots can lead to stroke. Many of the young people who suffer a stroke after a COVID-19 diagnosis have few (and sometimes no) risk factors normally associated with stroke.
Sometimes these strokes don’t occur for several weeks after a COVID-19 diagnosis, and it’s impossible to predict who might be at risk. For patients recovering from COVID-19 and a stroke, there is the added challenge of an impaired cardio-respiratory system. “Not only are we dealing with strength, motor, and balance deficits that go along with stroke, we also have to work around respiratory issues, tracheostomies, and other complications,” Kinzinger says. Stroke recovery is physically and mentally challenging anyway, and these complications can increase recovery time.
“When someone has a stroke and they are under 50, their whole life is uprooted,” he says. “A lot of people have younger kids or spouses, they may have a career or they’re going to school, so it’s just such a different phase of life than someone who is older.”
Authors: Michael Merschel, American Heart Association News
Two years into the pandemic, researchers have learned a lot about how COVID-19 affects people with heart disease and stroke survivors. But like the coronavirus itself, what everyone needs to know keeps evolving.
“You can’t assume that what was true three months ago is true now,” said Dr. James de Lemos, a cardiologist at UT Southwestern Medical Center in Dallas. Thanks to the omicron variant, “it’s a fundamentally different pandemic than it was at Thanksgiving.”
Early data suggests omicron causes less severe illness but spreads more easily than its predecessors. So heart and stroke patients need to protect themselves, starting with understanding that COVID-19 still is a threat to their health.
“Early on, we recognized that the risk was higher for those with pre-existing cardiovascular disease,” said Dr. Biykem Bozkurt, a cardiologist at Baylor College of Medicine in Houston. According to the Centers for Disease Control and Prevention, people with conditions such as heart failure, coronary artery disease and possibly high blood pressure may be more likely to get severely ill from COVID-19. So can people who have diabetes, are overweight or are recovering from a stroke.
SARS-CoV-2, the virus that causes COVID-19, also has been linked to increased risk of several cardiovascular conditions. According to a September 2021 report from the CDC, people with COVID-19 are nearly 16 times more likely to have heart inflammation, or myocarditis, than uninfected people. The report found about 150 cases per 100,000 people with COVID-19 versus about nine cases per 100,000 people without the virus.
In addition, an August 2021 study in the New England Journal of Medicine showed people with the coronavirus may have a significantly higher, albeit rare, risk of intracranial hemorrhage, or brain bleeding; heart attack; and having an arrhythmia, or abnormal heartbeat.
Researchers don’t have full data on omicron’s effects yet, Bozkurt said, but it’s still affecting people who are vulnerable. “And that’s why the hospitals right now are full.”
The risks of any one person having a severe problem from the new variant are relatively small, de Lemos said. “But the flipside is, given how many people are getting infected right now, the cumulative number of people with COVID-19 complications is still very large.”
De Lemos, who helped create the American Heart Association’s COVID-19 Cardiovascular Disease Registry, said omicron “is obviously wildly more infectious and able to evade the vaccine to some extent, although it does appear that the vaccine seems to prevent severe infections and hospitalizations.”
And overall, “we don’t know a ton about specifically why certain patients with heart disease do less well,” he said, although understanding has evolved over time.
In the beginning, de Lemos said, doctors feared the virus directly infected the heart muscle. “That doesn’t really appear to be the case,” he said.
Instead, it appears that in severe cases, the virus is inflaming the lining of blood vessels of the heart and increasing the likelihood of clotting in the smallest vessels, he said.
COVID-19 also can overwhelm the heart by making it work harder to pump oxygenated blood through the body as the lungs are overwhelmed.
But as they’ve learned more about the coronavirus, doctors have gotten better at fighting it. For example, de Lemos said, they now work proactively to treat blood-clotting disorders in hospitalized patients. And although researchers are working to understand lingering effects known as “long COVID,” it appears long-term implications for the heart look favorable.
“The vast majority of people who have mild COVID infections really appear to have nothing to worry about with their hearts,” he said. “That’s good news, I think, and doesn’t get emphasized enough.”
People with existing heart conditions or a history of stroke still need to protect themselves, and have many ways of doing so.
“Number one: Get vaccinated,” said Bozkurt, who has studied COVID-19 vaccine side effects. “And please, do get a booster.” Reports of rare cases of vaccine-related myocarditis, particularly in younger males, should not dissuade anybody with an existing condition. Most people with pre-existing cardiovascular disease are not young adult males, she noted. And regardless of age, the benefits from vaccines outweigh the risks.
Given how the vaccines don’t seem to be as protective against the spread of omicron, de Lemos said if you’re a heart disease or stroke patient, hunker down for the next several weeks until this wave passes, “and then you’ll be able to re-emerge.”
Patients should avoid indoor crowds, he said, and use a KN95 mask or, when possible, an N95 mask instead of cloth masks when being in a crowd is necessary.
Bozkurt said heart and stroke patients should keep in contact with their health care team and continue taking medications as prescribed. Anybody with symptoms that could be heart-related should seek care immediately. “Do not delay,” she said.
Both doctors said it was important to get information from reliable sources. Some false remedies promoted on social media can actually damage the heart, Bozkurt said.
De Lemos acknowledged that even from reliable sources, advice can shift. “I would say that the information is written in pencil, not in pen, because things are changing so fast.” It can be frustrating for him, even as a scientist, when experts disagree or alter their recommendations, but “that’s the way science goes.”
And even as COVID-19 “remains a bizarrely arbitrary virus in terms of who gets sick and who doesn’t,” he’s optimistic.
“Think about all the progress we’ve made in a year or two, and the remarkable effect of the vaccines, the fact that we have drugs” that should help keep people out of hospitals. Heart and stroke patients need to be extra careful right now, but “as frustrating as it is, we will not be in this situation forever. We really won’t.”
Editor’s note: Because of the rapidly evolving events surrounding the coronavirus, the facts and advice presented in this story may have changed since publication. Visit Heart.org for the latest coverage, and check with the Centers for Disease Control and Prevention and local health officials for the most recent guidance.
If you have questions or comments about this story, please email firstname.lastname@example.org.
Findings could give insight into long-term neurological symptoms of COVID-19
Date:July 5, 2022Source:NIH/National Institute of Neurological Disorders and Stroke
Summary:A new study describes the immune response triggered by COVID-19 infection that damages the brain’s blood vessels and may lead to short- and long-term neurological symptoms. The study examined brain changes in nine people who died suddenly after contracting the virus.
A study from the National Institutes of Health describes the immune response triggered by COVID-19 infection that damages the brain’s blood vessels and may lead to short- and long-term neurological symptoms. In a study published in Brain, researchers from the National Institute of Neurological Disorders and Stroke (NINDS) examined brain changes in nine people who died suddenly after contracting the virus.
The scientists found evidence that antibodies — proteins produced by the immune system in response to viruses and other invaders — are involved in an attack on the cells lining the brain’s blood vessels, leading to inflammation and damage. Consistent with an earlier study from the group, SARS-CoV-2 was not detected in the patients’ brains, suggesting the virus was not infecting the brain directly.
Understanding how SARS-CoV-2 can trigger brain damage may help inform development of therapies for COVID-19 patients who have lingering neurological symptoms.
“Patients often develop neurological complications with COVID-19, but the underlying pathophysiological process is not well understood,” said Avindra Nath, M.D., clinical director at NINDS and the senior author of the study. “We had previously shown blood vessel damage and inflammation in patients’ brains at autopsy, but we didn’t understand the cause of the damage. I think in this paper we’ve gained important insight into the cascade of events.”
Dr. Nath and his team found that antibodies produced in response to COVID-19 may mistakenly target cells crucial to the blood-brain barrier. Tightly packed endothelial cells help form the blood-brain barrier, which keeps harmful substances from reaching the brain while allowing necessary substances to pass through. Damage to endothelial cells in blood vessels in the brain can lead to leakage of proteins from the blood. This causes bleeds and clots in some COVID-19 patients and can increase the risk of stroke.
For the first time, researchers observed deposits of immune complexes — molecules formed when antibodies bind antigens (foreign substances) — on the surface of endothelial cells in the brains of COVID-19 patients. Such immune complexes can damage tissue by triggering inflammation.
The study builds on their previous research, which found evidence of brain damage caused by thinning and leaky blood vessels. They suspected that the damage may have been due to the body’s natural inflammatory response to the virus.
To further explore this immune response, Dr. Nath and his team examined brain tissue from a subset of patients in the previous study. The nine individuals, age 24 to 73, were chosen because they showed signs of blood vessel damage in the brain based on structural brain scans. The samples were compared to those from 10 controls. The team looked at neuroinflammation and immune responses using immunohistochemistry, a technique that uses antibodies to identify specific marker proteins in the tissues.
As in their earlier study, researchers found signs of leaky blood vessels, based on the presence of blood proteins that normally do not cross the blood brain barrier. This suggests that the tight junctions between the endothelial cells in the blood brain barrier are damaged.
Dr. Nath and his colleagues found evidence that damage to endothelial cells was likely due to an immune response — discovering deposits of immune complexes on the surface of the cells.
These observations suggest an antibody-mediated attack that activates endothelial cells. When endothelial cells are activated, they express proteins called adhesion molecules that cause platelets to stick together. High levels of adhesion molecules were found in endothelial cells in the samples of brain tissue.
“Activation of the endothelial cells brings platelets that stick to the blood vessel walls, causing clots to form and leakage to occur. At the same time the tight junctions between the endothelial cells get disrupted causing them to leak,” Dr. Nath explained. “Once leakage occurs, immune cells such as macrophages may come to repair the damage, setting up inflammation. This, in turn, causes damage to neurons.”
Researchers found that in areas with damage to the endothelial cells, more than 300 genes showed decreased expression, while six genes were increased. These genes were associated with oxidative stress, DNA damage, and metabolic dysregulation. This may provide clues to the molecular basis of neurological symptoms related to COVID-19 and offer potential therapeutic targets.
Together, these findings give insight into the immune response damaging the brain after COVID-19 infection. But it remains unclear what antigen the immune response is targeting, as the virus itself was not detected in the brain. It is possible that antibodies against the SARS-CoV-2 spike protein could bind to the ACE2 receptor used by the virus to enter cells. More research is needed to explore this hypothesis.
The study may also have implications for understanding and treating long-term neurological symptoms after COVID-19, which include headache, fatigue, loss of taste and smell, sleep problems, and “brain fog.” Had the patients in the study survived, the researchers believe they would likely have developed Long COVID.
“It is quite possible that this same immune response persists in Long COVID patients resulting in neuronal injury,” said Dr. Nath. “There could be a small indolent immune response that is continuing, which means that immune-modulating therapies might help these patients. So these findings have very important therapeutic implications.”
The results suggest that treatments designed to prevent the development of the immune complexes observed in the study could be potential therapies for post-COVID neurological symptoms.
This study was supported by the NINDS Division of Intramural Research (NS003130) and K23NS109284, Roy J. Carver Foundation, and the Iowa Neuroscience Institute.
Myoung Hwa Lee, Daniel P Perl, Joseph Steiner, Nicholas Pasternack, Wenxue Li, Dragan Maric, Farinaz Safavi, Iren Horkayne-Szakaly, Robert Jones, Michelle N Stram, Joel T Moncur, Marco Hefti, Rebecca D Folkerth, Avindra Nath. Neurovascular injury with complement activation and inflammation in COVID-19. Brain, 2022; DOI: 10.1093/brain/awac151