These nerve cells are often generated far from where they will eventually reside and function in the new brain, a migration that, while much investigated in animal models using chemical or biological tracers, has never been studied directly in humans. Until now.

In a new paper, published online April 20, 2022 in Nature, scientists at University of California San Diego School of Medicine and Rady Children’s Institute of Genomic Medicine describe novel methods for inferring the movement of human brain cells during fetal development by studying healthy adult individuals who have recently passed away from natural causes.

“Every time a cell divides into two daughter cells, by chance, there arise one or more new mutations, which leave a trail of breadcrumbs that can be read out by modern DNA sequencers,” said senior author Joseph Gleeson, MD, Rady Professor of Neuroscience at UC San Diego School of Medicine and director of neuroscience research at the Rady Children’s Institute for Genomic Medicine.

“By developing methods to read these mutations across the brain, we are able to reveal key insights into how the human brain forms, in comparison with other species.”

Although there are 3 billion DNA bases — and more than 30 trillion cells in the human body — Gleeson and colleagues focused their efforts on just a few hundred DNA mutations that likely arose during the first few cell divisions after fertilization of the embryo or during early development of the brain. By tracking these mutations throughout the brain in deceased individuals, they were able to reconstruct development of the human brain for the first time.

To understand the type of cells displaying these breadcrumb mutations, they developed methods to isolate each of the major cell types in the brain. For instance, by profiling the mutations in excitatory neurons compared with inhibitory neurons, they confirmed the long-held suspicion that these two cell types are generated in different germinal zones of the brain, and then later mix together in the cerebral cortex, the outermost layer of the organ.

However, they also discovered that the mutations found in the left and right sides of the brain were different from one another, suggesting that — at least in humans — the two cerebral hemispheres separate during development much earlier than previously suspected.

The results have implications for certain human diseases, like intractable epilepsies, where patients show spontaneous convulsive seizures and require surgery to remove an epileptic brain focus, said Martin W. Breuss, PhD, former project scientist at UC San Diego and now an assistant professor at the University of Colorado School of Medicine.

Breuss is co-first author with Xiaoxu Yang, PhD, postdoctoral scholar and Johannes C. M. Schlachetzki, MD, project scientist, both at UC San Diego; and Danny Antaki, PhD, a former postdoctoral scholar at UC San Diego, now at Twist Biosciences.

“This study,” the authors said, “solves the mystery as to why these foci are almost always restricted to one hemisphere of the brain. Applying these results to other neurological conditions could help scientists understand more mysteries of the brain.”

Co-authors include: Xin Xu, Changuk Chung, Guoliang Chai, Valentina Stanley, Qiong Song, Traci F. Newmeyer, An Nguyen, Beibei Cao, Jennifer McEvoy-Venneri and Brett R. Copeland, all at UC San Diego and Rady Children’s Institute for Genomic Medicine; Addison J. Lana, Sydney O’Brien, Marten A. Hoeksema, Alexi Nott, Martina P. Pasilla, Scott T. Barton, and Christopher K. Glass, all at UC San Diego; Shareef Nahas, Lucitia Van Der Kraan and Yan Ding, Rady Children’s Institute for Genomic Medicine and the NIMH Brain Somatic Mosaicism Network.

Funding for this research came, in part, from the Howard Hughes Medical Institute, the National Institute of Mental Health (grants MH108898, RO1 MH124890, R21 AG070462), the National Institute on Aging (grants RF1 AGO6106-02, R01 AGO56511-02, R01 NS096170-04) and the UC San Diego IGM Genomics Center (S10 OD026929).

Video: https://youtu.be/WfrvSYi9M6A

The results, published April 20 in Nature, may aid the development of a new generation of more effective neurological and psychiatric therapies with fewer side effects.

The new research takes a close look at glutamate, the most prevalent neurotransmitter in the brain. Glutamate binds to receptors on brain cells, which opens a channel into the cell, allowing ions to pass through to propagate an electrical signal.

“The way the brain works is through communication between neurons, and these are the main receptors which allow this communication,” says Alexander Sobolevsky, PhD, associate professor of biochemistry and molecular biophysics at Columbia and senior author on the paper.

Each receptor can bind up to four molecules of glutamate and produce four different levels of conductivity. Previous studies had linked binding to conductivity in a simple stepwise fashion, in which binding each additional glutamate molecule increased the conductivity another step.

While that explanation made sense, nobody had looked closely enough to confirm it. In the new work, the investigators combined a technique called cryo-electron microscopy with sophisticated data analysis to reveal the first detailed pictures of glutamate binding to its receptors.

“We actually carried out experiments in the conditions where we see all these intermediates, one glutamate and then two glutamates, three glutamates, and then it binds all four,” says Sobolevsky.

These images reveal that glutamate binds to the subunits of its receptor only in specific patterns. That overturns the prevailing view that each subunit binds glutamate independently and points toward new levels of complexity in neuronal signaling and drug responses.

Instead of straightforward stepwise transitions, Sobolevsky and his colleagues found that a glutamate molecule must bind to one of two specific receptor subunits before any glutamates can bind to the other two subunits. In addition, the conductivity levels of the receptor didn’t correlate directly to the number of glutamates bound to it; a receptor could have two or more glutamates attached but still only reach the first level of conductivity.

The results open an entirely new line of investigation, and the team is now probing how different accessory molecules on neurons affect the interaction. Learning more about the glutamate receptors’ specific activation states may aid the development of better drugs for conditions that involve glutamate receptors, such as depression, dementia, Parkinson’s disease, epilepsy, and stroke.

Video: https://youtu.be/IeQZTFMz5ek

The study was supported by the National Institutes of Health (R01 CA206573, R01 NS083660, R01 NS107253, R01 AR078814, R01 GM128195, and R01 AG065594) and the National Science Foundation (1818086, 1818213, and 1563291).

The study, published in npj Digital Medicine, analyzed sensor data on sleep, activity and heart rate from over 5,600 individuals. Among the findings, the team showed that the average resting heart rate of participants significantly increased the day following vaccination. The effect appeared to be stronger after the second dose of the Moderna vaccine, compared to the Pfizer-BioNTech vaccine, and more pronounced in younger individuals.

According to lead author Giorgio Quer, PhD, director of Artificial Intelligence at the Scripps Research Translational Institute, this study is a first step toward quantifying the physiological response to vaccination in individuals using commercial sensors.

“Investigating the physiological signals in the period around vaccination can help us better understand the variability of vaccine response between people, as well as the changes from an individual norm due to vaccination,” Quer says. “As these individual changes are due to a person’s initial immune response to the vaccine, they can potentially help guide future vaccine development to optimize their efficacy and safety, and allow for more precise, individualized vaccine regimens.”

The researchers drew their data from a larger project, called Digital Engagement and Tracking for Early Control and Treatment (DETECT) launched in March 2020, in response to the emergence and rapid spread of SARS-CoV-2, the virus that causes COVID-19. DETECT is a mobile-app research platform that allows participants to share physiological and behavioral data gathered through a fitness band or smartwatch, as well as manually entered symptoms, test results and vaccination status.

To determine whether consumer wearables could unearth digital biomarkers of vaccine-induced immune response, the scientists analyzed DETECT sensor data from two weeks before and after each vaccination dose. They compared post-vaccination changes to the participants’ resting heart rate, sleep and activity levels, to their baselines.

The analysis showed that the average resting heart rate of study participants increased significantly the day following vaccination, peaking two days post-vaccination, and returning to normal four days after the first dose and six days after the second. In addition to increases in resting heart rate being higher after the second dose of the Moderna versus the Pfizer-BioNTech vaccine, the study suggests that prior COVID-19 infection was linked with a significantly higher resting heart rate increase after the first vaccine dose relative to those without prior infection. This increase is consistent with an expected greater immune response for these individuals.

Findings also show that women experienced greater changes than men in resting heart rate in the five days following vaccination after the first dose, and that individuals under the age of 40 had higher changes in resting heart rate than older individuals, but only after the second dose.

Activity and sleep patterns appeared to be minimally affected by the first dose, but a significant decrease in activity and an increase in sleep relative to baseline were observed immediately after the second vaccine dose.

“While the link between physiological response and immune response still requires further investigation, digital tracking could provide a novel way to identify individuals who may not be responding optimally to the vaccine,” says Steven Steinhubl, MD, associate professor at Scripps Research and senior author of the study.

“DETECT really shows the power of wearable sensor data in furthering our understanding of multiple facets of COVID-19,” Quer says. “We hope to continue to expand this research by growing our study cohort further, which will allow us to delve into important questions regarding breakthrough infections and other areas that are currently poorly understood.”

The npj Digital Medicine article, “Inter-individual variation in objective measure of reactogenicity following COVID-19 vaccination via smartwatches and fitness bands” is authored by Giorgio Quer, Matteo Gadaleta, Jennifer Radin, Kristian Andersen, Katie-Baca-Motes, Edward Ramos, Eric Topol and Steven Steinhubl.

Funding for the research was provided by the National Center for Advancing Translational Sciences at the National Institutes of Health [UL1TR002550] and the National Institute of Allergy and Infectious Diseases [U19AI135995].

About DETECT

Since its launch, the DETECT (Digital Engagement and Tracking for Early Control and Treatment) study has enrolled over 40,000 participants nationwide. The data that has been shared has enabled scientists to gain valuable insights into various aspects of COVID-19 infection. Already, DETECT has led to several peer-reviewed publications that: show how wearable devices are capable of detecting COVID-19 infection; give valuable insights into long COVID; demonstrate how machine learning techniques can predict COVID-19 infection; and more. The team aims to develop tools to complement traditional public health interventions and bolster the ability to prevent and contain future pandemics. The DETECT study is ongoing. Visit detect.scripps.edu to learn more.

Cryptosporodium is a leading cause of diarrheal disease in young children around the world. The intestinal parasite contributes to childhood mortality and causes malnutrition and stunting. How a parasite like this one reproduces and completes its life cycle has significant impact on child health.

“It’s the product of parasite sex, that is infectious agent here, a spore, that is transmitted by contaminated water,” Striepen says. “So, if you break its ability to have sex, you would break the cycle of transmission and infection.”

In a new paper in PLOS Biology, Striepen and colleagues in his lab tread new ground in understanding how Cryptosporidium reproduces inside a host. Using an advanced imaging technique allowed the scientists to observe the entire lifecycle in the laboratory. They found the parasite completes three cycles of asexual replication and then directly switches to male and female sexual forms. Their observations refute an intermediate stage that was introduced in the 1970s and align well with the original description of physician and parasitologist Edward Tyzzer who discovered this pathogen more than a century ago.

“What we have shown contradicts what you see in most textbooks today, including the description at the Centers for Disease Control and Prevention website,” Striepen says. “It’s really a super simple lifecycle that is completed in a single host in three days and only has three characters: asexual cells, male cells, and female cells.”

Other parasites, such as the malaria parasite Plasmodium, a “cousin” of Cryptosporidium, have more complicated and lengthy paths to follow an overall similar life cycle. While Crypto completes its lifecycle in one host, most malaria parasites move between two: a mosquito, where the parasite’s sexual reproduction occurs, and a human, where its asexual replication occurs.

“Cryptosporidium is a great model to study parasite development; you can see analogous steps to what happens with the malarial parasite, but it’s much simpler because it all happens over only three days in one host, and we can observe it in simple cell cultures,” Striepen says.

In earlier work on Cryptosporidium, Striepen and colleagues had found that sexual reproduction appeared necessary for the parasite to move from one host to infect another but also to sustain itself in a host during chronic infection. Blocking developmental progression and parasite sex thus presents itself as a strategy to cure or prevent infection.

Cryptosporidium is a minuscule single-cell parasite that invades and reproduces within the cells of the intestine of its hosts. To get a closer look at what was happening, the researchers developed a live-cell microscopic imaging technique to track the progression of the parasite through multiple days in cell cultures. Using genetic engineering they added a fluorescent label to the nucleus of each parasite, allowing them to observe the replication of the parasite in real time and to distinguish its different lifecycle stages.

What they saw was the parasites “count to three,” says Striepen. Rather than responding to environmental cues, the parasites followed a rigid built-in plan. After infecting a culture, the parasite underwent three cycles of asexual reproduction. Each cycle took about 12 hours, during which the parasite established a home within the host cell and replicated itself resulting in eight new infectious parasites. Those were then released to infect surrounding host cells.

After these three waves of amplification, their fate changes abruptly, and they turn into either male or female gametes, or sex cells, in a process that also took about 12 hours. Tracking individual parasites and their offspring the researchers found no evidence for a specialized intermediate form assumed by many textbooks, demonstrating direct development.

Interestingly, the parasite appeared pre-committed to their future fate and carried that plan from one host cell into the next in a way not yet understood.

The researchers were intrigued to see that male and females arise from the infectious forms released from the same asexual parasites. “One of the really interesting aspects of sexual identity here is that it is not inherited and hard-wired in the genome but much more fluid,” Striepen says. “There’s an asexual cell that divides itself into genetically identical clones, and then those clones somehow become male or female on the fly, resulting in dramatically different cell shape and behavior.”

Future research will focus on the molecular mechanism of commitment to understand how this life cycle is programmed into the parasite’s biology. Understanding the life cycle of Cryptosporidium is critical in thinking about how to create a vaccine or therapy for the disease, Striepen says.

“How cells make decisions and execute developmental plans is one of the most fundamental questions in biology. Cryptosporidium offers a tractable system to better understand this mechanism in parasites. Hopefully we can gain insights that contribute to the understanding of cryptosporidiosis and malaria and lead the way to new urgently needed interventions for these important diseases.”

Boris Striepen is the Mark Whittier and Lila Griswold Allam Professor of Microbiology and Immunology at the University of Pennsylvania School of Veterinary Medicine.

Striepen’s coauthors were lab members Elizabeth D. English, Amandine Guérin, and Jayesh Tandel.

The study was supported by grants from the National Institutes of Health to Striepen and a postdoctoral fellowship from the European Molecular Biology Organisation to Guérin.

But they don’t work in all patients. And now a new study in Nature Immunology led by researchers from Penn’s School of Veterinary Medicine suggests a possible reason why: Not only can these drugs encourage the activity of cancer killing T cells, but they can, in some cases, also activate a population of regulatory T cells that serve the opposing function — to rein in that attack.

In the study, immunologists led by Penn Vet professor Christopher Hunter and doctoral student Joseph Perry discovered that blocking the activity of the checkpoint protein PD-L1, which interacts with a T cell receptor PD-1, enhanced the activity of a subset of T cells known as effector regulatory T cells, or effector Tregs. This intervention unexpectedly reduced the ability of mice to control a parasite infection.

The findings reveal a complexity to how the body “regulates the regulators” of the immune system, says Hunter. “Once you have those Tregs to control your T cell response, you also need to control them,” he says. “It’s like with a car. You have the ignition, the accelerator, and you also need a brake. PD-1 is a brake not only on killer T cells but also on Tregs.”

T cells may be best known for their roles in fighting infections and killing cancer cells. But the immune system also has several mechanisms in place to counterbalance those responses to prevent out-of-control inflammation that could damage healthy tissue. Tregs are one aspect of this balancing act.

“You can think of Tregs as the health and safety inspectors of the immune system,” Hunter says. “They’re really important, but sometimes, when you need to mount an immune response against a pathogen, you need them to take a back seat. When an infection is present, we see Treg levels crash so that an effector response can emerge. But we never understood what causes that crash.”

The researchers began to understand more after exploring an unanticipated finding. They discovered that, when the checkpoint protein PD-L1 was blocked, mice infected with Toxoplasma gondii were less effective at fighting off the parasite than mice with an uninhibited PD-L1. “That was the opposite of what we expected,” Hunter says, as the dogma would have suggested that blocking this checkpoint inhibitor would allow for a better effector T cell response against infection.

Digging into the surprising result, Perry, Hunter, and colleagues realized that it aligned with what some cancer researchers had recently reported. In certain cancers, the other groups had found, blocking PD-L1 led to worse outcomes, seemingly because of an increase in a population of Tregs that restrained killing of cancer cells.

When the Penn-led team looked in the context of a T. gondii infection, they discovered the signaling molecule interferon gamma turned on PD-L1, which precipitated a rapid decline in Treg numbers. A PD-L1 inhibitor mitigated this effect and stopped the Treg crash. This treatment alleviated the harmful effects of inflammation on mice but also impaired the ability of T cells to fight infection. Similarly, Tregs altered to lack PD-1, the receptor which with PD-L1 interacts, also led to increases in Treg activity.

“It seems that the effector T cell versus Treg ratio is really important,” says Hunter.

As the researchers began to learn more about how Tregs were activated and operated during an infection, they were curious about whether this pathway worked when animals were in a normal, healthy state. Just as there are different types of “regular” T cells, including CD8 and CD4 T cells and many more subdivisions besides, the new work underscores that there are subpopulations of Tregs as well, which have different roles in the body. In healthy, uninfected animals, the team found differences between the proteins expressed by different Treg populations, including some that expressed PD-1, which the researchers termed “effector Tregs.”

“These results taught us that there’s a large population of activated PD-1-positive Treg cells present as a normal part of everyday life that help limit the immune system,” Perry says.

“We think these PD-1 high cells are the most active Tregs,” says Hunter. “It’s a complex landscape, and it’s possible that some checkpoint inhibitor treatments have been inadvertently targeting these Tregs and not others, leading to unexpected outcomes.”

In ongoing investigations, Hunter, Perry, and colleagues continue to examine this pathway, as well as those involved in other immune checkpoints. The findings could have implications not only in refining cancer checkpoint inhibitor therapies but also in conceiving new strategies for treating autoimmune disease. “In that case you would want to increase the number of Tregs,” Hunter says. “Maybe we could think about ways to augment that arm of the immune system to treat inflammatory diseases.”

Christopher A. Hunter is the Mindy Halikman Heyer Distinguished Professor of Pathobiology at the University of Pennsylvania School of Veterinary Medicine.

Hunter’s coauthors are Penn Vet’s Joseph A. Perry, Lindsey Shallberg, Joseph T. Clark, Jodie Gullicksrud, Jonathan H. DeLong, Bonnie B. Douglas, Zachary Lanzar, Keenan O’Dea, Christoph Konradt, Jeongho Park, Daniel Grubaugh, Arielle Glatman Zaretsky, Igor E. Brodsky, and David A. Christian; Penn’s Perelman School of Medicine’s Andrew P. Hart; Merck & Co.’s Renee de Waal Malefyt; and Harvard Medical School’s Juhi R. Kuchroo and Arlene H. Sharpe.

The study was supported by the National Institute of Allergy and Infectious Diseases (grants AI125563 and AI41158).