Better ‘Mini Brains’ Could Help Scientists Identify Treatments for Zika-Related Brain Damage

UCLA researchers develop improved technique for creating brain tissue from stem cells

UCLA researchers have developed an improved technique for creating simplified human brain tissue from stem cells. Because these so-called “mini brain organoids” mimic human brains in how they grow and develop, they’re vital to studying complex neurological diseases.

In a study published in the journal Cell Reports, the researchers used the organoids to better understand how Zika infects and damages fetal brain tissue, which enabled them to identify drugs that could prevent the virus’s damaging effects.

The research, led by senior author Ben Novitch, could lead to new ways to study human neurological and neurodevelopmental disorders, such as epilepsy, autism and schizophrenia.

“Diseases that affect the brain and nervous system are among the most debilitating medical conditions,” said Novitch, UCLA’s Ethel Scheibel Professor of Neurobiology and a member of the Eli and Edythe Broad Center of Regenerative Medicine and Stem Cell Research at UCLA. “Mini brain organoids provide us with opportunities to examine features of the human brain that are not present in other models, and we anticipate that their similarity to the real human brain will enable us to test how various drugs impact abnormal or diseased brain tissue in far greater detail.”

For about five years, scientists have been using human pluripotent stem cells, which can create any cell type in the body, to develop mini brain organoids. But the organoids they produced have generally been difficult to use for research because they had highly variable structures and inconsistent cellular composition, and because they didn’t correctly mimic the layered structure of the brain and were too small — often no bigger than the head of a pin. They also didn’t survive very long in the laboratory and contained neural tissue that was difficult to classify in relation to real human brain tissue.

The organoids developed by Novitch’s group have a stratified structure that accurately mimics the human brain’s onion-like layers, they survive longer and have a larger and more uniform shape.

To create the brain organoids, Novitch and his team made several modifications to the methods that other scientists used previously: The UCLA investigators used a specific number of stem cells and specialized petri dishes with a modified chemical environment; previous methods used varying amounts of cells and a different type of dish. And they added a growth factor called LIF, which stimulated a cell-signaling pathway that is critical for human brain growth.

The researchers found critical similarities between the organoids they developed and real human brain tissue. Among them: The organoids’ anatomy closely resembled that of the human cortex, the region of the brain associated with thought, speech and decision making; and a diverse array of neural cell types commonly found in the cortex were all present in the organoids, and they exhibited electrical activities and network function, meaning they were capable of communicating with one another much like the neural networks in the human brain do.

The UCLA scientists also found that they could modify their methodology to make other parts of the brain including the basal ganglia, which are involved in the control of movement and are affected by neurodegenerative conditions such as Parkinson’s disease and Huntington’s disease.

“While our organoids are in no way close to being fully functional human brains, they mimic the human brain structure much more consistently than other models,” said Momoko Watanabe, a UCLA postdoctoral fellow and the study’s first author. “Other scientists can use our methods to improve brain research because the data will be more accurate and consistent from experiment to experiment and more comparable to the real human brain.”

When the team exposed the organoids to Zika, they discovered specifically how the virus destroys neural stem cells, the cells from which the brain grows during fetal development. Novitch’s team found that there are four specific molecules, called receptors, on the outer surface of neural stem cells; previous studies have indicated that the Zika virus could bind to these receptors and infect the cells. The researchers then mapped the changes that occur in the neural stem cells after Zika infection, presenting a clearer picture of how the virus infiltrates and harms fetal brain tissue.

Zika is associated with an unusually high incidence of fetal brain damage, so understanding how neural stem cells are affected by the virus could be an important new step toward a treatment.

The researchers tested several drugs on the Zika-infected organoids. They found three that are effective at blocking the virus’s entry into the brain tissue, including two that protected neural stem cells by preventing the interaction between the virus and entry receptors on the neural stem cells. In previous studies by Novitch and other UCLA colleagues, one of those drugs reduced brain damage in fetal mice infected with Zika.

“Many neurological diseases or conditions arise from defects in the way one neuron communicates with another or from the way an external factor, such as a virus, interacts with neural cells,” Novitch said. “If we can focus in at the level of cellular communication, we should be able to model those undesirable cellular interactions and counteract them with drugs or other therapies.”

The team plans to continue using its improved organoids to better understand human brain development and to learn more about autism spectrum disorders, epilepsy and other neurological conditions.

The experimental drugs used in the preclinical study have not been tested in humans or approved by the Food and Drug Administration for treating Zika in humans.

Gene Identified That May Provide Potential Therapy for Cerebral Cavernous Malformations

Researchers at University of California San Diego School of Medicine, with national collaborators, have identified a series of molecular clues to understanding the formation of cerebral cavernous malformations (CCMs). The study offers the first genome-wide analysis of the transcriptome of brain microvascular endothelial cells after KRIT1 inactivation. Findings were published September 28 in the Journal of Experimental Medicine.

“These mouse studies reveal a critical mechanism in the pathogenesis of cerebral cavernous malformations and point to the possibility of using angiogenesis inhibitors, such as TSP1 for potential therapy,” said Mark H. Ginsberg, MD, professor of medicine, UC San Diego School of Medicine.

CCMs are collections of enlarged and irregular blood vessels in the central nervous system (CNS), for which there is no drug therapy. The vessels are prone to leakage causing headaches, seizures, paralysis, hearing or vision loss, or bleeding in the brain. There are two forms of the condition: familial and sporadic, affecting 1 in 200 patients in the U.S. The current treatment for CCMs involves invasive surgery, however, surgery is not possible for all patients due to location of vascular lesions within the CNS.

The most common cause of familial cavernous malformations is mutations of KRIT1. The protein produced from this gene is found in the junctions connecting neighboring blood vessel cells. Loss of function mutations in KRIT1 result in weakened contacts between blood vessel cells and CNS vascular abnormalities as seen in CCMs.

“Inactivation of KRIT1 in endothelial cells causes a cascade of changes in the expression of genes that regulate cardiovascular development,” said Ginsberg. “What we learned is that reduced expression of a protein encoded by one of these genes, TSP1, contributes to the growth of CCMs. Loss of one or two copies of THBS1, the gene that encodes TSP1, makes a mouse model of the disease much worse. Conversely, administration of 3TSR, a fragment of TSP1, reduces lesions in this mouse model. This means that replacement of TSP1 by 3TSR or other angiogenesis inhibitors may be a preventative for CCMs or treatment of the disease.”

Neuroscientists Focus on Cell Mechanism That Promotes Chronic Pain

Researchers have discovered a new pain-signaling pathway in nerve cells that eventually could make a good target for new drugs to fight chronic pain.

The findings, published in the journal PLoS Biology by a UT Dallas neuroscientist and his colleagues, suggest that inhibiting a process called phosphorylation occurring outside of nerve cells might disrupt pain signals, and provide an alternative to opioid drugs for alleviating chronic pain.

Dr. Ted Price, the study’s co-author and associate professor of neuroscience in the School of Behavioral and Brain Sciences at UT Dallas, said the finding is significant.

“We found a key new signaling pathway that can be managed,” Price said. “Now we hope we can use the findings to discover a new drug.”

Phosphorylation is a biological process that occurs when a kinase — a type of enzyme — attaches a chemical called phosphate to a protein. This common process modifies proteins and their functions.

Phosphorylation was known to occur inside cells, but Dr. Matthew Dalva at Thomas Jefferson University, a co-author of the study, found that the process also occurs outside of cells, specifically nerve cells called neurons within the brain and spinal cord.

The new work shows that molecules called ephrins, which are bound in neuron membranes and include a portion that sticks out of the outer surface of the cell, are phosphorylated at those outward-facing sites. This extracellular phosphorylation event causes another type of protein, called NMDA receptors, to accumulate on the neuron’s surface.

Where those NMDA receptors gather is important: It’s at the synapse, the narrow space between two neurons where neurochemical signals are transmitted between cells.

NMDA receptors are critical to normal learning and memory development in the brain, but they also play a key role in controlling signals related to pathological pain. This type of pain is felt when there is no underlying cause, or when the pain continues long after the event.

“We know that the NMDA receptor plays a key role in neural plasticity for learning and memory. But it also plays a key role in learning or neural plasticity for pain,” Price said.

He said phosphorylating kinases appear to be the key factor for regulating the cell-signaling that occurs through NMDA receptors. Now that scientists understand that the phosphorylation process can occur both inside and outside the cell, they can research strategies to specifically block the process from happening outside the cell.

“We found this completely new type of signaling is what regulates the NMDA receptors. That was completely unexpected. The data are just extremely convincing,” Price said.

Price and his colleagues used isolated cells and animal models to specifically look at how this new signaling information applies to pain.

“We know that the extracellular phosphorylation event can drive pain, and we know if we reduce that process, it decreases pain,” he said. “Even after pain amplification has been established for a very, very long time, we can reverse extracellular phosphorylation very rapidly, and this reduces pain.”

Researchers were limited when they believed phosphorylation occurred only inside cells, Price said. Many drugs can’t move on in development because they are not able to cross cell membranes and, thus, are not able to get inside the cell. But he said discovering that extracellular phosphorylation also plays a key role in the processes associated with injury and pain makes it easier to develop drugs that can specifically target that process.

“If you don’t have to worry about all the kinases that are inside the cell, that limits the number of kinases that have to be targeted. Those extracellular kinases, which are a very distinct subset of that family of proteins, become a nice target to pursue,” he said.

The Price and Dalva labs are pursuing additional research grants to learn more about how this extracellular phosphorylation event works, identify the particular kinase involved and test it in pain models. With additional insight, they are hopeful that they can develop drugs capable of reversing chronic pain states in humans.

In addition to UT Dallas and Thomas Jefferson University scientists, researchers from the University of Arizona and New York University School of Medicine worked on the project.