Better Models for Brain Disease NEWS FEATURE Traditional Animal Models Have Had Limited Success Mimicking Mental Illnesses

NEWS FEATURE

Better models for brain disease NEWS FEATURE Traditional animal models have had limited success mimicking mental illnesses. Emerging technologies offer the potential for a major model upgrade.

Helen H. Shen, Science Writer

Starting with just a tiny chunk of skin, neuroscientist Flora Vaccarino tries to unlock mysteries hidden inside the brains of people with autism. By introducing certain genes to the skin cells, the Yale University School of Medicine researcher reprograms them to an embryo-like state, turning them into induced pluripotent stem cells (iPSCs); and with two more months of nurturing and tinkering, Vaccarino can guide the cells to develop into small balls of neural tissue akin to miniature human brains. Less than two millimeters across, these “cerebral organoids” don’t look or work exactly like full brains. But they contain many of the same cell types and un- dergo some of the same key developmental pro- cesses as fetal brains. Also key, the cells perfectly match the genetic makeup of the adults and children with autism who donated the original skin samples, allowing Vaccarino’s team to track the very beginnings of their disorder. Human brain tissue usually can’t be collected and studied until after death, and by then, it can be too late to glean important insights. “You don’t get to see the same person’s cells progressing through a series of steps and time points, like we do with these organoids,” Vaccarino explains. “The organoids are a very powerful system. You can actually change things and see what the outcome is going to be.” Neurons such as these, derived from the induced pluripotent stem cells of a Parkinson’s Predicting outcomes and, crucially, developing disease patient, are on the forefront of efforts to improve models for brain disease. psychiatric drugs has proven exceedingly difficult in Image courtesy of Cedric Bardy and Fred H. Gage (The Salk Institute, La Jolla, CA). recent decades. Inadequate animal models have been a major stumbling block, researchers say. First de- is the first time that we’ve been able to begin digging veloped in 2013 (1), cerebral organoids grown from deep into the causes and neurobiology of these human iPSCs—affectionately called minibrains by ” some—are one of several emerging technologies that disorders. are finally allowing researchers to make more sophis- ticated models of neuropsychiatric disorders. Ad- The Mouse Problem — vances in genomics are also helping to shed new For decades, traditional animal models commonly, — light on mental illnesses by pointing researchers to genetically engineered mice have allowed scientists ’ new gene targets. These, in turn, can be combined to manipulate the brain s cells, genes, and molecules, with recent precision gene-editing techniques to revealing some basic but important insights. Modi- make animal models that many researchers hope will fied mice, for example, have helped reveal how mis- more faithfully reproduce aspects of human diseases, folded versions of the α-synuclein protein gunk up such as Alzheimer’s, autism, and schizophrenia. the Parkinson’s-diseased brain and possibly injure “It’s a very exciting time,” says Guoping Feng, a neurons. Mice with mutations linked to Alzheimer’s neuroscientist at the Massachusetts Institute of Technol- disease have helped scientists examine how mis- ogy (MIT) in Cambridge, Massachusetts. “Between the folded amyloid-β protein collects into sticky plaques technology development and the genetic findings, this in the brain.

www.pnas.org/cgi/doi/10.1073/pnas.1605358113 PNAS | May 17, 2016 | vol. 113 | no. 20 | 5461–5464 Downloaded by guest on September 25, 2021 electrical properties or abnormal numbers of cell-to- cell connections (synapses) are thought to be involved in several psychiatric disorders, and synapse formation and function appear very similar between mice and humans. “There’s no such thing as schizophrenic behavior in mice, but if I can prove a particular gene causes the same synaptic defect in humans and in mice, and I can correct it in my model, then that has hope as a treatment,” says Feng.

Creating Better Copies Even with greater focus on basic biological mechanisms, researchers must know which genes and mutations to incorporate into models. This alone has proven challenging. But large-scale genomic studies of humans, facilitated by cheap DNA sequencing, are beginning to reveal new candidates, including some To study brain disorders in living human tissue, researchers grew this cerebral for schizophrenia, a disease whose genetic contribu- ’ organoid from stem cells derived from a healthy donor s skin sample. In this cross- tors have long perplexed scientists. section, the neurons are green, progenitors are red, and nuclei are blue. Image courtesy of Madeline A. Lancaster and Juergen A. Knoblich. Reproduced from ref. No single genetic abnormality accounts for a large 1, with permission from Macmillan Publishers: Nature, copyright (2013). number of cases of the disease. Instead, researchers have found many potential hits across different people, each with a relatively small effect on schizophre- But most current mouse models simply can’t cap- nia risk, says Pamela Sklar, chief of the psychiatric ture the genetic, cellular, or behavioral complexity of genomics division at Mount Sinai Hospital in New human psychiatric conditions, researchers argue. In York City. “The sample sizes are only now just getting many cases, researchers have struggled to clarify the large enough in schizophrenia to be able to do some genes and mutations required to replicate brain dis- fine mapping and localizing of genetic risk factors,” orders in mice. In others, scientists have been ham- says Sklar. pered by fundamental differences between mice and In 2014, the Psychiatric Genomics Consortium, an humans in brain structure and function. international research collaboration, announced their Without knowing precisely which molecular or ge- genome-wide scans of more than 150,000 people had netic defects to copy, researchers have tried to make found 108 regions associated with risk for schizo- animal models that at least mimic human psychiatric phrenia (3). And within the chromosomal region with symptoms. In one common test for depression-like the strongest links to schizophrenia risk identified so behaviors, for example, they measure how long mice struggle against being held upside down by the tail. far, a team led by researchers at Harvard Medical (Animals that give up sooner are typically judged as School reported in January that they had pinpointed a showing greater “despair.”) But such strategies have gene that could be a major contributor to the signal met with increasing skepticism, especially in light of coming from this area (4). The C4 (complement com- the lack of therapeutic breakthroughs. ponent 4) gene encodes a set of immune system “We can make models by challenging mice in dif- proteins, and the team found that the more people ferent ways and looking at their behavior, but it’snotat expressed one particular form of C4 protein, the all clear that these animals have the same disease that greater their risk of developing schizophrenia. In the we do,” says Fred H. Gage, a neuroscientist at the Salk neurons of newborn mice, researchers found that C4 Institute for Biological Studies in La Jolla, California. expression ramped up during the period when cell- Feng echoed these concerns in a 2015 commen- to-cell connections normally get pruned and refined. tary (2) in Nature Medicine, criticizing the over- When the researchers made mice with a disabled C4 interpretation of many current mouse models of gene, they saw decreased pruning, leading them to brain disorders, in particular those based primarily on speculate that overproduction of C4 could lead to early matching behavioral signs rather than known or sus- hyperactive pruning in people with schizophrenia. pected mechanisms. Among his caveats: mice lack a Beyond finding new genes to tweak, identifying well-developed prefrontal cortex, an area that in hu- the precise mutations in those genes that affect peo- mans is thought to mediate higher cognitive functions ple, and replicating them in mice will be equally im- and appears to play a role in disorders such as autism portant, says Feng. Many past studies have used and schizophrenia. “knockout” mice, which are designed to fully disable “I’m not saying mice are not useful for schizo- one or both copies of the gene of interest, to un- phrenia studies, but it’s important to understand the derstand a gene’s effect. But in reality, human patients limitations,” says Feng. Instead of relying on hard-to- often exhibit more subtle mutations that alter gene interpret mouse behaviors, Feng and others have ar- function rather than eliminate it entirely. “If you don’t gued in favor of modeling disease-related changes make the precise human mutation, you might be in basic neuronal properties. For example, aberrant studying the wrong disorder,” says Feng.

5462 | www.pnas.org/cgi/doi/10.1073/pnas.1605358113 Shen Downloaded by guest on September 25, 2021 Feng has been investigating how different muta- contact to communicate rather than to convey ag- tions in the Shank3 gene contribute to certain forms of gression. From a practical perspective, they also cost either schizophrenia or autism. In a paper published in less than macaques to maintain because of their January, Feng’s group created two lines of mice, each smaller size and the fact that entire families live to- carrying a human mutation associated with one of the gether in a single cage (9). conditions (5). Although the models showed some In February, scientists published the first behavioral neural deficits in common, mice with an autism-asso- descriptions of marmosets engineered to overexpress ciated mutation developed problems earlier in life, the methyl CpG binding protein 2 (MeCP2) gene (10); including weakened neuronal signaling in the stria- people with mutations in MeCP2 or extra gene copies tum, which is involved in certain repetitive or compul- develop syndromes that include autism symptoms. sive behaviors. Mice with a schizophrenia-associated The marmosets with extra copies of MeCP2 paced mutation, on the other hand, showed defects later obsessively in circles, showed decreased interest in on, such as reduced signaling in the medial prefrontal socializing with other marmosets, and produced noises cortex, a brain area related to social interactions and associated with anxiety. The monkeys do not mimic decision-making. all symptoms seen in humans with additional MeCP2 Although single genes like Shank3 offer scientists copies, such as seizures, but the authors propose that toeholds for studying certain aspects of autism and the animals could be useful models for studying human schizophrenia, generating mouse models with multi- brain disorders. ple mutations to more closely match human patients Okano and geneticist Erika Sasaki at Keio Univer- has been a significant challenge. With traditional ge- sity are currently studying genetically engineered netic engineering, producing just a few mice with a marmosets they created to model Rett syndrome, an single desired mutation requires making many gen- autism-related disorder. The team has also developed erations of animals; adding more mutations multiplies marmoset models of Parkinson’s disease, Alzheimer’s, the difficulty. and other conditions. At MIT, researchers, including But new precision gene-editing technologies, such Feng, are preparing to make genetically modified as CRISPR/Cas9 (6), are helping scientists to introduce marmosets to study brain disorders as well. mutations—even several at a time—directly into eggs or early embryos, to make genetically altered animals in a single generation [see “Core Concept: CRISPR Models are still models, and humans are the only perfect gene editing” (7)]. Researchers are still working to models for humans. boost efficiency and reduce unintended mutations —Guoping Feng with the emerging techniques. Even so, they are already much quicker and cheaper than old methods. Moreover, the advent of precision gene editing has Despite recent excitement about genetically engi- made primate models with custom mutations feasible neered primates, they are unlikely to overtake mice for the first time. as the dominant model for neuropsychiatric research. Ethical considerations curb their use, and monkeys still Coming Closer To Humans take more resources, years instead of months to raise, Although genetically modified monkeys are still in and produce fewer offspring than mice. “ early development, primate models are advancing Scientific limitations exist as well. Models are still quickly. Researchers in China reported the first mon- models, and humans are the only perfect models for ” “ keys created with custom mutations in 2014: a proof- humans, says Feng. We still have the same caveat ’ of-principle in cynomolgus monkeys (a type of ma- that we cannot diagnose [monkeys], just as we can t ” caque) possessing mutations in both an immune diagnose a mouse, with a psychiatric disorder. In the function gene and a metabolic regulatory gene (8). future, however, Feng thinks genetic monkey models Other genetically engineered monkeys are in the will become more powerful as human brain imaging works, and marmosets have attracted particular in- studies reveal electrical signatures of disease that can terest for studying disorders that disrupt social be- also be detected and analyzed in the primates. havior, such as autism. “One of the major advantages of monkeys is that Straight from the Source their brains are closer to humans in structure and Stem cells, meanwhile, are helping some neurosci- function, compared to mice,” says Feng. “They have entists to go beyond animal models altogether, with a very well-developed prefrontal cortex, and have “disease-in-a-dish” systems made from human cells. some higher cognitive functions that we cannot study iPSC technology, first described in 2006 (11), could in mice.” prove particularly helpful for studying the many brain “Studying social behavior in mice is very artificial,” disorders with complex or unknown genetics, allowing says Hideyuki Okano, a stem-cell biologist at Keio researchers to sidestep the guesswork of replicating University in Tokyo. “The marmoset has lots of human- all of the right mutations in animals. “Having cells di- like traits that are missing from the mouse and even rectly from patients who are diagnosed by physicians the macaque, such as a family structure.” Marmosets tells us we’re dealing with cells from humans that we typically live in units consisting of two parents and know have the disease,” says Gage. “It takes us closer their offspring, and like humans, they also use eye to examining the molecular basis of the disease.”

Shen PNAS | May 17, 2016 | vol. 113 | no. 20 | 5463 Downloaded by guest on September 25, 2021 In a 2014 study, Gage and his colleagues de- Notwithstanding excitement over human cell and veloped an iPSC-based model of bipolar disorder, organoid models, “it’s important to keep in mind how culturing neurons from the cells of six patients with the early days it is,” notes Arnold Kriegstein, director of the condition (12). The disease runs in families, but re- Developmental and Stem Cell Biology Program at the searchers have had trouble disentangling the web of University of California, San Francisco. Researchers are genetic and biological mechanisms behind it. This lack still grappling with standardizing methods for culturing of clarity has also made it hard to predict which people the brain cells, and trying to understand why different will respond well to treatment with lithium. batches of neurons—even those grown from the same Confirming what has been seen in animal models, donor—can produce different results, he says. Gage’s team found elevated electrical activity in the There’s also the question of how closely the labo- patient-derived neurons. They also found that lithium ratory-grown human neurons mimic real brain function reversed this defect in neurons grown from lithium (14). Current techniques only produce neurons that responders, but not in neurons from nonresponders. match very early human developmental stages. But Based on the results, Gage suggests that neuronal that hasn’t stopped some researchers from trying to hyperexcitability could be an important early cellular use iPSC-derived neurons to study Alzheimer’s dis- indicator of the disorder, and that iPSCs could lead to ease and other neurodegenerative disorders that ap- new in vitro methods of screening patients for drug pear late in life. Kriegstein predicts that over the next treatments. “We can begin to discover what is unique 5 to 10 years, researchers will resolve many fundamental about nonresponders, and try to diagnose ahead of questions about the technology, including learning to time whether or not a patient will be responsive,” he generate more mature neurons from iPSCs. says. “Usually, this takes years of trying.” “I think the way forward is going to take multiple Minibrains grown from iPSCs have already pointed levels of study. All these models are very useful if we Vaccarino to new leads in autism. In a 2015 study, she ask the right questions,” says Feng. Smarter mouse showed that cerebral organoids from the cells of models can provide convenient and powerful models people with one type of autism—a severe form in- for studying cellular and molecular defects, and ge- volving enlarged head size—overproduce inhibitory netically modified monkeys may be useful for studying neurons (13). These organoids approximated fetal neural circuits underlying social behavior and higher cerebral cortex development between 9 and 16 weeks, cognitive abilities, he points out. Stem cells could and her team traced the defect to overexpression of offer a new path for understanding how complex a gene called FOXG1 that regulates other genes in- genetic factors lead to abnormal neuron function in real volved in brain growth. In addition, the degree of human tissue. “As we combine these systems, this can change in gene expression correlated with autism se- lead to breakthroughs to understanding the neurobi- verity in their subjects. The researchers didn’tfindmu- ological basis of psychiatric disorders,” says Feng. tations in FOXG1 itself, but they are now looking for “This is a beginning of a great period for advancing other molecules that influence and are influenced by the understanding of brain disorders, especially in the FOXG1 expression. field of psychiatric disorders.”

1 Lancaster MA, et al. (2013) Cerebral organoids model human brain development and microcephaly. Nature 501(7467):373–379. 2 Kaiser T, Feng G (2015) Modeling psychiatric disorders for developing effective treatments. Nat Med 21(9):979–988. 3 Schizophrenia Working Group of the Psychiatric Genomics Consortium (2014) Biological insights from 108 schizophrenia-associated genetic loci. Nature 511(7510):421–427. 4 Sekar A, et al.; Schizophrenia Working Group of the Psychiatric Genomics Consortium (2016) Schizophrenia risk from complex variation of complement component 4. Nature 530(7589):177–183. 5 Zhou Y, et al. (2016) Mice with shank3 mutations associated with ASD and schizophrenia display both shared and distinct defects. Neuron 89(1):147–162. 6 Jinek M, et al. (2012) A programmable dual-RNA-guided DNA endonuclease in adaptive bacterial immunity. Science 337(6096): 816–821. 7 Dance A (2015) Core Concept: CRISPR gene editing. Proc Natl Acad Sci USA 112(20):6245–6246. 8 Niu Y, et al. (2014) Generation of gene-modified cynomolgus monkey via Cas9/RNA-mediated gene targeting in one-cell embryos. Cell 156(4):836–843. 9 Kishi N, Sato K, Sasaki E, Okano H (2014) Common marmoset as a new model animal for neuroscience research and genome editing technology. Dev Growth Differ 56(1):53–62. 10 Liu Z, et al. (2016) Autism-like behaviours and germline transmission in transgenic monkeys overexpressing MeCP2. Nature 530(7588):98–102. 11 Takahashi K, Yamanaka S (2006) Induction of pluripotent stem cells from mouse embryonic and adult fibroblast cultures by defined factors. Cell 126(4):663–676. 12 Mertens J, et al.; Pharmacogenomics of Bipolar Disorder Study (2015) Differential responses to lithium in hyperexcitable neurons from patients with bipolar disorder. Nature 527(7576):95–99. 13 Mariani J, et al. (2015) FOXG1-dependent dysregulation of GABA/glutamate neuron differentiation in autism spectrum disorders. Cell 162(2):375–390. 14 Sandoe J, Eggan K (2013) Opportunities and challenges of pluripotent stem cell neurodegenerative disease models. Nat Neurosci 16(7):780–789.

5464 | www.pnas.org/cgi/doi/10.1073/pnas.1605358113 Shen Downloaded by guest on September 25, 2021