NEWS FEATURE

Better models for brain diseaseTraditional 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, neuroscientistFlora Vaccarino tries to unlock mysteries hidden insidethe brains of people with autism. By introducingcertain genes to the skin cells, the Yale UniversitySchool of Medicine researcher reprograms them toan embryo-like state, turning them into induced plu-ripotent stem cells (iPSCs); and with two more monthsof nurturing and tinkering, Vaccarino can guide thecells to develop into small balls of neural tissue akinto miniature human brains.

Less than two millimeters across, these “cerebralorganoids” 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 perfectlymatch the genetic makeup of the adults and childrenwith autism who donated the original skin samples,allowing Vaccarino’s team to track the very beginningsof their disorder.

Human brain tissue usually can’t be collected andstudied until after death, and by then, it can be toolate to glean important insights. “You don’t get to seethe same person’s cells progressing through a seriesof steps and time points, like we do with these organo-ids,” Vaccarino explains. “The organoids are a verypowerful system. You can actually change things andsee what the outcome is going to be.”

Predicting outcomes and, crucially, developingpsychiatric drugs has proven exceedingly difficult inrecent decades. Inadequate animal models have beena major stumbling block, researchers say. First de-veloped in 2013 (1), cerebral organoids grown fromhuman iPSCs—affectionately called minibrains bysome—are one of several emerging technologies thatare finally allowing researchers to make more sophis-ticated models of neuropsychiatric disorders. Ad-vances in genomics are also helping to shed newlight on mental illnesses by pointing researchers tonew gene targets. These, in turn, can be combinedwith recent precision gene-editing techniques tomake animal models that many researchers hope willmore faithfully reproduce aspects of human diseases,such as Alzheimer’s, autism, and schizophrenia.

“It’s a very exciting time,” says Guoping Feng, aneuroscientist at the Massachusetts Institute of Technol-ogy (MIT) in Cambridge, Massachusetts. “Between thetechnology development and the genetic findings, this

is the first time that we’ve been able to begin diggingdeep into the causes and neurobiology of thesedisorders.”

The Mouse ProblemFor decades, traditional animal models—commonly,genetically engineered mice—have allowed scientiststo manipulate the brain’s cells, genes, and molecules,revealing some basic but important insights. Modi-fied mice, for example, have helped reveal how mis-folded versions of the α-synuclein protein gunk upthe Parkinson’s-diseased brain and possibly injureneurons. Mice with mutations linked to Alzheimer’sdisease have helped scientists examine how mis-folded amyloid-β protein collects into sticky plaquesin the brain.

Neurons such as these, derived from the induced pluripotent stem cells of a Parkinson’sdisease patient, are on the forefront of efforts to improve models for brain disease.Image courtesy of Cedric Bardy and Fred H. Gage (The Salk Institute, La Jolla, CA).

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But most current mouse models simply can’t cap-ture the genetic, cellular, or behavioral complexity ofhuman psychiatric conditions, researchers argue. Inmany cases, researchers have struggled to clarify thegenes and mutations required to replicate brain dis-orders in mice. In others, scientists have been ham-pered by fundamental differences between mice andhumans in brain structure and function.

Without knowing precisely which molecular or ge-netic defects to copy, researchers have tried to makeanimal models that at least mimic human psychiatricsymptoms. In one common test for depression-likebehaviors, for example, they measure how long micestruggle against being held upside down by the tail.(Animals that give up sooner are typically judged asshowing greater “despair.”) But such strategies havemet with increasing skepticism, especially in light ofthe lack of therapeutic breakthroughs.

“We can make models by challenging mice in dif-ferent ways and looking at their behavior, but it’s not atall clear that these animals have the same disease thatwe do,” says Fred H. Gage, a neuroscientist at the SalkInstitute for Biological Studies in La Jolla, California.

Feng echoed these concerns in a 2015 commen-tary (2) in Nature Medicine, criticizing the over-interpretation of many current mouse models ofbrain disorders, in particular those based primarily onmatching behavioral signs rather than known or sus-pected mechanisms. Among his caveats: mice lack awell-developed prefrontal cortex, an area that in hu-mans is thought to mediate higher cognitive functionsand appears to play a role in disorders such as autismand schizophrenia.

“I’m not saying mice are not useful for schizo-phrenia studies, but it’s important to understand thelimitations,” says Feng. Instead of relying on hard-to-interpret mouse behaviors, Feng and others have ar-gued in favor of modeling disease-related changesin basic neuronal properties. For example, aberrant

electrical properties or abnormal numbers of cell-to-cell connections (synapses) are thought to be involvedin several psychiatric disorders, and synapse formationand function appear very similar between mice andhumans. “There’s no such thing as schizophrenic be-havior in mice, but if I can prove a particular genecauses the same synaptic defect in humans and inmice, and I can correct it in my model, then that hashope as a treatment,” says Feng.

Creating Better CopiesEven with greater focus on basic biological mecha-nisms, researchers must know which genes and mu-tations to incorporate into models. This alone hasproven challenging. But large-scale genomic studiesof humans, facilitated by cheap DNA sequencing, arebeginning to reveal new candidates, including somefor schizophrenia, a disease whose genetic contribu-tors have long perplexed scientists.

No single genetic abnormality accounts for a largenumber of cases of the disease. Instead, researchershave found many potential hits across different peo-ple, each with a relatively small effect on schizophre-nia risk, says Pamela Sklar, chief of the psychiatricgenomics division at Mount Sinai Hospital in NewYork City. “The sample sizes are only now just gettinglarge enough in schizophrenia to be able to do somefine mapping and localizing of genetic risk factors,”says Sklar.

In 2014, the Psychiatric Genomics Consortium, aninternational research collaboration, announced theirgenome-wide scans of more than 150,000 people hadfound 108 regions associated with risk for schizo-phrenia (3). And within the chromosomal region withthe strongest links to schizophrenia risk identified sofar, a team led by researchers at Harvard MedicalSchool reported in January that they had pinpointed agene that could be a major contributor to the signalcoming from this area (4). The C4 (complement com-ponent 4) gene encodes a set of immune systemproteins, and the team found that the more peopleexpressed one particular form of C4 protein, thegreater their risk of developing schizophrenia. In theneurons of newborn mice, researchers found that C4expression ramped up during the period when cell-to-cell connections normally get pruned and refined.When the researchers made mice with a disabled C4gene, they saw decreased pruning, leading them tospeculate that overproduction of C4 could lead to earlyhyperactive pruning in people with schizophrenia.

Beyond finding new genes to tweak, identifyingthe precise mutations in those genes that affect peo-ple, and replicating them in mice will be equally im-portant, says Feng. Many past studies have used“knockout” mice, which are designed to fully disableone or both copies of the gene of interest, to un-derstand a gene’s effect. But in reality, human patientsoften exhibit more subtle mutations that alter genefunction rather than eliminate it entirely. “If you don’tmake the precise human mutation, you might bestudying the wrong disorder,” says Feng.

To study brain disorders in living human tissue, researchers grew this cerebralorganoid from stem cells derived from a healthy donor’s skin sample. In this cross-section, the neurons are green, progenitors are red, and nuclei are blue. Imagecourtesy of Madeline A. Lancaster and Juergen A. Knoblich. Reproduced from ref.1, with permission from Macmillan Publishers: Nature, copyright (2013).

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Feng has been investigating how different muta-tions in the Shank3 gene contribute to certain forms ofeither schizophrenia or autism. In a paper published inJanuary, Feng’s group created two lines of mice, eachcarrying a human mutation associated with one of theconditions (5). Although the models showed someneural deficits in common, mice with an autism-asso-ciated mutation developed problems earlier in life,including weakened neuronal signaling in the stria-tum, which is involved in certain repetitive or compul-sive behaviors. Mice with a schizophrenia-associatedmutation, on the other hand, showed defects lateron, such as reduced signaling in the medial prefrontalcortex, a brain area related to social interactions anddecision-making.

Although single genes like Shank3 offer scientiststoeholds for studying certain aspects of autism andschizophrenia, generating mouse models with multi-ple mutations to more closely match human patientshas been a significant challenge. With traditional ge-netic engineering, producing just a few mice with asingle desired mutation requires making many gen-erations of animals; adding more mutations multipliesthe difficulty.

But new precision gene-editing technologies, suchas CRISPR/Cas9 (6), are helping scientists to introducemutations—even several at a time—directly into eggsor early embryos, to make genetically altered animalsin a single generation [see “Core Concept: CRISPRgene editing” (7)]. Researchers are still working toboost efficiency and reduce unintended mutationswith the emerging techniques. Even so, they are al-ready much quicker and cheaper than old methods.Moreover, the advent of precision gene editing hasmade primate models with custom mutations feasiblefor the first time.

Coming Closer To HumansAlthough genetically modified monkeys are still inearly development, primate models are advancingquickly. Researchers in China reported the first mon-keys created with custom mutations in 2014: a proof-of-principle in cynomolgus monkeys (a type of ma-caque) possessing mutations in both an immunefunction gene and a metabolic regulatory gene (8).Other genetically engineered monkeys are in theworks, and marmosets have attracted particular in-terest for studying disorders that disrupt social be-havior, such as autism.

“One of the major advantages of monkeys is thattheir brains are closer to humans in structure andfunction, compared to mice,” says Feng. “They havea very well-developed prefrontal cortex, and havesome higher cognitive functions that we cannot studyin mice.”

“Studying social behavior in mice is very artificial,”says Hideyuki Okano, a stem-cell biologist at KeioUniversity in Tokyo. “The marmoset has lots of human-like traits that are missing from the mouse and eventhe macaque, such as a family structure.” Marmosetstypically live in units consisting of two parents andtheir offspring, and like humans, they also use eye

contact to communicate rather than to convey ag-gression. From a practical perspective, they also costless than macaques to maintain because of theirsmaller size and the fact that entire families live to-gether in a single cage (9).

In February, scientists published the first behavioraldescriptions of marmosets engineered to overexpressthe methyl CpG binding protein 2 (MeCP2) gene (10);people with mutations inMeCP2 or extra gene copiesdevelop syndromes that include autism symptoms.The marmosets with extra copies of MeCP2 pacedobsessively in circles, showed decreased interest insocializing with other marmosets, and produced noisesassociated with anxiety. The monkeys do not mimicall symptoms seen in humans with additional MeCP2copies, such as seizures, but the authors propose thatthe animals could be useful models for studying humanbrain disorders.

Okano and geneticist Erika Sasaki at Keio Univer-sity are currently studying genetically engineeredmarmosets they created to model Rett syndrome, anautism-related disorder. The team has also developedmarmoset models of Parkinson’s disease, Alzheimer’s,and other conditions. At MIT, researchers, includingFeng, are preparing to make genetically modifiedmarmosets to study brain disorders as well.

Models are stillmodels, and humans are the only perfectmodels for humans.

—Guoping Feng

Despite recent excitement about genetically engi-neered primates, they are unlikely to overtake miceas the dominant model for neuropsychiatric research.Ethical considerations curb their use, and monkeys stilltake more resources, years instead of months to raise,and produce fewer offspring than mice.

Scientific limitations exist as well. “Models are stillmodels, and humans are the only perfect models forhumans,” says Feng. “We still have the same caveatthat we cannot diagnose [monkeys], just as we can’tdiagnose a mouse, with a psychiatric disorder.” In thefuture, however, Feng thinks genetic monkey modelswill become more powerful as human brain imagingstudies reveal electrical signatures of disease that canalso be detected and analyzed in the primates.

Straight from the SourceStem cells, meanwhile, are helping some neurosci-entists to go beyond animal models altogether, with“disease-in-a-dish” systems made from human cells.iPSC technology, first described in 2006 (11), couldprove particularly helpful for studying the many braindisorders with complex or unknown genetics, allowingresearchers to sidestep the guesswork of replicatingall of the right mutations in animals. “Having cells di-rectly from patients who are diagnosed by physicianstells us we’re dealing with cells from humans that weknow have the disease,” says Gage. “It takes us closerto examining the molecular basis of the disease.”

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In a 2014 study, Gage and his colleagues de-veloped an iPSC-based model of bipolar disorder,culturing neurons from the cells of six patients with thecondition (12). The disease runs in families, but re-searchers have had trouble disentangling the web ofgenetic and biological mechanisms behind it. This lackof clarity has also made it hard to predict which peoplewill respond well to treatment with lithium.

Confirming what has been seen in animal models,Gage’s team found elevated electrical activity in thepatient-derived neurons. They also found that lithiumreversed this defect in neurons grown from lithiumresponders, but not in neurons from nonresponders.Based on the results, Gage suggests that neuronalhyperexcitability could be an important early cellularindicator of the disorder, and that iPSCs could lead tonew in vitro methods of screening patients for drugtreatments. “We can begin to discover what is uniqueabout nonresponders, and try to diagnose ahead oftime whether or not a patient will be responsive,” hesays. “Usually, this takes years of trying.”

Minibrains grown from iPSCs have already pointedVaccarino to new leads in autism. In a 2015 study, sheshowed that cerebral organoids from the cells ofpeople with one type of autism—a severe form in-volving enlarged head size—overproduce inhibitoryneurons (13). These organoids approximated fetalcerebral cortex development between 9 and 16 weeks,and her team traced the defect to overexpression ofa gene called FOXG1 that regulates other genes in-volved in brain growth. In addition, the degree ofchange in gene expression correlated with autism se-verity in their subjects. The researchers didn’t find mu-tations in FOXG1 itself, but they are now looking forother molecules that influence and are influenced byFOXG1 expression.

Notwithstanding excitement over human cell andorganoid models, “it’s important to keep in mind howearly days it is,” notes Arnold Kriegstein, director of theDevelopmental and Stem Cell Biology Program at theUniversity of California, San Francisco. Researchers arestill grappling with standardizing methods for culturingthe brain cells, and trying to understand why differentbatches of neurons—even those grown from the samedonor—can produce different results, he says.

There’s also the question of how closely the labo-ratory-grown human neurons mimic real brain function(14). Current techniques only produce neurons thatmatch very early human developmental stages. Butthat hasn’t stopped some researchers from trying touse iPSC-derived neurons to study Alzheimer’s dis-ease and other neurodegenerative disorders that ap-pear late in life. Kriegstein predicts that over the next5 to 10 years, researchers will resolve many fundamentalquestions about the technology, including learning togenerate more mature neurons from iPSCs.

“I think the way forward is going to take multiplelevels of study. All these models are very useful if weask the right questions,” says Feng. Smarter mousemodels can provide convenient and powerful modelsfor studying cellular and molecular defects, and ge-netically modified monkeys may be useful for studyingneural circuits underlying social behavior and highercognitive abilities, he points out. Stem cells couldoffer a new path for understanding how complexgenetic factors lead to abnormal neuron function in realhuman tissue. “As we combine these systems, this canlead to breakthroughs to understanding the neurobi-ological basis of psychiatric disorders,” says Feng.“This is a beginning of a great period for advancingthe understanding of brain disorders, especially in thefield 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-associatedgenetic loci. Nature 511(7510):421–427.

4 Sekar A, et al.; Schizophrenia Working Group of the Psychiatric Genomics Consortium (2016) Schizophrenia risk from complexvariation 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 editingtechnology. Dev Growth Differ 56(1):53–62.

10 Liu Z, et al. (2016) Autism-like behaviours and germline transmission in transgenic monkeys overexpressing MeCP2. Nature530(7588):98–102.

11 Takahashi K, Yamanaka S (2006) Induction of pluripotent stem cells from mouse embryonic and adult fibroblast cultures by definedfactors. Cell 126(4):663–676.

12 Mertens J, et al.; Pharmacogenomics of Bipolar Disorder Study (2015) Differential responses to lithium in hyperexcitable neurons frompatients 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 Neurosci16(7):780–789.

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