Uytengsu-Hamilton 22q11 Neuropsychiatry Research Awards Program

Treatments for the diverse symptoms associated with 22q11.2DS are lacking, in part due to poor understanding of the way in which the impacted genes function within specific cell types. This challenge is most pronounced in the brain. Fortunately, a tremendous advance recently occurred in our understanding of the human brain. Using cutting edge genomic methods (single-nucleus RNA sequencing, snRNAseq), scientists just published the first comprehensive atlas of cell types in the human brain. This atlas revealed remarkable complexity, including the finding of 461 unique cell types, plus evidence that even more cell types will be discovered as this work continues. We used this landmark data resource to map genetic risk for schizophrenia to cell types, and we discovered multiple novel cellular associations as well as discovering additional details regarding cell types previously linked to schizophrenia. Here we propose extension of this work to the 22q11.2 genes specifically. This cutting-edge approach has the potential to reveal the cellular basis of 22q11.2DS symptoms mediated by the brain. Most excitingly, due to the nature of the data that we will analyze, any discoveries emerging from this work will point directly to novel drug repurposing and drug development targets.

Most studies of 22q11.2 deletion syndrome (22q11DS) focus on using murine models or mono-cultures of human cells. Therefore, human neuronal phenotypes, especially neuronal migration and circuitry in the context of the unique 22q11.2 genomic deletion, remain poorly understood. Cortical circuit formation is dysregulated in murine models of 22q11DS, but compared to rodents, human interneurons undergo extended periods of development and migration, enhancing the complexity and cognitive capabilities of the human brain. Thus, studying interneuron migration in patient-derived cells is important to understanding this species-specific phenomenon and fully understand the complexities of how human neuronal circuitry is dysregulated in 22q11DS. To address this challenge, we propose a novel bioengineering platform to enhance the complexity of stem cell-derived neural assembloids as physiologically- and spatially-relevant in vitro models of interneuron migration in 22q11DS. We leverage a magnetic 3D-bioprinting technology (termed SPOT) to generate assembloids of precisely-controlled spatial orientation, constructing physiologically-relevant neural connections. In Aim 1, we will use SPOT to print three-part control and 22q11DS assembloids consisting of brain organoids from several distinct regions: cerebral cortex (hCO) medial ganglionic eminence (MGE)-like subpallium (hSO), and lateral ganglionic eminence (LGE)-like striatum (hStrO). In Aim 2, we leverage our three-part assembloids to study the effects of several 22q11DSassociated genes on interneuron migration. The proposed studies aim to address the lack of complex humanspecific models of 22q11DS. Ultimately, this transformational work will enable unprecedented molecular and functional studies into 22q11DS neuropathology, with the potential to yield new human-relevant targets for therapeutic intervention.

The 22q11.2 Deletion Syndrome (22q11.2 DS) predisposes individuals to high risk for developing neuropsychiatric disorders. To date, no effective therapies or preventative strategies have been identified to decrease or alleviate this risk. In a previous project funded by the MCHRI Uytengsu-Hamilton 22q11 Neuropsychiatry Award, we demonstrated mitochondrial and metabolic dysfunctions in excitatory neurons and their precursors using organoids, which are 3D human brain cell cultures obtained from individuals with 22q11.2 deletion syndrome. The defects we identified likely contribute to the abnormal formation of the brain connectivity and the increased risk for neuropsychiatric disorders. Moreover, we found a compound with translation potential that is able to rescue these defects. In this project we will use organoids to test inhibitory neurons, which are the other major neuronal type, and connect to excitatory neurons to modulate the brain connectivity. Importantly, we will test FDA-approved anti-psychotic and anti-inflammatory drugs, as well as psychedelics like LSD and Psilocybin for their potential to rescue the connectivity defects. Subsequently, we will perform extensive analyses to uncover how these compounds work at a molecular level. Building on our previous data, this project promises to further advance our understanding of the biology underlying the abnormal brain connectivity in 22q11.2 deletion syndrome and help us discover new FDA-approved or novel compounds with translational potential.

22q11.2 deletion syndrome (22qDS) is characterized by disrupted development and function of multiple organs, including the heart, gut, and brain, generally resulting in major impediments to quality of life. Brain-related challenges include variable levels of intellectual disability (ID), features of autism (ASD), and in roughly 25%, schizophrenia (SZ). Each of these challenges can, in brain disorders of individuals who do not have 22qDS, be related to abnormal development of neuronal synapses—the connections between neurons. Some studies of how deletion of the 22q11.2 region of one chromosome causes ID, ASD, and SZ have focused on neuronal aspects of synaptic dysfunction. However, a critical regulator of these connections has been relatively understudied, the brain macrophages also known as microglia. Microglia function to ingest cellular debris that would otherwise accumulate in the normal course of development. In addition, microglia are constantly surveilling their environment and secreting compounds that promote brain health, while also removing, or “pruning”, brain connections that are tagged for disposal as part of normal brain development. In pilot experiments, we find that human stem cell derived microglia-like cells that harbor the 22q11.2 deletion appear to be hyperreactive to inflammatory stimulation. We propose to test whether this hyperreactivity results in overexuberant pruning of synapses, a condition that could result in symptoms along the ID-ASD-SZ axis of dysfunction in neuronal connectivity, and that could be targetable with therapeutics. In addition, the platforms we employ could be modified for the relatively high-throughput development of these therapeutics.

The 22q11.2DS results from a 1.5 Mb (A-B region) or 3 Mb (A-D region) chromosomal deletion that removes 30 or 45 protein-coding genes, respectively. Both microdeletions are strongly associated with a high incidence of neuropsychiatric diseases, mainly schizophrenia. A similarly organized AB chromosomal region exists in the mouse, which led to the generation of mouse models of the deletion syndrome. However, these showed few or milder aspects of the clinical spectrum. Recent technological advances in molecular neuroscience now allow (1) the systematic investigation of multiple gene contributions to disease mechanisms and (2) the exit from animal models toward patient-centered biology using organoid models of the human brain. In this proposal, we will compare the relative contributions of the combined loss of expression of genes in the 22q11.2 region and the structural chromosome deletion itself that alters genome architecture.

We will use human stem cell (hiPSC)-derived brain organoids as a neurological disease model and focus on the 1.5 Mb (A-B region) microdeletion. We have already generated a hiPSC line with the 22q11.2 microdeletion and are in the process of generating a hiPSC line in which all expressed protein-coding genes of the 22q11.2 A-B region are inactivated. These hiPSC lines are both derived from a control hiPSC line so that the cleanest possible comparison can be made.

To achieve our main goal, we will generate organoids and perform state-of-the-art experiments to elucidate (1) genome-wide changes in gene expression (single-cell multiome experiments), (2) genome-wide changes in chromosome folding (HiC and 3C experiments), and (3) changes in the distribution of epigenetic marks (Cut&Tag experiments). Overall, clarification of molecular pathomechanisms will serve as a starting point for insights that will enable the development of innovative diagnostic tools and more effective treatments.

Treatments for the diverse symptoms associated with 22q11.2DS are lacking, in part due to poor understanding of the way in which the impacted genes function within specific cell types. This challenge is most pronounced in the brain. Fortunately, a tremendous advance recently occurred in our understanding of the human brain. Using cutting edge genomic methods (single-nucleus RNA sequencing, snRNAseq), scientists just published the first comprehensive atlas of cell types in the human brain. This atlas revealed remarkable complexity, including the finding of 461 unique cell types, plus evidence that even more cell types will be discovered as this work continues. We used this landmark data resource to map genetic risk for schizophrenia to cell types, and we discovered multiple novel cellular associations as well as discovering additional details regarding cell types previously linked to schizophrenia. Here we propose extension of this work to the 22q11.2 genes specifically. This cutting-edge approach has the potential to reveal the cellular basis of 22q11.2DS symptoms mediated by the brain. Most excitingly, due to the nature of the data that we will analyze, any discoveries emerging from this work will point directly to novel drug repurposing and drug development targets.

Structural heart defects, or congenital heart disease (CHD), are identified in almost 75% of those diagnosed with 22q11 Deletion Syndrome (DS). The exact cause of these defects is unknown. Moreover, the increased risk in the 22q11DS patient population has yet to be explained. Here, we will use human induced pluripotent stem cells derived from patients with 22q11DS to model the stage(s) of human heart development that we believe go awry in the disease setting. We’ll use recent technological advances in sequencing-based assays to identify the specific locations of DNA that are improperly regulated in those with CHD in the context of 22q11DS. Future analyses will assess if the genes and regions identified as disease-associated in our study harbor deleterious mutations that cause CHD in both 22q11DS and non-syndromic patients. This critical information will allow us to improve genetic counseling for families and bring us closer to developing targeted therapeutics that improve outcomes in this pediatric population for which there are currently no cures.

Sleep disturbance is common in 22q11.2DS, including restless sleep and insomnia.  So far, sleep, and its association with symptoms in children with 22q11.2DS is not well understood or documented.  We recently found distinct sleep characteristics in autism spectrum disorder (ASD), including longer slow wave sleep and shorter REM sleep.  Because ASD is common in 22q11.2DS, we believe we can utilize similar parameters to measure neurodevelopment in 22q11.2DS.  Performing sleep recording in the population of 22q11.2DS may be challenging due to sensory sensitivity, as we experienced in the ASD population.  To overcome these barriers to characterizing sleep in 22q11.2DS, we propose to use our newly developed disposable, wireless miniaturized frontal multi-channel EEG system with and without conventional polysomnography.  We believe our project can potentially impact the care of 22q11.2DS by providing an affordable, non-invasive method of monitoring brain development, enabling early detection of abnormality in brain development, and stratifying candidates for sleep intervention to improve outcomes.

Genome architecture—3D folding of our 2-meter-long genome into a 10-micron cell nucleus—can regulate gene expression by strategically positioning genes and their regulatory elements in 3D, and is implicated in many neurodevelopmental disorders. Dr Urban’s lab showed that 22q11.2 deletion leads to profound 3D genome changes in patient blood–derived cell lines. However, little is known about 3D genome aberrations at the single-cell level in the brain—in particular, how 22q11.2 deletion impacts 3D genome and transcriptome of different brain cell types. We will simultaneously profile single-cell 3D genome and transcriptome of 22q11.2DS mouse models and patient samples using our single-cell assay Dip-C. We recently developed a series of assays and algorithms, uncovering lifelong 3D changes in the normal human and mouse cerebellum. In this proposal, we will further develop these methods and apply them to the 22q11.2DS cerebellum.

Reduced activity of genes located in the 22q11.2 deletion leads to molecular and cellular dysfunction of neurons. Not all genes in the 22q11.2 deletion contribute to all phenotypes observed, not even all of these genes are properly expressed in neurons making it less likely that these genes would even play any role in the neuropsychiatric aspects of the disorder. Supporting this idea, we found that three of the 22q11.2 genes can restore specific aspects of the abnormalities found in neurons. The goal of this proposal is to further explore these abnormalities in intact mouse brain slices where much of the neuronal environmental context is preserved and in human neurons derived from 22q11.2 deletion syndrome patients. We will further assess whether single genes from the 22q11.2 deletion can restore dysfunctions of patient neurons. The identification of a single gene that can rescue specific abnormalities could provide an exciting strategy for therapeutic intervention.

Chromosome 22q11.2 deletion syndrome (22q), is characterized by an extreme heterogeneity in symptoms and penetrance including a significant occurrence of neurocognitive and psychiatric deficits. Currently, it is not clear which genes located in the deleted region or their dosage are responsible for the psychiatric symptoms commonly associated with 22q, including psychosis. Reliable prognostic biomarkers are not currently available. Thus, there is an urgent need to identify specific predictors of psychosis-proneness. This proposal will address this need by utilizing a longitudinal study design to identify and assess molecular biomarkers for predicting risk of psychosis-proneness even before the clinical symptoms appear. We aim to obtain measures of neurocognition and stress and identify molecular biomarkers which may serve as potentially strong predictors for psychosis risk. The identification of specific predictors of psychosis risk and/or protection could aid in prediction of psychosis onset in 22q, track changes during the early stages, and potentially aid in future development of pharmacological intervention.

Brain structural and functional deficits contribute to psychiatric symptoms in individuals with 22q11.2 deletion syndrome (22q11DS). Previously, we described the novel mechanism by which 22q11DS disrupts communication between auditory thalamus and auditory cortex. Remarkably, this deficit is restored to normalcy by antipsychotics. In recent studies, we also discovered in 22q11DS mice and patients a substantial deficit in the cerebellar paraflocculus (PF). We hypothesize that the dysfunctional cerebellar (PF)-thalamocortical auditory circuitry underlies psychotic symptoms in 22q11DS. First, we propose to prove causality between PF disruption, abnormal neuronal activity in the auditory cortex, and schizophrenia-like behaviors in 22q11DS mice, by state-of-the-art electrophysiological, chemogenetic, and 2-photon imaging tools. Second, we will elucidate specific cellular nodes disrupted in the 22q11DS cerebellar (PF)-thalamocortical auditory circuitry using neural circuit tracing. Third, we will build human assembloid models from cells carrying the 22q11.2 microdeletion to identify human-specific mechanisms of disrupted connectivity. Together, these experiments will reveal a previously unrecognized cerebellum-thalamocortical pathogenic mechanism of psychotic symptoms in 22q11DS. This may suggest a novel direction for developing modern therapeutic strategy to treat psychotic symptoms without devastating side effects of antipsychotics.

Adults with 22q11.2 deletion have a 20-fold higher risk of developing Parkinson’s Disease than healthy individuals. Clinically, these patients present with typical motor symptoms and respond well to dopamine replacement therapy, but show an earlier disease onset in their late 30s. In brain autopsies of cases with 22q11.2 deletions, loss of neurons and intracellular protein aggregates are found that are reminiscent of Parkinson’s disease. These clinical and neuropathological findings suggest overlapping pathways both for Parkinson’s disease and 22q11.2 deletion syndrome.

Our goal is to understand which specific genes in the 22q11 deletion region contribute to the neurodegenerative process. We will examine whether human induced pluripotent stem cell (iPSC)-derived neuronal networks from adults with 22q11.2 deletions show 1) changes in early oxidative stress markers, 2) altered neuronal network activity compared to control neuronal networks, and 3) test whether activation of a selected gene or groups of genes in the 22q11.2 deletion region can ameliorate phenotypes of early oxidative stress and restore neuronal network activity. Our multidisciplinary team developed a high-density neuronal network array with 16,000 electrodes to assess human iPSC-derived neuronal networks. To rescue function, we established gene engineering tools in cell lines from adults with 22q11.2 deletion to regulate gene expression within the 22q11.2 deletion region. 

The deletion in 22q11 is almost always surrounded by long stretches of DNA called SegDups (or LCRs). It is believed that these SegDups are what causes the deletion to happen in the first place. Also, we know that genes and other important sections of DNA sequence can be hiding in SegDups. Until recently it was impossible to study the DNA sequence of the SegDups on 22q11. We have developed a CRISPR-based technology (called CTLR-Seq) that now allows us to do so.

In this study, we want to build on CTLR-Seq, and look at what is happening inside the SegDups surrounding the 22q11 deletion, in stem cells and in different cell types of the brain. We think that there may be important differences between patients hiding there. We believe that the 22q11 deletion may interact with the rest of the genome through the SegDups. If we are correct, our study will give us clues as to why and how the 22q11 deletion is causing the symptoms we see in patients, and why these symptoms can be so different.

The study of brain-immune interactions has contributed to a paradigm shift in understanding of the causes of mental disorders and has provided new insights into their prevention and treatment. Notably, the brain vasculature functions to limit the influence of peripheral inflammation on the brain, so impairment of the brain vasculature could affect the development and outcome of neuropsychiatric disease.

Our team has found that weakness in the blood brain barrier (BBB) occurs in the 22q11.2DS. One 22q11.2 gene is a key component of junctions between vascular cells of the BBB, and several of the 22q11.2 deleted genes function in the mitochondria, from which much of the energy critical to BBB function is generated. In a nutshell, the BBB in 22q11.2DS appears to be leaky for circulating inflammatory mediators and immune cells, leading to an increase in neuroinflammation and behavioral deficits associated with neuropsychiatric disorders.

Here, we propose to use the 22q11.2 DS mouse model to test whether induced peripheral activation promotes neuroinflammation and related behavioral deficits in 22q11.2DS mice. Besides providing a compelling rationale to devise immune therapeutic strategies, the demonstration of a brain-immune link will be crucial for understanding the mechanisms driving disease processes in 22q11.2DS and mental illness at large.

22q11.2 Deletion Syndrome (22q11.2DS) is a genetic syndrome associated with increased risk of developmental neuropsychiatric disorders such as Autism Spectrum Disorders, schizophrenia and attention deficit hyperactivity disorder. These disorders, as well as 22q11.2DS, have been found to be associated with alterations in brain structure and function.

Brain mapping has shown that 22q11DS is characterized by asynchronous communication between brain areas. Hence, one of the keys to understanding neurobehavioral manifestations of 22q11DS, is to understand and decode the patterns of dysfunctional brain activity that characterize the genetic syndrome. Importantly, pinpointing the origin and significance of impaired brain function in 22q11.2DS can also guide the development of possible targeted therapies aimed to restore impaired circuit function.

Towards this goal, we designed a number of studies in both a mouse model and human 22q11DS patients, aimed at tracking the developmental trajectory of brain function and connectivity associated with this syndrome, before and after adolescence. Preliminary results in our laboratory have shown parallels between these trajectories in mice and human 22q11DS, corroborating the validity of our approach. A major goal of our research is to investigate the neural and genetic underpinnings of altered brain communication in 22q11DS. Here we will test the hypothesis that the cross-species dysfunction we found can be explained in terms of disruption of synaptic homeostasis. Our investigations will also provide means to identify specific genes within the 22q11.2 locus that may drive brain connectivity alterations in 22q11DS, providing opportunities for the identification of targets for novel therapies.

The 22q11.2 deletion is associated with a high risk for psychiatric disorders, such as schizophrenia and autism. However, it is largely unknown how this deletion affects human cells and their properties, and how it results in susceptibility for psychiatric disorders.

We propose to use stem cells derived from many individuals with 22q11.2 deletion to test how the deletion changes human brain cells through innovative experimental approaches. Specifically, we will examine how the deletion affects the morphology, or architecture, of brain cells (neurons), measuring several hundred morphological traits, including shape, size, texture, and many others. We will further measure the function of these cells by measuring their connections to each other, or synapses, and their electrical properties. We will thus generate rich datasets reporting on changes in cell shape and function then compare them with our existing data on gene expression changes in these cells. 

Psychosis is enormously problematic for patients, families, and caregivers and there are disappointingly only imperfect therapeutics. There are thought to be genetic aspects as well as environmental factors that drive the development of psychosis. Several epidemiologic features are known to be associated with the subsequent development of schizophrenia, the leading type of psychosis. These epidemiologic features can be conceptually tied together as early life stress and/or inflammation. People born with a deletion in 22q11.2 have an immune system that can be different, leading to increased infections, increased length of infections, and increased inflammatory disease; and the rate of psychosis in adults with 22q11.2 deletion syndrome is as high as 25%. Thus, there is a strong rationale for evaluating the immune system in more detail in people living with 22q11.2 deletion syndrome and schizophrenia. We hypothesize that the immunologic differences contribute to the high risk of schizophrenia in 22q11.2 deletion syndrome. Our study will examine inflammatory mediators in the blood and the packaging of the DNA in chromosomes. This packaging, known as epigenetics, represents a type of code separate from genetic sequence that dictates the behavior of cells and integrates environmental clues and overall health. By analyzing that epigenetic code, we can gain insight into the T cell features associated with psychosis. Successful completion of our study will lead to conceptualization of therapeutics directed at inflammation, infection, or T cell dysfunction. Our goal is risk mitigation through targeted modulation of the immune system.

Our goal is to advance gene therapeutics for 22q11.2 Deletion Syndrome (DS). A major limitation has been the development of adenoassociated viral vectors (AAV) that can efficiently transverse the blood-brain barrier (BBB) and disseminate throughout the cells in the brain. Patients with 22q11.2 DS produce less Claudin 5, a key protein in maintaining the structure and function of the BBB. We plan to develop BBB models of 22q11.2 DS patients, screen our AAV library and select novel vectors that provide enhanced gene transfer into cells that maintain the BBB. We will use these vectors to normalize Claudin 5 protein and restore BBB function.

A recent important finding in 22q11.2DS is that neurons derived from patient cells show a reduction in the strength of the electrical field across the neuron’s membrane. Our lab specializes in the development of methods for measuring neuronal electrical fields with a microscope, rather than with an electrode. We developed a fluorescent voltage indicator named ASAP3 that can measure the electrical field with high accuracy by changing the amount of light it emits. We will apply ASAP3 to confirm the abnormal transmembrane field in neurons derived from 22q11.2DS patient cells, and from a mouse model of 22q11.2DS. We will then perform screens of known neurologically active drugs to find those that can reverse the abnormality.

Proper cellular energy production by mitochondria is critical during human brain development. The 22q11.2 chromosomal region contains mitochondrial genes. Their deletion is suspected to induce energy production deficits in individuals with 22q11.2 deletion syndrome and contribute to functional alterations and increased risk for neuropsychiatric diseases. We will perform in-depth metabolomic profiling of developing brain cells carrying the 22q11.2 deletion, using human brain organoids and cutting-edge metabolomic assays. Through comparisons with normal cells, we aim to identify differences in energy production, which would point towards therapeutic targets as early as the fetal period and eventually decrease the risk for neuropsychiatric diseases.

There is a lack of knowledge which genes in the deleted region of chromosome 22 contribute to the disease phenotype in 22q11DS, or how to restore the genes to the physiological relevant levels to remedy the disease. Here we will use cutting-edge CRISPR tools to uncover gene contributions to the neuropsychiatric phenotypes associated with 22q11DS. We will further apply precision gene editing to restore expression of affected genes to physiological relevance and rescue the disease phenotype. This will help us identify more specific and effective treatments.

Patients with 22q11DS and comorbid psychosis have been found to be less responsive to dopamine-targeting antipsychotics and more prone to their potential adverse effects. There is a need for novel therapeutics targeting other neurotransmitters to reduce psychotic and cognitive symptoms in these patients. The aim of this study is to examine the efficacy of riluzole, a FDA-approved glutamate and γ-aminobutyric acid (GABA) modulating compound, for treatment of psychotic symptoms and cognitive impairment in 22q11.2DS patients. In addition, the effects of riluzole on the glutamate/GABA-balance will be examined to increase insight in the neurobiological underpinnings of psychotic and cognitive symptoms.

We recently discovered that in people with 22q11DS and mouse models, the cerebellar paraflocculus and flocculus (PF/F) are reduced in size, and that mouse models of 22q11DS are deficient in adjusting eye movements. Cerebellar dysfunction has long-been related to the development of psychiatric symptoms and abnormal cognition, but we still do not know which cerebellar structures or molecular mechanisms are involved. We will identify the gene(s) within the 22q11DS region that causes the PF/F reduction by screening mouse models. We will then test if 22q11DS patients carrying a deletion of this gene have smaller PF/F and are deficient in controlling their eye movements. Second, we will identify the cellular processes that lead to the smaller PF/F.

Our overarching goal is to develop a data-driven computational framework for robust identification of early biomarkers of risk for psychosis in 22q11 DS patients/children. We will do so by using a novel “Big Data” science approach combining exciting recent advances in deep learning neural networks (DNN) and our recent work on quantitative dynamic brain circuit analyses with a wealth of newly available large-scale open-source cross-sectional and longitudinal brain imaging and phenotypic data from consortia and repositories around the world.

We will establish a novel genome analysis methodology that will make it possible, for the first time, to resolve the currently impenetrable Segmental Duplication regions (SegDups, also Low-Copy-Repeats, LCRs) that are flanking almost all 22q11 deletions, and thus to determine the extent of the 22q11 deletion in 22q11DS patients at base-pair resolution.