Fragile X Syndrome (FXS), a leading genetic cause of autism, causes defective synaptic communication due to silencing of the FMRP gene by repeat expansion. Two major functions for the FMRP protein are RNA binding to modulate mRNA translation, and direct interactions with presynaptic voltage-gated calcium and BK channels. This second role has been poorly studied, but is proposed to control surface expression of these proteins. I will use new genetic toolkits in Drosophila to determine how synaptic levels of calcium and BK channels are set. Using intravital FRAP and channel photoconversion, I will measure delivery and turnover of these proteins at individual synapses during larval development in controls and mutants of the Drosophila FMRP homolog, dFXR. This analysis will determine how loss of FMRP disrupts presynaptic channel abundance that may contribute to synaptic dysfunction in FXS.

Social interactions with infected animals increase the risk of disease transmission. Therefore, animals change their social behavior according to the health status of interacting conspecific. For example, our laboratory has recently identified amygdalar neural circuits responsible for suppressing innate social behaviors toward sick partners. Intriguingly, exposure to a sick conspecific is also thought to prime the subject’s immune system in the absence of direct transmission. Such changes have been proposed to prepare the subject’s immune system to better respond to a potential attack by the same pathogen. However, how social interaction with sick individuals can lead to immune responses has not been explored. In this application, I propose to identify neuroimmune interactions that connect social exposure to sickness with priming of the immune system. This study will delineate the neuroimmune interactions that allow animals to minimize risks of infection through social communications.

I propose to construct a gene therapy method for delivering therapeutic cargo in specific cell where autism spectrum disorders manifests using two different strategies. In the first strategy, I will construct a CRISPRE RNA (crRNA) followed by trans-splicing ribozyme and the start codon deficient mRNA of gene of interest (GOI). Upon target RNA recognition by catalytic inactive Cas13, the trans-splicing ribozyme will splice mRNA of GOI into the target RNA achieving conditional expression only in the presence of target RNA in specific cell types defined by target RNA. In the second strategy, I will construct a crRNA array including two crRNAs separated by an inhibitory sequence that inhibits the natural crRNA array processing ability of Cas13a. Upon target recognition, the second crRNA will be released and trigger the knock down of GOI. Both the strategies will be tested in cell model first. The efficacy of gene therapy will be demonstrated by upregulating Ube3a, a key protein whose downregulation leads to Angelman syndrome, in neuron cells of mouse model. This new gene therapy strategy will limit the side effects and enhance the efficiency and advance the field of gene therapy and treatment of autism spectrum disorders.

Autism spectrum disorders (ASD) are a diverse family of neurodevelopmental conditions that lack any consistent biomarkers to date. The enzyme acetylcholinesterase, which degrades acetylcholine at cholinergic synapses, has been emerging as potential therapeutic target in ASDs, and could serve as synapse-specific biomarker for these disorders. However, probe technologies to correlate AChE activity and ASD remained elusive. This fellowship proposal therefore seeks to design and validate novel AChE-sensitive MRI contrast agents to probe cholinergic phenotypes in ASD models, facilitate therapy development, and ultimately establish a non-invasive diagnostic tool for clinical evaluation of autistic patients. Our approach will use a previously validated molecular mechanism developed in the Jasanoff lab to image brain enzyme activity in animal models. Furthermore, these new contrast agents will be designed to facilitate probing AChE activity via multimodal imaging in addition to MRI. This added feature will permit valuable integration of readouts obtained over a range of spatial scales, both in living subjects and postmortem tissue. In concert, this proposed work promises to yield an innovative and important tool for autism research, with the potential for future extension to evaluation of cholinergic function in human subjects.

Observational learning is the ability to learn about hidden states of the environment through observing others’ experiences. Although behavioral and neural signatures of observational learning have been reported, a mechanistic understanding of its underlying neural computations is lacking. Here, I formulate a computational hypothesis for the problem of observational learning based on the notion of prediction error and test this hypothesis in the non-human primate model by recording from multiple higher-order brain areas in a novel two-player decision-making task. I will use the behavioral and neural data to develop neural network models of observational learning and use model perturbations to characterize potential failure mechanisms that may underlie behavioral impairments in neuropsychiatric diseases such as autism.

Figuring out the desires of others is a crucial, yet complicated act of interpretation. Even seemingly straight-forward utterances like “Can you pass the salt?” can have different possible interpretations (hand the salt to you or am I physically able?). What’s more, our goals and desires factor into how we interpret the ambiguous utterances of others: If I don’t want to pass the salt, I might choose to interpret the question in an unintended way. Misunderstandings and social conflicts crop up when we have the wrong space of possible interpretations, or when the most likely interpretation conflicts with our own goals. Neurotypical children and individuals with autism may face particular challenges in these contexts; they may even be considered non-compliant or unhelpful, when they inadvertently interpreted someone’s request differently than what was expected. My project aims to better understand how we reason about other people’s goals and trade-off between our own and others’ needs to decide whether or how to comply with others’ requests. The insights derived from the models and experiments in this proposal will deepen our understanding and potentially provide avenues for intervention on both those with autism and their social partners to improve communication and social connection.

Individuals with autism can experience sensory overload, a state of panic induced by excessive sensory inputs such as repetitive sounds, light, and touch. In neurotypicals, the brain suppresses constant or repetitive stimuli. Sensory overload may result from a lack of this habituation. Our model of brain function suggests sensory input is carried by brain rhythms know as gamma, which are primarily found in the upper layers of the cortex. In contrast, alpha/beta rhythms are predominantly found in deeper layers and can suppress gamma rhythms. Deficient alpha/beta rhythms have been reported in autism. We hypothesize this deficient alpha/beta may be the cause of disrupted habituation and therefore sensory overload in autism. These theories of brain function are based on observed correlations and lack tests of causality that directly manipulate brain circuitry. We have developed a technique to selectively shut down neurons in upper or deep layers of cortex. We believe we can simulate sensory overload in rhesus monkeys by suppressing neurons in deep layers of rhesus monkeys. This is predicted to decrease alpha/beta activity and thus increase gamma activity and processing of sensory inputs. This will directly test a hypothesized mechanism of sensory overload, perhaps leading research towards improved symptom management.

Human Accelerated Regions (HAR) are recently identified DNA sequences that underwent unique changes in the human evolution. HARs can control gene expression and mutations in HARs are associated to the risk of neurodevelopmental disorders including autism. Despite of their importance, the function of HARs have remained largely uncharted. Here, I propose to develop a new method to screen the gene regulatory functions of hundreds of HARs simultaneously by assigning each HAR with unique barcodes, which can be read by sequencing. Using the platform of human brain organoids, human stem cell-derived 3D tissue cultures that resemble the embryonic brain, I will characterize the regulatory roles of HARs in brain development.
Finally, for candidate HARs with strong association to autism risk, I will use genomic editing to introduce specific mutation or delete the HAR sequence, and generate brain organoids to analyze the changes caused by the editing. Together, my proposed study will be a substantial technological advance for the field, and provide instrumental knowledge for better understanding human brain development and the etiology of autism.

Autism spectrum disorder is a common neurodevelopmental disorder that is highly heritable; now, via large scale genome sequencing initiatives, we are beginning to gain a clearer understanding for the genetic basis of the disorder. Despite this increase in genetic information, it is too costly and time consuming to validate each purported genetic mutation using traditional transgenic animal models and, even if proven, we have no means of correcting the gene mutation in patients in vivo. Advances in genome engineering have enabled precision gene editing but, to date, there is no tool able to seamlessly replace DNA in the postnatal human. One potential solution is to repurpose retrotransposons, an abundant and diverse class of naturally occurring genetic elements that efficiently insert large amounts of genetic material into their host—retrotransposons are so efficient that these insertion events compose over half of the human genome. My goal is to re-engineer this highly effective gene insertion machinery to mediate large-scale, site-specific genomic rearrangements in mammalian cells. Ultimately, a technology that efficiently inserts DNA into specific genomic loci could be used to rapidly study the effects of autism-associated genomic mutations, and may eventually be harnessed to reverse genomic mutations in patients with the disorder.

Currently, most methods exploring the CRISPR/Cas system for gene therapy are based on correcting genetic mutations in DNA through homology-directed recombination (HDR) or base editing. We chose to focus on RNA-based approaches because these methods do not generate breaks/nicks or changes in the DNA sequences, a significant safety concern for gene therapy. We propose to develop and test the effectiveness of Cas13-based RNA editing as a means of gene therapy targeting Rett Syndrome.

We propose a holistic and multi-scale molecular and structural interrogation of human induced pluripotent stem cell (iPSC)-derived cortical organoids at different stages of differentiation, with a particular interest in the cytoarchitecture of subplate and deep cortical layers, where Rett syndrome (RTT) related protein methyl-CpG-binding protein 2 (MeCP2) is highly expressed during early corticogenesis. Our study will be the first to dissect the neuronal diversity at single-cell resolution in various regions in the whole intact organoid culture, including subplate and deep cortical layers. In addition, we plan to generate cortical organoids from RTT patient-derived iPSC line harboring mutant MECP2 copy (MUT) and the isogenic control iPSC line (WT), respectively. We will employ the multi-scale volumetric phenotyping pipeline to characterize both WT and MUT organoids at different stages of differentiation. We aim to examine the molecular and cellular mechanisms of how the MECP2 mutation may be linked to corticogenesis.

Interacting with others is fundamental to everyday human life, and the importance of successfully perceiving and thinking about the social world is underscored by the devastating effects of impairment of these abilities in disorders such as Autism Spectrum Disorder (ASD). Given the importance of the social world, it is perhaps not surprising then that the brain contains a host of regions dedicated to interpreting social information (i.e., the “social brain”). Although the social brain is increasingly well studied in adults, almost nothing is known about how it develops. My project therefore aims to establish fundamental principles of social brain development using neuroimaging techniques in typically developing children. Understanding the typical development of the social brain is a fundamental step toward understanding how such development goes awry in developmental disorders like ASD.

Autism Spectrum Disorders (ASD) is characterized by impaired social communicational skills and restricted repetitive interests. One fascinating yet understudied phenotype of ASD is a rational, consistent and biasfree evaluation of information compared to healthy controls. While altered social cognition in ASD is typicallyconsidered a deficit, ASD Hyper-Rationality may confer distinctive strengths. A mechanistic understanding of ASD Hyper-Rationality may reveal how neurobiological differences in ASD relate to both difficulties and strengths. Here, I propose to elucidate the neurobiological basis of hyper-rationalism in ASD, by probing a putative reduction of the optimistic bias in ASD. The optimistic bias refers to the underestimation of one’s chances to experience a negative event, and overestimation of one’s chances for a positive event. TD individuals strikingly maintain their optimistic view by rejecting unfavorable future-related information, but ASD individuals integrate information equally, regardless of its favorability, displaying a lack of optimistic bias. I will use neuroimaging to test the hypothesis that differences in reward-related neural systems are related to more rational reasoning in ASD. This would be the first mechanistic link between ASD hyperrationality and altered neural processes.