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Principal Investigator: Natalie Hawken
Organization: UNIVERSITY OF CALIFORNIA LOS ANGELES
Fiscal Year: 2024
Award: $41,308
Funding agency: National Institute of Mental Health
PROJECT SUMMARY/ABSTRACT
Autism spectrum disorder (ASD) is a lifelong, common neurodevelopmental disorder (NDD) with high heritability
and complex genetic architecture. Known genetic risk factors, including large effect, de novo mutations that
cause approximately 10% of ASD cases, have their maximal expression and converge during early brain
development to impact neurogenesis and neuronal development. Yet, multiple analyses of post-mortem brain
from individuals with ASD have consistently identified pervasive microglial activation. Study of cortical
development in ASD has centered around neurons. So, there is a dearth of knowledge surrounding microglia
function in the developing brain in ASD, which is especially important because microglia colonize the developing
fetal cortex during the epoch during in which ASD risk genes converge. Here, I propose to leverage advances in
human stem cell (SC) culture, including the development and validation of 3D human cortical spheroid (hCS)
models of human brain development, to assess the impact of ASD genetic risk and neuronal-microglial
interactions on microglial function. Cell lines derived from control, non-ASD, SC lines and two SC lines with a
haploinsufficiency of high-risk ASD-associated genes (CHD8 and SCN2A) will be utilized to derive microglia and
hCS. Bulk transcriptomics, morphological analysis, and phagocytosis assays of microglia from SC lines
harboring high risk ASD-associated mutations will reveal how ASD genetic risk impacts microglia physiology,
independent of interactions with neuronal cell types (Aim 1). Transcriptomes will be mapped to the in vivo
developmental trajectory of microglia, and differential gene expression analysis will be performed on the ASD
microglia relative to controls. Perturbations to cortical neurogenesis in ASD will be assessed through single cell
RNA sequencing and immunohistochemical analysis of cell type and morphology in a 3D co-culture system of
human cortical spheroids (hCS) integrated with microglia to model fetal microglial colonization of the cortex (Aim
2). Cell type distribution, differential gene expression, and gene network analysis of ASD mutant hCS compared
to controls at early and late neurogenesis will be performed; immunohistochemistry of cell populations, synapses,
and microglial morphology will validate and extend transcriptomic findings. These experiments will help inform
ASD pathophysiology by elucidating how ASD genetic risk contributes to microglial activation and neuronal-
microglial signaling and provide a framework for testing other ASD risk mutations. This training plan will prepare
this applicant for a successful career as a physician-scientist studying neurodevelopmental disorders (NDDs),
via the following major goals: training in stem cell culture and functional genomics, rigor and ethics in scientific
thinking, professional development, and translation of research into the clinical setting. Working in a
collaborative, multidisciplinary laboratory specializing in functional genomic analysis alongside clinical training
and mentorship from physicians practicing in NDD clinics, this applicant is well-positioned to gain the skills
necessary for a productive scientific and clinical career in pediatric neurology and developmental neurobiology.
Terms: <0-11 years old><21+ years old><3-D><3-Dimensional><3D><ASD><Address><Adult><Adult Human><Affect><Assay><Astrocytes><Astrocytus><Astroglia><Autism><Autistic Disorder><Autopsy><Axon><Bioassay><Biologic Models><Biological Assay><Biological Models><Brain><Brain Nervous System><Cell Body><Cell Communication><Cell Culture Techniques><Cell Interaction><Cell Line><Cell Maturation><Cell Nucleus><Cell-to-Cell Interaction><CellLine><Cells><Cellular Morphology><Child><Child Youth><Children (0-21)><Classification><Clinic><Clinical><Co-culture><Cocultivation><Coculture><Coculture Techniques><Complex><Connector Neuron><Data Set><Developing fetus><Development><Diagnosis><Differential Gene Expression><Dysfunction><Early Infantile Autism><Encephalon><Ethics><Fetal Development><Foundations><Functional disorder><Gene Alteration><Gene Expression Monitoring><Gene Expression Pattern Analysis><Gene Expression Profiling><Gene Mutation><Gene Transcription><Genes><Genetic><Genetic Alteration><Genetic Change><Genetic Risk><Genetic Transcription><Genetic defect><Genetic predisposing factor><Goals><Heritability><Hortega cell><Human><Immunohistochemistry><Immunohistochemistry Cell/Tissue><Immunohistochemistry Staining Method><In Vitro><Infantile Autism><Intercalary Neuron><Intercalated Neurons><Interneurons><Internuncial Cell><Internuncial Neuron><Kanner's Syndrome><Knowledge><Laboratories><Lead><Maps><Measures><Mentorship><Methods><Microglia><Model System><Modeling><Modern Man><Morphology><Mutation><Nerve Cells><Nerve Impulse Transmission><Nerve Transmission><Nerve Unit><Network Analysis><Neural Cell><Neural Development><Neural Stem Cell><Neurocyte><Neurodevelopmental Disorder><Neuroimmune><Neurological Development Disorder><Neuronal Dysfunction><Neuronal Transmission><Neurons><Nucleus><Organoids><Pathogenesis><Pathologic><Pathway Analysis><Pattern><Pb element><Pediatric Neurology><Phagocytes><Phagocytic Cell><Phagocytosis><Phenotype><Physicians><Physiology><Physiopathology><Population><Position><Positioning Attribute><Productivity><Progenitor Cells><Proliferating><Protocol><Protocols documentation><RNA Expression><RNA Seq><RNA sequencing><RNAseq><Risk><Risk-associated variant><Role><Sampling><Scientist><Secondary to><Sensory><Single-Nucleus Sequencing><Social Interaction><Strains Cell Lines><Synapses><Synaptic><System><Systematics><Testing><Thinking><Tissue-Specific Differential Gene Expression><Tissue-Specific Gene Expression><Training><Transcript Expression Analyses><Transcript Expression Analysis><Transcription><Translational Research><Translational Science><Validation><adulthood><amebocyte><analyze gene expression><astrocytic glia><autism spectral disorder><autism spectrum disorder><autistic individuals><autistic people><autistic spectrum disorder><axon signaling><axon-glial signaling><axonal signaling><career><cell cortex><cell culture><cell cultures><cell morphology><cell type><child neurology><clinical training><compare to control><comparison control><cultured cell line><de novo mutation><de novo variant><developmental><developmental neurobiology><disease risk><disorder risk><ethical><excitatory neuron><experiment><experimental research><experimental study><experiments><fetal><functional genomics><gene expression analysis><gene expression assay><gene network><genetic architecture><genetic risk factor><genome mutation><gitter cell><glia signaling><glial activation><glial cell activation><glial signaling><global gene expression><global transcription profile><heavy metal Pb><heavy metal lead><high risk><human model><human progenitor><human stem cells><in vivo><individuals on the autism spectrum><individuals on the spectrum><individuals with ASD><individuals with autism><individuals with autism spectrum disorder><inherited factor><kids><mesoglia><microglial cell><microgliocyte><model of human><multidisciplinary><mutant><necropsy><nerve signaling><nerve stem cell><neural><neural dysfunction><neural precursor><neural precursor cell><neural progenitor><neural progenitor cells><neural signaling><neurodevelopment><neurodevelopmental disease><neurogenesis><neuron development><neuron progenitors><neuronal><neuronal development><neuronal progenitor><neuronal progenitor cells><neuronal signaling><neuronal stem cells><neuroprogenitor><neurotransmission><novel><pathogen><pathophysiology><people on the autism spectrum><people with ASD><people with autism><people with autism spectrum disorder><perivascular glial cell><postmortem><repetitive behavior><risk allele><risk gene><risk genotype><risk loci><risk locus><risk variant><sNuc-Seq><scRNA-seq><single cell RNA-seq><single cell RNAseq><single cell expression profiling><single cell transcriptomic profiling><single nucleus RNA-sequencing><single nucleus seq><single-cell RNA sequencing><single-nucleus RNA-seq><skills><snRNA sequencing><snRNA-seq><social role><spheroids><stem cells><synapse><thoughts><three dimensional><transcriptional profiling><transcriptome><transcriptome sequencing><transcriptomic sequencing><transcriptomics><translation research><translational investigation><validations><youngster>