Project Summary/Abstract
Copy number variations (CNVs) of the human 16p11.2 genetic locus, containing 29 coding genes, are associated
with a number of neurodevelopmental and psychiatric disorders. The deletion (16pdel) and duplication (16pdup)
variants of this region have poorly understood pleiotropic effects. Although autism is more common in patients
with deletions, and schizophrenia is more common in those with duplications, underlying mechanisms are not
clear. Several molecular pathways from the 16p11.2 region modulate neuronal differentiation, migration, axonal
development, and synapse formation, as well as energy and lipid metabolism. Studies of 16p-animal models
have suggested deficits in the KCTD13-RhoA pathway activation, neuronal migration, axonal development, and
behavior. In turn, disruption of ceramide homeostasis due to 16p11.2 CNVs at FAM57B locus altered lipid
abundance, cell membrane dynamics, synaptic protein expression, and synaptic transport, suggesting that
lipidome dysregulation could contribute to neuronal function and activity. However, the exact molecular
mechanisms underlying these neuronal dysfunctions in excitatory versus inhibitory neurons are lacking.
Moreover, contradictory results have been reported from different animal and human cell models. To address
this gap of knowledge, we developed human iPSC-derived neuronal models of 16p11.2 CNVs and demonstrated
that i) KCTD13 regulates RhoA pathway activation, and increased RhoA expression leads to hyperactivity of the
16pdel human cortical neuron networks, and ii) there are significant changes in key mitochondrial and lipid
enzyme transcripts, including decrease in FAM57B-mediated ceramide synthase expression, that directly
correlate with observed changes in the metabolome and lipidome. These data suggest that 16pdel leads to
complex metabolic disruptions and deficient ceramide expression that might contribute to the observed functional
neuronal network phenotypes. These data have led us to hypothesize that 16p11.2 CNVs cause dysregulation
of ceramide abundance in glutamatergic and GABAergic neurons that in turn promotes deficits in synaptic
development and function leading to network disorganization and hyperactivation. Here, we will investigate this
hypothesis and the effects of 16p11.2 CNVs on cortical neuron development and function in human iPSC-derived
2-dimensional excitatory-inhibitory neuron co-cultures and human iPSC-derived 3-dimensional forebrain
organoids. To reduce variability caused by different genotypic backgrounds, we will study CRISPR-Cas9 induced
16p11.2 CNV iPSC lines in addition to iPSC lines derived from patients and healthy controls. We will utilize state-
of-the-art molecular methodologies to uncover mechanisms underlying the synaptic dysfunction of the excitatory-
inhibitory neurons in 16p11.2 CNVs, including single cell transcriptional gene expression profiling and
lipidome/metabolome profiling. Finally, we will investigate the excitatory-inhibitory network function, connectivity,
and oscillation patterns with multi-electrode arrays and patch clamping. We anticipate that this study will uncover
new molecular targets related to cortical neuron dysfunction in 16p11.2 CNV disorders.