In rule-based modeling, molecular interactions are systematically specified in the form of reaction rules that serve as generators of reactions. This provides a way to account for all the potential molecular complexes and interactions among multivalent or multistate molecules. Recently, we introduced rule-based modeling into the Virtual Cell (VCell) modeling framework, permitting graphical specification of rules and merger of networks generated automatically (using the BioNetGen modeling engine) with hand-specified reaction networks. VCell provides a number of ordinary differential equation and stochastic numerical solvers for single-compartment simulations of the kinetic systems derived from these networks, and agent-based network-free simulation of the rules. In this work, compartmental and spatial modeling of rule-based models has been implemented within VCell. To enable rule-based deterministic and stochastic spatial simulations and network-free agent-based compartmental simulations, the BioNetGen and NFSim engines were each modified to support compartments. In the new rule-based formalism, every reactant and product pattern and every reaction rule are assigned locations. We also introduce the rule-based concept of molecular anchors. This assures that any species that has a molecule anchored to a predefined compartment will remain in this compartment. Importantly, in addition to formulation of compartmental models, this now permits VCell users to seamlessly connect reaction networks derived from rules to explicit geometries to automatically generate a system of reaction-diffusion equations. These may then be simulated using either the VCell partial differential equations deterministic solvers or the Smoldyn stochastic simulator.

Loss of UBE3A expression, a gene regulated by genomic imprinting, causes Angelman Syndrome (AS), a rare neurodevelopmental disorder. The UBE3A gene encodes an E3 ubiquitin ligase with three known protein isoforms in humans. Studies in mouse suggest that the human isoforms may have differences in localization and neuronal function. A recent case study reported mild AS phenotypes in individuals lacking one specific isoform. Here we have used CRISPR/Cas9 to generate isogenic human embryonic stem cells (hESCs) that lack the individual protein isoforms. We demonstrate that isoform 1 accounts for the majority of UBE3A protein in hESCs and neurons. We also show that UBE3A predominantly localizes to the cytoplasm in both wild type and isoform-null cells. Finally, we show that neurons lacking isoform 1 display a less severe electrophysiological AS phenotype.

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Loss of UBE3A expression, a gene regulated by genomic imprinting, causes Angelman Syndrome (AS), a rare neurodevelopmental disorder. The UBE3A gene encodes an E3 ubiquitin ligase with three known protein isoforms in humans. Studies in mouse suggest that the human isoforms may have differences in localization and neuronal function. A recent case study reported mild AS phenotypes in individuals lacking one specific isoform. Here we have used CRISPR/Cas9 to generate isogenic human embryonic stem cells (hESCs) that lack the individual protein isoforms. We demonstrate that isoform 1 accounts for the majority of UBE3A protein in hESCs and neurons. We also show that UBE3A predominantly localizes to the cytoplasm in both wild type and isoform-null cells. Finally, we show that neurons lacking isoform 1 display a less severe electrophysiological AS phenotype.Angelman Syndrome (AS) is a rare neurodevelopmental disorder that affects 1 in 15,000 individuals, and is characterized by severe seizures, intellectual disability, absent speech, ataxia, and happy affect 1 . AS is caused by loss of function from the maternally-inherited copy of UBE3A. UBE3A is regulated by tissue-specific genomic imprintingit is expressed exclusively from the maternal allele in neurons and is expressed biallelically in other cell types 2,3 . UBE3A encodes an E3 ubiquitin ligase that forms polyubiquitin chains to substrates, targeting them for degradation by the 26S proteasome 4 . In humans, there are three known UBE3A protein isoforms 5 , all of which include the Homologous to E6AP Carboxy Terminus (HECT) domain and are thus capable of functioning as an E3 ligase. Human isoforms 2 and 3 only differ from human isoform 1 by 23 and 20 amino acids, respectively, at their N termini 6 (Fig 1a). While there has been little published research examining the human protein isoforms in human cells, human isoforms ectopically expressed in mouse indicate that the isoforms likely have differences in localization and function 7-9 . Furthermore, it is currently unknown whether the different human isoforms have different abundances or functions. This knowledge is important as some therapeutic avenues currently being explored for AS involve delivery and expression of exogenous UBE3A transgenes using vector-based delivery 10 . For these approaches, knowledge of which of the protein isoforms need to be replaced in AS is essential.Recently, Sadhwani and colleagues published three cases of AS in two different families caused by missense mutations at the isoform 1 translational start site 11 . Interestingly, these patients present with milder phenotypes than AS patients with 15q11-q13 deletions or other UBE3A loss of function mutations, including normal gait and use of syntactic speech. This data, coupled with the fact that the human and mouse isoforms are not entirely conserved, illustrates the need to study the human UBE3A protein isoforms specifically, as they may play a role in both normal neuronal function and in disease. Here, we have used human embryonic stem cells (h...

Chromosome 15q11-q13 duplication syndrome (Dup15q) is a neurogenetic disorder caused by duplications of the maternal copy of this region. In addition to hypotonia, motor deficits, and language impairments, Dup15q patients commonly meet the criteria for autism spectrum disorder (ASD) and have a high prevalence of seizures. Here, we explored mechanisms of hyperexcitability in neurons derived from induced pluripotent stem cell (iPSC) lines from Dup15q patients. Maturation of resting membrane potential in Dup15q-derived neurons was similar to neurons from unaffected control subjects, but Dup15q neurons had delayed action potential maturation and increased synaptic event frequency and amplitude. Dup15q neurons also showed impairments in activity-dependent synaptic plasticity and homeostatic synaptic scaling. Finally, Dup15q neurons showed an increased frequency of spontaneous action potential firing compared to control neurons, in part due to disruption of KCNQ2 channels.Together these data point to multiple mechanisms underlying hyperexcitability that may provide new targets for the treatment of seizures and other phenotypes associated with Dup15q.