Hyperexcitability and translational phenotypes in a preclinical mouse model of SYNGAP1-Related Intellectual Disability.

Disruption of SYNGAP1 directly causes a genetically identifiable neurodevelopmental disorder (NDD) called SYNGAP1-related intellectual disability (SRID). Without functional SynGAP1 protein, individuals are developmentally delayed and have prominent features of intellectual disability, motor impairments, and epilepsy. Over the past two decades, there have been numerous discoveries indicting the critical role of Syngap1. Several rodent models with a loss of Syngap1 have been engineered identifying precise roles in neuronal structure and function, as well as key biochemical pathways key for synapse integrity. Homozygous loss of SYNGAP1/Syngap1 is lethal. Heterozygous mutations of Syngap1 result in a broad range of behavioral phenotypes. Our in vivo functional data, using the original mouse model from the Huganir laboratory, corroborated behaviors including robust hyperactivity and deficits in learning and memory in young adults. Furthermore, we described impairments in the domain of sleep, characterized using neurophysiological data collected with wireless, telemetric electroencephalography (EEG). Syngap1+/- mice exhibited elevated spiking events and spike trains, in addition to elevated power, most notably in the delta power frequency. For the first time, we illustrated primary neurons from Syngap1+/- mice displayed increased network firing activity, greater bursts, and shorter inter-burst intervals between peaks by employing high density microelectrode arrays (HD-MEA). Our work bridges in-vitro electrophysiological neuronal activity and function with in vivo neurophysiological brain activity and function. These data elucidate quantitative, translational biomarkers in vivo and in vitro that can be utilized for the development and efficacy assessment of targeted treatments for SRID.

Complex biophysical changes and reduced neuronal firing in an SCN8A variant associated with developmental delay and epilepsy.

Mutations in the SCN8A gene, encoding the voltage-gated sodium channel NaV1.6, are associated with a range of neurodevelopmental syndromes. The p.(Gly1625Arg) (G1625R) mutation was identified in a patient diagnosed with developmental epileptic encephalopathy (DEE). While most of the characterized DEE-associated SCN8A mutations were shown to cause a gain-of-channel function, we show that the G1625R variant, positioned within the S4 segment of domain IV, results in complex effects. Voltage-clamp analyses of NaV1.6G1625R demonstrated a mixture of gain- and loss-of-function properties, including reduced current amplitudes, increased time constant of fast voltage-dependent inactivation, a depolarizing shift in the voltage dependence of activation and inactivation, and increased channel availability with high-frequency repeated depolarization. Current-clamp analyses in transfected cultured neurons revealed that these biophysical properties caused a marked reduction in the number of action potentials when firing was driven by the transfected mutant NaV1.6. Accordingly, computational modeling of mature cortical neurons demonstrated a mild decrease in neuronal firing when mimicking the patients' heterozygous SCN8A expression. Structural modeling of NaV1.6G1625R suggested the formation of a cation-π interaction between R1625 and F1588 within domain IV. Double-mutant cycle analysis revealed that this interaction affects the voltage dependence of inactivation in NaV1.6G1625R. Together, our studies demonstrate that the G1625R variant leads to a complex combination of gain and loss of function biophysical changes that result in an overall mild reduction in neuronal firing, related to the perturbed interaction network within the voltage sensor domain, necessitating personalized multi-tiered analysis for SCN8A mutations for optimal treatment selection.

Hyperexcitability and translational phenotypes in a preclinical model of SYNGAP1 mutations.

SYNGAP1 is a critical gene for neuronal development, synaptic structure, and function. Although rare, the disruption of SYNGAP1 directly causes a genetically identifiable neurodevelopmental disorder (NDD) called SYNGAP1 -related intellectual disability. Without functional SynGAP1 protein, patients present with intellectual disability, motor impairments, and epilepsy. Previous work using mouse models with a variety of germline and conditional mutations has helped delineate SynGAP1's critical roles in neuronal structure and function, as well as key biochemical signaling pathways essential to synapse integrity. Homozygous loss of SYNGAP1 is embryonically lethal. Heterozygous mutations of SynGAP1 result in a broad range of phenotypes including increased locomotor activity, impaired working spatial memory, impaired cued fear memory, and increased stereotypic behavior. Our in vivo functional data, using the original germline mutation mouse line from the Huganir laboratory, corroborated robust hyperactivity and learning and memory deficits. Here, we describe impairments in the translational biomarker domain of sleep, characterized using neurophysiological data collected with wireless telemetric electroencephalography (EEG). We discovered Syngap1+/- mice exhibited elevated spike trains in both number and duration, in addition to elevated power, most notably in the delta power band. Primary neurons from Syngap1+/- mice displayed increased network firing activity, greater spikes per burst, and shorter inter-burst intervals between peaks using high density micro-electrode arrays (HD-MEA). This work is translational, innovative, and highly significant as it outlines functional impairments in Syngap1 mutant mice. Simultaneously, the work utilized untethered, wireless neurophysiology that can discover potential biomarkers of Syngap1 RI-D, for clinical trials, as it has done with other NDDs. Our work is substantial forward progress toward translational work for SynGAP1R-ID as it bridges in-vitro electrophysiological neuronal activity and function with in vivo neurophysiological brain activity and function. These data elucidate multiple quantitative, translational biomarkers in vivo and in vitro for the development of treatments for SYNGAP1-related intellectual disability.

Hyperexcitability and translational phenotypes in a preclinical model of SYNGAP1 mutations.

SYNGAP1 is a critical gene for neuronal development, synaptic structure, and function. Although rare, the disruption of SYNGAP1 directly causes a genetically identifiable neurodevelopmental disorder (NDD) called SYNGAP1-related intellectual disability. Without functional SynGAP1 protein, patients present with intellectual disability, motor impairments, and epilepsy. Previous work using mouse models with a variety of germline and conditional mutations has helped delineate SynGAP1's critical roles in neuronal structure and function, as well as key biochemical signaling pathways essential to synapse integrity. Homozygous loss of SYNGAP1 is embryonically lethal. Heterozygous mutations of SynGAP1 result in a broad range of phenotypes including increased locomotor activity, impaired working spatial memory, impaired cued fear memory, and increased stereotypic behavior. Our in vivo functional data, using the original germline mutation mouse line from the Huganir laboratory, corroborated robust hyperactivity and learning and memory deficits. Here, we describe impairments in the translational biomarker domain of sleep, characterized using neurophysiological data collected with wireless telemetric electroencephalography (EEG). We discovered Syngap1 +/- mice exhibited elevated spike trains in both number and duration, in addition to elevated power, most notably in the delta power band. Primary neurons from Syngap1 +/- mice displayed increased network firing activity, greater spikes per burst, and shorter inter-burst intervals between peaks using high density micro-electrode arrays (HD-MEA). This work is translational, innovative, and highly significant as it outlines functional impairments in Syngap1 mutant mice. Simultaneously, the work utilized untethered, wireless neurophysiology that can discover potential biomarkers of Syngap1R-ID, for clinical trials, as it has done with other NDDs. Our work is substantial forward progress toward translational work for SynGAP1R-ID as it bridges in-vitro electrophysiological neuronal activity and function with in vivo neurophysiological brain activity and function. These data elucidate multiple quantitative, translational biomarkers in vivo and in vitro for the development of treatments for SYNGAP1-related intellectual disability.

Transcriptional reprogramming restores UBE3A brain-wide and rescues behavioral phenotypes in an Angelman syndrome mouse model.

Angelman syndrome (AS) is a neurogenetic disorder caused by the loss of ubiquitin ligase E3A (UBE3A) gene expression in the brain. The UBE3A gene is paternally imprinted in brain neurons. Clinical features of AS are primarily due to the loss of maternally expressed UBE3A in the brain. A healthy copy of paternal UBE3A is present in the brain but is silenced by a long non-coding antisense transcript (UBE3A-ATS). Here, we demonstrate that an artificial transcription factor (ATF-S1K) can silence Ube3a-ATS in an adult mouse model of Angelman syndrome (AS) and restore endogenous physiological expression of paternal Ube3a. A single injection of adeno-associated virus (AAV) expressing ATF-S1K (AAV-S1K) into the tail vein enabled whole-brain transduction and restored UBE3A protein in neurons to ∼25% of wild-type protein. The ATF-S1K treatment was highly specific to the target site with no detectable inflammatory response 5 weeks after AAV-S1K administration. AAV-S1K treatment of AS mice showed behavioral rescue in exploratory locomotion, a task involving gross and fine motor abilities, similar to low ambulation and velocity in AS patients. The specificity and tolerability of a single injection of AAV-S1K therapy for AS demonstrate the use of ATFs as a promising translational approach for AS.

Touchscreen cognitive deficits, hyperexcitability and hyperactivity in males and females using two models of Cdkl5 deficiency.

Many neurodevelopmental disorders (NDDs) are the result of mutations on the X chromosome. One severe NDD resulting from mutations on the X chromosome is CDKL5 deficiency disorder (CDD). CDD is an epigenetic, X-linked NDD characterized by intellectual disability (ID), pervasive seizures and severe sleep disruption, including recurring hospitalizations. CDD occurs at a 4:1 ratio, with a female bias. CDD is driven by the loss of cyclin-dependent kinase-like 5 (CDKL5), a serine/threonine kinase that is essential for typical brain development, synapse formation and signal transmission. Previous studies focused on male subjects from animal models, likely to avoid the complexity of X mosaicism. For the first time, we report translationally relevant behavioral phenotypes in young adult (8-20 weeks) females and males with robust signal size, including impairments in learning and memory, substantial hyperactivity and increased susceptibility to seizures/reduced seizure thresholds, in both sexes, and in two models of CDD preclinical mice, one with a general loss-of-function mutation and one that is a patient-derived mutation.

Gait as a quantitative translational outcome measure in Angelman syndrome.

Angelman syndrome (AS) is a genetic neurodevelopmental disorder characterized by developmental delay, lack of speech, seizures, intellectual disability, hypotonia, and motor coordination deficits. Motor abilities are an important outcome measure in AS as they comprise a broad repertoire of metrics including ataxia, hypotonia, delayed ambulation, crouched gait, and poor posture, and motor dysfunction affects nearly every individual with AS. Guided by collaborative work with AS clinicians studying gait, the goal of this study was to perform an in-depth gait analysis using the automated treadmill assay, DigiGait. Our hypothesis is that gait presents a strong opportunity for a reliable, quantitative, and translational metric that can serve to evaluate novel pharmacological, dietary, and genetic therapies. In this study, we used an automated gait analysis system, in addition to standard motor behavioral assays, to evaluate components of motor, exploration, coordination, balance, and gait impairments across the lifespan in an AS mouse model. Our study demonstrated marked global motoric deficits in AS mice, corroborating previous reports. Uniquely, this is the first report of nuanced aberrations in quantitative spatial and temporal components of gait in AS mice compared to sex- and age-matched wildtype littermates followed longitudinally using metrics that are analogous in AS individuals. Our findings contribute evidence toward the use of nuanced motor outcomes (i.e., gait) as valuable and translationally powerful metrics for therapeutic development for AS, as well as other genetic neurodevelopmental syndromes. LAY SUMMARY: Movement disorders affect nearly every individual with Angelman Syndrome (AS). The most common motor problems include spasticity, ataxia of gait (observed in the majority of ambulatory individuals), tremor, and muscle weakness. This report focused on quantifying various spatial and temporal aspects of gait as a reliable, translatable outcome measure in a preclinical AS model longitudinally across development. By increasing the number of translational, reliable, functional outcome measures in our wheelhouse, we will create more opportunities for identifying and advancing successful medical interventions.

Excessive Laughter-like Vocalizations, Microcephaly, and Translational Outcomes in the Ube3a Deletion Rat Model of Angelman Syndrome.

Angelman syndrome (AS) is a rare genetic neurodevelopmental disorder characterized by intellectual disabilities, motor and balance deficits, impaired communication, and a happy, excitable demeanor with frequent laughter. We sought to elucidate a preclinical outcome measure in male and female rats that addressed communication abnormalities of AS and other neurodevelopmental disorders in which communication is atypical and/or lack of speech is a core feature. We discovered, and herein report for the first time, excessive laughter-like 50 kHz ultrasonic emissions in the Ube3amat-/pat+ rat model of AS, which suggests an excitable, playful demeanor and elevated positive affect, similar to the demeanor of individuals with AS. Also in line with the AS phenotype, Ube3amat-/pat+ rats demonstrated aberrant social interactions with a novel partner, distinctive gait abnormalities, impaired cognition, an underlying LTP deficit, and profound reductions in brain volume. These unique, robust phenotypes provide advantages compared with currently available mouse models and will be highly valuable as outcome measures in the evaluation of therapies for AS.SIGNIFICANCE STATEMENT Angelman syndrome (AS) is a severe neurogenetic disorder for which there is no cure, despite decades of research using mouse models. This study used a recently developed rat model of AS to delineate disease-relevant outcome measures to facilitate therapeutic development. We found the rat to be a strong model of AS, offering several advantages over mouse models by exhibiting numerous AS-relevant phenotypes, including overabundant laughter-like vocalizations, reduced hippocampal LTP, and volumetric anomalies across the brain. These findings are unconfounded by detrimental motor abilities and background strain, issues plaguing mouse models. This rat model represents an important advancement in the field of AS, and the outcome metrics reported herein will be central to the therapeutic pipeline.

Deletion of a non-canonical regulatory sequence causes loss of Scn1a expression and epileptic phenotypes in mice.

BACKGROUND: Genes with multiple co-active promoters appear common in brain, yet little is known about functional requirements for these potentially redundant genomic regulatory elements. SCN1A, which encodes the NaV1.1 sodium channel alpha subunit, is one such gene with two co-active promoters. Mutations in SCN1A are associated with epilepsy, including Dravet syndrome (DS). The majority of DS patients harbor coding mutations causing SCN1A haploinsufficiency; however, putative causal non-coding promoter mutations have been identified. METHODS: To determine the functional role of one of these potentially redundant Scn1a promoters, we focused on the non-coding Scn1a 1b regulatory region, previously described as a non-canonical alternative transcriptional start site. We generated a transgenic mouse line with deletion of the extended evolutionarily conserved 1b non-coding interval and characterized changes in gene and protein expression, and assessed seizure activity and alterations in behavior. RESULTS: Mice harboring a deletion of the 1b non-coding interval exhibited surprisingly severe reductions of Scn1a and NaV1.1 expression throughout the brain. This was accompanied by electroencephalographic and thermal-evoked seizures, and behavioral deficits. CONCLUSIONS: This work contributes to functional dissection of the regulatory wiring of a major epilepsy risk gene, SCN1A. We identified the 1b region as a critical disease-relevant regulatory element and provide evidence that non-canonical and seemingly redundant promoters can have essential function.

Cyclin D2-knock-out mice with attenuated dentate gyrus neurogenesis have robust deficits in long-term memory formation.

Neurobehavioral studies have produced contradictory findings concerning the function of neurogenesis in the adult dentate gyrus. Previous studies have proved inconsistent across several behavioral endpoints thought to be dependent on dentate neurogenesis, including memory acquisition, short-term and long-term retention of memory, pattern separation, and reversal learning. We hypothesized that the main function of dentate neurogenesis is long-term memory formation because we assumed that a newly formed and integrated neuron would have a long-term impact on the local neural network. We used a cyclin D2-knock-out (cyclin D2-/-) mouse model of endogenously deficient dentate neurogenesis to test this hypothesis. We found that cyclin D2-/- mice had robust and sustained loss of long-term memory in two separate behavioral tasks, Morris water maze (MWM) and touchscreen intermediate pattern separation. Moreover, after adjusting for differences in brain volumes determined by magnetic resonance (MR) imaging, reduced dentate neurogenesis moderately correlated with deficits in memory retention after 24 hours. Importantly, cyclin D2-/- mice did not show deficits in learning acquisition in a touchscreen paradigm of intermediate pattern separation or MWM platform location, indicating intact short-term memory. Further evaluation of cyclin D2-/- mice is necessary to confirm that deficits are specifically linked to dentate gyrus neurogenesis since cyclin D2-/- mice also have a reduced size of the olfactory bulb, hippocampus, cerebellum and cortex besides reduced dentate gyrus neurogenesis.

Large-Scale Profiling of Kinase Dependencies in Cancer Cell Lines.

One approach to identifying cancer-specific vulnerabilities and therapeutic targets is to profile genetic dependencies in cancer cell lines. Here, we describe data from a series of siRNA screens that identify the kinase genetic dependencies in 117 cancer cell lines from ten cancer types. By integrating the siRNA screen data with molecular profiling data, including exome sequencing data, we show how vulnerabilities/genetic dependencies that are associated with mutations in specific cancer driver genes can be identified. By integrating additional data sets into this analysis, including protein-protein interaction data, we also demonstrate that the genetic dependencies associated with many cancer driver genes form dense connections on functional interaction networks. We demonstrate the utility of this resource by using it to predict the drug sensitivity of genetically or histologically defined subsets of tumor cell lines, including an increased sensitivity of osteosarcoma cell lines to FGFR inhibitors and SMAD4 mutant tumor cells to mitotic inhibitors.

Promoter element arising from the fusion of standard BioBrick parts.

We characterize the appearance of a constitutive promoter element in the commonly used cI repressor-encoding BioBrick BBa_C0051. We have termed this promoter element pKAT. Full pKAT activity is created by the ordered assembly of sequences in BBa_C0051 downstream of the cI gene encoding the 11 amino acid LVA proteolytic degradation tag, a BioBrick standard double-TAA stop codon, a genetic barcode, and part of the RFC10 SpeI-XbaI BioBrick scar. Placing BBa_C0051 or other pKAT containing parts upstream of other functional RNA coding elements in a polycistronic context may therefore lead to the unintended transcription of the downstream elements. The frequent reuse of pKAT or pKAT-like containing basic parts in the Registry of Biological Parts has resulted in approximately 5% of registry parts encoding at least one instance of a predicted pKAT promoter located directly upstream of a ribosome binding site and ATG start codon. This example highlights that even seemingly simple modifications of a part's sequence (in this case addition of degradation tags and barcodes) may be sufficient to unexpectedly change the contextual behavior of a part and reaffirms the inherent challenge in carefully characterizing the behavior of standardized biological parts across a broad range of reasonable use scenarios.

Receptor association and tyrosine phosphorylation of S6 kinases.

Ribosomal protein S6 kinase (S6K) is activated by an array of mitogenic stimuli and is a key player in the regulation of cell growth. The activation process of S6 kinase involves a complex and sequential series of multiple Ser/Thr phosphorylations and is mainly mediated via phosphatidylinositol 3-kinase (PI3K)-3-phosphoinositide-dependent protein kinase-1 (PDK1) and mTor-dependent pathways. Upstream regulators of S6K, such as PDK1 and protein kinase B (PKB/Akt), are recruited to the membrane via their pleckstrin homology (PH) or protein-protein interaction domains. However, the mechanism of integration of S6K into a multi-enzyme complex around activated receptor tyrosine kinases is not clear. In the present study, we describe a specific interaction between S6K with receptor tyrosine kinases, such as platelet-derived growth factor receptor (PDGFR). The interaction with PDGFR is mediated via the kinase or the kinase extension domain of S6K. Complex formation is inducible by growth factors and leads to S6K tyrosine phosphorylation. Using PDGFR mutants, we have shown that the phosphorylation is exerted via a PDGFR-src pathway. Furthermore, src kinase phosphorylates and coimmunoprecipitates with S6K in vivo. Inhibitors towards tyrosine kinases, such as genistein and PP1, or src-specific SU6656, but not PI3K and mTor inhibitors, lead to a reduction in tyrosine phosphorylation of S6K. In addition, we mapped the sites of tyrosine phosphorylation in S6K1 and S6K2 to Y39 and Y45, respectively. Mutational and immunofluorescent analysis indicated that phosphorylation of S6Ks at these sites does not affect their activity or subcellular localization. Our data indicate that S6 kinase is recruited into a complex with RTKs and src and becomes phosphorylated on tyrosine/s in response to PDGF or serum.

Protein kinase C phosphorylates ribosomal protein S6 kinase betaII and regulates its subcellular localization.

The ribosomal protein S6 kinase (S6K) belongs to the AGC family of Ser/Thr kinases and is known to be involved in the regulation of protein synthesis and the G(1)/S transition of the cell cycle. There are two forms of S6K, termed S6Kalpha and S6Kbeta, which have cytoplasmic and nuclear splice variants. Nucleocytoplasmic shuttling has been recently proposed for S6Kalpha, based on the use of the nuclear export inhibitor, leptomycin B. However, the molecular mechanisms regulating subcellular localization of S6Ks in response to mitogenic stimuli remain to be elucidated. Here we present data on the in vitro and in vivo phosphorylation of S6Kbeta, but not S6Kalpha, by protein kinase C (PKC). The site of phosphorylation was identified as S486, which is located within the C-terminal nuclear localization signal. Mutational analysis and the use of phosphospecific antibodies provided evidence that PKC-mediated phosphorylation at S486 does not affect S6K activity but eliminates the function of its nuclear localization signal and causes retention of an activated form of the kinase in the cytoplasm. Taken together, this study uncovers a novel mechanism for the regulation of nucleocytoplasmic shuttling of S6KbetaII by PKC-mediated phosphorylation.