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Translational research is needed to discover pharmacological targets and treatments for the diagnostic behavioral domains of neurodevelopmental disorders (NDDs), including autism spectrum disorders (ASDs) and intellectual disabilities (IDs). One NDD, associated with ASD and ID, is Angelman Syndrome (AS). AS is a rare genetic NDD for which there is currently no cure nor effective therapeutics. The genetic cause is known to be the loss of expression from the maternal allele of ubiquitin protein ligase E3A ( UBE3A ). The Ube3a maternal deletion mouse model of AS reliably demonstrates behavioral phenotypes of relevance to AS and therefore offers a suitable in vivo system in which to test potential therapeutics, with construct and face validity. Successes in reducing hyperexcitability and epileptogenesis have been reported in an AS model following acute treatment with lovastatin, an ERK inhibitor by reducing seizure threshold and percentage of mice exhibiting seizures. Since there has been literature reporting disruption of the ERK signaling pathway in AS, we chose to evaluate the effects of acute lovastatin administration in a tailored set of translationally relevant behavioral assays in a mouse model of AS. Unexpectedly, deleterious effects of sedation were observed in wildtype (WT), age matched littermate control mice and despite a baseline hypolocomotive phenotype in AS mice, even further reductions in exploratory activity, were observed post-acute lovastatin treatment. Limitations of this work include that chronic lower dose regimens, more akin to drug administration in humans were beyond the scope of this work, and may have produced a more favorable impact of lovastatin administration over single acute high doses. In addition, lovastatin’s effects were not assessed in younger subjects, since our study focused exclusively on adult functional outcomes. Metrics of gait, as well as motor coordination and motor learning in rotarod, previously observed to be impaired in AS mice, were not improved by lovastatin treatment. Finally, cognition by novel object recognition task was worsened in WT controls and not improved in AS, following lovastatin administration. In conclusion, lovastatin did not indicate any major improvement to AS symptoms, and in fact, worsened behavioral outcomes in the WT control groups. Therefore, despite its attractive low toxicity, immediate availability, and low cost of the drug, further investigation for clinical study is unwarranted given the results presented herein.

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 (ID), motor impairments, and epilepsy. Over the past two decades, there have been numerous discoveries indicating 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 that was 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 that primary neurons from Syngap1 +/− mice displayed: 1) increased network firing activity, 2) greater bursts, 3) and shorter inter-burst intervals between peaks, by utilizing 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.

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 Na V 1.1 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 Na V 1.1 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.

Mature neocortical pyramidal cells functionally express two sodium channel (Na V ) isoforms: Na V 1.2 and Na V 1.6. These isoforms are differentially localized to pyramidal cell compartments, and as such are thought to contribute to different aspects of neuronal excitability. But determining their precise roles in pyramidal cell excitability has been hampered by a lack of tools that allow for selective, acute block of each isoform individually. Here, we leveraged aryl sulfonamide-based molecule (ASC) inhibitors of Na V channels that exhibit state-dependent block of both Na V 1.2 and Na V 1.6, along with knock-in mice with changes in Na V 1.2 or Na V 1.6 structure that prevents ASC binding. This allowed for acute, potent, and reversible block of individual isoforms that permitted dissection of the unique contributions of Na V 1.2 and Na V 1.6 in pyramidal cell excitability. Remarkably, block of each isoform had contrasting—and in some situations, opposing—effects on neuronal action potential output, with Na V 1.6 block decreasing and Na V 1.2 block increasing output. Thus, Na V isoforms have unique roles in regulating different aspects of pyramidal cell excitability, and our work may help guide the development of therapeutics designed to temper hyperexcitability through selective Na V isoform blockade.

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.

The human APOBEC superfamily consists of eleven cytidine deaminase enzymes. Among them, APOBEC3 enzymes play a dual role in antiviral immunity and cancer development. APOBEC3 enzymes, including APOBEC3A (A3A) and APOBEC3B (A3B), induce mutations in viral DNA, effectively inhibiting viral replication but also promoting somatic mutations in the host genome, contributing to cancer development. A3A and A3B are linked to mutational signatures in over 50% of human cancers, with A3A being a potent mutagen. A3B, one of the first APOBEC3 enzymes linked to carcinogenesis, plays a significant role in HPV-associated cancers by driving somatic mutagenesis and tumor progression. The A3A_B deletion polymorphism results in a hybrid A3A_B gene, leading to increased A3A expression and enhanced mutagenic potential. Such polymorphism has been linked to an elevated risk of certain cancers, particularly in populations where it is more prevalent. This review explores the molecular mechanisms of APOBEC3 proteins, highlighting their dual roles in antiviral defense and tumorigenesis. We also discuss the clinical implications of genetic variants, such as the A3A_B polymorphism, mainly in HPV infection and associated cancers, providing a comprehensive understanding of their contributions to both viral restriction and cancer development.

Mature neocortical pyramidal cells functionally express two sodium channel (Na V ) isoforms: Na V 1.2 and Na V 1.6. These isoforms are differentially localized to pyramidal cell compartments, and as such are thought to contribute to different aspects of neuronal excitability. But determining their precise roles in pyramidal cell excitability has been hampered by a lack of tools that allow for selective, acute block of each isoform individually. Here, we leveraged aryl sulfonamide-based molecule (ASC) inhibitors of Na V channels that exhibit state-dependent block of both Na V 1.2 and Na V 1.6, along with knock-in mice with changes in Na V 1.2 or Na V 1.6 structure that prevents ASC binding. This allowed for acute, potent, and reversible block of individual isoforms that permitted dissection of the unique contributions of Na V 1.2 and Na V 1.6 in pyramidal cell excitability. Remarkably, block of each isoform had contrasting—and in some situations, opposing—effects on neuronal action potential output, with Na V 1.6 block decreasing and Na V 1.2 block increasing output. Thus, Na V isoforms have unique roles in regulating different aspects of pyramidal cell excitability, and our work may help guide the development of therapeutics designed to temper hyperexcitability through selective Na V isoform blockade.

Neurodevelopmental disorders (NDDs) are a large group of profound, debilitating, and lifelong conditions that impact the normal development of the central nervous system. Autism spectrum disorders, developmental delay, intellectual disability, and seizure disorders are among the clinical presentations of NDDs. Although a large population of patients live with the devastating symptoms of NDDs, no corrective therapies are currently available, stemming from the complexity of etiologies and broad range of conditions. To broaden our understanding of the mechanisms that contribute to NDDs and advance the discovery and development of therapeutics, we used preclinical mouse models to establish clinically relevant outcome measures and identify potential drug candidates. Chapter 1 introduces several strategies that address the challenges of developing therapeutics for NDDs and provides rationale for studies detailed in subsequent chapters. Chapters 2 and 3 investigate one approach proposed in the introduction: drug repurposing. Following earlier successes of repurposed drugs for other NDDs, these chapters focus on preclinical evaluation of several potential small molecule therapeutics for treating Angelman Syndrome (AS); a disorder caused by the absence of the protein UBE3A in the brain. We performed comprehensive behavioral pharmacology batteries, tailored to AS, using an AS mouse model and wildtype littermates. We treated both genotypes with one of three separate compounds, paxilline, LB-100, and lovastatin, compared to vehicle. We found that paxilline and LB-100 did not exert positive rescue effects in the motor or cognitive behavioral domains. However, lovastatin treatment of AS mice exhibited an improvement to motor ability when we assessed gait using an automated treadmill system, providing promising evidence that this treatment could have potential in the clinic. Chapter 4 examines in vitro and in vivo techniques for assessing potential drug candidates for the treatment of AS. We utilized the highly objective and translational touchscreen operant chamber task for assessing cognition and the whole-body plethysmography (WBP) task for assessing pulmonary physiology and applied these methods to the AS mouse model, compared to WT, sex-matched littermates. To better inform our future behavioral pharmacology experiments, we established two in vitro assays to assess the structure and electrophysiological properties of primary neurons derived from the AS mouse model. Chapter 5 applies the lessons we learned from our research in Angelman Syndrome to another NDD, SYNGAP1-related intellectual disability (SYNGAP1-ID). SYNGAP1-ID is a disorder caused by the reduction of SynGAP1 protein in the brain, resulting in symptoms such as intellectual disability, hyperactivity, and epilepsy. We performed comprehensive behavioral characterization of a Syngap1 mutant mouse model to understand the clinically relevant phenotypes and to identify translational biomarkers. Hyperactivity, increased electroencephalographic (EEG) signal and disruptions to sleep were observed in the mutant mouse, offering clinically relevant targets for future preclinical therapeutic testing. In addition, we utilized the in vitro electrophysiological assay of primary neurons established in Chapter 4 to confirm similar hyperexcitability in both cultured neurons and recordings directly from the surface of the brain of the Syngap1 mouse model, bridging the gap between in vitro and in vivo assays of neuronal function. Taken together, this body of research increases our understanding of the complex nature of NDDs and highlights the progress toward improving preclinical biomarkers to aid in the discovery and development of therapeutics for NDDs.

Mature neocortical pyramidal cells functionally express two sodium channel (Na ) isoforms: Na 1.2 and Na 1.6. These isoforms are differentially localized to pyramidal cell compartments, and as such are thought to contribute to different aspects of neuronal excitability. But determining their precise roles in pyramidal cell excitability has been hampered by a lack of tools that allow for selective, acute block of each isoform individually. Here, we leveraged aryl sulfonamide-based molecule (ASC) inhibitors of Na channels that exhibit state-dependent block of both Na 1.2 and Na 1.6, along with knock-in mice with changes in Na 1.2 or Na 1.6 structure that prevents ASC binding. This allowed for acute, potent, and reversible block of individual isoforms that permitted dissection of the unique contributions of Na 1.2 and Na 1.6 in pyramidal cell excitability. Remarkably, block of each isoform had contrasting-and in some situations, opposing-effects on neuronal action potential output, with Na 1.6 block decreasing and Na 1.2 block increasing output. Thus, Na isoforms have unique roles in regulating different aspects of pyramidal cell excitability, and our work may help guide the development of therapeutics designed to temper hyperexcitability through selective Na isoform blockade.

Background Mutations in the SCN8A gene, encoding the voltage-gated sodium channel NaV1.6, lead to various neurodevelopmental disorders. The SCN8A p.(Gly1625Arg) mutation (Nav1.6G1625R) was identified in a patient diagnosed with developmental epileptic encephalopathy (DEE), presenting with moderate epilepsy and severe developmental delay. Methods We performed biophysical and neurophysiological characterizations of Nav1.6G1625R in Neuro-2a cells and cultured hippocampal neurons, followed by computational modeling to determine the impact of its heterozygous expression on cortical neuron function. Findings Voltage-clamp analyses of Nav1.6G1625R demonstrated a heterogeneous mixture of gain-and loss-of-function properties, including reduced current amplitudes, a marked increase in the time constant of fast voltage-dependent inactivation and a depolarizing shift in the voltage dependence of inactivation. Recordings in transfected cultured neurons showed that these intricate biophysical properties had a minor effect on neuronal excitability when firing relayed on both endogenous and transfected NaV channels. Conversely, there was a marked reduction in the number of action potentials when firing was driven by the transfected mutant Nav1.6 channels. Computational modeling of mature cortical neurons further revealed a mild reduction in neuronal firing when mimicking the patients heterozygous Nav1.6G1625R expression. Structural modeling of Nav1.6G1625R and a double-mutant cycle analysis suggested the possible formation of pathophysiologically-relevant cation-p interaction between R1625 and F1588, affecting the voltage dependence of inactivation. Interpretation Our analyses demonstrate a complex combination of gain and loss-of-function changes resulting in an overall mild reduction in neuronal firing, related to a perturbed interaction network within the voltage sensor domain.Competing Interest StatementThe authors have declared no competing interest.Footnotes* we revised the title, reformated the abstract, and corrected several typos in the figures