Vagal sensory neurons mediate the Bezold-Jarisch reflex and induce syncope.

Visceral sensory pathways mediate homeostatic reflexes, the dysfunction of which leads to many neurological disorders1. The Bezold-Jarisch reflex (BJR), first described2,3 in 1867, is a cardioinhibitory reflex that is speculated to be mediated by vagal sensory neurons (VSNs) that also triggers syncope. However, the molecular identity, anatomical organization, physiological characteristics and behavioural influence of cardiac VSNs remain mostly unknown. Here we leveraged single-cell RNA-sequencing data and HYBRiD tissue clearing4 to show that VSNs that express neuropeptide Y receptor Y2 (NPY2R) predominately connect the heart ventricular wall to the area postrema. Optogenetic activation of NPY2R VSNs elicits the classic triad of BJR responses-hypotension, bradycardia and suppressed respiration-and causes an animal to faint. Photostimulation during high-resolution echocardiography and laser Doppler flowmetry with behavioural observation revealed a range of phenotypes reflected in clinical syncope, including reduced cardiac output, cerebral hypoperfusion, pupil dilation and eye-roll. Large-scale Neuropixels brain recordings and machine-learning-based modelling showed that this manipulation causes the suppression of activity across a large distributed neuronal population that is not explained by changes in spontaneous behavioural movements. Additionally, bidirectional manipulation of the periventricular zone had a push-pull effect, with inhibition leading to longer syncope periods and activation inducing arousal. Finally, ablating NPY2R VSNs specifically abolished the BJR. Combined, these results demonstrate a genetically defined cardiac reflex that recapitulates characteristics of human syncope at physiological, behavioural and neural network levels.

Sensorimotor Integration Supporting Perception Requires Syngap1 Expression in Cortex.

Perception, a cognitive construct, emerges through sensorimotor integration (SMI). The molecular and cellular mechanisms that shape SMI within circuits that promote cognition are poorly understood. Here, we demonstrate that expression of the autism/intellectual disability gene, Syngap1, in mouse cortical excitatory neurons promotes touch sensitivity required to elicit perceptual behaviors. Cortical Syngap1 expression enabled touch-induced feedback signals within sensorimotor loops by assembling circuits that support tactile sensitivity. These circuits also encoded correlates of attention that promoted self-generated whisker movements underlying purposeful and sustained object exploration. As Syngap1 deficient animals explored objects with whiskers, relatively weak touch signals were integrated with relatively strong motor signals. This produced a signal-to-noise deficit consistent with impaired tactile sensitivity, reduced tactile exploration, and weak tactile learning. Thus, Syngap1 expression in cortex promotes tactile perception by assembling circuits that integrate touch and whisker motor signals. Deficient Syngap1 expression likely contributes to cognitive impairment through abnormal top-down SMI.

Syngap1 regulates experience-dependent cortical ensemble plasticity by promoting in vivo excitatory synapse strengthening.

A significant proportion of autism risk genes regulate synapse function, including plasticity, which is believed to contribute to behavioral abnormalities. However, it remains unclear how impaired synapse plasticity contributes to network-level processes linked to adaptive behaviors, such as experience-dependent ensemble plasticity. We found that Syngap1, a major autism risk gene, promoted measures of experience-dependent excitatory synapse strengthening in the mouse cortex, including spike-timing-dependent glutamatergic synaptic potentiation and presynaptic bouton formation. Synaptic depression and bouton elimination were normal in Syngap1 mice. Within cortical networks, Syngap1 promoted experience-dependent increases in somatic neural activity in weakly active neurons. In contrast, plastic changes to highly active neurons from the same ensemble that paradoxically weaken with experience were unaffected. Thus, experience-dependent excitatory synapse strengthening mediated by Syngap1 shapes neuron-specific plasticity within cortical ensembles. We propose that other genes regulate neuron-specific weakening within ensembles, and together, these processes function to redistribute activity within cortical networks during experience.

A simple and robust cell-based assay for the discovery of novel cytokinesis inhibitors.

Cytokinesis is the last step of mitotic cell division that separates the cytoplasm of dividing cells. Small molecule inhibitors targeting either the elements of the regulatory pathways controlling cytokinesis, or the terminal effectors have been of interest as potential drug candidates for the treatment of various diseases. Here we present a detailed protocol for a cell-based cytokinesis assay that can be used for the discovery of novel cytokinesis inhibitors. The assay is performed in a 96-well plate format in 48 h. Living cells, nuclei and nuclei of dead cells are identified by a single staining step using three fluorescent dyes, followed by rapid live cell imaging. The primary signal is the nuclei-to-cell ratio (NCR). In the presence of cytokinesis inhibitors, this ratio increases over time, as the ratio of multinucleated cells increases in the population. The ratio of dead nuclei to total nuclei provides a simultaneous measure of cytotoxicity. A screening window coefficient (Z`) of 0.65 indicates that the assay is suitable for screening purposes, as the positive and negative controls are well-separated. EC50 values can be reliably determined in a single 96-well plate by using only six different compound concentrations, enabling the testing of 4 compounds per plate. An excellent test-retest reliability (R 2 = 0.998) was found for EC50 values covering a ~1500-fold range of potencies. Established small molecule inhibitors of cytokinesis operating via direct action on actin dynamics or nonmuscle myosin II are used to demonstrate the robustness, simplicity and flexibility of the assay.

Re-expression of SynGAP protein in adulthood improves translatable measures of brain function and behavior.

It remains unclear to what extent neurodevelopmental disorder (NDD) risk genes retain functions into adulthood and how they may influence disease phenotypes. SYNGAP1 haploinsufficiency causes a severe NDD defined by autistic traits, cognitive impairment, and epilepsy. To determine if this gene retains therapeutically-relevant biological functions into adulthood, we performed a gene restoration technique in a mouse model for SYNGAP1 haploinsufficiency. Adult restoration of SynGAP protein improved behavioral and electrophysiological measures of memory and seizure. This included the elimination of interictal events that worsened during sleep. These events may be a biomarker for generalized cortical dysfunction in SYNGAP1 disorders because they also worsened during sleep in the human patient population. We conclude that SynGAP protein retains biological functions throughout adulthood and that non-developmental functions may contribute to disease phenotypes. Thus, treatments that target debilitating aspects of severe NDDs, such as medically-refractory seizures and cognitive impairment, may be effective in adult patients.

SYNGAP1 heterozygosity disrupts sensory processing by reducing touch-related activity within somatosensory cortex circuits.

In addition to cognitive impairments, neurodevelopmental disorders often result in sensory processing deficits. However, the biological mechanisms that underlie impaired sensory processing associated with neurodevelopmental disorders are generally understudied and poorly understood. We found that SYNGAP1 haploinsufficiency in humans, which causes a sporadic neurodevelopmental disorder defined by cognitive impairment, autistic features, and epilepsy, also leads to deficits in tactile-related sensory processing. In vivo neurophysiological analysis in Syngap1 mouse models revealed that upper-lamina neurons in somatosensory cortex weakly encode information related to touch. This was caused by reduced synaptic connectivity and impaired intrinsic excitability within upper-lamina somatosensory cortex neurons. These results were unexpected, given that Syngap1 heterozygosity is known to cause circuit hyperexcitability in brain areas more directly linked to cognitive functions. Thus, Syngap1 heterozygosity causes a range of circuit-specific pathologies, including reduced activity within cortical neurons required for touch processing, which may contribute to sensory phenotypes observed in patients.

Species-conserved SYNGAP1 phenotypes associated with neurodevelopmental disorders.

SYNGAP1 loss-of-function variants are causally associated with intellectual disability, severe epilepsy, autism spectrum disorder and schizophrenia. While there are hundreds of genetic risk factors for neurodevelopmental disorders (NDDs), this gene is somewhat unique because of the frequency and penetrance of loss-of-function variants found in patients combined with the range of brain disorders associated with SYNGAP1 pathogenicity. These clinical findings indicate that SYNGAP1 regulates fundamental neurodevelopmental processes that are necessary for brain development. Here, we describe four phenotypic domains that are controlled by Syngap1 expression across vertebrate species. Two domains, the maturation of cognitive functions and maintenance of excitatory-inhibitory balance, are defined exclusively through a review of the current literature. Two additional domains are defined by integrating the current literature with new data indicating that SYNGAP1/Syngap1 regulates innate survival behaviors and brain structure. These four phenotypic domains are commonly disrupted in NDDs, suggesting that a deeper understanding of developmental Syngap1 functions will be generalizable to other NDDs of known or unknown etiology. Therefore, we discuss the known molecular and cellular functions of Syngap1 and consider how these functions may contribute to the emergence of disease-relevant phenotypes. Finally, we identify major unexplored areas of Syngap1 neurobiology and discuss how a deeper understanding of this gene may uncover general principles of NDD pathobiology.

Improved Scalability of Neuron-Based Phenotypic Screening Assays for Therapeutic Discovery in Neuropsychiatric Disorders.

There is a pressing need to improve approaches for drug discovery related to neuropsychiatric disorders (NSDs). Therapeutic discovery in neuropsychiatric disorders would benefit from screening assays that can measure changes in complex phenotypes linked to disease mechanisms. However, traditional assays that track complex neuronal phenotypes, such as neuronal connectivity, exhibit poor scalability and are not compatible with high-throughput screening (HTS) procedures. Therefore, we created a neuronal phenotypic assay platform that focused on improving the scalability and affordability of neuron-based assays capable of tracking disease-relevant phenotypes. First, using inexpensive laboratory-level automation, we industrialized primary neuronal culture production, which enabled the creation of scalable assays within functioning neural networks. We then developed a panel of phenotypic assays based on culturing of primary neurons from genetically modified mice expressing HTS-compatible reporters that capture disease-relevant phenotypes. We demonstrated that a library of 1,280 compounds was quickly screened against both assays using only a few litters of mice in a typical academic laboratory setting. Finally, we implemented one assay in a fully automated high-throughput academic screening facility, illustrating the scalability of assays designed using this platform. These methodological improvements simplify the creation of highly scalable neuron-based phenotypic assays designed to improve drug discovery in CNS disorders.

An interactive framework for whole-brain maps at cellular resolution.

To deconstruct the architecture and function of brain circuits, it is necessary to generate maps of neuronal connectivity and activity on a whole-brain scale. New methods now enable large-scale mapping of the mouse brain at cellular and subcellular resolution. We developed a framework to automatically annotate, analyze, visualize and easily share whole-brain data at cellular resolution, based on a scale-invariant, interactive mouse brain atlas. This framework enables connectivity and mapping projects in individual laboratories and across imaging platforms, as well as multiplexed quantitative information on the molecular identity of single neurons. As a proof of concept, we generated a comparative connectivity map of five major neuron types in the corticostriatal circuit, as well as an activity-based map to identify hubs mediating the behavioral effects of cocaine. Thus, this computational framework provides the necessary tools to generate brain maps that integrate data from connectivity, neuron identity and function.

Publisher Correction: An interactive framework for whole-brain maps at cellular resolution.

In the version of this article initially published online, Daniel Fürth was not listed as a corresponding author. The error has been corrected in the print, PDF and HTML versions of this article.

Neuroepigenetic Regulation of Pathogenic Memories.

Our unique collection of memories determines our individuality and shapes our future interactions with the world. Remarkable advances into the neurobiological basis of memory have identified key epigenetic mechanisms that support the stability of memory. Various forms of epigenetic regulation at the levels of DNA methylation, histone modification, and non-coding RNAs (ncRNAs) can modulate transcriptional and translational events required for memory processes. By changing the cellular profile in the brain's emotional, reward, and memory circuits, these epigenetic modifications have also been linked to perseverant, pathogenic memories. In this review, we will delve into the relevance of epigenetic dysregulation to pathogenic memory mechanisms by focusing on two neuropsychiatric disorders perpetuated by aberrant memory associations: substance use disorder (SUD) and post-traumatic stress disorder (PTSD). As our understanding improves, neuroepigenetic mechanisms may someday be harnessed to develop novel therapeutic targets for the treatment of these chronic, relapsing disorders.

Syngap1 haploinsufficiency damages a postnatal critical period of pyramidal cell structural maturation linked to cortical circuit assembly.

BACKGROUND: Genetic haploinsufficiency of SYNGAP1/Syngap1 commonly occurs in developmental brain disorders, such as intellectual disability, epilepsy, schizophrenia, and autism spectrum disorder. Thus, studying mouse models of Syngap1 haploinsufficiency may uncover pathologic developmental processes common among distinct brain disorders. METHODS: A Syngap1 haploinsufficiency model was used to explore the relationship between critical period dendritic spine abnormalities, cortical circuit assembly, and the window for genetic rescue to understand how damaging mutations disrupt key substrates of mouse brain development. RESULTS: Syngap1 mutations broadly disrupted a developmentally sensitive period that corresponded to the period of heightened postnatal cortical synaptogenesis. Pathogenic Syngap1 mutations caused a coordinated acceleration of dendrite elongation and spine morphogenesis and pruning of these structures in neonatal cortical pyramidal neurons. These mutations also prevented a form of developmental structural plasticity associated with experience-dependent reorganization of brain circuits. Consistent with these findings, Syngap1 mutant mice displayed an altered pattern of long-distance synaptic inputs into a cortical area important for cognition. Interestingly, the ability to genetically improve the behavioral endophenotype of Syngap1 mice decreased slowly over postnatal development and mapped onto the developmental period of coordinated dendritic insults. CONCLUSIONS: Pathogenic Syngap1 mutations have a profound impact on the dynamics and structural integrity of pyramidal cell postsynaptic structures known to guide the de novo wiring of nascent cortical circuits. These findings support the idea that disrupted critical periods of dendritic growth and spine plasticity may be a common pathologic process in developmental brain disorders.

Methamphetamine-associated memory is regulated by a writer and an eraser of permissive histone methylation.

BACKGROUND: Memories associated with drugs of abuse, such as methamphetamine (METH), increase relapse vulnerability to substance use disorder by triggering craving. The nucleus accumbens (NAc) is essential to these drug-associated memories, but underlying mechanisms are poorly understood. Posttranslational chromatin modifications, such as histone methylation, modulate gene transcription; thus, we investigated the role of the associated epigenetic modifiers in METH-associated memory. METHODS: Conditioned place preference was used to assess the epigenetic landscape in the NAc supporting METH-associated memory (n = 79). The impact of histone methylation (H3K4me2/3) on the formation and expression of METH-associated memory was determined by focal, intra-NAc knockdown (KD) of a writer, the methyltransferase mixed-lineage leukemia 1 (Mll1) (n = 26), and an eraser, the histone lysine (K)-specific demethylase 5C (Kdm5c) (n = 38), of H3K4me2/3. RESULTS: A survey of chromatin modifications in the NAc of animals forming a METH-associated memory revealed the global induction of several modifications associated with active transcription. This correlated with a pattern of gene activation, as revealed by microarray analysis, including upregulation of oxytocin receptor (Oxtr) and FBJ osteosarcoma oncogene (Fos), the promoters of which also had increased H3K4me3. KD of Mll1 reduced H3K4me3, Fos and Oxtr levels and disrupted METH-associated memory. KD of Kdm5c resulted in hypermethylation of H3K4 and prevented the expression of METH-associated memory. CONCLUSIONS: The development and expression of METH-associated memory are supported by regulation of H3K4me2/3 levels by MLL1 and KDM5C, respectively, in the NAc. These data indicate that permissive histone methylation, and the associated epigenetic writers and erasers, represent potential targets for the treatment of substance abuse relapse, a psychiatric condition perpetuated by unwanted associative memories.

Histone acetyltransferase cofactor Trrap maintains self-renewal and restricts differentiation of embryonic stem cells.

Chromatin states are believed to play a key role in distinct patterns of gene expression essential for self-renewal and pluripotency of embryonic stem cells (ESCs); however, the genes governing the establishment and propagation of the chromatin signature characteristic of pluripotent cells are poorly understood. Here, we show that conditional deletion of the histone acetyltransferase cofactor Trrap in mouse ESCs triggers unscheduled differentiation associated with loss of histone acetylation, condensation of chromatin into distinct foci (heterochromatization), and uncoupling of H3K4 dimethylation and H3K27 trimethylation. Trrap loss results in downregulation of stemness master genes Nanog, Oct4, and Sox2 and marked upregulation of specific differentiation markers from the three germ layers. Chromatin immunoprecipitation-sequencing analysis of genome-wide binding revealed a significant overlap between Oct4 and Trrap binding in ESCs but not in differentiated mouse embryonic fibroblasts, further supporting a functional interaction between Trrap and Oct4 in the maintenance of stemness. Remarkably, failure to downregulate Trrap prevents differentiation of ESCs, suggesting that downregulation of Trrap may be a critical step guiding transcriptional reprogramming and differentiation of ESCs. These findings establish Trrap as a critical part of the mechanism that restricts differentiation and promotes the maintenance of key features of ESCs.

DNA methylation changes associated with risk factors in tumors of the upper aerodigestive tract.

Cancers of the upper aerodigestive tract (UADT) are common forms of malignancy associated with tobacco and alcohol exposures, although human papillomavirus and nutritional deficiency are also important risk factors. While somatically acquired DNA methylation changes have been associated with UADT cancers, what triggers these events and precise epigenetic targets are poorly understood. In this study, we applied quantitative profiling of DNA methylation states in a panel of cancer-associated genes to a case-control study of UADT cancers. Our analyses revealed a high frequency of aberrant hypermethylation of several genes, including MYOD1, CHRNA3 and MTHFR in UADT tumors, whereas CDKN2A was moderately hypermethylated. Among differentially methylated genes, we identified a new gene (the nicotinic acetycholine receptor gene) as target of aberrant hypermethylation in UADT cancers, suggesting that epigenetic deregulation of nicotinic acetycholine receptors in non-neuronal tissues may promote the development of UADT cancers. Importantly, we found that sex and age is strongly associated with the methylation states, whereas tobacco smoking and alcohol intake may also influence the methylation levels in specific genes. This study identifies aberrant DNA methylation patterns in UADT cancers and suggests a potential mechanism by which environmental factors may deregulate key cellular genes involved in tumor suppression and contribute to UADT cancers.

Inactivation of the putative suppressor gene DOK1 by promoter hypermethylation in primary human cancers.

The DOK1 gene is a putative tumour suppressor gene located on the human chromosome 2p13 which is frequently rearranged in leukaemia and other human tumours. We previously reported that the DOK1 gene can be mutated and its expression down-regulated in human malignancies. However, the mechanism underlying DOK1 silencing remains largely unknown. We show here that unscheduled silencing of DOK1 expression through aberrant hypermethylation is a frequent event in a variety of human malignancies. DOK1 was found to be silenced in nine head and neck cancer (HNC) cell lines studied and DOK1 CpG hypermethylation correlated with loss of gene expression in these cells. DOK1 expression could be restored via demethylating treatment using 5-aza-2'deoxycytidine. In addition, transduction of cancer cell lines with DOK1 impaired their proliferation, consistent with the critical role of epigenetic silencing of DOK1 in the development and maintenance of malignant cells. We further observed that DOK1 hypermethylation occurs frequently in a variety of primary human neoplasm including solid tumours (93% in HNC, 81% in lung cancer) and haematopoietic malignancy (64% in Burkitt's lymphoma). Control blood samples and exfoliated mouth epithelial cells from healthy individuals showed a low level of DOK1 methylation, suggesting that DOK1 hypermethylation is a tumour specific event. Finally, an inverse correlation was observed between the level of DOK1 gene methylation and its expression in tumour and adjacent non tumour tissues. Thus, hypermethylation of DOK1 is a potentially critical event in human carcinogenesis, and may be a potential cancer biomarker and an attractive target for epigenetic-based therapy.

Mammalian Ino80 mediates double-strand break repair through its role in DNA end strand resection.

Chromatin modifications/remodeling are important mechanisms by which cells regulate various functions through providing accessibility to chromatin DNA. Recent studies implicated INO80, a conserved chromatin-remodeling complex, in the process of DNA repair. However, the precise underlying mechanism by which this complex mediates repair in mammalian cells remains enigmatic. Here, we studied the effect of silencing of the Ino80 subunit of the complex on double-strand break repair in mammalian cells. Comet assay and homologous recombination repair reporter system analyses indicated that Ino80 is required for efficient double-strand break repair. Ino80 association with chromatin surrounding double-strand breaks suggested the direct involvement of INO80 in the repair process. Ino80 depletion impaired focal recruitment of 53BP1 but did not impede Rad51 focus formation, suggesting that Ino80 is required for the early steps of repair. Further analysis by using bromodeoxyuridine (BrdU)-labeled single-stranded DNA and replication protein A (RPA) immunofluorescent staining showed that INO80 mediates 5'-3' resection of double-strand break ends.

Epigenetic mechanisms and cancer: an interface between the environment and the genome.

Although epidemiological studies support the role of environment in a wide range of human cancers, the precise mechanisms by which environmental exposures promote cancer development and progression remain poorly understood. Environmental factors have been proposed to promote the development of malignancies by eliciting epigenetic changes; however, it is only with recent advances in epigenetics and epigenomics that target genes and the mechanisms underlying environmental influences are beginning to be elucidated. Because epigenetic mechanisms may function as an interface between environmental factors and the genome, deregulation of the epigenome by environmental stressors is likely to disrupt different cellular processes and contribute to cancer risk. In addition, the early appearance and ubiquity of epigenetic changes in virtually all steps of tumor development and progression in most, if not all, human neoplasms, make them attractive targets for biomarker discovery and targeted prevention. At the cellular level, aberrant epigenetic changes associated with environmental exposures may deregulate key cellular processes (including transcriptional control, DNA repair, cell cycle control, and carcinogen detoxification), which can be further modulated by environmental stressors, thus defining not only the phenotype of the disease but also potential biomarkers. This review summarizes recent progress in our understanding of the epigenetic mechanisms through which environmental factors may promote tumor development, with a particular focus on human lung cancer.

Intensity-dependent constitutional MLH1 promoter methylation leads to early onset of colorectal cancer by affecting both alleles.

Constitutional epimutation is one of the causes for MLH1 gene inactivation associated with hereditary non-polyposis colon cancer (HNPCC) syndrome. Here we investigate MLH1 promoter hypermethylation in 110 sporadic early-onset colorectal cancer patients. Variable levels of hypermethylation were detected in 55 patients (50%). Importantly a reduced MLH1 gene expression was found in patients with high-level methylation, with the association of microsatellite instability (MSI) in their tumor cells. Such high-level methylation accounts for 7.4% of all patients included in this study. Furthermore, we found that in one case constitutional methylation affected both alleles, indicating a post-zygotic methylation dysregulation. Our findings suggest that constitutional epimutation is a mechanism underlying early-onset colorectal cancer, although it is involved in only a small proportion of patients, who require appropriate surveillance. Our findings provide further insight into the role of aberrant constitutional methylation in colon carcinogenesis and raise the question of whether prevalent low-level methylation constitutes a potential risk factor for cancer development.

DNA methylation changes associated with cancer risk factors and blood levels of vitamin metabolites in a prospective study.

Aberrant DNA methylation is a major epigenetic mechanism of gene silencing in a wide range of human cancers. Previous studies on DNA methylation typically used paired tumor and normal-appearing surrounding tissues from cancer-bearing individuals. However, genomic DNA isolated from surrogate tissues such as blood cells represents an attractive material that can be exploited in the discovery of biomarkers of exposure and tumorigenesis. Here we examined the association between lung cancer and DNA methylation patterns in a panel of candidate genes. We also investigated whether blood levels of vitamin metabolites modify DNA methylation levels in blood cells. To this end, we quantitatively determined DNA methylation levels in blood cells of nested cases and controls from a prospective study with well defined dietary habits and lifestyles. Multiple CpG sites in five genes (CDKN2A/p16, RASSF1A, GSTP1, MTHFR, and MGMT) that are frequent targets of hypermethylation in a variety of human malignancies were included in the analysis. While no clear association between DNA methylation patterns and the case/control status was found, with the exception of RASSF1A hypermethylation, methylation level changed according to serum levels of 1-carbon metabolites and vitamins B. Overall, folate was associated with increased methylation levels of RASSF1A and MTHFR and methionine was associated with decreased methylation levels of RASSF1A. The associations with folate were more pronounced among never smokers while the associations with methionine were more evident among ever-smokers. These results are consistent with the notion that blood levels of 1-carbon metabolism markers and dietary/lifestyle factors may modify DNA methylation levels in blood cells and that blood cells can be exploited for the discovery of epigenetic biomarkers of exposures, providing proof-of-principle on the use of blood samples in the context of prospective studies.