MeCP2 represses the activity of topoisomerase IIß in long neuronal genes.

A unique signature of neurons is the high expression of the longest genes in the genome. These genes have essential neuronal functions, and disruption of their expression has been implicated in neurological disorders. DNA topoisomerases resolve DNA topological constraints and facilitate neuronal long gene expression. Conversely, the Rett syndrome protein, methyl-CpG-binding protein 2 (MeCP2), can transcriptionally repress long genes. How these factors regulate long genes is not well understood, and whether they interact is not known. Here, we identify and map a functional interaction between MeCP2 and topoisomerase IIß (TOP2ß) in mouse neurons. We profile neuronal TOP2ß activity genome wide, detecting enrichment at regulatory regions and gene bodies of long genes, including MeCP2-regulated genes. We show that loss and overexpression of MeCP2 alter TOP2ß activity at MeCP2-regulated genes. These findings uncover a mechanism of TOP2ß inhibition by MeCP2 in neurons and implicate TOP2ß dysregulation in disorders caused by MeCP2 disruption.

NSD1 deposits histone H3 lysine 36 dimethylation to pattern non-CG DNA methylation in neurons.

During postnatal development, the DNA methyltransferase DNMT3A deposits high levels of non-CG cytosine methylation in neurons. This methylation is critical for transcriptional regulation, and loss of this mark is implicated in DNMT3A-associated neurodevelopmental disorders (NDDs). Here, we show in mice that genome topology and gene expression converge to shape histone H3 lysine 36 dimethylation (H3K36me2) profiles, which in turn recruit DNMT3A and pattern neuronal non-CG methylation. We show that NSD1, an H3K36 methyltransferase mutated in NDD, is required for the patterning of megabase-scale H3K36me2 and non-CG methylation in neurons. We find that brain-specific deletion of NSD1 causes altered DNA methylation that overlaps with DNMT3A disorder models to drive convergent dysregulation of key neuronal genes that may underlie shared phenotypes in NSD1- and DNMT3A-associated NDDs. Our findings indicate that H3K36me2 deposited by NSD1 is important for neuronal non-CG DNA methylation and suggest that the H3K36me2-DNMT3A-non-CG-methylation pathway is likely disrupted in NSD1-associated NDDs.

NSD1 deposits histone H3 lysine 36 dimethylation to pattern non-CG DNA methylation in neurons.

During postnatal development the DNA methyltransferase DNMT3A deposits high levels of non-CG cytosine methylation in neurons. This unique methylation is critical for transcriptional regulation in the mature mammalian brain, and loss of this mark is implicated in DNMT3A-associated neurodevelopmental disorders (NDDs). The mechanisms determining genomic non-CG methylation profiles are not well defined however, and it is unknown if this pathway is disrupted in additional NDDs. Here we show that genome topology and gene expression converge to shape histone H3 lysine 36 dimethylation (H3K36me2) profiles, which in turn recruit DNMT3A and pattern neuronal non-CG methylation. We show that NSD1, the H3K36 methyltransferase mutated in the NDD, Sotos syndrome, is required for megabase-scale patterning of H3K36me2 and non-CG methylation in neurons. We find that brain-specific deletion of NSD1 causes alterations in DNA methylation that overlap with models of DNMT3A disorders and define convergent disruption in the expression of key neuronal genes in these models that may contribute to shared phenotypes in NSD1- and DNMT3A-associated NDD. Our findings indicate that H3K36me2 deposited by NSD1 is an important determinant of neuronal non-CG DNA methylation and implicates disruption of this methylation in Sotos syndrome. Highlights: Topology-associated DNA methylation and gene expression independently contribute to neuronal gene body and enhancer non-CG DNA methylation patterns.Topology-associated H3K36me2 patterns and local enrichment of H3K4 methylation impact deposition of non-CG methylation by DNMT3A. Disruption of NSD1 in vivo leads to alterations in H3K36me2, DNA methylation, and gene expression that overlap with models of DNMT3A disorders.

miR-378-3p Knockdown Recapitulates Many of the Features of Myelodysplastic Syndromes.

Myelodysplastic syndromes (MDS) are clonal neoplasms of the hematopoietic stem cell that result in aberrant differentiation of hematopoietic lineages caused by a wide range of underlying genetic, epigenetic, and other causes. Despite the myriad origins, a recognizable MDS phenotype has been associated with miRNA aberrant expression. A model of aberrant myeloid maturation that mimics MDS was generated using a stable knockdown of miR-378-3p. This model exhibited a transcriptional profile indicating aberrant maturation and function, immunophenotypic and morphologic dysplasia, and aberrant growth that characterizes MDS. Moreover, aberrant signal transduction in response to stimulation specific to the stage of myeloid maturation as indicated by CyTOF mass cytometry was similar to that found in samples from patients with MDS. The aberrant signaling, immunophenotypic changes, cellular growth, and colony formation ability seen in this myeloid model could be reversed with azacytidine, albeit without significant improvement of neutrophil function.

Postmortem diagnoses of spinal ataxia in 316 horses in California.

OBJECTIVE: To determine period prevalences of postmortem diagnoses for spinal cord or vertebral column lesions as underlying causes of ataxia (spinal ataxia) in horses. ANIMALS: 2,861 client-owned horses (316 with ataxia [ataxic group] and 2,545 without ataxia [control group]). PROCEDURES: The medical records database of the University of California-Davis Veterinary Medical Teaching Hospital was searched to identify horses necropsied between January 1, 2005, and December 31, 2017. Results were compared between the ataxic and control groups and between various groups of horses in the ataxic group. Period prevalences were determined for the most common causes of ataxia. RESULTS: 2,861 horses underwent full necropsy, and the period prevalences for the most common definitive diagnoses for ataxia were 2.7% (77/2,861) for cervical vertebral compressive myelopathy (CVCM), 1.3% (38/2,861) for equine neuroaxonal dystrophy or equine degenerative myeloencephalopathy (eNAD-EDM), and 0.9% (25/2,861) for trauma; the period prevalence of ataxia of unknown origin was 2.0% (56/2,861). Horses in the ataxic group (vs the control group) were more likely to have been warmblood horses (OR, 2.70) and less likely to have been Arabian horses (OR, 0.53). In the ataxic group, horses < 5 (vs ≥ 5) years of age had greater odds of CVCM (OR, 2.82) or eNAD-EDM (OR, 6.17) versus trauma or ataxia of unknown origin. Horses in the ataxic group with CVCM were more likely Thoroughbreds (OR, 2.54), whereas horses with eNAD-EDM were more likely American Quarter Horses (OR, 2.95) and less likely Thoroughbreds (OR, 0.11). CONCLUSIONS AND CLINICAL RELEVANCE: Results indicated that breed distributions differed for horses with CVCM versus eNAD-EDM; therefore, breed should be considered in the clinical evaluation of spinal ataxia in horses.

DNMT3A Haploinsufficiency Results in Behavioral Deficits and Global Epigenomic Dysregulation Shared across Neurodevelopmental Disorders.

Mutations in DNA methyltransferase 3A (DNMT3A) have been detected in autism and related disorders, but how these mutations disrupt nervous system function is unknown. Here, we define the effects of DNMT3A mutations associated with neurodevelopmental disease. We show that diverse mutations affect different aspects of protein activity but lead to shared deficiencies in neuronal DNA methylation. Heterozygous DNMT3A knockout mice mimicking DNMT3A disruption in disease display growth and behavioral alterations consistent with human phenotypes. Strikingly, in these mice, we detect global disruption of neuron-enriched non-CG DNA methylation, a binding site for the Rett syndrome protein MeCP2. Loss of this methylation leads to enhancer and gene dysregulation that overlaps with models of Rett syndrome and autism. These findings define the effects of DNMT3A haploinsufficiency in the brain and uncover disruption of the non-CG methylation pathway as a convergence point across neurodevelopmental disorders.

Dopamine-induced interactions of female mouse hypothalamic proteins with progestin receptor-A in the absence of hormone.

Neural progestin receptors (PR) function in reproduction, neural development, neuroprotection, learning, memory and the anxiety response. In the absence of progestins, PR can be activated by dopamine (DA) in the rodent hypothalamus to elicit female sexual behaviour. The present study investigated mechanisms of DA activation of PR by testing the hypothesis that proteins from DA-treated hypothalami interact with PR in the absence of progestins. Ovariectomised, oestradiol-primed mice were infused with a D1-receptor agonist, SKF38393 (SKF), into the third ventricle 30 minutes prior to death. Proteins from SKF-treated hypothalami were pulled-down with glutathione S-transferase-tagged mouse PR-A or PR-B and the interactomes were analysed by mass spectrometry. The largest functional group to interact with PR-A in a DA-dependent manner was synaptic proteins. To test the hypothesis that DA activation of PR regulates synaptic proteins, we developed oestradiol-induced PR-expressing hypothalamic-like neurones derived from human-induced pluripotent stem cells (hiPSCs). Similar to progesterone (P4), SKF treatment of hiPSCs increased synapsin1/2 expression. This SKF-dependent effect was blocked by the PR antagonist RU486, suggesting that PR are necessary for this DA-induced increase. The second largest DA-dependent PR-A protein interactome comprised metabolic regulators involved in glucose metabolism, lipid synthesis and mitochondrial energy production. Interestingly, hypothalamic proteins interacted with PR-A, but not PR-B, in an SKF-dependent manner, suggesting that DA promotes the interaction of multiple hypothalamic proteins with PR-A. These in vivo and in vitro results indicate novel mechanisms by which DA can differentially activate PR isoforms in the absence of P4 and provide a better understanding of ligand-independent PR activation in reproductive, metabolic and mental health disorders in women.

Previously Identified Genetic Variants in ADGRL3 Are not Associated with Risk for Equine Degenerative Myeloencephalopathy across Breeds.

Equine neuroaxonal dystrophy/equine degenerative myeloencephalopathy (eNAD/EDM) is a neurologic disease that has been reported in young horses from a wide range of breeds. The disease is inherited and associated with vitamin E deficiency during the first two years of life, resulting in bilateral symmetric ataxia. A missense mutation (chr3:71,917,591 C > T) within adhesion G protein-coupled receptor L3 (ADGRL3) was recently associated with risk for EDM in the Caspian breed. In order to confirm these findings, genotyping of this missense mutation, along with the three other associated single nucleotide polymorphisms (SNPs) in the genomic region, was carried out on 31 postmortem-confirmed eNAD/EDM cases and 43 clinically phenotyped controls from various breeds. No significant association was found between eNAD/EDM confirmed cases and genotype at any of the four identified SNPs (P > 0.05), including the nonsynonymous variant (EquCab2.0 chr3:71,917,591; allelic P = 0.85). These findings suggest that the four SNPs, including the missense variant in the ADGRL3 region, are not associated with risk for eNAD/EDM across multiple breeds of horses.

Exaggeration of PFS by blinded, independent, central review (BICR).

BACKGROUND: Recent published studies have shown meaningful discrepancies between local investigator and blinded, independent, central review (BICR) assessed median progression-free survival (PFS). When the local review but not BICR shows progression, generally, no further assessments are carried out and patients are censored in the BICR analysis, leading to violation of the statistical assumptions of independence between censoring and outcome used in survival analysis methods. METHODS: We carried out a simulation study to assess methodological reasons behind these discrepancies and corroborated our findings in a case study of three BRCA-mutated ovarian cancer trials. We briefly outline possible methodological solutions that may lead to improved estimation of the BICR medians. RESULTS: The Kaplan-Meier (KM) curve for the BICR PFS can often be exaggerated. The degree of bias is largest when there is reasonably strong correlation between BICR and local PFS, especially when PFS is long compared with assessment frequency. This can result in an exaggeration of the medians and their difference; however, the hazard ratio (HR) is much less susceptible to bias. Our simulation shows that when the true BICR median PFS was 19 months, and patients assessed every 12 weeks, the estimated KM curves were materially biased whenever the correlation between BICR and local PFS was 0.4 or greater. This was corroborated by case studies where, in the active arm, the BICR median PFS was between 6 and 11 months greater than the local median PFS. Further research is required to find improved methods for estimating BICR survival curves. CONCLUSIONS: In general, when there is a difference between local and BICR medians, the true BICR KM curve is likely to be exaggerated and its true median will probably lie somewhere between the observed local and BICR medians. Presentation of data should always include both BICR and local results whenever a BICR is carried out.

The Progestin Receptor Interactome in the Female Mouse Hypothalamus: Interactions with Synaptic Proteins Are Isoform Specific and Ligand Dependent.

Progestins bind to the progestin receptor (PR) isoforms, PR-A and PR-B, in brain to influence development, female reproduction, anxiety, and stress. Hormone-activated PRs associate with multiple proteins to form functional complexes. In the present study, proteins from female mouse hypothalamus that associate with PR were isolated using affinity pull-down assays with glutathione S-transferase-tagged mouse PR-A and PR-B. Using complementary proteomics approaches, reverse phase protein array (RPPA) and mass spectrometry, we identified hypothalamic proteins that interact with PR in a ligand-dependent and isoform-specific manner and were confirmed by Western blot. Synaptic proteins, including synapsin-I and synapsin-II, interacted with agonist-bound PR isoforms, suggesting that both isoforms function in synaptic plasticity. In further support, synaptogyrin-III and synapsin-III associated with PR-A and PR-B, respectively. PR also interacted with kinases, including c-Src, mTOR, and MAPK1, confirming phosphorylation as an integral process in rapid effects of PR in the brain. Consistent with a role in transcriptional regulation, PR associated with transcription factors and coactivators in a ligand-specific and isoform-dependent manner. Interestingly, both PR isoforms associated with a key regulator of energy homeostasis, FoxO1, suggesting a novel role for PR in energy metabolism. Because many identified proteins in this PR interactome are synaptic proteins, we tested the hypothesis that progestins function in synaptic plasticity. Indeed, progesterone enhanced synaptic density, by increasing synapsin-I-positive synapses, in rat primary cortical neuronal cultures. This novel combination of RPPA and mass spectrometry allowed identification of PR action in synaptic remodeling and energy homeostasis and reveals unique roles for progestins in brain function and disease.

Adult Neurogenesis in the Female Mouse Hypothalamus: Estradiol and High-Fat Diet Alter the Generation of Newborn Neurons Expressing Estrogen Receptor α.

Estrogens and leptins act in the hypothalamus to maintain reproduction and energy homeostasis. Neurogenesis in the adult mammalian hypothalamus has been implicated in the regulation of energy homeostasis. Recently, high-fat diet (HFD) and estradiol (E2) have been shown to alter cell proliferation and the number of newborn leptin-responsive neurons in the hypothalamus of adult female mice. The current study tested the hypothesis that new cells expressing estrogen receptor α (ERα) are generated in the arcuate nucleus (ARC) and the ventromedial nucleus of the hypothalamus (VMH) of the adult female mouse, hypothalamic regions that are critical in energy homeostasis. Adult mice were ovariectomized and implanted with capsules containing E2 or oil. Within each hormone group, mice were fed an HFD or standard chow for 6 weeks and treated with BrdU to label new cells. Newborn cells that respond to estrogens were identified in the ARC and VMH, of which a subpopulation was leptin sensitive, indicating that the subpopulation consists of neurons. Moreover, there was an interaction between diet and hormone with an effect on the number of these newborn ERα-expressing neurons that respond to leptin. Regardless of hormone treatment, HFD increased the number of ERα-expressing cells in the ARC and VMH. E2 decreased hypothalamic fibroblast growth factor 10 (Fgf10) gene expression in HFD mice, suggesting a role for Fgf10 in E2 effects on neurogenesis. These findings of newly created estrogen-responsive neurons in the adult brain provide a novel mechanism by which estrogens can act in the hypothalamus to regulate energy homeostasis in females.

Estradiol Preferentially Induces Progestin Receptor-A (PR-A) Over PR-B in Cells Expressing Nuclear Receptor Coactivators in the Female Mouse Hypothalamus

Estrogens act in brain to profoundly influence neurogenesis, sexual differentiation, neuroprotection, cognition, energy homeostasis, and female reproductive behavior and physiology through a variety of mechanisms, including the induction of progestin receptors (PRs). PRs are expressed as two isoforms, PR-A and PR-B, that have distinct functions in physiology and behavior. Because these PR isoforms cannot be distinguished using cellular resolution techniques, the present study used isoform-specific null mutant mice that lack PR-A or PR-B for the first time to investigate whether 17ß-estradiol benzoate (EB) regulates the differential expression of the PR isoforms in the ventromedial nucleus of the hypothalamus (VMN), arcuate nucleus, and medial preoptic area, brain regions that are rich in EB-induced PRs. Interestingly, EB induced more PR-A than PR-B in all three brain regions, suggesting that PR-A is the predominant isoform in these regions. Given that steroid receptor coactivator (SRC)-1 and SRC-2 are important in estrogen receptor (ER)-dependent transcription in brain, including PR induction, we tested whether the expression of these coactivators was correlated with PR isoform expression. The majority of EB-induced PR cells expressed both SRC-1 and SRC-2 in the three brain regions of all genotypes. Interestingly, the intensity of PR-A immunoreactivity correlated with SRC-2 expression in the VMN, providing a potential mechanism for selective ER-mediated transactivation of PR-A over PR-B in a brain region-specific manner. In summary, these novel findings indicate that estrogens differentially regulate PR-A and PR-B expression in the female hypothalamus, and provide a mechanism by which steroid action in brain can selectively modulate behavior and physiology.

Transient early-life forebrain corticotropin-releasing hormone elevation causes long-lasting anxiogenic and despair-like changes in mice.

During development, early-life stress, such as abuse or trauma, induces long-lasting changes that are linked to adult anxiety and depressive behavior. It has been postulated that altered expression of corticotropin-releasing hormone (CRH) can at least partially account for the various effects of stress on behavior. In accord with this hypothesis, evidence from pharmacological and genetic studies has indicated the capacity of differing levels of CRH activity in different brain areas to produce behavioral changes. Furthermore, stress during early life or adulthood causes an increase in CRH release in a variety of neural sites. To evaluate the temporal and spatial specificity of the effect of early-life CRH exposure on adult behavior, the tetracycline-off system was used to produce mice with forebrain-restricted inducible expression of CRH. After transient elevation of CRH during development only, behavioral testing in adult mice revealed a persistent anxiogenic and despair-like phenotype. These behavioral changes were not associated with alterations in adult circadian or stress-induced corticosterone release but were associated with changes in CRH receptor type 1 expression. Furthermore, the despair-like changes were normalized with antidepressant treatment. Overall, these studies suggest that forebrain-restricted CRH signaling during development can permanently alter stress adaptation leading to increases in maladaptive behavior in adulthood.

Effect of an inhaled adenosine A2A agonist on the allergen-induced late asthmatic response.

BACKGROUND: Adenosine receptor activation is suggested to play a role in asthmatic airway inflammation. Inhibition of adenosine receptors may have an effect on the late asthmatic response (LAR) after allergen inhalation and this mechanism could offer a potential new treatment in asthma. METHODS: We evaluated the effect of an inhaled adenosine-(2A) (A(2A))-receptor agonist (GW328267X), 25 microg, in 15 nonsmoking atopic asthmatics who underwent an inhaled allergen challenge following twice daily treatment for 1 week in a double-blind, placebo- and fluticasone propionate (250 microg) controlled study. RESULTS: In contrast to fluticasone, treatment with the A(2A)-receptor agonist neither significantly protect against the allergen-induced early and late asthmatic reaction, nor the accompanying inflammatory response as measured by sputum total cell counts, number of EG2+ cells, and the concentration of interleukin-8 and eosinophil cationic protein. CONCLUSION: The inhaled A(2A)-receptor agonist, GW328267X, 25 microg does not affect the allergen-induced LAR or the associated inflammatory response in asthma.

Effect of encapsulation on absorption of sumatriptan tablets: data from healthy volunteers and patients during a migraine.

BACKGROUND: Some comparative trials of selective serotonin 1B/ID-agonists in migraine have reported -15% lower efficacy for sumatriptan tablets than that reported in placebo-controlled trials. OBJECTIVE: This study was designed to test the hypothesis that the encapsulation methods used to mask active drug may delay absorption of sumatriptan from dosing to 2 hours after dosing (the traditional end point in clinical trials of migraine treatment), an effect that may be enhanced by migraine-associated gastric stasis. METHODS: Two randomized, open-label, 2-way crossover trials were conducted to evaluate the absorption and bioequivalence of conventional 50-mg sumatriptan tablets and encapsulated 50-mg sumatriptan tablets in supine, fasted, healthy volunteers (Glaxo Wellcome protocol SUM40270) and supine patients experiencing a migraine (Glaxo Wellcome protocol SUM40268). Absorption was assessed by calculating the area under the plasma concentration-time curve from dosing to 2 hours after dosing (AUC2) and the times to first measurable plasma concentration, 10 ng/mL, 20 ng/mL, and maximum plasma concentration. Data for the AUC from time zero to infinity and maximum plasma concentration were used to assess standard bioequivalence, which is considered to occur when the 90% CIs for the geometric mean treatment ratios (test/reference) fall between 0.8 and 1.25. RESULTS: Study 1 included 26 healthy subjects (73% men, 27% women; mean age, 39.1 years), and study 2 included 30 patients with migraine (67% women, 33% men; mean age, 42.7 years). Sumatriptan absorption was delayed with the encapsulated tablet compared with the conventional tablet 0 to 2 hours after dosing, particularly during a migraine. AUC2 values with encapsulated sumatriptan compared with the conventional tablet were 21% lower in healthy volunteers (ratio of capsule/tablet, 0.79; 90% CI, 0.588-1.050) and 27% lower in patients experiencing a migraine (ratio of capsule/tablet, 0.73; 90% CI, 0.519-1.023). Standard bioequivalence was demonstrated in both healthy volunteers and patients experiencing a migraine. CONCLUSIONS: Encapsulation delayed absorption of sumatriptan 0 to 2 hours after dosing, particularly during a migraine. This delay in absorption of the encapsulated form may account for the lower efficacy of sumatriptan in some comparative studies.

ICTUS: un desconocido para la población

Introducción. Una de las causas que influyen en el retraso en la llegada al Servicio de Urgencias de las personas que han sufrido un ictus es la falta de reconocimiento de los signos y síntomas. Objetivo. Estudio piloto destinado a cuantificar el grado de conocimiento de la población acerca del ictus (terminología, factores de riesgo, manifestaciones y actitud), previo a campañas de información. Pacientes y métodos. Estudio poblacional mediante cuestionario cerrado, realizado por neurólogos de forma aleatoria sobre 100 usuarios del centro de salud Vall d Hebron. Resultados. El 9 por ciento de la población desconocen absolutamente la enfermedad. Un 42 por ciento tienen un buen conocimiento de la sintomatología y un 46 por ciento de los factores de riesgo. Sólo un 22 por ciento de la población tienen un buen conocimiento global de la enfermedad. Ante un ictus, el 97,8 por ciento solicitarían ayuda médica urgente, a través del 061 (46,2 por ciento ) y acudiendo directamente al hospital (50,5 por ciento). Si la sintomatología remitiese (AIT) se produce un giro hacia la atención primaria (59,3 por ciento). Un 98,9 por ciento reconocen el término embolia y un 1 por ciento ictus. Los mayores de 65 años reconocen peor la sintomatología y consideran menos urgente el ictus. Los encuestados con familiar afecto (50,5 por ciento) localizan la enfermedad con más precisión en el cerebro y tienden a acudir con más urgencia al hospital. Conclusiones. Menos de una cuarta parte de la población tiene un buen conocimiento de la enfermedad. El ictus lo consideran una urgencia a diferencia del AIT. La información sobre la enfermedad mejora teóricamente el tiempo de llegada a urgencias. Estos resultados han hecho rediseñar el cuestionario para llevar a cabo una segunda fase del estudio que permita generalizarlos en la población española [REV NEUROL 1998; 27: 943-7].(AU)