Active N6-Methyladenine Demethylation by DMAD Regulates Gene Expression by Coordinating with Polycomb Protein in Neurons.

A ten-eleven translocation (TET) ortholog exists as a DNA N6-methyladenine (6mA) demethylase (DMAD) in Drosophila. However, the molecular roles of 6mA and DMAD remain unexplored. Through genome-wide 6mA and transcriptome profiling in Drosophila brains and neuronal cells, we found that 6mA may epigenetically regulate a group of genes involved in neurodevelopment and neuronal functions. Mechanistically, DMAD interacts with the Trithorax-related complex protein Wds to maintain active transcription by dynamically demethylating intragenic 6mA. Accumulation of 6mA by depleting DMAD coordinates with Polycomb proteins and contributes to transcriptional repression of these genes. Our findings suggest that active 6mA demethylation by DMAD plays essential roles in fly CNS by orchestrating through added epigenetic mechanisms.

Lin28A Binds Active Promoters and Recruits Tet1 to Regulate Gene Expression.

Lin28, a well-known RNA-binding protein, regulates diverse cellular properties. All physiological functions of Lin28A characterized so far have been attributed to its repression of let-7 miRNA biogenesis or modulation of mRNA translational efficiency. Here we show that Lin28A directly binds to a consensus DNA sequence in vitro and in mouse embryonic stem cells in vivo. ChIP-seq and RNA-seq reveal enrichment of Lin28A binding around transcription start sites and a positive correlation between its genomic occupancy and expression of many associated genes. Mechanistically, Lin28A recruits 5-methylcytosine-dioxygenase Tet1 to genomic binding sites to orchestrate 5-methylcytosine and 5-hydroxymethylcytosine dynamics. Either Lin28A or Tet1 knockdown leads to dysregulated DNA methylation and expression of common target genes. These results reveal a surprising role for Lin28A in transcriptional regulation via epigenetic DNA modifications and have implications for understanding mechanisms underlying versatile functions of Lin28A in mammalian systems.

Lack of RAN-mediated toxicity in Huntington's disease knock-in mice.

Identification of repeat-associated non-AUG (RAN) translation in trinucleotide (CAG) repeat diseases has led to the emerging concept that CAG repeat diseases are caused by nonpolyglutamine products. Nonetheless, the in vivo contribution of RAN translation to the pathogenesis of CAG repeat diseases remains elusive. Via CRISPR/Cas9-mediated genome editing, we established knock-in mouse models that harbor expanded CAG repeats in the mouse huntingtin gene to express RAN-translated products with or without polyglutamine peptides. We found that RAN translation is not detected in the knock-in mouse models when expanded CAG repeats are expressed at the endogenous level. Consistently, the expanded CAG repeats that cannot be translated into polyglutamine repeats do not yield the neuropathological and behavioral phenotypes that were found in knock-in mice expressing expanded polyglutamine repeats. Our findings suggest that RAN-translated products do not play a major role in the pathogenesis of CAG repeat diseases and underscore the importance in targeting polyglutamine repeats for therapeutics.

Metabolic pathways modulate the neuronal toxicity associated with fragile X-associated tremor/ataxia syndrome.

Fragile X-associated tremor/ataxia syndrome (FXTAS) is an adult-onset neurodegenerative disorder that affects premutation carriers (55-200 CGG repeats) of the fragile X mental retardation 1 (FMR1) gene. Much remains unknown regarding the metabolic alterations associated with FXTAS, especially in the brain, and the most affected region, the cerebellum. Investigating the metabolic changes in FXTAS will aid in the identification of biomarkers as well as in understanding the pathogenesis of disease. To identify the metabolic alterations associated with FXTAS, we took advantage of our FXTAS mouse model that expresses 90 CGG repeats in cerebellar Purkinje neurons and exhibits the key phenotypic features of FXTAS. We performed untargeted global metabolic profiling of age-matched control and FXTAS mice cerebella at 16-20 weeks and 55 weeks. Out of 506 metabolites measured in cerebellum, we identified 186 metabolites that demonstrate significant perturbations due to the (CGG)90 repeat (P<0.05) and found that these differences increase dramatically with age. To identify key metabolic changes in FXTAS pathogenesis, we performed a genetic screen using a Drosophila model of FXTAS. Out of 28 genes that we tested in the fly, 8 genes showed significant enhanced neuronal toxicity associated with CGG repeats, such as Schlank (ceramide synthase), Sk2 (sphingosine kinase) and Ras (IMP dehydrogenase). By combining metabolic profiling with a Drosophila genetic screen to identify genetic modifiers of FXTAS, we demonstrate an effective method for functional validation of high-throughput metabolic data and show that sphingolipid and purine metabolism are significantly perturbed in FXTAS pathogenesis.

Molecular signatures associated with ZIKV exposure in human cortical neural progenitors.

Zika virus (ZIKV) infection causes microcephaly and has been linked to other brain abnormalities. How ZIKV impairs brain development and function is unclear. Here we systematically profiled transcriptomes of human neural progenitor cells exposed to Asian ZIKVC, African ZIKVM, and dengue virus (DENV). In contrast to the robust global transcriptome changes induced by DENV, ZIKV has a more selective and larger impact on expression of genes involved in DNA replication and repair. While overall expression profiles are similar, ZIKVC, but not ZIKVM, induces upregulation of viral response genes and TP53. P53 inhibitors can block the apoptosis induced by both ZIKVC and ZIKVM in hNPCs, with higher potency against ZIKVC-induced apoptosis. Our analyses reveal virus- and strain-specific molecular signatures associated with ZIKV infection. These datasets will help to investigate ZIKV-host interactions and identify neurovirulence determinants of ZIKV.

Genome-wide alteration of 5-hydroxymenthylcytosine in a mouse model of Alzheimer's disease.

BACKGROUND: Alzheimer's disease (AD) is the most common form of neurodegenerative disorder that leads to a decline in cognitive function. In AD, aggregates of amyloid ß peptide precede the accumulation of neurofibrillary tangles, both of which are hallmarks of the disease. The great majority (>90 %) of the AD cases are not originated from genetic defects, therefore supporting the central roles of epigenetic modifications that are acquired progressively during the life span. Strong evidences have indicated the implication of epigenetic modifications, including histone modification and DNA methylation, in AD. Recent studies revealed that 5-hydroxymethylcytosine (5hmC) is dynamically regulated during neurodevelopment and aging. RESULTS: We show that amyloid peptide 1-42 (Aß1-42) could significantly reduce the overall level of 5hmC in vitro. We found that the level of 5hmC displayed differential response to the pathogenesis in different brain regions, including the cortex, cerebellum, and hippocampus of APP-PSEN1 double transgenic (DTg) mice. We observed a significant decrease of overall 5hmC in hippocampus, but not in cortex and cerebellum, as the DTg mice aged. Genome-wide profiling identified differential hydroxymethylation regions (DhMRs) in DTg mice, which are highly enriched in introns, exons and intergenic regions. Gene ontology analyses indicated that DhMR-associated genes are highly enriched in multiple signaling pathways involving neuronal development/differentiation and neuronal function/survival. CONCLUSIONS: 5hmC-mediated epigenetic regulation could potentially be involved in the pathogenesis of AD.

Synthetic conantokin peptides potently inhibit N-methyl-D-aspartate receptor-mediated currents of retinal ganglion cells.

Retinal ganglion cells (RGCs), which are the sole output neurons of the retina, express N-methyl-D-aspartate receptors (NMDARs), rendering these cells susceptible to glutamate excitotoxicity, with implications for loss of normal RGC excitatory responses in disorders such as glaucoma and diabetic retinopathy. Therefore, antagonists that inhibit NMDAR-mediated currents specifically by targeting the GluN2B component of the ion channel have the potential to serve as a basis for developing potential therapeutics. The roles of peptidic conantokins, which are potent brain neuronal NMDAR inhibitors, were studied. By using patch-clamp whole-cell analyses in dissociated RGCs and retinal whole-mount RGCs, we evaluated the effects of synthetic conantokin-G (conG) and conantokin-T (conT), which are small γ-carboxyglutamate-containing peptides, on NMDA-mediated excitatory responses in mouse RGCs. Both conG and conT inhibited the NMDA-mediated currents of dark-adapted dissociated and whole-mount RGCs in a dose-dependent, reversible, noncompetitive manner. Inhibition of NMDA-mediated steady-state currents by NMDAR nonsubunit-selective conT was approximately threefold greater than GluN2B-selective conG or ifenprodil, demonstrating its potential ability to inhibit both GluN2A- and GluN2B-containing ion channels in RGCs. Because the extent of inhibition of NMDA-evoked currents by conG and the pharmacologic GluN2B-selective inhibitor ifenprodil were similar (40-45%) to that of the GluN2A-selective antagonist NVP-AAM0077, we conclude that the levels of GluN2A and GluN2B subunits are similar in RGCs. These results provide a novel basis for developing effective neuroprotective agents to aid in the prevention of undesired glutamatergic excitotoxicity in neurodegenerative diseases of the retina and demonstrate functional assembly of NMDARs in RGCs.

Characterization of GFP-tagged GnRH-containing terminalis neurons in transgenic zebrafish.

The terminalis nerve (TN) has been described in all vertebrate species, in which it plays important roles in animal behavior and physiology. In teleost fish, the TN is located in the olfactory bulb and in its nerve tract. Here, we report a study on the characterization of the TN cell development, axon projection and physiology in zebrafish (Danio rerio). We have generated several lines of transgenic zebrafish [Tg (GnRH-3::GFP)] that express GFP in the TN cells. The transgenes are expressed under the transcriptional control of the zebrafish GnRH-3 promoter. During development, the first GFP-positive TN cell was identified at approximately 34 h post-fertilization (hpf). By 38 hpf, several clusters of TN cells were identified in the olfactory bulb and olfactory nerve tract. In the olfactory bulb, the TN cells projected axons caudally. In the forebrain, some of the TN axons extended caudally, but most crossed the midline of the brain at the commissural anterior. In the midbrain, some of the TN axons extended dorsally towards the tectum, whereas other axons extended caudally, or extended ventrally to the optic nerve where they entered the neural retina. We also examined the cell membrane property of the TN cells. Using patch-clamp techniques, we recorded spontaneous and evoked action potentials from isolated TN cells. We examined the expression of glutamate receptors in the TN cells. The data shed light on the mechanisms of TN function in the nervous system and in the regulation of animal physiology.

Conantokins inhibit NMDAR-dependent calcium influx in developing rat hippocampal neurons in primary culture with resulting effects on CREB phosphorylation.

The effects of conantokin (con)-G, con-R[1-17], and con-T on ion flow through N-methyl-D-aspartate receptor (NMDAR) ion channels were determined in cultured primary rat hippocampal neurons. The potency of con-G diminished, whereas inhibition by con-R[1-17] and con-T did not change, as the neurons matured. Con-G, con-R[1-17], and con-T effectively diminished NMDA-induced Ca(2+) influx into the cells. A similar age-dependent decrease in con-G-mediated inhibition of the amplitude of spontaneous excitatory postsynaptic currents (sEPSCs) was observed, compared to con-R[1-17] and con-T. The effects of the conantokins on NMDA-induced cAMP response element-binding protein (CREB) phosphorylation in immature (DIV 9) and mature (DIV 16) neurons showed that, at DIV 9, con-G, con-R[1-17], and con-T inhibited NMDA-mediated P-CREB levels, whereas in DIV 16 neurons the conantokins did not inhibit overall levels of NMDA-induced P-CREB. In contrast, P-CREB levels were enhanced through inhibition of the protein phosphatases, PP1 and PP2B (calcineurin). This ability of conantokins to sustain CREB phosphorylation can thus enhance neuronal survival and plasticity.

Author Correction: Truncation of mutant huntingtin in knock-in mice demonstrates exon1 huntingtin is a key pathogenic form.

A Correction to this paper has been published: https://doi.org/10.1038/s41467-020-19873-9.

Truncation of mutant huntingtin in knock-in mice demonstrates exon1 huntingtin is a key pathogenic form.

Polyglutamine expansion in proteins can cause selective neurodegeneration, although the mechanisms are not fully understood. In Huntington's disease (HD), proteolytic processing generates toxic N-terminal huntingtin (HTT) fragments that preferentially kill striatal neurons. Here, using CRISPR/Cas9 to truncate full-length mutant HTT in HD140Q knock-in (KI) mice, we show that exon 1 HTT is stably present in the brain, regardless of truncation sites in full-length HTT. This N-terminal HTT leads to similar HD-like phenotypes and age-dependent HTT accumulation in the striatum in different KI mice. We find that exon 1 HTT is constantly generated but its selective accumulation in the striatum is associated with the age-dependent expression of striatum-enriched HspBP1, a chaperone inhibitory protein. Our findings suggest that tissue-specific chaperone function contributes to the selective neuropathology in HD, and highlight the therapeutic potential in blocking generation of exon 1 HTT.

Age-related DNA hydroxymethylation is enriched for gene expression and immune system processes in human peripheral blood.

DNA methylation (DNAm) has a well-established association with age in many tissues, including peripheral blood mononuclear cells (PBMCs). Compared to DNAm, the closely related epigenetic modification known as DNA hydroxymethylation (DNAhm) was much more recently discovered in mammals. Preliminary investigations have observed a positive correlation between gene body DNAhm and cis-gene expression. While some of these studies have observed an association between age and global DNAhm, none have investigated region-specific age-related DNAhm in human blood samples. In this study, we investigated DNAhm and gene expression in PBMCs of 10 young and 10 old, healthy female volunteers. Thousands of regions were differentially hydroxymethylated in the old vs. young individuals in gene bodies, exonic regions, enhancers, and promoters. Consistent with previous work, we observed directional consistency between age-related differences in DNAhm and gene expression. Further, age-related DNAhm and genes with high levels of DNAhm were enriched for immune system processes which may support a role of age-related DNAhm in immunosenescence.

Ten-Eleven Translocation Proteins Modulate the Response to Environmental Stress in Mice.

5-hydroxymethylcytosine (5hmC) is enriched in brain and has been recognized as an important DNA modification. However, the roles of 5hmC and its writers, ten-eleven translocation (Tet) proteins, in stress-induced response have yet to be elucidated. Here, we show that chronic restraint stress (CRS) induced depression-like behavior in mice and resulted in a 5hmC reduction in prefrontal cortex (PFC). We found that loss of Tet1 (Tet1 KO) led to resistance to CRS, whereas loss of Tet2 (Tet2 KO) increased the susceptibility of mice to CRS. Genome-wide 5hmC profiling identified the phenotype-associated stress-induced dynamically hydroxymethylated loci (PA-SI-DhMLs), which are strongly enriched with hypoxia-induced factor (HIF) binding motifs. We demonstrated the physical interaction between TET1 and HIF1α induced by CRS and revealed that the increased HIF1α binding under CRS is associated with SI-DhMLs. These results suggest that TET1 could regulate stress-induced response by interacting with HIF1α.

DNA N6-methyladenine is dynamically regulated in the mouse brain following environmental stress.

Chemical modifications on DNA molecules, such as 5-methylcytosine and 5-hydroxymethylcytosine, play important roles in the mammalian brain. A novel DNA adenine modification, N(6)-methyladenine (6mA), has recently been found in mammalian cells. However, the presence and function(s) of 6mA in the mammalian brain remain unclear. Here we demonstrate 6mA dynamics in the mouse brain in response to environmental stress. We find that overall 6mA levels are significantly elevated upon stress. Genome-wide 6mA and transcriptome profiling reveal an inverse association between 6mA dynamic changes and a set of upregulated neuronal genes or downregulated LINE transposon expression. Genes bearing stress-induced 6mA changes significantly overlap with loci associated with neuropsychiatric disorders. These results suggest an epigenetic role for 6mA in the mammalian brain as well as its potential involvement in neuropsychiatric disorders.

Zika virus directly infects peripheral neurons and induces cell death.

Zika virus (ZIKV) infection is associated with neurological disorders of both the CNS and peripheral nervous systems (PNS), yet few studies have directly examined PNS infection. Here we show that intraperitoneally or intraventricularly injected ZIKV in the mouse can infect and impact peripheral neurons in vivo. Moreover, ZIKV productively infects stem-cell-derived human neural crest cells and peripheral neurons in vitro, leading to increased cell death, transcriptional dysregulation and cell-type-specific molecular pathology.

Selective loss of orientation column maps in visual cortex during brief elevation of intraocular pressure.

PURPOSE: To compare the orientation column maps elicited by different spatial frequency gratings in cortical area 17 of cats before and during brief elevation of intraocular pressure (IOP). METHODS: IOP was elevated by injecting saline into the anterior chamber of a cat's eye through a syringe needle. The IOP was elevated enough to cause a retinal perfusion pressure (arterial pressure minus IOP) of approximately 30 mm Hg during a brief elevation of IOP. The visual stimulus gratings were varied in spatial frequency, whereas other parameters were kept constant. The orientation column maps of the cortical area 17 were monocularly elicited by drifting gratings of different spatial frequencies and revealed by a brain intrinsic signal optical imaging system. These maps were compared before and during short-term elevation of IOP. RESULTS: The response amplitude of the orientation maps in area 17 decreased during a brief elevation of IOP. This decrease was dependent on the retinal perfusion pressure but not on the absolute IOP. The location of the most visible maps was spatial-frequency dependent. The blurring or loss of the pattern of the orientation maps was most severe when high-spatial-frequency gratings were used and appeared most significantly on the posterior part of the exposed cortex while IOP was elevated. However, the basic patterns of the maps remained unchanged. Changes in cortical signal were not due to changes in the optics of the eye with elevation of IOP. CONCLUSIONS: A stable normal IOP is essential for maintaining normal visual cortical functions. During a brief and high elevation of IOP, the cortical processing of high-spatial-frequency visual information was diminished because of a selectively functional decline of the retinogeniculocortical X pathway by a mechanism of retinal circulation origin.

Spatial frequency-dependent feedback of visual cortical area 21a modulating functional orientation column maps in areas 17 and 18 of the cat.

The feedback effect of activity of area 21a on orientation maps of areas 17 and 18 was investigated in cats using intrinsic signal optical imaging. A spatial frequency-dependent decrease in response amplitude of orientation maps to grating stimuli was observed in areas 17 and 18 when area 21a was inactivated by local injection of GABA, or by a lesion induced by liquid nitrogen freezing. The decrease in response amplitude of orientation maps of areas 17 and 18 after the area 21a inactivation paralleled the normal response without the inactivation. Application in area 21a of bicuculline, a GABAa receptor antagonist caused an increase in response amplitude of orientation maps of area 17. The results indicate a positive feedback from high-order visual cortical area 21a to lower-order areas underlying a spatial frequency-dependent mechanism.

Characterization of voltage-activated ionic currents in the GnRH-containing terminalis nerve in transgenic zebrafish.

The terminalis nerve (TN) is in a class of cranial nerves that plays important roles in animal development, physiology and behavior. Here, we report a study on the characterization of voltage-activated ionic currents in GnRH-containing TN cells in zebrafish. The experiments were performed using acutely dissociated TN cells from the transgenic zebrafish Tg (GnRH-3::GFP). In the transgenic zebrafish, the TN cells express GFP under the transcriptional control of the zebrafish GnRH-3 promoter. In all of the GnRH-containing TN cells examined, we recorded both low-voltage-activated (LVA) and high-voltage-activated (HVA) calcium current (I(Ca)). The characteristics of the I(Ca) were similar to those described in other zebrafish cell types. However, the distribution patterns of the currents in the GnRH-containing TN cells were different in comparison to the distribution of the currents in other cell types. In addition, we characterized TTX-sensitive sodium current (I(Na)) and 4AP-sensitive and TEA-resistant potassium current (I(K)). The characteristics of voltage-activated I(Na) and I(K) in the GnRH-containing TN cells were similar to those described in other zebrafish cell types. Together, the data from this study revealed the electrophysiological properties of the GnRH-containing TN cells, thereby providing insight on the regulatory mechanisms of TN-signaling in animal physiology.

Cloning and spatial and temporal expression of the zebrafish dopamine D1 receptor.

Dopamine plays important roles in the regulation of central nervous system (CNS) development and functions. In vertebrates, two families of dopamine receptors, collectively known as dopamine D1 and D2 receptors, have been identified. Recently, dopamine receptors have been targeted by pharmacological and therapeutic studies of neurological disorders, such as Parkinson's disease. Here, we report a study on the molecular characterization of dopamine D1 receptor in zebrafish (Danio rerio). We cloned the full-length cDNA of a zebrafish dopamine D1 receptor, designated as drd1. The sequence of drd1 shares high homology to the sequences of dopamine D1 receptors in mammalian, amphibian, and other fish species. drd1 is expressed in the CNS. The first drd1 expression was observed at approximately 30 hours postfertilization, at which time the expression was seen in the developing diencephalon and hindbrain. In developing retinas, the expression of drd1 was detected in the inner nuclear layer with the exception of the marginal zones. In adult retinas, drd1 expression was detected in most cell types in the inner and outer nuclear layers as well as ganglion cell layer. Differential expression of drd1 in developing and adult retinas may play various roles in regulating visual system functions.

Differential expression of voltage-activated calcium currents in zebrafish retinal ganglion cells.

We report a study on the characterization of voltage-activated calcium currents (I(Ca)) in retinal ganglion cells (RGCs) and the topographic distribution of RGCs that express different types of I(Ca) in zebrafish retinas. In acutely isolated zebrafish RGCs, both high-voltage-activated (HVA; peak activation potential +7.4 +/- 1.1 mV) and low-voltage-activated (LVA; peak activation potential -33.0 +/- 1.2 mV) I(Ca) were recorded. HVA I(Ca) were recorded in all of the tested RGCs, whereas LVA I(Ca) were recorded in approximately one-third of the tested cells. In RGCs that expressed both HVA and LVA I(Ca), the two currents were readily separated by depolarizing the cell membrane to different voltages from different holding potentials. Among RGCs that expressed LVA I(Ca), some cells expressed large LVA I(Ca) (up to 130 pA), whereas others expressed small LVA I(Ca) (approximately 20 pA). RGCs that expressed large and small LVA I(Ca) were designated as class I and class II cells, respectively, and RGCs that expressed only HVA I(Ca) were designated as class III cells. The topographic distribution of cell classes was similar in various areas of the retina. In the nasal-ventral retina, for example, class III cells outnumbered class I and class II cells by 10.8- and 2.6-fold, respectively. In the temporal and dorsal retinas, the density of class III cells slightly decreased, whereas the density of class I and class II cells increased. The differential expression of I(Ca) in RGCs may correlate with the development and function of the retina.