Brain-specific Expression Research Articles

Bioinformatic analysis of brain-specific miRNAs for identification of candidate traumatic brain injury blood biomarkers

ABSTRACT Background Detection of brain-specific miRNAs in the peripheral blood could serve as a surrogate marker of traumatic brain injury (TBI). Here, we systematically identified brain-enriched miRNAs, and tested their utility as TBI biomarkers in the acute phase of care. Methods Publically available microarray data generated from 29 postmortem human tissues were used to rank 1,364 miRNAs in terms of their degree of brain-specific expression. Levels of the top six ranked miRNAs were then prospectively measured in serum samples collected from 10 Patients with TBI at hospital admission, as well as from 10 controls. Results The top six miRNAs identified in our analysis (miR-124-3p, miR-219a-5p, miR-9-5p, miR-9-3p, miR-137, and miR-128-3p) were enriched 70 to 320-fold in brain relative to other tissues, and exhibited dramatically greater brain specificity compared to several miRNAs previously proposed as biomarkers. Furthermore, their levels were elevated in serum from patients with TBI compared to controls, and could collectively discriminate between groups with 90% sensitivity and 100% specificity. Interestingly, subsequent informatic pathway analysis revealed that their target transcripts were enriched for components of signaling pathways active in peripheral organs involved in common post-TBI complications. Conclusions The six candidate miRNAs identified in this preliminary study have promise as blood biomarkers of TBI, and could also be molecular contributors to systemic physiologic changes commonly observed post-injury.

3H]Dopamine Uptake through the Dopamine and Norepinephrine Transporters is Decreased in the Prefrontal Cortex of Transgenic Mice Expressing HIV-1 Transactivator of Transcription Protein.

Dysregulation of dopamine neurotransmission has been linked to the development of human immunodeficiency virus (HIV)-associated neurocognitive disorder (HAND). To investigate the mechanisms underlying this phenomenon, this study used an inducible HIV-1 transactivator of transcription (Tat) transgenic (iTat-tg) mouse model, which demonstrates brain-specific Tat expression induced by administration of doxycycline. We found that induction of Tat expression in the iTat-tg mice for either 7 or 14 days resulted in a decrease (∼30%) in the V max of [3H]dopamine uptake via both the dopamine transporter (DAT) and norepinephrine transporter (NET) in the prefrontal cortex (PFC), which was comparable to the magnitude (∼35%) of the decrease in B max for [3H]WIN 35,428 and [3H]nisoxetine binding to DAT and NET, respectively. The decreased V max was not accompanied by a reduction of total or plasma membrane expression of DAT and NET. Consistent with the decreased V max for DAT and NET in the PFC, the current study also found an increase in the tissue content of DA and dihydroxyphenylacetic acid in the PFC of iTat-tg mice after 7 days' administration of doxycycline. Electrophysiological recordings in layer V pyramidal neurons of the prelimbic cortex from iTat-tg mice found a significant reduction in action potential firing, which was not sensitive to selective inhibitors for DAT and NET, respectively. These findings provide a molecular basis for using the iTat-tg mouse model in the studies of NeuroHIV. Determining the mechanistic basis underlying the interaction between Tat and DAT/NET may reveal novel therapeutic possibilities for preventing the increase in comorbid conditions as well as HAND. SIGNIFICANCE STATEMENT: Human immunodeficiency virus (HIV)-1 infection disrupts dopaminergic neurotransmission, leading to HIV-associated neurocognitive disorders (HANDs). Based on our in vitro and in vivo studies, dopamine uptake via both dopamine and norepinephrine transporters is decreased in the prefrontal cortex of HIV-1 Tat transgenic mice, which is consistent with the increased dopamine and dihydroxyphenylacetic acid contents in this brain region. Thus, these plasma membrane transporters are an important potential target for therapeutic intervention for patients with HAND.

Rbfox1 Is Expressed in the Mouse Brain in the Form of Multiple Transcript Variants and Contains Functional E Boxes in Its Alternative Promoters.

The RNA-binding protein RBFOX1 is an important regulator of neuron development and neuronal excitability. Rbfox1 is a dosage-sensitive gene and in both mice and humans, decreased expression of Rbfox1 has been linked to neurodevelopmental disorders. Alternative promoters drive expression of Rbfox1 transcript isoforms that encode an identical protein. The tissue- and developmental stage-specific expression of these isoforms, as well as the underlying regulatory mechanisms, are, however, unclear. Here, we set out to capture all of the Rbfox1 transcript isoforms and identify transcriptional mechanisms that regulate brain-specific Rbfox1 expression. Isoform sequencing identified multiple alternative Rbfox1 transcript variants in the mouse cerebral cortex, including transcripts with novel first exons, alternatively spliced exons and 3′-truncations. Quantitative RT-PCR determined the expression of the alternative first exons in the developing cerebral cortex and different subregions of the juvenile brain. Alternative first exons were found to be highly stage- and subregion specific in their expression patterns suggesting that they fulfill specific functions during cortex development and in different brain regions. Using reporter assays we found that the promoter regions of the two first exons E1B and E1C/E1C.1 contain several functional E-boxes. Together, we provide an extensive picture of Rbfox1 isoform expression. We further identified important regulatory mechanisms that drive neuron-specific Rbfox1 expression. Thus, our study forms the basis for further research into the mechanisms that ensure physiological Rbfox1 expression in the brain. It also helps to understand why, in patients with neurodevelopmental disorders deletion of individual RBFOX1 transcript isoforms could affect brain function.

A chromosomal connectome for psychiatric and metabolic risk variants in adult dopaminergic neurons

BackgroundMidbrain dopaminergic neurons (MDN) represent 0.0005% of the brain’s neuronal population and mediate cognition, food intake, and metabolism. MDN are also posited to underlay the neurobiological dysfunction of schizophrenia (SCZ), a severe neuropsychiatric disorder that is characterized by psychosis as well as multifactorial medical co-morbidities, including metabolic disease, contributing to markedly increased morbidity and mortality. Paradoxically, however, the genetic risk sequences of psychosis and traits associated with metabolic disease, such as body mass, show very limited overlap.MethodsWe investigated the genomic interaction of SCZ with medical conditions and traits, including body mass index (BMI), by exploring the MDN’s “spatial genome,” including chromosomal contact landscapes as a critical layer of cell type-specific epigenomic regulation. Low-input Hi-C protocols were applied to 5–10 × 103 dopaminergic and other cell-specific nuclei collected by fluorescence-activated nuclei sorting from the adult human midbrain.ResultsThe Hi-C-reconstructed MDN spatial genome revealed 11 “Euclidean hot spots” of clustered chromatin domains harboring risk sequences for SCZ and elevated BMI. Inter- and intra-chromosomal contacts interconnecting SCZ and BMI risk sequences showed massive enrichment for brain-specific expression quantitative trait loci (eQTL), with gene ontologies, regulatory motifs and proteomic interactions related to adipogenesis and lipid regulation, dopaminergic neurogenesis and neuronal connectivity, and reward- and addiction-related pathways.ConclusionsWe uncovered shared nuclear topographies of cognitive and metabolic risk variants. More broadly, our PsychENCODE sponsored Hi-C study offers a novel genomic approach for the study of psychiatric and medical co-morbidities constrained by limited overlap of their respective genetic risk architectures on the linear genome.

Brain Cytoplasmic RNAs in Neurons: From Biosynthesis to Function.

Flexibility in signal transmission is essential for high-level brain function. This flexibility is achieved through strict spatial and temporal control of gene expression in neurons. Given the key regulatory roles of a variety of noncoding RNAs (ncRNAs) in neurons, studying neuron-specific ncRNAs provides an important basis for understanding molecular principles of brain function. This approach will have wide use in understanding the pathogenesis of brain diseases and in the development of therapeutic agents in the future. Brain cytoplasmic RNAs (BC RNAs) are a leading paradigm for research on neuronal ncRNAs. Since the first confirmation of brain-specific expression of BC RNAs in 1982, their investigation has been an area of active research. In this review, we summarize key studies on the characteristics and functions of BC RNAs in neurons.

Comparative Genomics Identifies Putative Signatures of Sociality in Spiders.

Comparative genomics has begun to elucidate the genomic basis of social life in insects, but insight into the genomic basis of spider sociality has lagged behind. To begin, to characterize genomic signatures associated with the evolution of social life in spiders, we performed one of the first spider comparative genomics studies including five solitary species and two social species, representing two independent origins of sociality in the genus Stegodyphus. We found that the two social spider species had a large expansion of gene families associated with transport and metabolic processes and an elevated genome-wide rate of molecular evolution compared with the five solitary spider species. Genes that were rapidly evolving in the two social species relative to the five solitary species were enriched for transport, behavior, and immune functions, whereas genes that were rapidly evolving in the solitary species were enriched for energy metabolism processes. Most rapidly evolving genes in the social species Stegodyphus dumicola were broadly expressed across four tissues and enriched for transport functions, but 12 rapidly evolving genes showed brain-specific expression and were enriched for social behavioral processes. Altogether, our study identifies putative genomic signatures and potential candidate genes associated with spider sociality. These results indicate that future spider comparative genomic studies, including broader sampling and additional independent origins of sociality, can further clarify the genomic causes and consequences of social life.

Deep sequencing of small non-coding RNA highlights brain-specific expression patterns and RNA cleavage

ABSTRACTWith the advance of high-throughput sequencing technology numerous new regulatory small RNAs have been identified, that broaden the variety of processing mechanisms and functions of non-coding RNA. Here we explore small non-coding RNA (sncRNA) expression in central parts of the physiological stress and anxiety response system. Therefore, we characterize the sncRNA profile of tissue samples from Amygdala, Hippocampus, Hypothalamus and Adrenal Gland, obtained from 20 pigs. Our analysis reveals that all tissues but Amygdala and Hippocampus possess distinct, tissue-specific expression pattern of miRNA that are associated with Hypoxia, stress responses as well as memory and fear conditioning. In particular, we observe marked differences in the expression profile of limbic tissues compared to those associated to the HPA/stress axis, with a surprisingly high aggregation of 3´-tRNA halves in Amygdala and Hippocampus. Since regulation of sncRNA and RNA cleavage plays a pivotal role in the central nervous system, our work provides seminal insights in the role/involvement of sncRNA in the transcriptional and post-transcriptional regulation of negative emotion, stress and coping behaviour in pigs, and mammals in general.

Persistent Neuroinflammation and Brain-Specific Immune Priming in a Novel Survival Model of Murine Pneumosepsis.

Pneumonia is the leading cause of sepsis and septic shock. Patients who survive pneumonia are vulnerable to long-term complications including increased risk of neurocognitive dysfunction. This study investigated the immune response and long-term complications of a non-surgical mouse model of Klebsiella pneumoniae pneumosepsis with antibiotic treatment. Pneumosepsis resulted in acutely enhanced expression of inflammatory cytokines, chemokines, and damage-associated molecular patterns in the brain and spleen. Despite resolution of infection, murine pneumosepsis survivors demonstrated a deficit in exploratory locomotor behavior at 2 weeks. This was associated with brain-specific persistent inflammatory gene expression and infiltrating myeloid cells in the brain. The brain inflammatory response was also primed in response to secondary challenge with lipopolysaccharide. The findings of this study demonstrate behavioral and inflammatory outcomes that parallel observations in other models of sepsis, but that have not previously been described in antibiotic-treated pneumonia models, highlighting a common pathway to the development of chronic brain dysfunction in sepsis survival.

Induction of chemokine CCL3 by NF-κB reduces methylmercury toxicity in C17.2 mouse neural stem cells

Methylmercury is an environmental pollutant that shows selective toxicity to the central nervous system. We previously reported that brain-specific expression of chemokine CCL3 increases in mice administered methylmercury. However, the relationship between CCL3 and methylmercury toxicity has not been elucidated. Here, we confirmed that induction of CCL3 expression occurs before pathological change by methylmercury treatment was observed in the mouse brain. This induction was also observed in C17.2 mouse neural stem cells before methylmercury-induced cytotoxicity. In addition, cells in which CCL3 was knocked-down showed higher methylmercury sensitivity than did control cells. Moreover, activation of transcription factor NF-κB was observed following methylmercury treatment, and methylmercury-mediated induction of CCL3 expression was partially suppressed by knockdown of p65, an NF-κB subunit. Our results suggest that NF-κB plays a role in the induction of methylmercury-mediated CCL3 expression and that this action may be a cellular response to methylmercury toxicity.

M79 - GENOME-WIDE DNA METHYLATION SIGNATURES OF AGGRESSION RISK IN CHILDREN

Background Child abuse is a highly prevalent public health problem and a risk factor for numerous psychopathological disorders. Early physical abuse and neglect have been associated with an increased risk of childhood conduct disorders. However, not all children exposed to abuse develop aggression later in life, suggesting interplay between genetic and environmental factors in the predisposition to aggression. Methods This study investigated genome-wide DNA methylation changes associated with aggressive behavior in children. A total of 105 9–15 years old children of European ancestry were included in the study (56% female, 12 ± 1.8 mean of age). The Illumina Infinium 450 K Methylation BeadChip array was used to conduct the methylation study using the genomic DNA extracted from saliva samples. Aggression was evaluated using the Child Behavior Checklist (CBCL) Scores ranged from 50–87 (mean: 57 ± 10.5), with 39% of the sample meeting the clinical cut-off (≤ 63). We conducted a linear mixed effects model to examine the main effect of trauma, main effect of DNA methylation and an interaction term, and adjusted for age, sex, cell composition, and 10 principal components (PCs) estimated using Barfield et al. method. Significance was set to 5.0×10-7 to correct for multiple comparisons. Results A total of eight methylation sites were genome-wide significant (GWS) for the DNA methylation term after adjusting for age, sex, cell type composition and 10 PCs. From these sites, cg18438793 (p=2.58×10-7) located within the hippocalcin like 4 (HPCAL4) gene is of importance due to its role in neuronal development and calcium signaling and brain-specific gene expression. Further, expression of this gene is induced after fear conditioning in the lateral amygdala of mice. Additional sites (p value range: 10-10 – 10-7) are involved in axon guidance, myelination and transcriptional regulation. Discussion This study increases our understanding of the epigenetic changes associated with aggression in children. By identifying predictors of aggression in early life, this study may provide novel tools to improve treatment and preventive interventions for aggression risk in children.

ADAR3 expression is an independent prognostic factor in lower-grade diffuse gliomas and positively correlated with the editing level of GRIA2Q607R

BackgroundRNA editing by adenosine deaminases acting on RNA (ADARs) converts adenosines to inosines (A-to-I) in RNA, that alters gene expression and generates protein diversity. Dysregulation of A-to-I editing has been found associated with a number of nervous system diseases. However, the role of ADAR3, a brain specific high expression adenosine deaminase, in gliomas has rarely been investigated. In this study we illuminated the clinical significance and molecular features of ADAR3 in patients with glioma.Methods309 glioma samples from Chinese Glioma Genome Atlas were enrolled into this study. In validation sets, 601 glioma samples in TCGA, 410 glioma samples in REMBRANDT and 258 glioma samples in GSE16011 were obtained. Relationships between ADAR3 expression and prognosis-related genomic alteration, outcome and gene ontology analysis were investigated. Moreover, the characteristic of GRIA2Q607R editing in gliomas has been investigated. Graphpad Prism 5.0, SPSS 16.0 and R language were used to perform statistical analysis and graphical work.ResultsADAR3 expression was down regulated along with glioma grade progression in CGGA dataset. ADAR3 was characteristically highly expressed in neural subtype and IDH1/2 mutant preference. Moreover, high expression of ADAR3 predicted a better prognosis in lower-grade glioma (LGG) patients and multivariate analysis suggested ADAR3 expression was an independent prognostic indicator. The results of the three other validation datasets showed similar findings. Bioinformatics analyses suggested that ADAR3 may play a role in the malignant transformation of glioma cells by affecting cell proliferation, angiogenesis or cell adhesion. Furthermore, the editing level of GRIA2Q607R was significantly correlated with ADAR3 expression.ConclusionsOur study demonstrated the clinical and molecular characterization of ADAR3 in glioma development and progression. ADAR3 expression was negatively associated with tumor malignant in the overall glioma patients. And it was a favorable independent prognostic indicator of LGG patients. ADAR3 appeared to act as a tumor suppressor in glioma cells. Therefore, ADAR3 represented a potential therapeutic target and useful prognostic factor for glioma patients.

Deciphering brain-specific transcriptomic expression of Ezr, Rad and Msn genes in the development of Mus musculus

Ezrin, Radixin and Moesin (ERM) are critical membranous component involved in cross-linking of actin filaments. Moesin (Msn) is recognized as a pivotal protein involved in regulation of cell signalling events associated with the maintenance of epithelial integrity, actin organization and polarity. Radixin (Rad) is known to cell-to-cell adherens junction as a barbed end-capping protein whereas ezrin (Ezr) is recognized at cell adhesion, motility, apoptosis and phagocytosis. The current study for the first time reports the transcriptional and RNA secondary structural variations among brain-specific ERM genes. Firstly, we analyzed brain-specific transcriptomic expression in selected embryonic and postnatal developmental stages (E10.5, E14.5, E18.5, P0.5, P3.5, P5.5, P10.5 and P20.5) of Mus musculus. Among designated developmental stages, Ezr has highest fold difference in early embryonic and postnatal stages (E10.5, P0.5 and P5.5). Rad showed a similar pattern of high expression especially at embryonic stages (E10.5 and E18.5) and postnatal (P0.5 and P5.5), however, Msn exhibited non-significant fold differences in comparison to controls leading to its curial role in development. Furthermore, computational prediction of ERM coding mRNA transcripts, reveals compact and less dynamic Msn secondary structure and pseudoknots configurations, in contrast to Ezr and Rad. Conclusively, transcriptomic levels are greatly associated with compact base pairing organization of its secondary structures. These findings open a new domain to understand the occurrence of ERM-specific cytoskeleton proteins during developmental stages.

Experimental Cerebral Ischemia Affects the Expression of Circular RNA Genes of Metabotropic Glutamate Receptors mGluR3 and mGluR5 in Rat Brain

Circular RNAs (circRNAs) are a new RNA type that show predominantly brain-specific expression pattern in mammals. Their individual representatives, acting as competitive endogenous RNAs, can bind microRNAs and contribute to the protection of functional transcripts. In the model of transient cerebral ischemia in rats, we studied the expression of mGluR3 and mGluR5 glutamate metabotropic receptor genes (Grm3 and Grm5). These genes are important participants in the metabolic pathways associated with neurosignaling. In the present study, we found that the rat Grm3 and Grm5 genes, in addition to mRNA, encode circRNAs, which are conserved in humans and rodents. In subcortical brain structures of rats containing a lesion focus, the level of these circRNAs is more stable than that of the corresponding mRNA. Using STarMirDB database analysis, the distribution of the microRNA binding sites along the mRNA molecules of human GRM3 and GRM5 genes which are homologous to the corresponding rat genes was elucidated. It has been revealed that sufficiently large number of binding sites is located within exons, which are also part of conservative circRNAs. The results may indicate the functional role of the circRNAs of the investigated genes as competitive endogenous RNAs in the response of brain cells to ischemia.

Effects of bisphenol A (BPA) on brain-specific expression of cyp19a1b gene in swim-up fry of Labeo rohita

Estrogen regulates numerous developmental and physiological processes and effects are mediated mainly by estrogenic receptors (ERs), which function as ligand-regulated transcription factor. ERs can be activated by many different types endocrine disrupting chemicals (EDCs) and interfere with behaviour and reproductive potential of living organism. Estrogenic regulation of membrane associated G protein-coupled estrogen receptor, GPER activity has also been reported. Bisphenol A (BPA), a ubiquitous endocrine disruptor is present in many household products, has been linked to many adverse effect on sexual development and reproductive potential of wild life species. The present work is aimed to elucidate how an environmentally pervasive chemical BPA affects in vivo expression of a known estrogen target gene, cyp19a1b in the brain, and a known estrogenic biomarker, vitellogenin (Vg) in the whole body homogenate of 30 days post fertilization (dpf) swim-up fry of Labeo rohita. We confirm that, like estrogen, the xenoestrogen BPA exposure for 5–15 days induces strong overexpression of cyp19a1b, but not cyp19a1a mRNA in the brain and increase concentration of vitellogenin in swim-up fry. BPA also induces strong overexpression of aromatase B protein and aromatase activity in brain. Experiments using selective modulators of classical ERs and GPER argue that this induction is largely through nuclear ERs, not through GPER. Thus, BPA has the potential to elevate the levels of aromatase and thereby, levels of endogenous estrogen in developing brain. These results indicate that L. rohita swim-up fry can be used to detect environmental endocrine disruptors either using cyp19a1b gene expression or vitellogenin induction.

Peculiarities of non-coding RNA functioning in the norm and cerebral ischemia

To date, it has been shown that not only information RNAs, but also various types of non-coding RNA, are involved in the transcriptome reaction in ischemia. In particular, non-coding RNAs can perform important protective functions, acting as competitive endogenous RNAs. They interact with microRNA, which negatively affects the expression of many mRNAs, and neutralize their activity. The particular interest is circular RNAs, which belong to the non-coding RNAs and can most effectively perform the functions of competitive RNAs. Circular RNAs demonstrate the increased resistance to exonucleases and the predominant brain-specific expression pattern, which may indicate their particular importance in this tissue as neuroprotective agents. This review demonstrates the most recent data on the structure and features of the functioning of noncoding RNAs, which indicate the important role of circular RNAs in cell in the norm and ischemia conditions.

RNA/DNA co-analysis from bloodstains on aged polyvinyl-alcohol gloves prepared for securing evidence from the hands of victims of fatal gunshot injuries.

In contrast to cumulative techniques (e.g., tape-lift) for qualitative gunshot residues (GSR) analysis, topographic methods are commonly applied to preserve the integrity of evidence from a shooter's or victim's hand in cases of gun-related crimes. Topographic sampling techniques employing adhesive foils, latex, or the polyvinyl alcohol (PVAL) method enable unambiguous sampling of biological and non-biological trace material while preserving its spatial distribution and relation to each other. The PVAL method in particular allows for a topographically veridic and quantitative conservation of traces of GSR and biological stains that are embedded in the PVAL glove, because it completely removes these traces from the hand. The present study investigated the success rates of STR profiling and the detection of blood and brain-specific gene expression from minimal traces of blood splatter as well as parallel to the positive detection of gunshot residues embedded in 17 PVAL gloves taken from the hands of deceased persons in the context of homicide cases in the period between 1996 and 2003. The water-soluble PVAL matrix is shown to be fully compatible with successful STR profiling and the detection of blood- and brain-specific miRNA expression, even after up to 20years of storage, demonstrating that this sampling technique offers advantages compared to other more simplistic sampling methods like taping.

Inflammation, Stem Cells, and the Aging Hypothalamus.

The aging hypothalamus has been hypothesized to play a key role in vertebrate aging. Increased inflammation in microglial cells during aging reduces the number of neural stem cells (NSCs) in the mediobasal region of the hypothalamus. Ectopic brain-specific expression of SIRT1 and localized hypothalamic expression of a dominantly acting nuclear factor kappa-light-chain-enhancer of activated B cells (NF-κB) inhibitor to microglia or to NSCs extend life span in mice. NSCs in the hypothalamus secrete miRNA-containing exosomes and gonadotropin-releasing hormone (GnRH) that oppose aging-associated neurological and skeletomuscular dysfunction. These results suggest that stem cells are not mere repositories of future differentiated cells, but can also be active physiological effectors. Development of drugs that attenuate microglial inflammation and/or promoted maintenance of NSC viability may have significant utility for extending health and life span.

The evolution of duplicate gene expression in mammalian organs.

Gene duplications generate genomic raw material that allows the emergence of novel functions, likely facilitating adaptive evolutionary innovations. However, global assessments of the functional and evolutionary relevance of duplicate genes in mammals were until recently limited by the lack of appropriate comparative data. Here, we report a large-scale study of the expression evolution of DNA-based functional gene duplicates in three major mammalian lineages (placental mammals, marsupials, egg-laying monotremes) and birds, on the basis of RNA sequencing (RNA-seq) data from nine species and eight organs. We observe dynamic changes in tissue expression preference of paralogs with different duplication ages, suggesting differential contribution of paralogs to specific organ functions during vertebrate evolution. Specifically, we show that paralogs that emerged in the common ancestor of bony vertebrates are enriched for genes with brain-specific expression and provide evidence for differential forces underlying the preferential emergence of young testis- and liver-specific expressed genes. Further analyses uncovered that the overall spatial expression profiles of gene families tend to be conserved, with several exceptions of pronounced tissue specificity shifts among lineage-specific gene family expansions. Finally, we trace new lineage-specific genes that may have contributed to the specific biology of mammalian organs, including the little-studied placenta. Overall, our study provides novel and taxonomically broad evidence for the differential contribution of duplicate genes to tissue-specific transcriptomes and for their importance for the phenotypic evolution of vertebrates.

Reactivity of anti-HNK-1 antibodies to branched O-mannose glycans associated with demyelination

Human natural killer-1 (HNK-1) epitope, a highly-expressed glycan in the nervous system, is critical for normal synaptic plasticity and spatial learning. HNK-1 epitope modifies N-glycans on several neural glycoproteins, and also modifies O-mannosyl glycans. A branching enzyme for O-mannosyl glycans (GnT-IX, Core M2 synthase) exhibits brain-specific expression, and the product core M2 glycans are also limited to the brain. In a previous study, we showed that cuprizone-induced demyelination increased HNK-1-capped core M2 glycan expression, while GnT-IX deficiency ameliorated demyelination, suggesting that these glycans could be useful diagnostic markers for demyelination status and act as therapeutic targets. Nevertheless, a lack of appropriate detection tools hampered further analysis of HNK-1-capped O-mannosyl glycans. In the present study, we chemoenzymatically synthesized HNK-1-capped core M2 glycans for antibody production, and confirmed that the resulting immune sera reacted with HNK-1-capped core M2 glycans. We then examined several HNK-1-related antibodies, including the Cat-315 antibody, for reactions with HNK-1-capped core M2 glycans. Finally, we confirmed the increased HNK-1 epitope expression in demyelinated brains of cuprizone-fed mice.

Automated deep-phenotyping of the vertebrate brain.

Here, we describe an automated platform suitable for large-scale deep-phenotyping of zebrafish mutant lines, which uses optical projection tomography to rapidly image brain-specific gene expression patterns in 3D at cellular resolution. Registration algorithms and correlation analysis are then used to compare 3D expression patterns, to automatically detect all statistically significant alterations in mutants, and to map them onto a brain atlas. Automated deep-phenotyping of a mutation in the master transcriptional regulator fezf2 not only detects all known phenotypes but also uncovers important novel neural deficits that were overlooked in previous studies. In the telencephalon, we show for the first time that fezf2 mutant zebrafish have significant patterning deficits, particularly in glutamatergic populations. Our findings reveal unexpected parallels between fezf2 function in zebrafish and mice, where mutations cause deficits in glutamatergic neurons of the telencephalon-derived neocortex.