Neuronal extracellular microRNAs miR-124 and miR-9 mediate cell-cell communication between neurons and microglia.

In contrast to peripheral macrophages, microglia in the central nervous system (CNS) exhibit a specific deactivated phenotype; however, it is not clear how this phenotype is maintained. Two alternative hypotheses were postulated recently: (a) microglia differ from peripheral macrophages being derived from the yolk sac (YS), whereas peripheral macrophages originate from bone marrow (BM); (b) microglia acquire a specific phenotype under the influence of the CNS microenvironment. We have previously shown that microglia express miR-124, which was also induced in BM-derived macrophages co-cultured with a neurons. We here investigated the possibility of horizontal transfer of the neuron-specific microRNAs miR-124 and miR-9 from primary neurons to microglia/macrophages. We found that after incubation with neuronal conditioned media (NCM), macrophages downregulated activation markers MHC class II and CD45. Neither cultured adult microglia nor YS- and BM-derived macrophages demonstrated intrinsic levels of miR-124 expression. However, after incubation with NCM, miR-124 was induced in both YS- and BM-derived macrophages. Biochemical analysis demonstrated that the NCM contained miR-124 and miR-9 in complex with small proteins, large high-density lipoproteins (HDLs), and exosomes. MiR-124 and miR-9 were promptly released from neurons, and this process was inhibited by tetrodotoxin, indicating an important role of neuronal electric activity in secretion of these microRNAs. Incubation of macrophages with exogenous miR-124 resulted in efficient translocation of miR-124 into the cytoplasm. This study demonstrates an important role of neuronal miRNAs in communication of neurons with microglia, which favors the hypothesis that microglia acquire a specific phenotype under the influence of the CNS microenvironment.

Chromosomal-Level Assembly of the Asian Seabass Genome Using Long Sequence Reads and Multi-layered Scaffolding.

We report here the ~670 Mb genome assembly of the Asian seabass (Lates calcarifer), a tropical marine teleost. We used long-read sequencing augmented by transcriptomics, optical and genetic mapping along with shared synteny from closely related fish species to derive a chromosome-level assembly with a contig N50 size over 1 Mb and scaffold N50 size over 25 Mb that span ~90% of the genome. The population structure of L. calcarifer species complex was analyzed by re-sequencing 61 individuals representing various regions across the species' native range. SNP analyses identified high levels of genetic diversity and confirmed earlier indications of a population stratification comprising three clades with signs of admixture apparent in the South-East Asian population. The quality of the Asian seabass genome assembly far exceeds that of any other fish species, and will serve as a new standard for fish genomics.

Correction: Chromosomal-Level Assembly of the Asian Seabass Genome Using Long Sequence Reads and Multi-layered Scaffolding.

[This corrects the article DOI: 10.1371/journal.pgen.1005954.].

Mouse chromocenters DNA content: sequencing and in silico analysis.

BACKGROUND: Chromocenters are defined as a punctate condensed blocks of chromatin in the interphase cell nuclei of certain cell types with unknown biological significance. In recent years a progress in revealing of chromocenters protein content has been made although the details of DNA content within constitutive heterochromatin still remain unclear. It is known that these regions are enriched in tandem repeats (TR) and transposable elements. Quick improvement of genome sequencing does not help to assemble the heterochromatic regions due to lack of appropriate bioinformatics techniques. RESULTS: Chromocenters DNA have been isolated by a biochemical approach from mouse liver cells nuclei and sequenced on the Illumina MiSeq resulting in ChrmC dataset. Analysis of ChrmC dataset by the bioinformatics tools available revealed that the major component of chromocenter DNA are TRs: ~ 66% MaSat and ~ 4% MiSat. Other previously classified TR families constitute ~ 1% of ChrmC dataset. About 6% of chromocenters DNA are mostly unannotated sequences. In the contigs assembled with IDBA_UD there are many fragments of heterochromatic Y-chromosome, rDNA and other pseudo-genes and non-coding DNA. A protein coding sfi1 homolog gene fragment was also found in contigs. The Sfi1 homolog gene is located on the chromosome 11 in the reference genome very close to the Golden Pass Gap (a ~ 3 Mb empty region reserved to the pericentromeric region) and proves the purity of chromocenters isolation. The second major fraction are non-LTR retroposons (SINE and LINE) with overwhelming majority of LINE - ~ 11% of ChrmC. Most of the LINE fragments are from the ~ 2 kb region at the end of the 2nd ORF and its' flanking region. The precise LINEs' segment of ~ 2 kb is the necessary mouse constitutive heterohromatin component together with TR. The third most abundant fraction are ERVs. The ERV distribution in chromocenters differs from the whole genome: IAP (ERV2 class) is the most numerous in ChrmC while MaLR (ERV3 class) prevails in the reference genome. IAP and its LTR also prevail in TR containing contigs extracted from the WGS dataset. In silico prediction of IAP and LINE fragments in chromocenters was confirmed by direct fluorescent in situ hybridization (FISH). CONCLUSION: Our data of chromocenters' DNA (ChrmC) sequencing demonstrate that IAP with LTR and a precise ~ 2 kb fragment of LINE represent a substantial fraction of mouse chromocenters (constitutive heteroсhromatin) along with TRs.

LINE-related component of mouse heterochromatin and complex chromocenters' composition.

Chromocenters are interphase nuclear landmark structures of constitutive heterochromatin. The tandem repeat (TR)-enriched parts of different chromosomes cluster together in chromocenters. There has been progress in recent years in determining the protein content of chromocenters, although it is not clear which DNA sequences underly constitutive heterochromatin apart from the TRs. The aim of the current work was to find out which DNA sequences besides TRs are involved in chromocenters' formation. Biochemically isolated chromocenters and microdissected centromeric regions were amplified by DOP-PCR, then cloned and sequenced. Alignment to Repbase, the mouse reference genome and WGS databases separated the sequences from both libraries into three groups: (1) sequences with similarity to pericentromere mouse major satellite; (2) sequences without similarity to any repetitive sequences; (3) sequences with similarity to long interspersed nuclear elements (LINEs). LINE-related sequences have a disperse pattern distribution on chromosomes predicted in silico. Selected clones were used for fluorescent in situ hybridization (FISH). The 10 clones tested hybridized to chromocenters and centromeric regions of metaphase chromosomes. These clones were used for double FISH with four known cloned TRs (satDNA, satellite DNA) and a probe specific for the sex chromosomes. The probes bind various chromocenters' regions without overlapping; so, FISH results reveal a complex chromocenter composition. We mapped 18 LINE-derived clones to the RepBase L1 records. Most of them grouped in a ∼2-kb region at the end of the second ORF and 3' untranslated region (UTR). So, even the limited number of the clones allows us to determine the region of the L1 element that is specific for heterochromatic regions. Although the L1 full-length probe did not hybridize at detectable levels to the heterochromatic region on any chromosome, the 2-kb fragment found is definitely a part of these regions. The precise LINE ∼2-kb fragment is the component of mouse and human constitutive heterochromatin enriched with TRs. The method used for amplification of the probes from two sources of the heterochromatic material uncovered the enrichment of a precise fragment of LINE within chromocenters.

Platelets promote epileptic seizures by modulating brain serotonin level, enhancing neuronal electric activity, and contributing to neuroinflammation and oxidative stress.

The drugs currently available for treating epilepsy are only partially effective in managing this condition. Therefore, it is crucial to investigate new pathways that induce and promote epilepsy development. Previously, we found that platelets interact with neuronal glycolipids and actively secrete pro-inflammatory mediators during central nervous system (CNS) pathological conditions such as neuroinflammation and traumatic brain injury (TBI). These factors increase the permeability of the blood-brain barrier (BBB), which may create a predisposition to epileptic seizures. In this study, we demonstrated that platelets substantially enhanced epileptic seizures in a mouse model of pentylenetetrazole (PTZ) -induced seizures. We found that platelets actively secreted serotonin, contributed to increased BBB permeability, and were present in the CNS parenchyma during epileptic seizures. Furthermore, platelets directly stimulated neuronal electric activity and induced the expression of specific genes related to early neuronal response, neuroinflammation, and oxidative phosphorylation, leading to oxidative stress in neurons. The intracranial injection of physiological numbers of platelets that mimicked TBI-associated bleeding was sufficient to induce severe seizures, which resembled conventional PTZ-induced epileptic activity. These findings highlight a conceptually new role of platelets in the development of epileptic seizures, and indicate a potential new therapeutic approach targeting platelets to prevent and treat epilepsy.

Fresh evidence for major brain gangliosides as a target for the treatment of Alzheimer's disease.

Although it was suggested that gangliosides play an important role in the binding of amyloid fragments to neuronal cells, the exact role of gangliosides in Alzheimer's disease (AD) pathology remains unclear. To understand the role of gangliosides in AD pathology in vivo, we crossed st3gal5-deficient (ST3-/-) mice that lack major brain gangliosides GM1, GD1a, GD3, GT1b, and GQ1b with 5XFAD transgenic mice that overexpress 3 mutant human amyloid proteins AP695 and 2 presenilin PS1 genes. We found that ST3-/- 5XFAD mice have a significantly reduced burden of amyloid depositions, low level of neuroinflammation, and did not exhibit neuronal loss or synaptic dysfunction. ST3-/- 5XFAD mice performed significantly better in a cognitive test than wild-type (WT) 5XFAD mice, which was comparable with WT nontransgenic mice. Treatment of WT 5XFAD mice with the sialic acid-specific Limax flavus agglutinin resulted in substantial improvement of AD pathology to a level of ST3-/- 5XFAD mice. Thus, our findings highlight an important role for gangliosides as a target for the treatment of AD.

Cyclic AMP Pathway Suppress Autoimmune Neuroinflammation by Inhibiting Functions of Encephalitogenic CD4 T Cells and Enhancing M2 Macrophage Polarization at the Site of Inflammation.

Although it has been demonstrated that cAMP pathway affect both adaptive and innate cell functions, the role of this pathway in the regulation of T-cell-mediated central nervous system (CNS) autoimmune inflammation, such as in experimental autoimmune encephalomyelitis (EAE), remains unclear. It is also unclear how cAMP pathway affects the function of CD4 T cells in vivo at the site of inflammation. We found that adenylyl cyclase activator Forskolin besides inhibition of functions autoimmune CD4 T cells also upregulated microRNA (miR)-124 in the CNS during EAE, which is associated with M2 phenotype of microglia/macrophages. Our study further established that in addition to direct influence of cAMP pathway on CD4 T cells, stimulation of this pathway promoted macrophage polarization toward M2 leading to indirect inhibition of function of T cells in the CNS. We demonstrated that Forskolin together with IL-4 or with Forskolin together with IL-4 and IFNγ effectively stimulated M2 phenotype of macrophages indicating high potency of this pathway in reprogramming of macrophage polarization in Th2- and even in Th1/Th2-mixed inflammatory conditions such as EAE. Mechanistically, Forskolin and/or IL-4 activated ERK pathway in macrophages resulting in the upregulation of M2-associated molecules miR-124, arginase (Arg)1, and Mannose receptor C-type 1 (Mrc1), which was reversed by ERK inhibitors. Administration of Forskolin after the onset of EAE substantially upregulated M2 markers Arg1, Mrc1, Fizz1, and Ym1 and inhibited M1 markers nitric oxide synthetase 2 and CD86 in the CNS during EAE resulting in decrease in macrophage/microglia activation, lymphocyte and CD4 T cell infiltration, and the recovery from the disease. Forskolin inhibited proliferation and IFNγ production by CD4 T cells in the CNS but had rather weak direct effect on proliferation of autoimmune T cells in the periphery and in vitro, suggesting prevalence of indirect effect of Forskolin on differentiation and functions of autoimmune CD4 T cells in vivo. Thus, our data indicate that Forskolin has potency to skew balance toward M2 affecting ERK pathway in macrophages and indirectly inhibit pathogenic CD4 T cells in the CNS leading to the suppression of autoimmune inflammation. These data may have also implications for future therapeutic approaches to inhibit autoimmune Th1 cells at the site of tissue inflammation.

A chromosome-level genome assembly of the Asian arowana, Scleropages formosus.

Asian arowana (Scleropages formosus), an ancient teleost belonging to the Order Osteoglossomorpha, has been a valuable ornamental fish with some varieties. However, its biological studies and breeding germplasm have been remarkably limited by the lack of a reference genome. To solve these problems, here we report high-quality genome sequences of three common varieties of Asian arowana (the golden, red and green arowana). We firstly generated a chromosome-level genome assembly of the golden arowana, on basis of the genetic linkage map constructed with the restriction site-associated DNA sequencing (RAD-seq). In addition, we obtained draft genome assemblies of the red and green varieties. Finally, we annotated 22,016, 21,256 and 21,524 protein-coding genes in the genome assemblies of golden, red and green varieties respectively. Our data were deposited in publicly accessible repositories to promote biological research and molecular breeding of Asian arowana.

The Asian arowana (Scleropages formosus) genome provides new insights into the evolution of an early lineage of teleosts.

The Asian arowana (Scleropages formosus), one of the world's most expensive cultivated ornamental fishes, is an endangered species. It represents an ancient lineage of teleosts: the Osteoglossomorpha. Here, we provide a high-quality chromosome-level reference genome of a female golden-variety arowana using a combination of deep shotgun sequencing and high-resolution linkage mapping. In addition, we have also generated two draft genome assemblies for the red and green varieties. Phylogenomic analysis supports a sister group relationship between Osteoglossomorpha (bonytongues) and Elopomorpha (eels and relatives), with the two clades together forming a sister group of Clupeocephala which includes all the remaining teleosts. The arowana genome retains the full complement of eight Hox clusters unlike the African butterfly fish (Pantodon buchholzi), another bonytongue fish, which possess only five Hox clusters. Differential gene expression among three varieties provides insights into the genetic basis of colour variation. A potential heterogametic sex chromosome is identified in the female arowana karyotype, suggesting that the sex is determined by a ZW/ZZ sex chromosomal system. The high-quality reference genome of the golden arowana and the draft assemblies of the red and green varieties are valuable resources for understanding the biology, adaptation and behaviour of Asian arowanas.

Primary analysis of repeat elements of the Asian seabass (Lates calcarifer) transcriptome and genome.

As part of our Asian seabass genome project, we are generating an inventory of repeat elements in the genome and transcriptome. The karyotype showed a diploid number of 2n = 24 chromosomes with a variable number of B-chromosomes. The transcriptome and genome of Asian seabass were searched for repetitive elements with experimental and bioinformatics tools. Six different types of repeats constituting 8-14% of the genome were characterized. Repetitive elements were clustered in the pericentromeric heterochromatin of all chromosomes, but some of them were preferentially accumulated in pretelomeric and pericentromeric regions of several chromosomes pairs and have chromosomes specific arrangement. From the dispersed class of fish-specific non-LTR retrotransposon elements Rex1 and MAUI-like repeats were analyzed. They were wide-spread both in the genome and transcriptome, accumulated on the pericentromeric and peritelomeric areas of all chromosomes. Every analyzed repeat was represented in the Asian seabass transcriptome, some showed differential expression between the gonads. The other group of repeats analyzed belongs to the rRNA multigene family. FISH signal for 5S rDNA was located on a single pair of chromosomes, whereas that for 18S rDNA was found on two pairs. A BAC-derived contig containing rDNA was sequenced and assembled into a scaffold containing incomplete fragments of 18S rDNA. Their assembly and chromosomal position revealed that this part of Asian seabass genome is extremely rich in repeats containing evolutionarily conserved and novel sequences. In summary, transcriptome assemblies and cDNA data are suitable for the identification of repetitive DNA from unknown genomes and for comparative investigation of conserved elements between teleosts and other vertebrates.

New types of mouse centromeric satellite DNAs.

Genomic databases do not contain complete sequences of the centromeric regions. We created a pUC19-based library of DNA fragments from isolated chromocentres of interphase nuclei. In this library we have found major satellite (MaSat) and two new satellite sequences - MS3 and MS4. The computer analysis of MS3 and MS4 sequences by alignment, fragment curved state and search for MAR motifs in comparison with the mouse major and minor satellite (MiSat) DNA has shown them to be new satellite fragments. Southern blot of MS3 and MS4 with total DNA digested by restriction enzymes shows the ladder characteristic of satellite DNA. 2.2% of the total DNA consists of MS3, the monomer of which is 150 bp long. The MS4 monomer is 300 bp long and accounts for 1.6% of the total DNA. On metaphase chromosomes MS3 and MS4 are located at the centromeric region. FISH analysis of L929 nuclei during the cell cycle showed relative positions of MaSat, MiSat, MS3, and MS4. All mapped satDNA fragments except MaSat belong to the outer layer of the chromocentres in the G0/G1 phase. MS3 is likely to be involved in the centromere formation. The mouse genome contains at least four satDNA types: AT-rich (MaSat and MiSat), and CG-rich (MS3 and MS4).