Ketone Bodies as Metabolites and Signalling Molecules at the Crossroad between Inflammation and Epigenetic Control of Cardiometabolic Disorders.

For many years, it has been clear that a Western diet rich in saturated fats and sugars promotes an inflammatory environment predisposing a person to chronic cardiometabolic diseases. In parallel, the emergence of ketogenic diets, deprived of carbohydrates and promoting the synthesis of ketone bodies imitating the metabolic effects of fasting, has been shown to provide a possible nutritional solution to alleviating diseases triggered by an inflammatory environment. The main ketone body, ß-hydroxybutyrate (BHB), acts as an alternative fuel, and also as a substrate for a novel histone post-translational modification, ß-hydroxybutyrylation. ß-hydroxybutyrylation influences the state of chromatin architecture and promotes the transcription of multiple genes. BHB has also been shown to modulate inflammation in chronic diseases. In this review, we discuss, in the pathological context of cardiovascular risks, the current understanding of how ketone bodies, or a ketogenic diet, are able to modulate, trigger, or inhibit inflammation and how the epigenome and chromatin remodeling may be a key contributor.

Grape ASR-Silencing Sways Nuclear Proteome, Histone Marks and Interplay of Intrinsically Disordered Proteins.

In order to unravel the functions of ASR (Abscisic acid, Stress, Ripening-induced) proteins in the nucleus, we created a new model of genetically transformed grape embryogenic cells by RNAi-knockdown of grape ASR (VvMSA). Nuclear proteomes of wild-type and VvMSA-RNAi grape cell lines were analyzed by quantitative isobaric tagging (iTRAQ 8-plex). The most significantly up- or down-regulated nuclear proteins were involved in epigenetic regulation, DNA replication/repair, transcription, mRNA splicing/stability/editing, rRNA processing/biogenesis, metabolism, cell division/differentiation and stress responses. The spectacular up-regulation in VvMSA-silenced cells was that of the stress response protein VvLEA D-29 (Late Embryogenesis Abundant). Both VvMSA and VvLEA D-29 genes displayed strong and contrasted responsiveness to auxin depletion, repression of VvMSA and induction of VvLEA D-29. In silico analysis of VvMSA and VvLEA D-29 proteins highlighted their intrinsically disordered nature and possible compensatory relationship. Semi-quantitative evaluation by medium-throughput immunoblotting of eighteen post-translational modifications of histones H3 and H4 in VvMSA-knockdown cells showed significant enrichment/depletion of the histone marks H3K4me1, H3K4me3, H3K9me1, H3K9me2, H3K36me2, H3K36me3 and H4K16ac. We demonstrate that grape ASR repression differentially affects members of complex nucleoprotein structures and may not only act as molecular chaperone/transcription factor, but also participates in plant responses to developmental and environmental cues through epigenetic mechanisms.

Lysines Acetylome and Methylome Profiling of H3 and H4 Histones in Trichostatin A-Treated Stem Cells.

Trichostatin A ([R-(E,E)]-7-[4-(dimethylamino) phenyl]-N-hydroxy- 4,6-dimethyl- 7-oxo-2,4-heptadienamide, TSA) affects chromatin state through its potent histone deacetylase inhibitory activity. Interfering with the removal of acetyl groups from lysine residues in histones is one of many epigenetic regulatory processes that control gene expression. Histone deacetylase inhibition drives cells toward the differentiation stage, favoring the activation of specific genes. In this paper, we investigated the effects of TSA on H3 and H4 lysine acetylome and methylome profiling in mice embryonic stem cells (ES14), treated with trichostatin A (TSA) by using a new, untargeted approach, consisting of trypsin-limited proteolysis experiments coupled with MALDI-MS and LC-MS/MS analyses. The method was firstly set up on standard chicken core histones to probe the optimized conditions in terms of enzyme:substrate (E:S) ratio and time of proteolysis and, then, applied to investigate the global variations of the acetylation and methylation state of lysine residues of H3 and H4 histone in the embryonic stem cells (ES14) stimulated by TSA and addressed to differentiation. The proposed strategy was found in its simplicity to be extremely effective in achieving the identification and relative quantification of some of the most significant epigenetic modifications, such as acetylation and lysine methylation. Therefore, we believe that it can be used with equal success in wider studies concerning the characterization of all epigenetic modifications.

Comparative Proteomic Analysis of Histone Modifications upon Acridone Derivative 8a-Induced CCRF-CEM Cells by Data Independent Acquisition.

The lead compound acridone derivative 8a showed potent antiproliferative activity by inducing DNA damage through direct stacking with DNA bases and triggering ROS in CCRF-CEM cells. To define the chromatin alterations during DNA damage sensing and repair, a detailed quantitative map of single and coexisting histone post-translational modifications (PTMs) in CCRF-CEM cells affected by 8a was performed by the Data Independent Acquisition (DIA) method on QE-plus. A total of 79 distinct and 164 coexisting histone PTMs were quantified, of which 16 distinct histone PTMs were significantly altered when comparing 8a-treated cells with vehicle control cells. The changes in histone PTMs were confirmed by Western blotting analysis for three H3 and one H4 histone markers. The up-regulated dimethylation on H3K9, H3K36, and H4K20 implied that CCRF-CEM cells might accelerate DNA damage repair to counteract the DNA lesion induced by 8a, which was verified by an increment in the 53BP1 foci localization at the damaged DNA. Most of the significantly altered PTMs were involved in transcriptional regulation, including down-regulated acetylation on H3K18, H3K27, and H3K122, and up-regulated di- and trimethylation on H3K9 and H3K27. This transcription-silencing phenomenon was associated with G2/M cell cycle arrest after 8a treatment by flow cytometry. This study shows that the DIA proteomics strategy provides a sensitive and accurate way to characterize the coexisting histone PTMs changes and their cross-talk in CCRF-CEM cells after 8a treatment. Specifically, histone PTMs rearrange transcription-silencing, and cell cycle arrest DNA damage repair may contribute to the mechanism of epigenetic response affected by 8a.

Sulfo-NHS-SS-biotin derivatization: a versatile tool for MALDI mass analysis of PTMs in lysine-rich proteins.

The discovery of PTMs in proteins by MS requires nearly complete sequence coverage of the detected proteolytic peptides. Unfortunately, mass spectrometric analysis of the desired sequence fragments is often impeded due to low ionization efficiency and/or signal suppression in complex samples. When several lysine residues are in close proximity tryptic peptides may be too short for mass analysis. Moreover, modified peptides often appear in low stoichiometry and need to be enriched before analysis. We present here how the use of sulfo-NHS-SS-biotin derivatization of lysine side chain can help to detect PTMs in lysine-rich proteins. This label leads to a mass shift which can be adjusted by reduction of the SS bridge and alkylation with different reagents. Low intensity peptides can be enriched by use of streptavidin beads. Using this method, the functionally relevant protein kinase A phosphorylation site in 5-lipoxygenase was detected for the first time by MS. Additionally, methylation and acetylation could be unambiguously determined in histones.

Environmental conditions elicit a slow but enduring response of histone post-translational modifications in Mozambique tilapia.

This study sheds new light on the timescale through which histone post-translational modifications (PTMs) respond to environmental stimuli, demonstrating that the histone PTM response does not necessarily precede the proteomic response or acclimation. After a variety of salinity treatments were administered to Mozambique tilapia (Oreochromis mossambicus) throughout their lifetimes, we quantified 343 histone PTMs in the gills of each fish. We show here that histone PTMs differ dramatically between fish exposed to distinct environmental conditions for 18 months, and that the majority of these histone PTM alterations persist for at least 4 weeks, irrespective of further salinity changes. However, histone PTMs respond minimally to 4-week-long periods of salinity acclimation during adulthood. The results of this study altogether signify that patterns of histone PTMs in individuals reflect their prolonged exposure to environmental conditions.

Ketogenic diet administration to mice after a high-fat-diet regimen promotes weight loss, glycemic normalization and induces adaptations of ketogenic pathways in liver and kidney.

OBJECTIVE: The ketogenic diet (KD), characterized by very limited dietary carbohydrate intake and used as nutritional treatment for GLUT1-deficiency syndromes and pharmacologically refractory epilepsy, may promote weight loss and improve metabolic fitness, potentially alleviating the symptoms of osteoarthritis. Here, we have studied the effects of administration of a ketogenic diet in mice previously rendered obese by feeding a high fat diet (HFD) and submitted to surgical destabilization of the medial meniscus to mimic osteoarthritis. METHODS: 6-weeks old mice were fed an HFD for 10 weeks and then switched to a chow diet (CD), KD or maintained on a HFD for 8 weeks. Glycemia, ß-hydroxybutyrate (BHB), body weight and fat mass were compared among groups. In liver and kidney, protein expression and histone post-translational modifications were assessed by Western blot, and gene expression by quantitative Real-Time PCR. RESULTS: After a 10 weeks HDF feeding, administration for 8 weeks of a KD or CD induced a comparable weight loss and decrease in fat mass, with better glycemic normalization in the KD group. Histone ß-hydroxybutyrylation, but not histone acetylation, was increased in the liver and kidney of mice fed the KD and the rate-limiting ketogenic enzyme HMGCS2 was upregulated - at the gene and protein level - in liver and, to an even greater extent, in kidney. KD-induced HMGCS2 overexpression may be dependent on FGF21, whose gene expression was increased by KD in liver. CONCLUSIONS: Over a period of 8 weeks, KD is more effective than a chow diet to induce metabolic normalization. Besides acting as a fuel molecule, BHB may exert its metabolic effects through modulation of the epigenome - via histone ß-hydroxybutyrylation - and extensive transcriptional modulation in liver and kidney.

The dCypher Approach to Interrogate Chromatin Reader Activity Against Posttranslational Modification-Defined Histone Peptides and Nucleosomes.

Bulk chromatin encompasses complex sets of histone posttranslational modifications (PTMs) that recruit (or repel) the diverse reader domains of Chromatin-Associated Proteins (CAPs) to regulate genome processes (e.g., gene expression, DNA repair, mitotic transmission). The binding preference of reader domains for their PTMs mediates localization and functional output, and are often dysregulated in disease. As such, understanding chromatin interactions may lead to novel therapeutic strategies, However the immense chemical diversity of histone PTMs, combined with low-throughput, variable, and nonquantitative methods, has defied accurate CAP characterization. This chapter provides a detailed protocol for dCypher, a novel approach for the rapid, quantitative interrogation of CAPs (as mono- or multivalent Queries) against large panels (10s to 100s) of PTM-defined histone peptide and semisynthetic nucleosomes (the potential Targets). We describe key optimization steps and controls to generate robust binding data. Further, we compare the utility of histone peptide and nucleosome substrates in CAP studies, outlining important considerations in experimental design and data interpretation.

Epigenetic regulation in the shoot apical meristem.

Epigenetic mechanisms form the basis of cellular memory, developmental decisions, and the cellular immune system that defends against transposons and viruses. Organs develop from the shoot apical meristem (SAM) to shape the plant's areal phenotype, and stem cells in the SAM serve as a functional germline. While many details on the regulation of stem cell pool size, organ initiation, and patterning at the meristem periphery are known, we know surprisingly little about the molecular characteristics of SAM cells, including their epigenome and how it changes during development. Here, we summarize information on epigenetic regulation of selected genes necessary for stem cell maintenance. As recent evidence suggests that SAM stem cells might be a hotspot of transposon activation, we discuss this aspect of epigenetic control in the meristem and speculate on mechanisms that maintain the flexibility of SAM stem cells in response to developmental or environmental cues.

Genome-wide occupancy reveals the localization of H1T2 (H1fnt) to repeat regions and a subset of transcriptionally active chromatin domains in rat spermatids.

BACKGROUND: H1T2/H1FNT is a germ cell-specific linker histone variant expressed during spermiogenesis specifically in round and elongating spermatids. Infertile phenotype of homozygous H1T2 mutant male mice revealed the essential function of H1T2 for the DNA condensation and histone-to-protamine replacement in spermiogenesis. However, the mechanism by which H1T2 imparts the inherent polarity within spermatid nucleus including the additional protein partners and the genomic domains occupied by this linker histone are unknown. RESULTS: Sequence analysis revealed the presence of Walker motif, SR domains and putative coiled-coil domains in the C-terminal domain of rat H1T2 protein. Genome-wide occupancy analysis using highly specific antibody against the CTD of H1T2 demonstrated the binding of H1T2 to the LINE L1 repeat elements and to a significant percentage of the genic regions (promoter-TSS, exons and introns) of the rat spermatid genome. Immunoprecipitation followed by mass spectrometry analysis revealed the open chromatin architecture of H1T2 occupied chromatin encompassing the H4 acetylation and other histone PTMs characteristic of transcriptionally active chromatin. In addition, the present study has identified the interacting protein partners of H1T2-associated chromatin mainly as nucleo-skeleton components, RNA-binding proteins and chaperones. CONCLUSIONS: Linker histone H1T2 possesses unique domain architecture which can account for the specific functions associated with chromatin remodeling events facilitating the initiation of histone to transition proteins/protamine transition in the polar apical spermatid genome. Our results directly establish the unique function of H1T2 in nuclear shaping associated with spermiogenesis by mediating the interaction between chromatin and nucleo-skeleton, positioning the epigenetically specialized chromatin domains involved in transcription coupled histone replacement initiation towards the apical pole of round/elongating spermatids.

Epigenetic mechanisms regulating COVID-19 infection.

Coronavirus disease 2019 (COVID-2019) outbreak originating in December 2019 in Wuhan, China has emerged as a global threat to human health. The highly contagious SARS-CoV-2 infection and transmission presents a diversity of human host and increased disease risk with advancing age, highlighting the importance of in-depth understanding of its biological properties. Structural analyses have elucidated hot spots in viral binding domains, mutations, and specific proteins in the host such as the receptor angiotensin-converting enzyme 2 (ACE2) and the transmembrane protease serine 2 (TMPRSS2) to be implicated in cell entry and viral infectivity. Furthermore, epigenetic changes that regulate chromatin structure have shown a major impact in genome stabilization and maintenance of cellular homoeostasis and they have been implicated in the pathophysiology of the virus infection. Epigenetic research has revealed that global DNA methylation along with ACE2 gene methylation and post-translational histone modifications may drive differences in host tissue-, biological age- and sex-biased patterns of viral infection. Moreover, modulation of the host cells epigenetic landscape following infection represents a molecular tool used by viruses to antagonize cellular signalling as well as sensing components that regulate the induction of the host innate immune and antiviral defence programmes in order to enhance viral replication and infection efficiency. In this review, we provide an update of the main research findings at the interface of epigenetics and coronavirus infection. In particular, we highlight the epigenetic factors that interfere with viral replication and infection and may contribute to COVID-19 susceptibility, offering new ways of thinking in respect to host viral response.

LC-MALDI-TOF ISD MS analysis is an effective, simple and rapid method of investigation for histones characterization: Application to EBV lymphoblastoid cell lines.

This contribution is the result of our progressive engagement to develop and to apply a top-down liquid chromatography (LC) matrix-assisted laser desorption/ionization (MALDI) time-of-flight (TOF) (LC-MALDI-TOF) analysis for the histone post-translational modifications (PTMs) and variants characterization, mainly in order to provide comprehensive and fast results. The histone post-translational modifications and the differential expression of the histone variants play an essential role both in the DNA packaging mechanism in chromosomes and in the regulation of gene expression in different cellular processes, also in response to molecular agents of environmental origin. This epigenetic mechanism is widely studied in different field such as cellular differentiation, development and in the understanding of mechanisms underlying diseases. The characterization of histone PTMs has traditionally performed by antibodies-based assay, but immunological methods have significant limits, and today systems that use mass spectrometry are increasingly employed. We evaluated an in-source decay (ISD) analysis for the histone investigation on human lymphoblastoid cells, and by this approach, we were able to identify and quantify several PTMs such as the di-methylation in the lysine 20 and the acetylation in the lysine 16 in H4 and the mono-methylation, di-methylation and trimethylations at K9 of the histone H3.1. Moreover, we detected and quantified in the same H2B spectrum the prevalent H2B 1C/2E type but also the minor H2B 1D, 1M and 1B/1L/1N, 1O/2F, 1J/1K variants. In this work, we show that MALDI-ISD represents an excellent methodology to obtain global information on histone PTMs and variants from cells in culture, with rapidity and simplicity of execution. Finally, this is a useful approach to get label-free relative quantitative data of histone variants and PTMs.

Biosynthesis of Fusapyrone Depends on the H3K9 Methyltransferase, FmKmt1, in Fusarium mangiferae.

The phytopathogenic fungus Fusarium mangiferae belongs to the Fusarium fujikuroi species complex (FFSC). Members of this group cause a wide spectrum of devastating diseases on diverse agricultural crops. F. mangiferae is the causal agent of the mango malformation disease (MMD) and as such detrimental for agriculture in the southern hemisphere. During plant infection, the fungus produces a plethora of bioactive secondary metabolites (SMs), which most often lead to severe adverse defects on plants health. Changes in chromatin structure achieved by posttranslational modifications (PTM) of histones play a key role in regulation of fungal SM biosynthesis. Posttranslational tri-methylation of histone 3 lysine 9 (H3K9me3) is considered a hallmark of heterochromatin and established by the SET-domain protein Kmt1. Here, we show that FmKmt1 is involved in H3K9me3 in F. mangiferae. Loss of FmKmt1 only slightly though significantly affected fungal hyphal growth and stress response and is required for wild type-like conidiation. While FmKmt1 is largely dispensable for the biosynthesis of most known SMs, removal of FmKMT1 resulted in an almost complete loss of fusapyrone and deoxyfusapyrone, γ-pyrones previously only known from Fusarium semitectum. Here, we identified the polyketide synthase (PKS) FmPKS40 to be involved in fusapyrone biosynthesis, delineate putative cluster borders by co-expression studies and provide insights into its regulation.

Chromatin Immunoprecipitation (ChIP) to Study DNA-Protein Interactions.

Chromatin immunoprecipitation (ChIP) is a method used to examine the genomic localization of a target of interest (e.g., proteins, protein posttranslational modifications, or DNA elements). As ChIP provides a snapshot of in vivo DNA-protein interactions, it lends insight to the mechanisms of gene expression and genome regulation. This chapter provides a detailed protocol focused on native-ChIP (N-ChIP), a robust approach to profile stable DNA-protein interactions. We also describe best practices for ChIP , including defined controls to ensure specific and efficient target enrichment and methods for data normalization.

An immune epigenetic insight to COVID-19 infection.

Severe acute respiratory syndrome coronavirus-2 is a positive-sense RNA virus, a causal agent of ongoing COVID-19 pandemic. ACE2R methylation across three CpG sites (cg04013915, cg08559914, cg03536816) determines the host cell's entry. It regulates ACE2 expression by controlling the SIRT1 and KDM5B activity. Further, it regulates Type I and III IFN response by modulating H3K27me3 and H3K4me3 histone mark. SARS-CoV-2 protein with bromodomain and protein E mimics bromodomain histones and evades from host immune response. The 2'-O MTases mimics the host's cap1 structure and plays a vital role in immune evasion through Hsp90-mediated epigenetic process to hijack the infected cells. Although the current review highlighted the critical epigenetic events associated with SARS-CoV-2 immune evasion, the detailed mechanism is yet to be elucidated.

Histone post-translational modifications in Silene latifolia X and Y chromosomes suggest a mammal-like dosage compensation system.

Silene latifolia is a model organism to study evolutionary young heteromorphic sex chromosome evolution in plants. Previous research indicates a Y-allele gene degeneration and a dosage compensation system already operating. Here, we propose an epigenetic approach based on analysis of several histone post-translational modifications (PTMs) to find the first epigenetic hints of the X:Y sex chromosome system regulation in S. latifolia. Through chromatin immunoprecipitation we interrogated six genes from X and Y alleles. Several histone PTMS linked to DNA methylation and transcriptional repression (H3K27me3, H3K23me, H3K9me2 and H3K9me3) and to transcriptional activation (H3K4me3 and H4K5, 8, 12, 16ac) were used. DNA enrichment (Immunoprecipitated DNA/input DNA) was analyzed and showed three main results: (i) promoters of the Y allele are associated with heterochromatin marks, (ii) promoters of the X allele in males are associated with activation of transcription marks and finally, (iii) promoters of X alleles in females are associated with active and repressive marks. Our finding indicates a transcription activation of X allele and transcription repression of Y allele in males. In females we found a possible differential regulation (up X1, down X2) of each female X allele. These results agree with the mammal-like epigenetic dosage compensation regulation.

Histone Post-Translational Modifications and CircRNAs in Mouse and Human Spermatozoa: Potential Epigenetic Marks to Assess Human Sperm Quality.

Spermatozoa (SPZ) are motile cells, characterized by a cargo of epigenetic information including histone post-translational modifications (histone PTMs) and non-coding RNAs. Specific histone PTMs are present in developing germ cells, with a key role in spermatogenic events such as self-renewal and commitment of spermatogonia (SPG), meiotic recombination, nuclear condensation in spermatids (SPT). Nuclear condensation is related to chromatin remodeling events and requires a massive histone-to-protamine exchange. After this event a small percentage of chromatin is condensed by histones and SPZ contain nucleoprotamines and a small fraction of nucleohistone chromatin carrying a landascape of histone PTMs. Circular RNAs (circRNAs), a new class of non-coding RNAs, characterized by a nonlinear back-spliced junction, able to play as microRNA (miRNA) sponges, protein scaffolds and translation templates, have been recently characterized in both human and mouse SPZ. Since their abundance in eukaryote tissues, it is challenging to deepen their biological function, especially in the field of reproduction. Here we review the critical role of histone PTMs in male germ cells and the profile of circRNAs in mouse and human SPZ. Furthermore, we discuss their suggested role as novel epigenetic biomarkers to assess sperm quality and improve artificial insemination procedure.

Histone Native Chromatin Immunoprecipitation.

Chromatin immunoprecipitation (ChIP) is becoming the standard method to study genome-wide distribution of histone variants and histone posttranslational modifications (PTMs). In this chapter, we describe a detailed native ChIP protocol and downstream procedures for the preparation of DNA libraries for next-generation sequencing. Compared to cross-linked ChIP, "native" ChIP has been shown to produce occupancy pattern data of histone PTMs and histone variants, with higher resolution and higher signal to noise ratio. We further present an adaptation of this protocol to perform native ChIP from as low as 50,000 cells.

Identification of novel post-translational modifications in linker histones from chicken erythrocytes.

Chicken erythrocyte nuclei were digested with micrococcal nuclease and fractionated by centrifugation in low-salt buffer into soluble and insoluble fractions. Post-translational modifications of the purified linker histones of both fractions were analyzed by LC-ESI-MS/MS. All six histone H1 subtypes (H1.01, H1.02, H1.03, H1.10, H1.1L and H1.1R) and histone H5 were identified. Mass spectrometry analysis enabled the identification of a wide range of PTMs, including N(α)-terminal acetylation, acetylation, formylation, phosphorylation and oxidation. A total of nine new modifications in chicken linker histones were mapped, most of them located in the N-terminal and globular domains. Relative quantification of the modified peptides showed that linker histone PTMs were differentially distributed among both chromatin fractions, suggesting their relevance in the regulation of chromatin structure. The analysis of our results combined with previously reported data for chicken and some mammalian species showed that most of the modified positions were conserved throughout evolution, highlighting their importance in specific linker histone functions and epigenetics. BIOLOGICAL SIGNIFICANCE: Post-translational modifications of linker histones could have a role in the regulation of gene expression through the modulation of chromatin higher-order structure and chromatin remodeling. Finding new PTMs in linker histones is the first step to elucidate their role in the histone code. In this manuscript we report nine new post-translational modifications of the linker histones from chicken erythrocytes, one in H5 and eight in the H1 subtypes. Chromatin fractionated by centrifugation in low-salt buffer resulted in two fractions with different contents and compositions of linker histones and enriched in specific core histone PTMs. Of particular interest is the fact that linker histone PTMs were differentially distributed in both chromatin fractions, suggesting specific functions. Future studies are needed to establish the interplay between PTMs of linker and core histones in order to fully understand chromatin regulation. A protein sequence alignment summarizing the PTMs found to date in chicken, mouse, rat and humans showed that, while many of the modified positions were conserved between these species, the type of modification often varied depending on the species or the cellular type. This finding suggests an important role for the PTMs in the regulation of linker histone functions.

Post-Translational Modifications of Histones in Vertebrate Neurogenesis.

The process of neurogenesis, through which the entire nervous system of an organism is formed, has attracted immense scientific attention for decades. How can a single neural stem cell give rise to astrocytes, oligodendrocytes, and neurons? Furthermore, how is a neuron led to choose between the hundreds of different neuronal subtypes that the vertebrate CNS contains? Traditionally, niche signals and transcription factors have been on the spotlight. Recent research is increasingly demonstrating that the answer may partially lie in epigenetic regulation of gene expression. In this article, we comprehensively review the role of post-translational histone modifications in neurogenesis in both the embryonic and adult CNS.