Is 5-Hydroxymethylcytosine a Suppressor or Activator in Epigenetic Marks?

poster abstractAlcohol has been observed to have teratogenic effects on humans and mice during different stages of embryonic development. These effects can be condensed under fetal alcohol spectrum disorder (FASD), exhibiting a variety of signs from growth retardations to neurobehavioral aberrations. Despite better understanding of several potential mechanisms, the question of how alcohol, as an environmental factor leads to brain growth delay in FASD remains elusive. DNA methylation is key to development and tissue specification. Studies have suggested that alcohol may alter gene expression by affecting DNA and histone methylation. Previous studies have demonstrated that 5-methylcytosine (5mC), a DNA methylation mark, is associated with histone 3 lysine-9me3, (H3K9me3) to play a role in gene repression. Recently another methylation mark, 5hydroxylmethylcytosine (5hmC), was found to prevail in the nervous system. However, its function has not been clear. Global analysis suggests that it is a transition of demethylation leading to transcription. The study will first identify its association with histone 3 lysine-4me3, (H3K4me3) a transcriptional activator in gene expression, and then study the 5hmC under influence of alcohol exposure. This study will utilize both an in vivo model—the vapor chamber, and an in vitro model—the embryonic culture system to address this question. Embryos were exposed to alcohol (400mg/dL, 88mM) from the beginning of embryonic day (E) 8 for 6hrs, harvested at E10, and processed for immunohistochemistry. Compare the DNA methylation marks, and histone modification marks to see if the spatial and/or temporal distribution has been affected by alcohol exposure. It is expected that in the alcohol-treated embryos, an overall retardation of embryonic growth, delayed neural tube formation, and altered expression of epigenetic markers will be observed. This study could indicate that alcohol, through alteration of DNA and histone methylation is a potential mechanism underpinning brain growth delay in FASD

Ionizing Radiation Affects Epigenetic Programming in Young Adult Mice

Humans are exposed to low and mild doses of radiation frequently, ranging from the natural environment to medical procedures like x-ray and CT scans. Ionizing radiation of various doses has been known to cause not only cellular and genomic changes, but specific neurological systems such as the limbic system have been indicated to be particularly vulnerable. Here, we demonstrated that epigenetics is also altered by radiation. Epigenetics is a subtle chemical coding above the gene, which plays a critical role in brain development, and downstream can cause the onset of cognitive aberrations and other neurological impairments. How radiation as an external environmental factor causes epigenetic changes is not clearly understood. DNA methylation, including 5-methylcytosine (5M) and 5-hydroxymethylcytosine (5-hmC) have been shown to either suppress or activate gene transcription and as such are key epigenetic players. To elucidate the role of radiation in epigenetic outcomes, we examined epigenetic, phenotypic and transcriptional markers via immunohistochemistry, in the hippocampus and cortex. In this study C57BL/6 mouse (postnatal day 21 (P21)) began a 4-week radiation treatment of various doses totaling (2Gy-4.5Gy) via global head targeting CT exposure. We found a loss of 5M and 5-hmC as well as transcriptional markers within regions of the hippocampus and cortex. There was a significant decrease in cell proliferation in the hippocampus- specifically, in the region responsible for adult neurogenesis. The cingulate cortex (a region adjacent to the hippocampus) also exhibited dramatic alterations in several epigenetic and transcriptional markers, indicating the vulnerability of the limbic system in radiation exposure. Understanding the mechanism by which ionizing radiation affects epigenetic programming will provide insight into the transmissibility of external factors to biological systems. Additionally, this work can aid the development of protective strategies against the harmful risks associated with radiation exposure

Ionizing Radiation Affects Epigenetic Programming in Adolescent Mice

poster abstractHumans are exposed to low and mild doses of radiation frequently, ranging from the natural environment to medical procedures like x-ray and CT scans. Ionizing radiation of various doses has been known to potentially cause not only cellular but also genomic changes. Here, we demonstrate that epigenetics is also altered by the radiation. Epigenetics is a chemical coding above the gene, which plays critical roles in brain development, cognitive aberrations and other neurological impairments. How radiation, as an external environmental factor, causes epigenetic change is not understood. DNA methylation, key in epigenetics, including 5-methylcytosine (5mC) and 5-hydroxymethylcytosine (5hmC) have been shown to either suppress or activate gene transcription. To aid in elucidating the role in which radiation affects epigenetic outcomes, we examined the effects of radiation on both epigenetic and phenotypic markers within the hippocampus. In this study we treated, via x-ray C57BL/6 mice, postnatal day (P) 21 with various doses (2Gy-4.5Gy) of radiation coupled with varying frequencies (0.5 Gy x 4, 1.5 Gy x 3, or 4.5Gy x 1) during a 4-week period. We used immunohistochemistry staining with cell proliferation, transcription and epigenetic markers. We found loss of 5mC in the sub-granular layer of the dentate gyrus (DG) in the upper and lower arms. Likewise a loss of 5hmC in the sub-granular layer of the DG, as well as in the cornu Ammonis (CA) layers 1 and 2. There was also loss of a transcriptional activation marker within the DG of the hippocampus. Furthermore, decreased cell proliferation in the adult neurogenesis in the hippocampus was found. Exposure to ionizing radiation altered the normal epigenetic profile of the mice. Understanding the mechanism by which ionizing radiation affects epigenetic programming will provide insight into how to develop protection against the potentially harmful risks associated with radiation exposure

Mechanisms Regulating the Association of Protein Phosphatase 1 with Spinophilin and Neurabin

Protein phosphorylation is a key mediator of signal transduction, allowing for dynamic regulation of substrate activity. Whereas protein kinases obtain substrate specificity by targeting specific amino acid sequences, serine/threonine phosphatase catalytic subunits are much more promiscuous in their ability to dephosphorylate substrates. To obtain substrate specificity, serine/threonine phosphatases utilize targeting proteins to regulate phosphatase subcellular localization and catalytic activity. Spinophilin and its homologue neurabin are two of the most abundant dendritic spine-localized protein phosphatase 1 (PP1) targeting proteins. The association between spinophilin and PP1 is increased in the striatum of animal models of Parkinson's disease (PD). However, mechanisms that regulate the association of spinophilin and neurabin with PP1 are unclear. Here, we report that the association between spinophilin and PP1α or PP1γ1 was increased by CDK5 expression and activation in a heterologous cell system. This increased association is at least partially due to phosphorylation of PP1. Conversely, CDK5 expression and activation decreased the association of PP1 with neurabin. As with dopamine depletion, methamphetamine (METH) abuse causes persistent alterations in dopamine signaling which influence striatal medium spiny neuron function and biochemistry. Moreover, both METH toxicity and dopamine depletion are associated with deficits in motor control and motor learning. Pathologically, we observed a decreased association of spinophilin with PP1 in rat striatum evaluated one month following a binge METH paradigm. Behaviorally, we found that loss of spinophilin recapitulates rotarod pathology previously observed in dopamine-depleted and METH-treated animals. Together, these data have implications in multiple disease states associated with altered dopamine signaling such as PD and psychostimulant drug abuse and delineate a novel mechanism by which PP1 interactions with spinophilin and neurabin may be differentially regulated

Proteomic Analysis of the Spinophilin Interactome in Rodent Striatum Following Psychostimulant Sensitization

Glutamatergic projections from the cortex and dopaminergic projections from the substantia nigra or ventral tegmental area synapse on dendritic spines of specific GABAergic medium spiny neurons (MSNs) in the striatum. Direct pathway MSNs (dMSNs) are positively coupled to protein kinase A (PKA) signaling and activation of these neurons enhance specific motor programs whereas indirect pathway MSNs (iMSNs) are negatively coupled to PKA and inhibit competing motor programs. An imbalance in the activity of these two programs is observed following increased dopamine signaling associated with exposure to psychostimulant drugs of abuse. Alterations in MSN signaling are mediated by changes in MSN protein post-translational modifications, including phosphorylation. Whereas direct changes in specific kinases, such as PKA, regulate different effects observed in the two MSN populations, alterations in the specific activity of serine/threonine phosphatases, such as protein phosphatase 1 (PP1) are less well known. This lack of knowledge is due, in part, to unknown, cell-specific changes in PP1 targeting proteins. Spinophilin is the major PP1-targeting protein in striatal postsynaptic densities. Using proteomics and immunoblotting approaches along with a novel transgenic mouse expressing hemagglutainin (HA)-tagged spinophilin in dMSNs and iMSNs, we have uncovered cell-specific regulation of the spinophilin interactome following a sensitizing regimen of amphetamine. These data suggest regulation of spinophilin interactions in specific MSN cell types and may give novel insight into putative cell-specific, phosphatase-dependent signaling pathways associated with psychostimulants

Correction: Baucum II, Anthony J. et al. Proteomic Analysis of the Spinophilin Interactome in Rodent Striatum Following Psychostimulant Sensitization. Proteomes 2018, 6, 53

The author wishes to make the following corrections to the methods section of their paper [...]. Erratum for Proteomic Analysis of the Spinophilin Interactome in Rodent Striatum Following Psychostimulant Sensitization. [Proteomes. 2018

The association of spinophilin with disks large-associated protein 3 (SAPAP3) is regulated by metabotropic glutamate receptor (mGluR) 5

Spinophilin is the most abundant protein phosphatase 1 targeting protein in the postsynaptic density of dendritic spines. Spinophilin associates with myriad synaptic proteins to regulate normal synaptic communication; however, the full complement of spinophilin interacting proteins and mechanisms regulating spinophilin interactions are unclear. Here we validate an association between spinophilin and the scaffolding protein, disks large-associated protein 3 (SAP90/PSD-95 associated protein 3; SAPAP3). Loss of SAPAP3 leads to obsessive-compulsive disorder (OCD)-like behaviors due to alterations in metabotropic glutamate receptor (mGluR) signaling. Here we report that spinophilin associates with SAPAP3 in the brain and in a heterologous cell system. Moreover, we have found that expression or activation of group I mGluRs along with activation of the mGluR-dependent kinase, protein kinase C β, enhances this interaction. Functionally, global loss of spinophilin attenuates amphetamine-induced hyperlocomotion, a striatal behavior associated with dopamine dysregulation and OCD. Together, these data delineate a novel link between mGluR signaling, spinophilin, and SAPAP3 in striatal pathophysiology

Ontogeny of midazolam glucuronidation in preterm infants

Purpose: In preterm infants, the biotransformation of midazolam (M) to 1-OH-midazolam (OHM) by cytochrome P450 3A4 (CYP3A4) is developmentally immature, but it is currently unknown whether the glucuronidation of OHM to 1-OH-midazolam glucuronide (OHMG) is also decreased. The aim of our study was to investigate the urinary excretion of midazolam and its metabolites OHM and OHMG in preterm neonates following the intravenous (IV) or oral (PO) administration of a single M dose. Methods: Preterm infants (post-natal age 3-13 days, gestational age 26-34 4/7 weeks) scheduled to undergo a stressful procedure received a 30-min IV infusion (n=15) or a PO bolus dose (n=7) of 0.1 mg/kg midazolam. The percentage of midazolam dose excreted in the urine as M, OHM and OHMG up to 6 h post-dose was determined. Results: The median percentage of the midazolam dose excreted as M, OHM and OHMG in the urine during the 6-h interval after the IV infusion was 0.44% (range 0.02-1.39%), 0.04% (0.01-0.13%) and 1.57% (0.36-7.7%), respectively. After administration of the PO bolus dose, the median percentage of M, OHM and OHMG excreted in the urine was 0.11% (0.02-0.59%), 0.02% (0.00-0.10%) and 1.69% (0.58-7.31%), respectively. The proportion of the IV midazolam dose excreted as OHMG increased significantly with postconceptional age (r=0.73, p <0.05). Conclusion: The glucuronidation of OHM appears immature in preterm infants less than 2 weeks of age. The observed increase in urinary excretion of OHMG with postconceptional age likely reflects the combined maturation of glucuronidation and renal function

Proteomic Analysis of the Spinophilin Interactome in Rodent Striatum Following Psychostimulant Sensitization

Glutamatergic projections from the cortex and dopaminergic projections from the substantia nigra or ventral tegmental area synapse on dendritic spines of specific GABAergic medium spiny neurons (MSNs) in the striatum. Direct pathway MSNs (dMSNs) are positively coupled to protein kinase A (PKA) signaling and activation of these neurons enhance specific motor programs whereas indirect pathway MSNs (iMSNs) are negatively coupled to PKA and inhibit competing motor programs. An imbalance in the activity of these two programs is observed following increased dopamine signaling associated with exposure to psychostimulant drugs of abuse. Alterations in MSN signaling are mediated by changes in MSN protein post-translational modifications, including phosphorylation. Whereas direct changes in specific kinases, such as PKA, regulate different effects observed in the two MSN populations, alterations in the specific activity of serine/threonine phosphatases, such as protein phosphatase 1 (PP1) are less well known. This lack of knowledge is due, in part, to unknown, cell-specific changes in PP1 targeting proteins. Spinophilin is the major PP1-targeting protein in striatal postsynaptic densities. Using proteomics and immunoblotting approaches along with a novel transgenic mouse expressing hemagglutainin (HA)-tagged spinophilin in dMSNs and iMSNs, we have uncovered cell-specific regulation of the spinophilin interactome following a sensitizing regimen of amphetamine. These data suggest regulation of spinophilin interactions in specific MSN cell types and may give novel insight into putative cell-specific, phosphatase-dependent signaling pathways associated with psychostimulants