Contrasting brain patterns of writing-related DTI parameters, fMRI connectivity, and DTI-fMRI connectivity correlations in children with and without dysgraphia or dyslexia.

Based on comprehensive testing and educational history, children in grades 4-9 (on average 12 years) were diagnosed with dysgraphia (persisting handwriting impairment) or dyslexia (persisting word spelling/reading impairment) or as typical writers and readers (controls). The dysgraphia group (n = 14) and dyslexia group (n = 17) were each compared to the control group (n = 9) and to each other in separate analyses. Four brain region seed points (left occipital temporal gyrus, supramarginal gyrus, precuneus, and inferior frontal gyrus) were used in these analyses which were shown in a metaanalysis to be related to written word production on four indicators of white matter integrity and fMRI functional connectivity for four tasks (self-guided mind wandering during resting state, writing letter that follows a visually displayed letter in alphabet, writing missing letter to create a correctly spelled real word, and planning for composing after scanning on topic specified by researcher). For those DTI indicators on which the dysgraphic group or dyslexic group differed from the control group (fractional anisotropy, relative anisotropy, axial diffusivity but not radial diffusivity), correlations were computed between the DTI parameter and fMRI functional connectivity for the two writing tasks (alphabet and spelling) by seed points. Analyses, controlled for multiple comparisons, showed that (a) the control group exhibited more white matter integrity than either the dysgraphic or dyslexic group; (b) the dysgraphic and dyslexic groups showed more functional connectivity than the control group but differed in patterns of functional connectivity for task and seed point; and (c) the dysgraphic and dyslexic groups showed different patterns of significant DTI-fMRI connectivity correlations for specific seed points and written language tasks. Thus, dysgraphia and dyslexia differ in white matter integrity, fMRI functional connectivity, and white matter-gray matter correlations. Of clinical relevance, brain differences were observed in dysgraphia and dyslexia on written language tasks yoked to their defining behavioral impairments in handwriting and/or in word spelling and on the cognitive mind wandering rest condition and composition planning.

Modulation of DNA synthesis by muscarinic cholinergic receptors.

Acetylcholine muscarinic receptors are a family of five G-protein-coupled receptors widely distributed in the central nervous system and in peripheral organs. Activation of certain subtypes of muscarinic receptors (M1, M3, M5) has been found to modulate DNA synthesis in a number of cell types. In several cell types acetylcholine, by activating endogenous or transfected muscarinic receptors, can indeed elicit cell proliferation. In other cell types, however, or under different experimental conditions, activation of muscarinic receptors has no effect, or inhibits DNA synthesis. A large number of intracellular pathways are being investigated to define the mechanisms involved in these effects of muscarinic receptors; these include among others, phospholipase D, protein kinases C and mitogen-activated-protein kinases. The ability of acetylcholine to modulate DNA synthesis through muscarinic receptors may be relevant in the context of brain development and neoplastic growth.

Activation of mitogen-activated protein kinase by muscarinic receptors in astroglial cells: role in DNA synthesis and effect of ethanol.

Mitogen-activated protein kinase (MAPK) can be phosphorylated by mitogens binding to G-protein-coupled receptors and is considered a major pathway involved in cell proliferation. In this study, we report on the activation of MAPK by muscarinic acetylcholine receptors in astroglial cells, namely the 1321N1 human astrocytoma cell line, primary rat cortical astrocytes, and fetal human astrocytes. Carbachol caused a rapid and transient phorphorylation of MAPK (ERK1/2) in all cell types, with an increase in MAPK activity, without changing the levels of MAPK proteins. Human astrocytoma cells were used to characterize the effect of carbachol on MAPK. Experiments with M2- and M3-receptor subtype-selective antagonists, and with pertussis toxin, indicated that the M3 subtype is responsible for activating MAPK in glial cells. Pretreatment of cells with the protein kinase C (PKC) inhibitor bisindolylmaleimide I, or downregulation of PKC by 24-h treatment with the phorbol ester TPA inhibited carbachol-induced MAPK activation. Additional experiments with PKC alpha- or PKC epsilon-specific compounds indicated that the epsilon isozyme of PKC is primarily involved in MAPK activation by carbachol. Chelation of calcium also inhibited MAPK activation by carbachol. Two MEK (MAPK kinase) inhibitors inhibited carbachol-induced DNA synthesis but only at concentrations that exceeded those sufficient to block carbachol-induced MAPK activation. Ethanol (< or =200 mM) had no effect on MAPK when present alone and did not affect carbachol-induced MAPK activation under various experimental conditions, although it inhibits carbachol-induced DNA synthesis at low concentrations (10-100 mM). These results suggest that activation of MAPK by carbachol may be necessary but not sufficient for its mitogenic effect in astroglial cells, and that does not represent a target for ethanol-induced inhibition of DNA synthesis elicited by muscarinic receptors.

Effects of alcohol on immediate-early gene expression in primary cultures of rat cortical astrocytes.

Ethanol is a potent inhibitor of muscarinic receptor-mediated proliferation in glial cells. Glial proliferation has been suggested as a major target of ethanol neurotoxicity during development, leading to the microencephaly that is a predominant feature of fetal alcohol syndrome. As part of an attempt to understand the mechanism of ethanol's inhibitory effects on muscarinic receptor-mediated proliferation, this study investigated the effects of ethanol on the expression of the immediate-early genes (IEGs), c-fos and c-myc, whose induction is thought to be an essential first step in the initiation of the mitogenic program. Unexpectedly, ethanol had no inhibitory effect on c-fos and c-myc mRNA expression induced in primary rat cortical astrocytes by the mitogens carbachol, histamine, or tetradecanoyl phorbol 13-acetate; rather, a modest potentiation of IEG expression was observed in the presence of 25 to 100 mM ethanol. Control experiments showed that ethanol alone was capable of IEG mRNA induction, with 100 mM ethanol inducing IEG mRNA levels comparable with those induced by 100 ng/ml of tetradecanoyl phorbol 13-acetate; as measured by [3H]thymidine incorporation, however, 25 to 100 mM ethanol had no effect on proliferation. Experiments with the protein kinase C inhibitor bisindolylmaleimide and the Ca2+ chelators BAPTA and EGTA indicated that this IEG induction by ethanol was not mediated by protein kinase C or Ca2+. A possible explanation for this ethanol-induced IEG expression in the absence of a proliferative effect might be found in the increasing number of studies showing IEG involvement (especially that of c-myc) in apoptosis.

Effects of organophosphate exposure on muscarinic acetylcholine receptor subtype mRNA levels in the adult rat.

Repeated exposure to organophosphorus (OP) insecticides results in a decrease of muscarinic acetylcholine receptors (MRs) in the central nervous system. OP-induced MR down-regulation in vivo is modeled by chronic in vitro exposure to muscarinic agonists. Many studies, both in vivo and in vitro, indicate that the treatment-induced decrease in MR number is accompanied by a decrease in the mRNA levels of specific MR subtypes. In this study, the in vivo effects of OP exposure on the mRNA levels of three MR subtypes (m1, m2, and m3) were examined in brain tissue and in peripheral mononuclear cells, which express the m3 subtype. Adult male Sprague-Dawley rats were orally administered disulfoton (2 mg/kg/day) for 14 days, and a subset of exposed animals was allowed to recover for 28 days. This treatment caused a 28% and 81% decrease, respectively, in [3H]-QNB binding and acetylcholinesterase activity in the cortex, similar to that observed in previous studies; after recovery, these levels had returned to 99% and 90%, respectively, of controls. There was a significant decrease in m1 mRNA levels in hippocampus (23%) after disulfoton treatment, while no change was observed in the cortex, corpus striatum, medulla, or cerebellum. The m2 subtype mRNA was significantly decreased in both hippocampus (24%) and medulla (19%), but not in cortex, striatum, or cerebellum. m3 mRNA levels were significantly decreased in cortex (10%), but no change was observed in hippocampus, medulla, cerebellum, or lymphocytes. After recovery, no differences in m1 or m3 mRNA levels were observed in any tissue examined, whereas the decrease in m2 mRNA in the hippocampus remained significant (29%). These results indicate that OP exposure can differentially regulate mRNA levels for MR subtypes in different brain areas, and suggest that m2 muscarinic receptors in the hippocampus are most affected by this treatment.

The DNA damage-inducible gene DIN1 of Saccharomyces cerevisiae encodes a regulatory subunit of ribonucleotide reductase and is identical to RNR3.

The sequence of the DIN1 gene of Saccharomyces cerevisiae is identical to RNR3, a gene encoding a DNA damage-inducible regulatory subunit of ribonucleotide reductase. Two sequence elements located upstream of DIN1 (RNR3) are homologous to putative DNA damage regulatory elements in the promoter of the reductase catalytic subunit gene, RNR2. The transcript start sites for DIN1(RNR3) have been localized, and induction by different agents has been compared with other DNA damage-regulated genes.