Enhanced tyrosine hydroxylase activity induces oxidative stress, causes accumulation of autotoxic catecholamine metabolites, and augments amphetamine effects in vivo.

In Parkinson\u27s disease, dopamine-containing nigrostriatal neurons undergo profound degeneration. Tyrosine hydroxylase (TH) is the rate-limiting enzyme in dopamine biosynthesis. TH increases in vitro formation of reactive oxygen species, and previous animal studies have reported links between cytosolic dopamine build-up and oxidative stress. To examine effects of increased TH activity in catecholaminergic neurons in vivo, we generated TH-over-expressing mice (TH-HI) using a BAC-transgenic approach that results in over-expression of TH with endogenous patterns of expression. The transgenic mice were characterized by western blot, qPCR, and immunohistochemistry. Tissue contents of dopamine, its metabolites, and markers of oxidative stress were evaluated. TH-HI mice had a 3-fold increase in total and phosphorylated TH levels and an increased rate of dopamine synthesis. Coincident with elevated dopamine turnover, TH-HI mice showed increased striatal production of

Measurement of Spine Density in Mouse Models of Hypodopaminergia

Dopamine (DA) is a key catecholamine neurotransmitter involved in motor control, cognition, and neuroendocrine regulation. Reduced DA transmission is associated with Parkinson’s disease, depression, and anhedonia. An overexpression of the dopamine transporter in mice (DAT-tg) results in a 40% reduction in extracellular DA, and can be classified as a genetic model of hypodopaminergia. Reserpine treatment depletes extracellular DA, and is a pharmacological model of hypodopaminergia. The aim of this study was to determine morphological and proteomic changes to medium spiny neurons (MSNs), which receive dopaminergic input, as a consequence of reduced DA transmission. To achieve this, MSNs were fluorescently labelled using a diolistics method and immunofluorescence. There were no observable changes to morphology or proteomic profile of MSNs in DAT-tg animals. Reserpine treatment resulted in reduced spine density in MSNs. DAT-tg animals may present a level of DA depletion that is below the threshold to induce morphological changes to MSNs.MAS

Selective Rescue of NMDA Receptor Function in Dopaminergic Neurons of NMDA Receptor Deficient Mice

Dysfunction in both the dopaminergic and glutamatergic systems has been implicated in the pathophysiology of schizophrenia. Schizophrenia is associated with a state of subcortical hyperdopaminergia, and NMDA receptor (NMDAR) hypofunction. The goal of our study is to assess which behavioural deficits present in NMDAR deficient mice are due to lack of NMDAR specifically in dopamine neurons. GluN1 knockdown (GluN1KD) mice express low levels of NMDARs resulting in global NMDAR hypofunction, leading to schizophreniaâ like behavioural endophenotypes, and an altered dopamine system. Schizophrenia-like behaviours observed in GluN1KD mice could be a result of the dysfunction of the dopamine system in these animals. To address this issue, a new mouse line was generated allowing for selective rescue of NMDARs in dopamine neurons using Cre recombinase. These rescue mice (GluN1DATâ Cre) have a global reduction of NMDARs except in dopaminergic neurons where a BAC transgene under the dopamine transporter promoter expresses Cre recombinase. In Aim 1, we assessed the neurochemistry and homeostasis of the dopamine system by measuring tyrosine hydroxylase activity and dopamine metabolism. In GluN1DAT-Cre mice, tyrosine hydroxylase activity was reduced compared to WT mice, and unchanged compared to GluN1KD mice. Additionally, in these rescue mice, dopamine catabolism was increased compared to both WT and GluN1KD mice indicating that neurochemical deficits are not improved by rescuing NMDARs in dopaminergic neurons. In Aim 2, we evaluated which schizophrenia-relevant behaviours result from lack of NMDARs in dopaminergic neurons. We assessed the following behavioural paradigms: locomotor activity, repetitive behaviours, sensorimotor gating, working memory, cognitive flexibility and executive function, sociability, and conditioned learning. GluN1DAT-Cre mice were fully rescued in sensorimotor gating and Pavlovian approach learning, and partially rescued in locomotor activity and repetitive behaviours. Unexpectedly, the rescue animals were more impaired in conditioned place preference compared to GluN1KD mice. Finally, similar behavioural abnormalities were observed in GluN1KD and GluN1DAT-Cre mice in working memory, cognitive flexibility and executive function, sociability, and fear conditioning compared to WT mice. These results indicate that rescuing NMDAR function selectively in dopaminergic neurons does not normalize neurochemical dopamine dysfunction, and ameliorates some, but not all the schizophrenia-relevant behaviours observed in GluN1KD mice.Ph.D.2018-12-19 00:00:0

Enhanced tyrosine hydroxylase activity induces oxidative stress, causes accumulation of autotoxic catecholamine metabolites, and augments amphetamine effects in vivo

In Parkinson\u27s disease, dopamine-containing nigrostriatal neurons undergo profound degeneration. Tyrosine hydroxylase (TH) is the rate-limiting enzyme in dopamine biosynthesis. TH increases in vitro formation of reactive oxygen species, and previous animal studies have reported links between cytosolic dopamine build-up and oxidative stress. To examine effects of increased TH activity in catecholaminergic neurons in vivo, we generated TH-over-expressing mice (TH-HI) using a BAC-transgenic approach that results in over-expression of TH with endogenous patterns of expression. The transgenic mice were characterized by western blot, qPCR, and immunohistochemistry. Tissue contents of dopamine, its metabolites, and markers of oxidative stress were evaluated. TH-HI mice had a 3-fold increase in total and phosphorylated TH levels and an increased rate of dopamine synthesis. Coincident with elevated dopamine turnover, TH-HI mice showed increased striatal production of

Clinical validation of cutoff target ranges in newborn screening of metabolic disorders by tandem mass spectrometry: A worldwide collaborative project

PURPOSE:: To achieve clinical validation of cutoff values for newborn screening by tandem mass spectrometry through a worldwide collaborative effort. METHODS:: Cumulative percentiles of amino acids and acylcarnitines in dried blood spots of approximately 25-30 million normal newborns and 10,742 deidentified true positive cases are compared to assign clinical significance, which is achieved when the median of a disorder range is, and usually markedly outside, either the 99th or the 1st percentile of the normal population. The cutoff target ranges of analytes and ratios are then defined as the interval between selected percentiles of the two populations. When overlaps occur, adjustments are made to maximize sensitivity and specificity taking all available factors into consideration. RESULTS:: As of December 1, 2010, 130 sites in 45 countries have uploaded a total of 25,114 percentile data points, 565,232 analyte results of true positive cases with 64 conditions, and 5,341 cutoff values. The average rate of submission of true positive cases between December 1, 2008, and December 1, 2010, was 5.1 cases/day. This cumulative evidence generated 91 high and 23 low cutoff target ranges. The overall proportion of cutoff values within the respective target range was 42% (2,269/5,341). CONCLUSION:: An unprecedented level of cooperation and collaboration has allowed the objective definition of cutoff target ranges for 114 markers to be applied to newborn screening of rare metabolic disorders. © 2011 Lippincott Williams & Wilkins