Maria Grazia Alessandrì

Arginine:Glycine Amidinotransferase Deficiency: The Third Inborn Error of Creatine Metabolism in Humans

Arginine:glycine amidinotransferase (AGAT) catalyzes the first step of creatine synthesis, resulting in the formation of guanidinoacetate, which is a substrate for creatine formation. In two female siblings with mental retardation who had brain creatine deficiency that was reversible by means of oral creatine supplementation and had low urinary guanidinoacetate concentrations, AGAT deficiency was identified as a new genetic defect in creatine metabolism. A homozygous G-A transition at nucleotide position 9297, converting a tryptophan codon (TGG) to a stop codon (TAG) at residue 149 (T149X), resulted in undetectable cDNA, as investigated by reverse-transcription PCR, as well as in undetectable AGAT activity, as investigated radiochemically in cultivated skin fibroblasts and in virus-transformed lymphoblasts of the patients. The parents were heterozygous for the mutant allele, with intermediate residual AGAT activities. Recognition and treatment with oral creatine supplements may prevent neurological sequelae in affected patients.

Striatal dopamine metabolism in monoamine oxidase b-deficient mice : A brain dialysis study

Abstract : We have studied striatal dopamine (DA) metabolism in monoamine oxidase (MAO) B‐deficient mice using brain microdialysis. Baseline DA levels were similar in wild‐type and knock‐out (KO) mice. Administration of a selective MAO A inhibitor, clorgyline (2 mg/kg), increased DA levels and decreased levels of its metabolites in all mice, but a selective MAO B inhibitor, l‐deprenyl (1 mg/kg), had no effect. Administration of 10 and 50 mg/kg l‐DOPA, the precursor of DA, increased the levels of DA similarly in wild‐type and KO mice. The highest dose of l‐DOPA (100 mg/kg) produced a larger increase in DA in KO than wild‐type mice. This difference was abolished by pretreating wild‐type mice with l‐deprenyl. These results suggest that in mice, DA is only metabolized by MAO A under basal conditions and by both MAO A and B at high concentrations. This is in contrast to the rat, where DA is always metabolized by MAO A regardless of concentration.

A damage to locus coeruleus neurons converts sporadic seizures into self-sustaining limbic status epilepticus

Various studies demonstrated that the neurotransmitter norepinephrine (NE) plays a relevant role in modulating seizures; in particular, a powerful effect consists in delaying the kindling of limbic areas such as the amygdala and hippocampus. Given the rich NE innervation of limbic regions, we selected a sensitive trigger area, the anterior piriform cortex, to test whether previous loss of noradrenergic terminals modifies sporadic seizures in rats. The damage to locus coeruleus terminals was produced by using the selective neurotoxin N‐(‐2‐chloroethyl)‐N‐ethyl‐2‐bromobenzylamine (DSP‐4, 60 mg/kg i.p.). In intact rats, bicuculline (a GABA‐A antagonist, 118 pmol) microinfused into this area produced sporadic seizures, while in rats previously injected with DSP‐4, bicuculline determined long‐lasting self‐sustaining status epilepticus. In intact rats, sporadic seizures were accompanied by a marked increase in norepinephrine release in the contralateral piriform cortex, while in locus coeruleus‐lesioned rats this phenomenon was attenuated. While bicuculline‐induced sporadic seizures were prevented by the focal infusion of amino‐7‐phosphonoheptanoic acid (AP‐7, a selective NMDA antagonist), or 1,2,3,4‐tetrahydro‐6‐nitro‐2,3‐dioxo‐benzo[f]quinoxaline‐7‐sulphonamide (NBQX, a selective non‐NMDA antagonist), status epilepticus obtained in norepinephrine‐lesioned rats was insensitive to AP‐7 but was still inhibited by NBQX. By using fluorescent staining for damaged (Fluoro‐Jade B) and intact (DAPI) neurons, as well as cresyl violet, we found that rats undergoing status epilepticus developed neuronal loss in various limbic regions. This study demonstrates a powerful effect of noradrenergic terminals in regulating the onset of limbic status epilepticus and its sensitivity to specific glutamate antagonists.

Effects of Pretreatment with N-(2-Chloroethyl)-N-Ethyl-2-Bromobenzylamine (DSP-4) on Methamphetamine Pharmacokinetics and Striatal Dopamine Losses

Abstract : We recently demonstrated that pretreatment with N‐(2‐chloroethyl)‐N‐ethyl‐2‐bromobenzylamine (DSP‐4) exacerbates experimental parkinsonism induced by methamphetamine. The mechanism responsible for this effect remains to be elucidated. In this study, we investigated whether the exacerbation of chronic dopamine loss in DSP‐4‐pretreated animals is due to an impairment in the recovery of dopamine levels once the neurotoxic insult is generated or to an increased efficacy of the effects induced by methamphetamine. We administered different doses of methamphetamine either to DSP‐4‐pretreated or to intact Swiss‐Webster mice and evaluated the methamphetamine‐induced striatal dopamine loss at early and prolonged intervals. As a further step, we evaluated the striatal pharmacokinetics of methamphetamine, together with its early biochemical effects. We found that previous damage to norepinephrine terminals produced by DSP‐4 did not modify the recovery of striatal dopamine levels occurring during several weeks after methamphetamine. By contrast, pretreatment with DSP‐4 exacerbated early biochemical effects of methamphetamine, which were already detectable 1 h after methamphetamine administration. In addition, in norepinephrine‐depleted animals, the clearance of striatal methamphetamine is prolonged, although the striatal concentration peak observed at 1 h is unmodified. These findings, together with the lack of a methamphetamine enhancement when DSP‐4 was injected 12 h after methamphetamine administration, suggest that in norepinephrine‐depleted animals, a more pronounced acute neuronal sensitivity to methamphetamine occurs.

Clonidine suppresses 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine-induced reductions of striatal dopamine and tyrosine hydroxylase activity in mice.

Abstract: Recent findings have shown that excitatory amino acid may be involved in 1‐methyl‐4‐phenyl‐1,2,3,6‐tetrahydropyridine (MPTP) toxicity. At the same time, evidence is accumulating that the endogenous nor‐adrenergic system plays a protective role in MPTP‐induced striatal dopamine (DA) depletion and nigral dopaminergic cell death. Recently, α2‐adrenoceptors located on glutamatergic axons have been shown to inhibit glutamate overflow. In this study, we evaluated the effects of an α2‐agonist (clonidine) and an α2‐antagonist (yohimbine) on MPTP‐induced striatal DA depletion and tyrosine hydroxylase activity reduction. We show that clonidine is able to prevent the neurotoxicity of MPTP in mice. To exert this effect, clonidine (0.5 mg/kg) must be administered at least twice (30 min before and 30 min after MPTP). Administration of another α2‐agonist (detomidine, 0.3 mg/kg) attenuated the neurotoxicity induced by MPTP. We provide evidence that the protective effect obtained with clonidine was not due to decreased striatal content of 1‐methyl‐4‐phenylpyridinium (MPP+). We also show that yohimbine, which is a classic α2‐adrenoceptor antagonist with low affinity for imidazoline receptors, produced by itself an enhancement of MPTP toxicity and was able to block the protective effect of clonidine. These data raise the possibility that α2‐adrenoceptor may modulate the susceptibility of the nigrostriatal dopaminergic pathway to neurotoxicity.

Noradrenergic Modulation of Methamphetamine-Induced Striatal Dopamine Depletion

ABSTRACT: Noradrenergic (NE) neurons belonging to the locus coeruleus (LC), much more than the A1 and A2 NE areas, are lost in Parkinsons disease (PD). In this study, we reproduced the selective pattern of NE loss involving axons arising from the LC using the selective neurotoxin N‐(‐2‐chloroethyl)‐N‐ethyl‐2‐bromobenzylamine (DSP‐4) (50 mg/kg). In these experimental conditions, we investigated whether NE loss potentiates methamphetamine‐induced striatal dopamine (DA) depletion in mice and in rats. Administration of a moderate dose of methamphetamine to C57B1/6N mice or Sprague‐Dawley rats produced only a partial striatal DA depletion 7 days after drug administration. Pretreatment with DSP‐4, in both animal species, significantly enhanced methamphetamine‐induced striatal DA depletion. Administration of a lower dose of methamphetamine did not decrease striatal DA levels when injected alone, but produced a significant decrease in striatal DA when given to DSP‐4‐pretreated rodents. Moreover, we found that agents reducing the noradrenergic activity (i.e., the alpha‐2 agonist clonidine) enhanced, whereas alpha‐2 antagonists decreased, methamphetamine toxicity. Enhancement of methamphetamine toxicity did not occur if the noradrenergic lesion was produced 12 hr after methamphetamine administration. By contrast, exacerbation of methamphetamine toxicity in NE‐depleted animals was accompanied by increased extracellular DA levels measured with brain dialysis and by a more severe acute DA depletion measured in striatal homogenates.

Modulation of dihydroxyphenylacetaldehyde extracellular levels in vivo in the rat striatum after different kinds of pharmacological treatment

We recently identified the direct product of dopamine (DA) by monoamine-oxidase (MAO) activity, dihydroxyphenylacetaldehyde (DOPALD) in the trans-striatal dialysate. Based on these findings, in this work, we directly measured the variations in DOPALD levels after various kinds of pharmacological treatment in rat striatal extracellular fluid. Using both reversible and irreversible MAO inhibitors, we found that MAO-A inhibition suppressed, whereas MAO-B inhibition did not modify DOPALD levels in the dialysate. The vesicular DA uptake blocker Ro 4-1284 led to an increase in extracellular DA and DOPALD, whereas the increase in extracellular DA obtained after administration of the plasma membrane DA uptake blocker GBR-12909 occurred without concomitant changes in DOPALD extracellular levels. Microinfusions of DA through the dialysis probe or systemic administration of L-DOPA increased striatal DOPALD to a greater extent compared with other DA metabolites, both in intact and in 6-hydroxydopamine (6-OHDA)-lesioned striatum. This study indicates that the direct product of MAO activity within the rat striatum derives from the activity of the isoenzyme MAO-A. The assay of DOPALD, together with DOPAC, represents a reliable tool to measure directly, in freely moving animals, DA oxidative metabolism. As recent studies have shown that microinfusions of exogenous DOPALD might induce cell death, pharmacological modulation of DOPALD levels might also be relevant for an understanding of the mechanisms involved in DA neurotoxicity.

Brief and repeated noise exposure produces different morphological and biochemical effects in noradrenaline and adrenaline cells of adrenal medulla

Exposure to stressful stimuli is known to activate the peripheral sympathetic nervous system and the adrenal gland. In this study, we evaluated the effects of single or repeated bouts of exposure to a readily measurable stressful stimulus (loud noise) on the catecholamine content and ultrastructure of the rat adrenal medulla. In particular, we measured tissue levels of dopamine, noradrenaline, adrenaline and metabolites. In parallel studies, we evaluated the fine ultrastructure of catecholamine cells, including a detailed study of catecholamine granules and a morphometric analysis of adrenaline and noradrenaline medullary cells. Animals were exposed either to a single (6 h) session of loud (100 dBA) noise, or to this noise stimulus repeated every day for 21 consecutive days. There was a marked correlation between biochemical indexes of catecholamine activity and the ultrastructural morphometry of specific catecholamine granules. Exposure to loud noise for 6 h induced a parallel increase in dopamine, noradrenaline, adrenaline and their metabolites, a polarization and an increased numerical density of noradrenaline and adrenaline granules in the cells. After repeated noise exposure, noradrenaline levels were significantly higher than in controls, and adrenaline decreased significantly. In addition, adrenaline cells also exhibited ultrastructural alterations consisting of wide homogeneous cytoplasmic areas and large, pale vesicles.

Arginine and glycine stimulate creatine synthesis in creatine transporter 1-deficient lymphoblasts.

Creatine transporter 1 (CT1) defect is an X-linked disease that causes severe neurological impairment. No treatment has been available for this condition so far. Because the transport of creatine (Cr) precursors Gly and Arg is not affected in this disorder, we tested the possible corrective effect of these two amino acids on Cr depletion in lymphoblasts lacking the transporter. Substrates enriched with Arg or Arg plus Gly increased the concentration of intracellular Cr in affected cells as well as in control cells. The greatest effect was obtained with 10 and 15 mM Arg and 10mM Arg plus Gly. These results encourage an in vivo trial with Cr precursors in CT1 defect.

Effect of metadoxine on striatal dopamine levels in C57 Black mice

Abstract— In the present study, we examined the effect of metadoxine on striatal levels of dopamine, 5‐hydroxytryptamine (5‐HT) and their metabolites in male C57 Black mice. Striatal content was assayed after systemic administration of metadoxine ranging from 1 μg kg−1 to 500 mg kg−1. Striatal dopamine increased 1 h after treatment with metadoxine (150 mg kg−1), but the most notable effect was obtained 24 h after the drug administration. At this time a plateau was reached; the two major metabolites of dopamine showed the same trend. Seven days after metadoxine administration, striatal dopamine approached the control values. Over the same time intervals, striatal 5‐HT increased to a lesser extent and 5‐hydroxy‐indoleacetic acid did not differ significantly from controls. Striatal dopamine increased significantly at a dose of 250 μg kg−1 up to a dose of 1 mg kg−1 metadoxine; no further increment was observed between 1 and 500 mg kg−1 metadoxine. Administration of each component at doses equimolar to 1 mg metadoxine showed that pyridoxine produced only a mild increase in striatal dopamine compared with controls. We suggest that the metadoxine‐induced striatal dopamine increase is obtained by increasing synthesis of dopamine.