Increased susceptibility to kainic acid-induced seizures in Engrailed-2 knockout mice.

The En2 gene, coding for the homeobox-containing transcription factor Engrailed-2 (EN2), has been associated to autism spectrum disorder (ASD). Due to neuroanatomical and behavioral abnormalities, which partly resemble those observed in ASD patients, En2 knockout (En2(-/-)) mice have been proposed as a model for ASD. In the mouse embryo, En2 is involved in the specification of midbrain/hindbrain regions, being predominantly expressed in the developing cerebellum and ventral midbrain, and its expression is maintained in these structures until adulthood. Here we show that in the adult mouse brain, En2 mRNA is expressed also in the hippocampus and cerebral cortex. Hippocampal En2 mRNA content decreased after seizures induced by kainic acid (KA). This suggests that En2 might also influence the functioning of forebrain areas during adulthood and in response to seizures. Indeed, a reduced expression of parvalbumin and somatostatin was detected in the hippocampus of En2(-/-) mice as compared to wild-type (WT) mice, indicating an altered GABAergic innervation of limbic circuits in En2(-/-) mice. In keeping with these results, En2(-/-) mice displayed an increased susceptibility to KA-induced seizures. KA (20 mg/kg) determined more severe and prolonged generalized seizures in En2(-/-) mice, when compared to WT animals. Seizures were accompanied by a widespread c-fos and c-jun mRNA induction in the brain of En2(-/-) but not WT mice. Long-term histopathological changes (CA1 cell loss, upregulation of neuropeptide Y) also occurred in the hippocampus of KA-treated En2(-/-) but not WT mice. These findings suggest that En2(-/-) mice might be used as a novel tool to study the link between epilepsy and ASD.

Connexin 26 Expression in Mammalian Cardiomyocytes.

Connexins are a family of membrane-spanning proteins named according to their molecular weight. They are known to form membrane channels mediating cell-cell communication, which play an essential role in the propagation of electrical activity in the heart. Cx26 has been described in a number of tissues but not in the heart, and its mutations are frequently associated with deafness and skin diseases. The aim of this study was to assess the possible Cx26 expression in heart tissues of different mammalian species and to demonstrate its localization at level of cardiomyocytes. Samples of pig, human and rat heart and H9c2 cells were used for our research. Immunohistochemical and molecular biology techniques were employed to test the expression of Cx26. Interestingly, this connexin was found in cardiomyocytes, at level of clusters scattered over the cell cytoplasm but not at level of the intercalated discs where the other cardiac connexins are usually located. Furthermore, the expression of Cx26 in H9c2 myoblast cells increased when they were differentiated into cardiac-like phenotype. To our knowledge, the expression of Cx26 in pig, human and rat has been demonstrated for the first time in the present paper.

CYP 2E1 mutant mice are resistant to DDC-induced enhancement of MPTP toxicity.

In order to reach a deeper insight into the mechanism of diethyldithiocarbamate (DDC)-induced enhancement of MPTP toxicity in mice, we showed that CYP450 (2E1) inhibitors, such as diallyl sulfide (DAS) or phenylethylisothiocyanate (PIC), also potentiate the selective DA neuron degeneration in C57/bl mice. Furthermore we showed that CYP 2E1 is present in the brain and in the basal ganglia of mice (Vaglini et al., 2004). However, because DAS and PIC are not selective CYP 2E1 inhibitors and in order to provide direct evidence for CYP 2E1 involvement in the enhancement of MPTP toxicity, CYP 2E1 knockout mice (GONZ) and wild type animals (SVI) of the same genetic background were treated with MPTP or the combined DDC + MPTP treatment. In CYP 2E1 knockout mice, DDC pretreatment completely fails to enhance MPTP toxicity, although enhancement of MPTP toxicity was regularly present in the SVI control animals. The immunohistochemical study confirms our results and suggests that CYP 2E1 may have a detoxifying role.

Cytochrome P450 and Parkinson's disease: protective role of neuronal CYP 2E1 from MPTP toxicity.

Elucidation of the biochemical steps leading to the 1-Methyl-4-Phenyl-1,2,3,6-Tetrahydropyridine (MPTP)-induced degeneration of the nigro-striatal dopamine (DA) pathway has provided new clues to the pathophysiology of Parkinson's Disease (PD). In line with the enhancement of MPTP toxicity by diethyldithiocarbamate (DDC), here we demonstrate how other CYP450 (2E1) inhibitors, such as diallyl sulfide (DAS) or phenylethylisothiocyanate (PIC), also potentiate the selective DA neuron degeneration in C57/bl mice. In order to provide direct evidence for this isozyme involvement, CYP 2E1 knockout mice were challenged with MPTP or the combined treatment. Here we show that these transgenic mice have a low sensitivity to MPTP alone, similarly to the wild type SVI, suggesting that it is likely that transgenic mice compensate for the missing enzyme. However, in these CYP 2E1 knockout mice, DDC pretreatment completely fails to enhance MPTP toxicity; this enhancement is instead regularly present in the SVI control animals. This study indicates that the occurrence of CYP 2E1 in C57/bl mouse brain is relevant for MPTP toxicity, and suggests that this isozyme may have a detoxificant role related to the efflux transporter of the toxin.

Species differences in the role of excitatory amino acids in experimental parkinsonism.

The present review discusses species differences in relation to the effects produced by the neurotoxin 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP); in particular, it focuses on recent evidence regarding the role of excitatory amino acids in experimental parkinsonism. The main aim of the review is to provide a phylogenetic perspective which may serve as a useful tool to study Parkinson's disease in rodents. Excitotoxicity might represent the final common pathway on which the actions of different neurotoxins, selectively directed towards nigrostriatal dompaminergic neurons, converge. This is clearly demonstrated in methamphetamine- and 6-dihydroxy-dopamine-induced parkinsonism. The role of excitotoxicity in the mechanism of action of MPTP is less clear. Although there are several species differences for MPTP it is possible to obtain in mice the same effects induced in MPTP-treated primates by combining acetaldehyde or diethyldithiocarbamate with MPTP administration. When mice are administered these combined treatments, the onset of experimental parkinsonism can be prevented using the same pharmacological agents (i.e. glutamate N-methyl-D-aspartate antagonists) that are effective in primates.

Selective lesion of the nigrostriatal dopaminergic pathway by MPTP and acetaldehyde or diethyldithiocarbamate.

We have previously reported that diethyldithiocarbamate and acetaldehyde enhance MPTP toxicity in mice (Corsini et al. 1986). Here we show that these drugs enhance the depletion of dopamine in the striatum and markedly increase MPTP-induced death of DA neurons in the substantia nigra. This enhancement of MPTP toxicity is specific for the nigro-striatal DA pathway and no recovery occurs, at least for four months after the treatment. Rats, although they show an MPTP-induced acute syndrome similar to the that induced in mice by the combined treatments, appear to be insensitive to both MPTP alone or to combined treatment with diethyldithiocarbamate or acetaldehyde. The selectivity of the permanent bilateral lesions of the nigro-striatal pathway make mice treated with acetaldehyde or diethyldithiocarbamate and MPTP a simple and reliable model for parkinsonism.

(+)MK-801 prevents the DDC-induced enhancement of MPTP toxicity in mice.

In order to reach deeper insight into the mechanism of diethyldithiocarbamate (DDC)-induced enhancement of MPTP toxicity in mice, MK-801, a non-competitive antagonist of NMDA receptors, has been used as a tool to study the role of excitatory amino acids. In agreement with previous reports, (+)MK-801 did not significantly affect either striatal dopamine (DA) or tyrosine-hydroxylase (TH) activity in MPTP-treated animals. On the contrary (+)MK-801, but not (-)MK-801 significantly reduced the DDC + MPTP-induced fall in striatal DA and TH activity. A similar preventing effect on DA metabolites (DOPAC and HVA) and HVA/DA ratio was observed. The number of TH+ neurons in the substantia nigra (SN) of (+)MK-801-pretreated mice was not significantly different from that of control animals, indicating that this treatment specifically antagonized the extensive DDC-induced lesion of dopaminergic cell bodies in this brain area. (+)MK-801 treatment did not affect the DDC-induced changes of striatal MPP+ levels, suggesting that the observed antagonism of MK-801 against DDC is not due to MPP+ kinetic modifications. Pretreatment with the MAO-B inhibitor, L-deprenyl, or with the DA uptake blocker, GBR 12909, completely prevented the marked DA depletion elicited by DDC + MPTP within the striatum. Both treatments also protected from the fall in DA metabolites and TH activity as well. This indicates that DDC-induced potentiation is dependent upon MPP+ production and its uptake by the dopaminergic nerve terminals. All these findings suggest that NMDA receptors play a crucial role in the DDC-induced enhancement of MPTP toxicity.

Role of excitatory amino-acids in diethyldithiocarbamate-induced cell death in mesencephalic cultures.

The effects of diethyldithiocarbamate (DDC) and DDC plus glutamate on mesencephalic cell cultures were investigated. DDC 10 microM was toxic for cell cultures as assessed by observation under a phase-contrast microscope and the drop in [3H]dopamine uptake. Moreover, DDC 1 microM greatly potentiated cell death induced by glutamate 10 and 50 microM. (+)MK801, a selective non-competitive antagonist of NMDA receptors, completely prevented the toxicity of the two neurotoxins.

L-deprenyl fails to protect mesencephalic dopamine neurons and PC12 cells from the neurotoxic effect of 1-methyl-4-phenylpyridinium ion.

L-Deprenyl, a monoamine oxidase (MAO)-B inhibitor, appears to slow down the progression of Parkinson's disease. While inhibition of MAO-B activity can account for some of the effects of this substance, the basis by which L-deprenyl slows the progression of the disease remains controversial. In recent years, a new mechanism of action has emerged that may explain the ability of L-deprenyl to increase neuronal survival. L-deprenyl has been reported to modify gene expression and protein synthesis in astrocytes and PC12 cells. In this study, we tested the ability of L-deprenyl to protect mouse mesencephalic cells from the toxicity of the 1-methyl-4-phenyl pyridinium ion (MPP+). We exposed mouse mesencephalic cell cultures to L-deprenyl (10 microM) and, 24 h later, to MPP+ (2.5 microM). On the fifth day after L-deprenyl and MPP+ exposition, cells were washed free of drugs, and the following day they were tested for dopamine uptake, intracellular dopamine content and tyrosine hydroxylase immunoreactivity. The experiments were performed either in the presence or in the absence of glia. It was found that L-deprenyl pretreatment failed to achieve any protection against MPP+ toxicity. The fall in dopamine uptake and intracellular dopamine content, and the diminution of tyrosine hydroxylase immunoreactivity observed in cells pretreated with L-deprenyl and then given MPP+ were not significantly different from the values observed in cells treated with MPP+ alone. Additional experiments performed in PC12 cells, confirmed the failure of L-deprenyl to abolish the toxicity of MPP+. Our data seem to be at variance with previous reports demonstrating that the MAO-B inhibitor L-deprenyl protects dopaminergic neurons against MPP+ toxicity [12,20]; furthermore they do not support alternative mechanisms of action of L-deprenyl against MPP+ toxicity.

beta,beta'-Iminodipropionitrile-induced persistent dyskinetic syndrome in mice is transiently modified by MPTP.

Chronic administration of iminodipropionitrile (IDPN) is known to produce a persistent dyskinetic syndrome. Recent neurochemical reports seem to point out the dopaminergic system as having an important role in mediating IDPN syndrome. In order to identify a possible role for the nigrostriatal dopaminergic pathway in determining at least some aspects of the IDPN-induced dyskinetic syndrome, we used the neurotoxin, 1-methyl, 4-phenyl,1,2,3,6-tetrahydropyridine (MPTP), as a tool for investigating which aspects of the IDPN-related syndrome could be due to enhanced dopaminergic activity in the neostriatum. In mice made permanently dyskinetic with IDPN, MPTP administration produced dramatic and biphasic effects on all behavioral patterns characteristic of the dyskinetic syndrome. Six weeks after the syndrome occurred, IDPN failed to produce any change in striatal DA levels with respect to controls. By contrast, IDPN seems to reduce striatal levels of extraneuronal metabolites of DA. These data suggest that the activity of the nigrostriatal dopaminergic pathway does not play a leading role in the maintenance of IDPN-related syndrome. The transient modification of all behavioral parameters immediately after MPTP administration could be explained by acute effects of MPTP on other dopaminergic areas which are not permanently lesioned by this neurotoxin, or by the acute effects of MPTP on the release of other neurotransmitters.

Stereoselective inhibition of muscarinic receptor subtypes by the eight stereoisomers related to rociverine.

The chemical structure corresponding to 1-hydroxy[1,1'-bicyclohexyl]-2-carboxylic acid 2-(diethylamino)-1-methylethyl ester has the classical profile of ester-type antimuscarinic drugs. The presence of three chiral carbons leads to eight stereoisomers and the substitutions on the cyclohexyl ring generate cis-isomers (1, named rociverine) and trans-isomers (2). The aim of this study was to determine the binding pattern of the eight stereoisomers and two derived compounds, (1S,2S)-1-hydroxy[1,1'-bicyclohexyl]-2-carboxylic acid 2-(dimethylamino)-1-ethyl ester (3) (1S,2S)-1-hydroxy[1,1'-bicyclohexyl]-2-carboxylic acid (S)-2-(diethylamino)-1-methylethyl ester methyl iodide (4), at the five cloned muscarinic receptors stably expressed in chinese hamster ovary cells, in order to define how stereochemical modifications could affect the affinity. Our data showed that cis-stereoisomers exhibited higher variations in affinity than trans-stereoisomers. Among the cis-stereoisomers, those with the (1R,2R) configuration showed considerably higher affinities (up to 240-fold) than those with the (1S,2S) configuration. The (1S,2S) configuration was important for binding selectivity; this was confirmed also by the use of the two additional compounds.

Apomorphine has a potent antiproliferative effect on Chinese hamster ovary cells.

Apomorphine is a potent non selective agonist at the D1 and D2 dopamine receptors acting both pre- and post-synaptically. In this report we describe a novel function of apomorphine, independent from its dopaminergic activity. Apomorphine inhibits Chinese hamster ovary (CHO)-K1 cell proliferation in a dose-dependent manner. The EC50 of apomorphine-induced inhibition of CHO-K1 cell proliferation determined by cell counting was 3.24 +/- 0.07 microM. Remarkably, the dose-response curve obtained by measuring the incorporation of [3H]thymidine was practically identical to the previous one giving an EC50 of 3.52 +/- 0.04 microM. The dopaminergic antagonists SCH23390 and spiperone at a concentration of 10 microM (well beyond their Kd values for the dopamine D1- and D2-like receptors respectively) were not able to antagonize the effect of apomorphine on CHO-K1 cell proliferation. Apomorphine exerts its effect early during incubation; CHO-K1 cells exposed to apomorphine for a period as short as 1 h and then allowed to grow for three days were significantly reduced in number with respect to untreated control cells. After four hours of exposition to apomorphine (10 microM) the antiproliferative effect was similar to that seen when this compound was present in the bath for all three days. Concentrations of apomorphine higher than 10 microM induced cell death, and the colony was completely destroyed at 50 microM. Cytometric analyses showed a significant accumulation of CHO-K1 cells in the G2/M phase.

Dopamine agonists and analogues have an antiproliferative effect on CHO-K1 cells.

Epidemiological studies have shown a reduced incidence of cancer in Parkinson's disease. Since nearly all parkinsonian patients with clinical impairment are treated with L-beta-3,4-dihydroxyphenylalanine (L-DOPA) and dopamine (DA)ergic agonists, a possibility exists that these therapeutic agents can influence the risk of cancer. We studied the antiproliferative effect of these therapeutic agents (and substances structurally correlated) on Chinese hamster ovary (CHO)-K1 cell growth. Among the compounds tested, apomorphine proved to be the most potent inhibitor of CHO-K1 cell growth, with an EC(50) of 3.35 +/- 0.12 micro M. The apomorphine analogues, apocodeine and hydroxyethylnorapomorphine, were less active as inhibitors of CHO-K1 cell growth. The activity of DA, 6-hydroxydopamine (6-OHDA), phenylethylamine (PEA), L-DOPA and bromocriptine as antiproliferative was one order of magnitude lower than that of apomorphine while pergolide was ineffective. To test whether or not the oxidative potential of these compounds was important for their antiproliferative effect, several antioxidants were assayed. Among them glutathione (GSH) and dithiothreitol (DTT) were effective in reversing the anti-proliferative effect of apomorphine, DA, 6-OHDA and PEA, conversely they did not work with bromocriptine. GSH and DTT are sulphydryl-reducing agents; while their effect could explain the efficacy against apomorphine, DA and 6-OHDA, it is difficult to understand why they should have any effect on PEA as this substance does not react with sulphydryl groups. The oxidative potential as a mechanism of action was also questioned by the results obtained with dihydrorhodamine 123, a probe that changes its fluorescent emission wave when oxidized. None of the compounds, with the exception of 6-OHDA, had any effect on the fluorescent emission wave of the probe at the maximal concentrations used to inhibit CHO-K1 cell growth. At concentrations five times higher, apomorphine and DA generated reactive oxygen species but PEA and bromocriptine did not. These data demonstrate that the antiproliferative effect of these compounds is not due to their oxidative potential, but another mechanism must be postulated.

Quercetin inhibits the sulfation of r(-)-apomorphine in human brain.

The first aim of this investigation was to study the sulfation of R(-)-apomorphine in human brain. The second aim was to investigate the inhibition of R(-)-apomorphine sulfation by quercetin in human brain. R(-)-apomorphine is hereafter referred to as apomorphine. Apomorphine sulfation was measured in 5 brain specimens; 3 derived from the frontal cortex and 2 derived from the temporal cortex. The rate of apomorphine sulfation was 5.6 +/- 4.3 pmol/min/mg. The activities of SULT1A1 and SULT1A3, which were also measured in these samples, were 11 +/- 9.1 and 2.6 +/- 1.7 pmol/min/mg, respectively. The rate of apomorphine sulfation correlated with the activity of SULT1A1 (r = 0.989; p = 0.002) and SULT1A3 (r = 0.973; p = 0.005). Apomorphine sulfotransferase followed Michaelis-Menten kinetics, the Km (mean +/- SD) and Vmax values (mean +/- SD) of which, measured in 5 brain samples, were 32 +/- 7.3 microM and 8.9 +/- 7.9 pmol/min/mg, respectively. Quercetin was a potent inhibitor of apomorphine sulfation with an IC50 value, measured in 5 brain samples, of 16 +/- 2.3 nM. The inhibition mechanism of quercetin using apomorphine sulfation in 5 brain samples was mixed, non-competitive with a Ki and Kies (mean +/- SD) of 16 +/- 4.1 and 87 +/- 37 nM, respectively (p = 0.008). The intrinsic clearance value of apomorphine (mean +/- SD) was 247 +/- 170 ml/min/mg(-1) and was decreased to 100 +/- 85 ml/min/mg(-1) (p < 0.01) in the presence of 25 nM quercetin. In conclusion, apomorphine is sulfated in human brain. Sulfation might reduce the level of apomorphine in human brain and be a factor limiting the effect of this drug. Quercetin is a potent inhibitor of apomorphine sulfation and may inhibit the sulfation of apomorphine in human brain in vivo.

Effects of lithium and rubidium on the differentiation of mononuclear cells.

Rubidium ions added at 0.5 mEq to liquid cultures of human mononuclear cells stimulated the differentiation of monocyte and particularly of granulocyte cell lines.

Effects of thiobenzamide on the rat immune system.

Thiobenzamide (TB) is a thiono-containing compound endowed with liver-damaging properties and promoting ability on liver carcinogenesis. When administered in a single dose to normal as well as to adrenalectomized rats, this compound induced a striking thymus cortex involution without relevant effects on the morphological features of other lymphoid organs such as spleen and lymph nodes. The proximal TB metabolite TB-S-oxide (TBSO) shared these effects with the parent compound, whereas the terminal metabolite benzamide (BA) was ineffective. The effect of TB on thymus was found to be dose- and age-dependent. Furthermore, acute TB treatment 12h before priming with the T-dependent antigen sheep erythrocytes impaired the secondary antibody response. In addition, TB administration affected not only cell-mediated immunity (as evidenced by a decreased delayed hypersensitivity response) but also mitogen-induced proliferation of blood lymphocytes. On the contrary, the chemotactic response of polymorphonuclear leukocytes obtained from TB-treated rats was unchanged.

Automated extraction and classification of dynamic metrical features of morphological development in dissociated Purkinje neurons.

The morphological development of in vitro single cerebellar Purkinje cells obtained from wild type P1 CD1 mice was assessed through a dedicated non-invasive technique based on image processing algorithms and multivariate analysis. Image processing algorithms were implemented to extract metrical features characterizing cell structure and dendritic arborization from sequential optical micrographs. Quantitative morphological features were analyzed in order to identify relevant metrical characteristics common to Purkinje cells in wild type P1 CD1 mice. Cell arborization was found to be characterized by a high fractal dimension and the directionality and level of complexity were shown to be key features for cell morphology classification, as underlined using a three-way PCA analysis.